If the form of Equation 5 is desired (and this ie really easier to work with), the procedure is straightforward. Two values af R are involved: R1 (pertaining to +1 - +’) and.&*(pertaining to +’-+J. R*
= &@I
+ Rr@a
The factor j does not appear, for it is incorporated in the constants M and N in the manner described previously in this paper. The constants e and B,, as always, must be calculated from gasexpansion-corrected retention volumes. This may be done conveniently by obtaining the necessary effective flow ratea
from Equation 2 using the j-corrected M and N valuea. LITERATURE CITED
(1) Dal Nogare, S., Langloie, W. E., ANAL.CHFiM. 32, 767 (1960). (2) Fryer, J. F., Habgood, H. W., Harris, W.E.,Ibid., 33, 1515 (1961). (3) Giddings, J.. C., J . Chromatog. 4, 11 (1960). (4) Giddings, J. C., Proceedings of GC Symposium, p. 41, Michigan State University, East Lansing, June 1961.
(5) Habgood, H. W., H a m , W. E., ANAL. CHEM.32, 450 (1960). (6) Handbook of Chemistry and Phpica,
39th ed., p. 2045, Chemical Rubber Publishin Co., Cleveland, 1955. 17) Hoae. J.. Rev. 81%.Imts. 32. 1 . (1960.’ (8) JameB, A. T., Martin, A. J. P., Biochem. J . 50, 679 (1952). (9) Partington, J. R., “An Advanced Treatise on Physical Chemistry,” Vol. I, p. 847, Longmans, Green I% Co., London, 1949. (10) Rowan. R.. ANAL. CHEM.33. 510
8.
I
.
‘ (igsi). (11) Sad, A. S., Proceedings of GC S pogium, p. 65, Michigan State niversity, East Lansing, June 1961.
r
RECEIVEDfor review March 8, 1962. Accepted June 1, 1962.
Effect of Substituents on Relative Retention Times in Gas Chromatography of Steroids B. A. KNIGHTS and G. H. THOMAS Department of Anatomy, Medical School, University of Birmingham, Birmingham 7 5, England
b A number of steroids have been chromatographed on columns coated with QF-1-0065 (a fluorinated silicone). The results have been expressed as the logarithm of the retention times relative to cholestane (log r). Evidence is presented to show that the log r values can be estimated from the additive contributions of the individwl substituents together with that of the steroid nucleus to which they are attached. The log r contribution for a substituent depends not only upon its chemical nature, but also on its position in the molecule and its stereochemical configuration.
Dmmo
the past two years the separation of steroids by gas chromatography has been accomplished successfully by a number of workers (9, 6-11, 13-28). The substances analyzed include in addition to derivatives of androstane and pregnane, vitamins Dz and DB @3), corticosteroids (17), Cn to C2g sterols @) and their methyl ethers (6), sapogenins (18), bile acid esters as their trifiuoroacetates (go), and estrogens as their acetates (92). Further, gas chromatography has been used to determine the concentration of cholesterol and squalene in blood (14) and the 17-ketosteroids in urine (9). Lipsky and Lrtndowne (13) studied the effect of both polar and nonpolar stationary phases on the relative retention times of androstane and pregnane derivatives. Horning, Vanden Heuvel, and Haahti have also used a number of phases of differing polarity. These range from the nonpolar methylsilicone gum SE30,through a variety of 1046
ANALYTICAL CHEMISTRY
polyesters to the fluorinated silicone QJ?-1-0065 (16). The latter had a remarkable &ty for ketones compared with alcohols. Thus, 5a-pregnane-3,20dione had relative retention time, 5.93, (cholestane = LO), whereas 3&20@dihydroxy-5a-pregnane had relative retention time, 1.94, and the corresponding 3-hydroxy-%ketone, 2.98. Using SE-30,the relative times were 0.72, 0.67, and 0.67, respectively, and for neopentylglycol succinate 7.20, 6.47, and 6.52. The most polar stationary phase for diols and diones so far used, appears to be ethyleneglycol isophthalate (10, 11). Mixtures of this ester in varying proportions with SE-30 have been used to obtain intermediate retention times without reducing the relative separation between steroids (10). In view of this, QF-1-0065 appeared to be a suitable stationary phase for studying the effect of substituents on the relative retention times of steroids. The results of this investigation are presented. EXPERIMENTAL
A Pye argon gas chromatograph with strontium-90 ionization detector was used. Chromatography was effected on a 4-foot column, 6/sAnch i.d., packed with acid washed Celite 545 (85-100 mesh) coated with 6% QF-l0065. The temperature of the column was 250’ C. The gas flow rate was 55 ml. per minute (inlet pressure 16 p.s.i. argon), Samples were introduced as solids from the end of a glass rod and were not preheated or flash-heated. The time of emergence was measured from the negative air peak, and the relative retention times (r) were calculated using cholestane as standard. The efficiency of the column was about
1600 theoretical plates (for androst-4ene3,17dione). Where the same compounds have been investigated, the results agree well with the values recorded by VandenHeuvel, Haahti, and Horning (16) using 1% QF-1-0065 as the stationary phase at 195-202’ C. RESULTS AND DISCUSSION
Relationship between Structure and Relative Retention Time of Steroids. The concept developed by Bate-Smith and Westall (1) that RM values for the components of a molecule contribute additively to its paper chromatographic mobility, has been applied only recently to steroid analysis (3-6,19). Clayton (7) has demonstrated that the retention time r of a polysubstituted steroid in which intramolecular group interactions are negligible can be expressed as r =rnXk,Xkr,Xk
where r, is the retention time of the unsubstituted nucleus and k..s,c . are group retention factors for a series of noninteracting groups a t positions, a,b,c . . of the nucleus. We wish to show that the results obtained for gas chromatography of steroids are also amenable to this type of quantitative treatment, and that the logarithm of the retention time of a steroid is made up of the additive contributions of the substituents together with that of the steroid nucleus to which they are attached. To demonstrate the accuracy with which such values can be determined, the log r contributions for the methyl group at C-10 were calculated by subtracting from the log r values of the compounds listed in Table I, the log r values of the corresponding 19-nor-
..
.
Table I.
Log r Contributions for C-10 Methyl Group
Compound 178-Hydroxyandrost-4en-3-one 17&H droxy-17amett ylandrost4en-3-one 17&Hydroxy-l7aethinylandrost-4en-3-one Pregn-4-ene-3,20&one 20a-Hydroxypregn4-en-3-one 20p-H ydroxypregn-
4-en-3-one
r
Log r (GlO
Methyl)
4.24
0.08
4.30
0.06
3.92
0.07
8.62
0.07
6.63
0.07
5.91
0.07
steroids. Table 11shows the change in log r accompanying conversion of a.'3equatorial hydroxyl to a 3-ketone. The consistency of these results suggests that noninteracting substituents have both constant and characteristic log r values. To illustrate the usefulness of such data, the relative retention times for 208hydroxy-5a-pregnan-3ne and 36-hydroxy-5a-pregnan-20-one were calculated from the log r values for 5apregnane-3j3,206diol (0.31) and 5apregnane-3,2Odione (0.77), respectively, by the appropriate addition or subtraction of the increment 0.27. The calculated values agreed to within 8% of the observed relative retention times
relative retention times are close to the calculated values for 116-hydroxyandrose4-ene-3,17-dione (12.9) and androst&ene-3,11,17-trione (12.0), respectively. The values for these two 17ketosteroids were obtained using the log parameters for an 116-hydroxyl and an 11-ketone in the androstane series, Table 111. Comparison of the relative retention times of 21-hydroxy- and 118,2ldihydroxypregn-4-ene-3,2Odione indicates that the latter compound also undergoes thermal decomposition, otherwise it would be necessary to postulate a negative log r increment for the 116hydroxyl group. However, its relative retention time is much greater than that
Table 111.
Substituent 11-Ketone
11FHydroxyl 1la-Hydroxyl l2a-Hydroxyl 17a-HydrOxyl
l6Methyl
Table II. Change in log r Contributions for 3-Equatorial Hydroxyl --j 3-Ketone
Compound 5a-Chol~tan-38+1 5&Choleatan-3a-ol Sa-Androstan-38-01 5&Androstan-3~-01 3a-H droxy-5& andrrostan-17-one 5@-Pre an-3a-ol 3a-Hy%oxy-5& pregnan-20-one 3&Hydroxy-5apregnane11,20dione
A Log
r
3.03 2.67
i-
0.26 0.28 0.28
0.54 0.50
0.21
2.23 0.83
0.28 0.29
2.76
0.29
6.96
0.26
Log r Values for Ketone, Hydroxyl, and Methyl Substituents Log r (SubstitCompound r uent) 5&Androstane-3,11,17-trione 7.41 0.24 0.79 0.48 5a-Pregnan-1 l-one Sa-Pregnane3,l l,2O-trione 12.42 0.32 58-Pregnane-3,l l,20-trione 11.10 0.32 17.40 0.30 Pregn-4ene-3,11,20-trione 3a,l1~-Dihydroxy-5&androstan-17-one 4.19 0.27 0.71 0.43 5a-Pregnan-1l&ol 1la-Hydrox regn4ene-3,20-dione 17.65 0.31 3a,12a-Dihgroxy-58-pregnan-20-one 4.31 0.20 3&17~-Dihydroxypregn-5-ene-20-one 4.19 0.19 17arHydroxypregn-4ene3,u)-dione 13.00 0.15 16~-Methylpregn-4ene-3,20-dione 8.44 -0.01 16&Methylpregn-4-ene-3,2O-dione 8.52 -0.01
(16). Vicinal Group Effects. The considerable variation in the parameters for an 118-hydroxyl and an 11-ketone given in Table 111, demonstrates that the log r contribution for a substituent can be affected by neighboring groups. Similarly, the negative contribution for a 16-methyl group is probably a reflection of the change in contribution of the 20-ketone due to steric hindrance rather than that of the methyl group itself. Thermal Decomposition of Corticosteroids. The data in Table I11 are useful for determining the nature of the products obtained on gas chromatography of corticosteroids. The decomposition of these compounds to 17-ketosteroids on SE-30 a t 222' C . has been reported by VandenHeuvel and Homing (17). The same behavior is observed using QF-1-0065 as the stationary phase. The relative retention times of some hydroxy-progesterones are given in Table IV. The value for 17a,21- dihydroxypregn - 4 - ene - 3,20dione is much lower than would be expected and coincides with the observed relative retention time (6.96) for androst-kne-3,17dione. A similar thermal breakdown of 116,17a,21-trihydroxypregn-kne-3,2O-dione and of 17a,21dihydroxypregn-4-ene - 3,11,20trione is suggested by the fact that their
calculated for 1lfi-hydroxyandrost-4ene-3,17dione, so that in this case the transformation evidently does not involve pyrolysis of the side chain to a 17-ketosteroid. Resolution of Stereoisomers. The change in log T for differences in configuration is small compared with the effects described above. When the relevant comparisons are made, $?-compounds are always eluted ahead of their 5a-isomers, and the degree of seDaration of the two is influenced bv thk nature of the substituent at C-5
Table V.
Table IV. Relative Retention Times of Some Corticosteroids
Compound dione 118121-Dihydroxypregn-4-ene-
3,20-dione 17a 21-Dihydrox ypregn-sene3,20-dione
18.3 17.4 7.14
17a,21-Dihydroxypregn-4-ene-
3,11,20-tnone 1Is, 17?,21-Trihydroxypregn4 ene-3,2O-dione
11.2 12.33
Effect of Substituents at C-3 on Resolution of 5a- and 5p-Steroids
Substituent %Ketone
Compound Sa-Choleatan-3-one 5a-Androstan-3-one Ba-Androstane-3,17-dione 5a-Pregnane-3,20-dione
3-Equatorial hydroxyl
5a-Cholestan-3&01 5a-Androstan-36-01 5a-Pregnane-38,2Oa-diol 5a-Pregnane38,20&diol 3&Acetoxy-5a-choletane 3&Acetoxy-5a-androstane
5a-Pregnane-3,11,20-trione
%Equatorial acetate
7
21-Hydroxypregn-4-ene-3,20-
38,20a-Diacetoxy-5arpregnane 38,208-Diacetoxy-5a-pregnane
5.46 1.02 4.72 5.90
-0.04 -0.04 -0.04 -0.04
12.42
-0.04
3.03 0.54 2.25 2.02 4.36 0.79 5.06 4.71
-0.05 -0.04 -0.03 -0.04
VOL. 34, NO. 9, AUGUST 1962
-0.08
-0.07 -0.08 -0.08
1047
(Table V). The marked effect of acetylation of a C-3 equatorial hydroxyl on the Alog r value for a change in configuration at (3-5, shows the potentialities of esterification for separating isomers. These preliminary results illustrate that data obtained from gas chromatography of steroids can be interpreted in the same systematic way as RM values in paper chromatography. The ability, for instance, to predict the approximate relative retention times of compounds should greatly increase the effectiveness of gas chromatography in its application to the identification of steroid metabolites. ACKNOWLEDGMENT
We thank Sir Solly Zuckerman for hie interest and encouragement. Steroids were kindly provided by I. E. Bush (Birmingham University), C. Djerksi (Stanford University), D. D. Evans (Parke Davis & Co.), E. Forchielli (Worcester Foundation), J. Fried (Squibb Institute for Medical Research), and W. Klyne (Westfield College). We
also thank the London Rubber Co. for funds to support a University Research Fellowship for one of us (B.A.K.) and the Caroline Harrold Research Fund for a grant to cover the purchase of a gas chromatograph. LITERATURE CITED
(1) Bate-Smith, E. C., Westall, R. G., Biochim. Biophys. Acta 4, 427 (1950). (2) Beerthuis, R. K., Recourt, J. H., Nature 186,372 (1960). (3) Brooks, S. G., Hunt, J. S., Long, A. G., Mooney, B., J. Chem. SOC.1957, 1175. (4) Bush, I. E., Biochem. SOC.Symp. 18, 1 (1960). (5) Bush, I. E., “Chromatography of
Steroids,” Pergamon Press, London,
1961. (6) Clayton, R . B., Nature 190, 1071 (1961). (7) Zbid:, 192, 524 (1961). (8) Eglinton, G., Hamilton, R. J., Hodges, R., Raphael, R. A., Chem. & Znd. (London) 1959, 955. (9) Haahti, E. 0. A., VandenHeuvel, W. J. A,, Homing, E. C., Anal. Biochm. 2,182 (1961). (10) Zbid., 2, 344 (1961). (11) Haahti, E. 0. A., VandenHeuvel,
W. J. A,, Horning, E. C., J . Org. Chem.
26, 626 (1961).
(12) Kabasakalian, P., Basch, A., ANAL. CHEM.32, 458 (1960). (13) Lipsky, 8. R., Landowne, R. A., Ibid., 33,818 (1961). (14) O’Neill, H. J., Gershbein, L. L., Zbid., p. 182. (15) Sweeley, C. C., Homing, E. C., Nature 187. 144 (1960). (16) VandenHeuvej, W.’ J. A., Haahti, E. 0. A., Homing, E. C., J. Am, Chem. SOC.83, 1513 (1961). (17) VandenHeuvel, W. J. A., Homing, E. C., Biochem. Biophys. Res. Commun. 3, 356 (1960). (18) VandenHeuvel, W. J. A., Homing, E. C., J. Org. Chem. 26, 634 (1961). (19) VandenHeuvel, W. J. A,, Homing, E. C., Sato, Y., Ikekawa, N., Zbid., p. 628. (20) VandenHeuvel, W. J. A,, Sjovsll, J., Homing, E. C., Biochim. Biophya. Acta 48, 596 (1961). (21) VandenHeuvel, W. J. A,, Sweeley, C. C., Homing, E. C., J. Am. Chem. Soc. 82, 3481 (1960). (22) Wotiz, H. H., Martin, H. F., J. Biol. Chem. 236, 1312 (1961). (23) Ziffer, H., VandenHeuvel, W. J. A.,
Haahti, E. 0. A., Homing, E. C., J. Am. Chem. SOC.82, 6411 (1960).
RECEIVED for review November 13, 1961. Accepted April 30, 1962.
identification of Carboxylic Acids in Alkyd and Polyester Coating Resins by Programmed Temperature Gas Chromatography G. G. ESPOSITO and M. H. SWANN Coating and Chemical laboratory, Aberdeen Proving Ground, Md.
,A gas chromatographic procedure is proposed for identifying dicarboxylic and monocarboxylic acids present in alkyd and polyester coating resins. The method was effectively used to identify 19 of the most frequently encountered acids used in the production of synthetic resins. The technique involves transesterification of the resin with lithium methoxide to form methyl esters and subsequent separation by programmed temperature gas-liquid chromatography (PTGLC) on polar and nonpolar columns and identification by their relative retention.
A
of mono- and dicarboxylic acids are used in the manufacture of alkyd and polyester resins where they finally occur as esters, either partially or completely reacted with a variety of polyhydric alcohols. The importance of these acids in synthetic resin production has resulted in the development of a number of analytical methods. ChemWIDE VARIETY
1048
m w r n c A i CHEMISTRY
ical and instrumental methods for nine dicarboxylic acids are discussed in Parker excellent reviews by Jones (4, (6),and Shreve (7). In general, the chemical methods required, a prior separation of the potassium salts of the dicarboxylic acids by nonaqueous saponification which is somewhat timeconsuming. The ultraviolet and infrared spectroscopic methods were limited primarily to aromatic type acids and none of the methods possessed the broad scope necessary for the range of acids encountered. A microscopic method (2) is available but is not suitable for dicarboxylic acid mixtures. Since the advent of gas chromatography, a number of analytical methods applying this technique to coating analysis have appeared. Zielinski, Moseley, and Bricker (8) presented a detailed method to characterize oils used in coating resins. A gas chromatographic method (6) was devised for the identification of fatty acids in vegetable oils using Apiezon L and polyester columns; only those dicar-
boxylic acids that are formed during fatty acid degradation studies were included. More recently (1) the principle of programmed temperature gasliquid chromatography (PTGLC) was used to identify the polyols present in alkyd and polyester resins and is an excellent companion method to this one for examining coating materials. Because the separation of the methyl esters of fatty acids has been studied extensively, emphasis was placed in this work on the separation of acids not found in drying oils. For gas-liquid chromatography (GLC) analysis, the material being examined must have a sufficiently high vapor pressure. In the case of polymeric materials, analyses have been conducted on products of pyrolysis and volatile derivatives. Methyl esters of carboxylic acids are congruous for GLC studies, and a rapid, general transesterification technique applied directly to resin samples was used to prepare the materials for chromatographic separation here. The method
