saved. The total time required for a determination is therefore usually less than 30 minutes. The results obtained on a wide variety of materials are shown in Table IV. As most of the above materials did not dissolve in the reaction mixture, the results cannot be considered as quantitative. However, they give a reasonably good indication of levels of alkoxy group contents and in many cases could provide rapid and reliable means of identification of certain of the above materials, especially woods. I n the case of wood, the methoxy groups are derived almost entirely from lignin; the procedure could therefore provide useful information as t o
the purification degree of paper pulp and lignin content of cooking liquors and by-products in the productions of paper and pulp. As a further check on the method and a possible application to the determination of free alcohols, the effect of diluting the hydriodic acid with water in the reaction mixture on the results was tested. The values obtained with methanol and ethanol (Table V) indicated t h a t methanol could be converted to the iodide a t a reasonable rate even with a solution containing 34% HI (3 ml. 57% HI 2 ml. water); ethanol required higher concentration of HI (46% or 4 ml. 57% HI 1 ml. water), but the addi-
+
+
tion of concentrated sulfuric acid increased considerably the efficiency of conversion. The procedure is therefore potentially suitable for the detection and determination of alcohols in dilute solutions. LITERATURE CITED
(1) Haslam, J., A. R., Analyst ( 2 ) Kratzl. K., Chem. 89, 618
Hamilton, J. I3., Jeffs,
83, 66 (1958).
Gruber, K., Monatsh. (1958). (3) hlartin, F., Vertalier, S., XVth International Congress on Pure and Applied Chemistry (Analytical Chemistry), Lisbon, September 8 to 16, 1956. (4)Miller, D. L., Samsel, E. P., Cobler, J. G., ANAL.CHEW33,677 (1961). RECEIVEDfor review June 29, 1962. Accepted August 29, 1962.
Acylated Cyclodextrins as Stationary Phases for Comparative Gas Liquid Chromatography HERMANN SCHLENK, J. L. GELLERMAN, and D. M. SAND The Hormel Instifute, Universify o f Minnesota, Austin, Minn. b 0-Cyclodextrin acetate, propionate, butyrate, and valerate have been used as stationary phases for gas liquid chromatography (GLC) of cyolefins, alcohols, aldehydes, esters, aldehyde-esters, and diesters. The retention times of homologs follow the rule of logarithmic linearity. The shifts of retention times of a compound from phase to phase facilitate its classification and tentative identification. Esters of fatty acids having one, two, or three side methyl groups have been chromatographed and the phases compared for separation of such isomers. The heat stability of (3-cyclodextrin esters makes them very suitable for comparative GLC.
T
HE advantage of gas liquid chromatography (GLC) with phases of different polarity has been pointed out in numerous cases where mixtures of different chemical types or closely related compounds were to be separated and tentatively identified. Retention time is regulated, in part, by the relat i r e polarities of sample and stationary phase. Therefore, shifts of retention times of a compound on different polarity phases are characteristic for that compound and its class. Conclusions dran n from these data are particularly important for those components of mixtures which are not more fully characterized. The more subtle differentiation of closely related compounds, like isomers, is not as obviously connected with polarity but still is aided b y com-
parative chromatography where superpositions on one phase may be resolved by another. It is of advantage in comparative GLC to use phases t h a t are heat stable, so that column performances are reproducible and constant. Further advantages of heat-stable phases are in better exploitation of the sensitivity of detectors and in preparative GLC. The stationary phases which are commonly used in high temperature GLC are polymers-Le., neither their melting points nor molecular weights can be defined precisely, and their preparation and quality are difficult to control. Accordingly, the heat stability of these phases may vary from batch t o batch and their performance may change considerably with time a t temperatures higher than 180" to 200" C. The use of cyclodextrin (CDX) esters for GLC of fatty esters has been reported and the stability of p-CDX acetate at 236" C. has been emphasized ( I O ) . This investigation has been extended t o cover the homologous series, p-CDX acetate to valerate. The stability of these carbohydrate esters was very satisfactory a t 220" C. in the analytical experiments reported here and a t 236" C. in preparative GLC. Because of their different polarities, these esters are promising phases for comparative chromatography. a-Olefins, alcohols, aldehydes, methyl esters, dimethyl esters, and aldehyde methyl esters have been chromatographed and their retention on the different phases has been
compared. Furthermore, the efficiency of the phases for separating isomeric fatty esters has been evaluated. Some results with p-CDX ester phases in preparative GLC have been outlined ( 5 ) , and the procedure will be detailed elsewhere (11). EXPERIMENTAL
Materials. T h e compounds to be chromatographed were obtained from commercial sources or from T h e Hormel Foundation, or were prepared in this laboratory. Aldehydes and aldehyde esters were obtained b y ozonization reduction of authentic unsaturated f a t t y esters. Some branched C17 acid methyl esters are listed in Table I with references to the method of their preparation.
Table I.
Branched C l i Acid Methyl Esters
2-Methylhexadecanoate 3-Methylhexadecanoate 7-Methylhexadecanoate 14-Methylhexadecanoate 15-Methylhexadecanoate
3,6-Dimethylpentadecanoate 2,14-Dimethylpentadecanoate 3,6,13-Trimethyltetradecanoate 9,lO-Methylenehexadecanoate
p-CDX and its esters were prepared essentially as described by French for the carbohydrate and its acetate ( 3 ) . b-CDX propionate and butyrate were purified by repeated crystallization from ethanol. The valerate was used VOL. 34, NO. 12, NOVEMBER 1962
0
1529
as a brittle resin after removal of valeric acid from the crude ester b y pouring its concentrated acetone solution into water, and by heating the dried material in vacuum. Crystals appeared, embedded in the resin, after several months, but a final melting point cannot be given yet. Difficulties were encountered in preparing pure p-CDX acetate of the reported melting point of 201" C. ( 3 ) : as the material is often converted into a form melting at 223" C. in the course of purification. The forms are equally suitable as stationary phasee. All preparations yielded correct combustion values and, according to their infrared spectra, they do not contain free hydroxyl groups. It is essential for their stability that they be free of acid. Such contamination can be detected and quantitatively determined by titrating the crude esters in acetone solution m-ith aqueous KOH. Besides analyses and melting points, heat tests are expedient for evaluating the purity and stability of the compounds to be used as phases. The heat test is carried out by immersing a micro test tube with about 100 mg. of p-CDX ester into a silicone bath a t 230" C. The temperature is raised to 245" C. in 15 minutes, during which time pure esters stay virtually colorless. Solvents of crystallization like ethanol, toluene, acetone, and ethyl acetate evaporate without causing decomposi-
Table
II.
I
a!
,~ +
8-CDXpropionate,
C106Hl64056
8 - C D X butyrate, C126H196058 8-CDX valerate, C1&38066 0 Two different forms.
BUTYRATE
x
.
I
12
10
14
8
I8
20
CARBON ATOMS IN A C I D S
tion, while organic acids cause rapid discoloration. Table I1 lists the 0-CDX esters, their formulas, melting points, and carbonoxygen and hydrogen-oxygen ratios for evaluation of their relative polarity. Columns. T o enable practical comparison of phases at one temperature, columns of different sizes and percentages of phase on carrier were prepared. Ten grams of p-CDX acetate were dissolved in 50 ml. of acetone and mixed with 40 grams of Chroniosorb R,30 to 60 mesh (20% coating). Most of the solvent was distilled off on a rotary evaporator a t 170" to 80" C.
P-Cyclodextrin Esters
Ratios Mp., "C. 201, 223" 169 125
Resin
c/o 1.50 1.88 2.25 2 63
H/O 2 00 2 75 3 50 4 25
Table 111.
Methyl ester
XI0
GLC of Unsaturated Esters on P-CDX Esters Equivalent chain length - Component resolution Acetate Propionate Butyrate Acetate Propionate Butyrate
16.80 (18.00) 18.55
14.25 (18.00) 18.15
13.20 (18.00)i 18.00
Linoleate
19.30
18.50
Linolenate
20.15 20.00 19.55
19.00 19.00 18.75
18.55 18.65
20.15 18,45
19.00 18.15
18.55 18,OO
(17.00)
(17.00)
17.60
17.25
Stearate Oleate
X*a
y-Linolenate
Linolenate Petroselinate n-Hept adecanoate 9,lO-Methylenehexadecanoate a
1,36
0.77
0 00
18.25 ,
1.92
1.45
1.24
i
2.03
1.80
1.66
)
0.75
1.01
1.39
1.57
0.74
0.50
')
(17.00)\ 17.10
Peaks arising from autoxidation of linolenate.
1530
ANALYTICAL CHEMISTRY
'
Figure 1. Retention times of normal saturated fatty acid methyl esters on p-CDX esters
2018 2312 2606 2900
C84H112066
ESTERS
00
Mol. weight 8 - C D X acetate,
I f.CYCLODEXTRlN
1
without suction. Evaporation was then continued, with heat at about 20 mm. and finally at 2-mm. pressure. An aluminum tube, 10 feet long and inch o.d., was packed under mechanical vibration with 33 grams of this material. The column was coiled and vibrated again. One-half gram of 8-CDX propionate was dissolved in 2.5 nil. of acetone and mixed with 2.0 grams of Chromosorb K, 60 t o 100 mesh (207, coating). The solvent was removed under the precautions described above. -In aluminum tube, 8 feet long and '/,inch o.d., vias packed nith 1.5 granis of the material. A colunin with p-CDX butyrate was prepared as described for the propionate, but required 1.9 grams of material (207, coating). 8-CDX valerate was applied a t a level of 15% to Chromosorb IT, 60 to 100 mesh, and 1.3 grams of the material packed into a column of 8-foot length and '/*-inch 0.d. (157, coating). All columns ivere tempered at 220" C. under a flow of He overnight. Their efficiencies expressed as number of theoretical plates for stearate ( I , 8) were, in the order of the above description, 1300. 2220, 3550, and 4550 a t 220" C. The heat stability of this type of phase has been evaluated previously by condensation of bleed material (10). Some further data nere determined with a column of 1/4-inch X 11-foot aluniinuni tubing, packed with 207, 8-CDX acetate on Chromosorb R, 35 to 60 mesh, which was operated a t 236" C. under a flow of 60 cc. of He per minute. The column was timed without an initial heating period and, after 20 hours, had the following characteristics with methyl stearate: retention time, 22.3 minutes; theoretical plates 1900; column resolution (8) for stearate-oleate, 1.54 -4fter use for an estimated 700 hours a t 236" C. the corresponding values were 20.7, 1950, and 1.51. The heat stability of p-CDX ester phases also became evident when a Hz flame detector functioned with these phases a t 220" C. for 16 months without requiring any cleaning. Furthermore, contaminants from the phases cannot be detected in fractions from preparative GLC a t 236" C. Chromatography. -4 Beckman GC-2 apparatus with a H2 flame detector was used. T h e temperature was 220' C. for all experiments; the gas flow was 75 cc. of H e per minute n-ith the p-CDX acetate column inch 0.d.) and 15 cc. of H e per minute with the other columns inch o.d.), the inlet pressure being 45 pounds in all cases. Mixtures containing about 1 wg. of each component dissolved in Skellysolve F gave suitable scale deflections of the recorder a t a n attenuation of 2 X lo2, without t h e base line showing unsteadiness or drifting. rl Brown 1-mv. recorder with a 1-second full span response n a s used a t a chart speed of 1 cm. per minute
P-CDX ACETATE
3,6,l3-TRI-,3,6-DI~,2,.14-DI;2-,3-,7-,15-,14-,n-C~~,
n
"
n
P ,
8
6
IO
12
14
16
18
RESULTS
The chromatographic data are presented as equivalent chain lengths (ECL) (9, I S ) . These values are little affected by amounts of phase, flow rate, or other operating conditions, and, therefore, reflect best the affinity of a compound for different phases, relative t o standards. The E C L of a compound is found graphically by referring its retention time t o the line connecting the log retention times of a homologous series of compounds n hich have been chromatographed under the same conditions. The methyl d e r s of iiorinal saturated fatty acids were chosen as reference for all compounds. Their log retention times plotted against number of carbon atoms are very close t o a straight line for all @-CDXester phases, as seen in Figure 1. The effect of aging upon retention times during the serial runs was ne11 within the limit of reproducibility of individual determinations, so that recalibrations m r e not necessary. The equivalent chain lengths are presented in Figure 2 for comparison of classes and phases. Some data are omitted from Figure 2 for clarity. The values for aldehyde esters on butyrate nearly coincide with the values on propionate. Yalues for dimethyl esters on butyrate are slightly above, and on propionate they are slightly below those for aldehyde esters on valerate. Aldehydes on valerate emerge too fast at 220" C. for precise measurement. The peaks of alcohols on valerate showed trailing; this can be greatly improved b y using commercial silanized Chromosorb.
40
Figure 3.
Table. I11 and IV list E C L of unsaturated esters and of isomeric heptadecanoates. As E C L does not indicate the quality of separations, the latter is expressed as column resolution (8) or component resolution (2) according to the term, 2Ay/(y, ybl. Ay is the distance between two peak maxima and ya and y b are the lengths of the base lines under the peaks, found by the usual triangulation. The values have been calculated either from peaks which were completely separated or from chromatograms of single substances by superposition (8). Actud chromatograms are illustrated
in Figure 3, where all the compouiids listed in Table IV Fyere mixed and chromatographed on 8-CDX acetate and valerate. Such mixtures have not occurred in our studies on fatty esters from natural sources, but n-heptadecanoate, 15-methyl- and 14-methylhexadecanoate, and similar mixtures had been encountered in fractions of fish oil esters.
+
Methyl ester 3.6.13-Trimethvltetradecanoate
DISCUSSION
The polarity of the phases decreases in the homologous series of p-CDX esters, while the retention times of normal saturated esters increase. The reversion of the lines for 8-CDX butyrate and valerate in Figure l is easily explained by the different percentages
GLC of Isomeric Heptadecanoates on /3-CDX Esters Acetate Propionate Butyrate __.____ Valerate ECLa R b ECL ECL R ECL R 14.i5
3,6-Dimethylpentadecanoate 15.15
__
2,14-Dimethvlpentadecanoate 15.25
2-JIeth~-lhesadecanoate
15.70
3-Methylhexadecanoate
16.00
7-Methylhexadecanoate
16.30
15-hlethylhexadecanoate
16.55
14-hlethylhexadecanoate
16.80
n-Heptadecanoate
00
GLC of isomeric C" acid methyl esters
X Valerate
Table IV.
I 50
MINUTES
Figure 2. Equivalent chain lengths of ru-olefins, alde hydes, alcohols, aldehyde esters, and diesters in reference to fatty acid methyl esters
0 p-CDX acetate
30
2@
CARBON ATOUS
Phases (as in Figure 1) 0 Propionate -k Butyrate
I --
Lp---
20 4
n-C,e
(17.00)
n-Octadecanoate (18.00) a Equivalent chain length. b Component resolution.
0.81 0.11 0.96 0.71 0.69 0.52 0.49 0.58 2.38
15.10 15,50 15.60 16.00 16,20 16.40 16.65 16.75 (17.00) (18.00)
1.07 0.32 1.21 0.67 0.60 0.85 0.49 0.95 3.77
15,20 15.GO 15.70 16.10 16.30 16.40 16.65 16.75 (17.00) (18.00)
1.66 0.60
0.91 0.93 0.i5
1.25 0.54 1.50 5.56
15.20 15.50 15.75 16.10 16.30 16.40 16.70 16.80 ( l i .00)
1.57 1.29 2.25 1.21 0.66 1.69 0.58
1.84 -~ 6.32
(18.00)
VOL. 34, NO. 12, NOVEMBER 1962
1531
Table V.
Comparative GLC
[Example: Peak ECL (acetate) 12.91 Diester ECL (acetate) = 12.9 can be ECL (propionate) of these compounds should be i E CL
Aldester
0
or-Olefin
C6
C5.25
c11.4
c 1 1 . 7
ClZ.9
CIS g
10.7 -2.2
9.8 -3.1
12.3 -0.6
11.8 -1.1
12.9
14.3 +1.4
of coating that had been applied. Figure 2 shows that ECLs, when plotted vs. chain lengths, are very close to straight lines for homologous series of compounds. I n accord with the identical increments of CH2 groups, the slopes are nearly equal t o that of the reference esters. A marked drviation from this slope was found only with aldehydes on butyrate. Hydrocarbons are the only compounds which emerge faster than the esters of corresponding chain length. Alcohols and aldehydes are eluted somewhat more sloivly, and dimethyl esters and aldehyde esters much more slowly than the fatty esters of comparable chain length. The ECL of a n olefin is highest on 8-CDX valerate-Le., the least polar phase-while the ECL of the other compounds is highest on acetate, the most polar of the phases. As expected, the equivalent chain lengths of a compound increase or decrease between these extremes in the order of the homologous phases. The only exception was found with aldehyde esters, where the sequence is ECL acetate>valerate>butyrate ?propionate. The increments of the ECL from phase t o phase are different for different type compounds, so that assignment of class and chain length can be made by comparative chromatography. Assume an unknown peak having ECL (acetate) 12.9. The scheme shown in Table V can be derived from Figure 2 for its tentative identification after recognizing the corresponding peak and determining E C L (propionate) in a chromatogram on 8-CDX propionate. Cornparatiye chromatography on more than two phases may further ascertain the conclusions. Fractionals, as listed in the first line of Table V, may occur with compounds of any class due t o unsaturation or isomerism, when chain lengths are derived from ECL. Some other knon-ledge about the sample or a microhydrogenation followed by GLC d l prevent misinterpretation due to unsaturation. In regard to branching, it appears t h a t with isomeric long-chain fatty esters the shifts of ECL from phase to phase usually are smaller than those due to functional groups. The line of log retention time us. 1532
Alcohol Aldehyde Ester
ANALYTICAL CHEMISTRY
...
chain length can be constructed precisely using three or four homologs to characterize a phase with respect t o that particular class. Characterization of phases by several classes of compounds permits more exhaustive analysis of a particular mixture and in this way contributes to the ultimate identification of components. The procedure of comparative chromatography is aided by the stability of 6-CDX ester columns and has become routine in this laboratory. During the synthesis of branched fatty esters, mixtures of compounds resulting from Kolbe electrolyses were analyzed in this manner. Aldehydes and aldehyde esters formed by ozonization of unsaturated fatty acid esters and subsequent hydrogenation were identified. A peak nei$ly detected in chroniatographing concentrates of unsaturated C17 esters of fish oil acids could be recognized as a compound more polar than the other fatty esters. It probably represents an autoxidation product (cf. Table 111, compound XI), rather than one of the isomeric CI7esters u-hich 11-ere the topic of that investigation. Separation according to unsaturation is best on the most polar phase, 8-CDX acetate. The separation of stearate and oleate is about equal t o that on hutanediol succinate, but it is not as good as on diethylene glycol succinate when compared in similar columns. Horn-ever, this can be compensated for by lengthening the column without losing the advantage of stability or, u-hen fractions are collected, of their purity. The less polar p-CDX ester phases are not suitable for separating stearate and oleate, but are efficient enough t o separate esters of higher unsaturation. I n addition, the ECLs in Table 111 show that superpositions on one phase-for example, n-nonadecanoate and linolenate on p-CDX propionate-are resolved in opposite sequences on acetate and butyrate. The elution times of highly unsaturated esters are long under the conditions which were chosen as standard here. Arachidonate and 4,7,10,13,16docosapentaenoate emerged from the propionate column after 102 and 198 minutes, respectively. The elution times from an 11-foot X ‘jr-inch
column, with 20% 0-CDX acetate on Chromosorb a t 236” C. and a flow of 50 cc. of He per minute, n ere 54 and 151 minutes for the same esters. However, the respective times were 30.5 and 69.5 minutes with the same column a t a flow rate of 120 cc. of He per minute with other conditions unchanged. Separation and resolution of homologous f a t t y esters are best on p-CDX valerate (Table ITr) and, accordingly, this phase is best for analysis of mixtures containing structural isomers. From the values for niethylhexadecanoates, it beconies likely that all isomers of this type have ECL within about one unit or less on the same phase, between extreme values of %methyl- and 14methylhexadecanoate. The only irregularity in the sequence of the five isomers in regard to the position of the methyl group is the reversion of 14and 15-methylhexadecanoates. This is in agreement with other reports ( I S ) . Multiple methyl branching lowers the ECL further. From Figure 3 it is seen that p-CDX valerate resolved the mixture of all isomers n-e had available. It would not, however, resolve more complex combination.. of iconiers under these conditions. ACKNOWLEDGMENT
We thank J. Chipault and G. 3Iizuno for the infrared spectra and G. Rajan and 11.Flom for preparations of cyclodextrin and its esterq. LITERATURE CITED
( 1 ) Desty, D. H., committee, in “Vapor
Phase Chromatography,” p. XIII, D. H. Desty, ed., Butterworth’s, London, 1957. (2) Farquhar, J. W., Insull, ’A7., Rosen, P., Stoffel, W., Ahrens, E. H., Xutr. Rev. 17, No. 8, 1, suppl. (1969). (3) French, D., Aduan. Carbohydrate Chem. 12, 189 (1957). (4) Greaves, W. S., Linstead, R. P.,
Shephard, B. R., Thomas, S. L. S., Weedon, B. C. L., J . Chenz. Soc. 1950,
3326. (5’1 Haines. T. H.. Aaronson. S..Gellerman, J. L., Schlenk, H., A’alure 194, 1282 (1962). (6) Horner, L., Spietschka, E., Chenz. Ber. 8 5 , 225 (1952). (7) Hunig, S., Lendle, W., Ibzd., 93, 909 (1960). (8) Martin, A. J. P., committee, in . Gas
Chromatography,” p. XI, D. H. Desty, ed., Butterworth’s, London, 1958. (9) Miwa, T. K., Mikolajczak, K. L., Fontaine, E. R., Wolff, I. 8.,- 4 ~ 4 ~ . CHEM.32, 1739-42 (1960). (10) Sand, D. M., Schlenk, H., Ibzd., 33,
1624 (1961). (11) Schlenk, H., Sand, D. &I., lbzd , 34, 1676 (1962). 112’1 Simmons. H. E.. Smith. R. D..’ J . ‘ Am. Chem. koc. 81,’4256 (1959). (13) Woodford, F. P., van Gent, C. XI., J . Lipid Res. 1, 188 (1960).
RECEIVEDfor review June 7, 1962. Accepted September 4, 1962. Work supported by research grants from the Xational Institutes of Health (RG-8824 and RG7917,) and by The Hormel Foundation.
