standard deviation of 0.003. The gold content determined by neutron activation was 0.019 ppm.
times more sensitive than atomic absorption procedures. When used with appropriate separations, the method is reasonably accurate and rapid. I t can serve as a routine method particularly valuable for geochemical investigations.
CONCLUSION
The rhodamine B fluorometric method has the requisite sensitivity to determine gold a t the low parts per billion level in rocks or in solution. This method is approximately 25
RECEIVED for review on January 19, 1968. Accepted March 28, 1968. Publication authorized by the Director, U. S. Geological Survey.
Correlation between Molecular Structure of Sterols and Retention Time in Gas Chromatography Nobuo Ikekawa and Reiko Watanuki Rikagaku Kenkyusho (The Institute of Physical and Chemical Research), Yamatomachi, Saitama-ken, Japan
Kyosuke Tsuda Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo
Kiyoshi Sakai Central Research Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo
NATURAL STEROLS with structures closely resembling each other can only be separated by gas chromatography; thus gas chromatography is a n essential technique for the study of sterols. I n our previous paper on the gas chromatographic behavior of sterols, we briefly reported the correlation between the structure of sterol and relative retention times in gas chromatography using SE-30 ( I ) . This report describes similar correlations in detail on various liquid phases. In 1962, Clayton reported the behavior of sterol methyl ethers on gas chromatography with diethylene glycol succinate (DGS) and discussed the correlation between the molecular structure and retention data (2). Knights reported a method for the characterization of sterol double bonds involving hydroboration of double bonds followed by gas chromatographic analysis (3). Relative retention times of several free sterols (4-9), acetates (5, 8, IO, 11) and trimethylsilyl ether (9,12,13)using different kinds of packing have been reported. But the relationship between the separation factor for double bond isomers and liquid phase has not been in-
(1) K. Tsuda, K. Sakai, and N. Ikekawa, Chem. Pharm. Buff. (Tokyo), 9,835 (1961). (2) R. B. Clayton, Nature, 190, 1071 (1961); 192, 524 (1961); Biochem., 1, 357 (1962). (3) B. A. Knights, J. Gas Cllromatog.,2, 160 (1964). (4) M. J. Thompson, W. E. Robbins, and G. L. Baker, Steroids, 2, 505 (1963). (5) W. E. Robbins, M. J. Thompson, J. N. Kaplanis, and T. J. Shortino, Steroids, 4, 635 (1964). (6) R. Fumagalli, P. Capella, and W. J. A. VandenHeuvel, Anal. Biochem., 10, 377 (1965). (7) E. Fedeli, A. Lanzani, P. Capella, and G. Jacini, J. Am. Oil Chemisrs’ Soc., 43, 254 (1966). (8) B. A. Knights, J . Gas Chromatog., 2, 338 (1964). (9) A. Rozanski, ANAL.CHEM., 38, 36 (1966). (10) J. W. Copius-Peereboom, J . Gas Chromatog., 3, 325 (1965). (11) G. Osske and K. Schreiber, Tetrahedron, 21, 1559 (1965). (12) W . W. Wells and M. Makita, Anal. Biochem., 4, 204 (1962). (13) P. Eneroth, K. Hellstrom, and R. Ryhage, Steroids, 6, 707 (1965); J . Lipid Res. 5, 245 (1964).
vestigated. In this report, retention times of 30 sterols as free alcohols determined o n polar and also nonpolar liquid phases are compared with separation factors. EXPERIMENTAL The samples used in this study were Cn,-sterols, C2*sterols, and Cn9-sterolsas listed in Table I. A Shimadzu Seisakusho Model GC-1C gas chromatograph equipped with a hydrogen flame ionization detector was used. The column consisted of U-type glass tube, 180-cm X 4-mm i.d. Column packings were prepared according t o Horning, Vanden Heuvel, and Creech (14) using Shimalite W (Shimadzu Co.), 80-100 mesh, as the support after washing with hydrochloric acid and silanization with dimethyldichlorosilane in toluene. Liquid phases used in this study were 1.5% SE-30 (methyl silicone gum, General Electric Co.), 1.5% SE-52 (methyl phenyl silicone containing 5 mole of phenyl groups, G.E.), 1.0% XE-61 (phenyl methyl silicone containing 35 mole of phenyl groups, G.E.), 1.5% QF-I (fluorinated alkyl silicone, Dow Chemical Co.), 1.5 XE-60 (nitrile silicone, G.E.), and 2.0 % N G S (neopentyl glycol succinate, Applied Science Lab.).
z
z
RESULTS AND DISCUSSION
The relative retention times of free sterols to cholestane on six kinds of packing are listed in Table I. The separation factors between double bond isomers in various positions obtained from Table I are shown in Table 11. Separation factors o n SE-52 and QF-1 are not shown in Table 11, because their values are comparable with that of SE-30 and XE-60, respectively. Gas chromatographic behavior of free sterol isomers on polar and nonpolar phases and also the kinds of phase which must be used for the separation of any pair of sterol isomers are shown in Tables I and 11. As can be seen
(14) E. C. Horning, W. J. A. VandenHeuvel, and B. G. Creech, “Methods of Biochemical Analysis” Vol. XI, D. Glick, ed., p 69, Interscience, New York, 1963. VOL. 40, NO. 7, JUNE 1968
1139
Table I.
Sterols [C27-~terols] Cholestanol Coprostanol Cholesterol A’-Choles ten-3/3-01 A8(14)-Cholesten-3/3-ol A 4-Cholesten-3/3-ol L5,2-Cholestadien-3P-ol Desmosterol A-’r 22-Cholestadien-3~-01 [C28-Sterols] Campestanol Campesterol (24a) L8(l4’-Ergosten-3/3-o1 A5~7-Campestadien-3/3-01
Brassicasterol (24P) As, 24-Ergostadieii-3/3-01 24-Methylenecholesterol A’] 2z-Ergostadien-3/3-ol Ergosterol (24P) [C29-Sterols] Stigmastanol P-Sitosterol(24cu) A7-Stigmasteii-3P-01 A5r7-Stigmastadien-3P-ol
Stigmasterol ( 2 4 ~ ~ ) Fucosterol Chondrillasterol(24P) a-Spinasterol(24a) A 4 z5-Stigmastadien-3$01 A’, 2~-Stigmastadien-3$-ol Ail
a
c
e f
2 2 25-Stigmastatrien-3/3-ol ~
1.5% SE-30 1.5% SE-52 1.0% XE-61 (PhSi) 1 . 5 z QF-1 1 . 5 % XE-60 2.0% NGS
Relative Retention Times of Sterols
All retention times relative to that of cholestane, 1.00 Position of XE-61c SE-30“ double bond SE-52* (PhSi) 1.96
0 0
1.79
5 7 8( 14) 14 5, 22 5, 24 7, 22
1.93 2.21 1.88 1.93 1.73 2.10 1.94
0
2.60 2.54 2.56 2.66 2.31 2.92 2.50 2.46 2.42
5 8( 14) 5, 7 5, 22 5, 24 5, 2408) 7, 22 5, 7, 22 0 5
3.33
7 5, 7 5, 22 5, 24(28) 7, 22 7, 22 5, 25 7, 25 7, 22, 25
3.23 3.66 3.46 2.81 3.23
3.21 3.23 3.10 3.60 3.04
Column temp, “C 210 215 218 193 221 220
in Table 11, the separation factors of double bond isomers are almost independent of carbon number, so that the retention times on any liquid phase of a sterol not mentioned in this table may be predicted using this data. Generally, the polar phases gave better separation than the nonpolar phases with some exceptions. The sterols having a double bond on Cs, C14,or c25 were eluted slightly before corresponding saturated sterols on nonpolar phases, but on polar phases, they were eluted after their saturated counterpart. Although C&)- and C2r(28)-stero1smay not be separated from the corresponding saturated sterol on SE-30, they can easily be separated on polar phases. Ab-Sterols and corresponding saturated compounds can only be separated on NGS. A’-Sterols exhibited a remarkably different retention time from corresponding As-sterols or saturated sterols. The separation factors associated with the double bond isomers of free sterols on NGS are almost the same as those for methyl ethers on DGS which are reported by Clayton (2). Since the A9(11)and A 16-sterols were eluted before saturated sterol on DGS, probably A 9 ( I 1 ) and A16 double bonds of free sterols may also shorten the retention time of corresponding 1 140
ANALYTICAL CHEMISTRY
QF-ld
XE-60’
NGSf
2.17 1.94 2.13 2.47 2.13 2.13 1.96 2.34 2.17
3.28 2.69 3.13 3.74 3.25 3.10 3.03 3.72 3.40
3.63 3.27 3.34 3.93 3.27 3.27 2.90 3.86 3.43
2.06 1.88 2.08 2.38 1.98 2.06 1.90 2.52 2.17
6.35 5.65 7.14 8.07 6.50 6.93 6.85 9.93 7.85
2.90 3.04 2.94
4.22 4.22 4.22 4.97 3.66 5.12 4.25 4.22 4.34
4.57 4.23 4.23 5.03 3.57 4.70 4.30 4.17 4.23
2.60 2.60 2.54 3.42 2.25 3.18 2.77 2.58 2.73
8.35 9.43 8.50 13.4 8.22 13.1 10.9 9.00 11.7
5.30 5.21 6.25 6.28 4.59 5.50 5.40 5.31 6.06 6.34 5.60
5.67 5.24 6.26 6.14 4.46 5.30 5.20 5.16 5.30 6.00 5.36
3.29
10.0 11.3 12.9 16.1 9.78 12.8 11.4 11.0 12.4 14.5 13.6
... 2.44 3,24 2.89 2.77 2.74 3.83 3.60 4.14
...
3.08 3.57 3.53
3.60 ...
... ...
Nzflow rate, ml/min. 80 70 60 100
100 100
3.33
3.62 4.16 2.75 3.44 3.32
3.34 3.32
3.83 3.29
Retention time of cholestane, min 8.6 8.0 5.7 5.1 9.2 2.2
saturated sterols. The same behavior of double bonds was also observed for triterpenes (15, 16). With the hopanezeorinane series, the compounds having double bonds in the side chain and at C3-C4 showed longer retention times than that of hopane, the corresponding saturated hydrocarbon. On the other hand, the compounds having double bonds in the ring system showed shorter retention times on both SE-30 and NGS. Magnitude of the effect of a double bond depends on its position in the molecule, but generally tetrasubstituted double bonds in the hopane series exhibit shorter retention times than trisubstituted double bonds on both phases. The double bond on C22characteristically shortened the retention time on both polar and nonpolar phases. The elution order of side chain double bond compounds was A?? < Ana < AZ4(?*)< A Z 4on SE-30 and also NGS, but A Z 5 and A24(28)-sterolsshowed similar retention times. These (15) N. Ikekawa, S. Natori, H. Itokawa, S. Tobinaga, and M. Matsui, Cliem. Pliarm. Birll. (Tokyo), 13, 316(1965). (16) N. Ikekawa, S. Natori, H. Ageta, K. Iwata, and M. Matsui, ibid., p. 320.
Table 11. Separation Factors SE-30 XE-61 0.98 0.95 1 .oo 0.98 0.97 0.98 1.13 1.14 1.10 1.18 0.99 0.96 1.00 0.99 0.98 0.94 1.02 1.18 1.18 1.04 0.90 0.97 0.91 0.88 0.87 0.91 0.87 0.91 0.86 0.87 0.85 0.88 0.88 0.87 0.89 0.84 1.19 1.09 1.21 1.15 1.01 0.98 1.05 1 .oo 0.96 1.16 0.98 1.02 1.34 1.29 1.26 1.29 1.10 1.22
isomers may only be separated on XE-61. Double bonds on CZ4(28)and C15can not be distinguished from corresponding saturated sterols on SE-30, but these were separated on XE-61 and NGS. When double bonds are isolated from each other, the effect on the retention time is additive, so that the structure of an unknown sterol can be inferred from the retention time. In conjugated double bonds, however, retention times were longer than expected on the basis of a simple additive effect. The retention factors associated with branching methyl groups on various phases are shown in Table 11. SE-30 is superior t o other phases for the separation of the isomers with carbon number having double bonds in the same position. With respect to the separation of cholestanol and coprostanol, XE-61 gave a greater separation factor than other phases. The isomers of Cz4-aand Cz4-Palkyl sterols, e.g.-campestanol and ergostanol-gave identical retention times on all phases. However, CZ4-aand /3 series compounds having a double bond at Ci might prove an exception in that chondrillasterol(248) and a-spinasterol(24a) showed different retention times on XE-61 and NGS. Thompson has also observed that 22-dihydroergosterol(24P) and 7-dehydrocampesterol(24a) showed different retention times on SE-30 and QF-1 (17). The a-isomer gave slightly shorter retention time than the p-isomer. The fact that a 20-isoster01 was eluted remarkably faster than the corresponding 20-normal sterol has already been reported ( I ) . It is now well established that gas chromatography is the most powerful technique devised for separation of the natural organic compounds such as terpenoids and steroids. However, a major problem still remaining is the precise identification of individual components of a complex mixture eluted from the chromatographic column. As one solution to this problem, the establishment of the relative retention times of the components on two or more stationary phases is neces(1 7) M. J. Thompson, private communication.
NGS 1.12 1.13 1.13 1.27 1.29 1.02 1.02 1.09 1.61 1.61 0.96 0.97 0.87 0.87 0.88 0.85 0.87 0.94 1.39 1.39 1.16 1.13 1.10 1.12 1.29 1.19 1.12
XE-60 1.01 1.00 1.01 1.15 1.10 0.96 0.98 1.00 1.31 1.27 0.91 0.91 0.87 0.80 0.92 0.92 0.83 0.86 1.21 1.22 1.06 1.03 1 .oo 1.06 1.24 1.25 1.10
sary. T o accomplish a more precise identification, direct combination of gas chromatography and mass spectrometry must be used. The application of the combination for the studies on sterols have been reported by Eneroth (13) and Knights (18). The correlation between the structure of sterols and retention time on various phases described in this report should be useful for the structural determination and identification of complex mixture of natural sterols by gas chromatography alone or in combination with mass spectrometry. Such structural correlations parallel in many respects other sterols and polycyclic compounds. For example, the effect of a double bond in the side chain of sterols is the same as in the triterpene cycloartenol and related compounds (19). Thus the correlation could greatly enhance the analytical value of gas chromatography as mentioned by Clayton (2). ACKNOWLEDGMENT
The authors are grateful t o Dr. W. Sucrow, Organic Chemistry Institute of Technischen Universitat Berlin, for samples of A7-stigmastenol, A5,2S-stigmastadienol, A7’25-stigmastadienol and Ai~22~25-stigmastatrienol, to Dr. M. J. Thompson, Agricultural Research Service, Beltsville, Maryland, for samples of campesterol, brassicasterol, A5,7-campestadienol sterol, and A5~7-stigmastadieno1,to Dr. K. Takeda, Shionogi Research Laboratory, Osaka, for samples of stigmastanol and A8(14)-stigmastenol, to Dr. S. Natori, National Institute of Hygienic Sciences, Tokyo, for sample of a-spinasterol, and t o Dr. A . Martellock, General Electric Co., for silicone polymer XE-61. This research was partially supported by the Hoan-sha Foundation. RECEIVED for review December 27, 1967. Accepted March 4, 1968.
(18) 9.A. Knights, J. Gas Chromatog., 5, 273 (1967). (19) N. Ikekawa, “Methods in Enzymology” Vol. 15, Steroid Metabolism, R. 9.Clayton, Ed., Academic Press, New York, in press. VOL. 40, NO,
7,JUNE 1968
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