.■.m 
 
 I 
 
 A 
 
THE DETERMINATION OF SUCROSE IN 
 PLANT EXTRACTS 
 
 BY 
 
 THOMAS ELIJAH HOLLINGSHEAD 
 
 THESIS 
 
 FOR THE 
 
 DEGREE OF BACHELOR OF SCIENCE 
 
 IN 
 
 CHEMISTRY 
 
 COLLEGE OF LIBERAL ARTS AND SCIENCES 
 
 UNIVERSITY OF ILLINOIS 
 
 1922 
 
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TABLIi OF GONTMTS 
 
 
 Page 
 
 Acknowledgement 
 
 Nature and Origin of the problem 
 
 1 
 
 Experimental 
 
 Preparation of Pure Sucrose 
 
 9 
 
 Purification of Glucose 
 
 9 
 
 Source of Asparagin 
 
 11 
 
 Comparison of the Defren's, 
 Ilunson- Walker , and Clerget 
 Methods of Sucrose Deter- 
 
 mina tkon. 
 
 12 
 
 Defren's Method 
 
 12 
 
 Lfims on- Walker Method 
 
 13 
 
 Clerget Method 
 
 13 
 
 Experiment I 
 
 14 
 
 Determination Copper 
 
 16 
 
 Experiment II 
 
 17 
 
 Experiment III 
 
 20 
 
 Conclusion 
 
 21 
 
 Summary 
 
 21 
 

 
 
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NATURE AlID QRIGII^ OE THE EKOBLSM 
 
 The v;ide variation hetween the sucrose values obtained by the 
 plariscopic and chemical methods is a source of difficulty encount- 
 ered by quite a number of workers in plant chemistry. This has 
 been especially true v;hen an effort was made to determine the 
 quantity of specific sugars, as sucrose, glucose and fructose. 
 Davis^ and his co-workers found differences of as much as eighty 
 per cent in the copper reduction and polariscopic values for 
 
 glucose and fructose in the mangold and potato; the polariscopic 
 
 2 
 
 value being the higher. Parkin also found the polariscopic 
 value to be higher than the chemical value in his work v;ith the 
 carbohydrates of the snow drop. Chemists working with quite a 
 variety of plant extracts and under different conditions have 
 reported similar results.^* ^ 
 
 Of all the sugars present in plant extracts it should seem 
 that sucrose might be determined most accurately, since it is a 
 non- reducing sugar and on hydrolysis in converted into glucose 
 and fructose, both of v/hich reduce Fehling's*^ solution which 
 offers a chemical method involving the determination of the increas( 
 in reducing sugar after hydrolysis. The fall in rotation after 
 hydrolysis offers a method for the determination of sucrose which 
 
 7 
 
 has been utilized in the method of Glerget 
 
 1. Jour. Ag. Science, _7, 349 (1915) 
 
 2. Biochem. Jour., _6, 18 (1911-12) 
 
 3. Brown and Morris, j. Ghem. Soc. , j^, 664 (1893) 
 
 4. Huncie and Englis, Thesis ( ) U. of Illinois 
 
 5. Browne, J. Am. Ghem. Soc. 451 (1906) 
 
 6. Bro;vn "Sugar Analysis” p.335, p. 389 
 
 7. Ibid p. 264 
 

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But as mentioned above the two methods do not check in the case 
 of plant extracts. The following is a typical analyses of leaf 
 
 4 
 
 extracts hy Lluncie • 
 
 _ Sucrose in IJilleg r ams 
 
 By Polarization Method 99.2 139.2 107.2 97.7 
 
 By Reduction Method 94.3 132.3 101.9 98.8 
 
 0 
 
 Davis assumes that the difference F;as caused hy certain 
 
 optically active nitrogenous impurities such as glutamine and 
 
 asparagin which are present, and are not prepititated hy basic 
 
 SI 
 
 lead acetate. Somers ^ and latter ^uisumhing and Thomas 
 thought tliat the auto-reduction of the Pehling’s solution might 
 introduce an error large enough to account for the difference 
 in the results of the two methods, hut the data from their 
 experiments shov/s that this error is quite insignificant. Somers 
 also stadiecT the effect of neutral salts, such as sodium acetate 
 and oxalate, upon the rotation of the invert sugar, hut found 
 them to have no effect. The presence and effect of such impur- 
 ities as Glutamine and asparigin which are present in plant 
 extracts has been noted hy Pellet^, Saillard^^, Ogilvie and 
 others. Ehrlich^^ pointed out quite early the presence of 
 large errors in the Clerget method due to the presence of amino 
 acid. The error that he refered to was due, however, chiefly 
 to the fact that amino compounds such as aparagine, aspartic 
 acid, glutaminic acid, leucine, isoleucine, and so forth, vary 
 
 7. Browne "Sugar Analysis p. 264 9. j^t. Sugar J., 17, 421(1915) 
 
 8. J. Agr. 3ci., ^,339, (1916) 10. Comp, r end. ~Tb 5 ,116 
 
 20. Somers and Englis, Thesis, 19 2() ll. Int. Sug. 14, 89 
 
 21. J.A.C.S. 43, 1503-1526 12. Z. deut. Zuckerind.53,809 
 
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 in optical activity depending upon the alkalinity and acidity of 
 
 the solution. This may he seen from the following table v/hich 
 
 gives the approximate specific rotations of several amino deriv- 
 
 13 
 
 atives in alkaline solution, in v/ater and hydrochloric acid. 
 
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 Isoleucine 
 
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 acidily of the solution it is only necessary to carry out the 
 
 hydrolysis by means of invertase, riBking both readings with 
 
 the solution neutral. Or, as suggested by Andrlik and Stanet^^ 
 
 both polarizations may be carried out in the presence of 
 
 hydrochloric acid and urea, depending upon the retarding 
 
 influence of the urea f or betaine) on the inversion of sucrose 
 
 15 
 
 with hydrochloric acid in the cold. Incidently, Browne offers 
 data to prove that the retarding action of the urea is not 
 sufficient in the case of substances rich in sucrose. If there 
 is no combination or reaction between the sucrose and amino 
 compounds the error, under the conditions stated above should 
 be constant, and not effect the difference in the direct and 
 
 13. Browne "Sugar Analysis" p. 270 
 
 14. Ibid, p. 271 
 
 Z. Suckerind. Bohmen, 417 
 
 15. Bro;vn "Sugar Analysis", p. 271 
 
- 4 - 
 
 and invert polarizations* This may perhaps be illustrated by 
 an equation: 
 
 D I = d 
 (Dia) - ( I±a)= d 
 
 in which D equals the direct polarization, I the invert polar- 
 ization, a the polarization of the amino compound which is a 
 constant, and d the difference in the tv/o polarizations. Unless 
 there is some reaction between the sugars and the amino compounds 
 thise equations should hold true, and the presence of amino 
 derivatives should not be the cause of difference in the values 
 obtained by the polarimetric and chemical methods as suggested 
 
 Q 
 
 by Davis and his co-workers in their work on the determination 
 
 of the glucose-fiructose ratio in mangolds* 
 
 15 
 
 Degener , a French chemist, found that asparagine may 
 introduce an error in the determination of sucrose in sugar 
 beet extract by plarization* He has shown that asparagin, 
 v;hich in neutral solution is ^ ightly levo-rotatory 
 becomses strongly dextrorotatory (Cf3 j;) 5 61.76 to 69.10t in the 
 presence of 10 per cent lead subacetate solution, every 0.1 
 per cent asparagin polarizing about the same as every 0*1 per 
 cent sucrose* To obviate this error the French chemists add 
 a drop of glacial acetic acid to the filtered sucrose solution 
 before polarizing* 
 
 As stated above, none of these considerations account for 
 the very large variations between the polariscopic and copper 
 
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 reduction methods. There is very evidently some greater source 
 of error, which may he due to some unstable compound formed by 
 the sugar and the amino substances, or possibilly to some 
 catalytic effect upon the reducing power of the glucose and 
 fructose formed by the hydrolysis of the sucrose. 
 
 Thacher says no nitrogenous groups of the protein type 
 have been found in combination v/ith sugars in glucosides. 
 Browne-^' mentions some animal substances, which are not of a 
 pure carbonhydrate nature, yielding glucose as one of their 
 hydrolytic products. Among these substances may be mentioned 
 different nucleo-proteids , various albumins and certain mucins 
 or mucoids. The chemistry of these products, hov/ever, is still 
 unsettled, and it is uncertain whether the sugar derived from 
 
 consists of glucose alone or a mixture of sugars. 
 
 22 
 
 Somers describes an. experiment to stady the effect of 
 asparagin on the polarization of invert sugar, which is as fol- 
 lows: "The asparagin was from a laboratory stock solution and 
 
 the apparatus used was a Schmidt and Haensch polariscope, 
 a half-shaded instrument, calibrated in Ventske degrees. The 
 asparagin and invert sugar solutions were mixed and polarized 
 in accordance with the following table 
 
 
 A 
 
 B 
 
 G 
 
 Asparagin 
 
 25 c.c. 
 
 25 c.c. 
 
 00 
 
 Invert Sugar 
 
 00 
 
 25 
 
 25 
 
 kVa ter 
 
 25 
 
 00 
 
 25 
 
 Polarization Reading 
 
 -.320 
 
 1.438 
 
 -1.060 
 
 16. Thatcher "Chemistry of Plant Life" , p.76 
 
 17. Browne "Sugar Analysis" p.S79 
 22. Somers and Englis, Thesis, 1920) 
 
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-7- 
 
 The mixed asparagin and invert sugar solution give a 
 greater negative rotatory value than that obtained by 
 adding the rotation values for the constituents determined 
 separately. This seems to indicate that there is some sort 
 of loose combination between the sugar and asparagin, and 
 it seems plausible that it may be responsible for at least 
 part of the disparity between the tv/o methods for the est- 
 imation of sucrose. He also made an attempt to repeat the 
 experiment v/ith a neutral solution of glutamic acid hydro- 
 chloride but there was such a development of color that it 
 was impossible to determine the polarization value of the 
 solution. 
 
 Stanek^® found that when he heated solutions of sucrose, 
 or invert sugar with sodium glutamate or aspartate or v/ith 
 asparagin at a temperature of one hundred- five to one 
 hundred- thirty degrees Centigrade that carbon dioxide was 
 liberated, the solution became acidic, and dark pigments were 
 formed which are almost completely precipitated with lead 
 acetate. Asparagin and aspartic acid being the most reactive. 
 
 Kaillard found that there was an actual reaction 
 between glucose and glycocoll when they were dissolved in 
 three or parts of water and warmed. The liquid slov/ly 
 assumed a characteristic yellow color, changing to a dark 
 
 18. J. Chem. Soc., 112 . I, 544 
 
 19. Comp. rend. 154 , 66 
 
rf 
 
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- 8 - 
 
 brown, follov/ed by the evolution of carbon dioxide. It has been 
 found that the carbon dioxide came from the carboxyl group of 
 the glycocoll. It v/as assumed that this loss of carbon dioxide 
 is accompanied by an union of the nitrogen v;ith the aldehyde carbo: 
 of the sugar; the glucose molecules forming part of the new 
 compounds must suffer dehydration resulting in the appearance of 
 double bonds or possibly rings. It v/as also shovm that other 
 amino acids work in a similar if not identical manner on glucose, 
 and other sugars. This seems to prove that sugars and amino 
 compounds can react under certain conditions to form new compounds 
 hence it seems plausible that they might have some effect upon one 
 another under ordinary pressure and room temperature. 
 
 The object of this investigation is to study the effect of 
 asparagin upon the rotatory power and reducing power of sugar 
 solutions. 
 

 * ^ 
 
 
 ;T"' 
 
 TJ 
 
 T / - 
 
 
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 ff,i lii. 
 
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 To QftififtiQi^pUii •d't rxJf vml^AiJridv^ rt^'l^ire. ^4 mpp; 
 
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 ■' '%. ' .'’ ’'0"i> . ... , 
 
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-9- 
 
 EKPEHIIvIMTAL 
 
 Preparation of Pure Sucrose ^ *2,5 
 
 Method of the International Commission for the Unification 
 of Sugar Analysis * 
 
 A hot saturated aqueous solution is prepared end the sugar 
 
 4 
 
 is precipitated v/ith absolute ethyl alcohol ; the sugar is 
 carefully spun in a small centrifugal machine and washed in the 
 latter with absolute alcohol. The sugar thus obtained is re- 
 dissolved in water, the saturated solution again precipitated 
 v/ith absolute alcohol and washed as above. The product of the 
 second crop of cry stalls is dried between blotting paper and 
 preserved in glass vessels for use. The miliisture still contained 
 in the sugar is determined and taken into account when weighing 
 the sugar which is to be used. 
 
 Sucrose prepared by the above method was found to contain 
 only 0.05% moisture and about 0.1% ash. 
 
 Purification of Olucose 
 
 The raw material is the commercial com sugar, "Gerelose", 
 manufactured by the Corn Products Refining Company, of New York. 
 
 Five hundred grams of the dry corn sugar are dissolved in 
 2.5 liters of cold water, about 20-30 grams of decolorizing 
 carbon, such as "eponite” or "norit”, are added and the mixture 
 is stirred for a few minutes. 
 
 1. U.S.Dept. Agri. ,Bur.Ghem. Bull. 73, p.58 
 
 2. J.A.M.G. 59 
 
 3. Sherman, Methods of Organic Analysis, p.91 
 5. Ibid, p.97 
 
 4. vV. A. Noyes, Organic Chem. for Laboratory, pp. 67-68 
 
r 
 
- 10 - 
 
 It is then heated to about 97® C., and niade just acid to litmus 
 with a few drops of phosphoric acid (H^PO^) and filtered through 
 an asbestos mat. The colorless filtrate is evaporated in a glass 
 flask under reduced pressure to a content of 70 to 75^ solids. 
 Glacial acetic acid is now added in the ratio of one part of 
 acid to one part of sirup. This Solution is placed in a jar, 
 seeded with pure glucose crystals, and stirred occasionally. 
 Crystallization v;ill commence almost immediately, v/hile the sol- 
 uition is still warm. It is well to stir in one more part of 
 acid as cry s tall iz.at ion proceeds to prevent the formation of a 
 solid mass of crystals. Crystallization is complete in a few 
 hours or over night. The thick mass of crystals is then filtered 
 from the mother liquor on a Buchner funnel fitted with a strong 
 filter paper, and v/ashed with glacial acetic acid. After draining 
 as dry as possible on the Buchner funnel, the crystals are washed 
 v/ith 95^0 alcohol and dried in a vacuum oven at 50® G., this 
 temperature may be raised to 70° C., in the course of one or 
 two hours . 
 
 Tv;o runs were made using this method. In the first the 
 yield was very low due to insufficient concentration of the 
 sirup before crys tallization. The Abbe® refractometer may be 
 used to advantage in estimating the total solids of the sirup. 
 
 The product of the first lun was not analyzed. In the second 
 run about 350 grams of dry pure glucose viexe obtained, which 
 upon analysis shov/ed 0.065 per cent moisture, end ash 0,199 fo, 
 
 8. Browne "Sugar Analysis” pp. 50-75. 
 

 ,',eM:"i:i ♦♦0' ':«,i:. ,^I 
 
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- 11 - 
 
 The product is colorless and its solution is colorless and 
 
 free from cloudiness* It is the anhydride, and is ^nixture of 
 
 the alpha and beta d-glucoses. The method works v;ell, and 
 
 practically the only difficulty encountered v/as the absolute 
 
 9 
 
 removal of all the acetic acid. 
 
 The Com Products Company are noMJ putting on the market 
 a purified glucose which is crystallized from v/ater, therefore 
 free from any acetic acid. The method they use is described 
 by Porst and Humford in the Llarch Journal of Industrial and 
 Engineering Chemistry, Vol. 14, No. 3, (1922). 
 
 Source of Asparagin 
 
 The asparagin used in this work was obtained from Herk 
 Company. It was the 1-asparagin v/hich has the following 
 formula and properties : 
 
 C- OH 
 
 I 
 
 CE- HH„ 
 
 VO 
 
 C - HHg 
 
 Asparagin or Amido- amino- succine 
 A ten percent HCl solution of asparagin, with the sodium 
 line, gives a rotation of 37.27? a neutral solution -6.14°; 
 and 16.4 grams in 100 c.c. ammonia solution gives a rotation 
 of -271°. 
 
 The material obtained from Merk in water solution gave a 
 
 rotation of aproximately -6°. 
 
 9. Hudson and Dale, J.A.G.S. 320 
 10. Abderhalden, Biochem. Eandlexikon, Yol.4, p597 
 
■r . ’ . • " • i*' ■ ■ * •'. '■ ’'■y.' >•:* ,"<;■; 
 
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- 12 - 
 
 Asparagine is only slightly soluble in water at ordinary temperat- 
 ures. 
 
 20.5° 
 
 G. 
 
 100 
 
 parts 
 
 of 
 
 water 
 
 will 
 
 dissolve 0.62 
 
 P* 
 
 Asparg 
 
 31.50 
 
 » I 
 
 1 I 
 
 1 1 
 
 1 1 
 
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 » 1 
 
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 1 1 
 
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 1 » 
 
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 70° 
 
 1 I 
 
 1 1 
 
 1 1 
 
 1 1 
 
 1 1 
 
 1 1 
 
 ” 2.25 
 
 1 I 
 
 1 1 
 
 It is considerably more soluble in acid or alkaline solution. 
 
 Co mparison of the lefrens « llunson- V/alker , and Qler^;et 
 Methods of Sucrose Determination. 
 
 Defren's Method 
 
 In Defren’s method, which adapted from O’Sullivan, Soxhlet’s 
 
 12 
 
 formula for Pehling’s solution is used: 15 c.c. of the copper 
 sulfate solution and 15 c.c. of the alkaline tartrate solution are 
 diluted with 50 c.c. of water in a 300- c.c. Erlenmeyer flask. 
 
 The latter is then immersed for 5 minutes in a boiling water bath, 
 w'hen 25 c.c. of the sugar solution are quickly run in from a 
 burette or pipette. The flask is replaced in the bath and heated 
 for exactly 15 minutes. The cuprous oxide is then filtered on 
 asbestos, washed with warm water, ignited and weighed as cupric 
 oxide, or it may be dissolved in nitric acid and determined vol- 
 ume trically. The amounts of glucose, maltose, lactose and inveit 
 
 sugar corresponding to different v/eights of cupric oxide are given 
 in tables. 
 
 11. Brov/ne, "Sugar Analysis" p. 425-6; J.A.G.S. 18, 751 
 
 12. Ibid, 389-90, 
 
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-13- 
 
 Ltimson-V/alker llriified Ivlethod^ ^ 
 
 Transfer E5 c.c. each of the copper and allcaline- tartrate 
 solutions (Soxhlet's formula) to a 400-c,c. Jena or Non- sol 
 beaker and add 50 c.c. of reducing stigar solution, or, if smaller 
 volume of sugar solution be used adA water to make the final 
 volume 100 c.c. Heat the beaker upon an asbestos gauze over 
 a Bunsen burner; so regulated that boiling begins in 4 minutes, 
 and continue the boiling for exactly 2 minutes* Keep the beaker 
 covered with a watch glass throughout the entire time of heating. 
 V/ithout diluting filter the cuprous oxide at once on an asbestos 
 felt in a porcelain Gooch crucible, using suction. V/ash the 
 cuprous oxide thorougly v;ith water at a temperature of about 
 60® C., then with 10 c.c. alcohol and finally with 10 c.c. of 
 ether. Dry for 30 minutes in a v;ater oven at 100® G., cool in 
 a desiccator and v/eigh as cuprous oxide. Tlie amiounts of glucose, 
 invert sugar, lactose or maltose corresponding to different 
 v/eights of cuprous oxide or copper are given in tables. 
 
 Qlerget Method 
 
 Dissolve 26 grarasoof the sugar in 60 c.c. of water, clarify 
 if necessary, dilute to 100 c.c., filter, and polarize at 20° 0. 
 
 The solution is then deleaded by means of anhydrous sodium or 
 ammonium carbonate, or disodium phosphate^^, followed by filtrat- 
 ion. To 50 c.c. of this solution 25 c.c. of v/ater are added, 
 then 5 c.c. of cone. HGl with constant shaking, and the solution 
 is made up to 100 c.c. at 20® Q., and allowed to stand over night 
 
 13. Brown "Sugar Analysis" p.426 15. Sherman "Organic Analysis"p.94 
 
 14. Ibid, 264-286 
 
 16. Englis and Tsang, J.A.G.S. M, 865 (1922) 
 
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-14- 
 
 at between 25^ and 30° Centigrade. Or the hydrolysis may be 
 
 17 
 
 carried out by use of Inver tase in stead of the acid, which 
 tends to decompose some of the fructose. Cool the solution 
 to 20° C. , mix the solution well and polarize, preferably in a 
 waterjacted 200 -mm. tuve with a wide tube through which the exact 
 tempeature of the solution at the time of polarization may be 
 determined. Since 50 c.c. of the sugar solution v/as diluted for 
 hydrolysis and finally made up to 100 c.c. this second reading 
 must be doubled to obtain the "invert polarization". The 
 difference between the first plarization and the "invert polar- 
 ization" multiplied by 100 and divided by the difference which 
 pure sucrose would show under the same conditions, gives the 
 percentage of sucrose; or 
 
 s > 100 {P - I) 
 
 142.66 - 0.5t 
 
 in which s equals percentage of sucrose, P the original polar- 
 ization, I the "invert polarization", and t the temperature in 
 degrees centigrate. 
 
 ■Experiment I 
 
 A sample containg 12.5 grams of pure glucose was analyzed 
 by the Defren’s , LIunson -Walker, and the polariscopic method. > 
 
 17. Sherman, J.A.C.S. 1566 
 
- .'Tain* xt:.- 
 
 V 
 
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-15- 
 
 D ef r en ’ s Me thod 
 
 
 
 
 
 I 
 
 II 
 
 111 
 
 C«C* NagSgOg 
 
 32.56 
 
 32.52 
 
 32.65 
 
 Y/t, of CuO 
 
 279.1 
 
 279 
 
 280 
 
 ’ * ’ ' Glucose 
 
 125.61 
 
 125.6 
 
 126.1 
 
 Per cent ’ ' 
 
 12.561 
 
 12.56 
 
 12.61 
 
 ifuns on- Walk er Me th o d 
 
 
 
 
 
 I 
 
 II 
 
 III 
 
 C.C. HagSgOg 
 
 35.53 
 
 34.1 
 
 55.22 
 
 Wt, of Gu 
 
 242.5 
 
 233.5 
 
 241.0 
 
 Wt. ' ’ Glucose 
 
 123.9 
 
 119.0 
 
 123.15 
 
 per cent ^ ’ 
 
 12.39 
 
 11.90 
 
 12.315 
 
 Polariscopic Method 
 
 
 
 
 Reading 
 
 37.7° V. 
 
 
 
 Per cent Glucose 
 
 12.44^ 
 
 
 
 Averages and Qomparison 
 
 or Results 
 
 
 
 Defren's Average-- 
 
 
 - 12.577;^ 
 
 
 Liunson- Walker ’ ’ 
 
 
 - 12.202/5 
 
 
 Polarimetric 
 
 
 - 12.44 fo 
 
 
 In comparison v\ith the polarimetric results the Defren 
 Method shows a difference of .137^ and the Munson- Walker 
 Method of *238 In using the Munson- Walker Method a good 
 
 deal of experience is required to tell just v/hen boiling 
 actually starts and to adjust the flame to start the boiling 
 exactly at the end of four minutes. This difficulty may 
 
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- i6- 
 
 accoimt for in part the poor checks obtained by the I.fimson- Walker 
 Me thod, 
 
 4. r. 18, 19, 20 
 Determination of Copper 
 
 The copper reduced in these experiments was determined by 
 a modification of tlie Low lodometric Method. The Cu.-0 is dissolved 
 
 Cj 
 
 on the Gooch by pouring over it 10 c.c. of nearly boiling 4N HNO^. 
 
 This is added very slov/ly while applying mild suckion to prevent 
 loss by splashing. Allov/ the copper nitrate and nitric acid to be 
 sucked down as completely as possible, then wash v/ith five or six 
 5 c.c. portions of boiling water. Boil for a few minutes to 
 remove the oxides of nitrogen, add 5 c.c. of saturated bromine 
 water and boil until until the excess bromine is expeSiled. Remove 
 the flask from the flame and add strong ammonia until a slight 
 excess is present. After boiling off the excess ammonia, add 10 
 c.c. of 30 fo acetic acid, which dissolves any copper oxide that has 
 been deposited. Cool to room temperature, add 3 grams of potassium 
 iodide and tirate the brov;n solution with standard sodium thiosulfat . 
 until nearly colorless, addiiig starch solution toward the last and 
 complete the tiration. In making the titration for the first 
 time one is bothered somevirhat by the fact fh&t the cuprous iodide 
 is of a light brovmi color. This difficulty is very much overcome 
 by keeping dovm the amount of thiosulfate solution which it is 
 necessary to use. 
 
 In standardizing^ the sodium thiosulfate solution a known amount 
 
 of metallic copper is used to begin with and a regular determination 
 18. Treadwell Hall, Vol.II, pp. 682-683 
 
 Browne "Sugar Analysis” pp.411^13{ various modification^ of method) 
 
 20. Peters, J.A.O.S. 422 
 
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-17- 
 
 is carried through. Tlie copper value of the solution is 
 
 calculated by dividing the grams of copper used by the number o£ 
 c.c. of solution used. 
 
 where A is grams of Copper used, t is c.c. of used, and 
 
 C.V. is the copper value of each c.c. of solution. 
 
 With a little experience just as good checks may be obtained 
 by this method as v/ith gravimetric methods, and it eliminates 
 a very great amount of weighing where a great many samples are 
 being run at the same time. 
 
 The only difference in the above method and that of low is 
 that in the above method 10 c.c. of 30^ acetic acid is used 
 instead of 3.5 c.c. of glacial acetic acid, as 3.5 c.c. is an 
 a'/'kward volume to handle. 
 
 Experiment II 
 
 The copper reduced by solutions of invert sugar, glucose, 
 sucrose, asparagine rn^v/ater solution, invert sugar in presence 
 of asparagine, glucose in the presence of asparagin, and sucrose 
 in the presence of asparagin v;as determined; also the rotatory 
 values for the above solutions. 
 
IjiPVIi 
 
-18- 
 
 Invert Sugar 
 
 
 I 
 
 II 
 
 c • c • Q ^ , 0 
 
 12.51 
 
 
 »vt. of 6u6 
 
 11.8 
 
 
 Invert Su^ar and Asparafiin 
 
 c.c. NaoSpOg 
 
 14.04 
 
 14.04 
 
 Wt. of UuO 
 
 12.00 
 
 12.00 
 
 Glucose 
 
 
 
 c.c. NapSpOct 
 Wt. of GuO 
 
 14.52 
 
 14.82 
 
 12.45 
 
 12.70 
 
 Glucose and Aspara^in 
 
 c.c. NapSpQ^ 
 
 14.54 
 
 14.67 
 
 Wt. of CuO 
 
 12.47 
 
 12.55 
 
 Sucrose 
 
 c.c. NapS.^O^ . 
 
 1.48 
 
 1.23 
 
 Wt. of Cu6 
 
 1.27 
 
 1.05 
 
 Sucrose and Aspara.qin 
 
 c«c* ITspSpO /2 
 Wt* of CuO 
 
 2.93 
 
 2.50 
 
 2.55 
 
 2.12 
 
 Asparagin 
 
 c.c. lTapS. 50 „ 
 
 i42 
 
 .46 
 
 Wt. of CuO ^ 
 
 .35 
 
 .38 
 
 Polarizations 
 
 
 Sucrose, 5;a, 20*^0., 400 ram. 
 
 tube 
 
 38.1° 
 
 Asparagin 2 /b(M 40 H soln. ) 28°G. , 400-mm. 
 
 tiibe- - 2.5° 
 
 Glucose lO/o, 280Q., 400-nm 
 
 tube 
 
 60 . 90 
 
 Sucrose 90 c.c., Aspar. 10 
 
 c .c . , 28^0 . , 
 
 400 mm 33.6° 
 
 Glucose and Aspar. (spoiled 
 
 by bacterial 
 
 action) 
 
 Invert Sugar plus 10 c.c. water, £8°C., 400-mm -4.0 
 
 Invert Sugar ’’ 10 c.c. Aspar. ’’ *' -2.2 
 
-19- 
 
 The results of j^xperiment II are very little value because of 
 insufficient blanlc determinations to enable one to come to 
 definite conclusions. 
 
 jj^xperiment III Copper Reduction 
 
 Blanlc s 
 
 c • c • NapCpOa 
 Wt. of CuO 
 
 I 
 
 .40 
 
 .34£ 
 
 II 
 
 .58 
 
 .356 
 
 Amiuonia soluti on containg 15 c,c. 
 in 100 c.c. as used for 
 Asparagine solvent 
 
 clc. NapSpOij; 
 Jt. of CuO 
 
 .£5 
 
 .£15 
 
 
 .£3 
 
 .197 
 
 
 Invert Su*R;ar 
 
 c.c. NagSgOg 
 2^t. of GuO 
 
 14.98 
 
 1£.85 
 
 III* 
 (16. £4) 
 (13.90) 
 
 14.90) 
 
 1£.78 
 
 IV* 
 
 (16.10) 
 ( 13.80) 
 
 Invert Sugar and ammonia 
 
 c.c. NapSpOa 
 St. of CuO 
 
 15.00 
 
 1£.87 
 
 III* 
 
 ( 15.38) 
 ( 13.19 
 
 15.15 
 
 1£.95 
 
 IV^ 
 
 (15.75) 
 
 (13.49) 
 
 Asparagin and ammonia 
 
 c.c. NapSoO„ 
 
 Wt. of CuO 
 
 £.5£ 
 
 £.£6 
 
 
 £.55 
 
 £.£8 
 
 
 Invert Sugar, Asparagin 
 and ammonia 
 
 16.40 
 
 14.0)5 
 
 (15.87) 
 ( 13.60) 
 
 13.45 
 
 11.50 
 
 ( 13.18) 
 (11. £9) 
 
 * Samples III and were run three days after I and II. No 
 reason for the increase in reducing power has suggested itself. 
 
 In this run a determination of the reducing povi/er in the 
 presence of ammonia was made because it was thought that the 
 ammonia toight possibly have a polmerizing effect on the sugars 
 which would effect their reducing power. The very poor checks 
 obtained in the case of the invertsugar, asparagin and ammonia 
 mixture make it impossible to draw any conclusions from this 
 experiment. 
 
I 
 
 
 ^ t * 
 
 vex. 
 
 i V. < ♦ .L 
 ; ‘.X^ 
 
 I . • f ‘t 
 
 W' - 1/ 4 i-.-. 
 
 O » . 
 
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 r ' < 
 
 
 
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- 20 - 
 
 ■^jjeriment III continued 
 
 iPolarizations 
 
 Invert Sugar, 26® 0., 400-mra. tube Q -10-2® V- 
 
 Asparagin in ammonia, 26® C-, 400-mra. tube - 8.0® V. 
 
 Invert Sugar and Asparagin, 26® G., 400-mm. tube 
 
 (equal amounts) - 9.2® V. 
 
 These results seem to indicate that the effect of the 
 asparagin on the polarization of the invert sugar is additive 
 only, and does not form any combination v/ith the sugar 
 changing the polarization value enough to explain the wide 
 differences obtained by Davis and other wrrhers v/ith plant 
 extracts- 
 
 Dxperiment III 
 
 In this experiment the asparagin was dissolved in sodium 
 
 carbonate rather than ammonia. The excess alkali was titrated 
 Vvlth ZQ'-/o acetic acid till the solution was just pink to phenol- 
 phthalein. 
 
 Invert Sugar and Asparagin 
 
 
 I 
 
 II 
 
 III 
 
 NapSpO-z c.c. 
 
 9.05 
 
 9.68 
 
 9.74 
 
 V7t. of GuO 
 
 7.75 
 
 8.30 • 
 
 8.35 
 
 Invert Sugar and Asparagin 
 
 Na„S^0„ c.c. 
 
 9.26 
 
 9.60 
 
 9.19 
 
 Yt. bf^GuO 
 
 7.92 
 
 8.22 
 
 7.82 
 
 Polarizations 
 
 Asparagin 20® G. , 400-mm tube .4® Y. 
 
 Invert Sugar, &iid as^AragiA’ -2.90 v. 
 
 Invert Sugar *3.00 V. 
 
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 OONGLUSION 
 
 Based upon the data obtained in these experiments the 
 effect of the asparagin upon the reducing power and polarization 
 values seems to additive only, and is not the chief cause for 
 the large variations in the sucrose determinations by the polar- 
 iscopic and chemical methods. 
 
 SULuiARY 
 
 1. Wide variation between the sucrose values obtained for plant 
 extracts have been observed by many chemists. 
 
 2. Somers eliminated the possibility that the variation was 
 due to the effect of neutral salts in solution, or to auto-reduc- 
 tion of the ffehlings solution. 
 
 3. Davis and his co-worhers attributed the variation to the 
 presence of amino compounds such as asparagin and glutamine, but 
 assuming that the proper neutrality of the solution was maintainec 
 this seemed improbable unless the amino substances entered into 
 combination with the sugars. 
 
 4. 2taneh and I-Iaillard prepared some compounds by the reaction 
 of sugars and amino substances by means of high temperature and 
 pressure. 
 
 5. Sucrose and Glucose were purified for use in the experimental 
 work of the problem. 
 
 6. A sugar solution was analyzed by the Defren’s, Ifunson-Wallcer , 
 and polariscopic methods dmd the results compared. 
 
 7. A modification of Low’s iodometric method for the determin- 
 ation of copper was used to determine the copper reduced. 
 

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- 22 - 
 
 8. The effect of asparagin upon the copper reducing power, 
 and rotatory value of sucrose, glucose and invert sugar was 
 studied v/ith the asparagin in ammonia solution and in sodium 
 carbonate solution and found to be of an additive nature and 
 insufficient to account for the large variations which are 
 observed between the sucrose values obtained by the chemical 
 and polariscApiciiliethods*