Chromatographic and spectroscopic characteristics of the selected stearic acid derivatives

MICROCHEMICAL

JOURNAL

Chromatographic

18,

207-2 14 ( 1973)

and

of the Selected

Spectroscopic Stearic

Acid

Characteristics Derivatives

J. SLIWIOK, T. KOWALSKA, J. ~ZEPA AND A. BIERNAT

Studies on the reaction of autoxidation of cis- and tvans-9-octadecenoic acids and their methyl esters are directly connected with the problem of identification of the received products (2, 3). Among these products one may name some stearic acid derivatives (epoxides, hydroxycompounds). Our endeavors* purposed to give chromatographic and spectroscopic characteristics of standard samples of stearic acid derivatives: cis- and trans-9,10-epoxystearic acids and their methyl esters, and erythro- and thrro-9,10-dihydroxystearic acids and their methyl esters. EXPERIMENTAL

A. Thin-Layer

METHODS

Chromatography

The glassplates (20 X 20 cm) were covered with a 0.3 mm thick Kieselgel H (E. Merck, Darmstadt) layer by means of a Shandon TLC equipment, then dried at room temperature for 24 hr and at last activated at 1 10°C for 30 min. Thus prepared plates were stored in a desiccator filled with P,O,. On these plates our samples were developed, with every sample in 5 ~1 (79 pg) of dimethylformamide solution. The mobile phase was composed of petroleum ether, dioxane and formic acid in a volume ratio of 76.5: 22.0: 1.5. The chromatograms were evoked with a 5% solution of ammonium molybdate in 20% sulfuric acid (2). The results are shown in Table 1. The examined compounds were in this way separated into four groups: epoxystearic acids, their methyl esters, dihydroxystearic acids and their methyl esters. Both groups of esters essentially differ from the acids in respect of their R, values. The AR, is higher in the case of epoxy- than dihydroxystearic compounds. This phenomenon was observed as a general regularity. Applying silicic acid (Calbiochem) (I) activated at 80°C for 1 hr as a chromatographic layer one separated cis- and trans-9,l O-epoxystearic acid methyl esters and erythro- and threw-9,l O-dihydroxystearic acids. Cis- and trcozs-9, IO-epoxystearic acid methyl esters 207

208

SLIWIOK

ET

AL.

were separated with the help of a mobile phase composed of petroleum ether, ethyl ether and formic acid in a volume ratio of 78.5 : 19.5 : 2. Separation of erythro- and threo-9, IO-dihydroxystearic acid was conducted with a mobile phase petroleum ether-ethyl ether (vol ratio, 45.5 : 54.2). B. Gas Chromatography

Higher fatty acids and some of their derivatives (epoxy-, hydroxyand other) cannot be directly determined by means of gas chromatography, but only as more volatile substances. The most frequently chosen way is esterification of acids with methanol or synthesis of trimethylsilyl derivatives. For analytical purposes 1% solutions of the discussed substances were prepared in dimethylformamide. Preparation of trimethylsilyl (TMS) derivatives: 0.03 g (about 1O-4 mole) of suitable methyl 9,10-dihydroxystearate was treated with 1 ml of mixture composed of 0.6 ml of pyridine (anhydrous, distilled over KOH), 0.2 ml of hexamethyldisilazane (HMDS) and 0.2 ml of trimethylchlorosilane (TMCS). This reaction was carried in a 5 ml flask. The contents of the flask was shaken for 15 min, then the precipitated ammonium chloride was decanted, and thus obtained trimethylsilyl diether of methyl 9, lo-dihydroxystearate was stored in a desiccator in an atmosphere of dry nitrogen, in small flasks with silicone membranes. The chromatographic analysis was run with the help of a Chrom-4 gas chromatograph produced by Laboratorni Pristroje (Czechoslovakia). The characteristics of columns and conditions of analysis: Column 1: 10% EGSS-X on Gas-Chrom Q 100/120 mesh, column length 200 cm, column diameter 0.3 cm, working temperature 180°C carrier gas Ar (flow rate 50 ml/min), FID. Column 2: 2% SE-30 on Chromosorb P-AW-DMCS 60/80 mesh, column length 300 cm, column diameter 0.3 cm, working temperature 250°C carrier gas Ar (flow rate 50 ml/min), FID. As it proceeds from the obtained results, the separation of methyl cis- and trans-9, IO-epoxystearates and silyl derivatives of methyl erythro- and threo-9,10-dihydroxystearates is better on the polar phase EGSS-X, which enables almost complete separation of these stereoisomers. One did not get the satisfactory results with methyl erythro- and threo-9, lo-dihydroxystearates on the polar phase EGSS-X, because their times of retention are too long and the obtained peaks are not suitable for analytical characteristics.

NUMBER VALUES OF

6.05 38.5 9.65 84.0

fl Analabs (USA). h A = kieselgel H, mobile phase: petroleum ether-dioxane-formic acid (vol ratio. 76.5:22.0: I .5); B = silicic acid, mobile phase: petroleum ether-ethyl ether-formic acid (vol ratio, 78.5: 19.5:2); C = silicic acid, mobile phase: petroleum ether-ethyl ether (vol ratio, 45.5:54.5).

-

9.12 6.21

9.34 10.4

-

-

2.94 2.72

I -

t,, St-Me

II<

SE-30

89.5

-

0.35 -

-

0.19 0.28 0.27

thrro-9, IO-Dihydroxystearic acid three-9,10-dihydroxystearate Methyl rrythro-9, IO-dihydroxystearate Methyl erythro-9,lO-dihydroxystearate ditrimethylsilyl ether Methyl thwo-9, IO-dihydroxystearate ditrimethylsilyl ether -

-40.5

18.9 -17.4

6.4

1 700 -4.65

t, (min)

tH St-Me

COMPOUNDS

59.5 58.4 39.7

43.5

7 0.26

- 8.7

tl, (mm)

t,

EXAMINED

EGSS-X

OF THE

F79 0.86

C

TIMES

0.39 0.88 0.87 0.16

1

RB

I

RETENTION

TABLE AND

trcrns-9, IO-Epoxystearic acid Methyl cis-9 ,I()-epoxystearate Methyl trww-9, IO-epoxystearate acid ery~kro-9,10-Dihydroxystearic

COEFFICIENTS

tY41

R,

MethylIO-Epoxystearic stearate (St-Me)acid cis-9,

Chemical compound”

THE

2 v,

B F: 5

b 0’

z3 z

2

210

.tiLIWIOK

C. Infrured

Absorption

ET AL.

Spectroscopy

The ir spectra of cis- and trans-9,10-epoxystearic acids and their methyl esters were run for the 0.25 mol/l solutions of these compounds in carbon tetrachloride. The measurements were performed on a double-beam UR-20 spectrophotometer of Zeiss-Jena manufacture (East Germany) in the range from 700 to 3800 cm-‘. In the measurements use was made of NaCl trays (thickness 0.15 mm) and NaCl prism. Comparing the spectra of cis- and truns-9,10-epoxystearic acids and their methyl esters one was concentrated on the position and shape of a band of symmetrical valency vibrations of oxirane ring (so called “respiration” of ring) and of a band of antisymmetrical valency vibrations of this ring. In the case of the discussed acids band of symmetrical valency vibrations of oxirane ring is badly separated from neighboring bands, especially from more intense one at 1287 cm-‘, ascribed to the valency vibrations of C-O formation. For both acids maximum of this band falls at about 1257 cm-‘. Therefore there is no practical difference in the position of this band, but with the trans-isomer one observes slightly better separation of the maximum (Fig. 1). Methyl esters of cis- and tram-9, IO-epoxystearic acid characterize with better separation of the band of symmetrical valency vibrations of oxirane ring from neighboring bands. In their case the discussed band appears at 1252 cm-’ (Fig. 2).

4400

4200

43cxl

f4oo Wavenumber

a FIG. 1. The position of band of symmetrical ir spectra of cis- (a) and rrans-9,10-epoxystearic

4400

mo

1300

4400

[cm-‘]

b valency vibrations acid (b).

of oxirane ring in the

STEARIC

f.700

4300

211

DERIVATIVES

-I

L ffO0

ACID

NO0

4400

kvenumber

fzm

4300

a

b

FIG. 2. The position

of band of symmetrical valency vibrations ir spectra of methyl esters of cis- (a) and trtrns-9. I O-epoxystearic

800

44oa

[cm-‘] of oxirane ring in the acid (b).

1 900

4000

8MJ

Wavenumber a FIG. 3a. The ir spectrum in the range 800-1000

000

mo

km-*] b

cm-’ for (,i.s-9,10-epoxystearic acid; the band of out-of-plane bending vibrations of associated O-H group (940 cm-‘) completely covers much weaker band of antisymmetrical valency vibrations of oxirane ring; (b) the ir spectrum in the range 800-1000 cm-’ for trtrns-9,10-epoxystearic acid: it shows badly separated bands of antisymmetrical valency vibrations of oxirane ring (896 cm-‘) and of out-of-plane bending vibrations of associated O-H group (940 cm-l).

212

SLIWIOK

ET AL.

The region of antisymmetrical valency vibrations of oxirane ring better differentiates spectra of the analyzed acids. In the case of cis9, IO-epoxystearic acid the band of antisymmetrical valency vibrations of oxirane ring is probably much weaker than the neighboring band of out-of-plane bending vibrations of O-H group associated with hydrogen bonds (0--H...O). In this circumstance it is not to be seen, covered with the O-H band with its maximum at 940 cm-‘. Only the lack of symmetry of the O-H band on the side of lower frequencies could confirm our assumptions. In the case of truns-9,10-epoxystearic acid the intensity of the band of antisymmetrical valency vibrations is of the same range as this of the band of out-of-plane bending vibrations of associated O-H group and in the ir spectrum one observes both m/m bands, though badly separated (Fig. 3). The ir spectra of methyl cis- and trans-9,10-epoxystearates in the range of antisymmetrical valency vibrations of oxirane ring are much simpler. It may be explained with the absence of the O-H band in esters. With methyl cis-9,10-epoxystearate one observes a weak absorption band at 887 cm-‘. which may be ascribed to antisymmetrical valency vibrations of oxirane ring. With methyl tvans-9,1 O-epoxystearate the absorption band of medium intensity is present at 896 cm-’ and it may be induced with antisymmetrical valency vibrations of oxirane ring (Fig. 4). The spectroscopic characteristics of erythro- and three-9,1 Odihydroxystearic acids and their methyl esters is far more difficult because of two reasons: they are insoluble in the solvents widely

L

Wavenumbtzr

a FIG. 4. The position of band of antisymmetrical valency vibrations of oxirane the spectra of methyl esters of cis- (a) and trans-9, IO-epoxystearic acid (b).

ring in

STEARIC

ACID

Wavenumber

lcm-‘I

a

Wavanumber C

213

DERIVATIVES

b

fCm+l d

FIG. 5. The ir spectra in the range 800-1350 cm-’ for: (a) rrlvrl2ro-9.10-dihydroxystearic acid. (b) 1/1~(,0-9.10-dihydroxystearic acid. Cc) methyl c,,:\tlrro-9,IO-dihydroxystearate, (d) methyl tllrro-9.10.dihydroxystearatc.

applied for ir purposes and in their case one observes cummulation of spectroscopic effects induced with the O-H groups of carboxyl and two vicinal hydroxyls in carbon chain. Therefore our considerations are confined to presentation of the “fingerprint” region in the ir spectra of analyzed substances. These spectra were run for the Nujol acids and their emulsions of ctythro- and threw-9, IO-dihydroxystearic methyl esters (Fig. 5) and are characteristic for every examined compound. The comparison of spectra shown in Fig. 5 permits some observations. Erythroand tlweo-9, IO-dihydroxystearic acids essentially differ in the range 900-970 cm-‘. It is the range of out-of-plane bending vibrations of dimeric hydroxyl groups. Diverse configuration of vicinal 9,10 hydroxyls in the case of both isomeric acids influences vibrations of different strength and character and it is reflected in the quoted range of spectra. In the same way structural diversities influence differences in the shape of absorption bands in the range 1240-1320 cm-‘, which is ascribed to the symmetrical and antisymmetrical valency vibrations of C-O formations, present in acids and alcohol groups. At last differences of the discussed spectra appear in the neighborhood of 1040 cm-’ ( 1048 cm-’ for er~~thro-isomer, 1038 cm-’ for three-isomer), which is probably induced with the deforma-

214

SLIWIOK

ET AL.

tive vibrations of O-H mainly vicinal) groups, which may give interand intramolecular associates. The above presented remarks generally concern methyl esters of isomeric acids as well. CONCLUSIONS

Thin-layer and gas chromatography proved to be efficient techniques in separation of the analyzed substances. Special attention should be payed to the separation of erythro- and threo-9,10dihydroxystearic acids by means of TLC and their methyl esters with GC. Owing to the TLC and GC one managed to separate methyl esters of cis- and tram-9,10-epoxystearic acids. Infrared absorption spectroscopy showed its usefulness especially to cis- and trans-9,10-epoxystearic acids and their methyl esters in the range of antisymmetrical valency vibrations of oxirane ring. The chromatographic and spectroscopic characteristics of the selected stearic acid derivatives enables further identificational investigations of autoxidation of oleic acid and its derivatives. SUMMARY Chromatographic and spectroscopic characteristics were determined for the following stearic acid derivatives: cis- and ~rans-9,10-epoxystearic acids and their methyl esters, and erythro- and threo-9,10-dihydroxystearic acids and their methyl esters. It will be helpful in the further identiticational investigations of the autoxidation process of oleic acid and its derivatives. REFERENCES I. Morris, L. J., Holman, R. T., and Fontell, K., Naturally occurring epoxy acids. I. Detection and evaluation of epoxy fatty acids by paper, thin-layer and gas-liquid chromatography. J. Lipid Res. 2, 68-76 (1961). 2. Sliwiok, J., and Kowalska, T., The application of thin-layer chromatography to the investigation of oxidation process in oleyl alcohol and oleic acid. Microchem. J. 16, 359-366 (1966). 3. Sliwiok, J. Kowalski, W., and Wasielewska, A., Chromatographic and spectroscopic investigations of the oxidation process in methyl oleate and methyl elaidate. Microchrm. .I. 17, 576-587 (1972).