Relation between structure and retention time of sterols in gas

Mar 17, 1971 - SE-30, QF-1, Hi-Eff 8BP, and PMPE. Sterols behave in a very predictable wayin gas chromatography, so that the retention time of most st...
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Table 11. Relative Volatility Values for Selected Pairs Of Steriods on Different Polyimide Phases CYZ,I (195 "C) (195 "C) Etioa2,i (215 "C) Stationary Androsterone/ cholanolone/ Cholestanol/ phase androstanediol androsterone cholesterol PZ-109 1.32 1.14 1.04 PZ-116 1.32 1.13 1.04 PZ-117 1,37 1.11 1.04 PZ-118 1.30 1.13 1 .os a*,,is the ratio of the adjusted retention times. (~2.1'

a

The stationary phases melt below 100 "C and can be heated to about 290 "C without a significant base-line drift. However, temperatures of 320-330 "C result in a sudden deterioration of the columns. There are little differencesin temperature stability among the individual polyimides, although PZ-117, perhaps because of a higher average molecular weight, is somewhat more stable than the others. The polyimides show a marked selectivity for "polar" groups of the steroid molecules as suggested by the CY^,^ values for androsteroneandrostanediol (see Table 11). The separation of androsterone and etiocholanolone as well as the difficulty resolved

cholestanol-cholesterol pair can be quite easily accomplished. Although their selectivity for the chosen pair of steroids is excellent when compared to other stationary phases commonly used in steroid analysis ( 2 3 , the differences between the individual polyimides are, as was true for hydrocarbons, indeed very small. The temperature stability, efficiency, and column selectivity suggest utility of these phases in the analysis of steroids. The separations of model mixtures of steroids are shown in Figure 4 and Figure 5. A chromatogram of a urinary steroid profile is shown in Figure 6. ACKNOWLEDGMENT

The authors express their thanks to T. E. Rushing for his assistance in obtaining the infrared spectra. We are also indebted to General Mills, Inc., for a generous sample of DDI-1410 diisocyanate. RECEIVED for review March 17, 1971. Accepted May 4, 1971. The authors (RGM and RDS) thank Pennzoil United, Inc., for permission to publish this paper. This study was supported in part by the Robert A. Welch Foundation (Grant NO. E-363). (23) M. Novotny and A. Zlatkis, J. Ciiromatogr., in press.

Relation between Structure and Retention Time of Sterols in Gas Chromatography Glenn W. Patterson Botany Department, University of Maryland, College Park, Md. 20742 Gas chromatographic relative retention times relative to cholesterol have been determined for ninety-two sterols and related compounds. Relative retention times were also obtained for the acetate derivatives VS. cholesterol acetate on four different columnsSE-30, QF-1, Hi-Eff 8BP, and PMPE. Sterols behave i n a very predictable way in gas chromatography, so that the retention time of most sterols can be calculated from their structures and the data provided. Gas chromatography can be very useful in the identification of unknown sterols because of this predictability. Although much GLC work has been accomplished using only one GLC column, it is apparent that at least three columns are necessary to even tentatively identify a sterol by gas chromatography. With the exception of sterols isomeric at C-24, all 92 of the sterols studied could be distinguished from each other on the basis of their retention times on these four columns.

IN THE PAST few years, the number of sterols known to science has increased dramatically. This has been largely due to our relatively new ability, through column and thinlayer chromatographic techniques, to separate and identify specific sterols from naturally-occurring mixtures, even when one makes up less than 1 % of the total sterol of an organism. Also isolated and identified with these techniques have been numerous tetracyclic triterpenoids and "methyl sterols" which have not generally been considered together with sterols in the past. Inasmuch as many of these compounds are now recognized to be precursors in the biosyn-

thesis of sterols, and they are certainly closely related structurally, it seems reasonable to treat them together in a study of the gas chromatographic characteristics of sterols. Since the early work of Beerthuis and Recourt ( I ) , it has been recognized that gas chromatography is an extremely powerful tool for separating and identifying sterols. Clayton observed (2) that not only could good separation be accomplished, but that the influence of substituents or other modifications of the sterol structure on retention time, could be described in a simple mathematical form. This made it possible to calculate rather precisely the retention time of a sterol not yet known, as long as data were available on the effect of each substituent of the molecule in addition to the basic sterol structure. However, even when an internal standard such as cholestane was used, agreement on relative retention times of sterols was not good from one laboratory to another. Even the excellent work of Ikekawa et al. (3) showed unexplained inconsistencies in separation factors. Many liquid phases have been used in the gas chromatography of sterols, but a value obtained with one liquid phase is of limited usefulness with another liquid phase. Also, a number of different sterol derivatives have been used for analysis, such as acetates (1) R. K. Beerthuis and J. H. Recourt, Nature, 186, 372 (1960). (2) R. B. Clayton, Biochemistry, 1, 357 (1962). (3) N. Ikekawa, R. Watanuki, K. Tsuda, and K. Sakai, ANAL. CHEM., 40, 1139 (1968).

ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971

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(4), methyl ethers (2), trimethylsilyl ethers (5-7), and free sterols ( I , 8). Internal standards also vary from laboratory to laboratory, sometimes depending upon the particular sterol derivative. With this lack of uniformity of methods, a worker wishing to use gas chromatography to identify a sterol must frequently obtain numerous known sterols for comparison under his particular conditions. This study was undertaken in an attempt to overcome some of the above problems by determining the gas chromatographic characteristics of a large number of sterols on several liquid phases useful for the separation of sterols.

separated from each other as the free sterols or sterol acetates. Analysis in this way makes it unnecessary to make derivatives solely for GLC analysis. Our standard method for the separation and identification of sterols by chromatography has involved: isolation of total free sterols from the nonsaponifiable lipid by digitonin precipitation; separation of 4,4’-dimethyl, 4-monomethyl, and 4-demethyl sterols by the method of Goad and Goodwin (12), and analysis of the free sterols on SE-30; and, acetylation of all sterols followed by AgNOs-silica gel chromatography (13), and GLC analysis of the acetates on all four GLC systems (Table I). Retention times are expressed relative to cholesterol acetate. Sterol acetates with a RRT (relative retention time) near that of cholesterol acetate were analyzed by injecting them into the column two minutes before or after injection of the internal standard. The retention times of two hydrocarbons are given in Table I, so that these data can also be expressed as retention indices (14). It is at once apparent that with few exceptions, the relative retention time of a sterol cs. cholesterol is identical to that of the sterol acetate us. cholesterol acetate. The exceptions are important when one considers their structure. When free sterols having one methyl at C-4 are examined, the RRT is 0.04 to 0.09 higher (approximately 3 to 4% higher) than the corresponding acetate value. Free sterols with two methyls at C-4 have RRT values 0.10 to 0.15 higher (approximately 6z to 8 higher) than the corresponding acetates, These data have been very helpful in the gas chromatographic identification of sterol biosynthetic intermediates, many of which contain one or two methyls at C-4. The only free sterols lacking methyl groups at C-4, which do not have the same RRT as the acetate, are sterols of the coprostane series (5p) and 3-a-hydroxy sterols. Each of these types of sterols also gives a higher RRT value as the free sterol than as the acetate. The separation factors between compounds differing by a single double bond are shown in Table 11. None of the four GLC systems gave total separation of A 5 sterols from the corresponding stanols, but they can be easily distinguished from each other except on Hi-Eff 8BP. The per cent difference in RRT between duplicate experiments is normally less than 1%. All four GLC systems separated Ai sterols from stanols and A5si sterols from stanols, but the separation was more complete on Hi-Eff 8BP and PMPE. The effects of cis and trans A 2 2 double bonds are similar on all liquid phases. Although they cannot be completely separated when cochromatographed, they are readily distinguished from one another, especially on SE-30 and PMPE. The effect of the A Z 2 in reducing the retention time of a sterol is lessened in a sterol with an eight-carbon side chain compared to that of a sterol with a nine- or ten-carbon side chain. The effect of the A 2 Zbond is consistent for a large number of compounds tested, regardless of the type of column. Only compounds containing a A Z 4 or A Z s bond, in addition to a AZ2, cause a change in the separation factor for the A22 double bonds. It has been reported that on the PMPE column, AZ2 sterols are eluted after the corresponding 22-dihydrosterols ( 1 9 , but the present work shows that A Z 2sterols are definitely eluted earlier on the PMPE column as well as all other known GLC columns.

EXPERIMENTAL

A Glowall Model 301 gas chromatograph equipped with an argon ionization detector and a Honeywell 12-inch recorder was used. Standard chart speed was 0.5 inch per minute. A coiled glass column 1.8 m X 3.4 mm was filled with column packings prepared according to Horning, VandenHeuvel, and Creech (9) using Gas Chrom Q, 100-120 mesh. The 1 % QF-1 packing was used as prepared by Applied Science Laboratories. Other liquid phases used in this study were SE-30 and Hi-Eff 8BP (cyclohexanedimethanol succinate) obtained from Applied Science Laboratories, State College, Pa., and PMPE (poly-M-phenoxylene) obtained from Varian Aerograph, Walnut Creek, Calif. Sterol acetates were prepared by heating the sterols with acetic anhydride for 30 minutes at reflux temperature. Sterols were obtained from four general sources : commercial samples, gifts from individuals (see acknowledgment), synthesized from available sterols, and isolated from Chlorella (10) in our laboratory.

z

RESULTS AND DISCUSSION

The relative retention times of sterol acetates to cholesterol acetate are shown in Table 1. Relative retention times of sterols compared to cholesterol ( I ) or cholesterol acetate (4) have been reported previously, but most data have been reported relative to cholestane (2, 3, 5, 6, 8). Retention times of steroids have also been reported relative to androstane and cholestane with data expressed as steroid numbers (11). The experience in our laboratory has been that a standard with a retention time much nearer to that of the compounds being analyzed is much more desirable. Small changes in operating conditions, especially temperature, between the time of the elution of the internal standard and the unknown, cause an undesirable variability in the relative retention time obtained. This can be largely overcome by using an instrument with a high degree of temperature control and by closing the time gap between the elution of the internal standard and the unknown. Sterols were analyzed as free sterols and as sterol acetates, because they gave satisfactory results on all columns. Another practical reason for doing this is that sterols are normally isolated from nature and (4) J. W. Copius-Peereboom, J. Gas Chromatogr., 3, 325 (1965). ( 5 ) W. W. Wells and M. Makita, Anal. Biochem., 4, 204 (1962). (6) P. Eneroth, K. Hellstrom, and R. Ryhage, J. Lipid Res., 5, 245 (1964). (7) P. Eneroth, K. Hellstrom, and R. Ryhage, Steroids, 6 , 707 (1965). (8) K. Tsuda, K . Sakai, and N. Ikekawa, Chem. Pharm. Bull., 9, 835 (1961). (9) E. C . Horning, W. J. A. VandenHeuvel, and B. G. Creech, “Methods of Biochemical Analysis: Vol. XI. D. Glick, Ed., Interscience, New York, N. Y.,1963, p 69. (10) P. J. Doyle, et a!., Phytochemistry, in press. (11) R. J. Hamilton, W. J. A. VandenHeuvel, and E. C. Horning, Biochem. Biophys. Acta, 70, 679 (1963). 1166

(12) L. J. Goad and T. W. Goodwin, Biochem. J., 99, 735 (1966). (13) H. E. Vroman and C. F. Cohen, J . Lipid Res., 8, 150 (1967). (14) B. A. Knights, J. Gas Chromafogr., 4, 329 (1966). (15) J. H. Beeson and R. E. Pecsar, ANAL.CHEM., 41,1678 (1969).

ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971

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Table I. Relative Retention Times of Sterol Acetatesa Gas chromatographic system HiSterol acetate Ai-Coprostenol Coprostanol AS( 14)-Coprostenol cis-22-Dehydrocholesterol Epicholestanol

SE-306

0.86(0.92) 0.86(0.93) 0.86(0.93) 0.87 0.91 (0.99) trms-22-Dehydrocholesterol 0.91 As 22,24-Cholestatrienol 0.94 0.99 ASS7'22-Cholestatrienol 1 .oo Cholesterol 1.03 Cholestanol 1.03 A8 14-Cholestadienol 14~~-Methyl-A~-cholestenol 1.04 As(Q)-Cholestenol 1.05 A5 2S-Cholestadienol 1.07 Desmosterol 1.09 1.09 AS,7-Cholestadienol 1.12 Brassicasterol 1.12 Ai-Cholestenol Ai ,'4.22-Ergostatrienol 1.13 1.13 Zymosterol I

I

I

14a-Methyl-AS, 22ergostadienol Pollinastanol 14a-methyl-Ai-cholestenol

~~~-Ergostatrienol Ergosterol Ai '22-Ergostadienol 24-Methylene cholesterol Lophenol AS( 14)-Ergostenol 14a-Methyl-A"2 2 ergostadienol Campesterol A5-Ergostenol 14a-Methyl-A8~ 24(28)ergostadienol A*,24(28)-Ergostadieno1 Aa(Q)14-Ergostadienol Campestanol A7,Q(ll)

I

14a-Methyl-A8-ergostenol

31-rior-Cycloartanol A8(Q)-Ergostenol 24-Dih ydrolanosterol 24R-As ,J-Ergostadienol

QF-lc 0.88 0.90 0.91 0.88 0.93 0.89 0.90 1.00 1.00 1.05 0.97 1.08 1.01 1.12 1.09 1.12 1.09 1.11 1.07 1.09

Eff 8BPd PMPE' 0.76 0 . 7 7 0.76 0.74 0.77 0.75 0.88 0 . 8 7 0.80 0.82 0.91 0.92 1.35 1.37 1.21 1.14 1.00 1 . 0 0 1.00 1.02 1.09 1.07 0.94 0.94 1.01 1.07 1.31 1.27 1.29 1.29 1.29 1.23 1.10 1.09 1.21 1.26 1.18 1.18 1.30 1.39

1.16 1.16 1.17 1.18 1.22 1.25 1.26 1.27(1.32) 1.28

1.17 1.23 1.21 1.11 1.22 1.20 1.28 1.25 1.23

1.04 1.16 1.17 1.24 1.44 1.32 1.43 1.32 1.26

1.01 1.17 1.16 1.28 1.33 1.37 1.39 1.32 1.27

1.29 1.30 1.30

1.30 1.26 1.29 1.32 1.29 1.32

1.25 1.29 1.29

1.30 1.33 1.33 1.34 1.34 1.35(1.39) 1.36 1.41 (1.52) 1.42 1.42 1.42 1.42 1.42 1.46 1.46 1.47(1.52)

1.37 1.29 1.24 1.35 1.38 1.40 1.29 1.50 1.45 1.45 1.32 1.32 1.40 1.58 1.42 1.53

1.29 1.47 1.38 1.32 1.20 1.22 1.38 1.08 1.60 1.60 1.32 1.32 1.76 1.63 1.63 1.31

Sterol acetate 24-Dih ydroobtusifoliol

4a-methyl-A8(9) '14ergostadienol 24-Methyl pollinastanol l4a-Met hyl-A7-ergostenol Lanosterol A6m7s2a-Stigmastatrienol 24S-AS, 7 , 22-Stigmastatrienol 4a-Met h yl-A8-ergostenol As, 2S-Stigmastadienol A7822'26-Stigmastatrienol Spinasterol Chondrillasterol 24-Methylene lophenol Cycloartanol AS( 4)-Stigmastenol Sitosterol Clionasterol Fucosterol 14-Stigmastadienol Stigmastanol 23-Demethyl gorgosterol 140-Methyl 24S-ABStigmastenol 28-Isofucosterol Cycloeucalenol As(g)-Stigmastenol

1.50(1.54) 1.54 1.33

1.23

l.SO(1.54) 1.51 1.52 1.54(1.66) 1.54 1.54 1.55(1.60) 1.55 1.57 1.58 1.58 1.60(1.65) 1.61 (1.71) 1.61 1.63 1.63 1.63 1.66 1.67 1.67

1.38 1.61 1.53 1.62 1.47 1.47 1.46 1.47 1.43 1.46 1.46 1.57 1.73 1.50 1.56 1.56 1.50 1.48 1.62 1.62

1.54 1.53 1.52 1.47 1.73 1.73 1.44 1.68 1.84 1.61 1.61 1.89 1.45 1.51 1.60 1.60 1.76 1.74 1.58 1.82

1.42 1.52 1.49 1.41 1.61 1.61 1.41 1.59 1.85 1.65 1.65 1.79 1.40 1.52 1.54 1.54 1.68 1.65 1.57 1.69

1.67 1.69 1.70(1.75) 1.70

1.66 1.55 1.82 1.56

1.49 1.85 1.84 1.59

1.43 1.79 1.70 1.65

1.71 1.73 1.73 1.75(1.87) 1.78 1.83 1.83 1.84

1.53 1.61 1.61 1.85 1.75 1.71 1.71 3.09

1.99 2.05 2.05 1.88 2.05 1.93 1.93 2.49

1.90 1.99 1.99 1.81 1.88 1.93 1.93 2.12

1.87(1.92) 1.67 1.88

1.69

1.88(1.94) 1.84 1.59

1.46

1.88 1.92(1.99) 1.92 1.94(2.08) 1.98(2.12) 2.06(2.21) 2.13(2.22) 2.22 2.50

2.24 1.67 2.55 1.87 1.90 1.79 2.31 2.51 4.24

AS(9),1 4 , 2 1 ( 2 8 ) .

Stigmastatrienol A7 2S-Stigmastadienol 24s-A7 26-Stigmastadienol I

8

1.34 1.46 1.44 1.30 1.23 1.27 1.33 1.13 1.69 1.69 1.34 1.34 1.74 1.68 1.59 1.46

Gas chromatographic system HiEff SE-3CP QF-IC 8BP' PMPE'

Cycloartenol A5,7-Stigmastadienol A'-Stigmastenol A7-Chondrillastenol Macdougallin 4a-Methyl-A8(Q)a 14stigmastadienol 4a, 14a-Dimethyl-24S-A8stigmastenol 28-f30-A?,24(28).

Stigmastadienol 4a-Methyl A8(g)-stigmastenol Peniocerol Cyclolaudenol 24-Methylene cycloartanol 24-Methyl cycloartanol Citrostadienol Gorgosterol Saringosterol

1.70 1.74 3.24 2.12 2.22 2.24 1.90 2.18 2.89

2.22 1.69 2.93 2.02 2.08 1.89 2.43 2:49 6.93

ASsi-Ergostadienol Stigmasterol Poriferasterol A', z a ( 28)-Ergostadienol 24-Methylene pollinastanol Ai-Ergostenol 0 jtusifoliol a Relative to cholesterol acetate, values in parentheses are for free sterols relative to free cholesterol. When no value is given, it is the same as the acetate value. * 3 z SE-30 on Gas Chrom Q, 244 "C, 20 psi, argon flow rate 150 ml/min, RRT of r~dotriacontaneand n-hexatriacontane,l.08 and 3.21, respectively. c 1 % QF-1 on Gas Chrom P, 231 "C, 20 psi, argon flow rate 50 mlirnin, RRT of rz-dotriacontane and n-hexatriacontane, 0.31 and 0.76, respectively. 3% Hi-Eff 8BP on Gas Chrom Q, 238 "C, 25 psi, argon flow rate 95 ml/min, RRT of n-dotriacontane and n-hexatriacontane 0.21 and 0.60, respectively. e 2% PMPE on Gas Chrorn Q, 250 "C, 20 psi, argon flow rate 95 ml/min, RRT of rz-dotriacontane and n-hexatriacontane, 0.20 and 0.57, respectively.

The 5p-A8(I4)sterol is eluted about the same rate as its corresponding stanol on all columns. The A*(14) sterol of the 5a series is eluted faster than the stanol on all columns. The configuration at carbon 5 has an obvious effect on the gas chromatographic characteristics of the A 8 ( I 4 ) double bond. The 5p configuration also nullifies the effect one would expect with A' sterols. Care must then be taken in

evaluating GLC results from members of the coprostane series. The A25 sterol is also eluted faster than the corresponding saturated sterol on SE-30 and QF-1, but it is eluted more slowly on Hi-Eff 8BP and PMPE. As with the AZ2 double bond, the retention characteristics of the A z 5 sterols are affected by an alkyl group at C-24 and additional unsaturation

ANALYTICAL CHEMISTRY, VOL. 43, NO. 10,A U G U S T 1971

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Table 11. Double Bond and Steric Separation Factors of Sterols Substit uent

Sterols compared

A5

A5/stanol(5a)

A7

A1/stanol (5p) A7/stanol(5a)

A5.7

A5*7/stanol (5a)

A8(14)/stanol (5p) A8(14)/stanol (5a) A8(8)*14/stanol (5a) A 25

Carbons in sterol

Carbons in side chain

SE-30

27 28 29 27 27 28 29 27 28 29 27 27 27 27 28 28 28 29 29 29 29 29 29 27 28 29 27 29 27 28 29 27 29 29 31 29 27 27 30 30 27 28 28 28 29 29 30 31 29 29 29 27 27

'8 9 10 8 8 9 10 8 9 10 8 8 8 8 9 9 9 9 9 10 10 10 10 8 9 10 8 10 8 9 10 8 10 10 9 10 8 8 8 8 8 9 9 9 9 9 9 9 10 10 10 8 8

0.97 0.97 0.97 1 .oo 1.09 1.09 1.09 1.06 1.06 1.06 0.87 0.91 0.91 0.86 0.86 0.86 0.86 0.85 0.86 0.87 0.87 0.86 0.91 1.02 1.02 1.02 1.00 0.96 1 .oo 0.99 0.99 1.07 0.95 0.95 0.94 0.99 1.09 1.08 I .09 1.09 1.03 0.97 0.97 0.97 0.97 0.97 0.98 0.96 1 .oo 1.04 1.03 0.84 0.88

in the side chain. An example is A5r25 cholestadienol, which does not at all resemble Az5 sterols with nine- or tencarbon side chains in its GLC characteristics. Sterols with A24(25) unsaturation are separated on QF-1 and SE-30, but are separated more completely on Hi-Eff 8BP and PMPE. Again, additional unsaturation in the side chain (such as a A22) changes the retention characteristics of the A24(25) double bond. The presence of a A24(28)double bond has little effect on the retention time of a sterol on QF-1 or SE-30, but the A 24(26) significantly increases the retention time on Hi-Eff 8BP and PMPE. Only slight differences are noted between sterols with nine- or ten-carbon side chains. When the rrans A24(28)double bond is present, as in 28-isofucosterol, the retention time on all GLC systems 1168

Gas chromatographic systems QF- 1 Hi-Eff 8BP 0.95 0.96 0.96 0.98 1.06 1.05 1.06 1.07 1.07 1.08 0.88 0.89 0.89 0.83 0.84 0.84 0.84 0.85 0.85 0.85 0.84 0.85 0.89 0.96 0.95 0.96 1.01 0.93 0.92 0.92 0.91 1.12 0.94 0.94 0.95 0.98 1.09 1.08 1.08 1.07 1.01 0.99 0.99 0.99 0.99 0.98 0.99 0.99 0.96 0.99 0.99 0.86 0.89

1 .oo 1.01 1.01 1 .oo 1.21 1.22 1.22 1.29 1.30 1.30 0.88 0.91 0.94 1.05 0.83 0.85 0.83 0.83 0.84 0.84 0.84 0.83 0.90 1.01 1.02 1.01 1.01 0.96 1.09 1.11 1.10 1.31 1.05 1.06 1.07 1.14 1.29 1.29 1.30 1.30 1.48 1.08 1.09 1.10 1.09 1.10 1.10 1.10 1.10 1.16 1.15 0.76 0.80

PMPE 0.98 0.98 0.98 1.04 1.23 1.23 1.23 1.20 1.21 1,20 0.87 0.92 0.93 1.06 0.84 0.83 0.84 0.84 0.84 0.86 0.86 0.86 0.93 1.05 1.05 1.05 1.01 0.97 1.05 1.05 1.05 1.27 1.03 1.03 1.04 1.12 1.29 1.30 1.30 1.29 1.49 1.08 1.08 1.07 1.07 1.07 1.07 1.06 1.09 1.16 1.16 0.73 0.80

is slightly greater than when the cis A24(28)double bond is present, as in fucosterol. The separation factors for various alkyl substituents of the sterol molecule are seen in Table 111. The reproducibility of data is good on all GLC systems. The effect of alkyl substituents in the side chain on GLC retention times is quite similar on both polar and nonpolar liquid phases. Regardless of which GLC system is used, a sterol with a C-24 methyl substituent requires from 1.28 to 1.31 times longer to be eluted. It should again be pointed out that the interaction between the A Z 2double bond and a C-24 alkyl substituent results in a different separation factor for a C-24 methyl group in A 2 2 sterols compared to 22-dihydrosterols. For example, on SE-30, the RRT of A5vZ2ergostadienol relative

ANALYTICAL CHEMISTRY, VOL. 43, NO. 10,AUGUST 1971

Table 111. Alkyl Substituent Separation Factors of Sterols Gas chromatographic systems

Carbons Substituent

Double bonds

in sterols 28/27

24-Methyl

29/28 31/30 28/27 29/27

24-Ethyl

30128 29/27 30128 29/28

29/28 30129 31/30 29/28 30129 29/28 30129

4-Methyl

14-Methyl

A7 AN9) AS(9)

29/28 31/30 30129 29/28 28/27 29/28 30129

A8(9).24(28)

A7 A7

28/27 29128

A7,22

4,4’-Dimethy1/4methyl

stanol

30129

to that of trans-22-dehydrocholesterol is 1.23, while the RRT of campesterol relative to that of cholesterol is 1.30. The same effect is seen in calculating the separation factor for the C-24 ethyl group. The presence of a 4a-methyl group does not increase the retention time as much as a C-24 methyl, possibly because of interaction between the 4a-methyl and the 3-hydroxy or 3-acetyl groups. This interaction makes possible the GLC method (described earlier) of distinguishing between 4,4-dimethyl, 4-methyl, and 4-demethyl sterols. Partial shielding by the polar 3-hydroxyl or 3-acetyl may also explain why the 4-methyl adds little to the RRT on the Hi-Eff 8BP and PMPE columns. The presence of a second methyl at C-4 increases the retention time of a sterol more on a nonpolar than on a polar liquid phase (19% and 24% for SE-30 and QF-1, respectively, us. 14% and 15% for Hi-Eff 8BP and PMPE, respectively). The effect of a 14a-methyl group on gas chromatographic

SE-30 1.23 1.23 1.30 1.30 1.30 1.30 1 ,29 1.30 1.29 1.28 1.30 1.54 1.63 1.63 1.63 1.61 1.61 1.63 1.62 1.27 1.26 1.26 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.16 1.16 1.13 1.13 1.13 1.12 1.14 1.13 1.14 1.13 1.14 0.99 0.98 0.98 1.04 1.04 1.03

QF-1

1.19

_

_

1.22 1.22 1.29 1.27 1.28 1.29 1.28 1.27 1.28 1.29 1.29 1.48 1.56 1.54 1.56 1.53 1.54 1.54 1.54 1.21 1.22 1.20 1.21 1.20 1.21 1.19 1.20 1.21 1.20 1 ,20 1.14 1.15 1.12 1.12 1.12 1.12 1.13 1.11 1.13 1.11 1.11 1.07 1.08 1.06 1.09 1.08 1.08

Hi-Eff 8BP 1.21 1.19 1.32 1.32 1.31 1.31 1.32 1.30 1.31 1.30 1.30 1.47 1.60 1.60 1.59 1.60 1.59 1.58 1.58 1.22 1.22 1.20 1.21 1.21 1.21 1.21 1.22 1.21 1.21 1.20 1.09 1.09 1.09 1.09 1.09 1.08 1.08 1.07 1.07 1.08 1.09 0.93 0.92 0.92 0.97 0.96 0.95

PMPE 1.18 1.17 1.29 1.29 1.29 1.30 1.29 1.28 1.28 1.28 1,29 1.43 1.54 1.53 1.53 1.54 1.52 1.54 1.54 1.21 1.20 1.21 1.19 1.18 1.18 1.19 1.19 1.19 1.19 1.19 1.04 1.04 I 04 1.03 1.02 1.03 1.02 1.02 1.02 1.03 1.05 0.88 0.87 0.88 0.92 0.91 0.91

1 .24

1.14

1.15

retention times was unexpected. On three columns, the sterol actually presence of a 14a-methyl group in a decreased the retention time. It is also apparent that the location of the nuclear double bond can alter the contribution of the 14a-methyl group to the RRT of the sterol. With A7 sterols, the 14a-methyl group increases the retention time on SE-30, as well as on QF-1. Additional separation factors can be calculated for other structural features, using data given in Table I, such as the contribution of a 3a-hydroxyl or acetyl group compared to the contribution of the normal 3p configuration, or the contribution of the 23-methyl or the cyclopropane ring of gorgosterol. Using the separation factors in Tables I1 and 111, we can also accurately calculate the probable relative retention times of scores of sterols which may not have been isolated or synthesized to date. Over a dozen previously unidentified sterols, thought to be intermediates in sterol biosynthesis in Chlorella, have been tentatively identified

ANALYTICAL CHEMISTRY, VOL. 43, NO. 10,AUGUST 1971

. 1169

~

in this way (16, 13,even before the usual methods of identification had been used. With the exception of sterols isomeric at C-24 in the side chain, each sterol in Table I can be distinguished from other sterols in Table I after analysis on the four GLC systems used in this work. Sometimes there were slight apparent differences in the retention times of sterols isomeric at C-24, but these differences were never as great as 1 %. Much GLC work has been done using only one column such as SE-30. If GLC is to be used as a tool in the identification of sterols, use of at least three columns seems to be required. In plants the most common sterol mixture is one which contains a C-28 sterol with a saturated side chain, a C-29 sterol with a saturated side chain, and a C-29 sterol with a A Z 2double bond (for example, campesterol, stigmasterol, sitosterol). Of the GLC systems used here, only the SE-30 column resolves this mixture. The QF-I column is unique with respect to 14a-methyl sterols. In scanning the list of sterols in Table I, a sterol with a higher relative retention time on QF-1 than on any other column, with very few exceptions, will be a 14a-methyl sterol. The Hi-Eff 8BP and PMPE columns differ from both the SE-30 and QF-1 in that they have a much stronger affinity for A24(25),424(28), and A5a7double bond systems, and less affinity for sterols with methyl groups at positions 4 or 14. These liquid phases differ from each other primarily in the degree of their affinities for A5f7 sterols and 14a-methyl sterols. Neither the Hi-Eff 8BP nor the PMPE can resolve A5 sterols from stanols as can an NGS column, but both columns have the advantage over most polar columns in their thermal stability. The PMPE column has been in this work (16) P. J. Doyle, Ph.D. Thesis, University of Maryland, 1970. (17) L. G. Dickson, Ph.D. Thesis, University of Maryland, 1971.

for extended periods at 250 "C without significant deterioration. Gas chromatography cannot replace any analytical method now used in the identification of sterols, but using available data, we can gain much more information about the structure of a sterol from gas chromatography than has been possible in the past. ACKNOWLEDGMENT

The author is grateful to: M. J. Thompson for samples of 47-coprostenol, coprostanol, A8(14)-coprostenol,cis-22dehydrocholesterol, trans-22-dehydrocholesterol, A59 2 , 4 - ~ h o lestatrienol, A58 25-cholestadienol,24S-A5,7-ergostadienol,and A6~7-stigmastadienol; to M. Barbier and A. Alcaide for pollinastanol, 31-nor-cycloartanol, 24-methyl pollinastanol, 24-methylene lophenol, and cyclolaudenol; to F. B. Mallory to I. A. Watkinson for A8(9), for 45~7~Z2-cholestatrienol; 14-cholestadienol; to D. H. R. Barton and D. N. Kirk for zymosterol, obtusifoliol, cycloartenol, and A75 2e-ergostadienol; to Merck and to G. R. Pettit for 14a-methyl-A7-cholestenol; Co. for 4 7 , 9 ( 1 1 ) , 22-ergostatrienol; to Carl Djerassi for lophenol, peniocerol, and macdougallin; to Wolfgang Sucrow for 45325-stigmastadienol,A7~2S-~tigma~tadienol, 47,22,25-~tigmastatrienol ; to J. A. Fioriti for A8(14)-stigmastenol, 28 iso47324(2s)-stigmastadienol,and A8!9) 4(28)-stigmastatrienol ; to G. J. Schroepfer for As-cholestenol; to Guy Ourisson for cycloeucalenol, and to F. J. Schmitz for gorgosterol and 23demethyl gorgosterol.

for review March 5 , 1971, Accepted May 4, 1971, This work was supported in part by Grant N ~ G - 7 0 - 3 from the National Aeronautics and Space Administration. This is Scientfic Article No. A1687, Contribution No. 4439 of the Maryland Agricultural Experimental Station. RECEIVED

Estimation of Chromatographic Peaks with Particular Consideration of Effects of Base-Line Noise P. C . Kelly' and W. E. Harris Department of Chemistry, University of Alberta, Edmonton 7, Alberta, Canada The precision of a measurement of a gas chromatographic peak depends on the intensity and character of the base-line noise, the shape and size of the peak, the prior information, and the method of estimation. An estimation method is presented that approaches the highest precision possible of any method used with a given gas chromatographic system. Unlike most other estimation methods, it can use all the information available to estimate the peak parameters, including prior information as well as information from the chromatogram. The base-line noise of many detectors can be statistically characterized by a powerdensity spectrum. In such systems, the estimation of peak parameters is most easily carried out in the frequency domain on the Fourier transform of the chromatogram. Although any mathematical model that can completely describe a peak is suitable for estimation purposes, a peak model that uses cumulants, which are related to moments, has certain mathematical advantages. 1 Present address, Dean and Kelly Analytical Chemists Ltd., 5920-149 Ave., Edmonton, Alberta.

1170

THEBASIC FUNCTION of an analytical instrument is to transform information about composition into a form that can be interpreted by the analyst. Many instruments present information in the form of a chart recording. This paper is concerned with translating the information contained in a recording into a comprehensible form. Specifically, techniques for the analysis of peaks obtained from an isothermal gas chromatographic system with a thermal conductivity detector are reported. The basic approach is applicable as well to all instrumental systems, particularly those giving the desired information in the form of a peak on a noisy base line or background. A true chromatographic peak may be defined as the mass or concentration of a solute flowing across a plane at the end of a column as a function of time. The size of a peak gives information about quantity ; the position and shape give information about the identity of the solute. In gas chromatography, size and position are usually given in terms of area and retention time. Area and retention time are

ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971