8s
ic Health, University of Pithburgh, Pithburgh, Pa.
internal diameter, with a disk-electrode and with 56 microcuries of Ra226, plated on E, stainless steel strip, as the source of ionizing radiation. The detector in the Chromalab instrument was 1 cm. in internal diameter, with a recessed tubular anode, a gauze diffuser, and 22.5 microcuries of Ra22e ing several functional groups. Comas the source of ionizing radiation. plete separations were made of mixThis detector was identical in design with that described recently by Lovetures such as cholestanol-cholestanone lock (Bj. and progesterone - 17a-hydroxyprogesCHROMATOGRAPHIC COLUMN,A glass terone on the basis of differences in column, U-shaped with a total length of polarity. Stereochemical change In 6 feet and internal diameter of 1/4 inch, the A/B ring system permitted the was pretreated with a 1% solution of separation of cholestane from coprodichlorodimethylsilane in chloroform, stane and epimers such as 5-androstenethen with methanol, t o reduce active 3&17a-diol and 5-androstene-3P,l7psites on the glass surface. The plug of diol have been distinguished. In many glass wool used in the flash evaporator was treated in a similar manner. When ~ icases, ~ n however, the rosolution of closely this precaution was taken, tailing of related steroids was incomplete with some of the polar steroids, noted in ~ ilicone ~ ~ columns. t e ~ The application of earlier studies, was practically elimithe bechnique of thin f i h s of stationary nated. The column was packed with liquid to polar columns containing 2 . 1 (w./w.) ~~ silicone gum (No. SE-30, polyesters, as described recently by Silicone Products Division, General ahti, VandenHeuvel, and Eorning Electric Co., Waterford, N. Y.) on acidand Lipsky and Eandowne (8), or a and alkali-washed, 80-100 mesh ar fluorinated silicone @),has greatly Chromosorb V, The support was prewashed with acid and alkali according extended the use of gas chromatogto the directions of Farquhar et al. (4). raphy for the qualitative determination The column packing was prepared by of steroids. As in the case of other the solution technique (7), using a 2.2% biological mixtures vhich have been (w./v.) solution of silicone gum in studied, it may be essential, with most toluene. Identical techniques were mixtures of steroids, to obtain separate used in the preparation of a glass coiled analyses on polar and nonpolar columns. column, 6 feet long with an internal 8ufficient information with respect to diameter of 1 / ~ inch, for use with the specific separations i s now available for Chromalab instrument. This column (Kontes Glass Co., Vineland, N. J.) the analyst to choose conditions best was packed by allowing the powder to r a particular problem. the use of gas c~rom&~ography trickle through a funnel into the injecting end of the column while pumping ative determina~~on of steroids with a high-vacuum pump on the outlet hag been explored insome detail, little atof the column. Both columns were tention has been directed to the problem conditioned initially a t 280" for 12 of ~ u a n t ~ t ~estimations. t~ve The preshours in an atmosphere of argon etnd ent study was undertaken to evaluate subsequently were operated a t 225' the use of gas ~ h r o ~ a t o g r a p hwith y an to 240' C. with argon inlet pressures of argon ionization detector €or quantita20~p.s.i.e ~ Solutions ~ ~ of~ steroids t ~ tive analysis. were prepared in suitable solvents (0.5~ to ~1.0%~ w,/v.). Injections of ~ X ~ ~ R I ~ ~ ~ 2.0 to 5.0 d. were delivered from a 1O-pl. Hamilton syringe ,(KO. 701, aratus. GABCHROMATOQRAPH. Hamilton Go., Wbittier, Calif.) through Colman Model 10 inetru8 silicone rubber septum into a boroa Chromalab instrument silicate glass wool plug inserted loosely (Qlowall Gorp., Qlenside, Pa.) were a t the top of the chromatographic used for this study. The argon column. This portion of the column ~ vionization e detector in the Barberserved as a ifash evaporating chamber Golman instrument WBB 2.5 cm. in
In the ~ ~ c r o d ~ ~ e of ~ ~steroids ~ n a t ~low o ~ (CIS to CN) and high (CW to C S ~ ) gas e h r o m a ~ o ~ r ~ a ~ hvariation y, in molecular weight components, For example, separations were made by the molar r e $ ~ o ~of~ ethe arg class of the androstane, pregnane, ionization d e ~ e ~ ~too various r stero cholestane, stigmastane, and diterpene has been noted. The observed molar series, although some overlap of the response 04 a iven steroid was classes was noted with steroids contaia~ e $ e r on n ~the ~ n ~ ~~~ m band e ~ nature I
on the detector. To apply the method to the quantitative d e ~ ~ ~ m i nof~ steroids in a mixture, the argon ~ o n detector ~ ~ must a ~be ~ ~~ l ~ i with each of the steroids present in the mixture.
several months reports three laboratories described tion of gas chromatography to the microdetermination of steroids (1, b,.fl). The analyses repor workers were a result of e technique to its practical limits. It was necessary, for example, to operate the columns near the upper limits of thermal stability of the stationary liquid phases, in order to obtain reasonable retention times. At these extreme t e ~ p e r a ~ ~ evidence r e s ~ of considerable thermal degradation of several steroids
tively thin coating of stationary liquid were equally effectiv mixtures of steroids ~ m p e r a t ~ r e and s within a shorter t h e afforded a practical solution tQthe rlier studies. The effectiveness firmed by Nicolaides (10). were excellent for the ~ ~ a ~ ~ t iaa t spectiion of ~ ~ x t u r ec so n ~ a i ~both n~
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Figure 1. Linear relation of concentration QC steroid and observedarea 0 A
Androstane Cholertane
0 d-Androstene-3,17dione A Cholesterol
and was maintained a t 280' to 270'. At higher temperatures, thermal changes were noticed with cholesterol as well as more highly oxygenated steroids (8, l a ) . Component Areas. To minimize errors which might be introduced by the injecting technique, multiple determinations were averaged for each experiment. Peaks were measured by computing the product of peak height and width a t half height. In the case of asymmetrical peaks, areas were also determined with a planimeter. Detector Calibration. Measurements were made in each instrument of a known mixture of methyl esters of long-chain, saturated fatty acids, The observed areas were directly proportional t o weight per cent within 2 to 3%. This test was considered to be a satisfactory indication of normal operation of the detectors prior to quantitative studies with the steroids. RESULTS AND DISCUSSION
Some time ago, while collecting qualitative data on steroids, it was observed that roughly equivalent quantities of various steroids did not produce uniformly equal areas. This result was surprising in view of the relatively uniform response of the argon ionization detector to various long-chain fatty acids, although Biittcher, Clemens, and van Gent had reported a variation in the molar response with shortchain (C, to &) fatty acids (a). This observation prompted a closer examination of the extent of variation with steroids; initially a comparison was made between cholestane and cholesterol. When equal quantities of these steroids were chromatographed a t several concentrations, the cholesterol peak was always smaller than that of cholestane. Averaging the results of multiple injections, the cholesterolcholestane ratio of areas was 0.70, using an applied potential of 600 volts on the detector. This ratio was not changed significantly when corrected for the difference in molecular weight. le
several steroids of the adrenocortical type such as cortisol, cortisone, and deoxycorticosteroneare known to undergo thermal rearrangements during gas chromatography (I$), it was not expected that simpler steroids such as cholesterol would be altered to a significant degree. The possibility that cholesterol was selectively adsorbed by the silicone column was also considered. The cholesterol peak was nearly symmetricaI, however, and this explanation seemed unlikely, though i t cannot be disregarded entirely since we have no conclusive evidence on this point. In view of the resuIts reported by Bottcher el at. with fatty acids (g), it was reasonable that a similar effect, variation in response of the detector with variation in oxygen content, might be observed with steroids. Accordingly, studies were made of the quantitative responses of a variety of steroids with widely differing structural features. With each of the steroids peak area increased linearly with concentration over a range from 1 to 20 Mg. (Figure 1). It was apparent from a comparison of the observed slopes in Figure 1 that sensitivity was not independent of the molecular species. The relative molar response of each steroid was calculated from the graph of area us. concentration and these data, shown in Table I, were used for a comparison of sensitivity and structure. Without exception, the presence of functional groups lowered the response significantly as compared to the polycyclic hydrocarbons cholestane and androstane. The iduence of total oxygen content may be observed by comparing the relative molar responses of cholestane (1.39), cholestan3P-01 (1.04), and allopregnane-3pj20pdiol (0.95), with 0, 1, and 2 oxygen atoms as alcohol groups. The same effect was noted with cholestane (1.39), cholestan-%one (1.10), allopregnane-8,20-dione (0.99), and allopregnane-3,Il,20-trione (0.84), in which the OXYgen atoms were entirely ia the form of
carbonyl groups. In the case of steroids with a conjugated carbonyl group, a direct comparison may be made between 4-choIesten-3-one (I .01) and 4-pregnene-3,20-dione (0.88),which contains an additional isolated carbonyl group. In general, the molar response was completely dependent on the total oxygen content, an effect isolated by comparing only groups of steroids in which the nature of the functional group was not varied. Of the various oxygen-containing functional groups which were studied, the ethers approximated most nearly the values obtained with hydrocarbons. Ketones generally gave EL somewhat higher response than the corresponding alcohols, as indicated by the results with cholestan-%one (1.10) and cholestan-38-01 (1.04),allopregnane-3,20-dione
Table 1. Relative Retention Times and Molar Areas of Various Steroids"
Relative RelaReten- tive tion Molar Time6 Area0
Steroid Hydrocarbons Androstane 0.08 1.44 Cholestane 0.56 1.39 Ethers 36-Methoxycholestane 0.92 1.25 3~-Methoxy-5-chole&ene 0.91 1.15 Saturated alcohols Cholestan-3p-oi 1.02 1.04 Allopregnane-3pj2Opdiol 0.39 0.95 Unsaturated alcohols 5-Cholesten-36-01 1. 00 1 .00 7-Cholesten3p-01 1.12d 0.90 Saturated ketones Cholestan-3-one 1.09 1.10 Pregnane-3,2O-dione 0.37 0.98 Allopregnen~3,20dione 0.41 0.99 Allopregnane-3,11,20trione 0.53 0.84 Unsaturated ketones 4-Cholesten-3-one 1.34 1.01 7-Cholesten-3-one 1 . W 0.93 PAndrostene-3,17dione 0.33 0.82 4Pregnene-3,20-dione 0. 50d 0.88 Mixed functional groups 5-Pregnen-38-01-20-one0 3gd 0.86 4Pregnen-17a-01-3,20dionee 0.71d 0.74 PPregnen-l7a,21-diol3,20-dionee 0.32 0.59 4-Pregnene-17~~~21diol-3,11,20-trione~ 0.39 0.34 a Operating conditions. 2.1% 8E-30, 228', 20 p.s.i., 2.5-cm. detector, 600 volts, 1 X 10-8 ampere full-scale. * Retention time of 5-cholesten-38-01, 29 minutes. Area produced b 5 cholestenW38-ol, 66.5 8 . mm./10-9 mag. Rsative retention times have not been reported reviously. Other values are in reasona&e agreement with published values (18, 14). 4 These adrenocortical steroids are thermally rearranged with loss o€ the side chain during flask vaporisation ( l a ) , VOL. 33, NO. 13, DECEMBER 1969
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(0.99) and allopregnane-3p,20j3-diol (0.95), and 7-cholesten-3-one (0.93) and 7-cholesten-3p-01 (0.90). This differcnce was small &s compared with the effect of changes in total oxygen content, but it was sufficient to create a spectrum of responses with steroids containing the same number of oxygen atoms. The molar response was also modified somewhat by the introduction of double bonds, especially in the B ring. For example, the response of 5cholesten-3P-01 (1.00) was lower than that of cholest~tn-3&oi(I&) but the decrease observed with 7-cholesten-3p-ol (0.90) was more marked. Similarly in the case of ketones, the relative molar response of 7-cholesten-&one (0.93), with a double bond in the E3 ring, was lower than that of 4-cholesten-%one (I Bl), which in turn was lower than that of the saturated ketone cholestan-3-one (1.10). An applied potential of 600 volts OA the detector was chosen for these initial experiments on the basis of previous studies of the suitability of the argon ionization detector for quantitative analyses of fatty acids as their
"I '
Figure 2. Effect of voltage on the molar response of cholestane methyl esters. In the latter case, very little variation in molar response was found with a variety of esters ranging from Cle to C24 and containing 0 to 4 double bonds. In view of the striking results obtained with the steroids,
omparison of Detector esponse to Various Steroids at Different Voltages" Relative Molar Rasponseb Steroid 400 volts 500 volts 600 volts 709 volts 800 voka 2.26 1.08 1.44 0.86 Androstane 1.32 1.42 0.97 1.39 0.95 Cholestane 1.00 I .00 1 .oo 1.00 1.00 5-Chdesten-3p-ol 0.96 1.05 1.01 0.87 1.26 PCboiesten-3-one 0.90 0.78 0.82 0.56 0.67 CAndrostene-3,17-dione 1.00 0.92 0.86 1.01 0.92 5-Pregnen-3P-ol-20-one Operating conditions identical with those given in Table I with exception of voltage. b Molar areas (sq. mm./lO-Q mole) produced by 5-cholesten-3&ol: 400 volts, 24.2; 500 volts, 41.8; 600 volts, 65.5; 700 volts, 122; 800 volts, 280.
...
onse of Various Steroids at Different Voltages" Relative Molar Responseb 800 volts 1000 volts 1250volts 1500 volts
Steroid Hydrocarbons 1.68 1.55 1.69 1.86 Cholestane Ethers 1.60 3&Methoxycholestane ... 1.50 1.51 3@-&fethoxy-5-cholestene ... 1.45 Esters 38-Acetoxychoiestane ... 1.07 1.41 ... Saturated alcohols 1.18 1.14 1.18 ... Cholestan-3p-ol 1.15 1.07 1.22 ... Allopregnane-3p,20p-diol Unsaturated alcohols 1.00 1.00 1.00 1.00 5-Cholesten-38-cl 0.97 0.96 0.96 7-Cholesten-3p-ol ... 0.55 0.84 Estriol Saturated ketones 1.46 ... 1.39 1.48 Cholestan-3-one 1.32 1.02 1.09 Allopregnane-3,20-dione ... 0.82 1.00 0.86 Allopregnane-3,Il,ZO-trione Ucsaturated ketones 1.19 1.19 1.03 1.42 4-Cholesten-3-one 1.29 1.14 ... 1.48 7-Cholesten-3-one 1.01 1.02 0.87 0.92 4hdrostene-3,17-dione Operating conditions. 2.1% SE-30,240', 20 p.s.i., 1-cm. detector, 1 X IOmnampere fuli-ocale. b Molar areas (sq. mm./l0-9 mole) produced by 5-cholesten-3p-ok 800 volts, 25.7; volta, 61.1; 1250 volts, 89.0; 1500volti3, 147.0. . . I
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ANALYTICAL CHEMISTRY
however, it was of interest to investigate the effect of different voltages on the molar response. The 2.5-cm. detector was used for this study and the operating temperature, flow rate of argon, and c o i m n packing were identical with the conditions of previous analyses. The results are summarized in Table 11. The relative molar response of each steroid was found to be dependent on the applied potential. The total molar area increased logarithmically with increased voltage, as shown in the case of cholestane (Figure 2). At any selected potential within a range from 400 to 800 volts the relative molar response of each steroid was consistently dependent upon the number and type of functional groups. The results suggested, however, that variations of molar response may be minimized a t higher potentials, since the differences were smallest a t 800 volts, with the exception of the hydrocarbons. This observation is in agreement with Lovelock's report (9) that strongly electroncapturing substances should be measured a t high applied potentials, using very low concentrations of sample. The signal-to-noise ratio with the 2.5cm. detector precluded studies a t still higher potentials, and a second instrument was chosen for further exploration of this point. The detector was 1 cm. in diameter and contained a tubular anode within a recessed Teflon cup. This detector was similar in design to that described recently by Lovelock (9). The results of a comparison of 16 different steroids a t four levels of a p plied potential are summarized in Table 111. As in the case of the larger detector, it was not possible to select a condition in which there was no effect of functional groups on the molar response. At the highest applied potential of 1500 volts, for example, a comparison of cholestane ( U s ) , cholestan-3-one (l.46), allopregnane-3,20-dione (1.32), and allopregnane-3,11,20-trione (1.00) indicated that the extent of variation was as great as that observed with the larger detector. As the potential was increased, the difference in response of ketones and alcohols became magnified, so that a t 1500 volts the relative molar areas of an alcohol such as cholesterol with one oxygen atom and alloprgnane-3,ll,2O-trionej with three oxygen atoms as ketones, were identical. The fact that a polyketone gave a lower response than cholesterol a t one potential and an equal response a t a second potential adds strength to the view that this phenomenon must be attributed to the detector rather than to partial decomposition of some steroids or to partial adsorption to the chromatographic column. A further comparison of the effects of various functional groups on the
molar response seems unwarranted, since the results were dependent on the applied potential chosen for study. As a further complication, the results obtained with two different detectors a t the same potential were similar but not identical (compare values at 800 volts in Tables I1 and 111), suggesting that the observed molar responses are also dependent to a certain extent on the design of the detector. I n this regard, the molar areas with the 2.5cm. detector were considerably greater than those obtained with the I-cm. detector. This observation may be due to differences in the amount of radioactive material in the two detectors. As a result of this study, several points may be made regarding quantitative microanalysis of steroids by gas chromatography employing the argon ionization process for detection. Since no set of operating conditions has been found by which the argon detector may be made to give uniform molar responses independent of structure, standards must be used for each steroid to be measured, using graphs such as those in Figure 1 for the conversion of observed areas into concentration. This method, though somewhat cumbersome, provides precise analyses, especially when the standards are determined daily. For example, it has been possible, in this laboratory, to measure accurately the cholesterol content of lipides isolated from human serum and from mixed animal-vegetable diets. By a similar procedure involving standards, Wotiz and Martin have determined urinary estrogens on a routine basis by gas chromatography (16).
Since the relative molar response for each steroid is also a function of the applied potential on the detector, it is necessary, after analyses have been made of the standards, to maintain a constant voltage throughout a given series of analyses. The use of a stepping switch is preferred for this purpose, since it is difficult to regulate the voltage in a reproducible manner with a continuously variable control. Aside from the problems of quantitation introduced by variations in molar response, interesting questions may be raised concerning the mechanism of the process. The most pronounced effect, that of decreasing sensitivity with increasing oxygen content, as well as secondary effects such as those observed with changes in the nature of the functional groups, may be attributable to recombination phenomena similar to those observed by Lovelock with an electron capture ionization detector (9). While the argon ionization and electron capture detectors are different in design and mode of operation, the effects with steroids may be related to differences in response shown in the electron capture detector with various halogen-containing materials. Although these studies have not been extended to other types of biological mixtures such as amino acids, urinary aromatic acids, and similar mixtures in which functional groups vary, it may well be that in all such cases, molar response will be dependent to a degree on the nature and number of functional groups. Hopefully, studies of such mixtures will provide further data from which may be obtained a more
complete understanding of this complexity of the argon ionization process and its relation to electron capture. LITERATURE CITED
(1) Beerthuis, R. K., Recourt, J. H., hrature 186,372 (1960).
(2) Bottcher, C. J. F., Clemens, 3. F.G.,
van Gent, C. M., J. C h r m a t o7.~ 3, 582
(1960). (3) Eglinton, G.,Hamilton, R. J., Hodges, R., Raphael, R. H., Chem. & Ind. (London) 1959,955. (4) Farquhar, J. W., Insull, William, Jr.,
Rosen, Paul, Stoffel, Wilhelm, Ahrena E. H., Jr., Nutrition Reus. 17, 8 (SUPP1.l (1960). (5) Haahti, E. 0. A., VandenHeuvel, W. J. A., Horning, E. C., J. Am. Chm. SOC.83,1516 (1961). (6) Haahti, E. 0. A,, VandenHeuvel, W. J. A., Horning, E. C., J. Org. Chem. 26,626 (1961). (7) Horning, E. C., Moacatelli, E. A., Sweeley, C. C., Chem. & Ind. (London) 1959, )sky, 751.S.R., Landowne, R.A,, ANAL. ( 8 ) Lil: _ . CHEM.33 818 (1961). (9) Lovelock J. E.,Ibid., 33, 162 (1961). (10) Nicolaides,. N.,. J. Chromatog. - 4,. 496 . (isso). (11) Sweeley, C. C., Horning, E. C., Nature 187, 144 (1960). (12) VandenHeuvel, W. J. A,, Horning, E. C., Bwchem. Biop&s. Research Communs. 3,356 (1960). (13) VandenHeuvel, W. J. A., Sweeley, C. C.. Horninn. E. C.. Ibid.,. 3.. 33 (1960)'. (14)VandenHeuvel, W. J. A., Sweeley, C. C.. Horninn. E. C., J . Am. Chem. Soc. 82,3481(ig66j. (15) Wotiz, H. H., Martin, H. F., J . Bi0Z. Chem. 236, 1312 (1961). I ,
RECEIVED for review November 14, 1960. Accepted September 13, 1961. Work supported by grants from the National Institute of Arthritis and Metabolic Diseases (A-4307) and the American Cancer Society.
uantitative P per Chro atogra phy f Plasma Amino Acids odification of: the Binitrophenylation Procedure of Levy CARL PERAlNO and ALFRED E. HARPER Department of Biochemistry, University of Wisconsin, Madison 6, Wis.
b A paper chromatographic procedure has been devised for the quantitative determination of 15 of the free amino acids present in blood plasma. The procedure involves the preparation of the dinitrophenyl derivatives of the amino acids, separation e f these derivatives by two-dimensional paper chromatography, elution of the derivatives from the paper, and mPasurement of the absorbances of the resulting solutions, using a spectro-
photometer. This method is sufficiently sensitive to permit accurate determination of the amino acids in 1 ml. of plasma, and it can readily b e adapted to the analysis of large numbers of samples. The samples need not b e desalted prior to analysis. Leucine and isoleucine appear as one spot on the chromatogram; methionine, tryptophan, histidine, arginine, and ornithine are not satisfactorily determined b y this procedure.
I
1954 Levy (8) described a method for the quantitative paper chromatography of free amino acids. The method involves the reaction of the amino acids with l-fluoro-2,4-dinitrobenzene (FDNB); separation of the yellow dinitrophenylated amino acids by two-dimensional paper chromatography; elution of the derivatives from the paper; and measurement of the absorbance of each derivative, using a spectrophotometer. Although the N
VOL. 33, NO. 13, DECEMBER 1961
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