noise allowance requirement, the computer should always go back to the same interval as the start of the peak. The last two columns in Table IV had only 5 counts as the noise allowance; the others had 10. This change had no significant effect on results. This change corresponds roughly t o doubling the sensitivity of a peak sensor in a no-memory system. The extreme sensitivity of certain shapes of peaks to such a change is illustrated in Figures 2 and 3. DISCUSSION
The results in Table IV indicate the performance of the system with rather noisy data. If the GC analysis were t o be repeated exactly, then, as has been shown by the accuracy of the storage and retrieval process, the new magnetic tape would differ from the first only within 0.1% or 1 count per interval, whichever is larger. The only other change would be a relocation of the noisiest intervals (a maximum first difference of 3 counts per interval in these tests) with respect to the chromatogram. However, even after the filtering action of a 2-second strip chart recorder, individual noise peaks in the base line correspond to more than 3 counts each (Figure 4). A representative variation of noise with respect t o the chromatogram should have been
obtained by the phasing changes during repeated playback. Accordingly, the range of variation in Table IV should be a good indication of the accuracy of the system on repeated analysis of the sample used. Since the computer programming will handle othrr shapes and sizes of peaks in the same way, i t can be concluded that the over-all accuracy is within 1% of true value for each peak a t the relatively high noise level of these data. If peaks were to be smaller with respect to this high noise, accuracy would fall off, as it would for manual interpretation. For less noisy experimental results, accuracy of the system would approach the 0.1% accuracy of the storage and retrieval system because of the principles discussed for square wave measurement (Figure 5). Only the phasing problem makes this type of accuracy estimation possible. Repeated playback of a data tape into a no-memory device would not have the same significance. I n fact, precision under these conditions should be extremely good if the playback and peaksensing amplifiers are performing consistently. APPLICATION OF SYSTEM T O OTHER COMPUTERS
Some computers have no provision for accepting data from an external
1-word memory as outlined in Figure 1. Also, some computers compute so rapidly that tying their speed to the tape-reading speed of this system would be impractical from a cost standpoint. I n either event, this system can be adapted by substituting a card or tape punch for the computer in Figure 1. The cards or tape will then contain the interval areas in a form that can be read a t the proper rate by the computer in question with conventional computer accessories. LITERATURE CITED
(1) Addison, L. M., Lane, L. H., 2. anal. Chem. 189, 80 (1962). (2) ANAL.CHEW33, 480 (1961). (3) Gardiner, K. W., International Gaa
Chromatography Symposium, Michigan State Gniv., June 1961, Proceedings, p. 225. (4) Heigl, J. J., MacRitchie, A. L., Symposium on Analytical Chemistry, University of Maryland, June 1962. ( 5 ) Infotronics Gorp., Houston, Tex., “CRS-1 Digital Chromatograph Integrator,” Bull. CRS-102 (1962). (6) Johnson, H. W., J r , Stross, F. H., ANAL.CHEJI.30,1586 (1958). ( 7 ) Ibid., 31, 1206 (1959). (8) Sawyer, D. T., Barr, J. K., Zbid., 34, 1213 (1962). RECEIVED for review November 19, 1962. Bccepted January 28, 1963. International Symposium on Advances in Gas Chromatography, University of Houston, Houston, Tex., January 21-24, 1963.
Quantitative Aspects of Gas Chromatographic Separations in Biological Studies E. C. HORNING, K. C. MADDOCK, K. V. ANTHONY, and W. J. A. VANDENHEUVEL Lipid Research Cenfer, Baylor Universify College of Medicine, Housfon, Texas
b This study was carried out to define the nature of several problems involved in establishing procedures for the determination of steroids and long-chain fatty acid methyl esters. Column packings which show very little adsorption of solutes are required for best results. It is necessary to use appropriate derivatives in some instances, and the sample size should be appropriate for the specific application. Questions relating to matters of instrument design, preparation of column packings, temperature-programmed operation, and choice of detection systems have been studied.
T
widespread use of gas chromatographic techniques in the isolation, identification, and estimation of substances of biochemical or biological HE PRESENT
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ANALYTICAL CHEMISTRY
interest is sufficient evidence of the value of this corpus of analytical methods in work in biology and medicine. However, experience with these procedures suggests that qualitative information may be obtained far more easily than quantitative data. The relative lack of published information about methods for establishing suitable conditions for quantification, and current differences in opinion about the degree of precision and accuracy which may be attained, have led to uncertainty in many laboratories about the future course of developments in this area. The experiments described in this paper were carried out to define the nature of several specific problems involved in quantification procedures for steroids and long-chain fatty acid methyl esters. The results indicate that satsifactory quantification may be obtained in a variety of applications, and some of the
causes of failure to obtain good results are illustrated by examples. The use of reference mixtures to evaluate new procedures and to validate day-to-day results also is the best way to estimate precision and accuracy in any given application. This practice should not be used t o determine “correction factors” which are used to convert basically inaccurate analytical data into “quantitative results.” Shortcomings arising from inadequate technique or poor instrument design are not likely to be overcome successfully by this approach. EXPERMENTAL
Instruments from three commercial sources were used. All required essential but easily carried out inodification in order to secure satisfactory quantita-
tive results. The effect of a temperature drop in the region of the connections between the end of the chromatographic columfi and the detector unit is described in the Discussion; the difficulty can be corrected by using a heated “bridge” O i by placing the detector unit in the column bath. Argon ionization and hydrogen flame ionization detection systems were used. Columns were of glass, either U-tube or coil, constructed so that the sample was injected directly into the heated chromatographic tube. The usual 1 to 2 inch glass wool plug was reduced to about 0.2 inch of silanized glass wool to hold the packing in place. Column packings were prepared by methods developed in this laboratory. Details of the procedures are given by Horning, T’andenHeuvel, and Creech, ( I O ) . The silanizing procedure was carried out with dichlorodimethylsilane in toluene. The material (Gas Chrom P, 100- 120-mesh) used for the preparation of acid-washed (concentrated hydrochloric acid) and silanized supports was size-graded before use by mechanical sieving. Coatings, all 1 weight yo,were applied by the filtration technique of Horning, Aioscatelli, and Sweeley (9). For comparative studies with F-60 on differently treated supports each glass column (all with the same dimensions) contained the same weight of packing. The liquid phases were F-60 (methyl p-chlorophenyl siloxane polymer, Dom Corning Corp.), QF-1 (fluoroalkyl siloxane polymer, Dow Corning Corp.), and EGSS-X (a diacid-diolsiloxane copolymer, Applied Science Laboratories). Samples in 1- to 3-pl. volume were injected with a Hamilton microsyringe. The d v e n t s were iso-octane, acetone, and tetrahydrofuran.
-1preliminary study was carried out to determine the best method for making observations n i t h poor columns. X h e n adsorption is a major p r o h l m , the repeated injection of samples usually leads to an observed increase in response during the course of the experiments This has led in some-‘ laboratories to the practice of presaturation (injection of a “soaking” amount of sample), so that subsequent responses will be a t or near the maximum letel likely to be observed for the system and samples in question. This practice was not used in this study. Instead, a systematic application of samples was used so t h a t the first application was that of the largest sample of the series. Successive samples were applied in order of decreasing size, and an entire set of observations was completed during a single experimental period (several hours). This method gave reproducible results, and i t was used for obtaining curves B and C in Figure 4 and B in Figure 5 . It was also used to determine curves A in Figures 4 and 5 , although in this in-
stance this was largely a matter of using a uniform esperiiiiental condition for comparison purposes rather than a necessary condition for obtaining reproducible results. Steroids used were purchased or prepared. For example, the trimethylsilyl ether of cholesterol was purified by sublimation, showed only one component during gas chromatography, and had a satisfactory elementary analysis (16).
Figures 7 to 9 show analytical separations of fatty acid methyl esters and methyl acetals from serum lipides of a normal individual. The lipide fractions were separated by thin-layer chromatography on silica gel G, and after elution a transesterification reaction was carried out in methanol-sulfuric acid. The separation methods and procedures for recovery from silica gel G will be published separately. The gas chromatographic conditions were as follows: 12-foot glass coil; 1% EGSS-X on Gas Chrom P (100- 120-mesh, acidwashed and silanized) ; nitrogen pressure 27 p.s.i. ; temperature-programmed separation; hydrogen flame ionization detector; samples. 5 to 10 pg. total in 0.3 to 0.5 pl. of iso-octane. The relative error was determined with fatty acid methyl ester reference standards (National Heart Institute and Hormel Institute). RESULTS A N D DISCUSSION
Instrument Design Problems. The use of a hot flowing gas i n a n analytical method presents a relatively new problem in instrument design, and t h e extent to which difficulties in quantification may arise because of plumbing and heating problems is roughly proportional to t h e evtent of deviation of the condition from atmospheric pressure and temperature. Very few difficulties are evident in work with compounds of relatively low molecular weight a t moderate temperatures, b u t when column temperatures above about 200” C. are required, several elements of design may affect the results. Wide variations in temperature along the column may result in “hot spots” which lead to localized deterioration of the column packing. This relatively unusual condition may be detected readily by determining the temperature a t several points in the column compartment under simulated operating conditions. If a n instrument with a “flash heating” zone for the chromatographic column is employed, the column packing should not extend into this region. Localized deterioration of the packing will usually occur if i t is subjected to flash heating temperatures. Alteration of the packing by thermal effects will lead t o unnecessary decomposition or adsorption of compounds under study.
X a n y gas chromatographic instruments are constructed with separate heating chambers for the detection unit and the chromatographic column. The “bridge” connecting these compartments is often not heated separately, although in some instruments a heated connecting block is present. A cool bridge may lead to poor quantitative relationships, apparently because of partial condensation of sample cornponents. This effect is rarely seen for compounds of molecular weight below about 300, and is not usually evident when large (5- to 10-pg.) sample3 are used. However, when steroid iainples containing less than about 0.5 pg. of each component are employed, this effect may emerge as a cause of erratic and inaccurate estimations. It is difficult t o estimate exactly the magnitude of errors which may be introduced in this way, but variations of 10 to 157, from the calculated value have been observed in work with sonie of our instruments for this reason. Corrective measures may be taken s i t h o u t difficulty; it is now our practice to use heated bridges or other devices to maintain a n aypropriate temperature for the connect. ing tube leading to the detector unit. When the detector unit is in the column compartment, effects of this kind have not been seen. A variety of designs of injection systems and chromatographic columns are in use. The present choice of materials is usually limited to glass, stainless steel, copper, or aluminum. Our experience has been limited to glass, treated with dichlorodimethylsilane or hexamethyldisilazane, since this is currently the most satisfactory material for applications requiring a relatively high degree of accuracy in quantitative work with compounds other than hydrocarbons and ethers. These matters involve properties of the substances under study as well as properties associated with a gas chromatographic system, and a n instrument which is unsatisfactory in one application may be suitable in a variety of others. I n many biochemical and medical studies the compounds under investigation are polyfunctional and often above about Czoin carbon content, and it is often necessary t o work with samples of a few micrograms or smaller. Under these circumstances i t is desirable to avoid difficulties arising from elementary problems in design or use of materials. Chromatographic Columns. The chroinatographic column is the site of the separation process and t h e majority of difficulties in quantification which arise in biological a n d biochemical applications are due t o inadequate technique in the preparation of t h e column. However, t h e nature of t h e compounds under study, VOL. 35,
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UNTREATED SUPPORT
CHOLESTERYL METHYL ETHER CHOLESTANE
CHOLESTEROL I
I
1
Figure 1 .
Gas chromatographic separation of mixture of steroids with 1 % F-60 column packing
Untreated 100- to 120-mesh diatomaceous earth support in 6-foot glass U-tube.
and the sample size, also must be taken into consideration whenever an attempt is made to improve the accuracy of a given procedure. The nature of this problem is illustrated in Figures 1to 5. Figure 1 shows a separation carried out with a column packing prepared in an ordinary way. A commercially available diatomaceous earth support was coated by the filtration technique (9) with 1% of silicone polymer F-60. The separation of cholestane, cholesterol methyl ether, and cholesterol was observed. Severe trailing \vas found for the ether and the result with cholesterol was disastrous; much of the compound was irreversibly adsorbed, the peak showed excessive trailing, and even the retention time was markedly changed from that usually observed. Any qualitative or quantitative observations made for hydroxyl-substituted steroids under these circumstances would be without value. The ideal support for a packed column iy free of active sites for the adsorption of the compounds under study; this circumstance has not yet been realized in practice, except perhaps for hydrocarbons and other nonpolar compounds. However, inactivated diatomaceous earth supports are now commonly used for the preparation of thin-film packings, and are the best that have been found so far for many applications. The earliest inactivation process was that of Horning, Noscatelli, and Sweeley (9). This employed dichlorodimethylsilane following the procedure of Howard and Martin (11) used earlier for preparing diatomaceous earth supports for reverse phase liquid partition chromatography columns. Since that time a number of silanizing procedures have been described. These include the modification of Holmes and Stack (8) employing dichlorodimethylsilane, the hesamethyldisilazane process of Purnell et al. ( I ) , and the Siliclad procedure of Dal Sogare ( 3 ) . These procedures decrease the surface area of the support and convert the surface to a hydrophobic one which is readily coated with
528
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ANALYTICAL CHEMISTRY
2 1 2' C., 13 p.s.i,; argon ionization detector (Ra source)
organic chemical phases. The value of
a silanizing process for the improvement of supports was recently demonstrated by Sawyer and Barr (16) for applications involving compounds of relatively low molecular weight. The fact that an untreated (not silanized) diatomaceous earth support may show adsorption effects can be demonstrated in many ways, yet adsorption is as much a property of the solute of the support, and it is not possible to categorize column properties except in terms of both the column and the substances under study. This is evident when Figure 2 is compared with Figure 1. The same column was used for both experiments; it is obvious that the trimethylsilyl ether of cholesterol is adsorbed by active sites on the support to a lesser extent than the methyl ether. The gas chromatographic properties of trimethylsilyl ethers resemble those of hydrocarbons as far as adsorption effects are concerned, and they are usually eminently satisfactory deriva-
tives for quantitative work on this account. Hydroxyl-substituted steroids represent an opposite extreme from hydrocarbons in terms of susceptibility to adsorption on supports. For this reason sterols may be used to determine the effectiveness of silanizing and other inactivation procedures. I n our experience, acid washing is necessary before the silanizing process is carried out, and acid washing alone may yield a satisfactory support for some applications. Figure 3 shows a separation of the same mixture used for Figure 1; the support was acid-washed with concentrated hydrochloric acid and coated with 1% polymer F-60. Some trailing is evident, but this is approximately the same for all components. We ascribe this effect to imperfect coating of the hydrophilic surface of the support by the nonpolar polymer. Our experience with columns made with acid-washed, silanized diatomaceous earth supports (10) indicates that
UNTREATED SUPPORT
CHOLESTERYL TMSi
..
MINUTES
Figure 2.
Gas chromatographic analysis of cholesteryl trimethylsilyl ether Same column and conditions as for Figure 1
ACID- WASHED SUPPORT CHOLESTEROL CHOLEST ERYL METHYL ETHER
y
CH OLES TAN E
i
I
30
15
0
MINUTES
Figure 3.
Gas chromatographic separation of mixture used for obtaining Figure 1
Column packing prepared with acid-washed diatomaceous earth support (100- to 120-mesh), contained 1% of F-60 liquid phase. Used in 6-foot glass U-tube in same instrument and under same condition as for Figure 1 Relative retention times for ether and sterol different from those of Figure 1. All peaks show trailing effect
these columns are satisfactory for quantitative work but are not ideal. Test procedures must be used to evaluate their behavior. Figures 4 and 5 show two relatively simple ways of evaluating adsorption effects by comparison of the behavior of a sterol (cholesterol) or a ketone (cholestane-3one) with a hydrocarbon (cholestane). Varying amounts of a mixture containing a fixed ratio of cholesterol to cholestane were used with three different column packings (in each case the stationary phace mas 1% F-60, but the treatment of the support differed) to provide the data given in Figure 4. The packing responsible for curve C was prepared by an inactivation process using trimethylchlorosilane. This packing is clearly inferior t o a packing prepared by acid washing alone, Fvhich gave curve R. The best packing in this group (curve A) waq prepared by
acid washing and silanizing of the support with dichlorodimethylsilane. However, even this relatively good packing is not quitable for use with cholesterol sample sizes below about 2 pg. A similar 3tudy for cholestane-3-one is shown in Figure 5 . I n this study i t Tvas assumed that the response of the argon ionization detector 1% as the same on a mass basis for tlie ketone and for cholestane. Cur\ e B n-as obtained with the same acid-n ashpd packing used to obtain curxe B in Figure 4, and A was obtained n i t h the same packing (acid-washed and silanized support) used for A in Figure 4. It is evident that the operation of acid washing alone, as a method of treatment of the support, does not lead to a satisfactory nonpolar packing for applicationq involving ketoor hydroxyderoids a t a n y level of sample size in thP range usually used r i t h
ionization detection systems. However, there is a marked difference in the behavior of cholestane-%one and cholesterol when a n acid-washed, silanized support is used. There is an essentially constant recovery of the ketone down to a sample size of about 0.1 pg., in contrast t o the end point of about 2 pg. observed for cholesterol. The result of these studies, as far as adsorption effects are concerned, may be summarized by the statement that in our experience the best support for quantitative work is an acid-washed, silanized diatomaceous earth support, and that "allowable" functional groups which permit, submicrogram samples to be used include the carbonyl and trimethylsilyl ether groups but not the free hydroxyl group. Two questions of practical importance remain to be answered: whether, with suitably prepared columns, i t is possible to obtain quantitative results in the analysis of polyfunctional compounds of biological interest, and how to define the sensitivity which may be obtained with retention of quantitative relationships. Each application brought under study presents a separate problem, but sufficient experience has been accumulated to provide several examples of good quantification. Table I contains anulytical results for a separation of androsterone, et'iocholanolone and tiehydroisoandrosterone by two procedures. The same functional groulx are present in each compoand, and the+ substances may be estimated eit'lic'r as the free steroids (7') or as the trimethylsilyl ethers (IS). The d u e s for free steroids were obtained with saml)le sizes falling in the plateau region shov n for cholesterol in Figure 4. Table I1 contain. analyt'ical results for a separation of edrone, estradiol, and estriol as triinethylsilyl ethers (15). In these instance., the mass response observed with :in argon ionization detector \vas the same for each compound within each group.
/
0
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2
3 4 5 MICROGRAMS
6
7
s
Figure 4. Variation of response ratio (ratio of peak areas) of cholesterol-cholestane mixture with sample size (micrograms of cholesterol) with three differently prepared column packings Liquid phase 1 % F-60. 6-foot glass U-tube used with argon ionization detection system. Diatomaceous earth support Gas-Chrom P, 100-1 20-mesh. A. Support acid-washed and silanized with dichlorodimethylsilane in toluene ( 7 0) 6. Support acid-washed and oven-dried (80'1 before coating C. Support acid-washed and silanized with trimethylchlorosilane in same fashion as for dichlorodimethylsilane
60 I
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I MICROGRAMS
I
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Figure 5. Variation of recovery of cholestane-3-one with sample size and with two differently prepared column packings Same columns as in Figure 4
VOL. 35, NO. 4, APRIL 1963
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0.02
0.04 0.06 0.08 0.10 SAMPLE SIZE-TMSi DER.
0.12
Figure 6. Variation of response ratio (ratio of peak areas) of cholesteryl trimethylsilyl ether (TMSi)-cholestane mixture with sample size of trimethylsilyl ether 1% QF-1 on acid-washed, silanized 100- 120mesh Gas-Chrom P, 6-foot glass spiral, 2 0 0 ' C.; 15 p.s.i. nitrogen, hydrogen flame ionization detector
Numerous other examples of quantitative procedures for steroids have been published: estimation of fecal sterols as the trimethylsilyl ethers by Wells and Makita ( I Q ) , of estrogens as the acetates by Wotiz and Martin (20) , and of 3,7,12-triketocholanic acid (as the methyl ester) by Ellin, Mendeloff , and Turner ( 4 ) . The limit of sensitivity with retention of quantitative relationships may be different for each application.
Table 1. Composition Data for a Mixture of Three 17-Ketosteroids by Two Gas Chromatographic Methods
TrimethylFree silyl steroids," ethers,*
Compound , calcd. yo 7% 770 Androsterone, 37.8 36.7 38.8 Etiocholanolone, 3 6 . 2 37.8 35 0 Dehydroisoandrosterone, 26.0 25.5 26.2 Average of three determinations by method of Haahti, VandenHeuvel, and Horning (7'). b Average of three determinations by method of VandenHeuvel, Creech, and Horning (18). 0
Figure 6 shows the result of an experiment in which small samples of a mixture of cholestane and the trimethylsilyl ether of cholesterol were carried through an analytical separation with a 1% QF-1column and a hydrogen flame ionization detector. The limiting sample size for quantification was found to be about 0.02 pg. of the trimethylsilyl ether. The limit of sensitivity for qualitative work alone was about one fifth of this value. These observations suggest that techniques which include presaturation of the column and correction factors based on irreversible adsorption on column packings are unnecessary when column packings are prepared by an appropriate procedure, appropriate derivatives are used, and the sample size is appropriate for the application. Detection Systems. It has been known for some time t h a t ionization detection systems do not give uniform responses with all organic compounds. It may therefore be necessary in some instances (when ionization detection systelns are used) to use a response correction factor in order to establish quantitative relationships. This problem was studied for steroids by Sweeley and Chang (17'). Unfortunately, there has been a tendency to assume that because the basic effect is well established it is also acceptable to determine a correction factor or a correction curve which may include such varied effects as column adsorption, condensation in a cold bridge area, and a detector effect in a single factor. The problem of variable detector responses can be separated from problems associated with the column packing only with the aid of a reference detection system based on a property other than ionization. The cross-sectional detector described by Lovelock (12) mag be used in this way, and the results of Lovelock and Simmonds (13) indicate clearly
that adsorption on column packings or other effects are often responsible for an ionization detector response which is lower than expected. This problem is illustrated by the data in Figure 5. Cholestane-%one gives a slightly lower response than cholestane (about 95% of the cholestane value) for a wide range of sample size. These data do not suggest that adsorption on the column packing is involved to a substantial extent; thermal transformation or some other form of loss (in the "flash heater," column, or detector unit) may occur to an eltent of about 5% in this instance. The effect cited by Sweeley and Chang (17) may also be involved. Because of effects of this kind, i t is our practice in quantitative work to use as an internal standard in specific applications a compound whose structural groups are related to those of the substances under analysis. I n current work in the estimation of urinary 17-ketosteroids, for example, epicoprostanol is used as the internal standard, and the reference substance is converted to a trimethylsilyl ether during the stage of derivative formation [in this application all steroids are converted to trimethylsilyl ethers ($1 18)1. The specific application usually determines the choice of detection system. Argon ionization detection systems are not suitable when large flow rat? changes occur during a determination, but the flame ionization detection system is usually suitable under these conditions (5). Temperature Programming. The time needed for a given separation may be reduced considerably when temperature programming is used inqtead of a n isothermni condition. It is usually possible to carry out a separation so t h a t the duration of time of elution of each component (peak width) is approvimately the ranie. and
Table (I. Composition of Estrone, 17-P-Estradio1, and Estriol Trimethylsilyl Ether Mixture with a PhSi Column"
Weight, Found,b Compound Yc 72 Estrone trimethylsilyl ether 36.6 36.5 17pEstradiol ditrimethylsilyl ether 25.2 25.5 Estriol tritrimethylsilyl ether 38.2 38.0 a By method of Luukkainen, VandenHeuvel, and Horning ( 1 6 ) ; b Calculated from individual peak area6 from typical gas chromatographic record. In separate studies precision of determination was found to be about 0.5%. This unusually high precision may be due to the fact that these derivatives give peaks of very nearly theoretical shape.
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ANALYTICAL CHEMISTRY
Figure 7. Gas chromatographic analyses of fatty acid methyl esters from human serum lecithins Column 12-foot X 4-mm. glass coil. Hydrogen flame ionization detection system. Column packing contained 1 of phase EGSS-X and support was 100- 120-mesh Gas-Chrom P, acid-washed Recorder chart speed shown was doubled and silanized. Carrier gas was nitrogen ( 2 7 p.s.i.1. for quantitative analytical work. Methyl esters are expected ones; major peaks in normal run are in order of increasing carbon number: palmitate, stearate, oleate, linoleate, a Czo triene, arachidonate
yo
and possibly different detection systems, a different problem is presented. Under temperature-programmed conditions the major long-chain components present in lipides invollved in human metabolism have been separated with a thin-film column containing 1% of polymer EGSS-X as a liquid phase. The conditions are suitable for mixtures containing compounds ranging from about Cl0 to Cz4 in carbon content. Results for three human lipide classes are shown in Figures 7 to 9. Unsaturated esters are eluted after the corresponding Saturated compounds; Figure 7 shon s this effect for serum lecithin fatty acid methyl esters. The palmitate-palmitoleate and stearate-oleatelinoleate separations resemble those usually seen under isothermal conditions with a polyester column. The separntion of arachidonate and a CZOtrienoic methyl ester is achieved without interference from the neighboring CZZsaturated ester. The sharp peaks seen in both frames in Figures 7 and 8 for methyl arachidonate are well suited for area measurements. Figure 8 shows the result of analyses of samples I containing methyl acetals. The CB and Ca methyl acetals are separated in the way usually seen with polyester columns. Figure 9 shows a scparation of a mixture which included Clot Clz, and Cp4 saturated components and also methyl nervonate (Cz, monoene). The time required for a scan which n-ould show a Clocomponent and ending with a C21 component is about 45 minutes.
CEPHALINS
!I
RELPTIVE C NUMBER MIN 0 - ’
T$MPIO0
Figure 8.
130
18
‘
20
22
24
30
15
160
45
190
220
Gas chromatographic analyses of fatty acid methyl
esters and fatty aldehyde methyl acetals from human serum
capha lins Instrument and conditions same as for Figure 7. Peaks seen immediately before palmitate and stearate ore CIS and CIS aldehyde methyl acetals
under thc:,e circumstances the calculation of niais relztionships from a recorder chart is usual!y easy. The ditficuities a.jsocinted with making calculations from a record shon-ing low late peaks are not, present. Unfortunately isothermal and temperatureprogrammed conditions are not interchangeable in the sense that the same column. and the same detection system, will gil e equivalent results under the two conditions. l\Iore often than not, a column suitable for >
> >
I
AN EARLIER REPORT from this laboratory ( 2 ) a systematic study was undertaken to determine the extent of the response elicited by certain model halogenated organic compounds from an electron capture detector when used in conjunction with the gas chromatographic technique. At that time, although only simple aliphatic halogenated substances were investigated, i t was theorized that this ancillary method (electron capture spectrometry) could be greatly extended by utilizing appropriate halogenated derivatives of more complex molecules to enhance significantly the limits of detection of these substances by this means. The important field of steroid analysis by gas chromatography was thought to provide N
