bead packings. It seems to leave little doubt as to thc validity of this measurement. The value of C, the mass transfer coefficient, is not nearly so reproducible, as is to be expected from the difficulties of measurement at the higher velocities. h-evertheless, the data lend strong support to tile existence of a mass-transfer resistance from velocity distribution. The lorn value
of C for the nonporous Microbead particles provides additional weight to this argument. LITERATURE C I T E D
(1) Deemter, J. J. van, Zuiderweg, F. J., Klinkenberg, A., Chem. Eny. Sci. 5, 271 (1956). ( 2 ) Giddings, J. C., Robinson, R. A,, ANAL.CHEM.34, 885 (1962).
(3) Kieselbach, R., Ibid., 33, 23 (1961). (4)Ibi& P. go& ( 5 ) Kieselbach, R., “Gaa Chromatography,” N. Brenner, J. E. Callen, M. D. Weiss, eds., p. 139, Academic Press, New Yorlr, 1962. (6) Knox, J. H., McLaren, L., ANAL. CHEM.35, 449 (19G3). (7) D.~ j 4 9 4o (lYG2).
‘.
RECEIVEDfor review May 21, 1963. Accepted July 12, 1963.
lonizatiorr Cross-Section Detector as a Reference Standard in Quantitative Analysis by Gas Chromatography P. G. SIMMONDS
and J. E. LOVELOCK
Lipid Research Center, Baylor University College o f Medicine, Houston, Texas
b The small volume ionization cross-section detector, although less sensitive than other ionization detectors, is absolute and therefore valuable in establishing new quantitative analysis by gas chromatography. This paper reports a preliminary exercise with this detector in the general problem of steroid analysis by gas chromatography. The results confirm the utility of empirical practices for column preparation.
T
of analysis by gas chromatography is sometimes hazarded by the loss of a substance between its injection onto the column and its arrival at the detector. This may be due to thermal decomposition, or to either temporary or permanent adsorption upon the surfaces of the apparatus. This problem is most f-equently encountered with relatively involatile polar substances for these require the use of high temperatures arLd lightly coated columns for their seprtration in reasonable times and the us(?of small sample loads. The development o ’ an accurate gas chromatographic mett od requires some means of comparing the quantity injected with that emerging from the chromatograph column. It is possible, although laborious, to do this by the direct collection and measurement of an eluted component by a static method such as weighing or r:tdioactive counting. Alternatively. given a n absolute detector, the quantity of any eluted component can be directly calculated knowing only the relevant molecular properties and the physical conditions of the measurement. The ionization cross-section detector is absolute in this sense and in its reccbntly developed, HE ACCURACY
small-volume form is well suited to the needs of gas chromatography (6). A remarkable development in gas chromatography techniques is the analysis of steroids made possible by the methods of Homing and his colleagues ( 2 ) . The possibility of thermal decomposition or loss by other causes with these complex substances and a t the high temperatures of the analysis has been recognized (3). With the steroids, the small sample loads which must be used and the severe conditions of the chromatographic separation make evaluation of the accuracy a formidable problem. This paper reports experiments using a small-volume ionization cross-section detector to measure precisely the recovery of steroids after their passage through a gas chromatography apparatus. I t s purpose is to draw attention to the value of this detector in such applications rather than to provide conclusive technical details on the methods of analysis of steroids by gas chromatography. Nevertheless, it con-
Figure 1. Small volume cross-section detector
ionization
firms that with the proper techniques the errors due to the loss of components are for must of the steroids acceptablv small. EXP€RIMENTAL
Apparatus. A modified Chromalab instrument (Glowall Corp., Glenside, Pa.) was used as the basic gas chromatographic unit. The detector oven was redesigned t o accommodate the cross-section detector and the signal from the detector was amplified by a Cary Model 31 vibrating reed electrometer. Output from this electrometer was connected directly to a POtentiometric recorder. The detector design and details of its construction are illustrated in Figure 1. The source is of thin stainless steel coated with a thin film of titanium or zirconium containing occluded tritium and may serve both as the radioactive source and as the chamber electrodes. The quantity of tritium was between 100 and 200 me. Two 6-ft., 3.4-mm. i.d. glass coiled columns were used. Column 1 was packed with 1% SE-30 gum rubber on Gas-Chrom P, 100 to 120 mesh (dpplied Science Corp.); Column 2 was packed with 1% fluoroalkyl silicone polymer (QF-1) on 60- to 80-mesh Gas-Chrom P. The columns and column materials mere prepared and packed according to a well established technique (4, 7 ) . The carefully prepared columns were conditioned a t 240’ C. for 48 hours in an atmosphere of argon. No further precautions were taken initially with the SE-30 column ( S o . 1) and it was used experimentally to observe minor relative losses of steroids under variable column conditions. Subsequently, this column R as treated with hexamethyldisilazane, this treatment being generally recognized as reducing the active sites. The QI71 column wasalso deactivated with hexamethyldisilazane. Uniform sample inV O L . 35,
NO. 10,
SEPTEMBER 1963
* 1345
w
p K Y 0
Y
0
a 0 u
a W
YI
*
e
'
4
Figure 2.
N
0
TIME (minutes)
Typical chromatogram
Gloss-coiled column, 6 foot, pocked with 100 to 1 2 0 mesh Chromosorb W Phase 1% SE-30. Column temperature 2 4 0 ' C. Quantity of eoch steroid 1 5 pg.
jection was achieved with a 10-p1. Hamilton syringe. Reagents. All steroids were prepared in suitable solvents such as acetone and tetrahydrofuran, 0.5 t o 1% (w./v.). A11 samples used gave only one peak by gas chromatography. The carrier gas was helium containing 3m o-/o
^P -..LL..-.IC\ VI I l l e b l l a l l e Id).
Conditions for Quantitative Measurements. The volumetric accuracy of a 10-pl. Hamilton syringe was calibrated by weighing the quantities of mercury delivered from fillings t o different indicated volumes. A systematic error of 2.5% for volumes ranging from 1 t o 6 pl. was observed and the reproducibility was within 1.9%. Measurements were made each day of the flow rate of the carrier gas and also for any change in the operating
*
t
conditions of pressure and temperature w i t h a flowmeter of the soap bubble type. I n all experiments a 0.5% (w./v.) solution of chrysene or of benz[alanthracene was used as a n internal standard. Steroid hydrocarbons werc occasionally uird a 4 wrondnrg- i n t c w d \tandad,. Peak arm m e ~ ~ u r r m e u tns ere ~ n a d r in ~ A V P Y A I nays. rlreas were coniputcd directly from the chromatogram as ~ w a kheight times nidth a t half height. Some chromatograms were photocopied and the areas of peaks measured by cutting out and weighing; this paper I\ as shown by separate calibration to have a weight per unit area consistent for different samples within =k0.570. Areas n ere also measured directly during the course of a chromatographic ceparation using a print-out electronic integrator. (Infotronics Corp., Houston, ?'ex.). Expression of the Results. l'lie current, I,, accompanying the presence of carrier g:ir alone within the drtcctor is given by t h e expression
where
P 7'
pressure in atmospheres temperature in degrees Absolute 11 = volume of carrier gas in which the peak is eluted, rm.3 Qs = molecular ionization crosb seution of S Qe = niolecular ionization cross section of the carrier gas Z,= molecular weight of S 1 = standing current in amperes A , = area of S in ampere seconds R = molar gas constant in cubic contimeters per gram molecule t = time in seconds for compound to pa,s through detectorLe., the peak nidth. I t is assumed that the pressure and temperature a t n hich the initial standing current is mtasured are maintained constant during any subsequent chromatography. K h e n the molar fraction of test substance is 1% or less within the detector, then the expression A,!!'. P . Q C in the denominator of Equation 3 is small and can be disregarded and, in consequence, for most practical purpohes Equation 3 reduces to
The observed change in current, A I , due to the presence of a vapor conctntration in molar fractions ( 2 ) is given by
where QA and Qe are the molecular ionization cross sections of the test compound and carrier gas, respectively. A11 of the d u e s of Equation3 1 and 2 are knoun or can be measured. The only unknonn, K . a constant related to the intensity of the radiation source and to the detector geometry, can be eliminated. The detector can therefore be con.idered absolute and a practical equation can be derived relating the peak area. of a substance S t o the mass, M e , which has passed through the detector as. follow
RESULTS AND DISCUSSION
The accuracy of the chromatographic procedure was determined by comparing the quantity of internal standard injec.ted with that recovered. The recoveries were calculated using Equation 4. For 15 pg. injected, the recovery mas 14.27 pg. 0.54 pg. An error due apparently to the loss of the internal standard had occurred. I n this preliminary investigation its mean magnitude was considered sufficiently small to justify the assessment of steroid losses, which were large by comparison, on the assumption that the recovery of the standard was complete. The calculation of recovery with all of the steroids
*
ANDROSTANE - 3 p-OL-I7 ONE
P
a
2
I
I
4 6 8 IO R E T E N T I O N T I M E (Minutes)
I
1
12
14
Figure 3. Effect of variable operating conditions on steroid losses Androstane A 4-Androstene-3,17-dione Conditions. Nonsilanized 170SE-30 0
1346
ANALYTICAL CHEMISTRY
401 L
2 I
A LLO P R E G N A N E - ~ P - ~ O W - D I O L
A
+
30
30
I
= =
4
6
t
81012 2 4 6 8 R E T E N T I O N TIME (Minutes)
IO 12 14 16 18
Figure 4. Effect of variable operating conditions on steroids with hydroxyl groups 0
+
2AOOC. 2200 c.
A 2000
c:Nonsilanized
Conditions.
1 % SE-30
40 r
Table 1.
Percentage Recoveries of Steroids Relative to Internal Standards
Steroid Androstane Bndrostane-17-one Androstane-3,17-dione l-A4ndrostene-3,17-dione 4-.4ndrostene-3,16-dione 4-rlndrostene-3,17-dione 1,4-.4ndrostadiene-3,17-dione 1,4,6-Androstatriene-3,17-dione
19-Xor-4-androstene-3,l 7-dL n i t
4-Androstene-3,11J17-trione
i - A - 5 5 L O P D (Micrograms)
Figure 5. Effect of varying sample load vs. steroid losses Allopregnone-3~,20a-diol Androstane-38-ol-17-one 4-Andrortene-3,17-dione 0 Androstane Conditions. Nonrihnized 1 % SE-30 0
A
+
tested, using Equal ion 4, would have been unduly laborious and hardly necessary in view of the, magnitude of the losses. Kevertheless, a proportion of the steroid recoveries were so calculated and found t o agree with the values determined from compar son \\ ith the internal standards. The percentage recoveries relative t o the internal standarcis are given in Table 1. It will be noticed that the losses obtained for steroids with different functional groups agree to a close approximation with results xeviously obtained and interpreted as a reduced detector response ( I , 6). The cross-section detector, unlike other ionization detectors, has a response t o different molecular Ppecies determined sdely by the sum of the cross section O F their constituent atoms. It is slight1.r more sensitive to oxygen-containing compounds, since this element has a largw ionization cross section than carbon or hydrogen (6). Pregnane&, 170,200-triol shows the greatest loss while ssturated hydrocarbons such as androstane and cholestane J i o w little or no loss. Also a progres>ive loss is noticed n i t h increase in hydro.tr.1 functions. On the nonselective SE-30 phase androst:ine-3, 17-dione and sndrostane-3a, 170-diol have similar retention times; howeIw, the diol shon-s mnsiderably greater loss than the dione. Similar results occur for 4-androstene:3,1l117-trione and 4-androstene-1 10-013 li-dione and for allopregnane-3.20{iione and allopregnaiie-3P,20a-dicl. From initial obser Jation it was concluded t h a t relative retention time, if indeed the phenomenon of adsorption on the column OCCUIS, may well affect loss of sample. I n s n attempt to obhewe a n y such occurxence a test sample
4-Androstene-1 lp-ol-3,17-diorir~ Androstane-&, 17p-diol Cholestane 2-Cholestene 4-Cholestene-3-one 3,5-Cholestadiene-7-one Cholesterol p-Cholest a n d * Cortisone (Androstene-3,1l,l'l-trione)= * Hydrocortisone (4-ilndrostene-1 lp-ol-3,17-dione)" * Reivhstein's substance S (4-Androstene-3,17-dione)" * Prednisone (1,4-Androstadiene-3,11,I7-trione)" * Prednisolone ( 1,4-Androstadiene-3,1 lp-01-17-trione)~ Testosterone Epitestosterone Estrone Dihydrotestosterone
Recovery relative to standard, yo 1% SE-30 1% QF-1 98.7 97.2 91.1 96.2 89.3 92.1 95.7 99.8 94.0 95.9 93.2 ss . 9 94.7 68 8 75.8 86.8 84.1 74.6 71.4 78.4 66.0 48.8 56.1 61.4 39.7 29.6 29.2 46.2 35.1 93.3
iillopregnane-3,ZO-dione
Allopregnane-3,11: 20-trione 75.5 4-Pregnene-3,l l,aO-trione 68.7 Allopregnane-3@,20a-diol 67.2 Pregnane-3a,17 a ,20a-triol 56.0 48.8 a Note that these compounds are not present as such on the column but suffer known decomposition, and exist as the compounds indicated in parenthesis. Decomposition would account for poor recovery. Table II.
Comparison of Stereoisomers
Percentage recoveries relative to androstane in Group I, and cholestane in Group I1 Group I Steroid Configuration Recovery, &: Androstane-3a-ol-17-one (,4/B trans, 3-OH-ax.) 91.4 ;Indrostane-3P-ol-17-one (A/B trans, 3-OH-eq.) 80.6 Etiocholan-3~-ol-17-one (A/B cis, 3-OH-eq.) 86.6 Etiocholan-3p-ol-17-one (A/B cis, 3-OH-ax.) 92.8 Group I1 Cholestan-3a-01 ( A B trans, 3-OH-ax.) 98.8 (A4/Btrans, 3-OH-eq.) 73.1 Cholestan-38-01 (A/B cis, 3-OH-eq.) 76.9 Coprostane-3a-01 Coprostane-3p-01 (BIB cis, 3-OH-ax.) 92 6
containing four steroids-androstane; androstane-30-01-17-one; 4-androstene3,17-dione; and allopregnane-30-20~~diol-and the internal standard benz[alanthracene was chromatographed on the SE-30 column prior t o silanizing, and a typical chromatogram is shown in Figure 2. The results of chromatographs run at different flow rates and temperatures are shown in Figures 3 and 4. Figure 3 > h o w a plot of retention time us. percentage loss for androstane and androstane-3,17-dione. The values indicated corer a temperature range of 200 to 240' C. It is at once apparent that the loss of the hydrocarbon androstane is linear Kith time but the corresponding dione is not. Over the range covered,
temperature appears to have a negligible effect. Figure 4 shows similar plots for androstane-3p-ol-17-one and allopregnane-3pl2Oa-diol. As before, the results indicate increasing loss with a slower flow rate; however, with these compounds the extent of loss is dependent upon temperature also. Possibly thermal decomposition has occurred. illso, some scattering of the points of the graphs may be due to the fact that injections of test substances were made sequentially, leading to a progressive saturation of the active sites within the column. The effect of varying sample load us. percentage loss is plotted in Figure 5. As would be expected the proportion lost is inversely related to the sample
VOL 35, NO. 10, SEPTEMBER 1963
1347
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 3~5 , 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. 3 5 , 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-
