size. The minimum sample consistent with the accurate measurement of errors due to losses was 6 fig. Although 5 fig. is the minimum sample load consistent with the accurate measurement of steroid losses this by no means determines the minimum detectable quantity, which for this dptpctor is 1 0 3 grams per second ( 5 ) . Two groups of stereoisomers were also chromatographed several times and the average results for percentage recovery related to structure are noted in TabIe 2. It is apparent that those steroids with the C3 hydroxyl equatorial configuration are recovered in lower yields than those with the Cs axial hydroxyl configuration. Several factors are probably involved in causing this effect, although the A/B cis and A/B trans arrangement appears t o have little effect upon the results. However i t is clear that small structural differences can profoundly affect the recovery of a steroid from the chromatograph column. It would appear t h a t the column is
the most likely site of sample losses, and that these probably occur by adsorption onto the so-called “inert support.” However this may be reversible since the very slow elution of adsorbed material would not give rise to a signal from the detector great enough t o be distinguished from drift or low frequency noise. With certain steroids thermal decomposition may occur, but below 235’ C. these losses should be small. Oxidation is yet another possible cause of losses. Preliminary evidence indicates that where the carrier gas was deliberately contaminated with 1% oxygen, relative losses were increased slightly, but no further investigation has been made. ACKNOWLEDGMENT
The authors express their estreme gratitude t o W. J. -4.VandenHeuvel and E. C. Horning for their valuable guidance and advice concerning
the chromatographic techniques scribed in this communication.
de-
LITERATURE CITED
( 1 ) Bloomfield, D. K.,
J. Chromatog.
9,
411, (1962). (2) Horning, E. C., Luukkainen, T., Haahti, E. 0. A., Creech, B. G., VandenHeuvel. W. J. A., “Recent Progress in Hormone Research,” G. Pincus ed., Vol. XIX, p. 57, Academic Press, New York, in press. (3) Horning, E. C., Maddock, K. C., Anthony, K. V., VandenHeuvel, W. J. A., ANAL. C H E 35, ~ 526 (1963). (4) Horning, E. C., VandenHeuvel, W. J. A., Creech, B. C ‘‘Methods of Biochemical Analysis,””D. Glick, ed., Vol. XI, Interscience, New York, 1963. (5) Lovelock, J. E., Shoemake, G. R., Zlatkis, A., ANAL. CHEN. 35, 460, (1963). (6) Sweeley, C. C., Chang, T., Ibid., 33, 1860 (1961). (7) VandenHeuvel, W. J. A., Creech, B. G., Horning, E. C., Anal. Biochem. 4,191 (1962). RECEIVEDfor review April 30, 1963. Accepted July 9, 1963.
Continuous Elemental Analysis of Gas Chromatographic Eff I uents Application to the Analysis of La beled Compounds FULVIO CACACE, ROMANO CIPOLLINI, and GlORGlO PEREZ Institute of Pharmaceutical Chemistry, University of Rome, Rome, Italy
b A technique i s described which allows the continuous elemental analysis and radiometric analysis of C’4-labeled compounds separated by gas chromatography. The substances emerging from the column are converted into COz and Hz, which are separated on an auxiliary column. From the ratio of the areas of the H?and COz peaks, the H:C ratio in each substance is deduced. The activity of the COzpeak is assayed by a 100-ml. flow ionization chamber or a 100-ml. internal flow proportional counter, depending on the specific activity of the sample. The method may be employed to determine the total activity in a given compound and its specific activity in one operation. Because of the separation of the H and C contained in each compound, the method shows considerable promise for simultaneous C14 and H3 assay in doubly labeled molecules.
A
has been developed recently for the continuous elemental analysis of volatile organic compounds emerging from a gas chromatographic column (3, 4). TECHNIQUE
1348
o
ANALYTICAL CHEMISTRY
According to this method, the effluents from the column are introduced into a reaction tube, where the organic compounds are quantitatively converted t o hydrogen and carbon dioxide. An ausiliary column, placed before the detector, separates the hydrogen from the carbon dioxide. Therefore, each compound produces in the chromatogram two peaks, due t o Hz and COZ, respectively; from the ratio of their areas, it is possible t o determine the H: C ratio in the empirical formula of a given compound. The method has proved useful in the identification of unknown peaks, by providing a characteristic parameterthe C:H ratio-for each eluted substance. I n addition, the method makes possible a form of elemental analysis for small amounts of volatile compounds (20 fig. t o 1 mg.) in a complex misture, by eliminating preliminary separation and purification. The present paper deals with the extension of this technique t o the quantitative analysis of C’L and H3-labeletl compounds separated by gas chromatography.
Both flow ionization chambers ( 6 4 ,
IS, 16-18) and proportional counters (9, 11, 14, BO) have been employed for the measurement of radioactive effluents, which have been, in many cases, directly introduced into the radioactivity detector. This approach is satisfactory for the analysis of gaseous hydrocarbons and other substances of low boiling point. However, t h e detector has t o be heated for the analysis of high boiling compounds, and the high temperatures necessary to prevent their condensation adversely affect the performances of both the ionization chamber and the proportional counter. I n addition, many types of organic compounds “poison” the proportional counter, impairing its efficiency and making its use impossible for precise quantitative analysis, unless elaborate precautions are employed ( 1 ) . The troubles arising in the high temperature operation of the radioactivity detectors are eliminated by combustion techniques (6, 12, 1 9 ) , involving the conversion of the eluted compounds into COz, which can be counted at room temperature. Re-
r--------1
perature and connected to a second detector for the quantitative measurement of the C02 and HZ produced in the combustion. The output of both the thermal conductivity cells is fed into two potentiometric recorders (Leeds and Northrup Co., Speedomax Model G) e uipped with electronic integrators Erba Co., Model 62). A mixture of He and Nz (about 5050 by volume) is employed as carrier gas. J t shows approximately the same sensitivity for H2 and COz and gives peaks of opposite polarity. The a m o u t of each compound injected into the combustion tube is kept below 0.1 mg. by proper operation of the four-way stopcock. The flow rate in the reaction tube, and therefore in the dimethylsulfolane column, is regulated to about 2 to 3 liters per hour, t o ensure a quantitative conversion. The apparatus is calibrated by injecting separately equal volumes of H? and Cot, the ratio of the areas under the peaks being employed as a calibration factor. The quantitative combustion of the organic samples is also occasionally checked by injecting a fixed volume of pure methane-or some other suitable hydrocarbon--and coniparing the areas of the resulting CO? and H2 peaks with those obtained by injecting the same volume of pure hydrogen and carbon dioxide. The radiometric analysis of the effluents can be performed with a flow ionization chamber or an internal flow proportional counter. JT7he11 the former detector is employed, the gases leaving the chromatograph are diluted with nitrogen, before being introduced into a 100-ml. flow ionization chamber, connected with a Cary Model 31 vibrating reed electrometer. The dilution with nitrogen is regnlated by a precision needle valve and the total flow rate through the chamber is maintained a t the standard value fnr which the chamber has been cnlibrated ( 7 ) . When a flow proportional counter is employed, methane is used to dilute the effluents from the gas chromatograph, in the ratio of 10 to 1. The small Phanges in this ratio which may occur in a series of analyses do not affect the plateau of the counter.
(8.
13
Figure 1 . 1. 2. 3. 4. 5. 6.
7.
a.
9. 10. 11. 12. 13. 14. 15.
Schematic diagram cf apparatus Gas from carrier gas supply Pressure-regulaicw valve Rotameter Gas-injection system Liquid-inlection system Separation column Four-way Teflon stopcock Thermal conductivity detector Thermostatic chamber Reaction tube Auxiliary dimethylsulfolane column Thermal conductivity detector Counting gas supply Radioactivity detector Soap film flowmeter
cently similar methods have been suggested for the analysis of tritiated samples (3, 19). I n all the techniques previously described the gases arising from the combustion of the lai3eled molecules are directly counted, mithout preliminary purification. Thcrcfore, combustion products such as hnlogrns, and nitrogen, and sulfur oxides, which are known to be effertive “poisons” for the proportional counter, even in traces (IO),are likely to be introduced into the detector with the C1402or the tritiated hydrogen. Sinre the continuous elemental analysis of the effluents involves. as a necessary step, the separation on a suitable column of the HI and C 0 2 produced from the combustion of the eluted compounds. its app1ic:ttion to the radiometric analysis of .,he effluents ensures that the radioactivity detector is supplied with Hf and COz subjected to a rigorous gas chrorr atograpliic purification and therefore free from traces of unwanted combustion by-products. The preliminary C :H analysis of the labeled compounds promises the additional advantage that, since the amount of carbon (or hydrogen) contained in a given substance n a y be directly deduced from the area of the corresponding C02 (or H2) peak, the specific activity of the substance can be measured together with its tctal activity in one operation.
U-shaped stainless steel columns and a standard thermal conductivity cell. T h e length and the size of the column, its stationary phase, the operating temperature, and the carrier gas flow rate are chosen according to the sample being analyzed. A four-way Teflon stopcock is inserted between the outlet of the column and the detector, within the oven of the unit and, in its normal position, allows the effluents from the column to enter the detector, thus giving a conventional chromatogram. By turning the stopcock, a suitable “slice” of a peak (or, if desired, the entire peak) may be introduced into the combustion tube (4), which converts the nrganic compounds into 112 and COz. The use of the stopcock is necessary to supply the reactor with a sharp slice of each compound, since the combustion of broad or poorly resolved peaks makes it difficult to resolve the resulting COz and Hf. The separation is carried out by means of a stainless steel coluinn (8 meters long, 4 mm. in i.d.), packed 11 ith 33% dimethylsulfolane on 20130mesh firebrick, operated a t room tem-
EXPERI,WENTAL
Apparatus and Procedure. The apparatus employed is shown in Figure>. The sample is introduced with a Hamilton microsyringe or a calibrated gas s a m d e r into a C. Erba Co. Model gas chromatograph, equipped wit11
u
TIME
Figure 2. mixture
Gas chromatographic separation of
four-component
Recorded b y first thermal conductivity detector
VOL. 35, NO. 10,
SEPTEMBER 1963
1349
ET1 IV I , ACETATE
TIME
Figure 3. Figure 2
Elemental analysis of all compounds in sample of
Given b y thermal conductivify cell placed after combustion tube. recorder reversed after elution of each peak
Table I.
Continuous Elemental Analysis of Gas Chromatographic Effluents Theoret. Av. exptl. Rel. std. No. of
Compound Toluene Cyclohexane Cyclohexene I -Propanol Methylcyclohexane 1-Heptane Isoheptane Benzene Isooctane Ethyl ether Ethyl acetate Table II.
Polarity o f
H: C rat,io
value
dev., %
detns.
1.14 2.00 1.67 2.67 2.00 2.29 2.29 1.00 2.25 2.50 2.00
1.14 2.01 1.66 2.66 2.00 2.25 2.27 1.00 2.25 2.47 1.9s
1.8 2.0 0.6 1.9 2.0 3.1 0.4 3.0 2.2 2.4
4 50 18 13 8 3 2 2 4 4 1
...
Simultaneous Elemental Analysis of All Componen!s of a Liquid Sample Theoret. Av. exptl. Rel. std. 3-0.of
Mixture Ethyl ether Methyl acetate Methyl acetate Ethyl acetate Methyl acetate Propyl acetate Ethyl ether llethyl aretstc Ethyl acetate Ethyl ether Methyl acetate Ethyl acetate Propyl acetate
H: C ratio
value
dev., %
detns.
2.50 2.00 2.00 2.00 2.00 2.00 2.50 2 00 2.00 2.50 2.00 2.00 2.00
2.49 1.99 2.06 1.98 2.06 2.04 2.46 1.96 1.9s 2.46 2.06 1.98 1.96
1.2 2.0
7
... ... ...
1
...
1.2 1.5 3.0
... ... ... ...
1
P i1
i'
El ~
I
i i
I
I iC
._________
(b)
~
ill7Ut"S
Figure 4. Response to radioactive CO, peak of a 100-ml. flow ionization chamber (a)and a 100-ml. proportional counter ( b ) ,connected in series
1350
ANALYTICAL CHEMISTRY
A stainless steel proportional counter, of the type described by Rowland and Wolgang (14, 202, having a sensitive volume of 100 ml., has been used in the present work. The counter is connected to a 4-kv. stabilized high voltage supply and to a precision ratemeter. Since the efficiency of both the radioactivity detectors depends on the flow rate, which may be affected by the elution of a peak from the column (16), a soap film flommeter is employed to determine the flow rate a t the moment a peak passes through the sensitive volume of the detecror. The outrmt of the electrometer. or the ratemeter,- is fed into a potentiometric recorder, Leeds and Torthrup Co. Speedomax Model G. The total activity of a compound is calculated from the area of the corresponding peak. I n addition, the counts from the proportional counter can be directly stored and registered with a simple, manually operated, integrator mounted in the ratemeter. RESULTS A N D DISCUSSION
Mass Analysis. TKOtypical records obtained in t h e analysis of it fourcomponent synthetic mixture arc shown in Figures 2 and 3. Figure 2 reproduces the record obtained from the thermal conductivity cell placed before the combustion tube; the record can be used in the conventional m y for the qualitative or quantitative analysis of the sample and clearly shows the portion of each peak injected into the reaction tube. The elemental analysis of the four components, as obtained from the cell placed after the dimethylsulfolane column. is illustrated in Figure 3. Tho rcsults of the carbon-hydrogen anal\-sis on the components of a numher oi synthetic mixtures are summarized in Tables I and 11. I t is apparent that the technique employed allows the determination of the H:C ratio in all the components of the sample, or in a single compound of interest, with a relative error lower than 3y0 for a single measurement. I n a series of determinations on the same compound the relative standard deviation was found to be below 3%. Since tht. combustion of 1 mole of the original compound generally produces several iuoles of C 0 2 and HPand the detector i i operated at room temperature, tlir qcnaitivity of the method is relatively high: satisfactory analyses have been obtained using about 20 pg. of hydrocarbons. Radiometric Analysis of el4Labeled Compounds. The most widely employed detectors for the radiometric analysis of gas chromatographic effluents are the flow ionization chamber and the proportional counter, whose relative merits ha\ e been diqcuwed ( 2 ) . Their performances
~~
Table 111.
~
~~
~~~
~~
SimlJltaneous Elemental and Radiometric Analysis of C1*-Labeled Esters by Ionization Chamber Method
Calcd.
Found
SIg. Ca
Activit v peak areas, sq.
I
2 .oo
I1
2.00
111
3.00
2.06 2.06 2.06 2.02 1.98 1.98 2.02 1.98 2.04 2.02 2.06 1.98 2.10 1.94 1.94 1.98 1.98 2.00 1.98 1.98 2.02 2.04 1.98 2.00 2 .oo 1.96 1.96 2.00 1.94 2.02 2.02
0.97 1.01 1.38 1.29 2.03 2.45 1.04 1.50 1.69 1.56 1.60 0.95 0.90 1.35 0.40 0.43 0.37 1.36 1 .so 2.27 2.08 1.99 2.20 1.71 1.78 4.43 3.74 2.53 3.15 2.24 2.62
13.0 13.7 19.1 17.3 28.3 34.3 14.0 19.2 21 . 7 20.2 20.8 7.6 7.0 11.4 3.2 3.2 2.0 29 .o 41.5 49 . O 46.3 42.2 48.9 36.7 37.6 37.0 29.9 19.5 23.8 18.2 19.8
H: C ratio Compound Methyl acetate
Ethyl acetate
2.00
.\ctivity, mpc.c 58.5 61.7 86 .O 77.9 127.3 154.4 63 . O 86.4 97.7 90.9 93.6 34.2 31.5 51.3 14.4 14.4 13.1 130.5 186.8 220.5 208.4 189.9 220.1 165.2 169.2 166.5 134.6 87.8 107.1 81 . 9 89.1
Specific activity, mpc. per mg. Cd Calcd. Foynl 59.9 f 0 . 6 59.9 f 0 . 6 59.9 f 0 . 6 59.9 f0.6 59.9 f 0 . 6 59.9 f 0 . 6 59.9 f 0 . 6 59.9 i 0.6 59.9 f 0 . 6 59.9 f 0 . 6 59.9 f 0 . 6 35.7 1 0 . 4 3 5 . 7 =k 0 . 4 35.7 f 0 . 4 35.7 f 0 . 4 35.7 i0 . 4 3 5 . 7 i0.4 97.6 1 0 9 97.6 f 0 . 9 97 6 f 0 . 9 97.6 f 0 . 9 97.6 i 0 . 9 97.6 i 0 . 9 97.6 i 0 . 9 97.6 i 0.9 35,s f0.4 35.5 i 0 . 4 35.5 f 0 . 4 35.5 i 0 . 4 35.5 f 0 . 4 35.5 i 0 . 4
Rel. dev. from known, % 0 .7 2.0 4.0 0.8 4.7 5.1 L .2 3.8 3 .5 2.7 2.3 0.8 2.0 6 .4 0.8 6.2 0,8 1 .6 I .2 0 .5 2.6 i! . 2 2.4 1 .o 2.3 5 .Y 1.4 2.2 4.2 3.1 4 .2
60.3 61.1 62.3 60.4 62.7 63 . O 60.6 57.6 57.8 58 . 3 58 . 5 36,O 35 . 0 38,O 36 . O 33.5 354 : 96 0 98.8 97.1 100.2 9; . 4
100.0 96.6 95.1 37.6 36.0 34.7 34.0 36.6 34 .o
a From CO, peak area. b .411 values referred t o 10-mv. scale of electrometer with imput resistor of 10" ohms: areas corrected t o actual value of flow rate measured a t outlet of chamber during passage of peak. Standard flow = 18.0 liters per hour. c Values calculated from previous column, using: calibration factor 1 sq. cm. = 4.50 mpc., obtained from C1402standard. Specific activity determined by standard combustion procedure of rompound and assay in static ionization chamber.
Table IV.
Simultaneous Elemental and Radiometric Analysis of C14-Labeled Methyl Acetate by Proportional Counter Method
H: C ratio Compound SIethgl acetate
I
Calcd.
Found
hIg. 0
2 00
2.10 2 .oo 2.04 2.06 1 94 2 .oo 2.00 1.94 2.04 2.04 2.02 2 .00 2.00 1.92 2 .00 2 04 2.06
0.76 1.59 1.06 1.60 1.95 1.82 1.22 1.57 1.59 3.87 2 .97 3.52 3.74 3.76 5.20 3.76 3.60
2.00
0.60
2.02 1 .98 2.04 2.02 2.06
0.75 1.04 0.71 0.75 0 . -58 0.6G 0.82 0.7%
Activity peak area, sq. cm.6 9.40 19.52 13.43 19.33 23 .09 22.95 15.49 20.94 20.82 5.51 4.44 4.96 5.32 2.04 i .41 5.4.i 4.93 70.77 89.13 120.18 86.79 90.15 69.42 78 .33 94.89 85.17
Activity, mpcC 6.40 13.47 9.27 13.34 15.93 15.83 10.69 14.45 14.37 3 .8n 3.06 3.42 3.67 3.47 5.11 3.76 3.40 48.83 61 .v50 82.92 59.88 62.20 47,on 54 , 0 4 65.47 58.76
Specific activity. per mg. Cd-_ Calcd. Found
-mwc. __
8 . 6 0 i 0.04 8.60 f 0.04 8.60 f0.04 8.60 i0.04 8 . 6 0 f 0.04 8.60 f 0.04 8 . 6 0 f 0.04 8.60 i 0.04 8 . 6 0 i 0.04 0.97 i 0.02 0.97 f 0.02 0 . 9 7 i 0.02 0 . 9 7 f 0.02 0 . 9 7 f 0.02 0.97 i 0.02 0.97 f 0 . 0 2 0 . 9 7 =k 0 . 0 2 81 . 0 -Ir 0 . 5 8 1 . 0 i 0.5 81.0 i0 . 5 R l 0 f 0 5 81 0 i 0 ri 8 1 0 f 0 .i SI . 0 1 0 . 5 111.0 f 0 . 5 81 .o f 0 . 5
Hel. dev. from known, r. /c
8.54 8.47 8.74 8.34 8.17 8.70 8.76 9.20 9.04 0.98 1.03 0 97 0.0X
0.7 1 5 1 6 3 0 5 0 1 2 1 9 7 0 5 2 i n 6 2
0 .n2
5 2 10 31 3 1 0 5 1 2 1 6 4 1 2 3 2 .0
n.w 1 .oo
0.9-4
81 . 4 82 0 79.7 843 82 8'1 t i 81.9 7!LS 81 .ci
no 1 .o
1.1 1 5
0.7
Froiii COLpeak area. * All values referred t o 100 counts per second scale of ratemeter; areas corrected for actual value of flow rate. Standard flow rate = 19.0 liters per hour. c Values calrulated from previoiis column, using d i b r a t i o n factor I sq. crn = 0.69 mpc., obtained from C1402si andard. Slmific activity of snrnple determined by a stmdard comblistion 1)roceduie on swrnIJle and assay in btatic ionization cham1)er. J
VOL. 35,
NO. 10, SEPTEMBER 1963
1351
limited number of tritiated substances analyzed and the restricted range of specific activity employed make further investigation advisable, before the general value of the proposed method for the analysis of tritiated compounds may be fully assessed.
cuo,
ACKNOWLEDGMENT
Figure 5.
Simultaneous H3 and CI4 radiometric analysis
of doubly labeled ester
I t is a pleasure to thank Giordano Giacomello for helpful suggestions and discussions and Lucilla ltuffilli for several gas chromatographic analyses.
By flow ionization chamber LITERATURE CITED
have been directly compared in the present work by allowing a radioactive peak from the gas chromatograph to enter the two detectors connected in series and operated under a similar set of conditions. I n the experiment illustrated in Figure 4 the chamber and the counter had the same sensitive volume. The ratemeter driven by the counter was set a t a full scale of 100 c.P.s., with a time constant of 3 seconds. The electrometer, having a time constant around 2 seconds, was operated a t 10 mv. full scale, with an input ohms. The output resistor of 2 x of the electrometer and the ratemeter was fpd into two potentiometric recorders having the same chart speed and n idth. The records reproduced in Figure 4 leave no doubt that the proportional counter has a much better signal to noise ratio than the ionization chamber, and seems therefore the instrument of choice when low activity samples have to be assayed. On the other hand, the inherent ruggrdness and reliability of the chamber may be usefully exploited in the intermediate activity range, as demonstrated by simultaneous total and specific activity determinations, obtained with a 100-ml. chamber and summarized in Table 111. The relative error of a single determination of specific activity, which includes the errors from both mass and activity measurements, is generally below 5% for specific activities higher than about 20 mpc. per mg. of carbon. The relative error of a single determination of the total activity has been foiind to he qoinewhat l o w r , within 3y0 for a peak larger than about 20 mpc. The minimum activity detectable with
1352
ANALYTICAL CHEMISTRY
an unshielded 100-ml. chamber in a peak emerging 10 minutes after the injection is of the order of 1 mpc. Table IV summarizes the results obtained in the simultaneous elemental and radiometric analysis of CI4-labeled esters using a 100-ml. flow proportional counter, having a background around 150 c p.m. with a Zinch lead shield and a 95% efficiency for the C14 when eniployed as a static counter. I t s proportional plateau is not affected by the low concentrations of Hz and CO, in thP effluents The minimum activity detectable in a peak emerging 10 minutes after the injection is around 0.1 mpc. of c14.
Analysis of HS-Labeled and Doubly Labeled Compounds. Since the continuous elemental analysis of gas chromatographic effluents is based on t h e quantitative conversion of the hydrogen contained in a given coinpound into molecular hydrogen, the method can be applied to thf analysis of tritiated samples. I n addition, the separation of IIp and COz by the dimethylsulfolane column allows, in principle, the simultaneous determination of C14 and H3 in doubly labeled molecules. This feature of the method has been successfully tested by analyzing a few tritiated hydrocarbons and several doubly labeled esters. Figure 5 shows a typical record obtained in the radiometric analysis of H3-methyl acetate-2-C14, carried out with the flow ionization chamber. The results so far obtained show considerable promise for the extension of the propowd tcchnique to a precise quantitative analysis of tritiated and doubly labeled samples. However, the
(1) Ache, H. J., Herr, W., Thiemann, A., “Symposium on Chemical Effects of Nuclear Transformations,” Vol. 11, p. 111, Prague, October 1960. (2) Cacace, F., A7ucZeonics 19, No. 5, 45 11961). (3j Cacice, F., Cipollini, R., Perez, G., Science 132, 1253 (1960). (4) Cacace, F., Cipollini, R., Perez, G., Possagno, E., G&. Chirn. Ilal. 91, 804 (1961). ( 5 ) Cacace. F.. Guarino. A.. Inam-ul‘ Haq, Ann. Chim. (Rome) 50,k1.5 50, Q1,5(1960). ((6) 6 ) Cacace, F., Inam-ul-Haq, Ric.Sci. 30, 501 I i m-n-), 501 (1960). (7) Cacace, F., Inarn-ui-Haq, Science 131, 732 (1960). (8) Dobbs, H. E., J . Chromatog. 5 , 32 (1961). I1961 (9) Evans, J. B., Quinlan, J. E., Willard, J. E. Znd. Eng. Chem. 50, 192 (1958) ( I 0) Giascock, It. F., “Isotopic Analysis for Biochemists,” p. 109, Academic Press, S o w York, 1954. (11) Gordus, .4.A., Sauer, 31. C., ,Jr., Willard, J. E.. J . =Ini. Cheni. SOC.79. 3‘151 (1957). (12) James, -4.T., Piper, E. A,, J . Chromatoa. 5 . 263 f 1961). (13) lfasoh, I,. ‘H., hiitton, H. J., Bair, I,. R., Ibid. 2, 322 (1939). (11) Rowland, F S., Wolfgang, R., ANAL. CEIEM. 30, 903 (19.57). (1.5~Stocklin, G., Cacace, F., Wolf, A. P., Z . A mal. Chem. 194. 406 f 1963). (16) Tolbert, B. M., “Advances ’in Tracer Methodology,” Vol. I, p. 175, Plenum Press, Sew York, 1962. (17) Tolbert, 13. M., Univ. California, Bio\ -
organic Chemistry Quart. Rept. UCRL-
8457, 3 (1958). (18) Wilzbach, K. E., Riesz, P., J . Phys. Chem. 62, 6 (1958) (19) Winkelman, I., Karmen, A., AKAL. CHEM.34, 1067 (1962). (20) Wolfgang, R . , Mackav, C. F., Nucleonics 16, No. 10, 69 (1958).
RECEIVED for review February 6, 1963. Accepted May 13, 1963. Work performed
under the auspices of the Comitato ?;mionale per 1’Energia Nucleme ( C X E S ) . Two of t,he anthors (C. R. and 1’. G.) thank the Consiglio Xazionale delle Iiicerche (CXR) for a grant.
