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Comparative studies of liquid scintillation counting of aqueous 14C samples
Comparative
Studies
of
of Aqueous
Liquid
Scintillation
Counting
’ “C Samples’
Liquid scintillat,ion count’ing is the most important recent development in the application of radioisotopes to agriculture and medicine. Ik&opment of small-volume internal sample counting techniques has greatly simplified the measurement of weak beta-emitting isotopes in biological materials. Both acid and base, such as alcoholic KOH, Hyamiie, and other organic; amines, have been utilized for the digestion or solubilization of tissues (l-4). Counting of respiratory CO, in liquid scintillation spectrometers also rticeivcd attention. Organic bases which are miscible with scintillation solution arc commonly used for t,rapping CO, (5-7). Recently, inhibition of t,he oxidizing system cwu~edby volatile organic bases or sol~t~nts was wportcd (:S), and the use of aqueous KUH or NaOH as a trapping agent for quantitative absorption of CO, and mixed directly iu Bray ‘Sscintillation Folution has been suggested. Recently WC mark tlircct, corrqwison of three commonly used watermiscible eolwnt systems 19-I.1’1 for counting IYSlabeled carbonate! glucose, RIMI benzmc either scpnwtely or in combination. Table 1 shows the counting efficiency of thc~ three ‘“(1 samplo,i:when counted individually and corrcvted with int’ernal standardization in these solvent systems in the presenceof various amount,uof mater, or organic bases. A Packard model 314E,S liquid seintillatioll spectrometer was used with windovlsettings at 100-1000 ant-I optSitnumhigh voltage for each solvent system. Equal arzkountsof Iabelcd ~implr were used in all comparative rneasurcmcnt8s.It, is clear that’, withi~l B given solvent, system, the counting efficiency varies between samples; and within a given sample, the counting efficiency iy c.lcp,cndtAnt upon the amount of wat.(tr or other solubilizing agents. The infiuencc of w:der on counting diciency
seems to t)r! more
pronounce(l in both Bray and Yrockop solvent systems than in toluene,’ Triton X-100 system. In general, the counting efficiency of all samples under various condition!+was considerably lower with Bray solution than with others. Pa both Bray antI Prockop syetems there was definitely a decreasing trencl of’ counting efficiency for both benzene and glucose: samples RF the wat,cr content increased. This it: presumably due to the quenching effect’ of mwtcr. How(b’i:er, with labeled carbonate samples ’ ‘rt~c1inic~:l.i I’nlIr~r90, 2871,Ort~prlllSt,a,te Agricultural
Experiment
Station.
239
SHORT COMMUNICATIONS
Counting
Efficiency
TABLE 1 of Aqueous 14C Samples in Three Solvent. Systems Prockop
Bray J&O content’,
48 8 44.4
61.4 61.9 61.8
64.7 --
36.2
61 7
63.0
49.0
62.3 61 2
64 5
50. !1 45.1 45.0 52.5 53. i 57.6
51.1 52.5 53.4
34.2 46.1 46.4
57.0 57.0 54.9
57.2 50. 1 44.0
54.6 52.6 39.6
33.T 32.T 40.4 39.2
53.5 57.6 57.8
CO,
0
40.2 40.4 39.5 38.7 37.6 36.6
49.7
34.5
47.1
30.5
45.2
45.5 51.9 50.9
43.3 44.x 47.5
5 Y0 Hyamine 5 Y. Soluene 5%NCS
60.2 57.x 55.1
60.8 59 3 58.11 56.2 54.7 54.3
zene Glucose
1 2 3 4 5
Ben-
Glllcose
Nfb-
X-100 -. -.--.-_
NasCO:i
Benzeue
Ben-
07 !V
Tul~~ene/Triton
zene
S%Glrlcose
COZr
Each vial contained 10 ml counting medium, and 5880 dpm benzene, 2390 dpm glucose, or 4890 dpm NaLX&, and was standardized with benzoie acid as internal standard. AI1 labeled solutions were measured with a 100 ~1 Hamilton syringe and each sample was counted for 10 minutes. Soluene 100 sample solubilixer purchased from Packard Instrument, Company, Downers Grove, 111.; NCS solubilizer pltrehased from Amersham/Searle Corporation, Arlington Height,s, Ill.
the changes of counting efficiency were not correlated to the amount of water in the system. The presenceof NaOH or organic basesin the scintillation also affected the counting efficiency and the degree of effect seemedt,o depend upon t’he nature of the labeled compound as well as the solvent system. Figure 1 presents the effect of NaOR on t#hc counting efficiencies of benzene and sodium carbonate in these solvent systems in terms of the volume of NaOH solution added and its concentration. The counting efficiency of labeled benzene in toluene/Triton X-100 medium seemed to be very stable in all concentrations of YaOH and various volumes tested. However, there was serious reduction of counting efficiency for benzene in Prockop medium, and t,his reduction was dependent. on the volume as well as the concentration of NaOH, In Bray medium, a less severe reduction of counting efficiency for benzene by NaOH was also observed and the Ievel of reduction was the same bet,ween 0.1 to 2.0 1Y NaOH. With 6 iV NaOH solution there seemedto be an increasing trend with the increasing volume of the base. With the counting of carbonate, severe decreases were noted with all concent8rationsof NaOH in both Bray and Prockop mediums. In addition to the influences of concentrat’ion and volume of NaOH in the counting system, the time beween the preparation of the counting vial and the counting is also an important;
240
50
I
‘3ray
) Prockop
60
SODIUM
[ Trlton
HYDROXIDE
0
X-100
SOLUTION,ml
TABLE 2 Liquid Scintillation Counting (dpm,l of a Carbonate Sampie (48W dpm) in Threw Solvent, Systems and Standardized wit)h Benzoic Acid as Tnt’ernd Sandard ------ __-----.__-. ‘rolaene/Trii,c,t~ Htq Yrockop x-100
Addition 0.5 ml HZ0 0.5 ml 0.1 3’ NaOH 0.5 ml 0.5 N NaOH 0 5 ml X.0 :%-NaOH 0.5 ml 2.0:\: N&H 0.5 ml 6.0 11;NaOH 0.5 ml Hyamine 0.5 ml Soluene 0.5 ml NCS 0.5 ml I M NaOH + 4% Cab-O-Sil
Immediately
Aft,er 24 ht
Immediately
After
4670 4960
37% 4060
4310 45x0
‘l710 2490
4540 4660 4210 4520 5540
4070 33%) 39%0 3X60
4500 4320 :5910 3060 :sxxo
2480 ‘2360 2620 3590
50’20
:36X0
5140
4060 3950
Each vial contained 10 ml counting medium and after addition of internal sbnndttrd.
24 hr
Immrdiately
After 24 hr
4r30
4901)
4860 4X80 4870
4i30
4,500
1030
:1070 5090
5020 4700 BOO
4160
4,531 4400
and ww cwwtetl
__----___---for 10 minlltes
hefore
+ + +
91.9 97.6 72.9 102.9
+ 5.0 (5)” 31 3.4 (6) -E-5.6 (4) + 3.0 (5)
Bray
24 hr __-__s. 7.6 (51 ~fi:3.6 (6’) zk 9.0 (4) + 8.1 (5j
of Aqueous
After
67.5 94.6 52.6 79.1
Counting
99.8 104.8 102.2 102.0
IO.5 +_ 4.5 f 3.6 -t 2.7
Immediately (5) (6) (4) (5)
iz + + -t
After 91.7 99.1 95.8 88.5
Prockop -
Alkali
--. !)4.1 97.6 65.7 91.5
& + i I:
25.6 (6) 4.9 (6) 7.6 (4) 28.2 (5)
Immediately
F 24.1 (6) 14.9 (6> 2 3.9 (4) I 10.9 (5)
24 hr
Systems
of labeled compounds
50.2 94.7 33.4 59.7
_..__I
After
X-100
Toluenefl’riton -
Solvent
in Three
(2 :V Na0H)
with benzoic acid. Amount
3.7 (5) 4.7 (sj 4.0 (4) 14.7 (5)
24 hr
TABLE 3 14C Samples in Presence of Strong
Q Mean &I standard deviat’ion (number of deterrr.inations). All vi& were standardized used in each viaI: 2730 dpm glucose, 3190 dpm xerine, or 4500 dpm succinate.
Glucose Glucose Serine Succinate
0.5 mI ___ N&H Immediately
Sample
ScintilIation
of Liquid
Accuracy
5 2 s 5 $ v;
8 s
8
ifi
SHORT
243
COMMGKICATXOXF
ACKNOWLEDGMENT This investigation was supported the National lnstitutrs of Health.
in part by U. S. P. H. S. Grant ES-00141 from
REFERENCES 1. PETROW.
C. P., PATT, H. H.. ANLI K~.~IR,P. P., In.ter~z. J. Appl.
Radiatiow
Isotope
16, 599 (1965). 2. DULCIXO,
J., Bosco,
R., \'ERLT, R. G., .IND M.4rsrs.
J. I~., C&L C&m. Acta 8, 58
(1963). 3. MEaDE, R.. C.. AND SITGLITZ, R.> Intern. J. Appl. Rudtition Isotopes 13, 11 (1962). 4. HANSEN, D. L., AND BUSH, E. T.. A~nl. Biochem. 18, 320 (1967). 5. EDWARDS, B., AND KITCHENER. J. ,4.. Intern. J, Appl. Rntlintion Isotopes 16, 445 (1964). 6. WOELLER, F. H., Anal. B&hem. 2, 508 (1961). 7, BOGGIOLINI, M., AKD BICGEL, M. H., A&. Biochem.. 14, 290 (1966). 8. DUNCOMMBE,W. G., ASD RISING, T. J., Anal. Biochwr~. 30, 275 (1969). 9. BRAP, G. A., Ad. Rio&em.. 1, 279 (1660). 10. PROCKLOP,D. J.. AND EBERT, P. S,, Anrrl. Bioch.em. 6, 263 (1963). 11. PATTERSON, M. S., .~ND GREEN, R. C., Awl. Chem. 37, 854 (1965). 12. MOHIN, R. J., AND CARRION. M., Lipi& 3, 349 (1968). I.
I'.
Af0sTA~h
ELIZEBETH
S. C.
FALLIN
FANG
Deparbment of Agticulbural Chemistry Oregon State University CorvalZk, Oregon $7531 Rrceir~rJ Jnnrcary %, 1970
Ammonia Reagent
Modification
Determination: and
tnterfering
Compound,s’
The phenol-hypochlorite reaction for the determination of ammonia is rapid and easy, and requires only two solutions (1, 2). Weatherburn (2) recently reported optimum reagent concentrations and time-temperature color development conditions for the procedure. Using his procedure (2) to determine the ammonia concentration in biological saSmples,we ob’ Contribution No. 105, Department of Biochemistry, Kansas Agricultural ment Station, Manhattan, Kansas 66502. This investigation was supported by Natioral Institutes of Health Grant FR07036.
Experiin part
Studies
of
of Aqueous
Liquid
Scintillation
Counting
’ “C Samples’
Liquid scintillat,ion count’ing is the most important recent development in the application of radioisotopes to agriculture and medicine. Ik&opment of small-volume internal sample counting techniques has greatly simplified the measurement of weak beta-emitting isotopes in biological materials. Both acid and base, such as alcoholic KOH, Hyamiie, and other organic; amines, have been utilized for the digestion or solubilization of tissues (l-4). Counting of respiratory CO, in liquid scintillation spectrometers also rticeivcd attention. Organic bases which are miscible with scintillation solution arc commonly used for t,rapping CO, (5-7). Recently, inhibition of t,he oxidizing system cwu~edby volatile organic bases or sol~t~nts was wportcd (:S), and the use of aqueous KUH or NaOH as a trapping agent for quantitative absorption of CO, and mixed directly iu Bray ‘Sscintillation Folution has been suggested. Recently WC mark tlircct, corrqwison of three commonly used watermiscible eolwnt systems 19-I.1’1 for counting IYSlabeled carbonate! glucose, RIMI benzmc either scpnwtely or in combination. Table 1 shows the counting efficiency of thc~ three ‘“(1 samplo,i:when counted individually and corrcvted with int’ernal standardization in these solvent systems in the presenceof various amount,uof mater, or organic bases. A Packard model 314E,S liquid seintillatioll spectrometer was used with windovlsettings at 100-1000 ant-I optSitnumhigh voltage for each solvent system. Equal arzkountsof Iabelcd ~implr were used in all comparative rneasurcmcnt8s.It, is clear that’, withi~l B given solvent, system, the counting efficiency varies between samples; and within a given sample, the counting efficiency iy c.lcp,cndtAnt upon the amount of wat.(tr or other solubilizing agents. The infiuencc of w:der on counting diciency
seems to t)r! more
pronounce(l in both Bray and Yrockop solvent systems than in toluene,’ Triton X-100 system. In general, the counting efficiency of all samples under various condition!+was considerably lower with Bray solution than with others. Pa both Bray antI Prockop syetems there was definitely a decreasing trencl of’ counting efficiency for both benzene and glucose: samples RF the wat,cr content increased. This it: presumably due to the quenching effect’ of mwtcr. How(b’i:er, with labeled carbonate samples ’ ‘rt~c1inic~:l.i I’nlIr~r90, 2871,Ort~prlllSt,a,te Agricultural
Experiment
Station.
239
SHORT COMMUNICATIONS
Counting
Efficiency
TABLE 1 of Aqueous 14C Samples in Three Solvent. Systems Prockop
Bray J&O content’,
48 8 44.4
61.4 61.9 61.8
64.7 --
36.2
61 7
63.0
49.0
62.3 61 2
64 5
50. !1 45.1 45.0 52.5 53. i 57.6
51.1 52.5 53.4
34.2 46.1 46.4
57.0 57.0 54.9
57.2 50. 1 44.0
54.6 52.6 39.6
33.T 32.T 40.4 39.2
53.5 57.6 57.8
CO,
0
40.2 40.4 39.5 38.7 37.6 36.6
49.7
34.5
47.1
30.5
45.2
45.5 51.9 50.9
43.3 44.x 47.5
5 Y0 Hyamine 5 Y. Soluene 5%NCS
60.2 57.x 55.1
60.8 59 3 58.11 56.2 54.7 54.3
zene Glucose
1 2 3 4 5
Ben-
Glllcose
Nfb-
X-100 -. -.--.-_
NasCO:i
Benzeue
Ben-
07 !V
Tul~~ene/Triton
zene
S%Glrlcose
COZr
Each vial contained 10 ml counting medium, and 5880 dpm benzene, 2390 dpm glucose, or 4890 dpm NaLX&, and was standardized with benzoie acid as internal standard. AI1 labeled solutions were measured with a 100 ~1 Hamilton syringe and each sample was counted for 10 minutes. Soluene 100 sample solubilixer purchased from Packard Instrument, Company, Downers Grove, 111.; NCS solubilizer pltrehased from Amersham/Searle Corporation, Arlington Height,s, Ill.
the changes of counting efficiency were not correlated to the amount of water in the system. The presenceof NaOH or organic basesin the scintillation also affected the counting efficiency and the degree of effect seemedt,o depend upon t’he nature of the labeled compound as well as the solvent system. Figure 1 presents the effect of NaOR on t#hc counting efficiencies of benzene and sodium carbonate in these solvent systems in terms of the volume of NaOH solution added and its concentration. The counting efficiency of labeled benzene in toluene/Triton X-100 medium seemed to be very stable in all concentrations of YaOH and various volumes tested. However, there was serious reduction of counting efficiency for benzene in Prockop medium, and t,his reduction was dependent. on the volume as well as the concentration of NaOH, In Bray medium, a less severe reduction of counting efficiency for benzene by NaOH was also observed and the Ievel of reduction was the same bet,ween 0.1 to 2.0 1Y NaOH. With 6 iV NaOH solution there seemedto be an increasing trend with the increasing volume of the base. With the counting of carbonate, severe decreases were noted with all concent8rationsof NaOH in both Bray and Prockop mediums. In addition to the influences of concentrat’ion and volume of NaOH in the counting system, the time beween the preparation of the counting vial and the counting is also an important;
240
50
I
‘3ray
) Prockop
60
SODIUM
[ Trlton
HYDROXIDE
0
X-100
SOLUTION,ml
TABLE 2 Liquid Scintillation Counting (dpm,l of a Carbonate Sampie (48W dpm) in Threw Solvent, Systems and Standardized wit)h Benzoic Acid as Tnt’ernd Sandard ------ __-----.__-. ‘rolaene/Trii,c,t~ Htq Yrockop x-100
Addition 0.5 ml HZ0 0.5 ml 0.1 3’ NaOH 0.5 ml 0.5 N NaOH 0 5 ml X.0 :%-NaOH 0.5 ml 2.0:\: N&H 0.5 ml 6.0 11;NaOH 0.5 ml Hyamine 0.5 ml Soluene 0.5 ml NCS 0.5 ml I M NaOH + 4% Cab-O-Sil
Immediately
Aft,er 24 ht
Immediately
After
4670 4960
37% 4060
4310 45x0
‘l710 2490
4540 4660 4210 4520 5540
4070 33%) 39%0 3X60
4500 4320 :5910 3060 :sxxo
2480 ‘2360 2620 3590
50’20
:36X0
5140
4060 3950
Each vial contained 10 ml counting medium and after addition of internal sbnndttrd.
24 hr
Immrdiately
After 24 hr
4r30
4901)
4860 4X80 4870
4i30
4,500
1030
:1070 5090
5020 4700 BOO
4160
4,531 4400
and ww cwwtetl
__----___---for 10 minlltes
hefore
+ + +
91.9 97.6 72.9 102.9
+ 5.0 (5)” 31 3.4 (6) -E-5.6 (4) + 3.0 (5)
Bray
24 hr __-__s. 7.6 (51 ~fi:3.6 (6’) zk 9.0 (4) + 8.1 (5j
of Aqueous
After
67.5 94.6 52.6 79.1
Counting
99.8 104.8 102.2 102.0
IO.5 +_ 4.5 f 3.6 -t 2.7
Immediately (5) (6) (4) (5)
iz + + -t
After 91.7 99.1 95.8 88.5
Prockop -
Alkali
--. !)4.1 97.6 65.7 91.5
& + i I:
25.6 (6) 4.9 (6) 7.6 (4) 28.2 (5)
Immediately
F 24.1 (6) 14.9 (6> 2 3.9 (4) I 10.9 (5)
24 hr
Systems
of labeled compounds
50.2 94.7 33.4 59.7
_..__I
After
X-100
Toluenefl’riton -
Solvent
in Three
(2 :V Na0H)
with benzoic acid. Amount
3.7 (5) 4.7 (sj 4.0 (4) 14.7 (5)
24 hr
TABLE 3 14C Samples in Presence of Strong
Q Mean &I standard deviat’ion (number of deterrr.inations). All vi& were standardized used in each viaI: 2730 dpm glucose, 3190 dpm xerine, or 4500 dpm succinate.
Glucose Glucose Serine Succinate
0.5 mI ___ N&H Immediately
Sample
ScintilIation
of Liquid
Accuracy
5 2 s 5 $ v;
8 s
8
ifi
SHORT
243
COMMGKICATXOXF
ACKNOWLEDGMENT This investigation was supported the National lnstitutrs of Health.
in part by U. S. P. H. S. Grant ES-00141 from
REFERENCES 1. PETROW.
C. P., PATT, H. H.. ANLI K~.~IR,P. P., In.ter~z. J. Appl.
Radiatiow
Isotope
16, 599 (1965). 2. DULCIXO,
J., Bosco,
R., \'ERLT, R. G., .IND M.4rsrs.
J. I~., C&L C&m. Acta 8, 58
(1963). 3. MEaDE, R.. C.. AND SITGLITZ, R.> Intern. J. Appl. Rudtition Isotopes 13, 11 (1962). 4. HANSEN, D. L., AND BUSH, E. T.. A~nl. Biochem. 18, 320 (1967). 5. EDWARDS, B., AND KITCHENER. J. ,4.. Intern. J, Appl. Rntlintion Isotopes 16, 445 (1964). 6. WOELLER, F. H., Anal. B&hem. 2, 508 (1961). 7, BOGGIOLINI, M., AKD BICGEL, M. H., A&. Biochem.. 14, 290 (1966). 8. DUNCOMMBE,W. G., ASD RISING, T. J., Anal. Biochwr~. 30, 275 (1969). 9. BRAP, G. A., Ad. Rio&em.. 1, 279 (1660). 10. PROCKLOP,D. J.. AND EBERT, P. S,, Anrrl. Bioch.em. 6, 263 (1963). 11. PATTERSON, M. S., .~ND GREEN, R. C., Awl. Chem. 37, 854 (1965). 12. MOHIN, R. J., AND CARRION. M., Lipi& 3, 349 (1968). I.
I'.
Af0sTA~h
ELIZEBETH
S. C.
FALLIN
FANG
Deparbment of Agticulbural Chemistry Oregon State University CorvalZk, Oregon $7531 Rrceir~rJ Jnnrcary %, 1970
Ammonia Reagent
Modification
Determination: and
tnterfering
Compound,s’
The phenol-hypochlorite reaction for the determination of ammonia is rapid and easy, and requires only two solutions (1, 2). Weatherburn (2) recently reported optimum reagent concentrations and time-temperature color development conditions for the procedure. Using his procedure (2) to determine the ammonia concentration in biological saSmples,we ob’ Contribution No. 105, Department of Biochemistry, Kansas Agricultural ment Station, Manhattan, Kansas 66502. This investigation was supported by Natioral Institutes of Health Grant FR07036.
Experiin part