Lipdhenwocanetmiracestcan® p 4 gu? i Schnanateare east died sate oe de aediii KS Baba Ne a eee : eee =" ¥ he a ee, Te a A ae ey =—~ BR te a a tty asa : Sais } ath ND Seo er fof $ ‘| au % ae a CS ey] ‘ ; Mien) ni aS a oP eae iy Noone Ny wh aM x" i wy Ts, jo oa Ww a so 4 e 4b aes eee ae ee 8 a oe aK : re vip ae Sel ed a a ,, ay ca . anh , ‘a ae ae ey hh Seite ie allt Wer De ee ae A ; fe Pai Ate anes Rat eerie Nias x 3 ANOUSTRIAL- RESEARCH 10 eee - LABOHATORIES 2201 New York Aye. N. W. - WASHINGTON, D. G. . ie , Jeo | Sud) ta hal) 4 Ye ol ok % © Raymond Pettibon RESEARCH LIBRARY THE GETTY RESEARCH INSTITUTE JOHN MOORE ANDREAS COLOR CHEMISTRY LIBRARY FOUNDATION ; Le TAN Tog. nat a _ a. i *) v J“ ; Sh 2 a + — ‘ ; . ‘ * ‘ ‘ a | } | , we ' : td ‘ A . 2 L e bh a a A! ’ = oa . a" > ¥ : hay * 0) [ J a = LP ® . 4 ‘ 7 ® 7 - — za: a = 7 SK } iS MASS + —_ @ f SEG Nahe E> e rc Tue Use or Guue 1n AnciENT Ecypt (aBour 1400B.C.). This illustration is taken from “The Life of Rekhmara” by Percy HE. Newberry (London, 1900), plate XVII, and is the sculpture referred to by Wilkinson (see p. 14). It occurs on the walls of the tomb of Rekhmara at Thebes. Newberry states (p. 36): “In the third row are shown the carpenters, wood carvers, cabinet makers, and painters. The carpenters saw rough wood up into planks and prepare it for the cabinet makers, who are repre- sented making chairs, boxes, a slender wooden column with lotus-bud capi- tal, and an elaborate shrine inlaid with ivory and precious woods. . . . To the right of this shrine, beyond the men making a chair or couch, are two ae working with glue which is being heated in a pot over a charcoal rere The portion of the engraving showing the glue pot heating, is given, partially in the original brilliant colors, by Rosellini, “Monumenti dell’ Egitto e della Nubia,” Vol. 2, Monumenti civili, plate XLVI. GLUE AND GELATIN BY JHROME ALEXANDER AUTHOR OF ‘‘COLLOID CHEMISTRY, AN INTRODUCTION ’’ - American Chemical Society Monograph Series BOOK DEPARTMENT The CHEMICAL CATALOG COMPANY, Ine. 19 EAST 24vrH STREET, NEW YORK, U.S. A. 1923 ; a t * - te oe ’ = AS i ial a ; a? Vege +?) 3 ¥ i, . ‘ x te xe 4 ty » € , ‘ ‘ ae ‘ wh 7 aan , % i 5 . pees . E . ‘ 2 yaa} Ps CopyriGHT, 1923, By The CHEMICAL CATALOG COM All Ragkhe Reserved Press of (7 7) ae J. J. Little & Ives Company New York, U. S. Ai v i THE GETTY RESEARCH INSTITUTE LIBRARY GENERAL INTRODUCTION American Chemical Society Series of Scientific and Technologic Monographs By arrangement with the Interallied Conference of Pure and Applied Chemistry, which met in London and Brussels in July, 1919, the American Chemical Society was to undertake the pro- duction and publication of Scientific and Technologic Mono- ‘graphs on chemical subjects. At the same time it was agreed that the National Research Council, in cooperation with the American Chemical Society and the American Physical Society, should undertake the production and publication of Critical Tables of Chemical and Physical Constants. The American Chemical Society and the National Research Council mutually agreed to care for these two fields of chemical development. The American Chemical Society named as Trustees, to make the necessary arrangements for the publication of the mono- graphs, Charles L. Parsons, Secretary of the American Chemical Society, Washington, D. C.; John E. Teeple, Treasurer of the American Chemical Society, New York City; and Professor Gellert Alleman of Swarthmore College. The Trustees have arranged for the publication of the American Chemical Society series of (a) Scientific and (b) Technologic Monographs by the Chemical Catalog Company of New York City. The Council, acting through the Committee on National Policy of the American Chemical Society, appointed the editors, named at the close of this introduction, to have charge of securing authors, and of considering critically the manuscripts prepared. The editors of each series will endeavor to select topics which are of current interest and authors who are recognized as author- ities in their respective fields. The list of monographs thus far secured appears in ‘the publisher’s own announcement elsewhere in this volume. 3 4 GENERAL INTRODUCTION The development of knowledge in all branches of science, and especially in chemistry, has been so rapid during the last fifty years and the fields covered by this development have been so varied that it is difficult for any individual to keep in touch with the progress in branches of science outside his own specialty. In spite of the facilities for the examination of the literature given by Chemical Abstracts and such compendia as Beilstein’s Handbuch der Organischen Chemie, Richter’s Lexikon, Ostwald’s Lehrbuch der Allgemeinen Chemie, Abegg’s and Gmelin-Kraut’s Handbuch der Anorganischen Chemie and the English and French Dictionaries of Chemistry, it often takes a great deal of time to coordinate the knowledge available upon a single topic. Consequently when men who have spent years in the study of important subjects are willing to coédrdinate their knowledge and present it in concise, readable form, they perform a service of the highest value to their fellow chemists. It was with a clear recognition of the usefulness of reviews of this character that a Committee of the American Chemical Society recommended the publication of the two series of mono- graphs under the auspices of the Society. Two rather distinct purposes are to be served by these mono- graphs. The first purpose, whose fulfilment will probably render to chemists in general the most important service, is to present the knowledge available upon the chosen topic in a readable form, intelligible to those whose activities may be along a wholly different line. Many chemists fail to realize how closely their investigations may be connected with other work which on the surface appears far afield from their own. These monographs will enable such men to form closer contact with the work of chemists in other lines of research. The second purpose is to promote research in the branch of science covered by the mono- graph, by furnishing a well digested survey of the progress already made in that field and by pointing out directions in which investigation needs to be extended. To facilitate the attainment of this purpose, it is intended to include extended references to the literature, which will enable anyone interested to follow up the subject in more detail. Ii the literature is so voluminous that a complete bibliography is impracticable, a critical selection will be made of those papers which are most important. GENERAL INTRODUCTION , 5 The publication of these books marks a distinct departure in the policy of the American Chemical Society inasmuch as it is a serious attempt to found an American chemical literature with-. out primary regard to commercial considerations. The success of the venture will depend in large part upon the measure of cooperation which can be secured in the preparation of books dealing adequately with topics of general interest; it is earnestly hoped, therefore, that every member of the various organizations in. the chemical and allied industries will recognize the impor- tance of the enterprise and take sufficient interest to justify it. AMERICAN CHEMICAL SOCIETY BOARD OF EDITORS Scientific Series: — Technologic Series: — WiuuiAM A. Noyss, Editor, Harrison E. Howe, Editor, GILBERT N. Lewis, C. G. Drricx, LAFAYETTE B. MENDEL, WILLIAM HOosKINS, ArTHuR A. NOoyYEs, F. A. Lipsury, JULIUS STIEGLITZ. ArtTHuR D. LitTTLe, C. L. REEsE, C. P. TOWNSEND. American Chemical Society MONOGRAPH SERIES Other monographs in the series of which this book is a part now ready or in process of being printed or written. Organic Compounds of Mercury. By Frank C. Whitmore. 397 pages. Price $4.50. Industrial Hydrogen. By Hugh S.. Taylor. 210 pages. Price $3.50. The Chemistry of Enzyme Actions. By K. George Falk. 140 pages. Price $2.50. The Vitamins. By H. C. Sherman and S. L. Smith. 273 pages. Price $4.00. The Chemical Effects of Alpha Particles and Electrons. By Samuel C. Lind. 180 pages. Price $3.00. Zirconium and Its Compounds. By F. P. Venable. Price $2.50. The Properties of Electrically Conducting Systems. By Charles A. Kraus. Price $4.50. The Analysis of Rubber. By John B. Tuttle. Price $2.50. The Origin of Spectra. By Paul D. Foote and F. L. Mohler. Price $4.50. Carotinoids and Related Pigments. By Leroy S. Palmer. Price $4.50. Thyroxin. By E. C. Kendall. The Properties of Silica and the Stlicates. By Robert B. Sosman. Coal Carbonization. By Horace C. Porter. The Corrosion of Alloys. By C. G. Fink. Piezo-Chemistry. By L. H. Adams. Cyanamide. By Joseph M. Braham. Liquid Ammonia as a Solvent. By E. C. Franklin. Wood Distillation. By L. F. Hawley. Solubility. By Joel H. Hildebrand. Organic Arsenical Compounds. By George W. Raiziss. Jos- eph L. Gavron. Valence, and the Structure of Atoms and Molecules. By Gil- bert N. Lewis. Shale Oil. By Ralph H. McKee. Aluminothermic Reduction of Metals. By B. D. Saklatwalla. The Chemistry of Leather Manufacture. By John A. Wilson. Absorptive Carbon. By N. K. Chaney. Refining Petroleum. By George A. Burrell, e¢ al. Extraction of Gasoline from Natural Gas. By George A. Burrell. The Animal as a Converter. By H. P. Armsby and C. Robert Moulton. Chemistry of Cellulose. By Harold Hibbert. The Properties of Metallic Substances. By Charles A. Kraus. The Structure of Crystals. By Ralph W. G. Wyckoff. The CHEMICAL CATALOG COMPANY, Inc. 19 EAST 24TH STREET, NEW YORK, U.S. A. PREFACE The manufacture of glue and gelatin is important not only because of the magnitude of the industry itself, but also because these products are essential to the production of many others, thus making it a key-industry. Furthermore glue and gelatin are typical jelly-forming colloids and they have been used in numberless experiments on and investigations into the nature and behavior of colloids. As a consequence there is an embar- rassing wealth of publications from which to draw material for a monograph. For the same reason, a discussion of the behavior of glue and gelatin involves the consideration of many moot points in colloid chemistry, and gives the subject an interest rather broader than the title would indicate. The theoretical aspect has been treated more at length than has been usual with books on glue, for a more complete under- standing of the nature of a product must in the end be useful to its makers and users. Where opinions vary, the different views are given, often in the very words of their principal advocates. Nor have I withheld my own views in such cases. In the technical sections elaborate descriptions of well-known apparatus have been avoided, because new forms and modifica- tions are continually appearing, and any one can have the latest particulars from their manufacturers. Such descriptions use space to no good purpose, and may give a reader at some later date the erroneous idea that the machines described are then the best of their kind. However the principles involved in manufacture, testing, and use, have been particularly stressed. On the other hand many excellent papers have been omitted or referred to but briefly, some because the points involved were treated adequately by others, some because they are beyond the scope of this book, and some perhaps because of inadvertence or inaccessibility. No attempt has been made to pass on ques- tions of priority, and the fact that a certain author is quoted as expressing certain views, does not necessarily imply that he was the first to express such ideas. For the benefit of those 7 8 PREFACE who wish to look further into such papers, reference may be made to the bibliographies and indexes mentioned below. I am indebted to many authors for books and reprints, and to a still larger number for scattered items of information. In all cases I have striven to make due acknowledgment in the text for material used, and to refer wherever possible to the original sources of information. JEROME ALEXANDER. Bibliography. An extensive bibliography on Glue, comprising about 150 titles, was published by Rudolf Ditmar in the Kolloid Zeitschrift, 1906, Vol. 1, p. 80. Another bibliography of about the same extent was published by Robert H. Bogue in Chemical and Metallurgical Engineering, 1920, Vol. 23, No. 5. The First Re- port of the British Adhesives Research Committee contains a descriptive bibliography of gelatin (75 pp.) by T. Slater Price, which gives a resumé of much important work. The indexes of the principal chemical, physical, biological, and technical journals may be consulted, and it must be remem- bered that many experiments with gelatin are apt to be found in papers or texts indexed under such headings as “Proteins,” ‘“Col- loids,” ‘Jellies,’ “Diffusion,” etc. The collective indexes of Chemical Abstracts, of the Journal of the Society of Chemical Industry, and the indexes of the Kolloid Zeitschrift will be found particularly useful. CONTENTS PAGE Cuapter 1. Introduction . . faa heli 19 Definitions. —Philology Trees ewetie and gela- tin industry in the United States.—Statistics of glue and gelatin industry in various countries. CHAPTER 2. eeeiicn of Gelatin among the Proteins, and the Nature of the Force ‘Binding fogstner its Con- stituents . . eke 229 Classification of Bens _ ary etree — Conju- gated proteins.—Derived proteins—Molecular struc- ture. CHaprter 3. Chemistry, Physical Chemistry and Colloidal Chemistry of Gelatin and Glue . . . . . 30-50 Chemical structure.—Loeb’s Theory.—Ash-free gelatin. —Fischer’s views.—Molecular weight.—Crystallization of gelatin. CuHaptTer 4. Chemistry, Physical Chemistry and Colloidal Chemistry of Gelatin and Glue... . ... 51-67 Is gelatin a distinct chemical entity ?—The significance of hydrogen ion concentration.—Titration curve of gel- atin. Cuapter 5. The Structure of Gelatin Solutions and Gela- , eee eliieds 6 60-5 Tt hy sie Pate Pag a ate 68-86 The Ultramicroscopic Ey ates CHapter 6. The Influence of Various Factors on the Swelling of Gelatin . .. pS eo ees 87 Lyotrope or Hofmeister series. MET oanane Theory. — Thermal expansion of gelatin. ee 7. The Viscosity of Glue and Gelatin Solutions 98-112 ° Influence of added substances on viscosity. CHapter 8. Collagen or Ossein . . .. . . . 1138-120 -Chondrigen, chondrin and mucin. — 9 10 CONTENTS PAGE Cuaptrer 9. The Effect of Tanning Substances on Glue ANG UAREIA GING ta pas ec. Se ete Tannin.—Chrome.—Organiec substances——Bichromates. —Silicie acid—Alum.—Iron.—Other salts—The halo- gens.—Formaldehyde. CuaptTer 10. Chemical Examination of Glue and Gela- 151s CCU MR EMEC rar TNT ee Hydrogen ion concentration.—Total acidity.—Ash.— Determinations involving nitrogen.—Diffusible nitro- gen test.—Reactions of gelatin.——Detection of glue and gelatin in various chemical products——Gold number. —A,. O. A. C. methods. CuaptTer 11. Technology of Glue and Gelatin Tek. 151-172 Glue stock.—Treatment of glue stock—Apparatus and methods.—Bone stock.—Hide and sinew stock.—Wash- ers—Tanned stock.—Boiling apparatus and methods. —Open kettle or tank.—Pressure tank.—Clarification, bleaching and evaporation of dilute liquors.—Antisep- tics—Chilling—Cutting, spreading and drying.—Blow down processes.—Percentage yields of glue stock. Cuapter 12. The Testing and Grading of Glue and Gela- tin 2 ee 4 rr Jelly strength—Viscosity or Running Test.—Water absorption test——Hygrometric test.—Melting point.— Setting point.—Strength test——Laboratory test series. —Standard glues——Smith’s Polariscopic method.— Moisture.—Ash-Recording tests. CuapTer 13. Uses of Glue and Gelatin. . . . . 200-215 Adhesive.—Sizing and stiffening —Compositions.—Col- loidal protector or colloidizer—Miscellaneous.—W ood joints.— Veneers.—Paper boxes.—Leather belting.—Siz- ing.—Printers’ rollers, etc.—Photography.—Inhibiting crystallization. — Bacteriology. — Formogelatin. — Gelatin as a food.—Food vs. technical gelatin, Cuapter 14. Fish Glue and Fish Isinglass . . . 216-223 Origin —Properties of fish glue—vVarieties of isinglass. —Properties of isinglass.—Uses of isinglass. AppENnpDIx—Test Methods of the National Association of Glue and Gelatin Manufacturers >. > Spies AuTHOR INDEX . . ) .°.) woe Supyect INDEX .. ..° : 5%) 204 09 See GLUE AND GELATIN Chapter 1. Tntroduction. Definitions. Glue is an organic colloidal substance of varying appearance, chemical constitution and physical properties, obtained upon drying the solutions resulting from boiling with water properly prepared animal matter such as skin and bone. The jellies which form on chilling soups, stews, boiled chicken and the like, represent very impure glue solutions. Glue appears in commerce in a wide variety of forms and colors, some of which are commonly, but erroneously, believed to be criteria of quality. The colors range through all shades of white, brown and yellow, and it may be transparent, translu- cent, or opaque. Gelatin colored red with aniline or vegetable coloring matter is used as a top dressing for cold meats, and specially colored glue compositions are used for making paper pads or blocks, ete. In European countries glue is usually marketed in the form of oblong cakes about 3x6 inches (Cologne shape) or in sheets about 10 inches square (French glue). “Scotch” glue comes in very thick cakes, about 6x10 inches, with a string through one end. In America the bulk of the glue is used in ground form, though considerable is sold in thin, broken flakes, and ' some is made in the form of “noodles” or “ribbons,”’ which spe- cial forms have no advantage other than a higher cost. Pow- dered glue is also used principally in mixing with whiting to make calsomine. For Eastern countries there is made a form known as “bazaar glue,” which consists of a poor quality of glue in square sticks about 8 inches long and 1 inch in section. Gelatin is made from bones and skin or hide fragments, 11 12 GLUE AND GELATIN selected, cleaned and treated with especial care so that the result- ing product is cleaner, purer and generally clearer and lighter in color than glue. Glue is in fact impure gelatin, and any glue possessing suitable strength and appearance may be termed gelatin, although of course all gelatins are not suitable for food purposes. It is surprising that such authoritative reference books as the Standard Dictionary and Murray’s Oxford Dictionary perpetu- ate the popular error that hoofs and horns yield glue. Hoofs and horns consist of keratin and are always removed by the glue maker, although of course the feet of animals and the interior bony support of the horn (horn pith) yield glue or gelatin (e.g. calves’ foot jelly). Philology. The word glue has been traced back to the unused Latin verb qluere, meaning to draw together, and the form glus, glue, was used by Ausonius. It is allied to the Latin gluten, glue, and glutus, tenacious (cf. the English gluten, glutinous), to the Greek gloios, mud, gum, and to the old French glu, birdlime. Birdlime is a sticky substance which exudes from the holly tree, and is used for snaring birds. In this word lime has the signifi- cance of the German leim, glue, being in no way related to lime, calcium oxide. The word gelatin comes from the Latin verb gelare, to congeal (cf. the Latin gelu, frost, and the English chill, gel, gelid, jelly). Thus Virgil in describing the awe inspired in the Trojans by the Cumzan Sybil, says: ... Gelidus Teucris per dura concurrit ossa tremor . The word gelatin came into the English through the French gélatine, from the Italian gelatina. Gelatin is often termed glutin by chemists, a practise that should be abandoned, owing to the similarity of this word to gluten, the composite protein of rye, wheat, etc. Gelatin is often erroneously termed “isinglass,” the confusion being due to the fact that gelatin under the name “patent isinglass’” came into use as a substitute for the true isinglass (see p. 219) and resembles it in appearance and working properties. Popular error goes so far as to apply the term INTRODUCTION 13 isinglass to the mineral mica, which also appears in thin, trans- parent, flexible sheets. Since the essential meaning of glue is that which draws or sticks together, while gelatin means essentially that which gela- tinizes, it is but natural that, in popular parlance, the use of these words have, by similitude, been extended to many sub- stances which are not glue or gelatin at all. Thus solutions of gums, dextrins, converted starches, etc., are often called glues, the modifying adjective “vegetable” being generally used. Sili- cate of soda is sometimes termed ‘‘mineral glue,” solutions of rubber, pitch and the like are called “marine glue,” and those of casein are called “casein glue.’’ Several varieties of sea weed, including Gelideum corneum?‘ or agar agar, form, when cleaned and dried, a cord-like product having enormous gelatinizing power, which appears in commerce under various names, such as vegetable or Japanese gelatin, vegetable or Japanese isinglass, Chinese moss, gelose, etc. Our well-known fruit jellies, when pure, contain no gelatin, their gelatinization being due to a jelly-forming carbohydrate known as pectin. The correct uses of the terms above discussed are not always academic. They are often of importance in determining the operation of tariffs and other legislation. Thus the regulations of the Official Southern and Western Freight Classification (Rule 14, section 2): “Fiberboard and Pulpboard used in making Fiberboard or Pulpboard boxes, without frames, must be three- ply or more, all plies firmly glued together .. .” The ruling of the chemist in charge was that the word “glue” meant glued with animal glue. This narrow construction of the rule still stands, although boxes meeting the tensile and other require- ments are passed, even if they are not glued with animal glue. Historical. Many of our most important discoveries have come as the result of some keen mind noting an incidental or accidental result. Though we may doubt the correctness of Charles Lamb’s story as to the origin of roast pig, discovery of British gum and dextrin is said to have followed the observation that some starch, which had been roasted or torrified during a fire in a 1Gelidium gracilaria yields a similar product. 14 GLUE AND GELATIN Manchester warehouse, yielded a sticky, gummy solution when wet with water. In all probability the discovery of glue grew out of the fact that stews, especially those containing bones or skins, yield a sticky solution and gelatinize when cold. It is to Egypt that we must look for the oldest record of the use of glue, the dis- covery of which evidently antedates the Exodus, as may be seen from the following quotation taken from Wilkinson.? “Among the many occupations of the carpenter, that of veneer- ing is noticed in the sculptures of Thebes, as early as the time of the third Thothmes, whom I suppose to be the Pharaoh of the Exodus; and the application of a piece of rare wood of a red colour, to a yellow plank of sycamore or other ordinary kind, is clearly pointed out. And in order to show that the yellow wood is of inferior quality, the workman is represented to have fixed his adze carelessly in a block of the same colour, while engaged in applying them together. Near him are some of his tools, with a box or small chest, made of inlaid and veneered wood, of various hues; and in the same part of the shop are two other men, one of whom is employed in grinding something with a stone on a slab, and the other in spreading glue with a brush. _ “Tt might, perhaps, be conjectured that varnish was intended to be here represented; but the appearance of the pot on the fire, the piece of glue with its concave fracture, and the workman before mentioned applying the two pieces of wood together, satis- factorily decide the question, and attest the invention of glue* 3,300 years ago. This is not, howéver, the only proof of its use at an early period, and several wooden boxes have been found in which glue was employed to fasten the joints.” The manufacture of violins and similar musical instruments during the Middle Ages and Renaissance, especially in Italy, indicates that glue was known and used at that period, and there are indications that early painters used glue size in preparing their canvases. *Sir John Gardner Wilkinson, ‘““Manners and Customs of the Ancient Hgyp-. tians,” John Murray, London, 1879, Vol. 2, pp. 198-199. 3 Rosellini seems to think that the application of color is here represented ; but the presence of the pot, containing the brush, upon the fire, will scarcely admit of this, though the figure grinding on the slab might appear to strengthen his conjecture. He has placed this subject with the painters of Beni-Hassan, but it is at Thebes. Pliny ascribes the invention of glue to Dedalus, as well as of the saw, the axe, the plumb-line, and the auger. (Plin., vii, 56.) INTRODUCTION 15 Murray (New Oxford Dictionary) gives a number of refer- ences to the early use of the word glue by English writers. Thus in the “Squire’s Tale” (line 174), Chaucer (about 1386), in describing the wonderful brass horse on which a royal messenger appeared, says: “The horse of brass that may not be remewed, It stant as it were to the ground yglewed.” Further in Lanfranc’s “Chirurgeon” (about 1400) it is stated (p. 185): “As it were two bordis weren ioyned togidere with cole or with glu.” Glue and gelatin, like most other manufactures of early days, were produced by individual artizans for their own use, and even to-day some paper and textile mills boil their own glue size from rawhide cuttings. From these somewhat primitive methods, the real glue and gelatin industry emerged about the beginning of the nineteenth century. In France the industry started in the vicinity of Lyons, and for many years these factories were the most important of their kind in Europe. During the Napo- leonic era extravagant claims were made as to the food value of gelatin, and probably this was one reason why the industry was fostered. ) Germany apparently appreciated the importance of the manu- facture of glue and gelatin as a key industry, for a German company organized in 1895 with three plants, expanded until in 1912 it controlled the output of seventeen plants, and had also factories in Austria, Russia, Belgium, Switzerland and France. The Glue Industry in the United States. According to Rufus W. Powell?‘ it is very difficult to obtain exact information about the glue manufacturing industry of the United States prior to 1860; but those long in the business re- ported that outside of regular glue manufacturers, a great many tanners boiled up their own stock in open kettles. The prin- cipal factories seem to have been in the vicinity of Boston, New York, Philadelphia and later on Cincinnati. One of the pioneer factories was at Marblehead, Mass., and probably secured its stock from the tanneries at Salem and Lynn. Peter Cooper’s 4“Glue Statistics,’ Brooklyn, 1893, 16 GLUE AND GELATIN factory was on Newtown Creek, Long Island, now in the Bor- ough of Queens, New York City. Powell gives the following table based upon reports given the Glue Manufacturer’s Association, 1887-1888, which accounts for the commencement of the 92 factories then reporting. 1830 1840 1850 1860 to 1870 to 1880 to Location of Before to to to 1870 1880 1887 Factories 1830 1840 1850 1860 Hide Bone Hide Bone Hide Bone New England ... 26 2 1 1 3 4 6 3 2 3 1 Middle States ... 35 2 1 1 1 8s — 7 3° -10 2 Western States ..24 — — — 2 5 1 6 2 6 2 Pacific Coast ... 7 —2 — — — 1 — 3 — 3 — GOL ance eee 4 2 2 6 18 7. 239 rb ery. 5 The Census of 1880 showed that there were then 82 plants pro- ducing glue as a principal or by-product. They employed 1,801 hands and a capital of $3,916,750. Powell estimates that the total production of glue in the United States for the fiscal year 1886-1887 was 38,032,000 lbs., of which 27,743,000 lbs. was from hide, fur and neat’s-foot stock, and 10,289,000 lbs. was from bone, bone liquor, and pigs’ feet. Some idea of the range of glue prices during this early period may be gleaned from the following table abridged from Powell (prices given in cents per pound). 1844. 1888 1848 1860 1863 1867 1876 = 1887 1892 A WExGra) 2 os sana 40 39 37 60 38 25 23 Val Os cay Gah # 34 30 32 53 30 22 19 NGEDISe. acta ee 30 26 27 47 30 19 17 orryainy eg ac ayer 25 24 24 41 25 17 15 Aa Ripa eee 21 22 21 36 21 16 144% Les ic ay biaatn are 19 20 19 32 is 15 1 La aa ee ae aes 18 19 18 29 17 14 13 {he an Sn IPR irc 17 18 17 27 15 12 11 Lge ce sae ne ce 16 17 16 25 14 11 10 Lig NOS i aneeets — = — — — — 9 Orbaky hehe dare ase: 14 16 15 23 13 10 8 The total value of glue and gelatin produced in the United States for 1914 was $19,725,703, of which 40,844,650 lbs. valued at $3,088,764 was produced by the packing houses. The 1919 figures given below do not include 36,603,000 lbs. of glue valued at $4,489,774, produced by the meat-packing industry, but may include some gelatin. No separate figures are obtainable for gelatin, and that produced by the packers is included under a- INTRODUCTION heading “All other products.” 17 Various other industries inci- dentally produced glue to the value of $1,039,794. The United States Census gives the following tabulated in- formation regarding glue, not elsewhere specified: is Ra's = S 3 rh ec 8 Peso cee ois She S > SS as §3 > a) eS Year and State =SZES as United States TOLGrite Saw e vaca ee 62 4,264 16,979 lL 3 Sa oe eee 57 3,129 13,304 S01! Aa eee 65 3,265 15,596 RR tay ses 58 2,864 14,280 Deena, viese ss 61 1 618 6,806 Ee ee 62 | 697 4,912 (seer Bo eLs0r TSO oe ivatsiene be bees» 70 ~=6.860-~—«i1,051 PRbU ee is iss 62 875 — SL hes Feed ne eda 7 39) —— States, 1914 ADRES cals cs 9 968 3,316 IOUT. Tine « <.'e 3 ral 355 Massachusetts .. 11 563 1,481 New York..... 9 381 2,082 Pennsylvania ... 8 6519 1,628 All other States. 17 627 4,442 3 e ees on Sania 5 eS Sa Sar oe Wien we te Ra SS Serene ie SG wSs BEF 23 Ruston Gane es Sty SS iss So ee Sat ht eS eS SS 8 SS) SS Pas sks ses 8 eS ee Ss So ie eg el ales och aot at et SS 27,237 4,777 19,280 32,134 12,854 17,162 1,854 9,868 13,733 4,365 14,289 1,571 7,525 13,718 6,193 10,673 1529 6186 10035 3,849 6.144 685 3767 5,387 1,622 PB50 D610, 2,0Lk 4.270. 1759 3917 600 2786 4324 1,538 1,955 310 883 1,710 827 1,053 306 Doli ekg k SO 649 520 99 372 652 280 5652 614 2385 3,751 1,346 356 30 see 280 123 2,956 294 1,789 2,589 800 2,459 249 1,942 2,483 541 2320 290 1418 2029 611 S019" 2337. 1677 ~~ 2621 944 The United States Department of Commerce has kindly sup- plied the following figures regarding the imports and exports of glue, gelatin, and glue stock, which indicate that large amounts of foreign glues and gelatins are consumed here. 1921 2 Imports Pounds Dollars Pounds Dollars Gelatin (unmanufactured) 2,396,645 1,231,035 2,527,198 997 896 Glue and Glue Size...... 3,561,831 762,557 4,174,785 574,311 Hide cuttings, raw, and other Glue Stock...... 36,104,659 2.272847 25322414 1,149,883 Exports (domestic) SS! 5 eS ee ea 5,991,872 1,148,666 2,101,328 348,643 These figures indicate the well-known fact that different vari- eties of glue are made in different factories, and move according to their fitness for certain uses. 18 GLUE AND GELATIN Great Britain. There are 57 glue manufacturers and 21 gelatin manufacturers in the United Kingdom, but figures regarding production are not available. For 5 months ending May 31, 1921, the following figures were kindly furnished by Mr. L. E. Bernays, British Consul at New York. Cwts. Value,& Imports—Glue, Gelatin, and Size......... 33,666 226,912 - Exports—Gelatin «, 0s (62 a whe cee ee 1,676 33,673 Glue: and. ‘Size. ice fan ee 14,808 61,678 France. In France there are about 60 factories, the main centers being Paris, Lyons, Marseilles, Dijon, LaPallice and Nantes. Ap- proximately 3,000 workers are employed. Before the war France produced about 11,000 metric tons of pressured bone glue and gelatin, 4,000 tons from acid treated bone, and 3,000 tons from by-products and waste. About 100,000 tons of bones were em- ployed annually. The following figures were obtained through the United States Department of Commerce. 4 1918 Imports Exports Metric Value Metric Value tons (francs) tons (francs) Fish? glue fievaaeGht, wate ae Tia 2,020,200 118.1 3,070,600 Glue from bones and other anitis |Swasie > sien cee hee, 1,883 2,259,600 7,367 8,840,400 Gelatm in powder sheets, etc. 237.6 653,400 461 1,267,750 In 1918, 20 tons of fish glue were imported from the United States, and 9 tons were exported to that country. Exports of bone glue to the United States amounted to 414 metric tons, while exports of gelatin to the United States were approximately 54 metric tons. 1920 Imports Exports Metric Value Metric Value | tons (francs) tons. (francs) Fish. @lie vat eo eee 90.1 858,000 422.9 26,960,000 Glue from bone and _ other BM al Washe-tan, tome paler 1,266.8 5,882,000 4,394.6 21,755,000 Gelatin in powder sheets, etc. 83.6 - 1,484,000 765.8 4,545,000 INTRODUCTION 19 Germany. As before remarked, the glue and gelatin industry of Germany is a most important one, and has recently interested American capital. Besides supplying the large home market, an extensive export business is done. Figures are not at present obtainable. Belgium, Switzerland, Holland, and other countries also pro- duce glue and gelatin, and notable quantities are produced in Japan, Argentina, Canada and Australia. Chapter 2. The Position of Gelatin among the Proteins, and the Nature of the Forces Binding Together Its Constituents. Glue and gelatin belong to that large and important group of nitrogen-containing colloidal organic substances known as the proteins, which are found in nature as essential components of plants and animals and as products of their metabolism. More specifically they belong to the sub-group of proteins known as albuminoids by American and Continental chemists, -and as scleroproteins by the Chemical and Physiological Societies of England, because the group includes the substances which are the chief organic constituents of the animal skeleton and of the skin and its appendages, i.e. elastin (from tendon), collagen (from bone and hide), and keratin (from horn and hoof). This new use of the term albuminoid (literally albumin-like) must be distinguished from its now obsolete meaning, for in the past it was used as synonymous with “proteid,” and therefore at that time included albumin and its congeners as well as gelatin and allied substances. The term albuminoid thus replaces “pro- teoids,” which was at one time applied to “proteids” (now pro- teins) of the gelatin group. It is to be regretted that all scientists have not yet accepted the new meaning for the term albuminoid. Thus according to W. O. Atwater,t the American Association of Agricultural Colleges and Experiment Stations subdivide protein compounds into albuminoids, gelatinoids, and extractives. The first group (the albuminoids) includes white of egg, lean meat, casein, and wheat gluten; whereas the second group (the gelatinoids) in- cludes collagen and ossein from which gelatin is made. This confusion in terms is to be deprecated, and perhaps the best way to do would be to drop the term albuminoid entirely, sub- 1Farmer’s Bulletin 142, U. S. Dept. of Agriculture, reprint January, 1921, p. 4. 20 POSITION OF GELATIN AMONG THE PROTEINS — 21 stituting in its place the more descriptive term scleroprotein (proteins of the skin and skeleton). The position of the albuminoids or scleroproteins among the proteins may be seen from the following tabular classifications, which include also the products of hydrolysis. Classification of Proteins. The American Classification, adopted by the American Physiological Society and the American Society of Biological Chemists, is: I. StmpPLeE PROTEINS: Albumins—i.e. egg albumen; serum-albumin. Soluble in distilled water and in salt solutions; their acid and basic functions are almost equal, and they are salted out by saturation of their solutions with ammonium sulphate. Globulins—i.e. egg-globulin separated from egg white by dilution with distilled water; edestin from the seed of hemp (Cannabis Sativa). Insoluble in distilled water, but soluble in dilute solutions of strong acids or bases, or of inorganic salts. They are salted out by half satu- ration of their solutions with ammonium sulphate, or by complete saturation with magnesium sulphate. They are rather more acid than basic. Glutelins—i.e, glutenin from wheat; oryzenin from rice. In- soluble in distilled water or in dilute salt solutions, but soluble in dilute solutions of strong acids or bases. Prolamins—i.e. gliadin from wheat and rye; hordein from barley; zen from corn. Soluble in 70 to 90 per cent. alcohol, and in dilute solutions of strong acids or bases, but practically insoluble in distilled water. On hydrol- ysis they yield a large percentage of proline. | Protamines—i.e. salmine from salmon spermatazoa. The simplest natural proteins, usually found in combination. Predominantly basic substances soluble in water, and forming with acids compounds precipitated by alcohol. On hydrolysis they yield considerable diamino acids. Histones—i.e. the histone of hemoglobin which is there com- bined with the colored acid radicle hematin. Soluble in 22 GLUE AND GELATIN dilute solutions of acids or of strong bases, but precipi- tated from acid solutions by ammonia. Less markedly basic than the protamines. Albuminoids (Scleroproteins)—This large heterogeneous group is tentatively sub-divided as follows: (A)—CoLLAGENS OR JELLY-FORMING ALBUMINOIDS: (1) Collagen and gelatin; Dissolve more or less readily in from skins, bones, white fibrous connective tis- sue. drin; from permanent cartilages. boiling water, yielding solu- tions which gelatinize on cool- ing. Contain little or no sul- phur. Chondrigen and chondrin are really glycoproteins, but are mentioned here because they occur in glue and gelatin and in the materials from which (2) Chondrigen and sa they are made. (3) Isinglass; fishes. (4) Sericin (silk - gum) ; from silk. (B)—Fiproips: (1) Elastin; from elastic ligaments. (2) Fibroin; from silk and spiders’ webs. (C)—CHITINOIDS: (1) Chitin; from external shells of beetles, crabs or lobsters. (2) Chonchiohn; from shells of mussels and snails. _ (3) Spongin; from sponges. (D)—KezratTINs: (1) Keratin; from hoofs, | horns, feathers, hair, wool, etc. (2) Neurokeratin; from brains. from the swimming bladder of Undissolved by dilute acids, boiling water, or boiling very dilute alkali. Dissolved by stronger alkali. Contain no sulphur. Have high tensile strength. Insoluble in boiling water or in alkalis (spongin dissolves in concentrated alkali). Contain no sulphur. Insoluble in water, salt solu- tions, or dilute acids or al- kalis. Difficultly soluble in strong alkali. Contain sul- phur. POSITION OF GELATIN AMONG THE PROTEINS — 23 II. ConsuGatep ProTeiIns:—Protein combined with a non-pro- tein radicle termed the prosthetic group. Nucleoproteins—from nuclei of cells. Compounds of a pro- tein (acting as a base) with one of the nucleic acids (substituted phosphoric acids containing carbohydrate and nitrogenous radicles). Insoluble in distilled water; soluble in dilute alkalis, such solutions being precipitated by weak acids such as acetic acid and carbon dioxide. Glycoproteins—here the prosthetic group is (1) an amino- carbohydrate; (2) a polysaccharide derivative of gluco- samin or its acetylated derivatives; (3) chondroitin-sul- phuric acid. There are three subdivisions: (1) Mucins—from mucous, snail-slime, etc., yield extremely viscous solutions from which the mucin is precipitated by acetic acid. (2) Mucoids—i.e. ovomucoid from egg white, are not as viscous in solution as mucins, and are not precipitated by ‘acetic acid. (3) Chondroproteins—trom cartilage, amyloid tissue, etc., are insoluble in water. Their solutions in dilute alkali are precipitated by an excess of acetic acid or on neutraliza- tion with strong acids. The chondrovrtin-sulphuric acid they yield on hydrolysis is composed of one molecule of sulphuric acid with one molecule of chondroitin, itself a compound of glucosamin and glucuronic acid, which physically resembles gum arabic. ee reotcins—ie. casein. Predominantly acid proteins, yielding phosphoric acid on hydrolysis. Hemoglobins—i.e. hemoglobin, in which hematin, an iron- containing complex organic acid, is the prosthetic group, united with a histone-like predominantly basic protein globin. Lecithoproteins—here the prosthetic group is a phospholipin. It is questionable whether the phospholipin is chemically combined or is simply an adsorbed impurity. III. Derrivep PROTEINS: (A) Primary PROTEIN DERIVATIVES: (1) Proteans.—Insoluble products formed by the incipient 24 GLUE AND GELATIN action of water, very dilute acids or enzymes, e.g. myosan from myosin, edestan from edestin (Hawk). (2) Metaproteins—These result from further action of acid or alkali, are soluble in very weak acid or alkali but in- soluble in neutral fluids, e.g. acid metaprotein (acid al- buminate); alkali metaprotein (alkali albuminate). (3) Coagulated Proteins—lInsoluble products resulting from the action of heat or alcohol on protein solutions. (B) SeconpDARY PROTEIN DERIVATIVES: (1) Proteoses—Soluble in water; not coagulated by heat; pre- cipitated by saturation of their solutions with ammonium or zinc sulphate; e.g. protoproteose, deuteroproteose, gelatoses. (2) Peptones——These differ from proteoses in that they are not precipitated by saturating their solutions with am- monium sulphate, i.e. antipeptone, amphopeptone, gela- tones. (3) Peptides——These are really peptones whose structure is known; that is, the polypeptides of Fischer; e.g. glycyl- glycine, etc. | The classification of proteins adopted by the British Medical Association is as follows: Ae Lis SIMPLE PROTEINS: Protamines—e.g. salmine, clupeine. Histones—e.g. globin. Albumins—e.g. serum albumin. Globulins—e.g. ovoglobulin. Glutelins—e.g. glutelin. Alcohol soluble Proteins—e.g. zein. Scleroproteins—e.g. elastin. Phosphoproteins—e.g. casein. CONJUGATED PROTEINS: Glucoproteins—e.g. mucin. Nucleoproteins—e.g. nucleohistone. Chromoproteins—e.g. hemoglobin. POSITION OF GELATIN AMONG THE PROTEINS © 25 III. Propucts or ProTtrin HypRo.Lysis: Infraproteins—i.e. acid or alkali albuminates formed by gently heating albumins in acid or alkali. Insoluble in distilled water; e.g. acid albuminate. Proteoses—e.g. protoproteose. Peptones—e.g. antipeptone. Polypeptides—e.g. dipeptides. -'T. Brailsford Robertson ? criticizes both the American and the British systems of classification as being based upon variations in physical behavior which do not necessarily correspond to dif-. ferences in chemical structure, whereas on the other hand there are many proteins or protein-like substances whose intermediate characteristics make their inclusion in any group a more or less arbitrary matter. His criticism is well-founded. Molecular Structure. This naturally raises the question as to the nature of the combination which holds together the various amino-acids in the molecules of different proteins. From the complexity of the amino-acids yielded on the drastic hydrolysis of gelatin and allied proteins (see table on p. 30), it is obvious that the combination is by no means as simple as ordinary two dimen- sional formule on paper would indicate. The classical work of Emil Fischer on the polypeptides shows that with these rela- tively simple (as compared with proteins) compounds, the link- age takes place according to the scheme first suggested by Hof- meister: | R’ Atif ¢ That is, the COOH and NH, groups of different molecules (the molecules may themselves be alike or different) combine with the elimination of water. But this relatively simple conception can not be carried over literally to proteins. In a trenchant criticism of Wolfgang Pauli’s ‘‘Kolloidchemie der Eiweisskorper,’ Wolfgang Ostwald ° 2“Principles of Biochemistry,” p. 125. 8 Kolloid Z. 27, 143 (1920). 26 GLUE AND GELATIN quotes Emil Fischer as saying: “It can not with certainty be predicted whether a 20-polypeptid or a substance of like com- plexity of constitution would behave physico-chemically like albumin or not. It may happen sometimes, but not always. When things become so complicated the way they are consti- tuted is not so easily explained. They become so indefinite. .. . In the course of time I have built up ever larger molecules. ‘The colloid chemists would do well, like Perrin, to reverse this and ‘from relatively large particles come down to molecules.” We must approach the subject without bias or fixed precon- ceived theories, and with minds flexible enough to fit all the facts of Nature, even though some be recently discovered facts. The complexity, frangibility, and even the variability of the so-called chemical elements, are established facts. The radio- active elements are spontaneously decomposing. Rutherford has shattered nitrogen by the impact of a particles shot out from radium at a speed of about 10,000 miles a second. By positive ray analysis Aston and others have demonstrated the existence of isotopes of many elements.*’ We have awaked to a realization of the fact that, just as there is no sharp line of demarcation between colloidal solution and true molecular solution or dis- persion, so too no sharp line can be drawn between physical and chemical attraction. Shght variations may mean much, e.g., the decimal in the atomic weight of hydrogen 1.008 represents electrons. The astronomer Herschel remarked that “the perfect observer will have his eyes, as it were, opened, that they may be struck at once with any occurrence which, according to received theories, ought not to happen, for these are the facts which serve as clues to new discoveries.” If the attractive forces existing between atoms (or atomic groups) were entirely satisfied or balanced by their chemical combination consequent upon the principal electronic attractive forces, or forces of primary valence as they are called, then every chemical compound would behave like a perfect gas so far as concerns the factor a in the equation of van der Waals. But in all chemical compounds there exist residual attractions or stray Thus there are three kinds of chlorine (atomic weights 35, 37 and 389 respectively) and six kinds of mercury; consequently there are 18 different mereuric chlorides and 63 possible mercurous chlorides (Harkins). POSITION OF GELATIN AMONG THE PROTEINS — 27 fields of force, which exert a controlling influence upon what we ordinarily call the physical properties of the compound—its state (gaseous, liquid or solid), its cohesion, solubility, melting point, freezing point, dielectric constant, conductivity for heat and electricity, etc. ‘These residual attractions are responsible for adhesion, adsorption, and the mechanical strength of materials, and their effective range of action (of the order of 10° cm.) is usually much less than the diameter of a molecule. Practically all molecules (and even all atoms) are polar and exhibit dissymmetry. They therefore tend to orient themselves so that their attractive forces may reach an equilibrium. This is particularly evident in cases of adsorption at surfaces where, as Langmuir ® observes, the molecules usually orient themselves in definite ways in the surface layer, since they are held to the surface by forces acting between it, and particular atoms or atomic groups in the adsorbed molecule.® Where the residual attractive forces reach an equilibrium, the molecules (or atoms) become more or less regularly distributed in the space lattice, and the compound (or element) is crystal- line, and usually shows its regularity of orientation when exam- ined by the X ray spectrometer of Bragg and Bragg.” In many cases, however, this tendency towards definite orientation is never realized. This is especially so where there are large and cumbersome molecules involved, as with the proteins, and even with metals and alloys where quick chilling tends to preserve the random or haphazard distribution of the atomic groups.® The experiments of P. Scherrer ® with the X-ray spectrometer show that colloidal gold particles too small to be seen even in the ultramicroscope, nevertheless show the same space lattice as macroscopic gold crystals. Old specimens of silicic acid and stannic acid gels exhibit well marked crystal interference figures in addition to the characteristics of amorphous substances, prob- ably representing substances on the point of crystallizing. But typical organic colloids such as gelatin, albumin, casein, cellu- 5 J. Am. Chem. Soc., 40, 1363 (1918). ®See also Harkins, Clark and Roberts, J. Am. Chem. Soc., 42, 706 (1920). 7 Bragg and Bragg, ‘““X Rays and Crystal Structure,’ London, 1915. §See Jerome Alexander, “The Colloidal State in Metals and Alloys,” Trans. Am. Inst. Mining and Met. Eng., Vol. 69 (1921); presented at Columbus, O., meeting, October, 1920; Chem. Met. Eng., January, 1922. ® Nachr. Ges. Wiss. Géttingen, 96-100 (1918). 28 GLUE AND GELATIN lose, and starch appear to be amorphous. The colloid particles, therefore, probably consist of large individual molecules or of groups of irregularly oriented molecules. Considering the information at present available, it would appear that the forces binding the relatively simpler molecular units into a “molecule” of gelatin are largely what have here- tofore been considered “physical” forces. Perhaps the difficul- ties of nomenclature may be to some extent avoided if we adopt the suggestion of P. EH. Wells.?° (1) Electronic forces—maintain positive nucleus and negative or valence electrons in equilibrium as a single system. (2) Atomic forces—maintain two or more atoms in equi- librium as a single system. (3) Molecular forces—maintain two or more molecules in equilibrium as a single system. (4) Molar forces—maintain two or more masses in equilibrium as a single system. “Hach group of forces may be regarded as the residual fields of force remaining unsaturated in the smaller systems con- stituting the components of the system under consideration... . Molecular systems have lost so much of their discreteness that combinations of molecules do not follow the laws of definite and multiple proportions. In such phenomena as molecular association and surface structure, the discreteness of atomic con- stitution begins to give way to statistical continuity. More- _ over, even in these phenomena, the forces are relatively so weak that molecules are not usually regarded as permanently grouped together.” (Wells, loc. cit.) What are generally called “chemical forces” are the atomic forces in the above classification, whereas what are generally called ‘physical forces” are the molecular forces therein men- tioned. A careful consideration of the experimental facts of physical chemistry, i.e. ionization, hydrolysis, adsorption, differ- ential diffusion, association, and dissociation, clearly show that it is just as impossible to draw a sharp line of demarcation be- tween physical and chemical compounds as it is to separate by fixed lines the several primary colors of the spectrum. It must not be supposed that this difficulty of definition is a new matter. Thus in 1884 in discussing a paper on adsorp- 10 J, Wash. Acad. Sct. 9, 361 (1919). POSITION OF GELATIN AMONG THE PROTEINS 29 tion by John Uri Lloyd," Prof. Prescott. said: “It has been asked whether we should refer Prof. Lloyd’s results to chemical action within molecules or to those forces which may be classed under physical action. Now we do not know a great deal about these modes of force, or their essential nature; but I think we know this much, that there is no sharp line of demarcation between chemical action within the molecules, and the physical action between the molecules. They grade off into each other. We must look for the interference of adhesion in a great many operations called chemical.” The same view has been voiced by many others, and recently by P. P. von Weimarn,” although but a short while before, he had regarded the condensation gas — liquid — solid as a chemi- cal phenomenon. In conclusion it may be said that, while the formation of amino-acids and even of some of the more complicated poly- peptides is controlled by the action of atomic forces, with in- creasing complexity of constitution, molecular forces appear and eventually predominate. The view held by many chemists that all attraction must follow the same simple laws that govern the formation of simpler compounds, must be abandoned; for, as Poincaré remarked, Nature is not as simple as all that. Once we realize the fact that gelatin has a coarser physical, as well as a finer chemical structure, we may be able to under- stand its hydrolysis and degradation without having recourse to ingenious but weird purely chemical explanations or formulas which have no counterpart in the actual facts, although in some cases they may not be disproved by the present experimental evidence. 11See J. Am. Pharmaceutical Assoc. for October, 1916. 12 Kolloid Z. 28, 97 (1921). Chapter 3. The Chemistry, Physical Chemistry, and Colloidal Chemistry of Gelatin and Glue. Since the chemistry of gelatin is inseparably bound up with its physical chemistry and its behavior as a colloid, confusion only can result if an attempt be made to discuss these aspects separately... We have already considered the position of gelatin among the proteins, and the general nature of the forces hold- ing its constituent atomic groups together (Chapter 2). Let us now consider the more intimate structure of the gelatin “molecule.” Chemical Structure. The most important evidence we have regarding the chemical nature of gelatin, is given by the products of its hydrolysis. Skraup and von Biehler+ found that on hydrolysis with hydro- chloric acid, gelatin yields the following substances, all of which had been found by previous workers. Glycine—a-amino-acetic acid. CH,(NH,).COOH LLysine—oa-e-diamino-caproic acid. CH,.NH,(CH,),.CH(NH,) COOH Alanine—a-amino-propionic acid. CH,.CH(NH,).COOH Phenylalanine—f-phenyl-a-amino-propionic acid. C,H,.CH., CHiN.) COC Leucine—a-amino-isocaproic acid. CH, .({CH,),.CH(NH.) COGS Aspartic acid—amino-succinic acid. . COOH.CH,.CH(NH,) .COOH Glutamic acid—o-amino-glutaric acid. COOH.CH,.CH,CH(NH,) .COOH Histidine—f-imino-azole-a-amino-propionic acid. N — C.CH,.CH(NH,) .COOH 1 Monatshefte fiir Chemie 30, 476 (1909). 30 THE CHEMISTRY OF GELATIN AND GLUE 31 Arginine—o-amino-$-guanino-n-valeric acid. NH:C(NH,) .NH(CH,),.CH(NH,) .COOH Proline—a-pyrrolidine-carboxylic acid. H,C — CH, ie H,C — C.COOH bo eae NH Oxyproline—f-oxy-a-pyrrolidine-carboxylic acid. H,C — CH.OH Ene. H,C CH.COOH ~~ Ti NH Whether these amino-acids exist in the gelatin “molecule” as such, or are formed from the disintegration of larger molecules, cannot with certainty be decided at present. To see how the degradation products of gelatin compare with those of other albuminoids or scleroproteins, there is given the following table prepared mainly from “The Chemical Constitu- tion of the Proteins,” by R. H. Alders Plimmer: a o “ 33 s 3 ae <5 Stale a2 0 fe OM a Bly, a8 e b a alae: S 2 ar a ee AN eens Sess peer 8 eee > cs} eo} s¥ sea Se poets as he Bo eee Se poss | 8200 eS 0lC(USeOUCUES ge" 2 Sey Bamecde So SSCS. Se BEG Sa Seces 85 sk 82 se SSar. 825 Soe cam os ca. a ne Base MES Glycine ..... 25.5 16.5 1925 124 360 S025 208 0.4 Alanine ..... 8.7 0.8 3.0 Q. 21.0 23.4 6.6 1.2 OU are 0.0 1.0 — _ 0.0 — 1.0 Bik Leucine yaa 2.1 6.75 9.2 1.5 8 214 18.3 Isoleucine ... 0.0 0.0 — — — — — Phenylalanine 1.4 0.4 — 1.0 1.5 — 3.9 3.0 Tyrosine .... 0.01 0.0 — — 10.5 8.2 0.4 46 Serine ...... 0.4 0.4 — — 16 — — 0.7 Cystine ..... — — — — — — — 6.8 Proline. ..... 9.5 5.2 6.25 104 + 3.7 1.7 3.6 Oxyproline .. 14. 3.0 6.4 3.0 — — — — Aspartic Acid 3.4 06 — 12 + — — 2.5 Glutamic Acid 5.8 0.9 1.75 168 0.0 11.7 0.8 3.0 Lysine ...... 59 28 — 6.0 Kp (basic dissociation); in these words, it behaves like a very weak acid. Pure albumin consists 2J. Coll. Agric. Tokyo 5, 355 (1916). 3 “Colloids in Biology and Medicine,” p. 154. 3a In a recent paper (Kolloid Z. 28, 49 [1921]) entitled “Der Allgemeine Bauplan der Kolloide,’ Pauli expresses the view that with albumins the amino- acids chemically combined with each other, form neutral particles which receive ‘their chargé from one or a few ionizing amino-acids. Wo. Ostwald, J. Loeb, and many others, including the author, do not agree with all of these views of Pauli, which are given in some detail as they represent one of the purely “chemical” views of the behavior of gelatin. ‘This is what Winkelblech terms an internal salt, whence the anhydride NH Rex | is formed by the elimination of water. Co THE CHEMISTRY OF GELATIN AND GLUE 33 principally of electrically neutral particles, but forms acid and alkali salts which are strongly ionized. There exist NH,OH NH,Cl NH,OH Bat a R< COOH COOH COONa neutral albumin —_ acid albumin alkali albumin “That the albumin ions are responsible for the great internal friction is to be assumed from the investigations of E. Laqueur and O. Sackur on alkali-caseinates. The cause of this phe- nomenon is found in the strong hydration (water fixation, swell- ing) of the albumin ions.° According to Wo. Pauli and M. Samec the existence of polyvalent ions must be assumed in the case of acid and alkali albumin. Even assuming the smallest values for the molecular weight gf albumin, the quantities of acid or alkali found are so large that they indicate the fixation of several acid or alkali molecules. This offers a further ex- | planation of the marked increase in hydration produced by acids | and alkalis. The stability of an albumin solution and its pre- | cipitability, e.g. by alcohol, are directly proportional to the / number of albumin zons it contains. The circumstances here are | quite analogous to those with crystalloids. Ions tend to go into | solution and to form hydrates; the saturation concentration of | neutral particles is always less than that of ions. i “In this way we may explain the properties of strongly ionized pure acid and alkali albumin as contrasted with the slightly dis- sociated neutral albumin. 7 “How does this theory agree with the effect of neutral salts? Wo. Pauli explains it in the following way: NH,Cl NH,Cl Ki + NaNO,2R< + HNO, COOH COONa acid albumin neutral Na salt of salt acid albumin In this way was explained not only the increased number of free | H ions, which he demonstrated, but also the marked diminution | in the internal friction; because an amphoteric salt, in which | 5 That the hydration rather than the size of the protein ions is the cause of their non-filterable character, is the view expressed by T. B. Robertson (‘‘The Physical Chemistry of the Proteins,” p. 148, footnote), J. A. ? 34 GLUE AND GELATIN both anions and cations tend to ionize about equally, is but slightly dissociated. “The action of neutral salts on alkali albumin is different; it follows the following scheme: 2 ae Ri +kCl2R< + H,O ees COONa COONa_~ water © us) alkali albumin neutral complex si salt albumin yan salt ai ow “Accordingly, a complex albumin salt is formed to which a less ce amount of ionization may be ascribed than to alkali albumin. The action of salts of the alkaline earths follows this scheme: NH;OH se Gn" NH,NaNO, R< +3-NO, 2 R< Ca foe COONa Coos The replacement of the alkali ion in the hydroxyl of the amino group results in a weakly ionized complex salt. The effect on albumin of organic bases, which are often highly toxic, and of amphoteric electrolytes, have also been studied by H. Handovsky, and the results agree with the above scheme. “The conditions governing the action of neutral salts upon acid albumin are not sufficiently understood to warrant proposing a simple scheme.” In a footnote Bechhold remarks that it should not be assumed that only free terminal NH, groups are to be considered, since the work of Blasel and Matula on deaminized gelatin make it probable that interior NH, groups are involved. The method of Blasel and Matula * for deaminizing gelatin is as follows: To a solution of 200 grams of the purest commercial gelatin in 1 liter of warm water, is added 200 grams of sodium nitrite also dissolved in 1 liter of water. After cooling 140 grams of - glacial acetic acid is carefully added, and after standing 12 hours, the mixture is heated for two hours on a water-bath. The deaminized gelatin is then salted out by saturation with am- monium sulphate and purified by prolonged dialysis (2 weeks) against running distilled water. 5a Biochem, Z. 58, 417 (1914). THE CHEMISTRY OF GELATIN AND GLUE 35 The deaminized gelatin, although its free amino groups are all destroyed, still has almost the same acid-combining capacity as ordinary gelatin. This certainly indicates that something other than the chemical attraction of the free NH, groups is responsible for acid fixation. T. B. Robertson ° believes that the — CONH — groups within the molecule are responsible for the acid-and-base-combining capacity of the proteins. The — CONH — group may exist in the keto form, — CO — NH —, or in the enol form, = = N-—, On but neither analytic or synthetic methods are able to distinguish between the two forms. Robertson thinks that the enol form is most probable, since it can attach either acids or bases. D. Jordan Lloyd’ thinks that “a more probable explanation seems to be that under the action of acids gelatin goes into the keto-form, and under the action of bases to the enol-form. This view would conform with the observation that the free acid from sodium gelatinate differs in properties from the free base of gela- tin hydrochloride. It can also be harmonized with Dakin’s theory ® that the non-terminal groups in proteins go from the keto-form (1) to the enol-form (2) © (1) NH.CO— (2) NH.CO — es ye R.C—C—NH.CHR.COOH R.C=C—NH.CHR.COOH ae | 7 1s een 6) OH | with loss of optical activity under the action of bases at low temperatures.” Wintgren and Kriiger® believe that since proteins have more than one NH, group, it is only in dilute solutions that we find the type [GE] NH,* + H,Os [GE] NH,OH-+ Ht corresponding to NH, + H,O = NH,OH + H* 8 “Physical Chemistry of the Proteins,’ p. 24; ‘Principles of Biochemistry,” . 156. i 7 Biochem. J. 14, 154 (1920). 8 J. Biol. Chem. 18, 357 (1913). ® Kolloid Z. 28, 81 (1921). 36 GLUE AND GELATIN In higher acid concentration several amino groups take part in salt formation, so that ‘proteins have not one but several dis- sociation constants whose values decrease at. varying speeds.” Loeb’s Theory of Colloidal Behavior. Jacques Loeb 7° concludes from the results of a series of care- ful and ingenious experiments that it is the H-ion concentration of protein solutions which controls their behavior. “Proteins exist in three states, defined by their hydrogen ion concentration, namely (a) as non-iongenic or isoelectric protein, (b) metal pro- teinate (e.g. Na or Ca proteinate), and. (c) protein-acid salts (e.g. protein chloride, protein sulphate, etc.). We will use gela- tin as an illustration. At one definite hydrogen ion concentra- tion, namely 10%’ N, or in Sorensen’s logarithmic symbol at ec Yn gelatin can combine practically with neither anion nor cation of an electrolyte. At p,,>4.7 it can combine only with cations (forming metal gelatinate, e.g. sodium gelatinate), at p.,<4.7 it combines with anions (forming gelatin chloride, etc.) .” Loeb then describes experiments with powdered gelatin swollen in ice-cold water, showing that only those gelatins having Py, >4.7 can fix Ag from AgNO, or Ni from NiCl,; and only those having p,,<4.7 can fix Fe(CN),. “In this way it can be shown,” says Loeb, “that when the p,, is >4.7 gelatin can com- bine only with cations; when p,, is <4.7 it can combine only with anions, while at p,, 4.7 (the isoelectric point) 1t can com- bine neither with anion or cation. The idea that both ions influence a protein simultaneously is no longer tenable. “Tt follows also that a protein solution is not adequately de- fined by its concentration of protein but that the hydrogen ion concentration must also be known since each protein occurs in three different forms—possibly isomers according to its hydrogen ion concentration.” , Loeb claims that the direct chemical union of acids with 10 Science, N. S., Vol. 52, p. 449 (1920). : 11 An H+ concentration of 2 x 10-5 is expressed according to Sdrensen’s nota- tion as follows: p y (the H-ion concentration) =—log (2X 10>) =— (0.3-5) Sb (le ee) 6 THE CHEMISTRY OF GELATIN AND GLUE 37 gelatin is demonstrated by his experiments, which show that 3 times as many cc. of 0.1 N H,PO, are required to bring 100 cc. of 1 per cent. gelatin solution to a given p,,, as are required in the case of HCl or HNO,, twice the number of cc. of 0.1 N oxalic acid, and the same number of cc. of 0.1 N H,SO, (... in a strong dibasic acid, like H,SO,, both hydrogen ions are held with a sufficiently small force to be easily removed”). Bases show analogous results, and in conclusion Loeb makes the rather sweeping assertion—“The behavior of the proteins, therefore, contradicts the idea that the chemistry of colloids differs from the chemistry of crystalloids.”’ If all molecular forces are to be regarded as ‘“‘chemical,”’ then Loeb’s case is proved at the outset by definition. W. D. Bancroft ?? takes Loeb to task for drawing general con- clusions on the basis of experiments made with dilute solutions. Bancroft says: “Under the conditions of the experiments Loeb found that on the acid side of the isoelectric point only anions of neutral salts are taken up and the alkaline side of the iso- electric point only cations. Since the Hofmeister series calls for an effect due to both ions of a neutral salt on the swelling of gelatin, Loeb concludes that the Hofmeister series is a delusion and a snare. This does not follow at all. Loeb is working at such extreme dilutions that the specific effects of all ions but hydrogen and hydroxy! ions are practically negligible. In acid solutions only anions are taken up and in alkaline solutions only cations. Loeb recognizes the specific effect of iodine ions over chlorine ions in causing the liquefaction of gelatin, but he con- siders that liquefaction stands-in no necessary relation to swell- ing, an assumption which will be shared by few. With higher concentrations Loeb will undoubtedly get entirely different re- sults. His conclusions as to the existence of definite compounds depend on the assumption that he is dealing with true solutions and will fall with that assumption.” In reply to Bancroft, Loeb ** points out that “salt solutions up to grammolecular concentration were used without any indi- cation of validity of the Hofmeister series being found. Ban- croft will surely not maintain that solutions of neutral salts up to molecular concentration are so dilute that the effects of all 2 “Applied Colloid Chemistry,” p. 255 (1920). 33 “Proteins,” p. 110. 38 GLUE AND GELATIN ions except the hydrogen and hydroxyl ions are practically negligible. “The writer’s (Loeb’s) statement that the liquefaction of solid gelatin stands in no necessary relation to swelling is correct, since higher concentrations of acids or of salts like CaCl, diminish the swelling of gelatin while they increase its solubility. This is due to the fact that swelling and solution of gelatin in the presence of acid are functions of different variables, swelling in acid depending on the Donnan equilibrium, while the solution of gelatin depends on the same forces which are responsible for the solution of ordinary crystalloids in water (probably second- ary valency forces).” Because of the facts pointed out in Chapter 4, it seems to the writer that Bancroft’s criticism is well founded. The Donnan equilibrium is based on the assumption that there is complete ionization of the colloid salt (“gelatin chloride”) and the crystal- loid (HCl). As concentrations increase, the degree of dissociation and ionization diminish. Another assumption is that the jelly cation of “gelatin chloride” is not diffusible; but as D. Jordan Lloyd points out (loc. cit., p. 164) the ‘colloidal ion” is dif- fusible to some extent and must exert an osmotic pressure. C. R. Smith +* has shown that traces of impurities, especially Ca salts, exercise a potent influence on the water-absorbing ca- pacity of gelatin, and that this fact may vitiate many of Loeb’s conclusions which were based on experiments made with ash- containing gelatin. Smith points out that the increased swell- ing observed by Loeb in gelatin treated with sodium chlorid, etc. (all excess being washed out), 1s due to the fact that “these ’ electrolytes remove the repressing lime salts and leave a gelatin combined with sodium cations. . . . It is not surprising that cal- cium, magnesium, strontium, barium chloride, or magnesium sulfate produce no increased swelling, for they do not remove the ash, and they also leave combined bivalent cations which do not increase swelling as much as univalent cations. Loeb continued to treat gelatin with various salts, under the impres- sion that they were reacting with the gelatin. Only when using oxalates does he mention the formation of 'a white precipitate (obviously from the lime). He (January 20, 1919) ascribes the Increase in osmotic pressure of gelatin to an increase in the 4 J, Am. Chem. Soc. 43, 1850 (1921). THE CHEMISTRY OF GELATIN AND GLUE 39 number of particles, ionization not considered, but later stated that free hydrobromic acid represses the ionization of gelatin bromide and again that the physical properties of gelatin are dependent only on the number of gelatin bromide molecules formed. . . . Loeb’s figures for osmotic pressure obtained on in- completely purified gelatin are from 25 to 50 per cent.’ too low. The results of this paper, however, confirm many of his con- clusions.” Loeb in his reply to Smith* publishes the analysis by Dr. Hitchcock of his laboratory, of two random samples of the kind of gelatin he used. (Cooper’s gelatin purified by treatment with 0.0078 M acetic acid and washing with distilled water of p,, a little above 5.0). The results showed 0.001 per cent. of ash, with qualitive tests for Fe, Ca, and PO,, but negative tests for Cl and S8O,. From this Loeb concludes that the ash content of the gelatin he had been using for experiments on swelling, os- motic pressure, and viscosity, ‘might have been about 1 mg.” (per gram). “It was shown by the writer’s (Loeb’s) experi- ments that that amount of ash (which equals roughly a 0.000033 M solution of tricalcium phosphate) has no influence on the physical properties of the proteins, such as osmotic pres- sure swelling, viscosity, or potential difference.” It would be more convincing if Loeb had determined the ash of the particular specimens of gelatin that he actually used. He says that Smith’s criticism, that his results on the osmotic pres- sure and swelling of gelatin are vitiated by the use of ash-con- taining gelatin, does not apply to his more recent papers pub- lished during the last three years. Loeb states that the correct values for the osmotic pressure (of solutions containing 1 g. of. originally isoelectric gelatin in 100 cc.) are given in the May (1921) number of the Journal of General Physiology. ‘Former values were lower since the solutions contained less than 1 g. in 100 cc., usually 0.8 g., as was pointed out in a paper published in January, 1921, in the same Journal.” Ash-free Gelatin. C. R. Smith applied for a public service patent (Serial No. 390,253 dated June 19, 1920), and points out+** that J. Loeb 15 J, Am. Chem. Soc. 44, 214 (1922). wa J, Am. Leather Chemists’ Assoc., Oct., 1922. 40 GLUE AND GELATIN erred in‘ crediting Miss Field as the discoverer of ash-free gelatin. Ash-free gelatin looks like the ordinary kind, but a one per cent. solution soon becomes turbid. C. R. Smith’s method of preparing ash-free volun is here epitomized: Gelatin of the highest jelly strength (maximum mutarotation ratio 2.2), ground to about 16 mesh, is washed on a filter with cold (0° to 10°) 10 per cent. sodium chloride solution containing 5 ce. of concentrated hydrochloric acid per liter, until the wash- ings are free from lime. The acid is washed out with cold 1 per cent. sodium chloride solution, and then gradually weaker salt solutions are used. Distilled or conductivity water is finally -used to wash the gelatin until the washings show no chlorine. After dehydrating with cold 90 per cent. alcohol, the gelatin is then dried. “When powdered gelatin is washed with cold water alone, the readily diffusible calcium salts soon pass away until further washing becomes ineffective. If it is now washed with a solu- tion of sodium chloride, ammonium chloride, potassium bromide, or presumably any uni-univalent electrolyte, dialysis of the re- maining lime salts takes place immediately, probably because certain slowly diffusible, possibly colloidal, salts of calcium react with them to form readily diffusible salts. If the added electro- lyte is now washed out, any alkali combined with the gelatin is almost invariably left. Using sodium chloride, sodium carbonate is found in the ash. In order to insure the removal of this alkali as well as iron, heavy metals, etc., acidulated salt solution must be used. The removal of all calcium salts can be accomplished in an hour, but the removal of the hydrochloric acid requires several hours and the use of dilute salt solution until the remain- ing acid can be removed by water alone without excessive swell- ing. It is almost impossible to wash gelatin swollen to 40 or 50 volumes. As the last traces of acid are being removed, the gelatin (at 15°) shrinks to particles swollen to about 7 volumes. The removal of the last traces of acid is probably facilitated by the fact that the isoelectric point of gelatin is on the acid side *6 snare Gs free gelatin thus obtained when incinerated leaves no ash 16 Michaelis, ‘‘Die Wasserstoffion Konzentration” ; Patten and Kellems, J. Biol. Chem. 42, 363 (1920). THE CHEMISTRY OF GELATIN AND GLUE 41 other than the traces of sand when the original glue or gelatin contains such. When ashed with pure sodium carbonate, chlo- rides, sulphates, or phosphates cannot be detected. “Ash-free gelatin swells in water at 15° to about 7 or 8 vol- umes. If such a gelatin be melted and cooled, a clear, stable jelly is produced. If, however, a weaker jelly be prepared, syneresis takes place, with the production of a cloudy jelly. A 0.5 per cent. jelly will flocculate into jelly particles (probably swollen to 7 volumes) and can be filtered off completely from the extruded water, which shows no trace of gelatin. ‘““Ash-free gelatin forms sols or gels with a minimum tendency to remain dispersed. It is readily precipitated by alcohol with- out the presence of electrolytes. Traces of acids or alkalis increase the osmotic pressure and prevent its precipitation by alcohol. Gelatin thus peptized by traces of alkalis or acids in the presence of a large percentage of alcohol exhibits a marked resemblance to the metal suspensoids. Traces of electrolytes, for example those present in a drop of tap water, cause immedi- ate precipitation. (This indicates the protective action of hy- drolysis products or “impurities” usually present in gelatin. J. A.) Bivalent and trivalent ions are most effective in bringing about precipitation. “Ultimate analysis of this gelatin gave the following results:*7 Carbon Hydrogen Nitrogen Oxygen 50.47 6.75 17.53 25.25 50.56 6.87 17.53 25.04 50.52 6.81 i7cbe 25.15 “Moisture was determined by drying at room temperature over sulphuric acid to constant weight for several weeks. Heat- ing at 100° caused no further loss in weight. Moisture correc- - tion was applied to all figures. The carbon content was from -0.5 to 1.1 per cent. higher than that in published analyses made on ash-containing material, probably because the latter retained carbon or carbon dioxide which was not considered.” Sheppard, Elliot and Benedict 1 report that gelatin free from ash and hydrolytic products may be prepared by electrolyzing a 5 per cent. solution of commercial gelatin in a cell of electro- filtros for three to four weeks, the salts passing through the cell 7 Carbon and hydrogen determinations were made by Dr. D. H. Brauns. “aS. EH. Sheppard, Felix A. Elliot, and Miss A. J. Benedict, Science 46, 550 (1922). 42 GLUE AND GELATIN into the electrode chambers, and reducing the ash to about 0.10 per cent. This partially de-ashed solution is then precipitated by acetone, thus removing the hydrolysis products and _ still further reducing the ash to about 0.01 per cent. The gelatin thus purified is dissolved in conductivity water, chilled in sheets, and dried. They recommend it for all research work on gelatin, as well as for providing culture media which can be brought to any particular reaction with complete knowledge of the salts present. Fischer’s Views. Martin H. Fischer 8 says: “The measurable hydrogen and hydroxyl ion contents of different protein-water systems upon which such emphasis has been laid for the explanation of their stability are only observable in relatively dilute systems; the ion contents are not inherent to, or necessary for, the stabiliza- tion; they are accidental accompaniments incident to the solu- tion of some of the acidic and basic proteins in the excess of water and their hydrolysis with the production secondarily of an overplus of hydrogen or hydroxyl ions.” 1° While recognizing the formation of chemical compounds in protein-acid and protein-alkali systems, Fischer says (loc. cit.): “How inadequate for the understanding of the colloid-chemical behavior of such systems are the overplayed ‘stoichiometrical,’ ‘chemical,’. ‘electrical,’ hydrogen and hydroxyl ion notions, usually called upon to explain in some exclusive fashion all the changes observed, must be self-evident. “Stoichiometrical views cover only those parts of the whole problem which have to do with the quantities produced of dif- ferently hydrateable or soluble compounds; ‘chemical’ notions are no more adequate for the explanation of the problem than they are, at present, for the understanding of the whole problem of solution; electrical and ionic notions are hardly of service when it is remembered that the most stabile of these hydrated colloid systems are such as are composed of chemacally produced, 18 “Soaps and Proteins,” p. 214. This recalls the behavior of ferric chloride, dilute aqueous solutions of which slowly hydrolyze and deposit Fe(OH); from a weak solution of HCl. By pouring a few drops of strong ferric chloride solution into boiling water, the decomposition takes place instantaneously, and is evidenced by the intense color of the colloidal Fe(OH)s3, which however soon precipitates. J. A. THE CHEMISTRY OF GELATIN AND GLUE 43 really neutral compounds of protein with base or acid, provided only that not more water is present in the system than can be absorbed by the hydration capacities of the protein derivatives. Yet these colloid systems contain no quantities of either hydro- gen or hydroxyl ions measurable by ordinary laboratory means.” Taking up the case of gelatin specifically, Fischer *° says: “Dry gelatin absorbs water (to yield the system water-dis- solved-in-gelatin) and has a limited solubility in water (to yield the system gelatin-dissolved-in-water). Between these extremes and depending merely upon the relative amounts of gelatin and water present there lie the systems gelatin-solution dispersed in hydrated-gelatin (gel) or, with more water, hydrated-gelatin dispersed in gelatin-solution (sol). “What is the action of alkalis (or acids) upon these systems? “Under variously worded headings this problem has received much study. The effects of alkalis (and acids) upon the lower- most of the four systems may be found under the caption ‘swell- ing’ of gelatin in the presence of acids and alkalis; *! their effects upon the system gelatin-solution-in-hydrated-gelatin under the heading liquefaction and ‘solution’ of gelatin; 7? their effects upon the system hydrated-gelatin-in-gelatin-solution under studies in viscosity ; 7° their effects upon the system true solution of gelatin- in-water as studies on the ‘solubility’ of gelatin.2* What is the relationship between all these? “Tt is well to begin by inquiring into the relationship between the swelling of ‘soluble’ ‘neutral’ protein and its ‘solution.’ The notion that solution is but a continuation of swelling persists to this day.2° Investigation ?° of the problem, however, has 20 Loc. cit., p. 218 et. seq. 21See for example K. Spiro, Hofmeister’s Beitrdge 5, 276 (1904); Wolfgang Ostwald, Pfliiger’s Arch. 108, 563 (1905); M. H. Fischer, ‘‘Edema and Ne- phritis,” 3d ed., p. 75, New York, 1920, where references to earlier studies may be found. 2M. H. Fischer, Science 42, 223 (1915) ; Kolloid Z. 17, 1 (1915). 23 See for example the work of Hofmeister, Pauli, Hardy, von Schroeder, Handovsky, Schorr, ete., on the viscosity of liquid proteins (‘‘sols’’). 24M. H. Fischer, ‘‘Hdema and Nephritis,”’ 3d ed., p. 518. As of similar impart but upon other proteins may be cited some studies on wheat gluten. T. B. Wood and W. B. Hardy (Proc. Roy. Soc. London, Series B, 81, 38 (1908), the influence of acids, while F. W. Upson and J. W. Calvin (J. Am. Chem. Soc. 87, 1295 [1915]) studied its swelling under similar circumstances. 2>See for example Wolfgang Pauli, ‘‘Kolloidchemie der Hiweiss Korper,’’ p. 63, Dresden (1920). 26M. H. Fischer, Science 42, 228 (1915) ; Kolloid Z. 17, 1 (1915). 44 . GLUE AND GELATIN shown that this is not the case. The matter is easily proved by working with gelatin at concentrations and temperatures near its gelation or melting point. Since alkalis and acids increase hydration (increase swelling) the addition of these substances to a barely liquid gelatin-water mixture ought to stiffen it. As a matter of fact just the reverse occurs. By working with a stiff gelatin, a previously solid mixture is made to liquefy upon the addition of these substances. “The phenomena of swelling (hydration) and of ‘solution’ ** in such soluble protein gels as gelatin, while frequently assoct- ated, are therefore essentially different. Swelling 1s best under- stood as a change whereby the protein enters into phystco-chemi- cal combination with more of the solvent (water), as a change in the direction of greater solubility of the solvent in the pro- tein; ‘solution’ is best conceived of as a change in the direction of greater solubility (an increased degree of dispersion) of the colloid in the solvent. . . . (p. 220) under the influence of added alkali or acid the ‘neutral’ gelatin is converted into a basic gelatinate or gelatin chloride. These compounds, at the same concentration, are more soluble in water than the neutral gelatin and hence the liquefaction of these systems.” Fischer ?° then describes experiments showing that the addition of a neutral salt in increasing concentration to a previously liquid gelatin at first increases its viscosity to an optimum point (gelation) and then decreases it. Just as in the case of soaps, the salt becomes hydrated and, as salt-water, becomes emulsified in the hydrated basic (or acidic) gelatin. When salt is added beyond the optimum point, the salt-water becomes the external phase and the viscosity of the system falls. When enough salt is added the whole of the gelatin (as sodium gelatinate or as gelatin chloride and not as “neutral” gelatin) separates off in practically anhydrous form. Substantially Fischer’s views agree with those of Wo. Ostwald. However, Ostwald *° believes that beyond a certain critical point swelling passes over into solution, the spatial continuity of the two phases relative to each other being then destroyed. 27 Since there are many opinions regarding the nature of ‘solution,’ accurate definition of the term is not easy. We are here using the term in its broadest sense as covering everything, in the case of colloids, from their liquefaction point upwards to tbe accepted ‘‘true’’ solution of the physical. chemists. 28 Loc. cit., p. 221. *9 ‘Handbook of Colloid Chemistry,’ 2d ed., M. H. Fischer’s translation, p. 261. . THE CHEMISTRY OF GELATIN AND GLUE 45 Based upon ultramicroscopic evidence the writer agrees with Ostwald’s view. When the Brownian motion of particles be- comes sufficiently violent to carry them beyond each other’s range of molecular attraction, then the dispersion due to swell- ing begins to pass over into solution. With the proteins there seems to be no sharp line between swelling and solution, for slight thermal changes or mechanical action may produce sufficient dispersion of part of the swollen protein to produce a colloidal solution, which may later aggregate once more to a gel with larger motionless particles. Indeed with some, if not all salts, colloidal dispersion precedes true solution. Thus Alexander and Bullowa *° observed that sodium citrate, on going into solution, gave off streams of actively moving ultramicrons. Solution, then, results when the intramolecular adsorption of the solvent is powerful enough to force molecules or molecular groups beyond a certain critical distance from each other. (The greater the degree of dispersion of the particles, the more rapid the Brownian motion and the nearer the approach to true or molecular solution; and this is conditioned by temperature, pres- sure, protective substances, coagulators, etc. As dispersed parti- cles aggregate, the Brownian motion decreases sharply, groups about 1.1 u being nearly motionless. The closer particles are the less water they tend to adsorb. Therefore dilute jellies, when dried, take up more water than do concentrated jellies. Heating to 40° annihilates these differences for the time being, as the complexes are then broken down. The molecular groups con- stituting gelatin are so large and possess such a powerful idio- attraction, that it is not easy to separate them. Furthermore the degree of separation, that is, the size of the gelatin “mole- cule,” seems to depend upon circumstances, as may be seen by considering work on the molecular weight of gelatin. Molecular Weight of Gelatin. Wide differences of opinion exist regarding the molecular weight of gelatin. Various methods have yielded the following: Schiitzenberger and Bourgeois........... — 1,836 A cas cee he re — 900 PrmtreneanG TUE... wk eck eee — 839 %o J, Alexander and J. G. M, Bullowa, Arch. of Pediatrics 27, 18 (1910). 46 GLUE AND GELATIN Proctor and \Wilsons. 2. <5 ie. ss yawn — 768 Berrarigeesih cess come eee eae — 823 eee Biltz, Bugge and Mehler................ a rs See 1) J OTGANs LOVE aontes oes eee maser as — 10,300 Dak is, ov GAR OR EGR Sa ewes — 11,800 Jeo re a ae eee — 12,000 to 25,000 GoR. Sunith x, 58 ck roa ee ae about — 96,000 D. Jordan Lloyd *! estimates that the molecular weight of gela- tin is about 10,000 or some multiple of this figure. Her evidence, based on chemical grounds, is given below: Van Slyke’s*? analysis shows the following distribution of nitrogen in gelatin: Per cent. of total mtrogen Ammonia nNItTOZeN. .ciukeess diode sie o's soe ae 2.25 Melanine Me « Veen. gus Son tis ar giatereieosie, 6 cal ae 0.07 Cystine 00 ABTS MS ae See aw ee ee 0.00 Arginine PE: SME TRO i) TAPIA os ee 14.7 Histidine. 8 oe pip wien soa 6 0 wate Go che Otenne ee 448 Lysine (..e00 7. Sie ew eee oe ete es eee nen th tee 6.32 Mono-amino nitrogen -.0..2).0%4.55 4. «ssdies a 56.3 Non-amino - = proline + oxyproline.......... 14.9 Assuming 1 histidine grouping in the gelatin molecules there must be 3 histidine nitrogens. The percentage histidine value, 4.48, can be reduced to 3 by multiplying by the arbitrary factor 0.665; but this would yield figures corresponding to fractional (half) molecules for both ammonia and arginine. Assuming 2 histidine groupings, the factor becomes 1.33 and we have Ammonia nitrogen *:.<. 5.44.00 2.9 approximately 1 x Arginine bye Me Top Teak CSA 19.7 4X5 Histidine AER PE Ron: Rye 6.0 t be A Lysine CTE he ah el eg eee 8.4 s 2X4 Mono-sdming (ici Go.e eect 152 e 1 X 76 Proline + oxyproline nitrogen..... 20.0 oi 1 X 20 These figures indicate that 1 gelatin molecule contains 133x nitrogen atoms distributed thus: 3x Ammonia (amide) groupings 4x Lysine groupings 2x Histidine 76x Mono-amino : 5x Arginine i 20x Proline + oxyproline “ In the absence of other evidence, x may be taken as unity. But the total nitrogen in dry gelatin is 18.0 per cent. of the total dry weight, as is evident from the following analysis: 51 Biochem. J. 14, 166 (1920). 32 J, Biol. Chem. 10, 15 (1912). THE CHEMISTRY OF GELATIN AND GLUE 47 Per cent. N CON) es ee 18.3 —Annalen, 45, 63 (1848). Chittenden and Solley.......... 18.0 —J. Physiol. 12, 33 (1891). Sad 18.12— Berichte, 25, 1202 (1892). SUMRMM DCMS. les sk eee es 17.81—J. Exp. Med. 2, 117 (1897). Schiitzenberger and Bourgeois... 18.3 —Jahresbericht Thier-Chem. 1876, 30. Sadikoff (Kjeldahl method)..... 1747—Z. physiol. Chem. 37, 397 (1903). ‘c (Dumas’ (79 ) dak 2P 18.18—“ 6c 66 (73 73 “ce 18.0 + 2% “If 133 atoms of nitrogen form 18.0 per cent. of the weight of the gelatin molecule, then the lowest weight of gelatin which can act as a chemical individual must be HBP SL eaauy = x ue 10,344, or approximately 10,300. The error in the mean of the analyses given above falls within 2 per cent.; the error in van Slyke’s analyses is of the order of 1 per cent.; the total error in the com- puted value is therefore of the order of 3 per cent.” C. R. Smith ** working with highly purified ash-free gelatin, found at 35° an osmotic pressure of approximately 48 mm. of water for a concentration of 2 grams (1.78 dry) gelatin per 100 cc. water, and 95 mm. for 4 grams (3.56 dry) per 100 cc. On the assumption that the gas laws apply, this indicates for gelatin molecular weight of about 96,000. The Crystallization of Gelatin. P. P. von Weimarn ** claims to have crystallized both gelatin and agar. He maintained a very dilute solution of gelatin in aqueous alcohol at 60°—70° in a dessicator containing dry potas- sium carbonate which absorbs water vapor but not alcohol vapor. As the concentration of alcohol slowly increases, the gelatin separates out in “crystals.” C. R. Smith ** reports that this method failed in his hands. It is to be noted that this method, similar to that by which Hofmeister crystallized egg albumen, involves an extremely slow aggregation of the constituent particles of the gelatin, during which their aggregation tendencies may have opportunity to establish themselves. In this respect it is analogous to the 83 J, Am. Chem. Soc. 43, 1350 (1921). 34P. P. von Weimarn, “Grundziige der Dispersoid Chemie,’ 1911, p. 106. 34a J, Am. Leather Chemists’ Assoc., Oct., 1922. 48 GLUE AND GELATIN deposit of quartz crystals from silicious waters, and is free from the criticism that must attach to “crystallization” of colloids in the presence of electrolytes. For just as colloids exercise a powerful influence on crystallization ®® so too do crystalloids tend to give colloidal gels a definite form or orientation.*® Thus the results of S. C. Bradford,’ who claims to have crystallized gelatin in the presence of mercury salts, are open to doubt. So also are von Weimarn’s results, for he did not use ash-free gelatin. Even when gelatin slowly dries, between a slide and a cover glass for example, there seems to be registered an attempt towards orientation; dendritic forms -appear which have been described by Liesegang.*® Contrary to what is commonly believed, colloids do diffuse, albeit but slowly. Too little detailed reference is made to the classic work of Graham,*® who clearly brought out this feature. Thus he says *° that tannic acid passes through parchment paper about 200 times slower than sodium chloride; gum arabic 400 times slower. “The separation of colloids from ecrystalloids by dialysis is, in consequence, generally more complete than might be expected from the relative diffusibility of the two classes of substances.” At the outset of his paper, Graham says: “The range also in the degree of diffusive mobility exhibited by dif- ferent substances appears to be as wide as the scale of vapor tensions. ‘Thus hydrate of potash may be said to possess double the velocity of sulphate of potash, and sulphate of potash again double the velocity of sugar, alcohol, and sulphate of magnesia. But the substances named, belong all, as regards diffusion, to the more ‘volatile’ class. The comparatively ‘fixed’ class, as - regards diffusion, is represented by a different order of chemical substances, marked out by the absence of the power to crystal- lize, which are slow in the extreme. Among the latter are hy- drated silicic acid, hydrated alumina, and other metallic per- oxides of the aluminous class, when they exist in the soluble form; with starch, dextrine, and the gums, caramel, tannin, gela- 85 J, Alexander, Kolloid Z. 4, 86 (1909). %°R, EK. Liesegang, Kolloid Z. 7, 96 (1910). 87 Biochem J. 14, 91 (1920). 38 R. H. Liesegang, Kolloid Z. 7, 306 (1910). 39 Phil, Trans. Roy. Soc. London 151, 183-224 (1861). 40 Tbid., pp. 213-217. THE CHEMISTRY OF GELATIN AND GLUE 49 tine, vegetable, and animal extractive matters. Low diffusibility is not the only property which the bodies last enumerated possess incommon. They are distinguished by the gelatinous character of their hydrates. Although often largely soluble in water, they are held in solution by a most feeble force. They appear singu- larly inert in the capacity of acids and bases, and in all ordinary chemical relations. But, on the other hand, their peculiar physi- cal aggregation with the chemical indifference referred to, ap- pears to be required in substances that can intervene in the organic processes of life. The plastic elements of the body are found in this class. As gelatine appears to be its type, it is proposed to designate substances of the class as colloids, and to speak of their peculiar form of aggregation as the colloidal con- dition of matter. Opposed to the colloidal is the crystalline condition. Substances affecting the latter form will be classed as crystalloids. The distinction is no doubt one of intimate molecular constitution.” We should not be surprised that colloids can be crystallized, for we now know that all substances may exist in either the col- loidal or the crystalloidal state, depending upon conditions. Where molecular mobility is great and capable of self-expression, visible crystals are formed, whereas where crystallization is in- hibited, as with glass, soaps, chilled metals, etc., the colloidal state tends to appear and persist. E. Hatschek ** has described the peculiar properties of camphorylphenylthiosemicarbazide whose suddenly chilled 5 per cent. alcoholic solutions form col- loidal gels which gradually become crystalline. W. B. Hardy *# had similar results with azomethin. The effect of temperature on the aggregation of gelatin par- ticles is shown by C. R. Smith,** whose work indicates a difference between gelatin dried at above 35° and that dried at below 15°. Some idea as to the relative size of the groups in the case of 1 per cent. gelatin solution may be gained from the following table taken from H. Bechhold,** which shows in decreasing order, the sizes indicated by ultrafiltration experiments: 41 Kolloid Z. 11, 158 (1912). 4 Proc. Roy. Soc. 87, 29 (1912). 4.7. Am. Chem. Soc. 41, 185 (1919). 44“‘Colloids in Biology and Medicine,” p. 99. 50 GLUE AND GELATIN SUSPENSIONS Prussian Blue Platinum-sol (Bredig) Ferric oxide hydrosol Casein, in milk Arsenic sulphide hydrosol Gold solution, Zsigmondy’s No. 4, about 40 uu Bismon (Colloidal oxide), Paal Collargol (colloidal silver), von Heyden, 20 uu Gold solution, Zsigmondy’s No. 0, about 1-4 uu 1 per cent. gelatin solution. bismuth 1 per cent. hemoglobin solution mol. wt. about 16,000. Serum albumin. Diphtheria toxin. Protalbumoses. Colloidal silicie acid. Lysalbinic acid. Deutero-albumose A. Deutero-albumose B, mol. wt. about 2,400. Deutero-albumose C. - Litmus. : Dextrin, mol. wt. about 965. CRYSTALLOIDS. Gelatin lies in the heart of the colloidal zone, and it is interest- ing to compare its superior water-taking capacity with that of dextrin which has much smaller particles. Chapter 4. The Chemistry, Physical Chemistry and Colloidal Chemistry of Gelatin and Glue (Continued). Is Gelatin a Distinct Chemical Entity? The great variation in the analyses of gelatin, and the diversity in the results of experimenters, naturally raises the question as to whether gelatin is a distinct chemical entity. Most experi- ments have been made on gelatins which have been very loosely described, if indeed any description is given at all. Thus D. Jordan Lloyd used ‘‘Coignet’s Gold Label Gelatin” and Jacques Loeb used “Cooper’s Gelatin.” These descriptions, even though fortified by determinations of ash, and hydrogen ion concentra- tion, give no idea as to the chemical nature of the gelatin experi- mented with. Gelatin always contains considerable gelatoses and even some gelatones which are products of its own hydrolysis, but prac- tically no one reports what per cent. these are or even gives the jelly strength, or optical rotation (mutarotation) from which a rough idea as to their percentage might be figured out. It is safe to assert that no one has ever prepared and experimented with “chemically pure gelatin,” assuming that such a thing could be made. From the evidence at present available it seems that gelatin is not a definite chemical entity. W. M. Bayliss,t while making experiments on the action of trypsin on “Coignet’s Gold Label Gelatin,” made some pertinent observations. To a solution of gelatin, trypsin was added. Part was heated to 100° at once to prevent digestion and the other part was digested at 39° for some days. An equal amount of 79 KCI was added to both parts, and the change in conductivity noted— US ES EGS EY 8 1 6,760 gemmhos - Perret GIMPOlAUT fh oc. es less kee cca dese 6 “ (A gemmho is a reciprocal megohm.) ? 1Arch, des Scien. Biologiques, Vol. XI, Supplt., p. 261, St. Petersburg, 1904. 51 52 GLUE AND GELATIN Commenting on this Bayliss says: “Gelatin behaves differ- ently (from caseinogen) ; it has been mentioned already that the presence of gelatin in a solution does not, to any degree worth consideration, effect its electrical conductivity. The products of its digestion, on the contrary, diminish the conductivity of a solution containing electrolytes. In the undigested mixture, in fact, the conductivity was practically the sum of that of the gelatin mixture and that of the KCl; in the digested, on the con- trary, it was far less, owing to the influence of the non-electrolyte now showing itself in the usual way. So that, therefore, changes of some kind take place in gelatin during digestion by trypsin which tend to diminish, instead of increasing, any conductivity due to electrolytes. Amino-acids are conductors to a certain degree,” but their prop- erties as such will not suffice to account for the great increase in conductivity observed. ... It appears, however, that in the case of gelatin, amino-acids are not produced in any appreciable quantity, at all events not within the first two hours of the action of trypsin. We must look elsewhere then for the causes. One of these, viz., the effect of change of physical state, has already been mentioned. The point next suggesting itself is that concerning the inorganic constituents of these proteid and related bodies. The balance of evidence seems to be decidedly in favor of the view that these constituents are, if not actually in chemi- cal combination with the proteid molecule, in such a close state . of association as to be incapable of ionization. It has not been found possible hitherto to prepare an unaltered proteid _ free from ash.* No doubt a considerable amount is usually present as impurity which can be separated by prolonged dialysis.” It seems that besides being a body of variable constitution, gelatin carries impurities which are likewise variable in kind and amount, and which may exercise a potent influence on experi- ments made with it. Most experimenters are not sufficiently careful in defining the moisture content of the gelatin they use, so that doubt exists as to the true strength of the solutions they 2 Kohlrausch and Holborn, “Leitvermégen der Electrolyte,’ 1898. 3C, R. Smith has prepared ash-free gelatin, which is quite a different thing from isoelectric gelatin, for the latter may contain neutral ash-producing salts. Dhéré and Gorgolewski (Compt. rend. 150, 484, 1910), made a demineralized gelatin which was almost ash free. J, A. THE CHEMISTRY OF GELATIN AND GLUE 53 worked with. Thus J. Loeb* made no mention of the ash or moisture content of his gelatin; his calculations were based on 1 per cent. solutions while in reality he was probably using solu- tions of about 0.8 per cent.—an error of about 20 per cent. from this cause alone. Even in his recent papers and book, while he reports ash and moisture, he does not report the quality or strength of the gelatin he used. C. R. Smith * states that ‘working with an indefinite product of varying jelly strength and ash content, it is not surprising to find few reliable measurements of its physical and chemical properties.” Smith, however, states that recent work points to the combination of acids and bases with gelatin, although he does not exclude the possibility of adsorption, and concludes that “we are justified either by reason of correctness or con- venience in referring to gelatin chloride, sodium gelatinate, etc.” The mere fact that satisfactory “stoichiometric” compounds have been produced with gelatins containing all sorts of ash, degradation and other impurities, would seem to indicate that | approximately uniform free or active surfaces or electrostatic fields lie at the basis of the experimental phenomena. Such considerations as those strike at the very root of the experiments and especially of the conclusions of those who like H. R. Procter, J. A. Wilson and J. Loeb ® insist on the formation of definite salts of gelatin. If gelatin is not a definite chemical entity, it is not justifiable to speak of ‘gelatin chloride” and “sodium gelatinate.” By referring to Chapter 2, it will be seen how various runs from different kinds of glue stock may appear in the market as “gelatin.” The analytical results of R. H. Bogue® show that while gelatins and glues have roughly the same general degrada- tion products, considerable differences exist between those derived from hide stock and those derived from bone stock, and there are wide variations in gelatins derived from the same class of stock. The analyses are given herewith, together with analyses of sev- eral glue proteins which Bogue purified by fourfold precipitation with 95 per cent. alcohol. | See e.g. his address before the Harvey Society, Science, N. S. 52, 451 (1920) ; J. Gen. Physiol. 3, 89 (1920). 4a J. Am. Leather Chemists’ Assoc., Oct., 1922. 5 See ‘“‘Proteins and the Theory of Colloidal Behavior.” . ©Chem. Met. Eng. 23, 61 (1920). 54 GLUE AND GELATIN It should be pointed out that even these analyses do not show the full extent of the variations to be expected in gelatin, for we must also take into account differences in the degree of hydrol- ysis, which begins the moment the gelatin forms, and continues during its manufacture and may even continue while experiments are being made with it. (See Chapter VIII.) . Hipp Give ANALYSES (BoaueE) Figures show per cent. of total nitrogen in each fraction Aver- A, A, Hy; Hyg Hs HH, age AMIMONIS PINGS wae sires 5 163 1.89 3.20 2.15 244 249 2.90 Melanin iN c6 oe eee sett 0.53 0.50 0.74 0.53 060 0.63 0.59 Cyetiner nos .cce tees 0.00 0.00 0.00 0.00 0.00 Trace 0.00 Arginine&N oni 2eG a2. 13.27 1628- 13.76 13.72 13.50.1237 Pistidimes Nie ero zat 1.3) a0 3.19 3.0L 245 1.59 2.19 LVSINO SUN cha oe een 8.17 8.50 8.58 7.40 800° 8722 7.97 Amino N in filtrate... 58.87 55.17 55.00 57.90 58.02 56.10 56.84 Non-amino N in filtrate 17.00 15.53 1558 1526 15.24 1520 15638 Total regained ..... 100.78 99.17 100.05 10027 100.25 96.10 100.02 BonE GuuE ANALYSES Aver- By Bz Bs B, Be B, age Ammonia N-....... 4.43 4.49 4.57 4.49 448 5.04 4.55 Melanin’ Nevt.+.e2 ea 0.74 1.18 1.03 0.82 0.76 0.95 0.91 Cystane ANS) a2 are 0.00 0.00 0.00 0.00 0.00 Trace 0.00 Arginine Nurs. sce 13.32 1282 1828 12.74 1356 Seige Histidine N ........ 1.60 0.54 1.52 1.44 1.58 4.02 1.78 Tefsine oN, Bos oe as 7.18 8.23 7.18 8.57 9.42 9.13 8.28 Amino N in filtrate. 56.90 58.15 57.30 57.58 5430 5340 56.27 Non-amino N in fil- trate. eee 1621 1518 15.32 1436 1590 eit Total regained .... 100.38 100.59 100.20 99.80 100.00 100.40 100.21 PuririeD Protein, Fish GLUE AND IsiIneLAss ANALYSES H; Protein By Protein Fish Glue Isinglass AINMONIS aNgev: ees oe eer 1.33 Bey? 5.15 3.98 Melanin WN. eseereee se ae 0.78 0.74 Ee. 0.68 Crstinie aN 05 sca eee 0.00 Trace - Trace 0.00 Arginine ONO a in asa eee 12.61 10.96 13.80 14.20 Histidine sNoc seek eee ee 0.82 2.24 2.04 2.00 Lysine aNt got eee ae 8.34 8.60 8.58 6.06 Amino N in filtrate....... 60.00 58.05 60.20 58.65 Non-amino N in filtrate... 15.49 15.47 9.66 13.59 Total regained ......... 99.37 99.63 100.55 99.49 Bogue summarizes his conclusions as follows: “Hide and bone glues vary slightly in their chemical con- stitution on passing from grade to grade. This is interpreted to THE CHEMISTRY OF GELATIN AND GLUE 55 signify that as the boiling of a glue progresses some ‘foreign substances’ as chondridin, keratin, mucin, etc., become hydro- lyzed and enter the solution. These have no value in glue, and by adulteration lower the value of the product. “Hide and bone glues differ from each other in their chemical constitution. This is taken to signify that the protein complexes from which the glues are derived are different in the two cases, or that the ratio of the several constituents is different. “Glues of different stock within both hide and bone series show a difference in constitution, which is attributed to varia- tions in the protein complexes of the several stocks. “The differences between hide and bone glues are found in the protein fraction to a lesser extent, and in the proteose-peptone fraction to a greater extent than obtained in the whole glues. “Tf the purified protein from the highest grade animal glues may be considered as pure gelatin, then. it follows that isinglass is not a pure gelatin, or if the assumption be made that isinglass consists only of gelatin, then the purified animal glue protein contains impurities. “The lower the grade of a glue, the further is it removed in constitution from that of the purified protein, and, if this protein be assumed to consist only of gelatin, then the gelatin content of glues diminishes with the grade, and substance from which the hydrolytic products are obtained consists of gelatin in de- creasing amounts, as the grade decreases. “Fish glue corresponds more closely in its composition to low- grade bone glue than to any other. “Fish glue and isinglass show a fundamental difference from animal glues in their low ‘non-amino nitrogen of the filtrate’ (proline, oxyproline, and tryptophane).” S. E. Sheppard and 8. 8. Sweet * have shown that impurities exert a powerful influence on gelatin. They brought ash-free gelatin to different De values, and found that it showed maximum rigidity (jelly strength) at p,, 8, at all concentrations. The curves show a “shoulder near the isoelectric point (which they say is p,, 4.8) but no definite maximum, or minimum. The curves were greatly altered by traces of aluminum salts, enough to give as little as 0.01 per cent. of Al,O, on the dry gelatin 7 Science 46, 28 (1922). 56 GLUE AND GELATIN displacing the maximum on the alkaline side and producing a secondary maximum at p,, 5. S. E. Sheppard has just published ™ an interesting discussion of gelatin in photographic processes, and is actively investigating many collateral questions. He reports that experiments with F. A. Elliot show that with high and low p,, values gelatin shows perceptible hydrolysis even at 50° C. He also observes that “on washing out strong electrolytes from gelatin, it will tend to approach the isoelectric point, but if hydrolyzable sub- - stances are present, the gelatin will retain unequal amounts of the basic or acidic constituents, the excess depending upon conditions.” The Significance of Hydrogen Ion Concentration. The importance of H ion concentration (p,, value) is so stressed to-day, that a brief consideration of the principles in- volved in its determination will not be amiss. Pure water is slightly dissociated according to the eee =H, 2H’ + OH This is a reversible reaction, and therefore at any temperature an equilibrium is reached where the rate at which water mole- cules split into ions, equals the rate at which these ions recom- bine to form water. The Law of Mass Action® demands that the rate of ion formation depends upon the concentration of un- dissociated water (C H,0)? while the rate of ion recombination is proportional to the product of the concentrations of positive and negative ions (C ,,. XK Co,,-). Therefore Cio as Oe 4 OF Cy X Con or ——_._—- = a constant value callea' kh: Ci O 2 But with water under ordinary conditions the dissociation is so small that the amount of undissociated water may be Bene to remain constant. That is 2 X Coy =k X (Cyo) = K the constant of snes for water. — 7a J, Ind. & Eng. Chem. 14, 1025 (1922). §’ How far the Law of Mass Action may be applied to colloidal solutions is still an open question, THE CHEMISTRY OF GELATIN AND GLUE 57 By several methods the value of Khas been to be 10% at 2°; that is C,, = C,,, = 10%, which means that in pure water there is a concentration of 1/10,000,000 for both H and OH ions. Assuming that the Law of Mass Action continues to hold, if C,, 1s increased by addition of an acid or acid salt which disso- ciates in water, the value of C AE must diminish proportionately, since C,, XC, yy Must remain constant. Correspondingly, the ace of an Seat or an alkaline salt diminishes the number of H ions.® Therefore a solution is acid when C,,>10%, or C,,, <10", au alkaline when O10? or Cte >107. It must be remem- bered, however, that the deviation of H or OH ion concentra- tion from the value 107 depends upon the extent to which the added acid or basic substance is dissociated, and represents therefore not the total acidity or alkalinity but rather the aes reaction, 1.e. the degree of acidity or alkalinity. Thus while 7 HCl and 0 acetic acid will each neutralize equivalent quantities of — ” NaOH, their respective H ion concentrations at 10 18° are given in the following table from Michaelis." Degree of Normality Cy+ Equivalent p jy value MGhloo S005 tiaras 1.0 8.0 X 107 0.10 0.1 84 X10? 1.07 0.01 9.5 xX 10* 2.02 0.001 9.7 X 107 3.01 0.0001 98 10- 4.01 Acetic Acid ...... 1.0 43 X-10° aad 0.1 - 1.36 X 10° 2.87 0.01 A310 oe 0.001 1.36 X 10% 3.87 DTS |S ae 1.0 0.90 X 10% 14.05 0.1 0.86 X 10°* 13.07 0.01 0.76 X 10” 12.12 0.001 0.74 X 10 T1138 ®“'The discrepancies observed, especially in strong acids, between the ionic concentrations as measured by conductivity methods on the one hand and with the hydrogen electrode on the other, suggest that the quantity which we call hydrogen ion concentration may not actually represent the degree of normality of hydrogen ions in the solutions under test. Some have preferred to call this quantity ‘activity.’’’ Leeds and Northrup Co, Catalog 75 (1921), p. 6. 10 “‘Wasserstoffionen Konzentration.” 58 GLUE AND GELATIN As the table shows, HCl is a “strong” acid and acetic a “weak” acid, as measured by effective reaction. But since the H* and Cl part company and ionize more readily than do H* and CHCOO’, it is evident that the latter bond is less readily relaxed in aqueous solution; so that acetic acid is really stronger from this point of view. This means that the anion largely if not entirely controls the p,, value of an acid. Therefore even if as J. Loeb claims, the p,, value be the main factor controlling the swelling and general colloidal behavior of gelatin through the Donnan equilibrium, the wltxmate cause is to be found in the specific nature of the anions of acids or the cations of bases, because they control the H ion concentration (p,,). They thus form the raison d’étre of the Hofmeister series. Thomas and Baldwin! showed that salts change the Pass In the case of hydrochloric and sulphuric acids, sodium chloride . increases the p,,, whereas sodium sulphate decreases it. These measurements were made after waiting two days. Since it is Inconvenient to express and read H ion concentra- tions by products of the character given in the table above, Sgrensen proposed to perform the multiplication logarithmically and use the logarithm of the product after dropping the minus slgn. | . Thus an acidity 10 times as great as that of pure water (that is C,, = 10 X 10°‘) would be represented by log 10 + log 107 = 1 — 7 = — 6; and dropping the minus sign, p yz (Sorensen’s ex- pression) = 6. In hke manner an acidity 100 times greater than that of water would be C,,= 100 XK 107’ =2—7—=—45, ‘or eats Number of times H (or OH) ion concentra- PHY value tion exceeds that of pure water Le ee ee ee 1,000,000 Dn ha aig iatema ee 100,000 FE meet Sec 10.000 2 PA Gye Ee A NES He eo 1,000 Beis ep eiae pete do 100 acid side Geer ck ee ante 10 Po i EGS SF ae ae Q pure water .....07..4 eee pees ON ey 10 J OUR eS eet eee 100 alkaline side LOS eee ee eee 1,000 LTRS BOL eee 10,000 1D 490 2 beer 100,000 10a J, Am. Chem. Soc. 41, 1981 (1919). THE CHEMISTRY OF GELATIN AND GLUE 59 This table shows two facts which must not be forgotten; first that an increase in p,, means a decrease in H ion concentration, and second that the acidity does not decrease numerically with the p,, value but decreases in logarithmic ratio. It is obvious, therefore, in plotting experimental results, that the P,; values should be reconverted into true values, i.e. H-ion concentration, or else logarithmic paper should be used. If the p,, values are laid off numerically as abcissas or as ordinates, the resulting curve will be logarithmically compressed, and the presence of inflections or of cusps in the true curve may thereby be over- looked. In any event such curves give a wrong idea of the relative hydrogen ion concentrations." : The table shows another point of great interest which is often overlooked, which is that p,, = 4.7 (the isoelectric point of gelatin), represents an extremely slight acidity. Thus ordinary distilled water prepared in the laboratory still, has a p,, of about 5.5, but when boiled to expel the CO, absorbed, the reac- tion drops to Py =7 (neutrality). On the other hand reel has ap, = only 2.02. The most striking changes with gelatin occur between about Py 2 and Py + on the acid side, and Pz —9 and p,,—11 on the alkaline side.’ These represent comparatively weak acidity or alkalinity; but with a slightly ionized acid like acetic, so much acid must be used to produce a Pp , = about 2, that the specific solubilizing action of the acid on gelatin becomes very marked.1*? 1 Many of J. Loeb’s experimental results are given solely in erroneous curves of this character, and should be given in tabular form or the curves redrawn. For full details regarding apparatus for determination of H ion concentration, the formulas whereby the electrical readings are converted into Cy,, and the precautions to be observed, the reader is referred to standard texts, and espe- cially to W. M. Clark’s book, “The Determination of Hydrogen Ions,” Balti- more, 1920. 122Thus C. R. Smith found that 1 gram of air dry ash free isoelectric gelatin swelled in pure water to 7-8 ec., while the maximum acid swelling was 48 cc. and the maximum alkaline swelling was 30 ce. Low jelly strength gelatins give decreased swelling.—Smith, J. Am. Chem. Soc. 43, 1860 (1921). 1za See e.g. J. Loeb, ‘‘Proteins,”’ p. 80. 60 GLUE AND GELATIN The Titration Curve of Gelatin. Dorothy Jordan Lloyd and C. Mayes,'* in order to determine - the amount of HCl or NaOH which would combine with a cer- tain weight of gelatin, determined potentiometrically the dif- ferences between the H ion concentrations of 1 per cent. gelatin solutions containing different percentages of acid and alkali. The gelatin was Coignet’s Gold Label, reduced to Py = 46, ash between 0.00 and 0.06 per cent. Knowing the H ion concentra- tion of equally concentrated systems containing no gelatin, they calculated by the formula of Blasel and Matula‘™ the concen- trations of HCl removed by the gelatin from independent solu- tion. The Blasel and Matula formula is based on the erroneous assumption that the ionization of HCl is the same whether or not the normality of H and Cl are identical, so they made a correction for this, using the Cl ion concentration as a factor. But the value of the Cl concentration is based on the further assumption, which was also made by Procter and Wilson *° that “gelatin chlorid” is completely dissociated. This latter assump- tion is not supported by.the figures of Bugarsky and Lieber- mann, on Cl ion concentration.t** Jordan Lloyd and Mayes remark that this assumption is liable to lead to an increasing error with higher H ion concentration. | On plotting their results with the normality values of H ion concentration as abscissas and the amount of HCl fixed as ordinates, they obtained not a simple smooth curve, but one con- sisting of two, possibly three distinct regions, indicating that “up to a given concentration of hydrogen ions, a group of hydroxyl ions having approximately equal ionization constants is involved; beyond this concentration, and up to a second fixed value, a second group approximating to a second constant is involved; and beyond this again there is slight evidence of a third group. The factors required in order to bring the second — and possible third groups into conformity with the generalized statement of the law of mass action are not yet fully known.” 28 Proc. ROY AS 0G. By 93, .09NGLo22) 14 Biochem. Z. 58, 417 (1914). 1% Trans. Chem. Soc. 109, 307 (1916). iba Pfliiger’s Arch, 72, 51 (1898). THE CHEMISTRY OF GELATIN AND GLUE 61 Their results for the amount of NaOH fixed had greater experi- mental errors than did the determination of acid fixation, for the gelatin was probably attacked by the alkali in the presence of the spongy platinum of the electrode. The curve was made by using normality values of NaOH as abscissas and the amounts of NaOH fixed as ordinates. The curve rises abruptly, appar- ently seeking a maximum when OH- = about 0.005 N, but then begins to rise sharply again, giving a very steep curve. “Hence it is obvious that in alkaline solution gelatin does not behave simply as a weak acid dissociating in accordance with the law of mass action. It is possible that this abrupt rise accompanies some structural change of the protein molecule such as Dakin had shown to occur in strong alkaline solution.” 1° Jordan Lloyd and Mayes then discuss the mechanism for the fixation of ‘acid and alkali. While in solutions of HCl less than 0.02 N it is possible that gelatin may bind the acid by its free amino-groups, “with increasing concentration of acid, more acid is bound than can be accounted for on this hypothesis, and it is therefore necessary to consider what part the imino-nitrogen of the peptide linkage (— COHN —) could play. Robertson *" states that the acid binding properties of the proteins are not much increased by hydrolysis, and we have found that the reac- tion of a 1 per cent. solution of gelatin, which was found to be Py, = 1.8, had only changed to Py = 112 after 12 hours at 100° C. This change is of the same order as the experimental error of the method, nevertheless hydrolysis of the gelatin had occurred during the heating in the strong acid solution, as was shown by the fact that the gelling power had been destroyed. It seems, therefore, that the peptide linkage can function as an acid-binding group. ...It seems clear that some of the — COHN — groups can act as basic groups combining with acids. What role, if any, other groups (such as the hydroxyl groups of the hydroxy acids) in the molecule play in acid fixation is still unknown. It will be necessary to follow experimentally the fate of the chlorine ion before final decisions are possible. . . . The theory that proteins fix bases by means of their free carboxyl groups has given way on accumulation of evidence that there are not enough of the latter to explain the quantitative reactions. 16 J, Biol. Chem. 13, 357 (1912-13). wz “The Physical Chemistry of the Proteins.” 62 GLUE AND GELATIN ... The possibility of linkage at some of the hydroxy groups of the substituted amino-acids, serine and hydroxy-proline, is not to be ignored. Hydrolysis of gelatin by caustic soda has been shown to increase slightly its basic binding power, a fact which suggests that not all the — COHN — linkages are as potent as base fixers as the free —COOH — groups. Loeb?® has shown that bases react with gelatin at the same hydroxyl ion concen- tration in equivalent proportions. This fact shows that the reaction is lonic, and that the compounds formed are of the nature of ionizable salts. Loeb only worked with solutions whose alkalinity is less than p,, = 9. His experimental values correspond very closely to our values over the same range. . There is both qualitative and quantitative evidence to show that in the same protein (gelatin) the mechanism of fixing acids is different from that of fixing bases.” D. I. Hitchcock *** determined the combination of gelatin with hydrochloric acid. Using 1, 24%, and 5 per cent. gelatin solutions with varying acid content, he subtracted the ise values of the acid-gelatin solutions from those of solutions con- taining equal amounts of pure acid, the difference being con- sidered as indicating the amount of acid combined with the gelatin. He reports that about 0.00092 mol. of hydrochloric acid combine with 1 gram of gelatin between Pj, 1 and 2, but found no evidence of a discontinuous section in the titration curve, as did Lloyd and Mayes (vide supra). Oakes and Davis?® found a definite relationship between the grade of a gelatin and the amount of acid required to titrate it over the range Pj, 4.7 to p,, 3.5. To make this change in reac- tion they report as follows: Jelly Strength “Molecular Weight” Gelatin Py 423°C. cc.02M HCl of Gelatin er iste tee cee eres 59 Broce 1,319 Din Ne 5 get 430 2.85 1.753 Digg ee ake is whee 565 2.70 1,852 ae eels ose ee Ripa 800 2.48 2,016 MI ay Sean te Mex ag Rote op 1,025 2.40 2,083 J. Gen. Physiol. 1, 379, 487 (1919). a J, Gen. Physiol. 4, 7383-9 (1922). 19 J, Ind. Eng. Chem. 14, 706 (1922). THE CHEMISTRY OF GELATIN AND GLUE _ 68 They think these figures ‘indicate the order of magnitude of the molecular weight, and its progressive increase with the grade of gelatin,” although they state elsewhere in the same paper: “Since there is probably no gelatin that is not made up of a series of the products of hydrolysis of the original tissue, no gelatin can have what may be called molecular weight. What is determined is the mean molecular weight of all the various fractions making up the gelatin sample.” Nevertheless they believe that a (presumably chemical) compound is formed at the maximum point of the viscosity—p ,, curve. Since Oakes and Davis do not give the ash content of these gelatins and since lower grade gelatins often contain more ash than higher grades, it is probable that the variations in the above table are to some extent due to the ash. T. B. Robertson found that profound hydrolysis did not materially change the acid fixing capacity of a gelatin. Furthermore the increase in vis- cosity of isoelectric gelatin by the addition of acid, is readily accounted for by swelling of its complexes without assuming the formation of a gelatin “salt.” Oakes and Davis also state: “The difference in ash content of gelatin is, then, the main cause for the lack of agreement between classifying gelatins by vis- cosity and jelly strength measurements, and for a given ash content viscosity measurements may be substituted for jelly strength measurements.” ‘The importance of ash depends not only on its amount, but also upon its composition; in any event, as above indicated, it cannot be left out of consideration. The fact that profound hydrolysis as well as deaminization do not materially alter the acid and base fixing power of gelatin, is an indication that definite gelatin salts and metal gelatinates do not exist, a view further strengthened by the irregularities of acid and base fixation. While the theory that such com- pounds exist may be made to fit many of the experimental facts by assuming conveniently variable dissociation values, it seems more likely to the writer that the fixation of acid and alkali should be regarded as due to adsorption, the extent of which varies as the total free or effective adsorbing surfaces of the molecular complexes are changed by varying conditions, one of which is hydrogen ion concentration. How do H* ions or OH ions function when they produce 64 GLUE AND GELATIN initially an increased swelling of isoelectric gelatin which has already been swollen to its limit in isoelectric water of p,,=7? Even ash-free isoelectric gelatin swells considerably in pure water P yz — 7, proving that its “molecules” or molecular groups have considerable residual attraction, which enables a high-class air- dry gelatin to hold about 20 per cent. of water that can be driven off by drying at 110°. Obviously with an acid or alkali the ions are concentrated within the gelatin particles, for the concentra- tion of the external solution (unless in too great an excess) is diminished. The original view of W. B. Hardy 2° (also accepted by J. Perrin) was that “as the H and OH ions have by far the highest specific velocity the colloidal particle will entangle an excess of H ions in acid and thereby acquire a + charge and of OH ions in alkali and thereby acquire a — charge. These charges will decrease the surface energy of the particles and thereby lead to changes in their average size.24_ This would mean that, in the kinetic equilibrium existing, H (or OH) ions would accumulate within the “molecule” of gelatin, and cause its distension by electric repulsion along the lines suggested by Tolman and Stearns. Hardy ”? later regarded the H and OH ions as being held by chemical attraction, but pointed out that “though one may speak of the colloid particles as being ionic in nature they are sharply distinct from true ions in the fact that they are not of the same order of magnitude as are the molecules of the solvent, the electric charge which they carry is not a definite multiple of a fixed quantity and one cannot ascribe to them a valency, and their electrical relations are those which underlie the phenomena of electrical endosmose.”’ Even though Hardy ** express the view that proteins form salts with acid and alkalis, he expressly points out that “the reactions are not precise, an indefinite number of salts of the form (B)n BHA being formed where the value of n is deter- 2 J. Physiol. 29, 29 (1903). 21 Very likely the small size as well as the speed of these ions is also a factor and as H- is smaller and speedier than OH- we should not be surprised if we find that the minimum swelling of gelatin is not in pure water, but very slightly on the alkaline side of the isoelectric point. J. A. 22, J, Physiol. 22, 251 (1905) ; Proc. Roy. Soc. 79, 413 (1907). 2'T, B. Wood and W. B. Hardy, Proc. Roy. Soc. 81, 38 (1909). THE CHEMISTRY OF GELATIN AND GLUE _ 65 mined by conditions of temperature and concentration, and of inertia due to electrification of internal surfaces within the solu- tion.?* It seems, as Tolman and Stearns suggest, that the H (or OH) ions, adsorbed at the free surfaces of the micellular groups, dis- turb whatever balance exists at the isoelectric point, and the resulting -- (or —) charges at these free surfaces cause repul- sions which still further distend the gelatin. But how shall we account for the fact that after reaching a maximum at about p,, = 3, further additions of acid or alkali cause contraction again? In higher concentrations the “salting out” action of the acid or alkali dehydrates the gelatin and causes shrinking. A. Kuhn,?> who investigated the swelling of gelatin in over fifty aliphatic and aromatic acids, found that the swelling is controlled by four factors: A 1. Individual Swelling or Hydration. eure . | increasing B He a ae (Sol formation) i ociinenieadiin nein | of acid. C 4. Dehydration, or Flocculation. The maximum is determined by groups A and B, and is defined as the point where the increasing swelling or hydration due to rising acid concentration is overbalanced by sol formation and hydrolysis, which also increase at the same time.?° Since the forces which govern adsorption are molecular, i.e. due to residual atomic fields of force (see Chapter II, p. 28), it is natural that they should be greatly influenced by the chemical constitution of the molecular groups which form the gelatin “molecule.” Part of these residual fields are balanced in hold- ing the molecular groups together as the gelatin “molecule,” but the remaining moieties are free to attract and hold ions or groups *4 According to R. Keller (Kolloid Z, 27, 255 [1920]), the degree of dispersion exerts a vital influence on the chemical and electrical properties of colloids. Thus very dilute solutions of methylene blue move to the cathode despite addi- tion of alkali; but in somewhat coarser dispersion it moves partially or entirely to the anode. The reversal of charge of colloids is not to be expressed in terms of H ion concentration, for chemical combination of stoichiometric character hardly exists between colloids and ions whose weight and volume are roughly as 1,000,000,000 to 1. J. A. 25 Kolloidchem. Beihefte 14, 147 (1921). *6 Kuhn’s experiments must be repeated for they were made with gelatin con- taining 3.13 per cent. of ash. 66 GLUE AND GELATIN having an opposite charge, and thus “fix” acid, alkali, etc., in proportion to the effective free fields of the acid and alkali, which of course vary stoichiometrically; that is, according to their respective effective valencies. Therefore the fixation of acids and bases in their stoichiometrical ratios is no proof that a definite chemical compound has been formed, i.e. that the gelatin has combined stoichiometrically. Nor is it surprising that on the acid side of the isoelectric point, when gelatin has a net positive charge it should act as an acid and vice-versa. As Wo. Ostwald 2? puts it, in adsorption there comes into play not the stoichiometric mass, but the active mass, which means the sum of the chemically active surface layers. When true molecular or “crystalloid” dispersion exists, the ratio between these two is unity; but in colloidal aggregation the active mass is only a fraction of the stoichiometric mass, the value of the fraction depending on the size of the particles of the colloid, i.e. on its specific surface. Furthermore according to N. Schilow,”* adsorption of electrolytes depends not only on the sign of the adsorbent, but on the nature of the electrolyte and solvent, that — is on the collective properties of the system. He was able to reverse some adsorption series merely by small additions to the solvent. Furthermore the Donnan equilibrium and its consequences which J. Loeb 2° relies upon to prove the formation of definite chemical compounds, are just as well explainable on the basis of a kinetically balanced adsorption, as on the basis of “chemi- cal compounds” which hydrolyze. Loeb says (loc. cit., p. 63): “It can be stated as a result of all these titration experiments, that the ratios in which acids and bases combine with proteins are identical with the ratios in which acids and bases combine with crystalloids. Or, in other words, the forces by which gela- tin, egg albumen, and casein (and probably proteins in general) combine with acids and alkalis are the purely chemical forces of primary valency.” Now while the first statement is Justified by the fact that simple ions like Cl-, and Na* always combine according to their _valence fields of force, the second statement is unwarranted, and ** Kolloid Z. 80, 254 (1922). 22 Z. physik. Chem. 100, 425 (1922). 22 “Proteins.” _-THE CHEMISTRY OF GELATIN AND GLUE 67 is not putting the first statement in other words; for the second statement assumes that the gelatin also combines with primary valence forces, which is not the case. The gelatin compounds lack the precise and definite character connoted by the present meaning of the expression ‘chemical compound.” Chapter 5. The Structure of Gelatin Solutions and of Gelatin Jellies. The structure of jellies has long been a moot question, and is not yet settled. In an historical review Zsigmondy * says that the oldest theory assumed a porous structure for distensible bodies; water penetrating the pores, was held by capillary or by molecular attraction, and thus produced swelling. In 1858 Nageli? advanced his micellular theory in which dis- tensible bodies were assumed to be made up of tiny anisotropic crystal-like aggregations of molecules called micells, which retain their identity in solution. The micells are surrounded by a layer of water whose thickness is limited by the fact that the attraction of the micells for each other finally dominates the attraction of the micells for water. The swelling caused by the penetration of the water into the micellular mass thus reaches an equilibrium which may be shifted by changes in temperature, pressure, etc. Frankenheim * had also expressed similar views. O. Biitschli * advanced what is known as the honey-comb theory. His first experiments ° included soap solutions and emulsions of oils. By hardening gelatin jellies with alcohol or chromic acid, Biitschli was able to demonstrate microscopically a honey-comb structure; but it is probable the structures demonstrated are in this case artifacts produced by the action of the hardening agents ® used with the intention of rendering visible a structure 1R. Zsigmondy, ‘““The Chemistry of Colloids,” p. 68, trans. by E. B. Spear, 1927; 2C¢. von Nigeli and S. Schwendener, ‘‘Das Mikroscop” (2d ed.), Leipzig, 1877; C. von Nigeli, ‘‘Theorie der Girung,’’ Munich, 1879. 3**Die Lehre von der Kohision,”’ Breslau, 1835. 40. Biitschli, ‘“‘Ueber den Bau quellbarer Koérper usw.,’’ Géttingen, 1896; “Untersuchungen itiber Structuren,”’ Leipzig, 1898; ‘‘Untersuchungen itiber die Mikrostruktur kiinstlicher und natiirlicher Kieselsiiuregallerten,” Heidelberg, 1900. 5“ Untersuchungen tiber mikroscopische Schiume und das Protoplasma,’”’ Leip- zig, 1892. ° See H. Bechhold, ‘Colloids in Biology and Medicine,” trans. by J. G. M. Bullowa, Ch. XXIII, New York, 1919; also W. Pauli, ‘‘Der Kolloidale Zustand und die Vorgiinge in der lebendigen Substanz,”’’ Brunswick, 1902. 68 THE STRUCTURE OF GELATIN SOLUTIONS _ 69 already existing. Biitschli’s view was supported by G. Quincke,’ who stresses the effect of the surface tension existing between the “oleaginous” phase and a second phase richer in water. Quite similar is the view of W. B. Hardy,® who considered gelatin jellies to consist of two phases, one a solution of gelatin in water, the other a solution of water in gelatin. Van Bemmelen, though first leaning to the micellular theory of Nageli, later agreed with Biitschli, whose work indicated the following dimensions for the diameters (d) of the tiny cavities in silica-gels, and for the major limits of thickness of their cell walls (m): Substance d m US 1.45u 0.152-0.187 (from bamboo nodes) van Bemmelen’s silica gel.. 1.00u 0.27 Biitschli’s silica gel....... 1.50u 0.30u Zsigmondy °® and his pupil, W. Bachmann, hold what may be termed the fine grained theory, which does not materially differ - from that of Nageli*° Zsigmondy believes (loc. cit., p. 70) that upon the relatively gross heterogeneity of Biitschli there is super- imposed a much finer discontinuity. Bachmann (loc. cit., p. 99) found that gelatin benzolgels and alcogels show the same type of curves as van Bemmelen found in silicic acid gels, in the _.course and hysteresis cycle of their vapor pressure isotherm. | “The application of the theory of capillarity to solidified gelatin jellies. permits an approximate calculation of their vacant spaces. On the average they are from 30 to 100 times smaller than the honey-comb spaces made visible by Biitschli in such jellies by coagulators. Biitschli’s honey-comb structure, whose spaces of 700 to 800 wu are enormous when compared with the truly amicroscopic dimensions here concerned, can play no part as factors depressing the vapor tension, and are therefore not re- sponsible for the hysteresis cycle of a gel, which is one of its special characteristics.”” Bachmann therefore concludes that the real gel structure is much finer than Biitschli’s “honey-comb.” N. Sutherland + advanced what may be termed the semplar 7 Drude’s Annalen, 1902 and 1903. 8Z, f. phys. Chem. 33, 326 (1900) ; Proc. Roy. Soc. 66, 95 (1900). ®R. Zsigmondy, Physik. Z. 14, 1098 (1918). 10 See W. Bachmann, Kolloid Z. 23, 85 (1918); also Zsigmondy, “The Chem- isty of Colloids,” p. 127 et seq.; p. 224 et seq. 1a Proc. Roy. Soc. 79B, 130 (1907). 70 GLUE AND GELATIN theory. ‘The molecules link up by their atomic electric charges, forming a three-dimensional pattern or semplar, which is re- peated many times in each particle. | W. Moeller 7 considers gelatinization as a kind of crystalliza- tion, in which thread-like crystals traverse the jelly in every direction and thus form a net-like lattice of thread-like crystals. Based largely on microscopic and ultramicroscopic evidence, many investigators advance a similar view. Thus Bogue *? considers gelatin made up of ‘‘streptococcal threads” of molecules. It must be remembered, however, that the ultramicrons seen in the ultramicroscope, are only diffraction images of particles smaller than a wave length of light; they can therefore never be microscopically resolved. The cocci-like appearance is no criterion of the actual shape of the particles; and as Wo. Ostwald ** points out, according to the Frauenhofer-Babinet prin- ciple, “holes” reflect the same as discs, 1.e. both show as “grains” in the ultramicroscope. Bogue *** has elaborated his views on the structure of gels, besides reviewing several other theories, as well as studying the influence of electrolytes of varying H ion concentration, and of the valence of the combining ion upon gel strength, viscosity, swelling, foam, alcohol number and turbidity. He reports that the greatest opacity results from the largest aggregates of least swollen particles, which coincides with the view advanced on p. 78 as to the complex nature of gelatin particles, and with Alexander’s zone of maximum degree of colloidality. _ D. Jordan Lloyd +4 adopts and extends the general view of Hardy, and believes that a gelatin gel consists “of two phases, solid and liquid, and two chemical states of gelatin, viz. gelatin per se and gelatin in the form of soluble salts. Such gels there- fore are three component systems, the components being water, gelatin, and an acid (or base). ... The process of gelation is therefore pictured as follows: gelation will only occur on the cool- ing of a sol which contains in solution isoelectric gelatin, and gelatin salts in equilibrium with free electrolytes. As the sol 1 Kolloid Z. 28,11 (1918). | 122R, H. Bogue, Chem. Met. Eng. 23, 61 (1920). See also J. Am. Chem. Soc. 44, 1843 (1922). 1% Kolloid Z. 22, 80 (1917). wa J, Am. Chem. Soc. 44, 1842 (1922). 4 Biochem. J. 14, 165° (1920). THE STRUCTURE OF GELATIN SOLUTIONS = 71 is cooled the insoluble isoelectric gelatin is precipitated in a state of suspended crystallization and forms a solid framework throughout the system. The more soluble gelatin salts remain in solution, and by their osmotic pressure. keep the framework extended. Gels therefore are two-phase systems, the solid phase consisting of isoelectric gelatin, the liquid of gelatin in the salt form.” Isoelectric gelatin, therefore, where the contractile forces of the framework are unopposed, should be unstable and should squeeze out the liquid phase. Jordan Lloyd?> has pro- duced such a contracting clot of isoelectric gelatin and finds that it shows numerous small spheres about 0.5 uw in diameter, like the spherites described by Bradford.*® What Jordan Lloyd calls ‘‘suspended crystallization” is an indication of the protective or crystal-inhibitive action of a - portion of the gelatin solution, for isoelectric gelatin as Loeb 1” has shown is inert and insoluble in cold water. The facts sup- port the general view of E. Jordis,1® that electrolyte impurities are essential to the stability of gels. In the case of gelatin, ow- ing to its very high protective action (gold number), surpris- ingly minute percentages of hydrogen- or hydroxyl-ions are able to produce sufficient “gelatin salt”? to stabilize the essentially unstable isoelectric gelatin; and, as Jordan Lloyd’ observes, the purest water obtainable is still an electrolyte, and is alkaline to gelatin, and therefore will react with it to produce “salts.” The highly purified gelatin produced by Field+® was evidently not absolutely free from stabilizing substances, since its jelly was stable; it gave, however, an opaque jelly which was made trans- parent by traces of acid or alkali, and even by carbon dioxide absorbed from the air. The formation of a gel by Jordan Lloyd’s isoelectric gelatin would seem to indicate that, contrary to her contention, the for- mation of a gelatin salt is not necessary to the formation of a gel, even although the salt may be necessary to stabilize the gel when once formed. Hence only the dispersed substance and water are really essential to gel formation with gelatin, a typical 15 Biochem. J. 14, 584 (1920). 16 Biochem. J. 14, 91 (1920). 7 J. Loeb, J. Gen. Physiol. 1, 41 (1918). 18 JZ, Electrochem. 8, 677 (1902). 19 Ada M. Field, J. Am. Chem. Soc. 43, 667 (1921). 72 GLUE AND GELATIN “emulsoid,”’ as is the case with ferric hydroxide and other so- called suspensoids. H. G. Bennett *° believes that a jelly contains a continuous network of water under a great compression due to the con- tractile forces of surface tension. ‘The higher the degree of dis- persion of the particles, and the greater the concentration, the greater the proportion of the water present in the gel will be in the compressed state, and therefore the greater the viscosity, finally culminating in rigidity. Swelling is caused by the electro- static repulsion of similarly charged adsorbed ions, which repul- sion, however, diminishes more rapidly as swelling proceeds than does the contractile force above referred to. A balance is con- sequently reached, which is influenced by the “lyotrope” influence of dissolved substances that affect the compressibility of the water. Procter *! criticizes Bennett’s theory, and points out that his own theory of acid swelling (see p. 93) accounts quantitatively for the phenomena observed. He also makes the justified objec- tion that surface tension itself causes compression.?* Procter further observes that any existing compression cannot cause a large increase in viscosity, and that electrostatic repulsion is out of the question because the colloid particles are neutralized by the formation of an electrical double layer. The reason charged particles move in electrophoresis is that the layer is continuously displaced in a direction opposite to that of the motion of the particle. H. R. Procter ??* in speaking of the structure of gelatin jellies said: “True homogeneity can only be postulated of a hypotheti- cal fluid, and certainly not of any atomic structure, or even of the atoms themselves. The dilute solution of any substance must have considerable spaces of solvent between the molecules,” whereas “large organic chains may cohere without separation from the solvent, where the smaller and more definitely polar molecules of a crystalline substance would form rigid crystals in which only a definite proportion of solvent would be included 20 J, Soc. Leather Trades Chem. 2, 40 (1918). 217, Soc. Leather Trades Chem. 2, 73 (1918). 22The compression nevertheless does exist, although it is due not to surface tension, but to the very forces that produce surface tension, namely the specific attractions or residual fields of force of the atoms or molecules involved. 22a Seventh Int. Cong. of Appl. Chem., 1909. THE STRUCTURE OF GELATIN SOLUTIONS 73 as water of crystallization. The true issue is therefore not whether jellies have a discontinuous structure, but whether the network is so fine that the constituents are within range of each other’s molecular forces, or so coarse that these forces may be wholly or mainly neglected.” Procter does not here consider that both conditions may co- exist, and that the “solvent”? may in addition contain selected portions of a complex mixture. F. C. Thompson ?”” believes that gelatin solutions ‘consist of a network of solid gelatin, molecular, or at least extremely fine, with pure water in the interstices.” To this view a similar criticism applies as to that of Procter. An indication that the water in gelatin jellies is “available,” is found in Graham’s observation that diffusion occurs in jellies almost as freely as in pure water, and in Dumanski’s observa- tions 7° that the conductivity of electrolytes shows the same effect. Thompson, however, regards gelatin solutions stronger than 0.18 per cent. as. solids because they resist indefinitely a small shearing strain. Wo. Ostwald ?* believes that the process of swelling repre- sents the reverse of syneresis. “The coarser structure of the solid is, as it were, broken up; in other words, coarse aggregates are divided into the primary particles of which they are com- posed. As N. Gaidukow ** has found the ultramicroscopic parti- cles of a gel become smaller in the process of swelling, or at least lose their highly refractive character. But in the process of swelling there occurs another change which may, under certain circumstances, actually run counter to the increase in dispersion. The individual particles absorb the medium in which they are swelling; they become solvated. ‘This increases the size of the particles and so fluid droplets may be formed. ‘The two changes, in other words, the combination of increase in degree of disper- — sion with a change in a type of the dispersed substance from the side of the solid to that of the fluid, seem most characteristic of the process of swelling.” 22b J, Leather Trades Chemists’ 3, 209 (1919). 22e J, Physic. Chem. 60, 553 (1907). 23 “Theoretical and Applied Colloid Chemistry,’’ 1917, trans. by M. H. Fischer, p. 100. 24See N. Gaidukow, ‘‘Dunkelfeldbeleuchtung und Ultra-mikroskopie in der Biologie und in der Medizin,” Jena, 1910; Kolloid Z. 6, 260 (1910). 74 ) GLUE AND GELATIN Here Ostwald tacitly assumes the existence of a secondary structure in the larger particles of gels, which is also the view held by Zsigmondy.2> Experiments made by J. Alexander *° with karaya gum reduced to various degrees of fineness support this view; for increase in viscosity and, later on, gel formation, accompany the hydration and swelling of the gum fragments. “In fact, with hydrophile or emulsoid colloids, as the dispersed phase becomes less viscous by swelling, the golloids as a whole becomes more viscous. Viscosity may also increase as ultra- microns condense or aggregate, as is the case in cooling gelatin.” (Alexander, loc. cit.) Ostwald believes that gelatin gel is a two-phase liquid system. He says (loc. cit., p. 103): “In gels produced by swelling, I do not know of an instance in which the dispersed elements are solid or crystalline in character. They are, apparently, always liquid.” E. Hatschek 27 has shown mathematically that this theory is untenable. It should be borne in mind that no substance is inherently gaseous, liquid, or solid. These three classic states of matter depend upon the proximity to each other of the constituent atoms or molecules of the substance in question. This proximity con- trols the degree of their attraction, their state of aggregation or dispersion, and the extent or practical cessation of their kinetic motion. It in turn is controlled mainly by temperature, pres- sure, and atomic or molecular forces (‘“‘chemical” and “physical” forces), especially of solvents, which may render the state of aggregation permanent within certain limits. There is no sharp line to be drawn between liquids and solids, as may be seen in the case of a cooling gelatin solution; and although the transition from the liquid to the gaseous state seems abrupt, we have liquids of all degrees of mobility. Whether we regard a substance as solid or liquid will therefore depend upon the tests or criteria we fix for these states. Furthermore substances liquid when in mass, need not neces- sarily act so when dispersed in or adsorbed by another substance. Small mercury globules act like a soft “solid” metal, and col- loidal mercury acts like a suspensoid. Bridgman ?* has shown 23 Z,. physik. Chem. 98, 14 (1921). 276 J. Am. Chem. Soc. 438, 434 (1921). 7. Hatschek, Trans. Faraday Soc. 12, 17 (1916). 2p. W. Bridgman, J. Franklin Inst., March, 1914. THE STRUCTURE OF GELATIN SOLUTIONS = 75 that water under high pressures is solid even at ordinary tem- peratures, and there are many reasons for believing that the water adsorbed by gelatin and other colloids no longer acts as a mobile liquid. In considering gel formation we cannot neglect the kinetic factor. The ultramicroscope shows that as particles grow smaller their Brownian motion increases rapidly; so that there comes a point where a small increase in temperature will change the gel into a fluid. Stirring, rubbing or other mechanical separation will suffice, as may readily be demonstrated ultramicroscopically with agar gel, from which a sliding cover glass dislodges ultra- microns that assume active motion. The Ultramicroscopic Evidence. Let us now consider the ultramicroscopic evidence in the case of gelatin solutions and jellies. When warm, pure gelatin solutions appear practically homo- geneous, but on cooling there forms a submicroscopic or amicro- scopic heterogeneity, depending on concentration. The largest particles are seen in 0.5 to 1 per cent. solutions which set to weak jellies and show flocks of microns and submicrons. Below 0.1 per cent. and over 6 per cent., no ultramicrons are visible, al- though the polarization of the Tyndall beam proves the presence of amicrons. W. Menz”* followed ultramicroscopically the formation of gelatin gels. As a 0.5 per cent. gelatin solution cools, numerous submicrons appear and join to form flocks. Shortly before the submicrons appear the field becomes luminous, and the new phase appears suddenly in the form of tiny drops.*° Zsigmondy, under whose direction much of this work was done, states: ** “On warming, a motion on the surface of these drops can be seen and they either become invisibly small, or the con- tours gradually fade and the place where the drops were is now characterized for some little time by a glimmering zone. Evi- 2 Z. physik. Chem. 66, 129 (1909); P. P. von Weimarn, Kolloid Z. 4, 133 (1909) ; 6, 277 (1910) ; “Grundztige der Dispersoidchemie,’’ Dresden, 1911; W. Bachmann, Inaug. Diss. Géttingen, 1910; and von Lepkowski, Z. physik. Chem. 75, 608-614 (1911). 30 The converse of this is seen in the case of the digestion of partly coagulated egg albumen by pepsin. See J. Alexander, J. Am. Chem. Soc. 32, 680 (1910). ' 41“The Chemistry of Colloids,” p. 226. 76 GLUE AND GELATIN dence of the small diffusion in the liquid is afforded by the fact that on cooling, the drops may be obtained on the spot where they disappeared. In fact, two particles that were prevented from uniting by warming, may be so far restored that they may still unite after the cooling process has been carried out. In contradistinction to the case of gelatin, submicrons from crit- ical systems unite to form a homogeneous phase even after the cooling. The drops are circular, large, and have no such vari- ations in form as are so prominent in the case of gelatin par- ticles.” | The actual facts are probably by no means as simple as most of these explanations of gel formation assume. The structure of the dispersed phase of gels seems to be at least duplex, if not even more complicated, and seems to be controlled largely by an equilibrium between two opposing forces—(1) the attraction of the molecules or ultimate particles for each other, (2) the attrac- tion of the molecules or molecular groups for the dispersion medium, which is water in the case of hydrosols. Molecular aggregation proceeds to a certain point dependent mainly on temperature, pressure and chemical nature, and the primary particles thus formed unite to make larger aggregates or second- ary particles.2? The adjustment of this equilibrium takes time, which accounts for the fact observed by F. Stoffel ** that quickly chilled gelatin and slowly chilled gelatin exhibit different perme- ability to the same diffusing substance, but become equalized upon standing several days at room temperature. This slow annealing or hysteresis is evidently the consequence of a pro- eressive aggregation. It is only to be expected that gelatin solutions of varying concentration will yield gels of different interior structure, and therefore dry gelatins of different water absorbing capacity. Thus L. Arisz (loc. cit., p. 88) found that 10 and 20 per cent. jellies swell much more than 50 and 80 per cent. jellies, and W. D. Bancroft ** mentions unpublished experiments of Cartledge showing that 8, 16, 24 and 32 per cent. gelatin jellies, when dried to 96 per cent., each took up water at a different rate. “8See W. Mecklenberg, Mitt. K. Materialpriifungsamt. 37, 110 (1919); The Svedberg, Proc. Faraday Soc. (1920); Chem. Met. Eng. 24, 26 (1921). 33 Inaug. Diss. Ziirich, 1908. **W. D. Bancroft, “Applied Colloid Chemistry,” p. 250. THE STRUCTURE OF GELATIN SOLUTIONS 77 Gelatins made from dilute solutions are more bibulous,*® and swell more than those made from more concentrated solutions. Probably their “secondary” particles are smaller and more numerous, which means that they possess a greater “free” sur- face. Polariscopic Evidence. C. R. Smith ** has shown that at 35° gelatin in 3 g. per 100 ce. solution exhibits a specific rotatory power (a), of — 120.6° to — 123.5°, or of — 141° figured on a moisture- and ash-free basis. When cooled at 15° or below, (a), is found to be about — 272°, or — 313° on a moisture- and ash-free basis. The ratio between (a), at 35° and 15° is thus 2.21 to 1, and holds for the best grades of commercial gelatin whether made from bones, hides or Russian isinglass. Smith attributes this mutarotation to a thermo-reversible equilibrium Sol form A= Gel form B, which to him appears to be a bimolecular reaction, disturbed to some extent by some other reaction, possibly of a monomolecular nature, which takes place at the same time. Between 35° and 15° both forms co-exist, and levorotation, signifying increasing formation of the gel form B, closely parallels increase in viscosity. Davis and Oakes *** find that the transition point Sol form A = Gel form B lies between 38° and 38.1°, and by interpolation fix it at 38.03°. M. H. Fischer (private communication) considers this the transition realm of water in gelatin to gelatin in water. C. R. Smith *’ has studied polariscopically and by jelly strength tests, the effect of salts and acids on the sols gel equilibrium. ‘They change both the final state and the velocity with which it is reached. Sulphates displace the equilibrium toward the gel side between 15° and 35° C. and the maximum jelly strength is rapidly reached at low temperatures. Chlo- *>This may be one reason why some people prefer “thin cut’’ glues, another reason being eaSe of solution. 38 J. Am. Chem. Soc. 41, 185 (1919). 36a Clarke H. Davis and Harle T. Oakes, J. Am. Chem. Soc. 44, 464 (1922). 87 Private communication of unpublished work. | | | 78 GLUE AND GELATIN rides, bromides, and iodides lower the viscosity of gelatin and shift the equilibrium toward the sol side. Salts and even weak acids do not, full strength being reached if a low temperature (about 5° C.) is maintained for a sufficient time. Therefore equilibrium rotation with sulphates is larger than with pure water, whereas with iodides it is less. The facts resemble those met with in the dynamic allotropy of sulphur,?* and indicate the formation, with decreasing tem- perature, of larger molecular aggregates, which, however, need not be chemical compounds in the ordinary acceptation of the term. It is interesting also to note that L. Arisz®® found that the Tyndall phenomenon in gelatin increases in intensity with fall- ing temperature, and is also dependent on the previous history of the gelatin, which controls the size of its particles. In the case of colloidal metals, the attraction of the metal molecules for each other is so powerful that unless an adsorbed layer of protector or ions intervenes, the aqueous films are squeezed out, and there results a more or less water-free metal sponge, bathed in practically metal-free water. With most oxides and sulphides the mutual attraction of the molecules is not so great, probably because the main attractive forces have been satisfied in the formation of the chemical compounds in question, or the attraction for water is greater; and therefore the tendency to gel formation is greater. G. Varga * estimated that particles of stannic acid gel 12.7 wu in diameter contain only 1g their volume of massive stannic oxide, the remaining % being mainly water. The micellular complexes even of dry gelatin, contain a very large amount of water, the higher cae holding most. Complex Structure of Gelatin. With gelatin we have a mixture of large and highly polar molecules containing NH, and COOH groups, which probably form adsorption +! aggregates that constitute the primary parti- cles of gelatin. These primary particles have an interior struc- ture with extremely fine capillaries, because the residual attrac- 33 WwW. E. S. Turner, “Molecular Aggregation,” p. 92. 39. Arisz, Kolloidchem. Beihefte 7, 1 (1915). * Kolloidchem. Beihefte 11, 1 (1919). 41'The larger and more complex the reacting masses the more “physical” rather than “chemical” does the reaction appear to be. THE STRUCTURE OF GELATIN SOLUTIONS 79 tions of the constituent molecules are relatively weak and are unable to displace the adsorbed water films which make the colloid hydrous and hydrophile. It is furthermore probable that the “fluid” surrounding the primary particles is not pure water, but is an aqueous solution containing a larger portion of the more soluble constituents or those with smallest molecules.*? The primary particles form secondary groups in this “fluid,” which fills the larger capillary spaces produced thereby. In- crease in temperature, or the addition of certain salts (e.g. CaCl, NaNO,), cause dispersion again into smaller or primary particles which is mainly reversible; but continued or high temperature or the presence of much acid and especially alkali, seems to attack the primary groups and bring about what we term hydrol- ysis, a splitting up of the adsorption complexes into their con- stituent polypeptides, and ultimately degeneration into amino- acids.** S. E. Sheppard and F. A. Elliott 44 see no need of postulating a sub-microscopic but supermolecular structure in gelatin, at- tributing any “structure” to an environment impress. Since P. W. Bridgman by mere pressure produced a new black allotropic form of phosphorus, and demonstrated that the same very high pressures produced in water molecular aggregates which persisted for days, it is reasonable to assume the existence of a sub-microscopic but supermolecular structure in gelatin, at. the surfaces of which great compression is known to occur. In a technical paper on “Colloidal Fuels” S. E. Sheppard 4# made a suggestion as to the emulsoid colloid state, which was developed more fully in a letter to “Nature.” 44° The essential feature of the hypothesis put forward was that the micelles, or plurimolecular units of such colloid systems, are formed, and their growth and aggregation determined by “the orientation of “ Segregations of this kind are common, especially in soaps and alloys. See Jerome Alexander, “Colloidal State in Metals and Alloys,’ Trans. Am. Inst. Min. and Met. Eng., Vol. 64 (1920); Chem. Met. Eng., January, 1922. # W. Mecklenberg attributes the differences in the nature of stannic acid gels to variations in the size of their primary particles. 45. Am. Chem. Soc. 44, 873 (1922). F s4a, J, Ind. Hng. Chem, 13, 37 (1921). These remarks are taken from an advance copy of a paper entitled ‘The Interfacial Tension between Gelatin and Toluol,” by 8. E. Sheppard and §..8S. Sweet, to appear in J. Am. Chem. Soc. My thanks are due to Dr. Sheppard for his courtesy. 44b “The Nature of the Hmulsoid Colloid State,’ Nature, March 17 (1921), | a oe 80 GLUE AND GELATIN definite atom groups, entirely in the sense of the theory of molecular orientation due to structure proposed for surface and interfacial tension phenomena by W. B. Hardy,**¢ W. Harkins 444 and J. Langmuir.**¢ “The genesis of a micelle, as plurimolecular unit of a colloid system, may be regarded as a consequence of equilibrium, usually incomplete, between homochemical solution forces and hetero- chemical forces, the former tending to dissociate and decompose the chemical molecule, the latter resisting decomposition. In the case of proteins the most probable general type of linkage, according to R. H. A. Plimmer ** is of the form NH,.CHR.CO. (NH.CHR.CO),.NH.CHR | COOH when n refers to the degree of polypeptide condensation and R is an alkyl or other substituent group. On the hypothesis sug- gested here we may, imperfectly, represent the redistribution of | this in the presence of water for the polypeptide chain by © Aqueous Zone HOH HOH fe Fe o™ wf Z f aN 7 Fa < O H O H O C ——_——_ H C N C tae a fi wwe ws CHR (CHR) CHR (CHR) CHR Lipoid Zone pe In this the arrows indicate the direction of an imagined plane or intra-molecular interface 7 separating the hydrophile groups 5 2 which are consolute with water (in virtue of residual affinities tending to complete the amino and carboxyl groups), from the hydrophobe or hydrocarbon groups—CHR. Not only in one and the same protein molecule, but also to a variable extent between molecules, we may admit that this primary orientation leads to mutual attraction between water-soluble and 44c Proc. Roy. Soc. 81A, 610 (1912). 44d J, Am. Chem. Soc. 39, 354 and 541 (1917). ue J, Am. Chem. Soc. 38, 2221 (1916): . 44f “Chemical Constitution of the Proteins,’ II. p. 2. THE STRUCTURE OF GELATIN SOLUTIONS 81 water-insoluble groups respectively. Without any actual cleav- age of the molecule, we have orientation and a stratichemical field of force which is of a similar character, in essence, to crys- tallization, but results in incomplete instead of complete equi- librium. The hydrocarbon or lipoid atom groups will approach the fluid on the solid state according to molecular weights and constitution; hence the system may be likened, in one aspect, to a sub-molecular emulsion, the lipoid groups tending to form interconnected sheets of atom-groups necessarily permeable to water and water solutes, although mechanically developing a stress resisting rupture in virtue of the fields of attraction and repulsion induced. The micelles are the smallest plurimolecular _ units thus built up. “The following brief survey indicates the present status of the question. Somewhat destructive criticism of the foregoing hypothesis by J. W. McBain ‘#2 was shortly followed, in the same journal **" by N. K. Adam’s observations on mono-molecu- lar films of palmitic acid on water and aqueous alkali solutions. They confirmed the theory of the orientation of soap molecules in surfaces and micelles, suggested by Harkins, Davies and Clark.*4 Further, the structure of the soap micelle proposed by Adams was quite in accord with Sheppard’s suggestion that orientation determined the growth of the micelle. More recently, J. Loeb *4i has explained the stability of protein solutions and the difference between gel formation and precipitation by ref- erence to an orientation hypothesis of the protein molecule. Loeb’s “watery” groups and “oily” groups correspond respec- tively to the “hydrophile” and “hydrophobe” or “hydrocarbon”’ groups of Sheppard’s note. Finally, it may be noted that E. J. Witzemann, in an interesting paper *** considers that orientation at surfaces, as shown by soaps, is of less importance for proteins and polysaccharides. Generally, however, his argument sup- ports a chemical view of the biocolloids. “Whatever the increased consideration gained for the hypothe- sis by these contributions, it remains actually a working hypothe- sis, to be tested by definite consequences capable of experimental “zg Nature, loc..cit., p. 74. 4h [bid., April 28, 1921. “41 Loc. cit. 44j “Proteins and the Theory of Colloid Behavior,” p. 283 (1922). “4k J, Phys. Chem, 26, 201 (1922). 82 GLUE AND GELATIN verification. These consequences reach in two directions,—on ’ the one hand, the behavior of emulsoid colloids to their thermo- dynamic environment,*! on the other, fundamental chemical changes (oxidation-reductions, substitution, etc.). On the first count, the surface and interfacial tensions of emulsoid colloids . are of particular interest. It is known that on shaking weak solutions of gelatin, with immiscible solvents such as benzole, gasolene, toluene, etc., gelatin, still considerably hydrated, tends to be thrown out and aggregated as an interfacial layer.**™ “Tt appeared desirable to investigate this more fully, in par- ticular as a function of hydrogen ion concentration. The prop- erties of gelatin as an emulsifying agent for kerosene have been studied by H. N. Holmes and W. C. Child “ in relation to. (a) the surface tension of the gel-oil interface, (b) determination of whether or not gelatin is adsorbed to form a concentration layer around the oil droplets and (c) viscosity of the solution. The present investigation, while not at variance with their results, shows that in such studies the hydrogen-ion concentration may be a determining factor. This is to be expected, but the relation of the property to p,, in the present case is somewhat different from those instanced by Loeb and others. It will be remembered that in the cell protoplasm we have a complex lipoid-protein interface, so that the property in question is physiologically important, as also industrially, in relation to certain processes for preparing glue and gelatin.” Although the original polypeptides or amino-acids composing gelatin may have a limited power to crystallize, in mixtures we — are confronted with what appears to be a general tendency on the part of substances of different crystallization speed to inter- fere with each others normal crystallization. This tendency seems to be due to the fact that in crystallizing, all substances must pass into or through the colloidal zone where they are apt to be adsorbed by larger particles; it is exhibited by glasses and metals, by soaps, and by mixtures of fatty acids, and tends to keep the mixture in a state of fine aggregation. Some substances, assuming an iso-colloidal state, are able to 441 On the orientation theory, their electromagnetic environment. | 44m Winkelblech, Zeit. f. angew Chem. 13, 1753 (1900); ef. also W. Bancroft, “Applied Colloid Chemistry,” p. 260. 4in J, Am. Chem. Soc. 42, 2049 (1920). THE STRUCTURE OF GELATIN SOLUTIONS ~ 88 interfere with their own crystallization (auto-protection) .*® ‘Hardy found that 5-dimethylaminoanilo-3, 4-diphenylcyclo-1, 2 dione, upop cooling its solutions in organic solvents, gives gels which gradually become crystalline. (See also p. 49.) Fre- quently substances whose crystallization is interfered with, as- sume the form of tiny globulites, as is the case with lactose for example. Concentrated solutions of sucrose crystallize with difficulty and act somewhat like a “glue.” ‘The tendency toward crystallization is markedly inhibited by colloidal protectors such as glue, gum arabic, etc.*® From the evidence at present available, the following picture may be drawn of the formation of a gelatin gel from any ordi- nary warm solution of gelatin: (1) The hot solution contains adsorption complexes of poly- peptides (“gelatin molecules”) which possess residual unsatisfied free fields of force, and a powerful idioattraction. ‘These com- plexes with their adsorbed ions, are dispersed in a “fluid” con- taining, in still finer dispersion, hydrolysis products of the origi- nal gelatin complexes, and ions from the dissociation of salts (including “salts” of gelatin), acids, or alkalis; and also contain- ing the undissociated salts, etc. (2) As the temperature drops, and thermal agitation dimin- ishes, the gelatin “‘molecules” begin to aggregate. As these ageregations increase in size, their Brownian motion diminishes, until they finally form clustered, almost motionless, masses, which, if the concentration is not too small, are on all sides within the range of each other’s molecular attraction. That is, although they are actually separated by adsorbed aqueous films, they practically “link arms” to form what D. Jordan Lloyd calls a continuous solid phase. _ (3) The size of these molecular aggregations and the size of their tiny pores, will vary with the nature of what is adsorbed at their interfaces; that is, will vary with the nature of the “im- purities” present. Speed of chilling and tempering also exert a syneretic influence, just as they do.with metals. Viscosity and jelly strength will vary with particle size, there being a zone of maximum colloidal effect.*% 46See eg. W. B. Hardy, Proc. Roy. Soc. London (A), 87, 29 (1913); J. Alexander, J. Ind. € Eng. Chem. (19238). 46 J, Alexander, J..Soc. Chem. Ind. 28, 280 (1909). 46a See Jerome Alexander, J. Am. Chem. Soc. 484 (1921). 84 GLUE AND GELATIN (4) The remaining “fluid” or dispersing phase, which sur-. rounds the molecular complexes, will be in a state of kinetic equilibrium with the molecular complexes, so far as concerns particles or ions diffusible into the pores of the latter. This dispersing phase will contain most of the water, the highly solu- ble products, and those ions which are too large to enter the pores of the “solid” phase or which are unadsorbed.** (5) The adsorption of ions (especially hydrogen or hydroxyl ions) tends up to a certain point to separate the gelatin “mole- cules” constituting the molecular groups ** and thus enables the groups to take up more water. Beyond this point, “salting-out,” sol formation and hydrolysis predominate.*** ‘The same effect may also be accounted for on the basis of the Donnan theory.**> In a heterogeneous mixture of complex groups such as are found in gelatin solutions or jellies, it is very unlikely that there is any definite arrangement of molecules into threads, chains, or strings. Since molecular groups adjoin each other in every direc- tion, it is only natural that microscopic or even ultramicroscopic examination reveals what seem to be spheres, which, according to the focus of the instrument, may appear to form elongated groupings. ‘Those familiar with the limitations of optical instru- ments will understand that these apparitions are only diffraction images of irresolvable particles. In fact, Scherrer has shown with the X ray spectrometer that gelatin is truly amorphous.*® Comparing gel structure to a “pile of shot,” °° “anastomosing threads,” ®+ “thread-like crystals,” °* “streptococcal threads,” *8 hardly gives one a correct picture; for while the imagination may isolate such groupings from the mixture of molecular groups, they have no real existence. It is true that the polar nature of the molecules may tend to produce some kind of orientation, and that some chain-like structures may be formed; but in gen- eral the tendency does not establish itself, and the incidental 47 Some (eg. H. R. Procter, J. A. Wilson and J. Loeb) believe that gelatin forms definite salts and that a Donnan membrane equilibrium exists. 48Tolman and Stearns, J. Am. Chem. Soc. 40, 264 (1918). 48a A, Kuhn, Kolloidchem. Beihefte 14 (1921). 48b J. Loeb, ‘Proteins and the Theory of Colloid Behavior.” 49P, Scherrer, Nach. Ges. Wiss. Géttingen, 1918. 50 Bradford, Biochem. J. 12, 382 (1918). 3 T. B. Robertson, “The Physical Chemistry of the Proteins,” 1918, p. 302. 52 W. Moeller, Kolloid Z. 23, 11 (1918). 8 R, H. Bogue, Chem. Met. Eng. 23, 61 (1920). THE STRUCTURE OF GELATIN SOLUTIONS — 85 ‘formation of chains or threads is not an essential of gel forma- tion. Jelly formation occurs even in emulsions where there is no evidence of chain structure. In the welter of conflicting attractive forces and closely packed molecules, the weak residual attractive forces remain unsatisfied, so that there may be a state of stress which is a cause of elasticity of the jelly. With very ‘dilute solutions of gelatin, however, polar grouping in chains probably takes place to a considerable extent, as ultramicro- graphs of such solutions would indicate.** D. Jordan Lloyd ** has followed the action of hydrochloric acid, sodium hydroxide and sodium chloride on the gelling power of gelatin purified by dialysis at the isoelectric point. Taking the minimum quantity of gelatin required to produce a gel after standing at 15° for 48 hours, she found the minimum concen- tration of pure gelatin to be 0.8 percent. Hydrochloric acid lessens the gelling power, showing maximum reduction at P 5, 2-3, and again at higher acidity than p,, 0.7. Sodium hydroxide causes slight decrease between p,, 10-12, and above this prevents gelatinization. Though no simple relation between sodium chloride content and gel power was evident, neutral salts oppose the action of H ions. The fact that gelatin may be altered by “molecular bombard- ment” was shown by E. Miihlenstein,®> who exposed a gelatin layer 46 u thick to X rays from polonium, and found, after soak- ing in water and drying, a permanent depression of about 22 u in the exposed portion of the gelatin. As the depression is invisible prior to the soaking, it is not purely mechanical, but is evidently due to some change in molecular structure. A. Tian ** found that wave lengths of quartz-mercury ultra- violet light of 3,000 A which coagulate albumin, did not affect dry gelatin and only fluidified the jelly. Gelatin jellies, upon being strained, show double refraction, an evidence of anisotropic structure. 54 J. S. van der Lingen, J. Franklin Inst. 191, 651 (1921), finds that the pseudoisotropic layers in such anisotropic liquids as p-azoxyanisole, p-azoxy- phenetol, anisaldazine, and ammonium p-cyanobenzalaminocinnamate (Stumpf’s ester), do not possess a space-lattice, and show no evidence of being micro- crystalline. 54a Biochem. J. 16, 530-45 (1922). 5 Arch. sci. phys. nat. 2, 423 (1920). 58 Compt. rend. 151, 219 (1910). 86 GLUE AND GELATIN P. W. Bridgman’ subjected gelatin jelly to a pressure of. 9,000 kilos per sq. cm., and found no visible change except that the gelatin was cracked into rather large lumps, doubtless because at this pressure water freezes into one of the four varieties of pressure-ice, making the gelatin jelly so rigid that it could not accommodate itself to the shape of the containing vessel. The addition of alcohol to gelatin solutions dehydrates the micellular groups, converting the “emulsoid” gelatin into a “sus- pensoid”’ opalescent solution which shows ultramicrons.°® W. O. Fenn °° has studied the effect of electrolytes upon this change. 57 Private communication. 58 See e.g. O. Scarpa, Kolloid Z. 15, 8 (1914). 588 Proc. Nat. Acad. Sci, 2, 5384 (1916); J. Biol. Chem. 22, 279, 34, 141 and 415 (1918). Chapter 6. The Influence of Various Factors on the Swelling of Gelatin. Many factors influence the amount of water absorbed by dry gelatin, and also the speed with which the water is taken up. Among these are: The ratio of the free surface area to the volume; the hydrogen 10n concentration; * the temperature of the system; * the elastic modulus of the gelatin; * the ratio of the mass of the gelatin in the system to the mass of dissolved electro- lyte;® the previous history of the gelatin, upon which depends its internal structure; *® and the effect of unclassified substances like urea, pyridine and the amines. _ When gelatin swells in water the volume of the swollen gelatin is less than the combined volumes of the original gelatin and the absorbed water.’ Heat is developed by the absorption of water,® indicating that there is a compression or condensation of the water coincident. with its entrance within the capillary and molecular spaces of the gelatin. This “heat of swelling” is analogous to the heat developed when moisture is absorbed by superdried peas, starch or dextrin. H. G. Bennett ® attributes the heat liberated by relEAe gelatin 1’, Hofmeister, Arch. erp. Path. Pharm. 27, 395 (1890) ; Wo. Pauli, P/fliiger’s Arch. 67, 219 (1897) ; Spiro, ““van Bemmelen Festschrift,’ 1910, p. 261; M. H. Fischer, ‘““‘Das Oedem,’”’ Dresden, 1910. 2Chiari, Biochem. Z. $8, 167 (1911); H. R. Procter, J. Chem. Soc. 105, 313 (1914) ; J. Loeb, J. Gen. Physiol. 1, 41 (1918). ’ Procter and Burton, J. Soc. Chem. Ind. 35, 404. 4Procter and Wilson, J. Chem. Soc. 109, 307 (1916). 5D, Jordan Lloyd, Biochem. J. 14, 149 (1920). This seems to be an illus- tration of the Donnan equilibrium. J. A. 6éWw. R. Hardy, Proc. Roy. Soc. 66, 95 (1900); Arisz, Kolloidchem. Beihefte 7%, 1 (1915) ; see also W. D. Bancroft, ‘“‘Applied Colloid Chemistry,” p. 251, 1921. 7G. Quincke, Arch. f. d. ges. Physiol. 8, 332 (1870). 8H. Wiedemann and C. Ltideking, Wied. Ann. 25, 145 (1885). See also HE. Hatschek, “An Introduction to the Physics and Chemistry of Colloids,’ London, 19138, p. 55. ®°H. G. Bennett, ‘‘Animal Proteins,” p. 204, 1921. 87 ~/ 88 GLUE AND GELATIN (5.7 calories per gram of gelatin) to the compression of the absorbed water, and from the LeChatelier theorem predicts what is actually found—that gelatin swells best in cold water. ‘The faet of water compression determines the rigidity of the gel, and the changes in this compression of the continuous phase ?° determines the surface tension resultant which hinders swelling, and which is one of the two main factors fixing both the rate at which gelatin swells in water, and the final volume attained by the gel” (p. 209). The stiffening, shrinking and “salting out” action of sulphates, tartrates, etc., Bennett considers to be examples of “lyotrope” compression, while the contrary effect is exhibited by iodides, thiocyanates and urea, which may entirely inhibit gelatinization. In this latter respect Bennett’s “lyotrope series” is marred, for calclum, magnesium and zinc chlorides, as well as sodium and calcium nitrates also inhibit gelatinization. Increase in temperature causes an increased heat of swelling, which leads Wilson '” to the conclusion that the heat is due, not to swelling, but to chemical combination between the gelatin and a small portion of the absorbed water. The more likely explanation is that in warmer water gelatin swells more rapidly, . thus producing more heat per unit of time. For as Hofmeister ™ observed, the swelling of gelatin plates has a higher initial velocity, after which it proceeds more slowly to a maximum. Furthermore at higher temperatures the gel particles probably undergo a further dispersion resulting in more free surface. Thus Arisz** observed the following differences in water ab- sorption by gelatin with variation in temperature, the figures | given representing the weight of one gram of swollen gelatin at the time indicated. 1 Bennett here means the water is the continuous phase. But in a jelly it is probable that the dispersed phase is also ‘continuous.’ ‘Bancroft gives as an illustration of such condition a roll of wire fencing standing in the air—the wire is continuous but so also is the air. Strictly speaking, all matter consists of discrete particles. J. A. 1 The other factor, according to Bennett, is the ‘“lyotrope’ (Hofmeister series) effect of salts, etc., upon the compressibility of water. J. A. 2 J, A. Wilson, 8d Report on Colloid Chemistry, British Association A. S. (1920), p. 51. : 13, Hofmeister, Arch. f. Exper. Path. und Pharm. 27, 395 (1890) ; 28, 210 (1891). 1440, Arisz, Kolloidchem. Beihefte 7, 49 (1915). THE SWELLING OF GELATIN 89 Temperature 1st day 2ndday 38rdday 2° 10-— 10 10 — Les 10 10 10 20° 16 18 19 267 33 40 46 30° 35 within the first few hours followed by solution. The figures tabulated are approximate, as Arisz’ results are given in curves which show that most of the water is taken up within a few hours. Hofmeister and Wo. Ostwald have also pointed out the influ- ence of the shape of the piece of gelatin on the degree of swell- ing. Thin sheets not only swell more rapidly, but they also swell more than thick sheets.1° The amount of water absorbed by a piece of gelatin depends very materially upon the previous his- tory of the gelatin which influences its internal structure. (See p. 83, Chapter 5). Procter dried out (presumably at low tem- peratures) three jellies containing respectively 5, 10 and 20 per cent. of gelatin. Upon soaking these in cold water for seven days he found that they absorbed respectively 14.6, 7.7, and 5.8 times their weight of water. Arisz *® gives curves showing the influence of the age of a block of gelatin jelly upon its capacity to absorb water. Freshly prepared jellies absorb most water, which is an indication of the progressive aggregation of the gelatin particles in aging jellies (syneresis), with a concomitant diminution of free surface. Curiously enough, Arisz found, contrary to expectation, that a block of gelatin jelly swollen at 10° loses water when warmed to 20°; and a jelly first swollen at 20° actually swells faster when the temperature is reduced to 10°. These variations in water absorption are influenced by the previous gel history. Appar- ently two opposing factors are at work: Ist, heat tends to produce finer dispersion with greater water absorption; but, 2nd, the heat seems to relax the attraction of the gelatin for the water, probably because of increased kinetic activity. 1% Therefore a ground glue or gelatin would appear to absorb more water than the same product in flake form. Incidently this indicates a source of error in grading on the basis of ‘‘water-absorption,’ and a possible reason why some users who test on this basis, prefer thin cut flakes of glue or gelatin. 4¢ Hoc. cit., p. 57. 17 For further details and experiments on the intermittent swelling and drying of gelatin jellies, the reader is referred to the original paper of Arisz. * See also A. G. Brotman, J. Soc. Leather Trades Chem. 5, 226 (1921). 90 _ GLUE AND GELATIN In general gelatin swells more in the solution of any acid or any alkali than it does in pure water.‘® With very minute quantities of acid there is a slight diminution of swelling, the minimum with HCl being at a concentration of sai’ after n 90.9 swelling beyond that which occurs in pure water. Wo. Ostwald’s results show that both with HCl and KOH the swelling reaches which the curve rapidly rises, so that at there is already a | a maximum about aaF after which it slowly diminishes. According to Fischer (loc. cit., p. 29), if two like gelatin discs are simultaneously immersed, the one in pure water and the other | in x HCl, the superior degree of swelling of the latter is plainly visible at the end of six hours, and is still more marked after a day or two. Then the disc in the pure water is still somewhat yellow and cloudy, whereas the gelatin in the dilute acid is swollen so clear and hyaline, that it can hardly be seen at the bottom of the dish. Lyotrope or Hofmeister Series. Wo. Ostwald inclined to the belief that the swelling was exclu- sively a function of the H-ion concentration of the acid solution, whereas Fischer believes that it is determined by the concen- tration of the H-ions minus the effect of the particular anion concerned. M. H. Fischer’s experiments show that the order of acids in increasing the swelling of gelatin is as follows: HCl>HNO,>CH,COOH > H,S80,>H,BO, “The position of the ‘weak’ acetic acid between the ‘strong’ nitric and sulphuric acids (which two are about equally disso- ciated, and yield a higher concentration of hydrogen ions than the equinormal acetic acid) is by itself an argument against the explanation which considers only the concentration of hydrogen ions.”’ 7° 1K, Spiro, Beitrdége zur chem. Physiol. 5, 276 (1904) ; Wolfgang Ostwald, Pfliiger’s Archiv. 180, 563 (1905) ; M. H. Fischer, ‘“Hdema,” New York, 1910. 19 Fischer, loc. cit., p. 31.- These series of anions and cations are known as the lyotrope (or solution changing) series. They are also termed the Hofmeister series in honor of their discoverer. THE SWELLING OF GELATIN 91 Gelatin is so sensitive to the presence of acid, that highly puri- fied gelatin will swell less in conductivity water than in ordinary distilled water which contains CO,.”° With alkalis there is no initial decrease in swelling. Their order in increasing swelling is as follows: KOH>NaOH>Ca(OH),>NH,OH. Since at the concentrations employed, the dissociation of the first three of these alkalis is about the same, Fischer concludes that the swelling of gelatin in various alkalis 1s dependent upon the OH-ion concentration minus the effect of the cation. Thus calcium is more active in inhibiting swelling than is sodium, while potassium permits the greatest swelling.”* It is well known biologically that bivalent ions counteract the injurious effect of monovalent ions, which often act as poisons. The antitoxic action of polyvalent ions has been demonstrated by Jacques Loeb on the fertilized eggs of fundulus herocltus, a small fish, by R. 8. Lillie on the larval forms of arenicola, a sea annelid, and by Wo. Ostwald on gammarus pulex, the sand flea. Many animals which live in sea-water are killed by sodium chloride solutions isotonic with sea-water. Ostwald has also shown that the swelling of gelatin is much more power- fully depressed by polyvalent ions than by monovalent ions (MgCu(ic) >Sr>Ba>Ca>Mg>NH,>Na>K.2# For anions citrate >tartrate > phosphate >SO,>acetate> I>CNS>NO,>Br>Cl. Thus according to Bechhold ** 0.78 gram gelatin in 100 ce. of 0.05 n HCl swelled until it weighed 14.61 grams. In the pres- ence of 5 Potassium citrate it weighed only 2.84 grams, and in 4 J. Am, Chem. Soc. 43, 1350 (1921). *4a M. H. Fischer (private communication) believes that the position of NH, is uncertain, and that it probably is the last member of the series. °° H. Bechhold, “Colloids in Biology and Medicine” (J. G. M. Bullowa’s trans- lation), 1920. THE SWELLING OF GELATIN 93 the presence of > KCl it weighed about 7 grams. Bancroft *¢ points out that a large part of the decrease in swelling may be due to the diminished H-ion concentration of the solution, and not exclusively to the citrate ion. In determining the H-ion concentration of gelatin colori- metrically, use may be made of the well-known series of indi- eators described by Clark, Lubs and Acree.?* The limitations of such indicators must be borne in mind. Donnan’s Theory. HH. R. Procter 2* gives the following abstract of the theory of the swelling of gelatin in acids, evolved by himself and his pupils, chief among whom is J. A. Wilson: ”° “In equilibrium between a jelly and its external solution not only must all osmotic pressures be equally balanced, but as has been shown by Donnan,’? the electro-chemical condition must be fulfilled that the products of any pair of diffusible anions and cations common to both phases, must be equal. Thus with gela- tin chloride and free acid the chloridions multiplied by the hy- drions must be equal in the jelly and the external acid.*? On the other hand, the osmotic pressures depend not on the products but simply on the swm of diffusible particles present. In the external acid the numbers of hydrions and chloridions are obvi- ously equal, while in the jelly the chloridion of the gelatin chloride is added to the equal hydrion and chloridion concentra- tion of the free acid present, thus making the final concentrations of these ions in the jelly unequal. “Now, as the sum of two unequal factors is always greater than that of two equals giving the same product, or, geometrically the perimeter of a square is always less than that of any other 2W. D. Bancroft, ‘Applied Colloid Chemistry,” p. 254. 27 J. Am. Chem. Soc. 41, 1190 (1919). 23 Hirst Report on Colloid Chemistry, Brit. Assoc. Adv. Sci. (1917), p. 8. 27H. R. Procter and J. A. Wilson, J. Chem. Soc. 109, 305 (1916). M. H. Fischer (private communication) points out that rubber swells in benzol, nitrocellulose in ether-alcohol, and soaps in many organic solvents, although in such cases the existence of a Donnan equilibrium is precluded, for there is no dissociation. J. A. 80 Z. Hlektrochem. 17, 572 (1917) ; Donnan and Harris, Trans. Chem. Soc. 99, 1575 (1911). 31 This is known as a “Donnan equilibrium.” J. A. 94 GLUE AND GELATIN rectangle of equal area, and as the sides represent the osmotic pressure, while the area represents the product, it is clear that the two inequalities cannot at once be completely fulfilled, but in electro-chemical equilibrium the osmotic pressure must be in excess and the jelly must tend to swell unlimitedly and finally to dissolve. That it does not do so is a consequence of its col- loid nature, which depends upon cohesive attractions drawing the colloid particles together to polymerized masses or to a continuous network, and which consequently opposes swelling and solution, while the diffusible ions are held to the colloid ions by electro-chemical attractions, and, as they cannot escape from the jelly, tend to drag it apart and dilute it by absorption of the external acid, from which they expel a part of its acid concen- tration.” “The equilibrium is therefore a very complex one, but finally depends on the excess of internal osmotic pressure being bal- anced against the internal attraction or cohesion of the colloid particles, both ions and molecules. For mathematical discussion the reader must be referred to original papers by Procter and his pupils. It will, however, be obvious that as the external solution becomes more concentrated, the proportion of absorbed acid (or salt) is increased, while that of gelatin chloride is limited to the quantity of gelatin present. ‘The difference of concentration of hydrion and chloridion in the jelly is therefore diminished, and it contracts under the influence of its own in- ternal attractions. “Precisely similar considerations apply to the action of alkalis on gelatin. Jonizable salts are formed by combination of the base with the carboxyl group of the proteid, and the osmotic equilibrium is with the cation and OH instead of with the anion — and H. Neutral gelatin, as an amphoteric body, of course ionizes to a limited extent with water alone, and its dissociation constants are of the same order of quantity as those of the water with which it is in equilibrium. It. is, however, slightly stronger as a base than as an acid, and consequently its neutral point of minimum swelling is slightly on the alkaline side. This 82 The distinction between this view and that of Tolman and Stearn, J. Am. Chem. Soc. 40, 264 (1918), is rather finer than claimed by J. A. and W. H. Wilson, J. Am. Chem. Soc. 40, 8S6 (1918). J. A. THE SWELLING OF GELATIN ' 95 has important bearings on manufacturing practice,** the greatest flaccidity of the raw skin, which is required for the softest leather, being obtained in weakly alkaline liquids. “Tt has been pointed out by Donnan ** that in consequence of the unequal distribution of positive and negative diffusible ions which has just been described, the surface of an acid or alkaline jelly in equilibrium has necessarily an electrical charge or poten- tial, greatest at the maximum swelling, and such charges seem an essential of the colloid state.24* The surface is positive or negative according to whether the diffusible anion or cation is retained in the colloid. Thus gelatin and hide fiber are negative in alkaline and positive in acid solutions, and it will be shown later that this has an important bearing on the theory of leather manufacture. “Wilson *> has extended these facts to a general theory of col- loids and adsorption, showing that all surfaces must possess a potential due to unbalanced chemical forces on the surface; and therefore in a liquid containing electrolytes, must condense ions or particles of the one sign on its surface, and repel those of the opposite sign; and also showing that surfaces must therefore be surrounded with a film of liquid of different concentration to the bulk, to which the same considerations and equations are ap- plicable as to the adsorbed solution of colloid jellies.” Wilson and Kern * report that “gelatin, like collagen, shows two points of minimum swelling with change of hydrogen-ion concentration, one at P,,4-7 and the other at 7.7.” They suggest “that the two points of minimum represent the iso-electric points of the gel and sol forms of gelatin, respectively.” Since Wilson and Kern describe the gelatin they used simply as “high-grade gelatin” without mentioning anything about the percentage or composition of the ash, their experiments must be repeated with gelatin of known purity before their results or deductions can be accepted. Their double minimum may be due to the presence of some adsorbed impurity such as alumina; for alum is very commonly used in preparing the stock or in clarifying the 33 Procter here refers to leather manufacture. J. A. 34 Z, Hlektrochem, 17, 579 (1911). 84a See note 29 above. J. A. 3% J, A. Wilson, J. Am. Chem. Soc. 88, 1982 (1916). sa J, A. Wilson and EH. J. Kern, J. Am. Chem. Soc. 44, 2633 (1922). 96 GLUE AND GELATIN liquors, and as 8. E. Sheppard has shown ** the addition of alumina to an ash-free gelatin superimposed upon a maximum of elasticity at about p,, 8 to 9, a second maximum at about pa Jacques Loeb ** has also discussed the Donnan equilibrium in its relation to membrane potentials and osmotic pressure, and found that Procter’s formula is the correct expression for the Donnan membrane equilibrium, which he thinks determines swelling and viscosity as well as osmotic pressure and electric charge.*” Thermal Expansion of Gelatin. Alan Taffel** has shown that gelatin gels expand regularly with increasing temperature. The expansion curves resemble that of water, but are flatter in proportion to the concentration of the gel, but show no sudden inflection as does that of glass below its softening point. The expansion coefficients, as well as the specific volumes for any one temperature, are linear func- tions of the concentration of the gel. Variation in H-ion con- centration does not affect the expansion coefficient. Irrespective of dilution, one gram of gelatin always exhibits the same contraction at any one temperature. This contraction is 0.073 cc. per gram of gelatin at 15°, and 0.065 cc. at 32°; which indicates that only a fraction of the gel water contracts, the weight percentage being the same for gels up to 25 per cent. Gel contraction is not due to filling up of pores in solid gelatin by water. The curve expressing the relation between concen- tration and the calculated distance between particles, is an hyperbola, whereas the concentration setting-point curve ob- served by Sheppard and Sweet *® shows a double flexure, the rapid rise at 70 per cent. concentration being attributed to the fact that their very large molecular forces begin to come into play. Gelatin lowers the temperature of maximum density of water 35b §, E. Sheppard, S. S. Sweet, and Anber J. Benedict, J. Am. Chem. Soc. 44, 1857 (1922). 36 J, Gen. Physiol. 3, 667 and 691 (1921). 37 Loeb’s views are fully set forth in his book, ‘Proteins and the Theory of Colloidal Behavior,’’ New York, 1922. 38 J, Am. Chem. Soc. 121, 1971-84 (1922). 39 J, Ind. Eng. Chem. 13, 413 (1921). THE SWELLING OF GELATIN 97 by an amount directly proportional to its concentration ex- pressed in grams of gelatin per 100 grams of water. This lower- ing is shown to be due to the ordinary volume changes of dry gelatin with changing temperature, and the variations in con- traction on imbibition of gels at various temperatures. Chapter 7. The Viscosity of Glue and Gelatin Solutions. The importance of viscosity measurements as a means of fol- lowing changes occurring in colloidal solutions, has long been recognized. Thus Thomas Graham in his classic paper entitled “On the Properties of Silicic Acid and Other Analogous Col- loidal Substances”! says: “The ultimate pectization of silicic acid is preceded by a gradual thickening in the liquid itself. ‘The flow of liquid col- loids through a capillary tube is always slow compared with the flow of crystalloid solutions, so that a liquid-transpiration- tube may be employed as a colloidoscope. With a colloidal liquid alterable in viscosity, such as silicic acid, the increased resistance to passage through the colloidoscope is obvious from day to day. Just before gelatinizing, silicic acid flows like an Ciles The instruments used for measuring viscosity must depend upon the degree of accuracy desired and the time and quantity of the solution available. For most scientific investigations the Ostwald viscosimeter ? or that of Couette,® * have been used. The hour glass viscosimeter of H. A. Determan ® is very useful where only small quantities of fluid are available. The well-known Engler viscosimeter is also used, and any simple graduated pipette will serve.® All these depend upon the time required for the fluid to flow through a tube. The MacMichael viscosimeter? operates on the principle of measuring by the angular torque of a standardized wire, the force required to cause two surfaces, one cm. apart, to move 1Proc. Roy. Soc. London, June 16, 1864; also Pogg. Ann. 1238, 529 (1864). 2See Ostwald-Luther-Drucker, ‘“Handbuch fiir physik. chem. Messungen,” Vol. 3, p. 230, Leipzig, 1910. ; % 4H. Couette, Ann. de Chim., Ser. 6, 8, 685. A modified form is described by E. Hatschek, Kolloid Z. 12, 2838 (1913). °>H. Bechhold, ‘‘Colloids in Biology and Medicine,” p. 113. 6 See J. Alexander, J. Soc. Chem. Ind. 25, 158 (1906). 7™J. Ind. Eng. Chem, 7, 961 (1915). 98 VISCOSITY OF GLUE AND GELATIN SOLUTIONS 99 past each other at the rate of one em. per second, at the same time overcoming the internal friction of the liquid under test, against itself throughout the intervening space. Part of the ‘liquid moves with each surface, and the intervening layers shear past each other.’ It would unduly extend the limits of this book to enter into a general theoretical and mathematical discussion of viscosity, for as Hatschek observes,® in the present state of theory, all that can be deduced from viscosity measurements is that some change has taken place, the nature of which is either a matter for specu- lation or for empirical interpretation. Davis and Oakes ® report that “the viscosities of gelatin solu- tions of various concentrations at 40° conform to Arrhenius’ viscosity formula. A note of warning must be sounded, however, against the automatic acceptance of any formula expressing viscosity, with- out considering all the factors influencing the case in question. Mathematics is essential in solving problems, but we should remember that it is only a tool to work with. Granted certain postulates, it proceeds infallibly to direct or collateral conclu- sions. The danger in applying mathematics to chemical and physical problems is that, blinded by its logical perfection, we may accept erroneous postulates or neglect influential factors. Frequently, in Nature, unsuspected factors are discovered which compel us to revise our previous conclusions—for example, the recognition of the vitamines has rendered necessary a careful reconsideration of former experiments upon nutrition and a revi- sion of the conclusions based thereon.?° | Prof. Eugene C. Bingham *® points out the fact that viscosity as commonly reported, consists of several factors: 8 The manufacturers of the instrument, Eimer and Amend, New York, issue a descriptive circular in which an accuracy of within 5 per cent. is claimed, which suffices for commercial work. ®Hirst Report of the British Assoc. for the Ady. of Science, on ‘Colloid Chemistry and its Industrial Applications,’ 1917, p. 2. citrate >tartrate > acetate >chlo- ride>nitrate > bromide > iodide > ae > benzoate > 21Z, physik. Chem. 45, 75 (1908). 2 Kolloid Z. 2, 210 (1907). 23 Kolloid Z. 3, 84 (1908). 24 “Fandbook of Colloid Chemistry,” 2d ed., p. 169. 25 “Colloids in Biology and Medicine,” p. 162. VISCOSITY OF GLUE AND GELATIN SOLUTIONS 107 salicylate. According to Bechhold the action of cations is of smaller importance. The results of R. H. Bogue 2¢ on glues are summarized by him as follows: Practically all the substances added lowered the gel strength. Strong (9N) sodium hydrate had the greatest effect, followed by potassium iodide, strong (9N) sulphuric acid, sodium sul- phate, acetic acid and magnesium chloride. The effect of the others was small. The viscosity was raised constantly by magnesium chloride,. ° chloral hydrate, and sodium silicate. The viscosity was raised to a maximum, after which it fell more or less rapidly, by sodium hydrate, disodium phosphate, and acetic acid. | 2 zs The viscosity was lowered constantly by potassium iodide, sulphuric atid, phosphoric acid, and sodium sulphate. There was no appreciable effect on the viscosity due to sodium chloride and magnesium sulphate. Monosodium phosphate produced a sharp drop of one second at 0.1 per cent., followed by a sharp rise of 31% seconds at 0.5 per cent., after which it rose a little further and then dropped again. The disparities between von Schroeder’s results on gelatin and Bogue’s results on glue indicate perhaps a difference in ex- perimental procedure (temperature and time of heating before taking viscosity, etc.), or else a material difference in the be- havior of glue and gelatin, or perhaps a difference in ‘‘impurities”’ or hydrogen ion concentration. As Ostwald remarks in a foot- note, pure gelatin would, perhaps, show totally different results from those of von Schroeder. In any event it is obvious that the experimental facts need to be carefully redetermined, having © in mind all the variable factors which recent investigations have shown materially effect the properties of gelatin.. Thus the experiments of D. Jordan Lloyd ?* and J. Loeb ** were performed with gelatin containing about 0.1 per cent. of ash. Loeb’s fig- ures (loc. ¢it., p. 35) indicate that one sample of his gelatin contained mainly calcium and iron phosphates. As C. R. Smith 2° 2 Chem. Met. Eng. 23, 61 et seq. (1920). 27 Biochem. J. 14, 584 (1920). 28“‘*Proteins and the Theory of Colloidal Behavior,’ New York, 1922. 229 J. Am. Chem. Soc. 48, 1850 (1921). 108 GLUE AND GELATIN has shown how to prepare absolutely ash-free gelatin, much of the preceding work must be repeated. The view of H. R. Procter, J. A. Wilson and J. Loeb *° is that gelatin forms definite hydrolyzable salts, e.g. gelatin chloride with HCl and sodium gelatinate with NaOH. Loeb believes that the effect of acids, alkalis, and salts on the viscosity, swell- ing, osmotic pressure, and general behavior of gelatin is explain- able on the basis of the Donnan theory of membrane equilibria. Loeb (loc. cit., p. 204) concludes from his experiments that “it ~ seems that the viscosity of the solutions of proteins is primarily a function of the relative volume occupied by the protein in solution,” but that “the difference in the viscosity of solutions of gelatin and crystalline egg albumen cannot be ascribed to differences in the degrees of hydration of the individual protein ions since at the isoelectric point the protein is not lonized.” (Loeb’s measurements were made at or near this point.) Loeb believes that gelatin solutions contain submicroscopic particles of solid jelly, and that a Donnan equilibrium arises be- tween these and the surrounding solution. This equilibrium regulates the amount of water occluded by the submicroscopic particles of solid jelly floating in the gelatin solution, and the high viscosity of gelatin solutions is due to the presence of these swollen particles which increase the relative volume occupied by the gelatin in solution. Loeb here criticizes the theory of Wo. Pauli, who holds * that the viscosity of protein solutions depends primarily upon hydrated protein 1ons. Wo. Ostwald and M. H. Fischer likewise disagree with Pauli, but uphold the aggregation hypothesis which is condemned by Loeb. Strange to say, Ostwald, Loeb, and Fischer draw about the same picture of what happens in gelatin, although they differ as to the mechanism by which the result is brought about. | “The quantities of water which can be occluded in a Gate jelly of gelatin are enormous. If we assume the molecular 30 The views of H. R. Procter and J. A. Wilson are set forth in many journal articles, a good abstract of them being found in the “First Report on Colloid Chemistry and its Industrial Applications,” London, 1917, pp. 5 et seq. (by Procter), and the Third Report, London, 1920, pp. 48 et seq. The various journal articles of J. Loeb are collected in his book, ‘Proteins and the Theory of Colloidal Behavior,’”’ New York, 1922, in which his views and many experi- ments which he believes confirm them, are set forth at length. 31 ‘*Kolloidchemie der Hiweisskérper,’ Dresden and Liepzig, 1920. VISCOSITY OF GLUE AND GELATIN SOLUTIONS 109 weight of gelatin to be of the order of magnitude of about 12,000, a solid gel of 1 per cent. originally isoelectric gelatin contains over 60,000 molecules of water to 1 molecule of gelatin. It is out of the question that such masses of water could be held by the secondary valency forces of the gelatin and water mole- cules. . . . All the experiments described agree with the occlu- sion theory but not with the hydration theory.” ** Occlusion is believed by most physicists and chemists to be due to residual stray fields of force at the surfaces involved. H. Freundlich ** would probably call it capillarity, while some would call it absorption, adsorption, or sorption, the latter term being suggested by McBain ** as being free from theoretical assumption as to its cause. R. 8. Lillie ** observed that neutral salts depress the osmotic pressure of gelatin solutions, a result explained as due to aggre- gation consequent upon the precipitating action of the salts. But salts decrease the viscosity of gelatin solutions, and as Loeb properly concluded from some of his experiments that aggrega- tion increases the viscosity of gelatin ** he argues that the salts cannot cause aggregation. Loeb here entirely overlooks the zone of maximum degree of colloidality referred to on p. 101. Aggregation increases viscosity only up to a certain point or zone, after which further aggrega- tion may reduce viscosity. Below this zone the kinetic motion of the particles seems to be a controlling factor, while above it diminution in the free surface of the particles or in the amount of water they hold, tend to reduce viscosity. Thus karaya gum powder whose water-imbibing capacity has been diminished by heating, yields less viscous solutions than the original gum. The heated gum particles show inferior swelling because their con- stituent submicroscopic molecular groups remain more aggre- gated (that is less dispersed) ; more water remains “free” (unad- ‘sorbed or unoccluded) and the viscosity is therefore less than is the case with the unheated gum. A similar condition exists with minerals like clay, and emulsions like cream; their viscosity increases with subdivision of the dispersed phase. 82 Loeb, loc. cit., pp. 229-230. 33 “*Kapillarchemie,’’ Leipzig, 1909. 34 J. W. McBain, Phil. Mag. (6), 18, 916 (1909). 3% Am. J. Physiol. 20, 127 (1907). 36 J, Gen. Physiol. 4, 97 (1921-2) ; also loc. cit., pp. 16 and 114. 110 GLUE AND GELATIN The importance of the hydration (hydration, swelling, water occlusion) of secondary groups or micellular aggregates in in- creasing viscosity, is obvious, and depends upon the total free surface of these groups. Thus the atoms or molecular groups of metals draw together so powerfully, that the water films about their nascent colloidal dispersions are squeezed out; the dispersed phase is dehydrated and the viscosity very low, most of the water being in the dispersing phase. When the dispersed phase is highly hydrated as with gelatin, gum karaya, etc., the viscosity is high, most of the water being in the dispersed phase. With emulsions, increasing subdivision of the dispersed phase brings increase in active surface and in viscosity, although presumably the individual particles of dispersed oil are hydrated only at their exterior. Comparing these three types, the fact that the gelatin particles have interior surfaces and are at least duplex, becomes evident. With cooling gelatin, however, the zone of maximum colloid- ality or viscosity is approached from the opposite side, and we have an increase in viscosity due to aggregation of the dispersed phase. Sodium salts of the fatty acids also illustrate this ap- proach, as their solutions become more viscous and hold more water with increase in the molecular complexity of the fatty acid.27 From Bechhold’s table (see Chapter 3, p. 50) it may be seen thatthe particles in gelatin solution are of the order of 4 up, so that there is considerable latitude for increase in viscosity due to aggregation, before passing beyond the colloidal zone. Inter- esting results should be obtained by taking the viscosity of gelatin heated to 110° for various periods of time, for the results of Bogue *§ indicate that a rise followed by a fall in viscosity may be expected. M. A. Rakusin *® in a monograph entitled “The Animal Skin as an Amphoteric and Colloidal Protein” quotes the analytical results of von Schroeder and Passler *° showing that animal skins of diverse origin show a remarkable uniformity in ultimate analysis. ‘7 For further discussion of this view see J. Alexander, ‘‘The Zone of Maximum Colloidality. Its Relation to Viscosity in Hydrophile Colloids, Especially Karaya Gum and Gelatin.” J. Am. Chem. Soc. 43, 484 (1921). 88 Chem. Met. Eng. 23, 61 et seq. (1920). 8° Kolloidchemische Beihefte 15, 103-184 (1922). 40 Dingl. polytech. J. 287, Heft 11, 12 and 13 (1893). VISCOSITY OF GLUE AND GELATIN SOLUTIONS 111 Skin b. H N OSB CRVETOZC)) ce. ecko ees 50.47 6.46 17.76 OT. A ee 50.21 6.46 17.78 US edie Aa Se 50.02 6.43 17.67 ia. a ee He ae a 50.20 6.44 17.93 Od 49.90 6.31 17.84 Rhinoceros (average) ........ 50.19 6.37 18.04 OS Eire. ea ede «ars fuse ere 50.31 6.35 17.48 DOSS i ra 50.34 6.38 17.42 SON EO. gpk ble Si ree 50.19 6.49 17.05 PP NIIS Er cle seals acc tlee e's 50.14 6.37 17.38 LUGE ee a ne an 50.26 6.45 16.97 Ch. onl ieee 51.10 6.51 17.05 ON a a 49.91 6.35 17.72 The figures apply to water-free substance. In the case of the ox and rhinoceros the average refers to pieces of hide from dif- ferent portions of the animal. The gelatin was one sold by Gribler for bacteriological purposes. From these figures von Schroeder and Passler concluded that hide substance and gelatin represent a chemical individual, a conclusion in which Rakusin unwisely concurs, for in the light of our present knowledge of isomerism, stereoisomerism, poly- merism, tautomerism, etc., its danger would be obvious even in the case of substances far less complex than the proteins. With ossein and gelatin where we have an as yet undefined complex of polypeptides and amino-acids, the impossibility of such a conclusion becomes immediately manifest. Here again we have a striking instance of the desirability of refraining from rushing to frame a plausible theory which will fit a certain set of facts ‘within the limit of experimental error,” without considering other known experimental facts and the possibility of unsuspected but potent factors. In the case of the proteins colloidal protection is probably such a factor, and as Bismarck once said the things he most feared were the ‘‘im- ponderables.”’ Rakusin states (loc. cit., p. 110) that hide powder, in con- tradistinction to gelatin, contains a small quantity of sulphur that can be split off, for with lead or bismuth salts in the pres- ence of alkali it gives a slight precipitate. Md6rner *! attributed this sulphur to the presence of cystine, but Rakusin claims to have proven that “the sulphur in gelatin is fixed as chondroittin- “1 Oppenheimer, Handb. d. Biochem. d. Tiere u. des Menchen 1, 331 and 395 (Jena 1909).- i 112 GLUE AND GELATIN sulphuric acid which exhibits no protein reaction whatever, but reacts with barium chloride; its dextro-rotation distinguishes it from sulphuric acid.” From this it is evident that Rakusin used a gelatin containing chondrin or some similar impurity, and it leads one to suspect that the sulphur in hide powder may come from some impurity such as keratin, for example. Herzog and Adler # showed that both hide and gelatin exhibit an apparent negative adsorption—that is, they selectively adsorb the solvent leaving the solute more concentrated. Rakusin says that in dyeing hide a positive and negative adsorption occur simultaneously,** but this seems to be a rather recondite way of saying that the hide takes up both water and dye. He also asserts with great positiveness that the combination between dye and hide is chemical, but says that crystal violet, which washes out with alcohol, constitutes “a preliminarily inexplicable exception.” So too did methylene blue, which was dissolved out by both boiling water and by alcohol; and in this case Rakusin promises to repeat the experiment with the purest dye obtainable. Hide powder and gelatin were both dyed by methyl orange (dimethylanilin-azo-benzolsulphonic acid), but the adsorption product showed the curious anomaly of being reversible in boil- ing water but irreversible in 95 per cent. alcohol. Rakusin also discusses at length the tanning of hide by tannin, formaldehyde, aldoses, phenols of various kinds, and homologous substances, picric acid, naphthols, chinone, ‘“‘neradol,” and alum, iron, and chrome salts. For full details reference must be made to his monograph. Non-electrolytes in general have slight action on the viscosity of gelatin, although many of them materially influence its jelly strength and melting point. Bechhold and Zeigler report the following: M.Pliam- eG 10 per cent. gelatin. ........:6. 00008040 4s eee 31.66 +1 mol. grape sugar;...eeee 32.25 1D Sc anaes “ A-2 “ glycerin |... eee 32.17 LO gas «+2 “~ alcohol’ ““ urea )..5.4.. ae 260 Furfurol, rescorcinol, hydroquinone and pyrogallol also lower the apparent rcHitie point of gelatin. “ Kolloid. Z. 2, Suppl. II, 3 (1908). 43 See also M. Rakusin and G. Pekarskaja, J. Russ. Chem. Ges. 1917, 1899. Chapter 8. Collagen or Ossein. Collagen? (literally glwe-former) is, as its name indicates, the parent substance of glue and gelatin. It is a substance par- ticularly characteristic of mature vertebrates, and is found in all of them with the exception of the border-line Amphioxus lanceolatus, according to Hoppe-Seyler. This same investigator reports that jelly-forming tissue is practically never met with in invertebrates, although he found some in two cephalopods, Octopus and Sepiola (the devil-fish and the cuttle-fish). From this it is obvious how vitally collagen is involved in bone formation. In fact, bone consists essentially of tricalcium phosphate and calcium carbonate deposited in collagen which acts as a colloidal protector and inhibits their crystallization. H. Bechhold? has discussed some of the theories of ossification, and describes experiments of R. E. Liesegang, who simulated bone formation by allowing disodium phosphate and calcium chloride to diffuse toward each other in gelatin jellies. Pauli and Sameé found that serum albumen increases the solubility of calcium carbonate 475 per cent., and of calcium phosphate 90 per cent.; but with the cleavage products of albumin, the figures are reversed. Since human bone ash contains about 85 per cent. of Ca,(PO,), and 9 per cent. CaCO, Bechhold believes that the disposition of the bone salts is consequent upon or accompanies the disintegration of cells or tissues, which corresponds with the histological evidence. In pathological cases bone formation may be inhibited, as in rickets; or bone already formed may be destroyed, as in osteo- malacia. The disposition of lime is evidently closely bound up with variation in the protective action of the body colloids, too high a degree of colloidal protection working against deposition. 1Ossein is collagen derived from bones. a2“Colloids in Biology and Medicine,” p. 268. 3’ “Beitrag zur einer Kolloidchem. Theorie des Lebens,’”’ Dresden, 1909. 113 114 GLUE AND GELATIN Selective adsorption also seems to be a factor in bone formation, as well as in the allied phenomenon of calcification, of tubercles for example. The presence of a vitamine (possibly certain fatty acids) in foods is a factor, and sunlight also exercises an in- fluence. W. von Gaza‘ in dealing with the changes of tissue colloids in the healing of wounds, says that connective tissue cells have the specific property of forming collagen from simpler albumin- ous substances. This collagen is held in colloidal solution ini- tially because of the presence of the acid oxidation products of life (especially CO,). As the colloidal solution of collagen accumulates in the cell, oxidation diminishes and finally a re- versal of reaction occurs—neutrality instead of acidity. The formation of collagen is thus analogous to lignification, the for- mation of lignin from cambial sap, a phenomenon which has been investigated by Wislicenus.’ Collagen and lignin both func- tion as supporters and protectors, and are marked .by great stability under normal conditions of life. Even after death this stability persists as is evidenced by fossil bones and wood. After growth, collagen remains unchanged apart from col- loidal syneresis. In order that healing of a wound (i.e. in the skin) may take place, it 1s necessary that the paraplastic col- lagen be brought into a fluid or semi-fluid condition, for it is an old maxim that corpora non agunt, nisi fluida. Fluidification seems to be brought about by local accumulation of acid due to interruption of the normal circulation consequent upon the wound. Similarly collagen must first be swollen to a certain extent by hydrochloric. acid before pepsin will disintegrate it. It is not, however, attacked by leucocytes or by the tryptases of the tissues. R. H. A. Plimmer ® points out that pepsin attacks both gelatin and ossein (collagen), while trypsin attacks only gelatin. This is held to indicate that ossein has a “closed ring” or anhydride 4 Kolloid Z. 23, 1 (1918). 5 Kolloid Z. 6, 19 (1910). 6“Chemical Constitution of the Proteins,’ 2d ed., Part II, p. 11. A. W. Thomas and I. L. Seymour-Jones (article in press) have been able to attack collagen with trypsin under certain conditions. The action is most rapid at Py 5.9 and is not materially accelerated by soaking. Collagen without trypsin, slowly hydrolyzed at 40°, as was observed in a blank determination. Fine hide powder was attacked by trypsin much more rapidly than coarse hide powder, showing the great effect of surface. COLLAGEN OR OSSEIN 115 form, which only pepsin can open. A more likely explanation that in ossein the constituent molecular groups are more highly dehydrated and closer together than in gelatin, and this con- dition is relieved to some extent by the acidity essential to the activation of the pepsin. This is not inconsistent with the fact that, as Emil Fischer and Abderhalden have shown, the action of enzymes on the comparatively simpler polypeptides depends upon the configuration of the latter, as is shown in the case with sugars.‘ A. Ewald,® working with collagen derived from the tendon of the mouse, studied the shortening of its fibrils on heating in water, which is very marked if the collagen is first purified by tryptic digestion. ‘The behavior of collagen treated with formal- dehyde is so characteristic that F. C. Thompson thinks ® it may serve as a new qualitative test. At 93° C. such fibers shrink to one third of their original length, but regain half the loss upon soaking in cold water. On treating again at 69° C. they once more contract to one third; but their original length is completely regained by prolonged soaking in cold water. Hofmeister observed that on heating dry gelatin to about 130° it became insoluble, being reconverted into ossein; and he held that this indicates that ossein is an anhydride of gelatin. J. Alexander *° believes the insolubility consequent upon the re- moval of the protective aqueous films, the constituent molecules or particles of the gelatin approaching so close as to form an irreversible gel. An analogous condition exists with silica and with clay, where dehydration up to a certain point is reversible, after which the material will not hydrate or redisperse again within a reasonable time. Thomas and Kelly 1 report the isoelectric point of collagen as Py, = 9, whereas Porter 1°” reports it as 4.8. _ While the main sources of collagen are hide or skin and bones, it is found also in tendons or sinews, connective tissue, in the cornea and sclerotic coat of the eye, and in the scales of fishes. 7See S. B. Schryver, ‘‘Allen’s Commercial Organic Analysis,” 4th ed., Vol. 8, p. 469. 8Z, Physiol. Chem. 105, 115 (1919). 9«S C¢. I. Annual Report on Appl. Chem.,”’ 1919, p. 361. 10 “‘Allen’s Commercial Organic Analysis,” 4th ed., Vol. 8, p. 586. 10a J, Am. Chem. Soc. 44, 195 (1922). 10 J, Soc. Leather Trades Chem. 5, 259 (1921); 6, 83 (1922). 116 GLUE AND GELATIN W. 8. Ssadikow ™ has made experiments with native tendocol- lagen, which is a compact mass of fibrous structure. If dehy- drated by alcohol and carefully dried over sulphuric acid at room temperature and then up to 105°-180°, the fibrous struc- ture is not lost. The collagen does not become brittle and can- not be ground. Above 180° it browns and becomes brittle, the ‘“intracolloidal” water being driven off. If dried in the presence of its adsorption water, tendocollagen loses its white color and opacity, and assumes the hyaline form, which may also be pro- duced by heating it for some time with hot water on a hot water bath, or by treatment in the cold with weak caustic alkali or strong alkali sulphide. Resoaking the hyaline tendocollagen in water restores the fibrous condition, unless the hydrolytic influence has exceeded a certain limit, when the change becomes irreversible, i.e. soaking one month in 2 per cent. KOH, or treatment with a saturated solution of Na,S. On treatment with CS,, thionyltendocollagen is formed, which shows characteristic reactions and is quite re- sistant to hydrolysis. The amount of sulphur bound depends upon the previous treatment of the collagen, and varies from 0.12 to 3.05 per cent. Ssadikow also reported !* on the action of carbon bisulphide on gelatin and on ossein. To a hot solution of gelatin he added 1 per cent. of powdered caustic soda or calcium hydroxide, fol- lowed by a few ce. of carbon bisulphide. The mixture was then allowed to set. ‘The reaction was evidenced by the development of a brown color, the evolution of ammonia and hydrogen sul- fide, and the precipitation of thiogelatin (thioglutin). On slowly drying thioglutin at ordinary temperatures, an intensely red skin develops which is not soluble in hot water. When CS, acts on collagen in the presence of alkali, the alkali first causes an hydrolysis and the CS, then is taken up by the resulting products of hydrolytic splitting. This process Ssadikow calls “thiohydratation.” The amount of sulphur taken up de- pends upon concentration and time of action of the alkali, i.e. - upon the degree of hydrolytic splitting. Glutin (gelatin), thionylized by 5-10 per cent. solutions of C8, in alcohol, ether, benzene, etc., takes up from 0.32 to 0.40 per cent. 1 Kolloidchem. Beihefte, Vol. 1 (1911). 12 Loc. cit.; also Kolloid Z. 1, 193 (1907). COLLAGEN OR OSSEIN 117 sulphur (average =0.39 per cent.). The addition product usually shows a dark color with alkaline lead acetate, but always exhibits a characteristic “erythrin reaction” as follows: Thionyl- glutin dried at 110° is heated on a water bath for ten minutes with 1 per cent. chloracetic acid. The solution is filtered, cooled, and mixed with three volumes of strong alcohol and then neu- tralized with ammonia. A precipitate forms immediately or after a while, depending on the concentration of the thionyl- glutin; and on standing from two to twelve hours this precipitate develops a fine pink color, which starts at the top (probably because it is due to oxidation) and gradually deepens to brown. When CS, acts on highly degenerated gelatin the product is yellow and has a characteristic odor of mustard oil. The solu- tion of this thionylxanthoglutin (thionyl&glutin) in chloracetic acid is yellow, and shows the erythrin reaction markedly. This erythrin reaction is also shown to some extent by collagen which has not been thionylized. ‘‘Glutein” made from the nasal sep- tum cartilage of the pig bound from 0.61 to 1.89 per cent. sulphur depending on the degree of hydrolysis. Glutin brominated in ethereal solution adds as much bromine whether thionylized or not. Tanning with tannin or formalde- hyde does not interfere either, nor does treatment with methyl iodide or benzoyl chloride. Chondrigen, Chondrin and Mucin. These substances are apt to be met with in glues, but may be considered as impurities in the higher grades.1? Chondrigen occurs in hyaline cartilage, of which it forms the chief organic constituent. It is elastic, semi-transparent, insoluble in hot or cold water, and swells but slightly in water or dilute acetic acid. - Upon heating for some hours with water under pressure (120°) chondrigen yields chondrin, whose solutions gelatinize upon cool- ing. ‘Thus chondrigen, as its name indicates, is the parent sub- stance of chondrin, just as collagen is the parent substance of gelatin. 143 Mucin is a prolific source of foam in glue. It may be liberated from chondrin during manufacture of the glue. 118 GLUE AND GELATIN Allen gives the following procedure for preparing approxi- mately pure chondrin: Boil costal cartilage in water for a few minutes and after scraping off the perichondrium, boil with water at ordinary pressure for 24 hours, or under pressure (at 120° C.) for 3 to 4 hours. Filter the solution to remove elastin, cellular elements, etc., and then precipitate the chondrin with a large excess of alcohol, and wash the precipitate with alcohol and ether. Re-solution in hot water followed by precipitation with alcohol will still further purify it. The chondrin thus pre- pared is hard, transparent, odorless, tasteless, and insoluble in cold water. Hot water dissolves it, yielding on cooling a jelly of weaker strength than that given by the same percentage of gelatin. Some analyses of chondrin are given herewith: 6 H N O Ss VE MEta cise | One rs ow Vet en oh eaten 49.3 6.6 14.4 29.3 0.4 Fischer and Bodecker ............. 50.0 6.6 14.4 28.6 0.4 Schiitzenberger and Bourgeois ..... 50.16 6.58 14.18 29.08 0.0 Vonsiiehring 2%... v.c. wean ee dee a ee 47.74 6.76 13.87 31.04 0.6 Allen states: “It will be seen from the above figures that the elementary analysis of chondrin present considerable discrep- ancies, and suggest that the substance dealt with is not a defi- nite substance but is liable to variations in composition. The results obtained by Morochowetz, and confirmed by Landwehr, Krukenberg, and Morner, strongly support this view. Moro- chowetz found that on treating cartilage from various sources with lime- or baryta-water, a 0.5 per cent. solution of sodium hydroxide, or a 10 per cent. solution of common salt, mucin is dissolved out, and may be thrown down from the solution by acetic acid; while the substance left undissolved is readily con- vertible by boiling water into perfectly normal gelatin. Ac- cording to these observations, chondrin is a mixture of gelatin and mucin, while chondrigen is a mixture of collagen with mucin or hyalogen, the latter component masking its true nature.” This led the writer to remark ** that chondrin is probably an adsorption compound of simpler substances, ie. gelatin and mucin. The following table shows that chondrin behaves toward re- agents like a mixture of gelatin and mucin: ~ “Comm. Organic Analysis,’ 4th ed., Vol. VIII, p. 625. 15 Allen, loc. cit., p. 626. COLLAGEN OR OSSEIN 119 Gelatin Chondrin Mucin RENUDMItY Ys ory... Insoluble in cold water, alcohol, or ether. Same. Same. Soluble in hot water; solutions gelatinize on cooling. Same. Insoluble in hot Reaction with let Acetic Acid..... No ppt. Ppt.; insoluble ex- cept in large ex- | cess. Same. Mineral acids..... = No ppt, Ppt.; readily solu- ble in excess. Same. Watnic acid....... Ppt. Ppt. No ppt. Mercuric chloride.. Ppt, Ppt. No ppt. Lead acetate...... No ppt. Ppt. Ppt: Sy ee No ppt. Ppt. Pot. Boiling dilute min- eral acids....... No reducing sub- stance formed. Yield syntonin and reducing sub- stances. — Same. For further information regarding these substances, see Chap- ter II, p. 28, under Glycoproteins. By microchemical staining methods Morner found that tracheal cartilage had a collagenous network enclosing “chondrin balls.” By treatment with 0.1 to 0.2 per cent. HCl followed by treatment with 0.1 per cent. KOH he dissolved out the ‘‘chon- drin balls,’ and then with the aid of dilute acid or superheated water converted the network largely into typical gelatin. The “chondrin balls” proved to be a mixture of free chondroitic acid, and chondromucoid. This latter is the same as the chondromu- coid isolated from tendons by Gies and Buerger, and on decom- position it yields a protein fraction and chondroitic acid (chon- droitin sulphuric acid or cartilage acid), which when acted upon by acids gives free sulphuric acid and chondroitin. This latter substance yields acetic acid and chondrosin, which according to Hawk reduces Fehling’s solution even more strongly than dex- trose. Upon hydrolysis chondrosin is split into glucosamine and glycuronic acid. The cartilages, which are precursors of bone, are thus related 120 GLUE AND GELATIN to the mucins, and in fact free sulphuric acid has recently been found in the slime of snails. A. P. Mathews? points out the importance of this relation from the standpoint of evolution, for it shows a chemical relationship of mesodermal to ectodermal tissues. Thus chitin which forms the shells of arthropods (e.g. beetles) yields on hydrolysis acetic acid and glucosamine, and some sulphuric acid is also present. 16 “Physiological Chemistry,’ 3d ed., 1920. Chapter 9. The Effect of Tanning Substances on Glue and Gelatin. According to J. T. Wood! one of the earliest contributions to the chemistry of tanning was made by Humphry Davey on February 4, 1803, in a paper entitled “An Account of some Ex- periments on the Constituent Parts of certain Astringent Vegetables, and their Operation in Tanning.” ? Davey remarked: “The tanning principle in different vegetables, as will be seen hereafter, demands for its saturation different proportions of gelatin, and the quantity of the precipitate obtained by filtration is not always exactly proportional to the quantities of tannin and gelatin in solution, but is influenced by their concentration. Thus, I found that 10 grains of isinglass, dissolved in two ounces of distilled water, gave with solution of galls in excess, a pre- cipitate weighing, when dry, 17 grains, whilst the same quantity dissolved in six ounces of water produced, all other circumstances being similar, not quite 15 grains. With more diluted solutions, the loss was still greater; and analogous effects took place when equal portions of the same solution of isinglass were acted upon by equal portions of the same infusion of galls diluted in dif- ferent degrees with water, the least quantity of precipitate being always produced by the least concentrated liquor.” The amount of tannin precipitated by 100 parts of gelatin is reported by different authors as follows: I CR CEE. J's ca ec bc lee cee ss vecepecccevetsawdees — 85 Lipowitz (Jahresb. Forts. Chem., 1861, p. 624)............-. — 65 S. Rideal (“Glue and Glue Testing,” 1900, p. 111)......... —134 ee as oh fs aia a 0b vies ace Vie edb pease w oltnukis —135 IRM od pct diese wo chan cle) teeeas — 78 eM ee dn ae a's vis px he's s Wa vce os bs aaln die eaiele cd amen — 50 One cause for the discrepancies was found by Wood to be the fact that a definite excess of tannin is required to produce 1 J. Soc. Chem. Ind. 27 (1908). 2 Roy. Soc. London, Phil. Trans. 233 (1803). 121 122 GLUE AND GELATIN 4 maximum amount of precipitate. His results are given in the following table, which shows the amount of tannin precipi- tated by adding 1 gram of gelatin to varying quantities of 1-100 tannin solution. No. of cc.1% Tannin Tannin pptd. Tannin Solution Grams Grams 100. Fo. 35 oe 45 See oe eee 1 0.91 200. 6s caeh> vasa abies cere 2 1.50 OUO Vacs ve oka male hoe oa ee te 3 1.90 ADO SO, ois a aiae Se os ee ee 4 244 DOU ares 22a 9 a cee ee eens 5 2.28 GOO Ook. co haue 3 os ete Berea ee 6 2.36 LOO RAs EAST Ns pees oe 7 2.36 BOO fair shew arse eho eran ee eros 8 2.36 Wood also made a quantitative record of the well-known fact that the precipitate of gelatin with excess of tannin has a dif- ferent composition from the precipitate of tannin with excess of gelatin, and that a considerable amount of tannin may be re- moved from the latter by washing with hot water. He found that 100 parts of gelatin carried down 300 parts of tannin, of which 88 parts were given up upon washing with boiling water.* In conclusion Wood observes: “An examination of the facts shows that the combination of gelatin and tannin compound is — not of constant composition, nor a purely physical one, since it does not obey the solution laws, which require the concentra- tion of the tannin in the solution and the tannin in the gelatin to maintain a constant ratio.” It is therefore an error to con- sider “tannate of gelatin” as a definite chemical compound, for it is a typical adsorption compound, and as von Schroeder‘ has shown, the precipitation of gelatin by tannin follows the adsorp- tion isotherm. | The precipitation of gelatin by tannin is also a typical in- — stance of the precipitation of one colloid by another of opposite charge. Aqueous solutions of tannin are positively charged (negatively conducted), whereas gelatin is amphoteric, and as Ricevuto > has shown is not precipitated by tannin unless in the negative condition (positively conducted). Carefully dialyzed ’ Apparently the excess of gelatin exercises a protective action so that part of the tannin, and some of the gelatin as well, are washed away, probably in colloidal solution. * Kolloidchem. Beihefte 1, 1. 5 Kolloid Z. 3, 114 (1908). THE EFFECT OF TANNING SUBSTANCES 123 gelatin is not precipitated by tannin, nor is hide tanned by tannin unless it is on the acid side. When dry the gelatin-tannin compound forms a yellowish- brown, brittle mass which melts in boiling water to a tenacious sticky mass like bird-lime. In this state it may be drawn out or spun into fibers fine as a spider’s web, which have a metallic luster like silver slightly tinged with gold. When soaked in alum solution they acquire a blue tinge like polished steel. The gelatin-tannin compound is tasteless and does not yield tannin to alcohol, ether, or acetone. Prolonged boiling with water, especially in the presence of magnesia, decomposes it, probably because of the hydrolytic cleavage of the gelatin. Chrome. The chroming of gelatin does not affect its absorption of tannin, for a sheet of heavily chromed gelatin absorbs as much tannin as before chroming. This fact is not so strange as it seems if we remember that the chromium is absorbed only from basic solutions, and is apparently attracted to a different part of gelatin complex. It seems difficult, however, to reconcile with the fact that treatment with basic chromium salts renders collagen insoluble; indeed the progress of chrome tannage may be followed by immersing strips of the hide in boiling water, which causes distortion by converting any unchanged collagen » into glue. Chrome tannage appears to be the “mirror picture” of tannin tannage, positively charged gelatin being precipitated by -negatively charged colloidal chromium hydroxide. This view is supported by the experiments of Bancroft,’ who found that gelatin sheets took up chromic sulphate practically un- changed. If the gelatin containing chromic sulphate is washed repeatedly with boiling water, acid is slowly extracted together with some gelatin. By treating with dilute alkali, however, the acid may be removed without causing swelling or solution of the gelatin, which is combined with about 3.3 to 3.5 grams of Cr,O, per 100 grams of gelatin. | Since Namias showed that the tanning action of chrome alum ® No attempt will be made here to consider the question of the tanning of hides and skins, which is even more complicated than the tanning of gelatin. 7™“Applied Colloid Chemistry,” p. 229. 124 GLUE AND GELATIN was increased by adding alkali up to the point of precipitation of hydrous chromic oxide, Lumiére and Seyewetz made experi- ments with the green basic chromic sulphate of Recoura, and found it yielded a more insoluble gelatin than did a less basic solution. Excess alkali apparently produces such a high degree of dispersion of the chromic oxide, that little or no tanning occurs. Organic Substances. Besides tannin and basic chromium solutions, many other sub- stances are well known as tanning agents for gelatin; chief among them are alums, formaldehyde, and ferric salts. L. Meunier and A. Seyewetz ® report having obtained the precipi- tation of gelatin solutions with the following organic compounds: phenol, resorcin, orcine, hydroquinone, pyrocatechin, gallotannic acid, pyrogallic acid, p-amidophenol, chlorophenol, picric acid, monochlorhydroquinone (durol), R acid (disulfo-6 naphthol 2.3.6), G acid (disulfo-B-naphthol 2.6.8.), S acid (monosulfo- 6-naphthol 2.6.). From their results with various substituted quinones ® they conclude that the tanning action of a quinone in- creases in rapidity with decreasing power of penetration. The importance of this tanning action in the “hardening” or fixing of gelatin-coated photographic negatives or positives, must be at once manifest. ‘“‘Neredol,”’ the sulphonated phenol-formalde- hyde patented product of Stiasny, is largely used to tan leather. Bichromates. A large literature exists regarding the action of light on gela- tin containing bichromates.?° The tanning effect, which forms the basis of several photo- graphic reproduction and engraving processes, seems to depend upon the liberation of colloidal chromic oxide, for as 8S. J. Levites 1 has shown K,CrQO, is reduced to Cr,O, by most albu- minoids. A. and L. Lumiére and A. Seyewetz?* have shown 8 Collegium, 1908, No. 318, p. 195. ® Collegium, 1914, No. 531, p. 528. 10 See e.g. Eder, ‘‘Reaktionen der Chromsiiure und der Chromate auf organische Substanzen in ihren Beziehungen zur Photographie,” 1878. 1 Kolloid Z. 9,5 (1911). 12 Phot. Korresp, 6, 75, 192, 239 (1906). THE EFFECT OF TANNING SUBSTANCES 125 that the illuminated bichromate-gelatin differs from that tanned by basic chromium salts, the chromium oxide in the former con- sisting of two fractions. The first, which equals 3.5 per cent. of the bichromated gelatin, represents what is held by the tanned gelatin; the second varies with the time of illumination, and arises from the reduction of the bichromate by light in the presence of organic matter. The first fraction increases dispro- portionately to the illumination, and decreases with increasing concentration of bichromate. Silicic Acid. Colloidal silicic acid reacts with gelatin to form a co-silicate or colli-silicate of gelatin, whose composition varies with condi- tions of its formation. Graham states: 4? “When a solution of gelatin was poured into silicic acid in excess, the co-silicate of gelatin formed gave, upon analysis, 100 silicic acid with 56 gelatin, or a little more than half the gelatin stated above as found in that compound prepared with the mode of mixing the solutions reversed. The gallo-tannate of gelatin is known to offer the same variability in composition.” Alum. The action of alum, and other aluminium salts, in tanning gelatin, appears to be consequent upon their hydrolysis, colloidal alumina being formed and fixed by the gelatin, while the acid may be differentially washed or diffused out. The results of A. and L. Lumiére and A. Seyewetz ** are briefly: (1) Aluminium salts and nascent alumina raise the setting point of gelatin, the effect depending on the percentage of alu- mina present. 0.107 grams of Al,(OH), per 100 grams gelatin raises the gelatinization point 1°. (2) Alum has relatively a _. weak effect, as is to be expected; aluminium chloride has the greatest effect. (3) Irrespective of the kind of aluminium salt used, the settling point rises with increasing alumina content, up to about 0.64 grams alumina per 100 grams gelatin. Over this the setting point remains stationary and then falls. (4) The increase in the setting point varies with the concentration of the 1% Phil. Trans. Roy. Soc. London 151, 206 (1861). ; 1447. fiir wiss. Photographie 4, 360 (1906); Bull. Soc. chim. Paris 35, 676 (1906). 126 GLUE AND GELATIN gelatin solution. (5) Gelatin fixes a maximum of about 3.6 grams alumina per 100 grams gelatin, and gives up to the water the acid and salts with which the alumina was combined. It was concluded, therefore, that gelatin forms a definite chemical compound with alumina. Lumiére and Seyewetz also found that an excess of alkali or ammonia completely inhibited the tanning of gelatin by alumina, just as is the case with chrome salts. In reviewing this work, H. Freundlich *® expressed the view, in which the writer concurs, that it is more probable that a colloid complex is formed, rather than a chemical compound between hydroxide of aluminium and gelatin. The effects of selective absorption and differential diffusion are so great 1° that even potassium sulphate may be decomposed by percolat- ing its dilute solution through a column of sand, the alkali being held by the sand, while a dilute solution of sulphuric acid issues from the bottom. Gutbier, Sauer, and Schelling ‘” report that at ordinary tem- peratures a higher concentration of alum is required to raise the viscosity of bone glues than of hide glues. Alum lightens both glues, and at higher temperatures, if the solution is slightly acid, forms a precipitate, which is an adsorption compound and clari- fies better if it settles rapidly. On dialysing glue solutions the aluminium only is held back; the colloidal aluminium hydroxide and the H-ion concentration control the action of the alum, optimum conditions varying with kind of glue and with concen- tration. Hide glues seem especially sensitive to the’ action of an excess acidity, for with them more often than in bone glues, alum clarification causes hydrolysis resulting in foam and de- creased strength. The precipitate, however, seems to adsorb impurities; it removes part of the ash-producing substances and all the addéd acid; and the resulting solution contains but little Al. : Iron. The action of iron on gelatin is well known to gelatin manu- facturers, for rusty nets often produce an insoluble reddish- 18 Kolloid Z. 1, 157 (1906). 16 See J. Alexander, J. Am. Chem. Soc. 39, 84 (1917). % Kolloid Z. 30, 876-95 (1922). THE EFFECT OF TANNING SUBSTANCES 127 brown compound. According to Liippo-Cramer ** ferric chloride solutions precipitate gelatin. A 1 per cent. solution of gelatin mixes with the chloride without precipitation, but the color is darker than a solution of ferric chloride of the same concentra- tion, showing that the salt undergoes, in the presence of gelatin, an hydrolysis similar to that which it suffers on boiling. Iron alum also tans gelatin, but. not in the presence of an excess of alkali, which turns the gelatin dark but allows the iron to be washed out. The adsorbed ferric hydroxide can also be washed out by potassium citrate or oxalate, and by oxalic and other acids. Fifty cc. of 10 per cent. FeCl, + 50 cc. 10 per cent. gela- tin solution at 50° give a thick red-brown fluid which sets and can be remelted. Here the excess of gelatin evidently acts as a protective colloid to the iron-gelatin adsorption compound. In fact, according to Stiasny the unsatisfactory tanning action of iron-salts consequent upon their rapid and complete hydrolysis, is improved by the presence of soap, blood, albumen, gelatin, and similar colloidal protectors. Other Salts. Uranic salts (e.g. uranium nitrate) act similarly to ferric salts, and auric chloride has a particularly powerful tanning action, which it likewise exerts on the skin. Copper, silver, mercury and lead salts are powerfully fixed by gelatin, and even barium chloride undergoes a partial hydrolysis in its presence. Indeed, as Van Bemmelen’® has shown, colloids by their adsorptive action can effect a chemical decomposition of most salts. Thus if a red solution of thiocyanate of iron is added drop by drop to a 10 per cent. solution of gelatin, the ruby-red precipitate soon changes to the rust-brown color of ferric hydroxide. . Phosphomolybdic and phosphotungstic acids precipitate gela- tin, and its precipitate with picric acid is used for its detection in the Stokes method.?° The Halogens. The halogens have a powerful tanning action on gelatin. As far back as 1840, Mulder ** described the compound formed by 18 Kolloid Z. 1, 353 (1907). 19 Rec. Trav. chim. ‘Pays Bas 7, 37 (1888). 20 Analyst, 1907, p. 320. 21 Berzelius Jahresber. 19, 734; J. fiir Chem. 44, 489. 128 GLUE AND GELATIN treating gelatin with chlorine, and Allen and Searle ?? described a similar compound with bromine, while Hopkins and Brooks ** made like observation with respect to iodine. According to Rideal and Stewart 24 when chlorine is bubbled through 1 per cent. gelatin solution the liquid remains clear for a time and then froths, each bubble of gas becoming encased in a white pellicle. With an excess of chlorine the liquid becomes clear again and the gelatin forms a white granular precipitate which on washing and drying yields a pale yellowish-white pow- der, odorless, tasteless, and insoluble in water or alcohol, but soluble in alkalis. | Cross, Bevan, and Briggs ?> found that moist gelatin spread out very thin by immersing cotton yarn in its solution, combines with 15.4 per cent. of chlorine figured on air-dried gelatin. The © extremely stable substance resulting they regard as a gelatin chloramine; it is sensitive to antichlors, and when treated with sulphuric acid reverts to the original gelatin. This reaction is made the basis of a method for the detection and estimation of gelatin in tub-sized papers (loc. cit., p. 263). — Lumiere and Seyewetz 7° found that the best results with chlo- rine were obtained by adding, say, 10 grams of gelatin to 500 ce. of a saturated solution of chlorine, containing 50 grams of NaCl and held at 0° to drive back the ionization of hydrochloric acid. They found it much easier to use hypochlorites, 10 grams of gela- tin in thin sheets being rendered insoluble at room temperature by a solution of 100 grams of commercial sodium hypochlorite - and 2 cc. of HCl (21° Bé) in 400 cc. of water. They found that bromine acted similarly but more energetically, but were unable to render gelatin insoluble with iodine. Formaldehyde. The tanning action of formaldehyde, both in solution and as , a gas, has long been known and utilized. Acrylic aldehyde is said to act similarly, but acetic aldehyde acts only in the pres- ence of water. 2 Analyst, 1887, p. 258. J, Physiol. 22, 184. 24 Analyst, 1897, p. 228. 25 J. Soc. Chem. Ind. 27, 260 (1908). 2° Bull. Soc. Chim. (4) 11, 344 (1912). THE EFFECT OF TANNING SUBSTANCES 129 The maximum amount of formaldehyde fixed, when its 10 per * cent. solution acts on dry gelatin, is between 4.0 and 4.8 per cent.27 The “insoluble” formo-gelatin is decomposed by re- peated washing with boiling water, as well as by heating to 110°, and by cold 15 per cent. HCl. R. Abegg and P. von Schroeder 7° half filled a test tube with a 10 per cent. solution of gelatin which had a melting point of 36°, and after the gelatin had set, covered it with a 5 per cent. formalin solution which was allowed to act for 24 hours. The upper fully tanned layer was infusible, but crumpled at 85° with the development of a brown color. Lower layers showed melting points of 48°, 42°, and 37°, whereas the bottom layer was unaffected. They also found that the time of tanning (as determined by the time needed to reach the high- est melting point observed, 48°) varied inversely with the con- centration of the formaldehyde. H. Bechhold?® prepares ultra filters by impregnating filter paper with gelatin solutions of various strengths, allowing the gelatin to set, and then immersing the treated paper in 2-4 per cent. ice-cold formaldehyde. R. H. Bogue *° has examined into the effect of formaldehyde on various glues. He found that the viscosity increased directly as the amount of formaldehyde added; it decreases with rise of temperature up to 40°, after which it rises rapidly to the setting point. It also increases with time. On the other hand the jelly strength of glues is decreased proportionately to the amount of formaldehyde added, the effect being most marked in lower con- centrations and with weaker glues, some of which actually re- mained fluid. The higher the grade of glue and the higher its concentration, the less formaldehyde is required to produce “‘in- solubility.” Alums produce increased viscosities but have little or no effect on the jelly strength.*? 7 Lumiére and Seyewetz, Bull. Soc. Chim. 35, 872 (1906). 28 Kolloid Z. 2, 85 (1907). 22“Colloids in Biology and Medicine,” p. 97. 80 Chem. Met. Hng. 23, 61 et seq. (1920). 31 Chrome alum increases the melting point however. Chapter 10. The Chemical Examination of Glue and Gelatin. Hydrogen Ion Concentration, or pj, Value. Since recent investigations + have shown the great influence of the effective reaction (hydrogen ion concentration or p,, value) on the viscosity and jelly strength of glue and gelatin, its deter- mination by the electrometric or the colorimetric methods may form a necessary part of factory control.2 For colorimetric determination of p,,, the following indicators are used: Color change Indicator Py range acid-alkaline Solution strength Methyl Violet ........ O1to 32 green to blue 0.02% Thymol Blue .......... 12to 28 red to yellow 0.04% (lower range) Methyl Orange ....... 3.2to 44 red to yellow 0.02% Methyicdveds ue ee 44to 6.0 red to yellow 0.02% in 60% alcohol Fheucl Hedicse. ese 68to 84 yellowtored 0.02% Thymol Blue ......... 8.0to 96 yellow to blue 0.04% (upper range) Phenolphthalien ...... 8.3to10.0 colorless to red 0.05% in 50% alcohol The limitations of these indicators for gelatin are still to be determined, and in all cases the percentage ‘of gelatin present is a factor of importance. That is, a one per cent. solution may show a different p,, than a 5 per cent. solution. Patten and Johnson ** state that gelatin does not interfere with the deter- mination of Re colorimetrically. Bogue even recommended the determination of H-ion concen- — tration as part of the regular laboratory routine test of both glue and gelatin. He states*: “If the p,, value is 4.7, the viscosity, swelling, etc., are low, and the product nearly insoluble. On either side of this point * these properties increase very consid- 1J. Loeb, R. H. Bogue and others. * For details see W. M. Clark, ‘‘The Determination of Hydrogen Ions,” Balti- more, 1920. 2a J. Biol. Chem. 38, 179 (1919). 3.7. Ind. Hng. Chem. 14, 439 (1922). *This is the isoelectric point of gelatin. 130 CHEMICAL EXAMINATION OF GLUE AND GELATIN 131 erably, attaining their maximum on the acid side at Py, 3-5, and on the alkaline side at P,, 9-0. At greater acidity than p,, 3.5 or at greater alkalinity than P,, 9.0 these properties again de- crease. The ee value indicates, therefore, not only the reaction of the material, and the degree of acidity or alkalinity, but also the proximity of the substance to the points of maximum or minimum properties. ... One per cent. solutions are best in either case, and the results expressed in terms of P,; to the nearest tenth.” The experiments of Bogue and of Loeb were made with rather dilute solutions of glue gelatin. In actual practice the relatively small variations in the H-ion concentration of glues produce such slight changes in the viscosity of working solutions that they are barely perceptible with the viscosimeters in common use. Outside of moisture and ash which have already. been con- . sidered, chemical tests on glue are seldom made, unless they be tests to simulate working conditions where alum, formaldehyde or other substances are added to the glue. The reason is that most of those whose knowledge of. glue transcends laboratory experience, are agreed that the chemical tests proposed for glue do not enable one to form any trustworthy idea as to its practical value, and they are besides much more difficult, expen- sive, and time-consuming than the very satisfactory physical tests. In the case of food gelatin it is essential to make an exact estimation by chemical methods of arsenic, zinc, copper, lead, sulphur dioxide and ash. Chemical tests are required for gelatin intended for special uses, e.g. photography (see Chapter 18, p: 208). Acidity or alkalinity may be determined by titration. To estimate free acid, Kissling® soaks 30 grams of glue in 80 cc. of water for several hours and then drives over the volatile acid by a current of steam. When the distillate amounts to 200 cc., it is titrated with standard alkali, and titrate back the unused portion. Total Acidity. The total acidity of a glue may, of course, be determined directly by titration with 0.1 N NaOH solution, using phenol- 5 Chem. Z. 11, 691. 132, GLUE AND GELATIN phthalein or roseolic acid as an indicator. H,SO, may then be determined by a separate titration with 0.1 N I solution. For more accurate work phenol red (effective range p,,= 6.8 to 8.4) should be used as indicator. If the glue contains formaldehyde or other substances which react with iodine, H,SO, must be determined by acidifying with H,PO,, distilling off the SO, in a current of steam or CO,, and weighing it as BaSO,. In the case of bone glues a direct titration of the acids other than H,SO, may be made with 0.1 N NaOH, using as an indicator alizarin which, in the presence of at least 1 per cent. of glue, possesses the curious property of reacting only with strong acids and not with H,SO,.° Owing to legal restrictions the exact estimation of SO, and sulphates in gelatin is of great importance and will be referred to later. Determinations Involving Nitrogen. Clayton* considered the estimation of non-gelatinous sub- stances the best single chemical test for glue. To determine non- gelatinous substances, C. Stelling * dissolved 15 grams of glue in 60 ce. of water made up to 250 ec. with 96 per cent. alcohol, and after thorough shaking, evaporated to dryness 25-50 cc. of the fluid filtered off after standing six hours. Trotman and - Hackford ® separated the hydrolyzed from the’ non-hydrolyzed products by precipitating the former by saturation with zine sulphate and estimating them by the Kjeldahl method. H. J. Watson ?° does not regard the test as having any value. R. H. Bogue ** found that the ash and total nitrogen bore no consistent relation to the jelly strength of glues, but that the strongest glues showed the highest moisture content. Bogue made the following determinations on a series of hide and bone — glues and a few other glue products: ™ 6 Gutbier, Sauer and Brintzinger, Kolloid Z. 29, 180 (1921). 7J. Soc. Chem. Ind. 21, 670 (1902). 8 Chem. Z. 20, 461; Analyst 21, 289 (1896). ®J. Soc. Chem. Ind. 24, 1072 (1904). 10 J, Soc. Chem. Ind. 23, 1189 (1904). 11 Chem. Met. Eng. 23, 61 et seq. (1920). 2 For details regarding these methods see S. B. Schryver, ‘“‘Allen’s Comm. Organic Analysis,’ 4th ed., Vol. 8, pp. 467 et seq. Schryver recommends the addition of 2 ce. of diluted sulphuric acid (1 part concentrated acid to 4 parts water to each 100 ec. of mixed protein and sulphate solutions) for the protein precipitation, for which he used zine sulphate. Bogue, using magnesium sul- ee found maximum precipitation to occur with % ec. of dilute sulphuric acid. CHEMICAL EXAMINATION OF GLUE AND GELATIN 133 (1) Total nitrogen, by Kjeldahl’s method. (2) Protein nitrogen, from the precipitate formed on adding 50 cc. saturated magnesium sulphate solution to 50 cc. of water containing one gram of glue. (3) Protewn-proteose nitrogen, from the precipitate formed by saturating a similar glue solution with magnesium sul- phate. (4) Proteose nitrogen, difference between (3) and (2). (5) Amino mtrogen, from the filtrate of (3), using Sdrensen’s formaldehyde titration method. Bogue found that the temperature exercises a marked influence on protein precipitation, 3 to 8 per cent. more coming down at 17° than at 25°, but his determinations were made at 25° as this was more convenient.?® Bogue’s results are given in the following table: RELATION BETWEEN NITROGENOUS CONSTITUENTS AND JELL STRENGTH Amino Protein Proteose Peptone Acid Grade N N Nd N H, 92.2 6.3 1.1 0.4 H2 90.4 7.0 2.0 0.6 H; 86.2 12.0 14 0.4 Hide and fleshing Hy 84.6 12.4 26 0.4 ies ss » H; 78.7 16.0 45 0.8 H, 776 17.0 4.7 0.7 Hs 52.0 38.6 8.4 0.9 Bi 79.1 14.9 48 1.2 Bz 185 16.4 8.1 2.0 Bs 64.6 28.3 5.6 1.5 Bz 59.8 32.4 6.4 14 Bone glues ........ Bs 53.6 36.6 84 14 Bs 52.5 37.9 78 18 B; 48.2 40.1 10.1 16 Bs 36.8 47.1 12.5 2.3 9 31.5 50.6 14.8 3.0 Special Glues Russian isinglass.... He 91.0 44 4.5 0.1 Edible gelatin ..... Hz 87.8 11.3 0.7 0.2 Piso Ciie’...+>..-... Bo 33.4 42.3 21.9 24 Pressure tankage ... Bz 34.3 46.4 16.3 3.0 CG Bs 0.0 33.2 48.5 18.3 (Here 1 represents the highest grade and 9 the lowest.) 13 Samples secured by empirical precipitation methods of this character, ob- viously represent mixtures of substances of variable composition. 134 GLUE AND GELATIN These figures indicate that the jelly strength varies approxi- mately as the protein nitrogen determined by Bogue’s procedure. No consistent relation could be shown, however, between viscosity and nitrogenous constituents. The amino nitrogen is greater in bone than in hide glues, and tends to increase with decrease in jelly strength. Bogue also treated a number of glues having uniform jelly strength (grade), but different viscosities, with various concen- trations of magnesium sulphate from 50 per cent. down to 24 per cent. of saturation. Below that the precipitate was so finely subdivided and slimy that filtration was practically impossible. The results, given in the following table, show that there is no definite relation between viscosity and precipitate, except that SHowina Per Cent. or NitroceEN THRowN Down By VARYING PERCENTAGE SATURATIONS OF MAGNESIUM SULPHATE 50 36 30 28 26 24 Per Per Per Per Per Per No. Grade Jell Visc. Cent. Cent. Cent. Cent. Cent. Cent. Series 8...... 1 H. 65 456 844 692 453 — — — 2 HH, 65 472 873 G88 e4Gn — — — 3. Hy 66° 480. 870° -(saeeeee — — — 4°- Hy. 66 49.0" 852) \7i pee — — — 5 HH, 66 502. -852 68.005 — 346 286 6 H, 64 510. 849° GS0>saZz2 — 35.3 290 7 HH, 64° 540 S818. 645835503 — 348 30.0 RBs? 68924435 25 ioe — 22.3 — mas = Bs 3 2 OR 4a Rou — 36.3 — — — 10>). By ee Ue eee — 38.9 a — — il. By 705, 480). 762 — , 398 — — —_ 50 35 30 28 26 24 ‘Per Per Per Per Pers ree No. Grade Jell Visc. Cent. Cent. Cent. Cent. Cent. Cent. Series 4 ..... 1‘ Hs 64 45.4 818° 582 44355. — — 2 Hs 64 474 873 688 460 421 —_ — 3 Hs 64 506 740 .606 499 S458 — — 4 HH, 65 4462 854 629 473559905 — — 5 H, 65° 478 S872 65.1 46 — — 6 H, 65 480 756 576 44:7 ae — — ’ HH, 65 494 765 590 “4700 — — 8 H, 65% 490 722 590 445 42.0 — — 9 H, 65% 500 860 669 528 44.0 — — 10 Hy, 65% 502 852 664 521 487 — — 11 H+ 66 474 830 63.1 47,7 5a — — 12 Hit 66 486 870 (718) 40 7 — — 13 Ht 66 488 852 716 503 461 — — 14 Ht 66 492 862 643 522 468 — — 15 H.t 66 496 852 680° 517e= eee — — 146 Ht 66 498 8382 655 52.7 494 — —_ CHEMICAL EXAMINATION OF GLUE AND GELATIN 135 with about 30 per cent. saturation the precipitate varies as the viscosity. Bogue interprets these figures to mean that “if the jell strength be constant the viscosity will vary as the size of the protein molecule.” By the “molecule” he means ‘“‘a group which may not be subdivided except by chemical processes, as of hydrol- ysis, whereas the colloidal complex is established probably by electrical phenomena and the processes chemical condensation or hydrolysis are not involved.” Bogue also treated several glues having about the same vis- cosity, but varying jelly strengths, with 50 per cent. and 30 per cent. saturated magnesium sulphate. The precipitates showed the following percentage of nitrogen (Kjeldahl): 50 S30 ; No. Jell. Viscosity PerCent. Per Cent. RE Sas sa oles « 63 46.2 83.5 45.0 eee 64 45.8 84.2 46.2 Oo nee 66 46.0 84.9 46.7 0 LS 68 458 85.3 65:1 oS Nee eee 70 46.2 85.5 57.4 “Tt will be seen that at both 50.0 per cent. and 30.0 per cent. magnesium sulphate saturations, the nitrogen thrown down in the several precipitates varies directly as the jell strength, the viscosities being practically constant. This means then that at constant viscosity the jell strength will vary as the size of the protein molecule, as well as with the total amount of protein.” In the precipitation of purely empirical groups such as “gela- tin,” “gelatoses,’ and “gelatones” the larger molecular groups are for the most part thrown down more readily: but no quan- titative relations are deducible from the amount of the precipi- tate, for the various fractions exercise a varying protective influence on each other, which accounts for the observations of Haslam ** that part of any fraction remains in solution, while the precipitate may carry down part of a subsequent fraction.’® With precipitates of this character we are obviously dealing with molecular groups rather than with simple molecules. Upon investigating the crazing of glues, Bogue reports that this crackling up of the glue pieces is “due to an exceptionally great hydrolysis of the protein molecule and the consequent 14 J, Physiol. 32, 267 (1905) ; ibid., 36, 164 (1907). 13 The work of BH. Zunz shows the great variation in the protective action of various albumose fractions. 136 GLUE AND GELATIN inability of the resulting mixture to retain water above that minimum content below which crazing occurs.” His analytical results hardly justify this conclusion, for 7 crazed glues showed an average of 11.99 per cent. moisture, while with 7 firm glues of equal grade the average moisture was 11.91 per cent. Al- though in the firm glues the average protein nitrogen was higher and the average proteose and peptone nitrogen lower than in the crazed glue, still one crazed glue had the highest protein and the lowest proteose and peptone nitrogen. It is true that only very low-grade glues craze, but some other factors must be reckoned with, probably differences inherent in the original raw material, for glue is no more a definite chemical entity than is gelatin. Presence of an excess of a fraction having excessive syneresis, or diminution of a fraction having protective action against a syneresis, might possibly account for crazing. Diffusible Nitrogen Test. ' The British Adhesives Committee *** evolved this test, which, they say supplies an indication of the stability of glues towards water, and furnishes a rough measure of their tensile strengths, the stronger glues generally being low in diffusible nitrogen. The glue under test is made up into a jelly containing 2.1 grams of nitrogen in 75 cc. of water; this requires 15 grams of glue, approximately. The exact amount of glue is soaked over night in 75 cc. of water, heated to 37° C. for 2 hours, then to about 90° C. for 30 minutes (This procedure must produce marked hydrolysis. J. A.), and finally poured into a Petri dish 14 cm. in diameter, where it is allowed to set. One hundred cc. of water are now layered over the jelly and the dish is placed in a thermostat at 20° C. for 20 hours. The number of milli- grams of nitrogen per 100 cc. of the supernatant fluid, as deter- mined by Kjeldahl’s method, constitutes the diffusible nitrogen number. oe Since some of the constituents of the glue act as protectors to others and thus tend to peptize them (the Report even mentions this on p. 29), and since the value of the various hydrolysis products in this respect is not known, the investigation, to use the Committee’s own words, is ‘admittedly incomplete, and the a Wirst Report, p. 20 et seq., London, 1922. CHEMICAL EXAMINATION OF GLUE AND GELATIN 137 subject requires further study. Again, the test may lend itself to the study of the size of the hydrated gelatin aggregate under various conditions. The addition to a glue solution of salts that lower the surface tension of water will tend to reduce the size of the gelatin aggregate, and presumably, consequently, to in- crease the amount of diffusable nitrogen.” The test should also indicate the degree of hydrolysis. | The Committee tested the effect on joint or tensile strength and jelly strength of a series of sodium salts of organic acids, and also the effect of some sugars. Variations in viscosity com- plicate the joint tests, but sodium formate and salicylate ‘and the sugars markedly increase the joint strength. Reactions of Gelatin. *® Gelatin is totally insoluble in absolute alcohol, ether, chloro- form, benzene, carbon disulphide, and fixed and volatile oils. It is practically insoluble in ice-cold 10 per cent. alcohol, and precipitates as a white coherent, elastic mass if an excess of alcohol is added to its aqueous solution. The precipitate swells in cold water and may be redissolved as before. Fairbrother and Swan? tested the “solubility of gelatin in cold water, and report the following results given in grams per 100 cc.: 0.02 at 0°, 0.07 at 18.3°, and 0.10 at 22°. The solubility in solutions of hydrochloric, sulphuric, nitric and acetic acids and ‘in solutions of potassium and of sodium hydrates (concentra- tions varying from 0.2 to 5000 millimols per liter) were also determined. With acids the solubility passes through a mini- mum, rising then to about 0.2, after which solution gradually © occurred. With alkalis similar results were obtained, but there was no minimum. Neutral salts decreased the solubility, their effect being approximately in the order of the Hofmeister series. These experiments were done with Coignet’s Gelatin Extra analyzing 2.24 per cent. ash and having a P ,, Value in 1 per cent. solution of 5.6 at 20°. They should be repeated with ash-free gelatin properly freed from products of hydrolysis, for it is probable that only the hydrolysis products dissolve in cold water. 16 See also Chapter 9. Most of the reactions apply to the more or less impure gelatin known as glue. isa FY, Fairbrother and E. Swan, J. CCH. Soc. 121, 1273-44 (1922). — 138 GLUE AND GELATIN According to Zlobicki1® 0.5-0.8 grams of gelatin to 100 ce. of water causes a marked lowering of the surface tension of water, although further addition does not increase the effect. As has been pointed out by Victor Lehner ™ selenium oxychlo- ride readily dissolves glue and gelatin in the cold. This remark- able solvent likewise dissolves resins (natural and synthetic), rubber, shellacs, and asphalt. Solutions of gelatin in strong acetic acid do not gelatinize on cooling and are used as liquid glues. Warming with dilute nitric acid yields a liquid product, but strong nitric acid destroys gelatin, giving oxalic-acid and other substances. Gelatin may also be held in fluid condition by urea, zinc, magnesium, and calcium chlorides, sodium iodide and sodium and calcium nitrates, naphthalene sulphonate, etc. Gelatin is completely precipitated by saturating its aqueous solution with ammonium, zinc, or magnesium sulphates. Phos- phomolybdic and phosphotungstic acids also precipitate it, as ‘do tannin and sufficient quantities of mercuric chloride and of picric acid. The reaction with tannin is used to detect gelatin (or vice versa), a 0.02 per cent. solution of gelatin yielding a white or buff-colored precipitate which is insoluble in presence of an excess of tannin. Without such excess the precipitate tends to dissolve in pure water, especially if hot, apparently going into colloidal solution because an excess of gelatin acts as a protector or dispersing agent toward the tannin precipitate. Ruffin '* proposed to determine gelatin by precipitating it with tannin, and titrating the excess of tannin with iodine. The evidence shows, however, that the so-called “tannate of gelatin” is not a substance of definite composition. Thus H. Trunkel '® found that 1 gram of freshly dissolved gelatin is pre- cipitated by 0.7 grams tannin, but after standing 24 hours 0.4 ~ tannin will precipitate it. On rewarming the original condition returns. Any excess of tannin up to 3 parts per unit of gelatin is carried down, but upon washing the precipitate with alcohol, 97 per cent. of the tannin may be removed. Trunkel’s conclu- sion is that the gelatin tannin complex is an adsorption com- pound. ° 1b Bull. Acad. Sci. Cracovie, 1906, p. 497. 17 J, Am. Chem. Soc. 48, 29 (1921). 18 Chem. Z. 24, 567 (1900). 19 Biochem. Z. 26, 458 (1910). CHEMICAL EXAMINATION OF GLUE AND GELATIN 139 J. T. Wood ”° quotes much of the prior work on compounds of tannin and gelatin, going back to experiments of Humphry Davy,* who found that variable quantities of tannin were fixed by isinglass. Wood, using Coignet’s Gold Label Gelatin, found that the greatest amount of tannin which could be precipitated by 1 gram of air-dry gelatin was about 2.4 grams from a solu- tion containing about 6 grams of tannin, the volume of the solution after the addition of gelatin being 150 cc. Chromed gelatin absorbs just as much tannin as unchromed gelatin. Chromed hide powder is used for the assay of tanning materials. See Chapter 9 for the action of tanning BONERS: on cee and Chapter 8 for the effect of CS,. The halogens, chlorine, bromine and iodine react with Pane yielding insoluble compounds which form the basis of analytical methods. The chlorine compound described by Allen”? is a pale, yellowish-white powder, which is odorless, tasteless and imputrescible, and insoluble in water or alcohol, but soluble in alkalis. Yet Allen found that the bromine precipitation method could not be applied to commercial gelatin and glue, which ‘‘vielded results which at present are incapable of interpretation. The completeness of the precipitation of gelatin by bromine- water is affected by conditions not at present understood. In some cases the precipitation was very complete, while in other experiments, in which the conditions were but very slightly varied, much nitrogen remained unprecipitated.” Since glues and gelatins may vary considerably in composition and in their protective action toward the bromine compound, and since their “previous history” may affect their ability to form adsorption compounds, it is not surprising that Allen reported confusing results. | Platinic chloride and sulphate give precipitates with gelatin, and Crismer recommends an acid solution of chromic acid as a precipitant. Northrup ** followed the hydrolysis of gelatin by pepsin, trypsin, acid, and alkali, and found that the early action of the enzymes and alkali were similar but different from the action of 20 J. Soc. Chem. Ind. 25, 384 (1908). 2 Phil. Trans. 1808, p. 283. 2 “Comm. Organic Analysis,” 4th ed., Vol. 8, p. 591. 23 J. H. Northrup, J. Gen. Physiol. 4, 7 (1921). 140 GLUE AND GELATIN acid. Comparing the relative velocities of hydrolysis of dif- ferent peptide linkages, he found that trypsin would attack all those linkages that pepsin attacked, and some others besides. Linkages rapidly attacked by pepsin yielded only slowly to trypsin, while those most rapidly attacked by the enzymes yielded readily to alkali but slowly to acid. For a discussion of the cleavage products of gelatin, including those resulting from bacterial decomposition, see ‘Allen’s Com- mercial Organic Analysis,” 4th ed., Vol. 8, pp. 594 et seq. According to Seemann.** oxidising agents, like permanganates, yield with gelatin such products as: oxalan, NH,.CO.NH.- C,O,NH,; ammonium oxaminate, C,0,.NH,.NH,; ammonium oxalate; and oxalic, succinic, benzoic, butyric, acetic, and formic acids. Sometimes benzaldehyde, propionic and valerianic acids are produced. 3 | Detection of Glue and Gelatin. According to Allen® the property of gelatinizing on cooling is the only test from which the presence of gelatin in a complex animal liquid.can be safely inferred. To detect gelatin in ice-cream or in cream, the U. 8. Department of Agriculture use Stokes’ picrie acid method,?® which is as follows: Dissolve 5 grams of mercury in 10 grams of nitric acid (sp. gr. 1.42), and dilute to 25 times its bulk. To 10 ce. of this solution add 10 cc. of cream and 20 cc. of water, in order to precipitate all proteins except gelatin. If gelatin be present, the filtrate will give an immediate yellow precipitate with an equal part of a saturated aqueous solution of picric acid. The nitrate solution should give no turbidity with the picric acid solution. This test will detect one part of gelatin in 10,000 parts of water. To detect: gelatin in preserves, A. Desmouliére 27 takes 20 grams of the sample and precipitates the gelatin by gradually adding 100 ce. of 90 per cent. alcohol.27* After standing 2-3 hours, the supernatant fluid is decanted, the residue dissolved in hot water, and tested with picric acid and tannin which give pre- 24 Zentr. Physiol. 18, 285 41904). °° “Comm. Organic Analysis,’ 4th ed., Vol. 8, p. 592. 26 Analyst, 1907, p. 320. 7 Ann. Chim. anal. appl. 7, 201 (1902). “7a Gelatin is somewhat soluble in 90 per cent. alcohol, and the preserve usually contains water; therefore stronger alcohol should be used. Since gelatin precipitates most readily at the isoelectric point, the solution should have a py value of about 4.7. CHEMICAL EXAMINATION OF GLUE AND GELATIN 141 cipitates in the presence of gelatin. A confirmatory test 1s to add quick-lime, which evolves ammonia. 3 Henzold 28 proposed the following method for detecting gelatin. in foods: The specimen is boiled with water and the filtrate boiled with an excess of 10 per cent. potassium dichromate. If gelatin be present, a few drops of concentrated sulphuric acid produces a white flocculent precipitate, which gradually ag- glomerates at the bottom of the vessel. E. Schmidt 7° makes a reagent said to be sensitive for glue in the presence of ammonia, by acidifying Nessler’s reagent slightly with sulphuric acid. This gives a red precipitate which is filtered off, leaving a clear yellow solution which constitutes the reagent. Diirbeck *° uses a solution of thionin (a thiazin dye) to detect gelatin or agar in sausage. Agar gives a violet color, gelatin a deep blue. . Gold Number. Glue and gelatin possess such a powerful action as colloidal protectors, that a determination of the “gold number” after Zsigmondy’s method *! may demonstrate their presence, at least differentially. The “gold number” is the number of milligrams of a colloidal substance which just fails to prevent the color change (from bright red to violet) of 10 cc. of a colloidal gold solution upon the addition of 1 cc. of 10 per cent. NaCl. The colloidal gold solution is prepared as follows: One hundred and twenty ce. of distilled water, condensed in a silver worm, are placed in a 300-500 cc. Jena glass beaker. While heating there are added 2.5 cc. of a 0.6 per cent. solution of gold hydrogen chloride and 3-38.5 cc. of 0.18 N potassium carbonate solution, both of the highest purity. After boiling add promptly 3.5 cc. of dilute formaldehyde (0.3 cc. of commercial 40 per cent. formal to 100 cc. water). A bright red color should develop slowly, usually beginning as a brown or orange tint. Jena or equivalent glass should be used throughout, both in preparing the solutions and in making tests with them. 2 Z. offentl. Chem. 6, 292 (1900). 22 Farber Z. 24, 97. 30 Z, Nahr. und Geniissm. 27, 801. 31“Colloids and the Ultramicroscope,” New York, 1909; Z. anal. .Chem. 40, 697 (1901). 142 GLUE AND GELATIN The following table gives the gold numbers of some proteins according to Zsigmondy and Schryver: Substance Gold Number Gelatin acy oo ace een Ge ks cs el ee ee eee 0.005-0.01 Russian glue sic. c's sais su ce eee met eee ae 0.005-0.01 . [sitiglass’) os SON Oss ae ee re a 0.01 -0.02 Casein: (in ammonia) 0) ose agewe ss meee Prey rt Heg-globulin . . cass cscs Private communication. CHEMICAL EXAMINATION OF GLUE AND GELATIN 149 through CuSO, solution to eliminate sulphides. He distils over 200 to 250 cc. and catches it in 25 to 50 cc. x iodine solution. The former official method was to collect the distillate in standardized iodine solution, and then determine the excess of iodine by titration with standardized sodium thiosulphate. This method was severely criticized *®* and was abandoned. Parts of animals slaughtered under Government supervision as well as gelatin made therefrom and gelatin containing no added sulphur dioxide or sulphites, all showed apparent sulphur dioxide. C. Mentzel ** found in pure chopped meat from 0.0014 to 0.0021 _ per cent. of apparent sulphur dioxide equivalent to from 0.0054 to 0.0084 per cent. of sodium sulphite. When onions were added to the chopped meat, the percentage of apparent SO, was largely increased, probably owing to the presence of allyl sulphide. The substance responsible for apparent SO, in meat, gelatin, etc., is possibly the sulphur-containing protein cystine. It should be noted that, while meat contains about 70 per cent. of water (besides much fat), gelatin contains only about 15 per cent. of water, and in it all water-soluble substances would natu- rally be concentrated. Meat may absorb SO, from the atmos- phere,*® and Poetschke (loc. cit.) found the same condition with gelatin. Another possible source of error was pointed out by Baythein and Bohrisch,*® who observed that the limestone or marble used to generate CO, sometimes contains sulphides, and the gas should be washed with copper sulphate. From the results of the analysis of over 1,000 samples Poetschke reports as follows: *° SO, Content or SAMPLE Less than From 100 to Over 100 parts 500 parts 500 parts Year per millton per million per million Me ile eke sacks ss 19.65 44.87 35.48 ROMs as is bone t5 40 60.62 - 18.61 20.70 i Oe 66.64 18.92 14.44 RR dig ors vc cles i0 8 66.47 23.92 9.60 COON a re 87.90 9.16 2.94 BER as cs'tsseas s 48.27 42.65 9.08 36 J, Alexander, J. Am. Chem. Soc. 29, 783 (1907) ; E. Gudeman, J. Ind. Eng. Chem., Vol. I, No. 2 (1909) ; P. Poetschke, J. Ind. Eng. Chem., Vol. 5, No. 12, (1913). 31 Zeit. fiir Untersuch. Nahrungs und Genuss.. 11, 3820 (1906), 88 A, Kickton, Zeit. fiir Untersuch Nahrungs Genuss. 11, 324 (1906). 39 Zeit. fiir Untersuch Nahr. und Genuss. 5, 401 (1902). 40'The figures given are the percentage of the total number of samples. 150 GLUE AND GELATIN Poetschke also found free chlorine in the CO, which would cause error by oxidizing SO,; it is also removed by washing through copper sulphate: He prefers the use of iodine instead of bromine in the gravimetric method for determining SO,, and finds Gudeman’s steam distillation method shows no advantage. Some samples of gelatin contained hydrogen peroxide evidently added to oxidize the SO,.* As a result of these analytical uncertainties, traces of sulphur dioxide are disregarded in practically all jurisdictions. The fig- ures of Poetschke, including foreign as well as domestic gelatins, show a very creditable and successful effort on the part of gelatin manufacturers to comply with the official regulations. “Irving Hochstadter (U. S. Pat. 1,412,523, dated April 11, 1922), has pro- tected the process of bleaching foods (including gelatin) with SO., and then oxidizing any traces of this gas or of sulphites that may remain into sulphates by means of H2O, or other peroxide. ‘ Chapter 11. Technology of Glue and Gelatin. The technical operations in the manufacture of glue and gelatin may be grouped under the following heads: 1. Stock or raw material and its treatment prior to boiling. 2. “Boiling” or cooking, that is preparing a solution of glue or gelatin from the treated stock by the action of hot water. 3. Clarifying, bleaching, filtering, evaporating or otherwise treating the dilute glue liquor. Chilling the glue liquor to a jelly. Cutting, spreading and drying the jelly. Packing, breaking, or grinding the dried glue. Testing, grading, and selecting or blending the finished product. sb a The treatment of glue stock varies considerably, but the opera- tions of glue manufacture subsequent to boiling, are along the same general lines in most factories, although there are differ- ences in apparatus and in chemical and mechanical treatment. Special machinery may effect the consolidation of several, or the elimination of one or more of the above operations. ‘Thus, if the glue is dried on a heated rotating drum, the operations of chilling, cutting and spreading are eliminated, the glue liquor being fed directly to the roll or drum. G.,. Illert? describes a complete plant (which he claims can be operated by three men), in which a concentrated glue-foam is fed automatically to a drying roll, the dry glue passing directly thence to the packing apparatus. Glue Stock. The most motley array of raw materials find their way to the glue factory. They include slaughter-house refuse such as heads, feet and bones from the canning department; butchers’ refuse; 1“Die neuzeitliche Hinrichtung und der Betrieb einer Lederleimfabrik,’’ Chem. App. 8, 78 (1921). : 151 152 GLUE AND GELATIN dried bones (junk bone) which come largely from South America or India; bones from garbage; clean bone trimmings from button and handle manufacturers; horn pith (the cornellion or interior bony support of the horn); trimmings of hides and skins such as raw hide, calves’ pates, tannery trimmings and fleshings; shreds of rabbit, hare, nutria and other skins from the hatters’ fur cutting industry; sinews and pizzles. Glue stocks (and the glues which they yield) may be divided into four groups. 1. Bone stock. Besides the kinds above mentioned, this in- cludes: ossein, which is degreased granulated bone from which the soluble lime salts have been leached by acid; “spectacles” (knochen-brillen, dentelles), a variety of ~ ossein showing the round holes from which buttons have been cut; acidulated horn pith, a variety of ossein show- ing the original shape of the horn. Acidulated bone (ossein and horn pith) yield very high-grade glues and gelatins, fully equal to those produced from the finest hide stock. 2. Hide stock. This includes such curious things as old Turk- ish raw hide moccasins, lips and ears, hide bale wrappings, and discarded loom-pickers worn out from incessant impact of the shuttle. Drved-hide stock may have been limed be- fore drying; thus guaras negras is dried unlimed, while guaras blancas is dried limed hide stock from South Amer- ica. Wet or green hide stock is usually salted or limed to preserve it, but wet limed stock will not stand long trans- portation unless the temperature is low. 3. Sinew stock. Sinews are imported in dried state, but local sinew stock is usually shipped in green salted condition. With the imported dried sinew stock are often included dried bull’s pizzles. 4. Tanned stock. In recent years processes have been perfected for making glue from leather waste. Treatment of Glue Stock. Before describing in detail the steps in the treatment of glue stock prior to boiling, it should be borne in mind that with some stocks the complete. cycle of operations is unnecessary, while with other stocks special treatment is required. TECHNOLOGY OF GLUE AND GELATIN 153 Hide and acid leached bone stocks are limed, but rabbit skin stock is merely soaked. Many manufacturers cut or shred their hide stock so that it may be limed and extracted more readily. Sinews and pizzles are limed and treated like hide stock. The treatment of bones varies considerably, depending on the facilities of the plant and the products desired. The finest bone glues and gelatins are made by first converting the bone into ossein, which is accomplished by an acid leaching process (see p. 156). Many packers and slaughter houses aim to secure mainly “steamed bone,” and submit the fresh bone to one or two extrac- tions in a pressure tank whereby most of the grease and a large part of the glue are incidentally obtained. Plants operating on dried or junk bone usually granulate the bone and recover the remaining grease with volatile solvents, after which the glue is extracted. The poorest bone glues are usually made by the unskillful “boiling” of uncleaned and frequently unwashed garb- age or slaughter-house bone. Glues made from bones that have not been degreased, usually contain considerable grease. In general all salted stock is washed free from salt, limed stock, if old, is washed to get rid of old carbonated lime, and dried stock is soaked to soften and swell it. The glue manu- facturer must be on the alert to discover adulterations—thus a precipitate of barium sulphate is sometimes formed on hide stock to add to the weight. Even preservatives and disinfectants legitimately used may cause trouble; for example, arsenic is often used in curing hides, and the customs regulations of some countries require disinfection against anthrax, for which purpose bichloride of mercury, formaldehyde or sulphur dioxide may be officially prescribed. The following diagrammatic table shows the usual schemes of handling various stocks: Bone Stock. a. Wash and boil. Green, fresh or packer bone |b. Wash, grind, degrease and Refuse, town, or garbage bone boil. Dried or junk bone c. Wash, grind, degrease, acidu- Steamed bone late, lime, wash, neutralize and boil. Acidulated bone (ossein) Soak, lime, wash, neutralize and Acidulated horn pith boil. 154 GLUE AND GELATIN Hides and Sinew Stock. Green or fresh trimmings or fleshings, also the same stock | Wash, lime, wash, neutralize and partly limed boil. Salted hide or sinews Dried hide pieces or ned oem wash, lime, wash, neutral- (whether previously limed or se nny Nea not) Leather or Tanned Stock. Soak, detan, lime, neutralize and boil. Modern practice is to cut up hide and leather stock to render subsequent operations more rapid. Water Supply and Sewage Disposal in the Glue and Gelatin Factory. Whatever salts or other non-volatile impurities exist in the water with which the glue solution is made, will be found in more concentrated state in the dried product. Where such impurities accumulate in a steam boiler, they may be largely removed by blowing down the boiler from time to time; but there is no practical way of removing them from the glue solu- tion. | The importance of pure water in a gelatin factory, is, there- fore, obvious, and for especially pure products filtration or even distillation may be advisable. i An abundant supply of water is essential, as well as adequate provision for the disposal of the large volume of wash-water which may cause difficulty owing to the fact that at times it is - liable to putrefy. Putrid glue has a peculiarly nauseating odor, and the sewage from a glue factory requires even more con- sideration than that from a tannery. Methods and Apparatus for Preparing Glue Stock. Bone Stock. To remove metallic and other impurities which would otherwise cause costly damage to machinery or contami- nate the glue or gelatin, the bones are first sorted and passed before a powerful electro-magnet which removes bits of iron concealed by fat or dirt. The bones then pass to the crushing or granulating machines. In Europe heavy-toothed steel rolls driven by powerful spur gears are generally used for crushing, but high-speed percussion mills TECHNOLOGY OF GLUE AND GELATIN : 155 may be used providing the bone dust is sifted out. Stamp mills are also used. ea ak Bones are often given a preliminary boiling to remove the major portion of the grease. For the extraction of grease from the crushed bone, petroleum, benzine or naphtha is generally employed, although coal tar benzene, carbon tetrachloride, or carbon bisulphide may be used. According to H. G. Bennett, the petroleum fraction boiling at about 212° F. is now used in most British factories. All of it must be volatile at 280° F., and the last traces are blown out of the bones with steam at 80 lbs. pressure. L. Thiele? reports the use for bones of a mixture of 20 parts benzol, 60 parts toluol, and 20 parts xylol, and gives distillation tables of this and of three satisfactory benzines. A suitable benzine, according to Thiele, should have a sp. gr. of 0.745 and boil at about 100° C.; 99 per cent. should distil over at 180° C. and any balance between 130° and 140° C. Carbon tetrachloride has a low boiling point, gives light- colored grease and above all is non-inflammable; but its cost is high, special tin-lined apparatus is necessary, and the loss in solvent value is high. The use of naphthalene as a solvent has been patented, but it is not used. There are many types of apparatus suitable for extraction, and since improvements appear from time to time, the makers of such machinery should be consulted for latest details.* De- scriptions of extraction apparatus are also to be found in most standard books on chemical engineering. | The general principle on which most extraction systems work, is to have the solvent percolate through a granulated bone con- tained in a closed tank, the fat-containing solvent being caught in a still set at a lower level. The solvent vapors from the still are condensed and the fat-free solvent is again sent through the bone. When extraction is complete, the condensing solvent is diverted to a storage tank, and the solvent remaining in the bone is driven out by steam, a special separator being used to part the solvent and the condensed steam. The apparatus is then 2“Glue and Gelatine,”’ 2d ed., M. Jiinecke, Leipzig. 3 Nothing is to be gained here by burdening the book and the reader with a diagram of some selected form of extraction apparatus and details regarding the operation of its valves, etc. This would simply serve to perpetuate types which may soon become obsolete because something better has been discovered. Loony GLUE AND GELATIN opened, the extracted bone removed, and the solvent-free fat run off. Before boiling, the degreased bones are freed from dirt and adhering meat by being rotated in a large perforated screen or drum called a rattler or cleaner, in which they are polished by auto-attrition. Thiele reports the following average yield from raw bones: (Crushes DODGE lee. born wen ten eee 50.3 to 59.5 per cent. Bonetadusticy Pye eo. cate SG at ataw Bone aber tay ser eet ido eee HGiS Volts ic eco ee ee oo ie Horie ids oh eee ae ees 0.01 “ 0.04 Tennongs ate cok: donecte ees 0.19 "Fis SONS eis Meee sc eee ae ee ae 0.02 “ 0.10 The degreased bone has from 5 to 6 per cent. of glue and about 60 per cent. of calcium phosphate. The acidulation of the degreased bones is Hefei accomplished by dilute (about 8 per cent.) hydrochloric acid and takes place in large wooden vats, which may be subjected to intermittent rotation, or through which the acid solution is slowly circulated by pumps. The counter current system of circulation is used, and the time required for treatment varies with the nature and size of the bone, being from 8 to 10 days with occasional limits of 4 to 14 days, according to Bennett, and from 2 to 3 days, according to Thiele. Degreased bones are attacked more rapidly than steamed bone. According to Bogue ** American practice is to use from 2 to 5 per cent. hydrochloric acid, one pound of bone requiring roughly one pound of 22° Beaumé acid for com- plete extraction: In acidulation the main reactions are as follows: Ca,(PO,), + 4HCl = 2CaCl, + CaH,(PO,), (acid phosphate) - Ca,(PO,), + 6HCl = 3CaCl, + 3H,PO, Ca,(PO,), + 4H,PO, = 3CaH,(PO,), The acid phosphate is then precipitated by carefully adding milk of lime: CaH,(PO,), + CaO = Ca,H,(PO,), + H,O H,(PO,),-++ 2CaO = Ca,H, (PO,), 4a (Secondary reactions occur here to some extent.) To avoid an excess of lime which would cause a reformation sa “The Chemistry and Technology of Gelatin and Glue,” McGraw-Hill Book Co., 1922. TECHNOLOGY OF GLUE AND GELATIN 157 _of tricalcium phosphate, filtered samples of the acid liquor are tested from time to time with molybdic acid solution. When the acid phosphate and free phosphoric acid are both completely con- verted into Ca,H,(PO,)., a precipitate of ammonium phospho- molybdate no longer forms, and the addition of lime is stopped. Should an excess of lime be accidentally used a suitable quantity of acid liquor may be added to retrieve the error. The precipi- tate, Ca,H,(PO,)., is then washed free from calcium chloride in a filter press. Since the phosphate readily hydrolyzes, as little wash water as possible is used. It is known as “precipitated bone phosphate” and is largely used in the manufacture of bone china and fertilizers. The ‘acid phosphate” is used in making phosphate baking powder. The soft collagen, after washing and neutralization of the residual acid with lime water, may be made directly into glue or gelatin. If dried at a low temperature it yields commercial ossein. : Other acids than hydrochloric may be used to acidulate bone. Sulphurous acid is the one most employed, though phosphoric acid has been tried. Sulphuric acid is not suitable owing to the formation of insoluble calcium sulphate which blocks the process. In the process patented by Grillo and Schroeder bones are disintegrated by moist sulphurous acid gas or by liquid sul- phurous acid according to the equation: fae = SO, | H,O — Ca,H,(PO,), + CaS0,. Bones thus treated readily dissolve upon boiling or steaming, yielding a “mud” which forms a valuable fertilizer after the calcium sulphite is oxidized to sulphate. The Bergmann process for decalcifying bones is as follows: The degreased bones are placed in closed tanks and a solution of sulphurous acid is percolated through them, its strength being maintained by continuous additions of sulphurous acid gas. This leaves a thoroughly bleached ossein which is washed free of acid. ‘The leach liquor is heated in a lead lined tank, liber- ating free SO, for further use, and precipitating calcium phos- phate and calcium bisulphite. The bisulphite is decomposed by hydrochloric acid, freeing a further quantity of SO, for return to the process, so that all told only about 5 per cent. of SO, is lost. 158 GLUE AND GELATIN Dentelles consists of ossein made from button makers’ bone refuse. It is often called “spectacles” because the pieces of bone contain round holes which recall the appearance of eye-glasses. Prepared horn pith is a variety of ossein made from the cornel- lion or interior osseous core of the horn. Since it does not come in contact with flesh, and has a porous structure that renders easy its extraction, it produces high-grade gelatin. The acidu- lated horn pith of commerce keeps its original shape. Pieces of the skull bones are often left on, and if not properly leached, constitute “dead bone,” which adds to the weight but reduces the percentage of gelatin yielded. The yield from ossein is said | to vary from 65 to 85 per cent. Hide and Sinew Stock. The liming of hide, sinew and ossein stock has for its object the thorough swelling or “plumping” of the stock and the elimi- nation of mucin. The lime pits are square wooden or cement tanks about 4 feet deep, sunk in the ground like those of a tannery. The soaked and washed stock is thrown into a satu- rated solution of lime contained in the pits, and the stock is occasionally stirred up or transferred from one pit to another, with the aid of a long-handled fork, the lime solution being agitated, renewed or strengthened as often as necessary. ‘Thick hide pieces often have to remain in the lime vats several months before they are properly limed; thin fleshings or skivings lime much quicker, and in general liming proceeds more slowly in winter. In some plants to save labor the stock is pumped in and sucked out of the vats by large centrifugal pumps similar to those used in dredging operations, or else is handled by bucket chains. To shorten the time of liming, the hide pieces may be cut up or shredded. Furthermore caustic soda is often added to the lime liquor to “sharpen” it and produce a quicker swelling—too much must, of course, be avoided. With gelatin stock sodium peroxide is often used to produce a bleaching action at the same time. Washers. , To swell up and soften dried hide or sinew stock or to free it _ from salt or lime, it is treated in mechanical washers. TECHNOLOGY OF GLUE AND GELATIN 159 The type most popular in America is the “cone washer.” This consists of a heavy slatted, hollow cone about 5 feet long, that is rolled by a rotating arm around a shallow circular tank about 10 feet in diameter through which a current of water is passing. ‘The cone presses and kneads the stock and the agita- tion results in thorough washing by the water which enters at the center and runs off through perforated grids at the outside of the tank. In Europe smaller tanks with rotating paddles are in com- mon use. G. Illert * describes a series of washers having horizontal arms turning at 90-100 R.P.M., the hide stock being passed from one to the other by bucket elevators. A copious spray of water removes lime, etc., through the perforated sides and bottom and the washed stock is automatically fed to a press, where it is squeezed before dumping into the boiling tanks. A capacity of 10 tons per hour is claimed. After washing limed stock, it is usual to add some hydro- chloric or sulphuric acid to the last wash water and let the stock soak in it so that any remaining lime may be neutralized. This is readily determined by cutting open a piece of swollen stock and testing with litmus or phenolphthalein. Alum is also fre- quently added to the last wash water. The remaining acid is then washed out and the stock is trans- ferred to the cookers. Sulphuric acid usually clouds the glue by forming a small quantity of calcium sulphate. Hydrochloric acid forms calcium chloride which keeps the glue clear but which lowers the jelly strength if much be present, for whatever is - left in the stock remains in the finished glue. In making photo- graphic gelatin it is* particularly objectionable to have any quantity of salts left; calcium chloride is, of course, hygro- scopic. Tanned Stock. H. R. Procter® suggested that chrome tanned hide may be stripped of chrome for glue manufacture by a solution of Rochelle salt or other salt of an hydroxy-acid. M. C. Lamb 4Chem. App. 8, 78 (1921). 5 Soc. Chem. Ind. Annual Rept. on Appl. Chem., 1916, p. 232, 6 J. Soc. Chem. Ind. 38, 572A (1919). 160 GLUE AND GELATIN leaches the cleaned disintegrated chrome leather for 48 hours in a 15-40 per cent. solution of organic acids containing two or more hydroxyl groups, oxalic acid being preferred. The chrome is precipitated as hydroxide from the extract, and the regen- erated hide, after washing in weak alkali, is limed as usual to make glue. The results are said to be very satisfactory, which can hardly be the case with the drastic process of A. Wolff,’ who dissolves chrome leather waste in at least its own weight of 5 per cent. sulphuric acid. After removing the separated fat, the chrome is precipitated as hydroxide by lime, the lime re- moved and the filtrate dried for glue. S. R. Trotman ® dechromed hide for glue making by oxidizing the chrome to sodium chromate with sodium peroxide. W. Prager ® converts the basic chrome salt into the normal soluble salt by a 2 per cent. solution of sodium bisulphite. Lime and other bases have also been used to de-tan chrome leather,’® but the acid methods seem to be preferable. Boiling Apparatus and Methods. In the extraction, cooking or “boiling” process, the prepared stock is subjected to the solvent action of hot water or steam, whereby the swollen collagen is changed into gelatin or glue. Hofmeister regarded the change in a definite hydrolysis pro- ceeding in two stages according to the equations: Cro2HsgNsi08 + H,0 = Cy o2H15:N310 95 Collagen + water = Gelatin Cro2Hy51N3i025 ++ 2H,0 = C5sH5N1;02 ae Cur HroN,Or9 Gelatin + Water = Semiglutin + Hemicollin Considering the fact that the final disintegration products of gelatin are amino-acids, and that progressive heating results in progressive degradation, it is obvious that the much used term “hydrolysis” simply conceals our real ignorance of what actually does occur. In fact, Emmett and Gies claim that the process is one of molecular rearrangement and no hydrolysis at all. But even this does not seem to coincide with the experimental facts. ™J. Soc. Chem. Ind. 38, 331A (1919). 8 J. Soc. Chem. Ind. 30, 1462 (1911). ®J. Soc. Chem. Ind. 32, 501 (1918). 10 See e.g. German Patent 202,510, TECHNOLOGY OF GLUE AND GELATIN 161 With colloidal substances like gelatin, chemical changes are | so closely associated with physical changes, that-no line of sepa- ration can be drawn between the two. Indeed, the whole boil- ing process appears to be a gradual breaking up of colloidal adsorption complexes (or of large ‘molecules’ held together by residual molecular attractive forces), with accompanying changes in free surface and amount of water adsorbed. The higher the extraction temperature, and the longer the stock and liquor are exposed to it, the more rapidly these degen- erative changes proceed, and the lower in test and darker in _ color the glue will be. Therefore, it is desirable to extract the stock as quickly as possible, and at the same time to keep the extraction temperature low. Since these two factors oppose each other there is a wide range of possibilities. Usually with gelatin where color is important, the temperature is kept low, even though the extraction period is thereby lengthened. With glues, especially bone glues, higher temperatures are generally used. Since the stock dissolves quicker in pure water than in glue solution, attempts to produce too concentrated a lquor unduly protract the period of extraction. It is really a misnomer to call the extraction process ‘“‘boil- ing,” for the boiling temperature is seldom reached, except in _ pressure tanks, or when extracting residues. The temperatures generally used vary from about 70° to 90° C., depending upon the kind and condition of the stock. Too low a temperature, which would favor bacterial growth and decomposition, must be avoided. | Whereas slow circulation of the glue liquor (i.e. by rotary pumps) aids solution, unnecessary agitation is harmful as it tends to lower viscosity and jelly strength and cloud the liquor. Two types of kettles, or cookers, are used: (1) open tanks; (2) pressure tanks. Open Kettle or Tank. The open tank, used mainly for hide, sinew or ossein stock, usually consists of a rectangular or round wooden tub having a closed steam coil over which is placed a perforated or slatted false bottom of wood or iron, so as to leave a circulating space 162 GLUE AND GELATIN between the two. Upon the false bottom is placed a layer of excelsior or straw often topped with a thin layer of hair, thus forming a rough strainer, upon which the stock is thrown until it reaches within a foot or so of the top of the tank. Sufficient pure water is then added (almost enough to cover the stock) and steam is turned into the coil until the desired temperature is reached; whereupon the steam is cut down to minimum necessary to maintain this temperature. As a rule the first “run” or “boiling” is made at about 70° C. (158°), and subse- quent runs are made at progressively increased temperatures. During boiling the stock is occasionally “opened up” by | stirring with a long pole, so as to permit a more perfect circula- tion of the liquor, or a circulating “chimney” is provided. From time to time a sample of the liquor is tested by a hydrometer or by chilling it in a cup, and when a sufficiently concentrated “soup” is obtained, it is run off, fresh water is added, and another “run” or boiling is made. A boiling usually takes from 2 to 6 hours, depending on the nature of the stock and the tem- perature used. ‘The last run or wash water is extracted at boil- ing heat and is usually so weak that it is added to another kettle or to a stronger run, or else must be evaporated. The residual tankage is used for fertilizer. It may contain considerable grease or insoluble lime salts of fatty acids. In this event it is boiled with sulphuric acid to liberate the grease, which is then squeezed out in an hydraulic press. During boiling in open tanks, most of the grease contained in the stock rises to the surface and is skimmed off. With fleshing stock the yield of grease is heavy, often exceeding the yield of glue in the case of machine fleshings which take in relatively little of the hide or skin substance. . Pressure Tank. The pressure tank is largely used for extracting untreated or degreased bone stock, and acidulated bone. The tanks are ver- tical steel cylinders with convex ends, having large manholes at the top for filling, and at or near the bottom for discharging the spent bone. Hide glues made in pressure tanks are usually weak, and the tank is seldom used for hide stock. The bones may be boiled with water under a pressure of from TECHNOLOGY OF GLUE AND GELATIN 163 10 to 20 lbs.; or hot water may be allowed to trickle in from the top while steam enters from the bottom (English process) ; or the condensation of the steam may supply the necessary water. ‘The successive runs of glue obtained are more concen- trated than those yielded by open tanks, though they do not have so strong a jelly. They are drawn off from time to time through a perforated false bottom. The most modern practice is to work pressure tanks in gangs upon the counter-current principle. Pressure tanks with circulating mechanism for handling ground bone have also been patented. If bones are intended for making bone black, they are either degreased by volatile solvents or else they are given one light cooking to remove most of the fat and but little of the glue. If too much of the nitrogenous matter is removed, the bone black will be of poor quality. After extraction of the glue, the bones are dried in a rotary drum dryer, ground in a high-speed rotary: mill, and sold for fertilizer. Thiele reports the following analyses of bones from which the glue has been extracted. Bones previous extracted with benzine Boiled bones Steamed bones Vo eS a 8.54 to 9.25 10.81 10.79 to 12.18 Organic matter ........ 1/1074 19.53 25.97 22.48 “ 24.62 Ca and Mg carbonates.. 7.50 “ 8.74 6.28 6.33 “ 6.89 A IS Ses O02) "0.59 1.07 O86 25 Ca poospnate ......... Ghigo 00410 53.15 5d04 Fo Bia LO os Trace 0.27 Trace (OS CONE 38sto. 178 2.45 168 to 1.74 OST... ee 068 “ 1.05 1.91 164°“ 1.72 Equivalent in glue..... Gios. C06 10.06 9.10 “ 9.56 He also gives a detailed description of the operation of a diffusion battery of four pressure tanks in making bone glue (loc. cit., p. 50). He also describes (p. 98) a gelatin extractor with false bottom, in which the stock is treated with a spray of superheated water. He recommends glass enameled vessels for handling the gelatin liquors, which come off continuously and may be collected into one “run” or divided into various runs or fractions. The liquor resulting from the first extraction of the stock is known as the “first run,” that from the second extraction the “second run,” etc. The first run, having been subjected to less 164 GLUE AND GELATIN heat for a shorter time naturally yields the strongest glue. The various runs may be kept separate (known as ‘‘successive glues’’) or they may be mixed together and dried as one batch. The cheaper bone glue liquors are often mixed with hide glue liquors, the resulting glue being known in Germany as “misch-leim.” In America such mixtures are usually made by mixing the sepa- rately granulated glues, although factories producing both hide and bone glues often mix the liquors. Clarification, Bleaching and Evaporation of Dilute Glue or Gelatin Liquors. While the removal of relatively coarse particles from glue liquors is readily effected by a strainer or filter press, the high protective or deflocculative action of glue makes difficult the separation of any finely subdivided or colloidal matter, which renders the glue turbid. The methods of clarification, apart from simple straining, fall into three groups: 1. Mechamecal (settling or centrifugation). 2. Adsorptive (filter-mass or bone black). 3. Formation of precipitates (albumen, phosphoric acid, alum, etc.). About twenty years ago the writer was able to clarify gelatin liquor in an ordinary cream separator (DeLaval type) but the modern super-centrifuge (Sharples type) is, of course, much more efficient and is said to be much used in the United States for gelatin liquors. The use of paper or cellulose filter-mass, along the lines of modern brewery practice, and of bone black as per the methods used in sugar refining or glucose clarification, give excellent results; and they are widely used for gelatin. An old method of clearing liquors was to add blood or a solution of blood or egg albumen and then boil. The albumen in coagulating would carry down most fine turbidity; but the heating weakened the glue or gelatin, and gave a soapy smell to the gelatin. In the Grillo and Schroeder process the precipi- tate of calcium sulphite carries down most suspended matter, 11 Several varieties of filters are on the market, details regarding which may be obtained from the makers. TECHNOLOGY OF GLUE AND GELATIN 165 and the use of phosphoric acid is very common in clarifying gelatin liquors. If made from bone stock they may have sufi- cient lime to give the desired precipitate of calcium phosphate; otherwise milk of lime may be added as is done in the defeca- tion of sugar liquors. The glue liquor must be warm enough to let the precipitate settle, but the temperature must be kept as low as possible to avoid loss of strength. Phosphate of soda may be used, but has the disadvantage of leaving soluble sodium salts. Oxalic acid may. also be used, producing a precipitate of calcium oxalate. Alum or aluminum sulphate is the clarifying agent most used for glues. It seems to undergo hydrolysis in the presence of the colloid, and the nascent hydrate of alumina combines with the glue or the impurities giving a precipitate which can be settled or filtered out. Lambert?” gives the following details: “A bucketful of liquor, which should have a temperature of about 80° C., should be drawn from each vat, the necessary quantity of alum stirred in, and the contents thoroughly mixed in the mass, the heat at the same time being raised to 100° C. by means of a steam pipe. After boiling ten minutes the steam is turned off and the liquor allowed to settle, during which the heavier mineral and organic impurities fall to the bottom, while the lighter form a coagulated scum on the surface.” Such a procedure is obviously very injurious to the strength of the glue; in fact, as a general rule highly clarified glues are relatively weak, strength being sacrificed for appearance. Schwerin ** has patented a process for clarifying gelatin and glue liquors by electrodsmosis, but this method has not yet found general application. H. Fleck * describes a process for improving glue by precipi- tation with ammonium sulphate or sodium bisulphite, which removes some of the products of hydrolysis. He warned against the danger of boiling glue solutions. Of course, any grease rising to the surface of the glue liquors is skimmed off, though with cheap bone glues, when grease is 122“Glue, Gelatine and Their Allied Products,’ London, 1905. 23 “Kolloid Z. 20, 64 (1917), Ger. Pat. 293,188 (1918). 144“The Manufacture of Chemical Products from Animal Offal,’’ Brunswick, 1878. 166 : GLUE AND GELATIN low in price, as much grease as possible is kept in the glue liquor and helps to render the glue free from foam _ besides increasing the yield. For the bleaching of glue, sulphurous acid or bisulphites are most commonly used because of their cheapness. The stock may be bleached before boiling, or SO, gas or its solution may be added to dilute or to concentrated liquors. Sodium hydro- sulphite and analogous compounds (zinc hydrosulphite, sodium formaldehyde sulphoxalate) have certain advantages, but are more expensive. For bleaching gelatin liquors, hydrogen peroxide may be used, but it is preferable to bleach the stock before boiling, in which case sodium peroxide or sulphurous acid are also of service. Peroxides also oxidize sulphites to sulphates. — Evaporation. Many first or second run gelatin and glue liquors, as they come from the boilers, will set firmly enough to be chilled, cut, and dried; but in most cases it is necessary to evaporate or concentrate the liquors. The old style steam worm rotating in a trough-like tank has been superseded by the modern double or triple effect evaporator. It is not proposed to burden the reader with a detailed descrip- tion of the various types of vacuum evaporators, which are being continually improved; for the apparatus used to-day may be superseded by a superior form soon after publication of this book, if not before. The apparatus is well known, is described in many elementary books, and the various manufacturers are at all times ready to give the latest details. The film evaporators (Yaryan, Kestner, Lillie, and Blair- Campbell) are in high favor, since in them the glue liquors are but a short time under the action of the heat, and this is conducive to maintenance of strength. The degree of evaporation depends upon: - 1. The jelly strength of the original glue liquor; 2. Its initial concentration; 3. The temperature of the outside air. Weaker glues and warm weather naturally demand greater evaporation. TECHNOLOGY OF GLUE AND GELATIN 167 Antiseptics. Among the antiseptics commonly used in glues are zinc sul- phate (which tends to precipitate odor-producing decomposition products), boracic acid, borax, sulphurous acid and bisulphites, and formaldehyde (about 1-10,000). Beta-naphthol may be used in the limes if desired. Phenol (carbolic acid) has also been used on stock and in liquors, but the odor is very objec- tionable. Chilling. In most factories the glue liquors, after evaporation and other treatment, are poured into small rectangular galvanized iron pans and allowed to gelatinize, preferably in a chill room. Some- times the pans are allowed to stand in cold water. Many European factories have casting tables, which form the glue directly into cakes. These tables have hollow tops through which is circulated cold water or brine; the upper surface (of metal or glass) is divided into square or oblong recesses which are filled with the glue liquor and which often have a trade mark etched in, so that it appears on the glue cake. Many devices have been patented for chilling concentrated glue liquor into a continuous sheet, which is automatically spread upon nets. None of these have found general widespread use, and several have been tried and abandoned. One of the early patents was that of Peter Cooper-Hewitt (U.S. P. No. 11,426, issued 1894). Some of the European gelatin factories are said to employ such apparatus. In the United States the apparatus of Maurice Kind (U. 8S. P. No. 1,046,307, issued 1912) is em- ployed by several factories.1° This machine has an endless belt which passes through a refrigerating tunnel, and on which is formed a continuous sheet of jelly of the desired thickness. The continuous sheet of chilled jelly coming from the belt is automatically cut into sheets of the required size, and these sheets are automatically spread upon drying nets of the usual kind. Fifteen minutes after leaving the evaporator, the first sheets are ready to enter the drying room. A single unit of the Kind machine is said to have a capacity 15 See Arthur Lowenstein, Trans. Am. Inst. Chem. Eng. 10, 105 (1917). 168 GLUE AND GELATIN of 4300 lbs. of dry glue per day of 20 hours with a 16 per cent. jelly spread 14” thick. The unit occupies a space of about 85 feet by 10 feet, takes about 10 horse power, and requires about 10 to 12 tons of refrigeration. The advantages of such an automatic machine are obvious. Since gelatin solution is a particularly good culture medium, the more rapidly a glue jelly can be handled and dried, the less chance there is of contamination and bacterial decomposition. This applies especially to food gelatins which, because of the absence of antiseptics, are especially susceptible to the attack of bacteria. There is furthermore a big saving in labor, and more or less freedom from the effect of weather conditions. Glue and gelatin are frequently dried upon steam-heated rolls, which may rotate in a vacuum (Passburg system) or which may have a close-fitting hood forming a narrow channel through which a rapid blast of air carries off the evaporated moisture. Where vacuum or forced draft drying rolls are used, the chill- ing process is eliminated, for the concentrated glue liquor is fed to them direct. A recent German method (Ruf system) is to beat the thick glue liquor to a foam before feeding it to a steam-heated drum.® Cutting, Spreading and Drying. Where glue has been chilled on casting tables, the sheets of jelly are picked off by hand and spread upon the drying frames or nets. But where the glue has been chilled in pans or boxes, the large jelly blocks are removed (usually by dipping the pans for an instant in hot water) and cut into slices which are spread upon the nets. With large pans, the jelly blocks are first cut into several smaller blocks before slicing. | Any heavy impurities or particles of dirt-in the glue or gelatin liquor settle to the bottom of the jelly blocks, while grease and light particles float to the top. Therefore in most factories the blocks are sliced horizontally, and the ‘‘tops and bottoms” dried separately to make an inferior grade of goods, or else they are worked into another batch. If the jelly blocks are sliced ver- tically, the top and bottom impurities, if any, are distributed throughout the sheets. . 16 See G. Illert, Chem. App. 8, 78 (1921). TECHNOLOGY OF GLUE AND GELATIN 169 Most slicing contrivances have a grid of steel wires through which the jelly block is forced by a plunger; the distance be- tween the wires regulates the thickness of the slices. Or the blocks on a moving belt are carried against wires, some distance apart, and fixed at different heights. With hand cutters the jelly block is sunk below the surface of a table and raised inter- mittently by a ratchet;.between each successive elevation, a wire stretched on a bow-like handle is drawn through the block, which is thus cut into slices whose thickness depends on the number of teeth in ratchet. For very stiff jellies, knife cutters are sometimes used, but as a rule highly concentrated jellies show rough or wavy marks which detract from the appearance of the finished glue. The slices of jelly as spread on the nets vary from about 3 to 10” in width, to about 6 to 12” in length. A certain auto- matic chilling and spreading machine which was operated for a time in one plant, spread a strip the size of a whole net. Rib- bon glue is cut in strips about 114 to 2” wide and about 8” long. Noodle glue is cut in strips having a cross section about 1%” square. Bazaar glue is similar to noodle glue, but of about 1” cross section. The thickness of the finished glue depends upon two factors: the thickness of the jelly slice, and the concentration of the jelly. The sheets of glue suffer more or less distortion on dry- ing, and usually show the marks of the nets on which they were dried. In Japan nets of bamboo are evidently used, as some Japanese glues show distinctly. Cast glues are usually highly concentrated, and the cakes tend to hold their shape well. The so-called Scotch glue is dark and comes in cakes about 10x 12” with a loop of string through one of the long ends. Thin flake glue is usually made from jellies strong enough to be dried with little or no evaporation. But thinness of flake is no criterion of quality, since concentrated jellies of weak glues are often cut very thin to simulate this supposed ear-mark of quality. Gela- tins are usually quite thin cut, especially those which are to be put up in paper packages holding about 1 pound. The drying nets upon which glue and gelatin are dried, usually consist of rectangular wooden frames about 3 x 5 feet, upon which are stretched pieces of galvanized iron wire netting with a mesh resembling chicken wire fencing. Some factories use cotton or 170 GLUE AND GELATIN linen fish net, especially for gelatin, because if the zinc protec- tion cracks off the wire net, rust forms which injures the appear- ance of the product and may actually form insoluble brown flakes. The net frames may, of course, be made of metal, but each frame has a small block or leg in each corner so that when the nets are piled up into stacks there is a.space, usually about 1 inch, between the frames for air circulation. The stacks of nets are mounted upon wheeled tracks or bogies, which generally run on tracks, and pass on into the dry room. In the old days the dry room was simply an upper floor or loft with louvred sides, and the drying was left to the vicissitudes of the wind and weather. Warm weather would melt the glue on the nets or foggy weather would pit or mold it. In all modern plants the glue is dried by passing a current of warm air over the stacks packed in narrow alleys or tunnels. : Some prefer the forced draft or blower type of fan, whereas others prefer the exhaust or ventilating type which sucks the air through the tunnels. Since the latter type fan has a tendency to churn if pulling against much resistance, the positive pressure fan is generally used. The air entering the alleys is heated by passing it over banks or stands of steam pipes, any of the approved systems being used. Exhaust steam is utilized where possible. In order to prevent the air short circuiting and thus passing through the alley without taking up its quota of moisture from the glue, it is, of course, necessary that the stacks of nets fill the alley as completely as possible, and that the top, bottom, and sides of the alley be air-tight. Windows are usually provided, through which the temperature at various parts of the alley may be read from the outside. The alley itself may be straight or U shaped. The glue enter- ing the end furthest removed from the source of heat, first comes in contact with moist air that has suffered a drop in temperature because it has already evaporated water from the forward stacks. The stacks move progressively toward the pipe coils, near which they get their final “baking” before being removed from the alley. | Considerable experience and care are required to get the best results from a drying alley. Improper adjustment of the tem- TECHNOLOGY OF GLUE AND GELATIN 171 perature to the particular batch may cause the glue to melt and run through the nets or sink into the mesh, forming pendu- lous drops (“titted” glue), which are difficult to remove with- out damage to the nets. The formation of a very thin surface skin (skinning over) is a partial protection against these diffi- culties, but if the surface skin is too thick or formed too rapidly, it impedes further evaporation. The addition of a small quan- tity of formaldehyde to the concentrated glue liquor is said to facilitate the formation of a skin, besides acting as an antiseptic and giving a stronger jelly. Excessive drying must also be avoided, for besides diminishing the yield, it is apt to cause the glue to crack or check. | The time required to dry a batch of glue depends upon the concentration of the liquor, the thickness of the jelly slices, the strength or quality of the glue and the weather conditions. Good practice requires the drying to be completed as speedily as possible, usually within about 24 hours. Any plant where drying takes from 10 to 30 days, as claimed by E. Sauer,” must needlessly tie up an enormous amount of capital, product and apparatus. In England and on the Continent the prevailing practice is to pick the dried sheets or cakes off the nets and pack them into bags or other packages. In America the glue is simply dumped off by inverting the net, and the sheets are still further crushed by passing them through a rough breaker whence they issue in flakes (flake glue). A large percentage of glue in America is ground to about 8 to 16 mesh in high-speed percussion mills. At one time barrels were the leading packages here, but bags are coming into wider use, both for flake and ground glues. The different batches or boilings of glue and gelatin, which may amount to several hundred or even several thousand pounds, are as a rule kept separate for testing and grading. Where large lots of one grade are required, it is common practice to mix a number of boilings and then to test the mixture. According to Thiele the various brands of sheet gelatin, which are usually sold in 1-pound packages for food purposes, are selected as follows: 1% Kolloid Z. 17, 180 (1915). 172 GLUE AND GELATIN Average number of Run of Gelatin Brand or label ' sheets per lb. | -forileand 22) eee Non? plus ultfa:.<5 scene 285 Dae nats Bas Peaiien hates Maas Oo Gold extra and Gold....... 227-200 DO ALONG s Hele cutee eee Silvers se Ge a ee 180 tania oni eink soe ee CODDELe a des ss eee eee 136 Hielone ts). co. s ocd. eat Blacks. Pues edie. ean ee 90 The finished glue should be stored in a place of mean humidity and temperature. Excessive moisture is apt to cause the glue to soften and even be decomposed by molds or bacteria, whereas excessive heat and dryness tends to make the glue crack and check. According to E. Sauer ?§ if sheets of glue that have been stored for a long time in a very dry room are transferred to a very damp room, many sheets, often with a loud report, may burst into small pieces. The unequal absorption of water, which naturally takes place at first on the surface, produces a tension which forcibly relieves itself by breaking the sheet of glue. Low grade, highly hydrolyzed glue are especially apt to one or crack. Blow-down Processes. | Since the cutting, spreading and drying operations are expen- sive, and both time and space consuming, attempts are being made to “spray-dry” the concentrated glue and gelatin liquors along the lines now successfully used in making milk powder. If the cost can be gotten down, such processes should have a big future, especially for gelatin. Percentage Yield of Glue Stock. Since glue stock is for the most part a highly variable com- modity, definite yields can hardly be predicted without examina- tion; but the following table, prepared by a factory manager, gives the results of his experience with several varieties of stock: Percentage Yields Kind of Stock Glue Grease Green bones ........ ek ok ticks ee eee 10-12 8-10 Dry bones. 45 ue ee ee eee 14-16 5- 7 Green calf)... 2230 ee eee ae 18-22 3- 5 Green salted hide, 7720.5 i. ee 16-18 3— 5 Dry hide’ .2..s4. 25s aes Ses a oe Ce ee a Salted sinews ........ RE Par eect 18-22 5 ee i) Dry, SINCWS 3.44 un < chine leet eee ee 40-50 — Dry fleshings ... c.04.< 7st: » «sles es Ge 10-12 Wet. fleshings °. .i4.< 10 acer ein 8-12 12-15 Horse chide *(wet) i. Shoes ee eee 10-15 — Sheep ‘skin, (wet). 0\.. citen eee 6-10 . 10-18 % Kolloid Z. 17, 183 (1915). Chapter 12. The Testing and Grading of Glue and Gelatin. At the outset let it be emphasized that there is no single chemical or physical test which will satisfactorily gauge the value of a glue or gelatin for all purposes. Many authors have recommended individual tests and while these may have some value for special purposes, the wisest and safest way for the factory or sales manager is to run a series of connected tests which will grade the glue or gelatin against preceding lots of the same type, and thus render possible uniform deliveries to the consumer, whatever his business may be. This view was voiced by E. G. Clayton,’ who says: “In con- clusion the observations seem to show that, whilst it would be. rash to form a judgment on glue from a single test the evidence afforded by a number would be irresistible. Glue may be shown by certain tests to be suitable for one purpose, though less per- fectly adapted for another. ‘The expert’s wisest system appears to be, not to rely upon single short-cut tests of general quality but to employ a number of methods, including any having especial bearing on the present or prospective uses of the glue, and then to base his conclusions on a consideration of all the results together.” Before describing a connected series of general physical test methods which are, with more or less modification, used in glue ‘testing laboratories, let us review briefly some of the methods that have been proposed for testing and grading glue. Secrecy has been traditional in the glue trade, with the result that treas- ured methods may be inferior to other methods known to all. The references given are to books and papers published in the usual scientific journals. An exhaustive review attempting to fix priorities is not attempted. Some partially completed meth- ods of the National Association of Glue and Gelatin Manu- facturers are given in an Appendix. 1“The Technical Examination of Glue,” J. Soc. Chem. Ind. 21, 675 (1902). 173 174 GLUE AND GELATIN Jelly Strength. One of the first tests proposed was the consistency test of Lipowitz 2 which determines the relative capacity of the jelly for bearing a weight. ‘The instrument used ° consists of a saucer- shaped piece of tinned iron, 1 inch in diameter, having a thin metal rod soldered vertically to its concave side, the upper end of the rod being provided with a small metal funnel. The rod slips loosely in a perforated metal strip which supports the apparatus. The saucer is placed with its convexity next to the jelly, and shot gradually poured into the funnel until the saucer breaks through. The total weight required to rupture the jelly indicates the jelly strength. Of course, the determination must be made at a definite temperature, and with a definite concen- tration of glue. A thick top “skin” must be avoided. More recently R. Kissling? stressed the importance of jelly strength test, which is often called Kissling’s test. J. Fels® describes it as “correct as a comparative method,” and more recently R. H. Bogue® has found that this test alone would correctly gauge about 75 per cent. of all glues. It is also known as the “shot test,” the “jelly test,” and the “finger test,” the last from the fact that the relative jelly strength may be easily fixed by pressing the jellies with the finger tips. F. Davidowsky ? describes a jelly tester used by the factory at Hamborn. It consists ® of a hemispherical weight, bearing a vertical scale, which slides in a guide cylinder that rests on the jelly with a broad flat rim, and bears also an adjustable pointer to fix the zero point. According to K. Kieser® Davidowsky’s method is largely used in Germany to determine jelly strength. ‘This cor- responds to our “shot test” in all practical particulars. Many mechanical contrivances have been suggested to fix the jelly strength of glue. Some depend upon measuring the depres- sion produced by placing a definite weight on the jelly, without 2“Neue Chem.-tech. Untersuchungen,” Berlin, 1861, pp. 37-42. 3’ “Allen’s Comm. Organic Analysis,” 4th ed., Vol. 8, 607. 4*Chem. Z. 17, T26 (1898) ; ibid., 22, 450 (1898). 5 Chem. Z. 56 and 70 (1897). ®& Chem. Met. Eng., July, 1920. ™“Glue and Gelatin Manufacture,” 5th ed., p. 26. 8’ This instrument is practically the same as that described in Technical Note No. F-32 of the Forest Products Laboratory, Madison, Wisconsin, who will supply working drawings on request. ® Kolloid Z. 28, 186. ‘TESTING AND GRADING 175 breaking through. The jelly tester of E. S. Smith 2° consists of a pressure chamber whose bottom contains a thin elastic rubber diaphragm; attached is a rubber air bulb to produce pressure and a manometer to measure the pressure produced, that re- quired to produce a certain depression being taken as Jelly strength. EK. T. Oakes and C. E. Davis! described the jelly tester of A. Schweitzer, which consists of a balance, one arm of which carries a plunger below and a beaker above, so that on adding water to the beaker a definite compression of the jelly beneath the plunger may be produced. W. H. Low” has proposed a modification of Smith’s instru- ment. The Forest Products Laboratory 1? describes the con- struction and operation of a tester with a constant weight plunger, whose depression in the jelly is a measure of the jelly strength. All these methods ieolve re ae the jelly strength through the skin of uncertain strength and thickness which forms on the upper surface of the jelly. J. Alexander ** devised a jelly tester which avoids this disturbing factor by casting the jelly into truncated conical blocks in brass cups of definite size, and then removing the blocks and determining their resiliency. The trou- blesome skin is placed at the bottom, and a gradually increas- ing weight is applied to the top of the jelly block, until a definite compression of the jelly is produced. The jellies are cast in round brass caps 6 cm. high, 5.5 cm. in diameter at the open top and 5 cm. in diameter at the bottom, which is closed with a tight-fitting external friction cap. The truncated cones thus formed should be exactly 4.5 cm. high, the cups being filled only to that level. If the jellies do not push out readily on removing the cap, the closed cup may be dipped for an instant in hot water. After removal the jellies are placed 1 in a thermo- stat until they reach the desired temperature. S. E. Sheppard ** states that all modifications of the “shot test” 10U. S. Pat. 911,277; see J. Soc. Chem. Ind. 28, 252 (1909). toa J, Ind. d Hng. Chem. 14, T06 (1922). 11 J, Ind. Eng. Chem. 12, 255 (1920). 122 United States Dep’t of Agriculture, Madison, Wisconsin, in Technical Note F-32. #83. S. Pat. 882,731; see J. Soc. Chem. Ind. 27, 459 (1908). 14 Sheppard, Sweet, and Scott, J. Ind. Hng. Chem. 12, 1007 (1920). 176 GLUE AND GELATIN err because the stress applied affects both elasticity of the bulk and elasticity of the figure. For more exact results he devised a torsion dynamometer which measures the force required to twist to the breaking point jellies which had been chilled for 3 hours at 0° C. Above 10° the jelly strength begins to diminish rapidly, though no material change occurs till then. Using this instru- ment, Sheppard found that no simple relation holds between the concentration of gelatin and the jelly strength; and that jelly strength values determined for a single arbitrary concentration give a very arbitrary comparison of the jelly strengths, because the curves relating these values for different concentrations of commercial gelatins are not of a common family and often cut each other. He concludes that there is no definite relation be- tween the jelly strength at a given concentration, and the glue- joint or tensile strength of a dry glue joint.. While the H-ion concentration affects jelly strength, there is no simple relation between the two.t® C. R. Smith 1® describes a simple jelly-testing device which may be rigged up in any laboratory. An 80 mm. short-stemmed funnel accurately formed to a 60° angle, is closed at one end, and 120 grams of mercury are poured in, forming an upper sur- face 3 cm. in diameter. Over the mercury is layered 50 cc. of gelatin solution, which is allowed to set in a horizontal position (fixed by a spirit level) in a constant temperature bath at 10° C. The mercury is then run out, the funnel is connected with a water manometer, and a reduction pressure (6 dm. of water) is produced. The depression of the upper surface of the jelly produced by the suction is measured with a micrometer and indicates the jelly strength. Smith’s figures show, however, that the jelly strengths thus determined do not exactly parallel those estimated by his polariscopic method. This consists in finding the increment in the specific rotation of a 3 grams per 100 cc. solution between 35° C. and 10° C. Most commonly the jelly strength is determined by the finger test against standard samples, for this method is speedy and of sufficient accuracy for commercial purposes. Besides it fits in readily with other tests and does not. require any special ap- 18 Sheppard and Sweet, J. Am. Chem. Soc. 438, 589 (1921). 16 J, Ind. Eng. Chem. 12, 878 (1920). e TESTING AND GRADING 177 paratus. But it demands the possession of standard glues for comparison. ‘These, however, may be obtained by careful selec- tion, aided by any of the mechanical devices. Viscosity or Running Test. A viscosity determination in the form adopted by J. Fels ‘% is largely used in Germany.'® Fels originally proposed taking the viscosity of a 15 per cent. solution of glue at 30° C. with the Engler viscosimeter; but finding that some very high test glues would not flow at this temperature, later he 1® raised the testing temperature to 35° C. Fels’ test, as it is sometimes known, is therefore the viscosity of a 15 per cent. glue solution at 35°, as fixed by the Engler viscosimeter; and it is a single test of great importance. It is interesting to note that the temperature finally fixed by Fels is that recently shown by C. R. Smith ?° to be the one at which incipient gel formation begins, as is evidenced by the polariscope. R. H. Bogue *! has also recently recommended what he terms a “melting point” determination, which he says gives “a truer evaluation of product than by the use of the old and time-hon- ored methods.” Curiously enough Bogue’s method, though evolved independently after numerous experiments, is practi- cally the same as that of Fels. Bogue advises the use of the MacMichael viscosimeter?? to determine the viscosity (“melting point”) of a 30 to 100 solution of glue at 83° F. foe GC.) . It has been the general practice in glue testing laboratories in the United States to take viscosities with a simple pipette at temperature more closely approaching those at which the glue is ordinarily used, and with concentrations varying from 10 to 25 parts per 100 of water, depending on the strength of the glue. J. Alexander ?* adopted as standard a pipette of the fol- lowing dimensions: 7 Ohem. Z. 21, 56 and 70 (1897). 1% See J, Rudeloff, Mitt. K. Materialpriifungsamt 36, 2 (1918). 18 Chem, Z. 25, 23 (1901). 20 J. Am. Chem. Soc. 41, 1385 (1919); J. Ind. d Eng. Chem. 14, 485 (1922). 2 Ohem. Met. Eng. 23, July, 1920. 22See Winslow Herschel, J. Ind. Eng. Chem. 12, 282 (1920). 2 J. Soc. Chem. Ind. 25, 158 (1906) ; ‘“‘Allen’s Comm. Organic Analysis,” 4th ed., Vol. 8, p. 605. 178 GLUE AND GELATIN Capacity: Rio. ve Corie Si eee ster te ee 45 ce. of water at 80° C. - Internal diameter of effuent tube... 6 mm. External diameter of effluent tube... 9 mm. Over-all length of effluent tube..... 7em, Smallest diameter of outlet (about).. 1.5 mm. Outside diameter of bulb........... 3 cm. Dengthyor-pulbg acy soi nee eee 9.5 cm. Length of upper tube........ Detects 22 vGIn: This pipette should permit the efflux of 45 cc. of hot water at 80° C. from the bath in which the glue testing glasses are im- mersed, in exactly 15 seconds. The viscosities of glues vary greatly as will be seen from the table showing the viscosities and jelly strengths of the glues chosen as standards. Considerable care is necessary to make pipettes that will give concordant results. The size and form of the outlet hole, and the length and diameter of the effluent tube are the main factors controlling the time of delivery. The efflux hole is made by cutting the effluent tube square across, and holding it pendant in a Bunsen flame with constant rotation. As the glass softens — the hole gradually draws together, and after a few trials can be brought to the desired size. It is necessary to have the lower graduation point just about where the effluent tube joins the bulb, for with very viscous glues there might otherwise be much uncertainty due to dribbling of the last few drops. The time of efflux is taken with a stop-watch, and care must be used to see that no particles of paper, wood, dirt, undissolved glue, or glue slime clog the outlet even for an instant during a determination, at the conclusion of which the pipette is washed out with hot water from the bath. A refinement is to keep the pipette in a simple water jacket thermostat while running; in this case a glass stop-cock or a rubber tube and pinch-cock is used to control the pipette. They impede rapidity of work without corresponding increase in ac- curacy. More complicated viscosimeters like the Rideal-Slotte 7+ though more accurate than a simple pipette are not practical for routine work where speed is essential. The results of R. H. Bogue (loc. cit.) indicate that the viscosity test alone, would correctly classify about 75 per cent. of all glues—that is viscosity determined at about 60° C. As E. *4 J. Soc. Chem. Ind. 10, 615 (1891). Bogue (‘‘The Chemistry and Tech- nology of Gelatin and Glue,” p. 384) describes many of the various viscosimeters that have been suggested. TESTING AND GRADING 179 Sauer 7° points out, glue is not a pure substance, but may con- tain impurities having a viscosity of their own, or substances which materially affect the viscosity of the glue. He concludes that “viscosity measurement is no absolute method for deter- mining the quality of a glue, especially when dealing with products of different origin, which according to their raw ma- terials and methods of making may contain more or less foreign material and which therefore cannot be compared with each other. For practical purposes, however, it is important, espe- cially if certain other characteristics be taken into considera- tion.”” Sauer here speaks of Fels’ test at 35° C.; he says that viscosity 1s good for factory control purposes if foreign addi- tions, etc., remain unchanged. H. J. Watson,?® while placing most reliance upon jelly strength, found it necessary in many cases to take the viscosity as well, by Fels’ method. Trotman and Hackford 2” also regard jelly strength as the more reliable physical test, although they mistakenly place more reliance upon chemical tests. Water Absorption Test ** (Schattermann’s Immersion Test). A known weight of glue or gelatin is soaked 24 hours in water at room temperature. ‘The excess water is drained off, and the amount absorbed estimated by weight. High test glues absorb from 10 to 15 times their weight of water, weaker glues take up only 3 to 5 parts, and very weak glues may actually go into solution, forming a slime. Considering the many factors in- fluencing water absorption (see p. 87), it is obvious that this test can give only a crude approximation as to value. It is, of course, impossible to apply it to finely broken or ground glues or gelatins. | Hygrometric Test (Cadet’s Test). This practically obsolete test is based upon the amount of moisture absorbed by a glue exposed to damp air, and is even less reliable than the preceding. Where a glue or a glued article is to be exposed to a damp 2 Kolloid Z. 17, 1380 (1915). 26 J. Soc. Chem. Ind. 23, 1189 (1902). 27 J. Soc. Chem. Ind. 23, 1072 (1902). 28 Dingler’s J. 96, 119 (1845). 180 GLUE AND GELATIN climate, some form of hygrometric test, simulating conditions of service, is desirable. An unpublished report by E. Bateman and G. G. Town of the Forest Products Laboratory (U.S. Dep’t of Agriculture), Madison, Wisconsin, indicates that at about 30 per cent. moisture content glue becomes weaker than wood, and that above 33 per cent. moisture results in molding, against which no harmless preservative has been found. Hygroscopic salts greatly increase the water absorption, whereas tanning agents seem to decrease it. In Technical Note F-10 of the Forest Products Laboratory it is indicated that the moisture resistance of animal glues is proportional to the viscosity, jelly strength, and grade. High-grade glues absorb water more slowly. 5 Melting Point. Glue and gelatin jellies soften gradually when warmed, and show no sharp line between solid and liquid. The “melting point” will therefore depend upon definition, and is an uncertain factor. As a general rule it varies as the jelly strength. N. Chercheffsky 7° described a simple apparatus for determin- ing the melting point. A 250 cc. beaker is filled with refined paraffin oil into which is hung a wire with a horizontal end on which are threaded several small blocks of jelly. When these lose their rectangular form on warming, the melting point is read on a thermometer which hangs as close to them as possible. Increased accuracy is had by placing the oil beaker in a EM jacket which is gradually warmed. Cambon’s fustometer *° consists of a small brass cup which is held to a brass rod by a jelly made by dissolving 10 grams of the glue or gelatin under test in 40 cc. of water. The brass cup is hung at the surface of a beaker of water which is slowly warmed, and the melting point is taken as the temperature of the water when the brass cup drops off. A cane ferrule weighing about 7 grams will serve as the cup. A. W. Clark and L. Du Bois* propose to determine the per- centage of glue or gelatin which just maintains a solid jelly at 2 Chem. Z. 25, 413 (1901). *°Cambon and Bergmann, Monit. Scient., June, 1907; J. Soc. Chem. Ind. 26, 7038 (1907). 31 J, Ind. and Eng. Chem. 10, TOT (1918). mee SING AND GRADING — 181 10° C., but this is a troublesome method and the work of Shep- pard indicates that it is not dependable. C. F. Sammet ** deter- mines roughly the comparative melting point of glues by placing their jellies on an inclined brass plate, which is warmed by dipping one end in hot water. The weaker glues melt and slide down the plate more rapidly than the stronger ones. Bogue’s suggestion (loc. cit.) is to continue the low temperature viscosity curve so as to determine, by extrapolation, the point where the solution would cease to flow, i.e. the viscosity would reach infinity. H. Bechhold and J. Ziegler ** propose to determine the melting point of jellies by observing the temperature at which 5 grams of mercury breaks through. To prevent skin-formation from interfering with this test, it would seem wise to protect the upper surface of the jelly with a layer of oil or wax, which may be removed later on. J. Herold ** observed the temperature at which a thin tube containing jelly dropped from a thermometer about which the jelly had set. Sheppard and Sweet * describe an apparatus which serves to measure both melting point and setting point. An intermittent stream of air bells (bubbles), under constant pressure, is passed through the test solution whose temperature is lowered or raised by a surrounding bath. The setting point is fixed as the tem- | perature at which the bubbles cease to pass, and the melting point as the temperature at which they begin to pass. They also describe a simpler melting point apparatus consisting of an annular brass weight which slips over a thermometer and rests on the jelly by three equidistant wedge-shaped feet. The ther- mometer is centrally imbedded in a wide tube of jelly, the bulb being just below the surface, and the melting point taken as the temperature at which the weight sinks just above the feet. The results of Sheppard and Sweet show that melting point and setting point are not identical, and also that, as they had pre- viously found with jelly strength,** the concentration of the gelatin solution is a factor which may of itself change the order of grading. Furthermore the order obtained by melting or 82 J. Ind. and Eng. Chem. 10, 595 (1918). 33 Z, physik. Chem. 46, 110 (1906). 4 Chem. Z. 35, 93 (1910). 35 J, Ind. Hng. Chem. 18, 423 (1921). 86 J, Ind. Eng. Chem. 12, 1007 (1920). 182 GLUE AND GELATIN setting point determinations may not coincide with the order of grading according to jelly strength. Setting Point. The determination of the setting point or temperature of gelatinization, as proposed by K. Winkelblech *’ is just as diffi- cult and uncertain as that of melting point. The apparatus of Sheppard and Sweet has been referred to above. C. R. Smith likewise fixed the setting point by the air bubble method, using his polariscope tube. Strength Test, also called Shear Test, Joint Test, etc. Since glue is used very largely for gluing wood, and since its binding strength on wood is a good indication of what it will do on other service, it is only natural that a large number of strength tests have been described and recommended. ‘The trou- ble with them all is the great difficulty of obtaining concordant results because of the many variable factors involved. Some authors naively discard from their averages results that are from 100 to 300 per cent. too low, which shows that one single test may be very misleading. While impractical as a routine laboratory test in a glue factory, the strength test is valuable, and has been largely used to check other tests on glues for air- plane propellers, etc. R. H. Bogue (loc. cit.) points out one highly important source of error in this test, namely the joining pressure. He found that the strength of a glued joint varies directly as the joining pres- sure applied, up to about 1,000 lbs. per sq. in. Below 200 lbs. per sq. in. the variation is large, but beyond that it is small. S. Rideal ** tried to avoid the uncertainty due to the variability of wood, by using porcelain blocks. Further variable factors are the conditions of drying, the amount of glue spread, temperature and moisture content of the wood. The old method of the Konigliche Artillerie-Werkstatt at Spandau was radically defective in that the glue solution to be tested was first boiled down to % of its original weight, which naturally hydrolyzed the glue but indicated what it would do if 37 Z. angew. Chem. 19, 1260 (1906). 88 “Glue and Glue Testing.” TESTING AND GRADING 183 mishandled by workmen. Among the various other methods may be mentioned those of Rudeloff,?® who uses red beech blocks, and of A. H. Gill,’ who tests maple briquet-shaped blocks in a cement tester. Gill also tried unsuccessfully paper impregnated with glue in the Mullins paper tester which recalls the similar test of Setterberg,*! and briquets of sawdust, sand, fullers’ earth, ete., which recalls the work of pe ca bne ch o2 a used impreg- nated rods of plaster of Paris. P. A. Houseman ** used straight ee walnut wood. G. Hopp ** dissolved and redried the glue, cutting it then into strips of definite size which were tested for strength and stretch in a tensile machine. One hide glue showed an average tensile strength of 13,240 lbs. per square inch, while another glue showed 8,523 lbs. The method of the Forest Products Laboratory *° used in test- ing airplane glues is as follows: “Two blocks of hard maple, about 1 x 2.x 12 inches in size, are glued together lengthways along their flat grain, and after stand- ing about a week to dry out the glue, are each cut into four shear specimens having a glued area of 4 square inches. The shearing pressure to separate the blocks is then noted by a testing machine, and the percentage of wood torn out by the glue is estimated. If the failure occurs entirely in the glue, a measure of the strength of the glued joint is obtained, but if the failure is entirely or partly in the wood, as frequently happens, the full strength of the glue is not developed, and the test may have to be repeated, using stronger blocks. The same method has been used in securing data on the strength of wood in shear. Consequently when the strength of glue has been determined it can be compared with that of any wood whose average shearing strength is known. 39 Mitt. K. Materialpriif. 36, 2 (1918) ; J. Soc. Chem. Ind. 37, 743A (1919). 40 J. Ind. Eng. Chem. 7, 102 (1915). 41 Schewed. teknisk Tideskrift 28, 52 (1898) ; Chem. Z., 1898, p. 283. #2 Dingler’s J. 152, 204. #8 J. Ind. Hng. Chem. 9, 359 (1917). 44. J, Ind. Eng. Chem. 12, 356 (1920). “See their Bulletin No. 66, Washington, 1920; also Mech. Eng. 41, 382 (1919). Bogue originally started out to determine the melting point with a series of viscosities at successively lower temperatures, plotting the resultant curve, and taking the point of infinite viscosity as the setting point. This being very laborious, he found that the viscosity taken at 382°C. gave him a figure that was relative to the setting point so determined. This figure was therefore taken in subsequent investigations in lieu of the true setting point. 47 See First Report of the Adhesives Research Committee, London, 1922, p. 18. 186 GLUE AND GELATIN of an inch thick, are planed true on the flat face and then toothed with a toothing plane having 25 teeth per inch. The glue (concentration not stated) is soaked 24 hours at room temperature,*® heated a short time between 60° and 80° C., and allowed to cool to 60° before application. Two pieces of warm wood, which have remained several hours in a constant temperature oven at 35° C., are glued on the pre- pared surfaces by the warm glue solution applied by the finger, carefully avoiding the formation of air bubbles. The two glued surfaces are then placed together so as to form a one-inch over- lap, giving two square inches of glued area, and a pressure of 400 pounds per square inch is applied for 12 to 18 hours by tested box springs. After removal from pressure the joint stands three days and its breaking stress is then determined in an adapted form of the Avery or Buckton cement-testing machine, the mean of four tests being taken. The Committee comments that this test cannot in itself be regarded as an absolute criterion of the value of a glue, since a number of disturbing factors arise, such as variations in porosity of the wood, in the heating of the glue, in the temperature of the wood, the thickness of glue films, in atmospheric humidity and temperature, and in application of the test load. If these factors are carefully held in mind, and any obviously erroneous test rejected, the experimental error will generally not exceed 10 per cent. The British Engineering Standards Association have fixed the following standards for 414-inch test pieces: | Class Breaking Stress Use Propeller glues ...... 1,100 lbs. per sq. in.—Airscrew manufacture. CLOSB aie tal tere edo ee 1000 “ “ “ “ _Tmportant stress-bearing work. (Sianeo] lee cere. ene © 900 “ “ “ “ __No stress-bearing work. and under Laboratory Test Series. Many people mistakenly attempt to judge glue by its color, odor, clearness, fracture, shape, or thinness of flake, etc. Since glue is used for a multitude of different purposes, the use for 48 Obviously thick cake glue was mostly used, for ground glue soaks up within an hour or less. TESTING AND GRADING 187 which a glue is intended should always be borne in mind when submitting it to test or technical examination. Frequently spe- cial tests must be devised which simulate special conditions under which the glue is to be used, so that it is obviously impos- sible to include all tests in any ordinary laboratory series. There is here given, however, a connected series of tests which may be conveniently and quickly run. They cover practically all that is needed to gauge the value of a glue, especially when one has had practical experience with other lots of the same glue. Thin blown glasses about 314 in. high and 21% in. in diameter are convenient for making these tests. Twenty-five grams of each glue to be tested is broken into small pieces and soaked in 100 ce. of cold water until thoroughly softened. Thick sheet or flake glue should soak overnight in a cool place. With the glues under examination, there are at the same time soaked up a number of glues of known strength (standards) for tests of glue are preferentially comparative to avoid the great loss of time involved in fixing absolute conditions. It is desirable and convenient to use the standards described later (p. 190), since they cover the range of glues and gelatins ordinarily met with, and are familiar to most American manufacturers and dealers, and besides to many others. In cold weather or in testing high-grade glues, 20, 15 or even 10 grams of glue to 100 cc. of water may be used, providing un- - knowns and standards are treated alike in all respects. In warm weather low-grade glues must sometime be tested 30 to 100, unless ice is available to chill the jellies. Gelatins are usually tested from 3 to 10 grams to 100 cc. of water. When the glues are thoroughly softened, the glasses are placed in a simple rectangular water bath having a double bottom to prevent the glasses from being too close to the flame, and the temperature of the glues raised to 80° C., the contents being - meanwhile thoroughly stirred from time to time to insure com- plete solution. Insufficient soaking or stirring is apt to leave some undissolved glue, which vitiates the test. Upon complete solution, the following tests are made in the order given. 1. Reaction. This is determined by a strip of litmus paper which is then allowed to adhere to the right-hand edge of the test sheet. If the exact degree of acidity or alkalinity is desired a separate titration must be made. The degrees of acidity, alka- 188 GLUE AND GELATIN linity, foam, grease, and odor are conveniently noted on an arbitrary scale of 1 to 5; thus under acidity, 1 would mean prac- tically neutral, 2 would mean slightly acid, 3 would mean fairly acid, 4 would mean strongly acid, 5 would mean very strongly acid. 2. Odor. While the glues are being dissolved, or at any other convenient time during the test, the odor is noted. This gives some indication as to the stock from which the glue was made; in fact, the odor was once seriously proposed as a test of quality. Decomposition, though often masked by antiseptics or essential oils, is readily detected, for decomposed glue or gelatin has a peculiarly nauseating odor. Glues are rated as “sweet” (1) or “off” (2 to 5). The ordinary stock odor of a glue is not an objection, but with food gelatins freedom from all odor is . desirable. | 3. Viscosity. The viscosity is then taken on each sample and each standard, by running the hot glue solution from a pipette (previously warmed each time by the hot water of the glue bath which serves also to wash it out between determina- tions), noting the time of efflux with a stop-watch. Any con- venient pipette may be used, but Alexander’s standard pipette described on page 177 is of convenient size and shape. ‘The average time for a viscosity determination with it is about 40 seconds. Special caution must be used to see that nothing inter- feres, even momentarily, with the efflux of the glue solution. If anything clogs the pipette it must be cleaned and the viscosity run anew. 4. Grease. A flat camel’s hair brush is dipped in the glue solution, worked into a little aniline or pulp color on the corner of a piece of hard sized paper. The colored glue is then painted out upon the sheet, where whitish spots or “eyes” appear whose number is roughly proportionate to the amount of grease present. For an exact determination of grease a separate determination must be made. 5. Foam. Beat the glues rapidly with the glass stirring rod, using, say, 30 double strokes (across the diameter of the glass and back), and then note comparatively how the foam fades away or persists. With some glues the foam dies away speedily or even instantaneously, and such are rated 1 (foam free). ‘TESTING AND GRADING 189 This is especially the case with greasy glues. Other glues are rated from 2 to 5 depending upon the persistence of the foam. For more accurate estimation of foam, the glues may be re- heated after the tests are concluded, and agitated in a small bowl by an egg-beater, the foam being measured in mm. after the glues have been poured back into their glasses, or into gradu- ated cylinders. While very undesirable in a veneer glue, foam is advantageous in a gelatin used for making marshmallow confectionery. Trot- man and Hackford *° give a method for determining foam, which is similar to that described above. They found that peptones, overboiling, and alkali (which causes hydrolysis) produce foam. H. J. Watson *° found that foam was favored by free alkalis or alkaline earths, free acid, zinc compounds, overheating, and mucin (Rideal) .°* 6. Comparative Set. The glasses are now taken from the water bath and set aside to cool. Note is made of the order in which the jellies set, which is usually in the order of their jelly strengths. In warm weather, especially with glues of low strength or in weak concentrations, the glues are placed in cold or even iced water. 7. Jelly Strength or “Finger Test.” After the glue solutions have set or gelatinized, the glasses are arranged in order of the strength or firmness of their jellies. This is done by pressing the jellies with the middle finger or with two fingers, and noting their comparative resiliency. The unknown glues group them- selves as equals to or as stronger or weaker than the various _ standards used on the test. The standards should be so chosen that they cover the range of the glues on test, which may then, if necessary, be graded in between the standards. The difference between each standard “grade” is divided into ten “points,” and the differences fixed in increments of two points. The concentration and temperature of the glue jellies must be such as to permit the ready detection of small differences of jelly strength, which is difficult if the jellies are too stiff. With the finger test the personal equation is naturally a factor; 40 J. Soc. Chem. Ind. 25, 104 (1906). 50 J, Soc. Chem. Ind. 25, 209 (1906). 51 Technical Note No. F-9 of the Forest Products Laboratory deals with foamy glues. 190 GLUE AND GELATIN but given proper standards, it is speedy and sufficiently accurate for commercial purposes. Mechanical devices are apt at times to give erroneous results, particularly in attempting to. take the jelly strength under “absolute” conditions without the steadying effect of standards. Slight differences in temperature, time of set, and amount of evaporation or surface skin will make any instrument, no matter how perfect mechanically, give variable results. But with standards which are treated the same as the unknown glues, there is little chance of sertous error. Where glues are nearly alike in strength, their jellies may be broken up with the fingers to see the difference between them, if any. 8. Keeping Properties. The glasses are now allowed to stand at room temperature to see how the glues resist bacterial attack and mold. If it is necessary to know the keeping properties under certain conditions (i.e. at greater dilutions, at higher tem- perature, if mixed with color or other ingredients) these condi- tions must be simulated in a special test. 9. Appearance of Jelly. Practically all glues are turbid and a rough description clear, cloudy, or opaque will answer. Note should be made of any flocculent precipitate or sediment. “Opaque” usually means that some whitener has been added, 1.e. oxide of zinc. With gelatins the clarity of the jelly is usually a very im- portant matter, especially when they are intended for photo- graphic or table use. The clarity is best measured against other samples of gelatins. S. E. Sheppard *? has described a turbidi- meter which may be used to determine the degree of clarity of gelatin and other substances. Standard Glues. : As these form the fixed scale by means of which unknown glues are to be measured, their careful selection and preserva- tion in moisture tight packages is a matter of great importance. In the past, results of glue tests by various investigators have not been comparable because they used different glues, different methods, and had no standards of comparison. Although no official unanimity of standards exists even now, for nearly a century American manufacturers have been using oJ. Ind. Eng: Chem. 12, 167 (1920). TESTING AND GRADING 191 a series of loosely fixed standards based upon those established by Peter Cooper, the well-known philanthropist, who was an American manufacturer of glue. And about twenty years ago J. Alexander attempted to fix these standards** so that uni- formity might prevail and all use standards of the same strength. The table below gives sixteen arbitrarily established, nearly equidistant grades which cover the range of jelly strength usually met with. The jelly strengths were determined by Alexander’s jelly tester, and the viscosities by Alexander’s viscosity pipette, both previously described. The viscosity figures are based upon many years of laboratory experience with glues tested 25 grams of glue in 100 cc. of water; but since glues of the same jelly strength may vary greatly in viscosity, there is indicated in the table reasonable limits for such variation. Opposite each stand- ard is the corresponding Cooper grade, and also the grade re- cently suggested by Bogue, based upon the viscosity in center- poises of an 18 per cent. (dry basis) solution of glue, at 35° C.°4 (ay QQ So ~ aes = | OS e. a a9) 2 Sa oS S S a) oes Son ics) QS “2 QS io 373 5 $85 2a = ty Sef sec ae See SS. SS Ss OS wa EGU etetis: oa 40 12 — — — — HOU aa eh ace 34 8 — — _ —— RAG Saihe. 28 5 —— — } — — LBD esc 26 3 258 7.014 A Extra 12 A ea ae 25 1 236 6,691 No. 1 Extra 11 TIO ess 24 34. 214 6,067 1 10 RO ee es 23 34 192 5,443 lx * 9 ioe. 22 34, 170 4.820 1% 8 SICAL s «5 21 In 148 4,196 136 | 711 Pie Sean 20 % 126 3,572 1% 6 ct ene 19 % 104 2,948 158 5 DOF ess. 18 % 82 2,324 1% 4 TICES. aetna 17 yy, 60 1,701 1% a “| 1614 % _— -— 2 2 DAL, see 16 7A — -— — 1 AOE kee 'e 15% Y = — << — Water 15 * Called “fone cross.”’ 58 J, Soc. Chem. Ind. 25, 158 (1906). 54 These were kindly furnished by Dr. Bogue in a private communication. Their parallelism to the Cooper grades can be regarded only as approximate because of the variability between the jelly strength and viscosities of the standard samples. Hide glue grades are known as Hy, Hu, etc., bone glues as Bis, Bu, ete. 192 GLUE AND GELATIN In the old days, manufacturers or dealers would occasionally check their standards by mutual exchange or by purchasing some of a known grade in open market. Nothing has been pub- lished regarding the origin of the nomenclature of the Cooper grades, but from information received it is probable that they represent the distance that a certain weighted foot-rule would compress a certain bowl or vessel of glue jelly of known concen- tration and temperature. A weak glue allowed it to sink 2 inches, a strong glue only 1 inch; and the intermediate grades were measured in eighths of an inch. The instrument must have resembled the jelly tester described by Davidowsky (see p. 174). C. R. Smith’s Polariscopic Method.*® Smith’s own description follows: “In grading gelatins or glues polarize 3. g. per 100 cc. at 35° to 36° C. in a 2-dm. tube; cool a portion of the solution rapidly to 15° (or 10°) and transfer, before the sample has jellied, to a cold 1-dm. tube. This procedure avoids contractions in the jelly which may produce poor readings. If the samples need clarifica- tion, digest the solution with 5 cc. of light powdered magnesium carbonate at 30° to 40° C. for one hour or longer, and filter until clear, avoiding appreciable evaporation. Occasionally it has been found advantageous to add 0.10 g. of ammonium citrate to the filtrate to avoid the formation of insoluble calcium com- pounds, but this. does not appear to be necessary if the mag- nesium carbonate has been used in sufficient quantity and the digestion has not been too short. The procedure for clarifica- tion outlined has not been found to change the Pole results when applied to clear samples. “In place of a constant temperature bath the tubes can be placed in a large vessel of water in a portion of the ice-chest where the temperature ranges between 13° and 16° and left overnight. The next day the temperature can be controlled for 4 to 7 hours at 15+ 0.4°.. If a constant temperature bath is used the tubes may be read at once in the morning. “Considering a sample which polarizes —20.5° at 35° C. and — 40.0° at 15° C. in a concentration of 3 g. per 100 cc., 55 J. Ind. Eng. Ohem. 12, 878 (1920). The original must be consulted for tables of results on a large number of glues and gelatins, and for any further details desired. , TESTING AND GRADING 193 it is suggested that the strength be expressed as 19.5 pomts at 15° C., the increment in rotation in Ventzke degrees. Referring to Table II (omitted here) we see that a 25-point gelatin at 15° represents the maximum strength obtained. In factory control the jelly strength determinations can be made by the polariscope in the progress of the extractions, evaporation, or drying. The solutions are diluted to approximately 3 g. per 100 cc., con- trolled by rotations at 35° C. The jelly strength at 15° is determined as usual and calculations made by simple proportion to reduce rotations to the average basis of — 20.5° V. at 35° C. An actual test in factory control gave the strength of a first extraction as 17 points at 15° C.; after evaporation it was 10 points. The evaporated extract was mixed with some unevapo- rated material, bringing the strength to 11.5 points; after dry- ing it tested 11.6 points. These figures obviously represent poor extraction, and considerable loss of strength in the evaporator, but show no loss from bacterial action in drying. “Jelly strength tests made on samples direct and after in- cubation for 24 hours at 37° C. show little or no loss in strength of nearly sterile gelatins, while those in active state of decom- position show considerable loss with the development of bad odors. The following results illustrate this: fe ptsdl io CG aieole at 16. GC: Odor 95.0.3 49. Before After After NG. per 100 Cc. Incubation Evaporation Evaporation CS 2 ee are — 20.3 — 33.4 — 33.8 Sweet ee ee — 20.5 — 39.7 — 368 Bad oh ks ae — 20.3 — 35.6 — 316 Bad “The solutions were filtered through magnesium carbonate to clarify. The increase in rotation in No. 1 was probably due to evaporation and experimental error. The loss of jelly strength in Nos. 2 and 3 was quite pronounced, with correspond- ing production of disagreeable odors.” In a lot of bone glues the mutarotation at 15° V. varied from 10.4 to 24.9, and in a lot of hide and sinew glues, from 3.9 to ; . rotation 15° V. Ste 784° rotation 3° V. 1.55 to 2.14, with the hide and sinew glues from 1.20 to 2.15. The polariscope results checked with the jelly strengths, those showing the greatest mutarotation having the highest Jelly varied with the bone glues from 194 GLUE AND GELATIN strengths and requiring the smallest amounts to produce a jelly of certain standard strength. In addition to the foregoing laboratory series of tests, it is sometimes desirable to determine moisture and ash. Moisture. From two to three grams of glue or gelatin are roughly granu- lated and dried at 110° until constant in weight. If the product is commercially dry, the estimation of water is of no practical value, for it varies rapidly with atmospheric conditions, and any unusual percentage would at once register itself in reduced vis- cosity and jelly strength. R. H. Bogue ** found that the water content of air dry glues varies directly as the jelly strength, being 13.66 per cent. in a fairly strong hide glue and 10.68 per cent..in a weak bone glue. D. Jordan Lloyd *’ reports that a specimen of Coignet “Gold Label” gelatin had 20 per cent. of moisture removable by drying 6-8 hours in a hot air oven at 110°. Ash. The ash of gelatin is of importance for in it may be sought certain forbidden impurities. Besides, in some jurisdictions, the ash of gelatin, if above certain allowed percentages, is regarded as an adulteration. No manufacturer would intentionally raise the ash, for this would lower the test; but excessive ash may indicate careless manufacturing. The ash of glue runs usually between 3 and 4 per cent. Some bone glues contain considerable calcium phosphate, while hide glues are apt to have calcium sulphate or chloride resulting from neutralization of the lime used in preparing the stock. In the ash also appear various whitening agents such as zinc oxide, lead sulphate, or carbonate, chalk, clay, ete. | To estimate ash, place 2-3 grm. glue in a large platinum cru- cible and heat slowly, as the glue at first intumesces violently. Ash at a low redness, preferably in a muffle, using a few drops of nitric acid to insure the oxidation of all the carbon. Accord- ing to Kissling °* the ash of bone glue fuses in the bunsen burner, 5 Chem. Met. Eng. 28, 105 (1920). 5 Biochem. J. 14, 148 (1920). 88 Chem. Z. 11, 691 and 719. 195 TESTING AND GRADING sds SNUqVT SY LDUW9 Y PETTITT TTT onbedo soqye1edag Anos Ost & ie STE fen ae Os > Se Sec e a OOT Z — ry ae ae Ronit ar 08 6 i hee ee oS 09 GI oy eEs sag me imal aoe 0S a a “ pre ae a ors OF SI Zz may a a ? am Os OL VST T I I I I 06 8 SI I IT T G T SP LI 9T I AG I I i 09 ial SOT I G I IF G OS OT OT G I I I G OST § IG T I I if I OLT ¢ SSI I I I if G 91 Il Lt I I I I “Al ae 61 S91 T § i if G O9T I LG I I i I if COL 9 Vad re I IT I I 8G €I1 Yell G T “s AG I NN =< ™R 7 o~ 2 es < 2 = SUNOF FI AIAVOG YALVAA “09 OOT OL TAIN “sWU 0Z 799YY 789, A1oqL0gQnT eooee ee eo eee eee eee eecereceece eee ee ee ee ” OST-6I » O€I-8T 99 OOI-LT ” O8-9T ” 09-ST 0S-F1 ves eves ce es «6 wees piepueyg OF-EI “a Ulld[IIpe],, UBUIIES) A oi a ONS UTS, YSSUG (9u0q) Joays SuUOTGO UBULIIr (9u0q) Jeosys suOTGO yYsI[suy **(au0q) Jooys srenbs yous aie Yoo s UlessoO UNI puodveg Rhee on[s su0q 9speRis-YysIZ “anys suo0q sapvis-UUINIps/y LESSENS on[s su0q 9spe1s-MO'T sree s"onys eply epeis-ysty ‘**-on[s apry speis-wnIpefy abel en[s aspiy speis-Mo'T uamwadg fo uoudiwosaq 196 GLUE AND GELATIN is neutral and contains phosphoric acid and chlorides, while hide elue ash is infusible, alkaline, and generally free from phos- phates and chlorides. Since hide and bone glues are frequently mixed both in the liquor and dry form, it is obviously unsafe to draw any con- clusion from the ash as to the raw material used. The com- position of the ash usually depends upon the process used, and its estimation, in glue, unless for some special purpose, is not commercially necessary. Recording Tests. There is given on page 195 a specimen of a laboratory test sheet used in recording the connected series of tests just described. In 1898 Friman Kahr *® proposed a series of four tests de-: signed especially to grade glues for joining use. These were: 1. Adhesion =the weight of dry glue necessary to make up 100 lbs. of liquid having the proper viscosity or body at 60° for use in making joints. This figure varied from 60 lbs. in the case of the lowest grade glues to 29 lbs. with the highest grades. It is in effect a viscosity determination. 2. Economic value = adhesion figure x cost per pound. Thus Kahr’s figures showed 29 X 18 = $5.22 per 100 lbs. of liquid made with the highest grade of glue (cost 18¢ per pound) and 60 « 5144 = $3.80 per 100 lbs. of liquid made with the lowest grade (cost 544¢ per pound). 3. Cohesion or strength = crushing strength of the glue jelly at the concentration indicated under 1, and determined at 65° F. (about 18° C.). This was subsequently fortified by a joint strength test. 4. Congealing test = temperature at which the glue made up as indicated under 1, would gelatinize. This figure ran between 91° and 75° F., and gave some indication as to speed of set. One radical defect in this system is the latitude left open in fixing the “proper viscosity” under 1. But this is the method most consumers of glue follow—they find by rough trial about how much glue is needed to make up a ready-to-use solution and then figure its cost. Since many tests are needed to estab- lish the minimum amount of glue needed, they usually stop when 5° International Fisheries Congress, ‘Bergen, Norway, 1898 ; also a periodical, Glue, published by him at Hast Haddam, Conn. TESTING AND GRADING 197 an approximate figure is reached. In any event Kahr’s tests are practically intended for joint work, and cannot serve for a general laboratory method intended to indicate the value of glues for most ordinary uses. R. H. Bogue,®® after reviewing the various testing methods, proposes a system of evaluation based on measurement of the viscosity of an 18 per cent. dry basis (equal on the average to 20 per cent. of commercial glue) solution of glue in the Mac- Michael viscosimeter at a temperature of 32° to 35°, the latter being preferred since there is less change of viscosity with time. This is the one primary test; and Bogue’s figures show that it correctly classifies glues on the basis of their joining strength. Speaking of the jelly strength and viscosity at 60° which the primary test is to supersede, Bogue says: “They may, however, be of great value in secondary evaluation, i.e. in determining the adaptability of a given glue to a given service. For example, the jelly consistency would be of value in selecting glue for the printer’s rollers, and the rapidity of setting of the jelly as well as the. viscosity at working temperatures would be desirable data for the wood-working industry.” While Bogue’s primary test is the best single test of the value of a glue or gelatin for joint work, the fact that the similar test of Fels, proposed in 1897, has not met with general acceptation indicates that neither is_ self-sufficient. Bogue’s statement above shows that the secondary tests are essential.