chromatography and the total fecal sterols assumed to be the sum of the digitonide precipitated sterols plus the supernatant sterols as determined by gas chromatography (Table 111). Analysis of fecal bile acids obtained as the ether extract of acidic fecal lipids after the nonsaponifiable materials have been removed has been more difficult. The methodology is identical to that for sterols except a larger proportion, the equivalent of 0.5 to 2.0 grams of rat feces, is required. The bile acids are quantitatively methylated with ewess diazomethane and dissolved in benzene so that bile acid concentration is 5 to 25 mg. per ml. Cholestane standard is added to a concentration of 0.2 to 0.4 mg. per ml. Values obtained are frequently 10 to 20% lon-er than those obtained by the fluorimetric method of Levin, Irvin, and Johnston (4) applied to feces. Nevertheless, when
known amounts of bile acids are added to fecal extracts, they can be accounted for with the same 10% accuracy (Table 111). It seems, therefore, that the method may be more accurate than fluorimetry in this application to feces. There are few compounds which have retention times similar to bile acids so that interference is not a problem. The method, though empirical, may have wider application with biological substances. With C-21 steroids, for example, if one takes t h e data of Sweeley and Chang (6) and converts relative retention times and responses to androstane as a standard, a plot similar to Figure 5 may be drawn that encompasses allopregnane-3p,20p-diol, pregnane-3,20-dione, 4-pregnene-3,20dione, and 5-pregnen-3p-ol-2O-one, and comes reasonably close to other thermostable steroids. The same holds true for the data of Wotiz and Martin (7‘) if estrone acetate is used as the
standard for a homologous series of acetylated estrogens. LITERATURE CITED
(1) Bergman, IT., J . Bid. Chem. 132, 471 (1940). (2]‘Cook, R. P., Rattray, J. E. M. in Cholesterol,” R. P. Cook, ed.? p. 118, Academic Press, Sew York, 1938. (3) Horning, E. C., Moscatelli, E. A,, SReeley, C. C., Chem. & Ind. (London) 1959, 751. ( 4 ) Levin, S. J., Irvin, J. L., Johnston, C. G., ANAL.CHEM.33, 856 (1961). ( 5 ) Sjovall, J., Meloni, C. R., Turner, D. A., J . Lzpzd Research 2,317 (1961). (6) Sweeley, C. C., Chang, T., ANAL. CHEM.33,1860 (1961). ( 7 ) Wotiz, H. H., Martin, H. F., J . Biol. Chem. 236, 1312 (1961). RECEIVED for revierr January 12, 1962. Accepted April 2, 1962. Work completed during the tenure of an Established Investigationship of the American Heart Association. Work supported by grants from the Heart Societies of Portage County and Cleveland, Ohio.
Proportional Counter Assay of Tritium in Gas Chromatographic Streams J. K. LEE, EDWARD K. C. LEE, BURDON MUSGRAVE, YI-NO0 TANG, JOHN W. ROOT, and F. S. ROWLAND Department of Chemistry, University o f Kansas, lawrence, Kan.
b The assay of tritium b y gas proportional counting of the effluent from a chromatographic column has many advantages. Aside from the precautions common to all gas chromatography practice, the critical point for accurate assay i s maintaining constant efficiency of detection for tritium in various molecular forms. The variations in detection efficiency arise especially from coincidence losses a t high count rates and from composition changes in the operating counter gas during the passage o f a macroscopic peak. The behavior o f helium-methane and helium-propane flow mixtures i s discussed extensively.
G
CHROMATOGRAPHIC separation and immediate radioactive assay of the separated gaseous components form a powerful combination tool for the investigation of many radioactive systems (2, 5, 9). The rapid increase in the availability of tritium, and in our case, the interest in the reactions of recoil tritium atoms, has led to the development of this technique for special application to tritium-labeled molecules (9). The primary design limitation for tritium assay arises from the low energy of the beta particle (Eomax = 18 k. e. v.) emitted in its decay to AS
He3. The practical consequence of this low energy is that the tritiumlabeled molecule must be present in the counting gas; hence the gas-counting tube must be made to operate on the effluent gas from the gas chromatographic separation apparatus The chief advantages of operation of an internal gas proportional flow counter in this manner have been discussed previously (9). The purpose of the present study is to examine the most important factors that affect the accuracy and reproducibility of this method for the analysis of tritiated radioactive components in gas chromatographic streams. I n our laboratory, the technique has been most frequently applied to the analysis of mixtures of labeled hydrocarbons, but many other volatile tritiated molecules have also been examined. The general problems are common to this method of internal counting for other weak beta emitters, and for similar radioactivity measurements in general. This counting system has also handled successfully C14, Ar37, Xe133,and other beta activities. W t h strong beta or gamma-ray emitters, however, it is not necessary to run the effluent gases directly through the counter itself, and many of the problems encountered with tritium are thereby avoided ( 1 ) . Evans and Willard ap-
plied a n external counting technique sensitive to gamma radiation and hard betas for the measurement of halogen radioactivities ( 2 ) . SAMPLE INJECTION
Sample injections are made through a sample chamber bounded by two ground glass T-joints and equipped with a by-pass. The sample loop is filled on a vacuum line with the sample in the gaseous state, and the equipment is equilibrated n i t h carrier gas passing through the by-pass. The injections are then made b y simultaneous turning of the stop-cocks to substitute the sample loop for the by-pass. With relatively nonvolatile samples, a sample loop has been used that is sufficiently big to accommodate all the sample in the gas state. Errors of sampling involved in the operation of any gas chromatograph will also affect the radioactivity measurements. SPECIFIC RADIOACTIVITY A N D OBSERVED COUNTS
This technique of radioactive assay relies on measurement of the number of counts observed in a particular gas chromatographic peak as a measure of the specific radioactivity of the corresponding compound. I n relative VOL 34, NO. 7, JUNE 1962
741
30COL 5CO
T,
COdNTS
COUNTS
2:1
a m
2c
5 SECONDS
SECCU38
Figure 1 .
Passage of tritium activity peak, as recorded on brush recorder
assay of several components in a gaseous mixture, it is important that the proportionality factor relating observed counts to specific radioactivity be unchanged during the measurement of each of the peaks. I n absolute assay of a component, the proportionality factor itself needs to be known accurately. -4study of the reproducibility and accuracy of this method of gas proportional counting largely reduces t o a study of this proportional relation between specific activity and observed counts, and of the factors that can cause variations in it. The relationship between observed counts and specific radioactivity is a composite of the efficiency of the counter for detection of a decay event occurring within it and the average residence time for the labeled compounds in the active counter volume. Many of the frequent variables in detection efficiency (geometry, absorption, scattering) of a proportional counter are constant for gases within the active volume and a-ill be the same for a given radioactive isotope in all chemical forms as long as the macro characteristics of the flow gas (composition and flow rate) are unchanged. Since the de-
EXTERNAL
?a
SOLRCE
-
2Oml
i 3 G G L CCO
tection efficiency approaches unity for beta decays, moderate changes in the macro characteristics will ordinarily have imperceptible effects. The flow rate of the gas through a counter, and therefore the average residence time of gaseous molecules within the counter, can usually be controlled and measured with sufficient accuracy to reduce errors from this source well below the inherent errors of random counting statistics. Stabilization has been obtained in standard fashion by the inclusion of surge tanks for both helium and hydrocarbon gas in the gas flow line immediately following the high pressure regulators. ilbsolute calibrations have been performed through an intercomparison of the number of counts observed in this flow system and in internal gas proportional counting with a stationary counter mounted on a vacuum line (6). Wolf and Stocklin ( 7 ) have privately pointed out to us the large changes in actual flow rate from a chromatographic column that can occur during the emergence of a macroscopic peak. These effects are most important nith large, very sharp peaks, and the effect on the residence time in the
FLOW COUN'E?
? CH,T
: c
I
,
010
I00
50
15C
SECONDS
Figure 2. Resolution of neighboring peaks with different counting intervals
counter is reduced by the addition of the organic component. I n the measurements illustrated here, the effect on the residence time is less than 1%, anti normally less than the statistical deviation from random radioactive decay. APPARATUS
The apparatus of Wolfgang and Rowland (Qj was used FTith t'he folloning changes: (1) The proportional counters \vert? constructed of borosilicate glass with a silver mirror on the inside wall, and a tungsten center nire, with an active counter volume of 20 ml. (Roman Scientific Glass Co., Patchogue, K.Y.). Counters of similar construction with active volumes of 10 ml. and 85 ml.
EYTLRNAL No" SOURCE
20ml. FLOW COUNTER
Figure 3. mixtures
Count rate vs. voltage plateaus for CHd-He Flow rates (ml. per minute) A B
CH, CH4 He C CH4 He C' C3Hs He D CH4 He
742
ANALYTICAL CHEMISTRY
10-200 37) 51
;7)
Figure 4. rates
Plateaus for CH4-He
mixtures at high count
Flow rates (ml. per minute)
A
371
B
2 A1 ( 20j
C
CH, CH4 He CHI He
37
3 71
51 3 71 21;
have also been used. Similar results !{ere obtained using metal counters resembling those previously described (9). Various other designs for flow counters have been discussed by Wolfgang and 1lacKay (8). (2) The elapsed time for the accumulation of a fixed number of counts (10, 102, or lo3) is recorded as a mark on the moving chart paper of a writing oscillograph (Brush Electronic Co., Cleveland, Ohio), replacing the recording rate meter of reference (9). An alternate method of recording the data is to feed the output of the scaler into a digital printer. The actual recording of an activity peak (Figure 1; each interval = 100 counts) is ordinarily converted into the usual peak shapes by reading the number of counts in a fixed time interval, and plotting these numbers us. time. Figure 2 s h o m two plots from the same recording-the fixed time interval for curve -4is 20 seconds, and for curve B , 5 seconds. I t is obvious that the resolution of the plot is improved with the shorter intervals, and the time intervals are therefore chosen short enough t o give the maximum resolution available with the particular column. CHOICE O F COUNTING G A S MIXTURE
The composition of the gas mixture flou ing through the proportional counter will be determined by the characteristics of the particular experiment. The counting considerations are greatly simplified when the same gas serves adequately as the flow gas for chromatography and as the counting gas in the proportional counter. Gordus, Sauer, and Willard have successfully used methane gas in the separation of hydrocarbons in this fashion, but were unable to obtain any measurements of hydrocarbon mass peaks because of the similarity in thermal conductivity of the carrier gas and the compounds being analyzed (4). I n our experiments, we usually nished to record simultaneously any mass peaks, as well as the radioactivity, and have therefore used helium as the carrier gas for the chromatographic separation and thermal conductivity detection. Argon and hydrogen can also be used if the situation \Tarrants it. TF7e have used thermal resistivity detectors because our usual experiments involve sample injections of 1 cc. STP or more, and the macroscopic peaks of interest have been 10-4 cc. or larger. With these carrier gases, proper counter operation then requires the addition of an appropriate amount of a gas which n-ill make a suitable mixture for gas proportional counting. While many organic gases will provide such a suitable mixture, we have restricted our experimentation to methane and propane gases, both of which are quite satisfactory and available in high purity at reasonable cost. Propane is preferable to methane in most of our ap-
plications, as shown below, but a methane-helium mixture can be very useful for experiments requiring trapping of some volatile components from this stream. The two gas streams have been mixed through fritted disks on one gas stream emerging into a 1-inch diameter bulb inserted into the path of the other stream. The design of this bulb is not very critical, since the turbulence a t the mixing point is an effective aid in equilibration. VARIATIONS IN DETECTOR EFFICIENCY
The two most common causes for variation in counting efficiency of a gas mixture flowing through a proportional counter are: coincidence losses at high count rates; and macroscopic changes in the gas composition during the passage through the counter of a separated component. The change in counter efficiency can arise from the introduction of a gas which interferes with or even kills the porportional counting action of the gas, for example by electron attachment as with 0 2 , or more frequently, from gross alteration of the location of the counting plateau. An appropriate procedure for controlling such efficiency variations is described below. The necessity for accurate control over the composition of the gas mixture requires SI constant flow rate from the chromatograph column, This effectively limits chromatographic operation to constant temperature columns without a much more elaborate gas mixing system. PLATEAU CHARACTERISTICS
The location of the plateau in counting response us. voltage applied to the counter depends strongly on the composition of the gas mixture flowing through the counter. The plateaus obtained with various CHpHe ratios in our standard flow counter are shown in Figure 3, together with one C3H8-He mixture for comparison. Pure methane gives a n identical plateau for all flow rates up to 200 ml. per minute; at higher flow rates, turbulence begins to interfere with optimum counting response at the higher voltages. At the relatively low counting rate of 16,000 c.p.m., all plateaus behave normally and rise very slightly until going into discharge. Higher CH4/He ratios lengthen the plateau and a t the same time raise both the threshold and the plateau to higher voltages. The plateaus obtaincd with similar counters operated on individual gas fillings, rather than under flow conditions, showed very little difference in response to an internal tritium filling and to external radium or -“;az2 sources (6). The differences that were observed were primarily in the response of the counter
near the threshold and well below the plateau. Results obtained with these external radioactive sources are then assumed to be applicable for internal tritium activity. At higher rates, the plots of count rates us. applied voltage no longer give the flat plateau obtained with low count rates. The resolving time for these gas mixtures increases markedly with increasing applied voltage, especially for low CH4/He ratios, resulting in heavy coincidence loss at the higher voltages and a dip in the observed count rate. iit 2 X lo5 d.p.m., the count rate tis. voltage now exhibits a peak rather than a plateau. The observed count rate falls off steadily until discharge begins, as shown in Figure 4. The propane-helium mixture exhibits the same characteristics, but the falloff in count rate us. voltage is less severe, as shown in Figure 5 . The resolution times, as measured by the method of matched radioactive source (S), for the gas mixtures of Figures 4 and 5 are shown in Table I. Tracer quantities of other gases do not affect the macro counting characteristics of the alkane-helium mixtures. However, Figures 6, 7 , and 8 show the change in response of a flow counter to a fixed external source during the passage of large mass peaks of air and ethylene. The time lag of the minimum count rate after the maximum thermal conductivity represents the time interval in the flow system between the two detecting devices. The similarity in percentage reduction of count rates in Figures 6 and 7 , and Table 11, for count rates differing by a factor of 10, demonstrates that the effect of the gas in the mass peak is not a n increase in the resolving time of the counter, but an actual reduction in the counting efficiency. The hydrocarbon in Figure 8
Table 1. Resolving Times for AlkaneHelium Mixtures in Gas Proportional Counting
Voltage Voltage 2900
2950
3000 3050 3100 3200 3300
Resolvine Time,” Microseconds ’ MethanePropanehelium helium 0.,53 i 0.05
... ...
3 9 i0.4 12
13
3400 __.. 3500
+1
=t1
0 15 2 ’ 0 . 0 2 0.16 f 0.02 0.19 i 0 . 0 2 0.22 f 0.02 0.18
+ 0.02
0 24 i 0 02 0 65 f 0 05 3700 0 89 f 0 08 3800 a Resolving time determined for gas flow mixture of alkane flowing at 37 ml. per minute and helium flowing :tt 21 ml. per minute, by the method of matched
3600
sources.
VOL 34, NO. 7, JUNE 1962
743
'0' SCALE
A SCALE
I
L
E
SOURCE
x IO'
I
I 2300
/ /
// MOO
2500
4000
3500 VOLTAGE
Figure 5. Count rate vs. voltage plateaus for CSHs-He mixtures a t different court rates Flow rates (ml. per minute)
Table II. Percentage Loss in Counting Efficiency, Averaged over 2-Minute Passage Time for Macroscopic Peak
Percentage Loss At 15,000 At 190,000 c.p.m. c.p.m.
Sample Oxygen 36 37 Nitrogen 28 33 Ethylene 32 30 23 Propylene 22 9 Ethane 8 Flow rates: 37 ml. per minute of methane: 21 ml. per Ipinute of helium; 10-ml. STP samples used for each gas.
COUNTING EFFICIENCY THROUGH AIR
50'
DIMETHYLSULFOLANE
M1hu-E5
AFTER SAMPLE IYJECTlON
Figure 6. Counting efficiency of counter during passage of air peak, low count rate.
I:
Cans He
did not interfere as much with the counter efficiency, in part because the instantaneous impurity concentration does not get as high in the counter as during the passage of the air peaks. Large, sharp hydrocarbon peaks are quite capable of causing a substantial
i I2
Flow rates (mi. per minute)
CHI He
40'1 20
reduction in efficiency as shown later in some detail with methane. The actual counting loss registered in the passage of various compounds through the counter is shown in Table 111. For these measurements, the voltage was set at the beginning of the count rate plateau obtained with a 2 X lo5 c.p.m. source. KO chromatographic separation was performed in the comparisons of Table 11 and 111, so the macroscopic peaks all possessed approximately the same shape as they passed through the counter. K i t h the exceptions of oxygen and methyl iodide, which probably quench by electron attachment, none of the gases had very much effect on the counter operation with the propane-helium mixture despite the large quantities of gas involved. The other gases are primarily just changing the gas mixture, moving the plateau to slightly
MASS PEAK
COVNTING
50'
COLUMN
I
higher voltages, and decreasing the counting efficiency and number of counts observed. Although no data are s h o m here, the effect of mas5 peaks on counting efficiency becomes much more pronounced for loner alkanehelium ratios. The average counter efficiency for the passage of a 10-ml. S T P peak of a radioactive compound cannot be estimated from Table 111, for the peak in expected count rate nould then correspond exactly to the minimum in the counter efficiency. A double-humped peak is quite possible n hen the counter efficiency is decreasing more rapidly with change in gas composition than the rise in actual amount of radioactivity present in the active volume of the counter. More often the loss of efficiency merely shortens the radioactive peak in comparison n-ith the recordd mass peak.
E i F C I E Y C Y THROUGH CzH+ M A 5 5 PEAK DIMETWLSJLFOLANE
COLUMN
20
:
C P M X IO'
i
ID
-
I2
I4
16 MINUTES AFTER SAMPLE INJECTION
i
0
0.5
I8
Figure 7. Counting efficiency of counter during passage of air peak, high count rate
6
I8
744
ANALYTICAL CHEMISTRY
22
Figure 8. Counting efficiency of counter during passage of ethylene peak Flow rates (ml. per minute)
Flow rates (ml. per minute)
cH4 He
20
MINUTES AFTER SAMPLE INJECTION
cH4 He
401
20J
03
Table 111. Variation of Counter Efficiency during Passage o f Macroscopic Mass Peaks through Proportional Counter
___ _
w
2iOo
k3m
A50
loi-nrr
3 . CPU ' ,:;4
A \ :
RATIO
0;
B
3
21
7o-L WL'.'S LCS*
Counts Lost during Passage of Peak = Normal Counts in 3 Minutes minus Observed Counts during Passage of Macroscopic Mass Peaka hlethane- PropaneCompound helium helium Oxygenb 165,900 141 900 Kitrogenb 150, 400 5 io0 Ethyleneb 140,400 7 500 Propyleneb 101,100 i 800 Butene-lb 57 800 Kot measured Ethaneb 43 600 - 600 Propaneh 36 500 100 Methyl chlorideb 35,900 3,100 Methyl iodidec ... 149 300 ... 104,000 Ethyl iodidec Ethyl bromidec ... 2 ,000 Dichloro... < 100 methanec a All values are accurate to f1000. * Flow rates: alkane 37 ml. per minute; helium 21 ml. per minute. Sormal count rate: 196,000 c.p.m. Samples passed through 25 inches of '/,-inch polyethylene tubing, but not through any separating column. Sample size: 10 ml. of each gas, STP (about 20YGof total gas flon- at peak of xnacroscopic trace). Flow rates: propane 46 nil. per minute; helium 26 ml. per minute. Normal count rate: 198,000 c.p.m. for methyl iodide; 210,000 c.p.ni. for all others. Samples passed through a lo-foot, tritolyphosphate column held at 100" C. Sample size: 3 nil. of the vapor of the molecules at room temperature.
~-
zoo0
3500
-
73
oz
LOUITS CPOH
~
0
,
'
S3"QCL
d :/
,:o'
c'l; 1
42
; ( _I,
/#%"
,;
,/a
O X HlN--E
3061) VOLTS
,
1
I
,3150 VOLTS
~
Figure 9. Count rate vs. voltage plateau shapes at different count rates
~
~ - 3 2 5 C VOLTS
~
~
Flow rates (ml. per minute)
C3Hs He
1 3 3 5 3 VOLTS
~
43 251
2
~
rn,?
The prevention of significant variations in the efficiency of detection of the gna-proportional counter largely resolves itself into avoiding losses through changes in efficiency with composition, and coincidwce losses. The focus of such control lies especially on highly radioactive and/or very bharp chromatographic peaks, and the appropriate counting conditions should be chosen n ith respect to their possible occurrenw. The coincidence losses can be minimized by operating the counter near the beginning of the plateau in the region of shortest resolving times. C'onditioni are quite favorable over a considernbk voltage range for propanehelium f l o s mixtures. On the other hand, the composition changes in the counter n i t h the passage of a large macro peak normally substitute the impurity for some of the helium carrier gas, n i t h a resultant increase in the organic/heliuni ratio. -4s this mill raise the plateau to a higher voltage, a drop in efficiency \\ill be observed. The efficiency drop is most seTere, of course, if the plateau for the new gas mi.;turt l i v ahoi c t h c chosen operating C O L U M U 50 3 K 5 de4 FLOW RATE 2 5
TEMPERITvRE
20
CLI+ 8YJiCTED
Figure 10. Variations in counting efficiency during passage o f CHI peak as function of applied voltage
~
MINIMIZING COUNTER EFFICIENCY VARIATIONS
15
lo
~
counting efficiency is not necessarily linear with the amount of impurity gas. The counting efficiency is practically unaffected by mass peaks of 1 ml. STP (for oxygen, the loss is about 1%) or less, under these operating condit ions. The effect of a CH, macroscopic peak on the counter efficiency toward an external source is shown in Figures
Table IV. Variation of Counting Efficiency with Amount o f Oxygen in Macroscopic Mass Peak
voltage. Operation at voltages well above the "knee" of the plateau makes it much less likely that composition variations nil1 throw the instantaneous plateau above this voltage. The changes in counting efficiency in Figures 6-8 and Tables 1-111 are exaggerated compared to the usual errors involved in a n analysis, since most observed mass peaks are much smaller than 10 ml. STP. Furthermore, a. shown in Table IT', the change in
Volume Counts of Air Registered in Sample, in 3 Minutes M1. at through STP Mass Peak 0
Count Loss ( I!C 1000)
.561.6OO
;
547 600 499,300 405,400 15 336,400 Flow rates: 37 ml. per minute 21 ml. per minute of helium. 2
5 10
COLvMh m~/m-
17,000 65,300 159,200 228,200 of methane;
10' SmLICA GEL 45 mI/mn 78' C
FLOW R A T E TEMPERATURE
He.
24' C U U
0
p
1
I 'to
-._
NO
f2
5.4 f0.2
}
)57f2
COUNTING VOLUME AND RESIDENCE TIMES
1
1179
)
3Z
2
4.9 i0.3
0.8 f0.1 0 . 2 f 0.1
0 . 2 3Z 0 . 1 5 7 . 6 =?= 1 . 0
57.9 f 0 . 7
1
c
I f I
...
\C/
Dd
?f
1.4 f0.4
I
c
i
injected. The methane peaks came through completely in about 1 minute in these experiments. These data show that the loss is strongly dependent upon the voltage at which the measurement is made. The loss is much larger at the lowest voltage, 3060 volts, which is just at the beginning of the plateau in Figure 9. No diminution of the observed count rate is seen at 3350 volts. The comparative data for different count rates at 3060 and a t 3250 volts both show that the fractional loss is nearly the same, but slightly higher for the higher count rates. Counter operation at 3350 volts would avoid changes in efficiency during the passage of a large CH, peak, and a t the same time have only a small coincidence loss a t 105 c.p.m. and above.
Radiochemical Analysis of Separate Aliquots on Several Chromatographic Columns
HT CHST
182 & 4
1
0.7 f0.1
I
c/ c'
c-c-c-c c-c-c=c
32
f2
...
C
\
...
Y-c-c C c
\cy
c \c
C
16.2 f 0 . 4
i I
105. 64
...
30.2 i 0 . 7 15.6 f 0.5 5.6 f 0.6
10oe
) 4 4 f 2
...
106e
C
\
/
c=c
...
5.8 f0.3
6.7 f0.7
c-c=c
...
0.2 f 0.1
0.3 f 0.1
C \ C
I
/
...
Eight foot, S.G.; 83" C.; He, 10 lb.; 1.64 ml. per second. 24' C.; He, 20 lb.; 1.16 ml. per see. Fifty foot, Safrol; 0" C.; He, 30 lb.; 1.67 ml. per second. Eight foot, Narcoil; 20" C.; He, 10 lb.; 1.67 ml. per second. 6 The observed radioactivity from this column is normalized to the indicated figure in this column. a
* Fifty foot, D.M.S.;
746
ANALYTICAL CHEMISTRY
in Figure 10 us. the amount of methane
Another practical problem of some importance involves the optimum counting volume of the flon proportional counter and the closely related choice of gas flow rates. The most important considerations involved in these choices are as follows: The sensitivity of detection by a counter is directly proportional to the average residence time of a molecule in the counter. Seglecting the increase in the background activity and then doubling the size of a counter doubles the sensitivity a t constant flon rate. The resolving power of a proportional counter for neighboring peaks is closely related to the average residence time of the molecules. In general, separate peaks that are closer together in time than the residence time will not be resolved in the radioactivity measurements. The counter size is then chosen in accord with the relative desirability of increased resolution and increased sensitivity. On occasion, we have operated a small and a large counter in sequence in order to have both resolution in the early peaks and sensitivity in the later measurements.
~~~
Table VI.
' p 4 ; 000 14,700 7,200 151,000
c-c c-c c-c-c
j1.000 0.035 0.017 0.356
Silica Gel Ratio Counts 0,954 45,200 0.046 2,200 0.037 1, 740 0.017 820 0,357 16,900
Graphed in Figure 11.
[-
50'
CPM
Reproducibility of High Count Rates Observed in Separate Aliquots
Dimethylsulfolane" Counts Ratio
Peak HT CH,T
a
~
ti* - CH, PEAK SIMETHYLSULFOLANE
I ~
I MINUTE
Figure 15. 106 c.p.m.
-
Flow counter operation at
For a fixed helium flow rate from a particular gas chromatographic arrangement, a n increase in propane flow rate gives better counter characteristics, in terms of coincidence losses and constant counting efficiency, but reduces the average residence time. It is usually possible to choose a set of chromatographic conditions for analysis, under nhich the presence of macroscopic amounts of separated components in the normal gas mixture has a negligible effect on the operation and counting efficiency of the proportional counter, and the coincidence losses are less than the normal statistical fluctuation in observed counts. Our present counting arrangement utilizes a propane/helium ratio of about 1.8, and furnishes the alternate choice of either a 20-nil. or an 85-ml. counter to permit some adjustment in residence time for different chromatographic columns and separation conditions. The radioactivity measurement as a function of time from three such columns is shown in Figures 11 and 12. The agreement betn-een the peak shapes of a macroscopic mass measurement and the radioactivity assay is shown in Figure 13. These peak shapes need not be identical, for the radioactivity measurement is broadened since it is measured Over a Period of time from 10 seconds to 2 minutes, depending on flow con-
ditions, while the mass measurement is relatively instantaneous. .Ilso, the radioactivity measurement may also be broadened further by mixing in the counter itself. The agreement of shapes in Figure 13 indicates that the stream of radioactive gas flows rather uniformly through the counter. Under some circumstances, it is possible to check the radiochemical purity of a compound by determining whether the specific radioactivity is constant through the entire peak. I n such a comparison, care must be taken that the isotopic compounds have identical retention times. When the separation conditions are chosen correctly, good separations of isotopic molecules are obtainable as shown in Figure 14 for the isotopic hydrogen molecules, HT and DT. REPRODUCIBILITY A N D SENSITIVITY
A rather severe test of the reproducibility of the over-all counting arrangement is given b y the agreement between separate aliquots of one sample run on the same, or different, separation columns. The agreement obtained for a set of four aliquots on four separate columns is shown in Table V. Another test of reproducibility at high count rates is shown in Figure 15 and Table VI. The maximum count rate recorded in Figure 15 is lo6 c.p.m. (for a 5-second period) ; the agreement between the two aliquots shows t h a t no substantial coincidence loss occurred under these conditions. The data shown in the last two tables are illustrative of the accuracy obtainable with this type of apparatus with suitable control over the proportional counter operation. The background counting rate for the 20-ml. counter is about 40 c.p.m., and radioactivity peaks with 50 counts above background in 1 minute are readily detected. The accuracy of the assay is usually limited by t h e statistical variation until lo4 or more counts have been recorded in a peak. LITERATURE CITED
(1) Cacace, F., Nucleonics 19, No. 5, 45 (1961).
( 2 ) Evans, J. B., Willard, J. E., Chem. SOC.78,2908 (1956).
J. Am.
(3) Friedlander, G., Kennedy, J., "Nuclear and Radiochemistry," p. 265, Wiley, New York, 1959. (4) Gordus, A. A., Sauer, M. C., Jr., Willard, J. E., J . Am. Chem. SOC.79, 3284 (1957). ( 5 j Kokes, R: J., Tobin, H., Jr., Emmett, P. H., I bid., 77,5860 (1955). (6) Rowland, F. S., Lee, J. K., White, R., Oklahoma Conference, TID-7578,U. S. A. E. C., p. 39, 1959. ( 7 ) Wolf, A. P., Stocklin, G., Brookhaven Kational Laboratory, Upton, L. I., N . Y.. urivate communication. 1962 (8) Wolf$ang, -R., MacKay, ' C. 'F., Nucleonics 16, No. 10, 69 (1958). (9) Wolfgang, R., Rowland, F. S., ANAL. CHEM.30,903 (1958). RECEIVEDfor review October 4, 1961. Accepted March 21, 1962. Division of Analytical Chemistry, 137th Meeting, ACS, Cleveland, Ohio, April 1960. Research was supported by AEC Contract No. AT-(11-1)-407, and by Contract No. AF-19( 604)-4053 with the Geophysics Directorate of the U. S. Air Force.
Corrections Gas Chromatographic An a lysis of Aromatic Hydrocarbons at Atmospheric Concentrations Using Flame Ionization Detection I n this article by A. P. Altshuller and [ ~ ~ N A LCHEM. . 34, 466 (1962)], on page 471, Table 111, a chromatographic band immediately following o-xylene m-as identified as npropylbenzene. Although the identification is correct, styrene has a retention time indistinguishable from npropylbenzene on the column used. I n analyses made during the course of the photooxidation of auto exhaust this band decreased very appreciably. Such a degree of reactivity would be unusual for n-propylbenzene judging from other studies of the aromatic hydrocarbonnitrogen dioxide photooxidations. Consequently, it appears that most of the peak area is more properly associated with the more reactive styrene molecule.
C. A. Clemons
Determination of Chromium and Copper on Trimetallic Lithographic Printing Plates In this article by Rubin Shapiro and K. H. Colberg [ANAL.CHEM. 34, 435 (1962)], on page 435, column 3, line 29, 0.1% starch solution should read 1% starch solution. VOL. 34, NO. 7, JUNE 1962
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