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Conf.650507-I
THEORETICAL AND EXPERIMENTAL ASPECTS OF QUENCHING. VARIABLES
FROM BIOMEDICAL SAMPLES IN LIQUID SCINTILLATOR SYSTEMS
Harley H. Ross
Analytical Chemistry Division,
Oak Ridge Nat.onal Laboratory,
Oak 64.dge, Tennessee, USA

-LEGAL NOTICE -
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PATENT CLEARANCE OBTAINED. RELEASE The
THE PUBLIC IS APPROVED. PROCEDURES
ARE ON FILE IN THE RECEIVING 8601701,
*Research sponsored by the U. S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
1.
Introduction
The usual methods of samp3.e preparation for liquid scintillation counting
often result in systems that are considerably less than ideal from the stand-
point of counting efficiency. Several techniques have been investigated for
correcting for total quenching by an internal standard method /1-3/ or by an
approximation technique /4/. While work has been done on chemical quenching
15-6), little has been published on the general phenomenon of color quenching.
Some specific instances have been noted. Leffingwell, Riess, and Melville /7/
cbserved the effect o color on irin-59 systems containing the colored tris-l,
10-phenanthroline iron(II) chelate but noted an enhancerent or photon yield,
relative to their colorless standards, that was chelate-concentration dependent.
Baille /8/ found a difference in over-all magnitude between chemical quenching
and color quenching at higher concentrations, and Herbert /9/ investigateå the
effect of color in ootaining suitable background solutions for certain types
of biological materials. Helf and White /10/ studied the quenching effects of
organic nitrocompounds as a function of their ultraviolet absorption spectra
but did not suggest a way to correct for this other than to change the chemistry
of the counting system. Halvorsen /11/ used an extrapolation technique to cor-
rect for color quenching in tissue samples. However, this technique can be used
only for the same or similar samples.
In the present studies, two different approaches to determine the amount of
color quenching in a sample were examined. The first method illustrates a gen-
eral mathematical model for color quenching as a function of the spectrophoto-
metric properties of the system. The second provides a simple experimental
technique to evaluate color quenching directly.
tas
2.
Theory
2.1 Spectrophotometric methci
In order to evaluate mathematically the total color quenching in a system,
licht absorption of a multicomponent syster that follows Beer's law, for which
the expression for total absorbance takes this form, assuming some constant
light path:
A - 8201 + azoa + azº3 + ... en in
where
At = total measured absorbance
as molar absorptivity of indicated component
c = concentration of Indicated component
It is conventional in these systems to measure Ato obtain values of a from
independent data, and calculate the concentrations of the various components
by solving a series of simultaneous equations.
The total color quenching in a system can be represented by the following
integral:
-S (K)(a)an
where
Q - per cent color quenching
K - quenching coefficient
A = absorbance of solution
^ = wavelength
Since K and A are dependent upon wavelength, an approximate solution to
the integral may be expressed by this series:
»2-
Recent Cany) + (Kun And + (Kay! (Ang) + ... My
in which the subscripts indicate values at specific waveienctbs. To eval-
uate the various K's, it is necessary to prepare n different solutions, mea-
sure the absorbances of each solution at a different wavelengths, and deter-
mine the per cent quenching for each solution. These data cive n equations
in n unknowns. Obviously, 11 n is very large, a computer would be desirable.
Since any wavelength region can be divided into any number of segments,
the value chosen for n is purely arbitrary. As a gets smaller, the values of
K become more and more approximate and represent averages of all possible
values for the segment chosen.
In order to correct an observed counting rate for color quenching, the
following equation 2.6 used:
Rate corr - Rate obsd le
- 2.2 Isolated Internal Standard Method
The isolated internal standard technique is based on the absorption of
light photons in a color quenched liquid scintillator sample. However, in
contrast to the spectrophotometric method, the light source consists of a
small ampoule containing an unquenched liquid scintillator spiked with the
desired isotope. The photons emitted by the standard have the same intensity
and spectral distribution as those produced in the sample itself.
In practice, the isolated internal standard is used in the following
manner. After initial construction, the standard assembly is placed in a
vial containing a sample of pure liquid scintillator solution. The sample
is counted and the resulting activity becomes the unquenched standard count
(A). The standard 18 now ready for use. A sample that exhibits color quench-
ing is introduced into a liquid scintillator and counted in the usual way (A.).
- 30
-
.
...
.
.
.
.
The isolated internal standard assembly is placed in the sample and counted
again (A). Then:
AS - A = A
Ao - Age
and
where Q is the degree of color quenching. To correct the observed activity
of the sample for color quenching:
Ag (corrected) = A (obsd.)
-
1.Q
CS
It should be pointed out that different standards are required for each
1sotope counted. Standards of 14c, 36c1, 3H, and others are easily prepared.
3. Experimental
3.1 Apparatus and Reagents
Counting was done with a Packard Tri-Carb liquid scintillation spectro-
meter, Model 314-DC. Discriminator and high-voltage settings were adjusted
to give balance point operation in the red channel (10-50 volts). Spectral
measurements were made with either a Bausch & Lomb Spectronic-20 colorimeter
with matched 1/2-inch cells or a Cary recording spectrophotometer, Model 14,
with calibrated 1 cm. cells.
The scintillating medium consisted of a dioxane solution containing
IS
200 grams per liter of naphthalene, 7 grams per liter of 2,5-diphenyloxazole
(PPO), and 0.3 gram per liter of 1, 4-bis-(5-phenyl-2-oxazolyl)-benzene (POPOP).
A similar scintillating solution was prepared for the isolated internal stan-
dard except that 100 ml. of toluene label.ed with carbon-14, and having a spouin
fic activity of 4 x 10% d.p.m./m2. was used to "spike" the scintillator. Only
one milliliter of this solution was prepared. Analytical reagent grade and
WAS
scintillation grade chemicals were used whenever possible. Coloring agents
used were FIC type coal-tar Ayes, although not necessarily certified for such
use. Stock dye solutions were prepared in toluene; aliquots of these solutions
were used for individual measurements. The weight of dye used in each experi-
ment was kept small (< 50 HS.) to eliminate the possibility of chemical quench-
ing effects. .
3.2 Procedure
3.2.1 Spectrophotometric liethod. Complete visible spectra were
obtained for the dyes used. Aliquots of these dyes were added to unquenched
counting samples of c++ toluene and to samples of pure dioxane with the same
volume as the counting sample (20 m2.). The activities of the colored count-
ing saraples were determined and the per cent quenching noted. Absorbance
measurements were made on the dyed dioxane samples. The per cent quenching
values and the absorbence of the samples (at selected wavelengths) were used
to calculate quenching coefficients from equations derived in section 4.1.
MUUT
Once the quenching coefficients had been determined for the scintillator
system, it was possible to calculate the quenching of an unknown system from
absorbance measurements only.
Quenching coefficients were determined for various levels of activity
(0.1 uc. to 0.01 uc.) and at different instrument settings. No significant
variation in quenching coefficients was observed.
Absorption spectra of the dyes used are shown in Figure 1. The data
collected in the quenching measurements are shown in Figure 2.
.
.
OTR
3.2.2
Isolated Internal Standard Method. The isolated internal
standard is shown in Figure 3 and was constructed as follows. A small pyrex
tube was drawn to an ampoule with dimensions as shown in the figure. A 500-ui.
sample of the carbon-14 loaded scintillator was introduced into the ampoule,
which was cooled in liquid nitrogen and sealed with a hand torch. A six
millimeter hole was drilled into the center of a standard plastic cap used
on the 20-mi. sample vials; the ampoule was sealed into the hole with epoxy
cement end allowed to cure for 24 hours.
The first set of experiments was designed to compare the isolated inter-
nal standard technique with the spectrophoto metric method and the internal
standard method when only color quenching is present. To this end, 20 mi.
of scintillato: solution were pipetted into a sample vial and the isolated
internal .standard assembly was screwed into place. This sample was counted
for two minutes in the Tri-Carb instrument and the count in the red scaler
recorded (A). The standard assembly was removed, a small aliquot of desired
dye solution was added, the standard assembly was re-installed, and a second
: count was taken (A). The predicted degree of color quenching for this solu-
tion is then given by:
When the above measurements were completed, the dyed liquid scintillator
solution was examined spectrophotometrically to predict the degree of color
quenching. Then, the degree of color quenching was alternately measured using
the normal internal standard method with standard samples of carbon-14-toluene.
Another series of tests was designed to determine how well the proposed
method could separate the combined variables of color and chemical quenching.
Liquid scintiilator samples were prepared conte ning both a dye color quencher
and an acetone chemical quench. The predicted color quenching was determined
using the isolated internal standard and was then subtracted from the total
quenching observed with the usual internal standard method. This difference
represents the chemical quenching due to acetone alone.
4. Results and Discussion
4.1
Spectrophotometric Method
The quenching curves, as illustrated in Figure 2, are linear functions
until about 50% quenching is reached. The nonlinear deviations in the hicily
absorbing solutions of red and yeilow dye can be explained by the geometry of
the transmission system. When a small amount of color is present in the scin-
tillator vial, the emitted protons must travel through many different optical
path lengths. The system, in effect, exhibits an average path length. When
the absorbance of the solution is low, this average puth length remains con-
stant. As absorbance increases, a point is reached where some total absorna
tion takes place. When this occurs, the effective average path length changes
and deviations from linear response are observed. Practical applications in-
dicate that only the linear portions of the curves need be considered.
Because of the wavelength characteristics of the licht emitted in this
scintillator system, it was expected that the red and yellow solutions would
show the greatest quenching effect. That they do is shown in Figure 2. By
the same criteria, it was expected the yellow solutions would show the great-
est quenching. However, the particular spectra overlap for the yellow and
red dyes most probably accounts for the quenching curves obtained.
Since solutions of three different colors were used in this study, an
attempt to determine only three K-values was made, employing the wavelength
absorption maxima for the yellow (400 mu), red (520 mu), and blue (625 mu)
solutions. The equations to be solved took the form: .
. Qy - (K_)(4400) + (K5) (4520) + (Kg)(Y625)
V = (K4)(R400) + (Kg)(R520) + (Kg)(R625)
B = (K4/(B400) + (Kg)(B510) + (Kg)(R625)
ERNT
**
2.
..
hy
F
*
where.
Q = observed quenching by the designated solution, yellow, red, or blue
Y = absorbance of yellow solution at designated wavelength
R = absurbance of red solution at designated wavelength ·
B = absorbance of blue solution at designated wavelength
K1- quenching coefficient. at 400 mu
K - quenching coefficient at 510 mů
Ko= quenching coefficient at 625 inu
Attempts to solve this set of equations gave values oî K, which were
negatire and hence of no physical significance. Closer examinarion and
evaluation (by trial and error techniques) led to the conclusion that almost
no quenching resulted i'rom the blue absorption maximum at 625 mil, and that
the observed quenching of the blue solution was due to the small. peak at
420 mji and the absorbance at 520 mu. The value of Kg was too small to be
computed with the available precision of the data.
Assuming, that for all practical purposes, any absorption above 520 mu
did not contribute to the over-all quenching, a new set of equations involving
data at 400 mp, 455 m, and 510 mau was developed. The 455-mu region was
arbitrarily chosen, being half-way between the other two:
Qy' = (K_)(x400) + (K2)(Y455) + (K3)(4520)
Qy - (KL)(R400) + (K2)(R455) + (Kg)(R510)
By • (K2) (B400) + (K2)(B455) + (x3)(B520)
· where the terms have the same significance as before except that
K, - quenching coefficient at 400 mu
Kg = quenching coefficient at 455 mu
Kg - quenching coefficient at 510 mu
-8.
Solution of this set of equations gave the following values for the
quenching coefficients:
Ky = 80/unit A
Ką = 38962/unit A
Kz = 80%2/unit A
EA
From these values it was possible to estimate the degree of color
quenching for various systems by means of this relationship:
Qc = (KZ)(A400) + (K2)(4455) + (K2)(A510)
where
Q. = calculated color quenching
K7 = 80%a/unit
Kg = 38%Q/unit A
Kg = 80%Q/unit A
A = measured absorbance of solution at designated wavelength
The data in Table I compare predicted and observed quenching for several
colored systems.
Calculated and obseived values for the
factor are in good agree-
ment except in the case of methyl red. Continuing studies indicate that methyl
red is not stable in the scintillator solution at the concentrations used.
In the event that color quenching in a given system were to come from a
single, known material, evaluation of quenching coefficients would not be
necessary. Various amounts of this quenching material could be added to
liquid scintillator systems, the absorbance of each solution measured at any
convenient wavelength (preferably the wavelength corresponding to the absorp-
tion maximum), the amount of quenching determined, and a curve similar to one
in Figure 2 constructed. It would then be necessary only to measure the
absorbance of the unknown solution at the same wavelength and read the per
cent quenching directly from the graph.
Because the quenching effect is dependent upon the disintegration energy
of the particular radioactive tracer being counted /12), different quenching
co ellicients : will be obtained for different isotopes. Tracers with similar
disintegration energies should be similarly quenched. Becquse the mechanisms
of energy transfer are complex, different quenching coefficients may be found
for the same radioactive isotope in different liquid scintillator systems.
Thus, quenching coefficients are valid only for a specified tracer in a
specified scintillator system. Further, the calculated values reflect the
combined response efficiencies of the multiplier phototubes and associated
electronics of the particular counting equipment used.
4.2
Isolated Internal Standard Method
The data in Table II show that the isolated internal standard is in very
good agreement with both of the other methods. The results are not biased in
either direction and are independent of the spectral distribution of the color
quenching species and the absolute degree of quenching. The degree of varia-
bility between the three methods is vell within the range of error usually
observed in routine liquid scintillation procedures.
The results in Table III indicate that the proposed method can also be used
to evaluate the individual effects of color and chemical quenching. The series
usire a 0.5 ml. acetone quench shows excellent agreement between the directly
observed value and the values obtained by difference. However, the second
group using a 20 ml. quench shows that the values calculated by difference are
slightly lower than the standard sample. This is probably explained by assuming
that the combined color and chemical quenching effects have projected the system
into the region of non-linear quenching. Other workers /12,13/ have shown that
both color and chemical quenching have a non-linear region as a function of
quencher concentration. There is, therefore, every reason to assume that com-
bined quenching effecis will also have a nou-linear region above some minimum
value.
-20-
If color is the only type of quenching in a particular system, the isolated
internal standard technique can be used to correct any obsexved counting rate.
However, a more important application is the study of complex counting systems
to separate the effects of color and chemical quenching. In this respect,
the technique offers two important advantages over the spectrophotometric method.
The isolated internal stanicard is faster requiring only one additional measure-
ment. Also, expensive accessory equipment is unnecessary; an easily prepared
standard assembly that is stable indefinitely is all that is required.
4.2.1 Analysis of Liver, Blood, and Urine Samples. The isolated
internal standard technique was used to separate the quenching. variables in
C-14 samples of liver, blood, and urine. Portions of liver and blood were
digested in hyamine hydroxide (one ml., 50% in methanol) and then added to
20 ml. of the scintillation mixture. Urine samples were added directly and
without pretreatment. The samples were analyzed using the same general pro-
cedure. After initial counting, a standard amount of C-14 toluene was added
to each sauple and they were recounted. The amount of color and chemical
quenching was then calculated.
The results are shown in Table IV. The data indicate that, under the
conditions of the experiment, color quenching is the most important factor in
all three types of samples. In samples of liver and blood, chemical quenching
-
--
-
--
-
--
--
- -
is relatively small and somewhat constant. However, the chemical quenching
-
-
.
..
--
---
-
-
-
-
-
-
--
-
in urine samples is essentially equal to the color quenching. Also, there
is a high degree of variability in color quenching from sample to sample.
By using results of this type, evaluations of different methods of sample
preparation and different scintillation mixtures can be made to optimize the
counting efficiency of any sample.
-
-2.10
5. Bibliographical References
11/ Hayes, F. N., Intern. J. Appl. Radiation Isotopes 1, 46(1956).
12/ 0xita, G. T., Spratt, J., Leroy, G. V., Nucleonics 14, No. 3, 76(1956).
13/ Williams, D. L., Hayes, F. N., Kandel, R. J., Rocers, W. H., Ibid.,
1, 62(1956).
/4/. Helf, S. White, C. G., Shelly; R. N., Anal. Chem. 32, 238(1960.
15/ Brown, F. M., Furst, M., Kallmann, H., "Proceedings of the University
of New Mexico Conference on Organic Scintillation Detectors," U. S. At.
Energy Comm. Report TID-7612.
16/ Helmick, M., Atomlight pp. 6-7, February 1960.
. 17/ Leffingwell, T. P., Riess, R. W., Melville, G. S., U. S. Dept. Commerce
0.T.S., P.B. 148-081, 1960.
18/ Ballle, L. A., Atom).ight pp. 1-7, June 1961.
79/ Herberg, R. J., Anal. Chem. 32, 1468(1960).
/20/ Helf, S., White, C., Anal. Chem. 29, 23(1957).
/11/ Halvorsen, K., "Tritium in the Physical and Biological Sciences," Vol. I,
p. 313, International Atomic Enercy Agency, Vienna, 1962.
/12/ Guinn, V. P., "Liquid Scintillation Counting," Proceedings of Northwestern
University Conference on Liquid Scintillation Counting, C. G. Bell and
/13/ DeBersaques, J., Int. J. Appl. Rad. Isotopes 14, 173(1963).
w..
.
......
..............
.........-
............
....
..
..
..
.................
.
.
.
.
.
.......
....
w
Table I. Comparison of Calculated and Observed Quenching for Various Colored Substances
Dye
-
200 mAbsorbance
400 mg
45'; mu 510 mu
Calca.
Obsa.
Calca.
Obsd.
Bromcresol purple
41.9
1.72
Methylene blue
2.0
0.48
0.00
0.14
0.27
2.02
0.07
0.01
0.30
0.15
0.01
0.02
0.17
0.06
42.2
43.6
2.8
1.5
50.6
50.7.
1.75
1.02
2.02
1.47
Methyl red
36.2
1.57
32.1
33.7
1.47
FD and C yellow
dye + FD and
C blue dye
30.5
Methyl. orange
8.3
1.09
1..12
0.08
0.01
0.05
0.01
0.00
0.02
11.8
10.!
Azur II Fosin
2015
2.8
1.8
2.03
1.03
1.02
0.7
Table II. Various Methods of Color Quench Correction
Compared Using Different Dyes

NIG
Quencher
eine
I
Spectro.
Int. Sta.
Spectro.
Iso. Int. Sta
Bromcresol
Purple
0.144
0.299
Iso. Int. Std.
0.133
0.297
0.169
0.424
1.15
1.43
1.17
1.43
1.15
· 1.42
Int. Sta.
0.127
0.299
0.174
0.423
0.160
0.311
Yellow AB
0.183
0.448
1.21
1.20
1.22
1.81
1,73
1.74
1.17
1.2)
Quinoline
Yellow
0.148
0.319
0.164
0.311
0.114
1.19
1.45
1.45
0.099
1.11
Malachite
Green
1.13
1.47
1.14
1.28
0.120
0.216
0.246
0.249
1.33
1.33
Orange I
0.131
0.268
1.13
1.40
1.15
1.37
0.217
0.284
0.143
0.358
0.130
0.285
0.141
0.349
1.15
1.40
1.16
1.54
Oil Red XO
1.17
0.115
0.346
1.13
1.53
1.56
K
......
....
.....
......
......
.....15:
.4..
Table III. Separation of Color and Chemical Quenching Effects
Quencher(s)
.
1
(Total quenching)
1_1
0.233 1.30
0.406 1.68
Difference
(Chemical Quenching Q1--22)
1-Q3
1.30 (sta.)
0.248
1.33
Isolated Internal Std.,
(Color quenching)
1
I-2
0.000
0.158
1.19
0.132
1.15
Acetone (0.5 ml.).
+ Quinoline Yellos
+ Orange I
+ Oil Red XO
0.233
0.357
1.56
0.225
1.29
1.27
0.354
1.55
0.142
1.17
0.212
0.401
1.67
0.000
0.401
1.67: (sta.)
0.523
2..10
1.19
1.57
Acetone (1.0 mi.)
+ Quinoline Yellow
+ Orange I
+ Oil Red XO
0.362
0.365
0.495
0.161
0.130
0.133
1.98
1.15
.
1.57
1.61
..0.512
2.05
1.15
0.379
-
-- .
Table IV. Color and Chemical quenchine of Liver, Blood, and Urine
Sample No.*
(Chem. )
Liver
Q(Color),
13.6
21.1
4.
fw Nr
26.4
5.3
f m a
3.8.6
16.9
Blood
2.1
23.2
14.9
1.4
20.0
0.9
27.5
1.8
Urine
17.3
5.9
15.9
13.6
av F w or w
15.5
11.8
18.6
15.3
7.7
116.7
3.5.5
8.2
*C-14 Labeled
Figure 1
.
...
.
....
.....
.
.
....
.....
.
. . .-
-
.-.
--
..-
-
..
ABSORBANCE
0.& 1

350
YELLOW DYE
450
Absorption spectra of dyes
WAVELENGTH, MY
RED DYE
550
BLUE DYE
650
.
.
1.
-
ui.
*.
Figure 2
ena.
.
.
..

..
-.--
-
.
X
S
$ QUENCHING
.
--. rom
--
--
.
.
..
1
.1
.
.
-
-
-
-
-
-
-
-
- -
-
-
-
-
0.4
1.0
Yrit
0.6
0.8
ABSORBANCE
Quenching as a function of dye concentration
Red dye
+ Yellow dyo :
O Blue dyo
w
he
**:4,"
T
..
-
-
Figure 3. The Isolated Internal Standard
ORNL-DWG. 64-9033

EPOXY CEMENT

SCREW
CAP

60 mm
CARBON-14
LOADED
LIQUID
.
--
a
-...--
.
.
'
-
.
* ķ~5mm I.D.
* K~7 mm 0.D.
.
".
DATE FILMED
3/ 7 /65







L.
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-LEGAL NOTICE

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This report was prepared as an account of Government sponsored work. Neither the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the use
of any information, apparatus, method, or process disclosed in this report may not infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, or for damages resulting from the
use of any information, apparatus, method, or process disclosed in this report.
As used in the above, “person acting on behalf of the Commission” includes any em-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
such employee or contractor of the Commission, or employee of such contractor prepares,
disseminates, or provides access to, any information pursuant to his employment or contract
with the Commission, or his employment with such contractor,
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im
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END