A water-miscible, nonhazardous liquid scintillation cocktail

ANALYTICAL

BIOCHEMISTRY

120,

414-419 (1982)

A Water-Miscible,

Nonhazardous

Liquid Scintillation

Cocktail

THOMAS F. KELLOGG Isotope Center, Mississippi State University, Mississippi State, Mississippi 39762 Received October 8, 1981 The optimal way to count aqueous samples by liquid scintillation counting is in a homogeneous solution. Technical limitations have previously made this difficult. Triton X-100 is a water-miscible liquid scintillant which counts 14C with 80% efficiency and ‘H with 17% efficiency. It has a high flash point (over 300”F), is nonvolatile, and does not cause swelling or leaching when used in polyethylene vials. Liquid-scintillation counting cocktail using Triton X-100 as the sole scintillant (i.e., no toluene or xylene) does not have to be disposed of as a hazardous waste. The large aqueous sample capacity of a miscible cocktail, its safety, and ease of disposal make its use highly attractive for many applications.

Liquid scintillation counting (LSC)’ is the method of choice for counting weak betaemitting radionuclides such as 3H and 14C. With aqueous samples, solubility in the hydrophobic scintillators toluene, xylene, and pseudocoumin is a problem. Current approaches to solve this problem involve the use of (a) the water-soluble scintillator dioxane with its inherent chemiluminescence problems, (b) a second solubilizing solvent such as ethanol to form a monophasic solution with a limited water solubility, and (c) detergents such as Triton X-100 and X114 which may form emulsions and/or gels which may not yield valid external standard quench corrections (1,2). Systems employing toluene and xylene have the inherent problems of toxicity and flammability which make them hazards in the laboratory and, under Environmental Protection Agency regulations, must be disposed of as hazardous wastes. We have recently reported that Triton X-100 acts as a scintillant on Cerenkov counting systems (3). The scintillation capability of Triton X-100 was originally reported by Turner in 1969 (4). We have ’ Abbreviations used: LSC, liquid scintillation counting; PPO, 2,5-diphenyloxazole; bis-MSB, p-bis(o-methylstyryl)-benzene; TCA, trichloroacetic acid. 0003-2697/82/040414-06%02.00/O Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

414

investigated the properties of LSC cocktails using Triton X-100 detergent as the only scintillant and find that they have many superior characteristics over sol gel systems for counting aqueous samples. MATERIALS

AND METHODS

A Packard Model 3255 Liquid Scintillation Counter was used. Triton X-100 detergent was purchased from Sigma Chemical Company, and the 2,5-diphenyloxazole (PPO), p-bis(o-methylstyryl)-benzene (bisMSB), tritiated water, and 14C standards from Packard Instrument Company. The “C amino acid mixture was purchased from New England Nuclear, and standardized against the standard 14C toluene. Polyethylene vials, 20 ml nominal volume, with foillined caps were used. All cocktail and sample total volumes were constant at 10 ml. The sample compartment and samples were maintained at 13°C for counting. The adjustable discriminators were set to optimize efficiencies and SCR. Preset discriminators and gain settings for toluenebased cocktails will not yield optimum results with our cocktail. All reported efficiencies are the mean of three samples each counted to 40,000 total counts. Phase con-

A WATER-MISCIBLE. TABLE EFFECT

ON COUNTING

NONHAZARDOUS

OF 14C OF PPO

AT VARYING OF ADDED WATER Hz0

in Triton

0

10 Counting 71 72 76 75

74 75 81 77

5 7 9 11

tact was determined son (5).

LEVELS

X-100

(vol W)

20

30

efficiency 67 69 69 69

(%) 65 66 64 65

40 FIG. 1. Variation 100 cocktail with 58 58 57 57

by the method of Ben-

RESULTS

The optimal PPO concentration was determined with varying amounts of water and nitromethane (a strong chemical quencher) to be 8 g/liter (Tables 1, 2). In all experiments reported here the cocktail was 8 g/ liter PPO and 0.05 g/liter bis-MSB dissolved in 1 liter of Triton X- 100. Phase-contact studies (5) reveal that there are no significant deviations from true solution contact over a water concentration range of l-60% (Fig. 1). The cocktail and water formed a miscible solution in all proportions. The solutions were colorless, transparent, stable, and exhibited good ESR and SCR curves vs counting efficiency (see Figs. TABLE

415

SCINTILLANT

I

EFFICIENCY

CONCENTRATION

PPO (g/liter)

LIQUID

in phase-contact varying amounts

QUENCHER

2

0.0

3 5 7 9 11 13

75 77 78 79 79 78

l

.

in Triton (ml)

efficiency 67 69 71 71 70 70

l

.

0.14

0.04 Counting

*

.

NITROMETHANE

Nitromethane X-100 PPO (g/liter)

X-

2-5). There was no minimum amount of water necessary to form a monophasic cocktail as is true for the toluene-Triton cocktails. The counting efficiencies for tritiated water and a 14C amino acid mixture in water and common LSC samples are given in Tables 3 and 4 with the ESR, SCR, and figure of merit (% sample X efficiency). The 14C efficiency with 100% Triton X- 100 was 79%, the 3H efficiency was 17%. To determine the effect of salts on counting efficiency OS-ml samples of CeClz solutions were added to 9.5 ml of cocktail. The molar concentrations of CeCl, in the 0.5 ml of added solution were 0.5 to 5.0 M. This is the range of concentrations that would be typical of CeCl, density gradient centrifugations. The 3H efficiencies ranged from 14.2% for 0.5 M CeC& to 8.5% for 5.0~ CeClz solutions. The 14C efficiencies were from 78 to 77%, respectively. A similar study

EFFECT ON COUNTING EFFICIENCY OF 14C OF PPO CONCENTRATION AT VARYING LEVELS OF THE CHEMICAL

ratio for Triton of water.

(%) 23 40 46 45 42 42

OV

.4 EXTERNAL

STANDARD RATIO

FIG. 2. Percentage counting efficiency vs external standard ratio with increasing amounts of water added to Triton X-100 cocktail containing 14C amino acid mixture.

416

THOMAS

i

and 4.0%, respectively, and r4C counting efficiencies of 76.6, 55.8, and 49.8%, respectively. Chemiluminescence was noted with the NaOH containing cocktails. The Triton cocktail does not diffuse through polyethylene vials over a 2-month period as shown by no weight loss and does not cause the vial to swell.

. . .

01

I

2 3 4 SAMPLE CHANNEL RATIO

DISCUSSION

4 5

FIG. 3. Percentage counting efficiency vs sample channel ratio with increasing amounts of water added to Triton X-100 cocktail containing ‘Y amino acid mixture.

of NaCl salt solutions in solution volumepercentages from 10 to 60% with NaCl concentrations from 1 to 25% by weight indicated very little effect of NaCl concentration in this range. The counting efficiencies were effected by the volume-percentage of water but only marginally by the NaCl concentration. Addition of l-ml solutions of HCl from 0.6 to 10 M to 9 ml of cocktail gave 3H counting efficiencies of 12.1 to 6.0%, respectively, and 14C counting efficiencies of 76.3 to 67.7%, respectively. Additions of 1 ml of 1, 10, and 20% NaOH solutions by weight to 9 ml of cocktail gave 3H counting efficiencies of 8.8, 5.3,

4 w

.

p

z

The phase-contact experiment and visual examination of cocktails with varying amounts of samples indicate that Triton X100 is miscible with water in all proportions. This allows valid use of ESR quench correction techniques over the complete range of sample concentrations. Benson (5) as well as others (1,2) have shown that this is not always true of sol gel cocktails. Detailed examination of the 0.01 to 10% aqueous sample range indicate that there is no minimum amount of water necessary for homogeneity as is true for 30% Triton X- l OO-70% toluene cocktails. Samples of water, 1% HCl, 10% sucrose, 1% NaCl, and 1% NaOH all have maximum figures of merit for 14C in the 5060% sample range and for 3H in the 30% range. TCA solutions (10%) and serum and urine have figures of merit at 20-40% sample for 14C and lo-20% for tritium. The high figures of merit for aqueous samples combined with the miscibility of the sys-

20

f$

g

F. KELLOGG

. IO

. I

OlI

.

. I

.2

EXTERNAL

I

3

,4

STANDARD RATIO

FIG. 4. Percentage counting efficiency vs external standard ratio with increasing amounts of water added to Triton X-100 cocktail containing tritiated water.

SAMPLE

CHANNEL

RATIO

FIG. 5. Percentage counting efficiency vs sample channel ratio with increasing amounts of water added to Triton X-100 cocktail containing tritiated water.

A WATER-MISCIBLE,

NONHAZARDOUS TABLE

LIQUID

417

SCINTILLANT

3

EFFECT OF VARIOUS VOLUME-S OF SAMPLES ON ‘H COUNTING MERIT

Substance”

WITH TRITON

X-100,

Volumepercent

None

8 g/liter

PPO,

EFFICIENCIES, ESR, SCR, AND FIGURES AND 0.5 g/liter bis-MSB COCKTAIL

ESR

SCR

Percentage efficiency

OF

Figure of merit’

0.3856

1.282

16.8

Water

10 20 30 40 50

0.3278 0.2816 0.2274 0.1591 0.1063

1.204 1.162 1.128 1.093 1.076

12.0 9.59 7.05 4.57 3.28

120 192 212 183 164

TCA

10 20 30 40 50 60

0.2969 0.1994 0.0708 0.0138 0.0002 0.0000

1.188 1.121 1.081 1.052 1.038 1.045

10.5 6.6 2.8 1.4 1.0 1.0

105 132 84 56 1.0 1.0

HCl

10 20 30 40 50 60

0.3235 0.2736 0.1985 0.1033 0.0518 0.0161

1.215 1.158 1.330 1.089 1.066 1.154

11.9 9.1 5.5 4.3 2.5 1.5

119 182 165 172 125 90

10 20 30 40 50 60

0.3285 0.2835 0.2231 0.1610 0.0900 0.042 1

1.219 1.179 1.152 1.107 1.079 1.064

12.1 9.8 7.0 5.2 3.2 2.1

121 196 210 208 160 126

NaCl

10 20 30 40 50 60

0.3323 0.2856 0.2024 0.1589 0.0879 0.0395

1.226 1.174 1.098 1.105 1.081 1.064

12.3 9.6 6.6 4.9 3.1 2.1

123 192 218 196 155 126

NaOH

10 20 30 40 50 60

0.2614 0.2136 0.1922 0.1286 0.0682 0.0303

1.164 1.134 1.080 1.088 1.071 1.057

8.8 7.2 5.8 4.2 2.8 1.9

88 144 174 168 140 114

Serum

10 20 30 40 50 60

0.1336 0.0156 0.0059 0.0007 0.0002 0.0000

1.125 1.081 1.086 1.081 1.085 1.081

4.0 1.1 1.0 1.0 1.0 1.0

40 22 1.0 1.0 1.0 1.0

Urine

10 20 30 40 50 60

0.3045 0.2254 0.1008 0.0713 0.0136 0.0028

1.204 1.145 1.098 1.079 1.057 1.048

10.8 7.4 4.9 3.0 1.5 1.0

108 148 147 120 75 1.0

‘Substances used were: 5% trichloroacetic solution; 1% NaOH solution; human serum; * % Efficiency X % substance.

acid solution; 1% HCl and human urine.

solution;

10% sucrose

solution;

1% NaCl

418

THOMAS

F. KELLOGG TABLE

4

EFFECT OF VARIOUS VOLUMES OF SAMPLES ON 14C COUNTING MERIT

Substance’ None Water

TCA

USING TRITON

X-100,

VolumePercent

ESR

PPO,

EFFICIENCIES, ESR, SCR, AND FIGURE AND 0.5 g/liter bis-MSB COCKTAIL Percentage efficiency

SCR

OF

Figure of merit’

0.3810

1.286

78.7

10 20 30 40 50 60 70 80 90

0.3247 0.2808 0.2214 0.1528 0.0984 0.0478 0.0418 0.0018 0.0005

1.374 1.464 1.673 2.054 2.539 3.316 5.182 10.92 15.82

76.1 73.4 67.4 59.4 53.9 47.2 35.7 20.9 8.9

761 1470 2020 2380 2700 2830 2500 2670 800

10 20 30 40 50 60

0.3004 0.1897 0.1168 0.0121 0.0001 0.0000

1.441 1.786 3.197 5.443 13.825 32.225

71.6 62.2 46.6 33.9 19.4 8.3

716 1244 1398 1356 970 498

:: 30 40 50 60

0.3317 0.2730 0.2741 0.1345 0.0605 0.0913

1.371 1.505 1.846 2.214 3.012 4.450

753 1414 1887 2228 2440 2418

0.3326 0.2805 0.2311 0.1636 0.1002 0.0435 0.3226 0.2705 0.2548 0.1426 0.0784 0.0349 0.2902 0.2367 0.2067 0.1409 0.0753 0.0343

1.375 1.477 1.732 1.901 2.405 3.360

75.2 70.9 62.9 55.7 48.8 40.3 75.4 73.3 67.3 63.1 55.1 46.9 76.4 71.0 67.1 62.4 53.9 45.8

0.2003 0.0388 0.0239 0.0013 0.0003 0.0000 0.2632 0.1279 0.0919 0.0037 0.0002 0.0000

1.677 2.638 5.132 5.651 5.995 10.930 1.551 1.990 3.373 4.808 7.858 19.005

76.6 72.3 66.8 63.5 53.8 46.9 57.8 41.2 22.3 12.1 7.4 3.1 65.7 56.5 34.3 32.7 22.3 13.1

766 1446 2004 2540 2690 2814 578 824 669 484 370 186 657 1130 1029 1308 1115 786

HCl

Sucrose

8 g/liter

10 20 30 40 50 60

NaCl :8 30 40 50 60 NaOH

10 20 30 40 50 60

Serum

10 20 30 40 i:

Urine ;oo 30 40 50 60 a Substances used were: 5% trichloroacetic solution: 1% NaOH solution; human serum; ’ I Efficiency X % substance.

1.394 1.537 2.098 2.012 2.609 3.641 1.466 1.581 1.790 2.073 2.678 3.678

acid solution; 1% HCI and human urine.

solution;

10% sucrose

754 1466 2019 2524 2755 2814 764 1420 2013 2496 2695 2748

solution;

1% NaCl

A WATER-MISCIBLE,

NONHAZARDOUS

tern make this a very attractive cocktail for counting biological samples containing betaemitting radionuclides. The advantages are further strengthened by the compatibility of the cocktail with polyethylene vials. Toluene-containing cocktails cause problems of polyethylene vial swelling and diffusion of toluene through the vial walls. This results in fluor deposits in the vial walls and may lead to erroneous ESR values. The absence of the problems with Triton X-100 as the only scintillant allows use of polyethylene vials, which can be incinerated, thus precluding problems of disposal of empty glass scintillation vials. The tritium efficiency of 17% in Triton X100 cocktail is about one-half of the efficiency for 30% Triton X-100-70% toluene cocktails. However, these Triton-toluene cocktails have a minimum 0.5% water requirement to form monophasic samples and can often accept no more than 10% water without departing from acceptable phase contact, and therefore quench may not be correctable by ESR techniques (see Benson (5)). The 100% Triton X-100 cocktail maintains excellent phase contact with all proportions of water (Fig. 1). Thus the maximum figure of merit with acceptable phase contact is similar, or better, for 100% Triton X- 100 than for Triton-toluene cocktails. A major advantage of the 100% Triton X100-based cocktail is its nonflammability. Its flash point is over 300°F. There are no organic solvent fumes to contend with, an advantage from both an explosion and a health standpoint. Triton X-100 is not a hazardous waste as defined by the Resource Conservation and Recovery Act and therefore does not have to be disposed of under their regulations (6).

LIQUID

SCINTILLANT

419

This allows disposal down sanitary sewers if the maximum permissible radionuclide concentration is not exceeded (7). One of the major factors causing difficulty in commercial burial ground disposal of LSC wastes is the flammability and toxicity of current cocktails. Use of Triton X- 100 or similar materials as sole scintillators for LSC may overcome many of the objections. Triton X-100 at room temperature is fluid enough to dispense through repetitive pipetters. If the Triton X-100 is heated to approximately 50°C in a water bath or with electrically heated tape it is easier to pipet and mix with the sample. Care should be taken to mix cocktail and sample by swirling to avoid bubble formations. The vials should be allowed to cool before counting to avoid high background counts. REFERENCES 1. Kobayashi, T., and Maudsley, D. V. (1974) in Liquid Scintillation Counting, Recent Developments (Stanley, P. E., and Scoggins, B. A., eds.), pp. 189-205, Academic Press, New York. 2. O’Connor, J. L., and Bransome, E. D., Jr. (1980) in Liquid Scintillation Counting, Recent Applications and Developments. (Peng, C., Horrocks, D. L., and Alpen, E. L., eds.), pp. 245-258, Academic Press, New York, 3. Kellogg, T. F. (1982) In?. J. Appl. Radiat. Isotopes, in press. 4. Turner, J. C. (1969) Int. J. Appl. Radiat. Isofopes 20, 499. 5. Benson, R. H. (1980) in Liquid Scintillation Counting, Recent Applications and Developments (Peng, C., Horrocks, D. L., and Alpen, E. L., eds.), pp. 237-244, Academic Press, New York. 6. Hazardous Waste and Consolidated Permit Regulations (1980) Fed. Regist. 45, No. 98, 33,03333,588 (May 19, 1980). 7. Biomedical Waste Disposal (1981) Fed. Regist. 46, No. 47, 16,230-16,234 (March 11, 1981).