634 Analyst, October, 1967, Vol. 92, pp . 634-638

Thin-layer Chromatography of Polyglycerols

BY M. S. J. DALLAS AND M. F. STEWART ( Unilever Research Laboratory, The Frythe, Welwyn, Herts.)

A simple thin-layer chromatographic technique is described for separating commercial polyglycerol products into their various components. Two slightly different procedures have been developed, the first of which is suitable for carrying out separations according to molecular weight or chain- length, and the second for carrying out separations of branched and cyclic isomers of the lower molecular-weight polyglycerols.

POLYGLYCEROLS, which consist of chains of glycerol molecules joined by ether linkages, are prepared commercially by the alkali-catalysed thermal dehydration of glycerol. This process yields a mixture of components with varying chain-lengths, the average chain-length depending on the temperature and duration of heating. The usual methods of production control involve the measurement of a bulk property, e.g., the refractive index or hydroxyl value, and are thus of little value if detailed information about the composition of the mixture is required.

The fatty-acid esters of these polyglycerol mixtures constitute a versatile class of emulsi- fiers, covering the entire range of hydrophilic - lipophilic balance. Extensive feeding trials1 ,2

have shown that the esters are completely non-toxic and their use as food additives has been approved by the World Health Organisation and Food and Agricultural Organisation Expert Committee, and is permitted in the United Kingdom3 and the United States4 As the poly- glycerol esters are becoming more widely used, particularly in the food5 and pharmaceutical6 industries, it is desirable to have a method of characterising them. Although the degree of esterification and the nature of the fatty acids present can be determined by using routine procedures, satisfactory methods for the determination of the detailed composition of polyglycerol mixtures are not as readily available.

Because primary hydroxyl groups are more reactive than secondary groups, loss of water between the primary hydroxyl groups of two molecules to form a linear polyglycerol, I, may

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be expected to be the dominant reaction in the commercial preparation of polyglycerols. On the same basis, the formation of IIa (“branched” polyglycerol) should be less likely than that of I, and that of IIb less likely than that of IIa. The cyclic compound, IIIa, could be formed from IIa, but IIIb might be formed from either I or IIb. Before investigating any methods of analysis it was, therefore, necessary to prepare authentic samples of poly- glycerols of the types I, I1 and I11 to act as standards.

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DALLAS AND STEWART 635

The synthetic routes used in the preparation of the three diglycerols are described later. Type IIb would normally be expected to be formed only in very small amounts and

has not yet been synthesised in the pure form. As it is symmetrical there are no isomers of it, optical or otherwise, but it may be mentioned that there are two asymmetric carbon atoms (marked *) in type I, one in type IIa and two in types 111. Upon further condensation with glycerol, the linear diglycerol, I, may be expected to give mainly type I (linear) triglycerol, but diglycerol, IIa, whose primary hydroxyl groups are not equivalent, should give two isomeric branched triglycerols by condensation only of primary hydroxyl groups. Similar addition of a further glycerol unit to IIb, IIIa or IIIb would be straightforward, but addition of a fourth unit would be possible a t two different positions. Thus, even when addition by primary alcohol condensation only is considered, the number of possible isomers increases rapidly with increasing number of glycerol units. If the very minor components formed were not disregarded the analysis would be extremely difficult. The objective has therefore been to separate as many as possible of the normal linear polyglycerols, type I, and then most of the isomers of di- and triglycerol.

Paper chromatography has been quite extensively applied,' 3899910311 and the method of Zajic8 has given satisfactory results in this laboratory, but the greater speed, resolution and flexibility of thin-layer chromatography has made the latter technique worthy of careful attention. Good separa- tions have also been achieved here by using a gas - liquid chromatographic procedure, which is the subject of a separate publication.12

The thin-layer method of Seherl3 on kieselguhr impregnated with 0.66 per cent. sodium acetate has been found to be good, but not to be easily reproducible. A more reproducible method was therefore sought and has now been developed. The first of two similar procedures described below is designed to give a satisfactory separation of the higher linear polymers, and the second to give a satisfactory separation of the branched and cyclic isomers of the lower polyglycerols.

The chromatography of polyglycerols is similar to that of sugars.

ROUTES USED IN THE SYNTHESES OF THE REFERENCE COMPOUNDS-

Attention was first directed to the synthesis of the linear polyglycerols, I, because these were expected to be the major components of the polyglycerol mixtures. A survey of the literature revealed that, although various polyglycerol fractions had been isolated from commercial mixtures by fractional distillation of derivatives,l* 9 1 5 9 l 6 3 1 7 the only reported syntheses of individual linear polyglycerols by unambiguous routes were those of diglyceroll* and triglycerol.20 These syntheses involved the hydroxylation of 1-0-ally1 and 1,3-di-0- ally1 glycerol, respectively, and could not be readily extended to the synthesis of linear tetra- and higher polyglycerols.

We have found a series of reactions involving the condensation of a suitably protected P-toluenesulphonate with the sodium alcoholate of 1,2-isopropylidene glycerol, IV (R = Na), to be a convenient method of ascending the linear polyglycerol series.

Thus, linear 1,l'-diglycerol was synthesised by the condensation of IV (R = Na) with 1,2-isopropylidene-3-(~-toluenesulphonyl)-glycerol, IV (R = @-CH,.C6H,So2-), followed by removal of the protecting isopropylidene groups. This linear diglycerol was then converted into the derivative, V, by successive treatments with trityl chloride, benzyl chloride, hydro- chloric acid (to remove the trityl groups) and $-toluenesulphonyl chloride. The condensation of V (1 mole) with IV (R = Na) (2 moles), followed by removal of the protecting groups, yielded linear tetraglycerol. Linear hexaglycerol, octaglycerol, etc., are being prepared by subjecting the linear tetraglycerol to the same sequence of reactions. Similarly, linear tri- glycerol was synthesised by the condensation of IV (R = Na) (2 moles) with 1,3-di-(p-toluene- sulphonyl-2-0-benzyl glycerol (1 mole), followed by removal of the protecting groups. This series is being extended to the synthesis of linear pentaglycerol, heptaglycerol, etc.

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Iv V

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636 DALLAS AND STEWART : THIN-LAYER [Analyst, Vol. 92 Branched 1,2'-diglycerol, IIa, was prepared by the Woodward and Brutcher hydroxyla-

tion21 of 2-0-ally1 glycerol. The cyclic diglycerol, IIIa, which has been isolated from glycerol chlorohydrin preparations,22 was synthesised by using a modification of previously described methods.23 524

A detailed description of the experimental procedures used in the synthetic work will shortly be published elsewhere.

EXPERIMENTAL Of the two procedures described below, (b) is recommended for the separation of the

higher linear polyglycerols and (c), for the separation of the isomers of the lower polyglycerols (see Fig. 1).

PROCEDURE- (a) GeneraLCoat 20 x 20-cm glass plates with about an 0.25-mm layer of the relevant

adsorbent, leave them in a horizontal position for about 1 hour, then dry at 110" C for half an hour and store over anhydrous calcium chloride until required. Make up 2 per cent. w/v solutions of the test samples in ethanol and apply 1 or 2-p1 portions as small spots on a line 2 cm from the bottom edge of the chromatoplate. Make up the relevant developing solvent accurately and place a suitable amount in an all-glass tank, which should be lined with chromatographic paper. Develop the chromatograms by ascending technique at room tem- perature and allow 1 hour for the solvent to evaporate from the plate, before spraying with detecting reagent.

(b ) For separating higher linear polymers-Coat the plates with a slurry of 15 g of kiesel- guhr G (E. Merck, 8129) plus 15 g of silica gel (Whatman SG41) in 60ml of 0-5 per cent. aqueous sodium metabisulphite (Na2S,05) and dry as described in (a) above. Apply the samples as described above and develop the chromatogram in ethyl acetate - isopropyl alcohol - acetone - methanol - water (50 + 15 + 15 + 4 + 16). The development over 15 cm has normally taken 75 to 100 minutes, depending upon the grade of adsorbent and the temperature. A second development in the same direction gives a moderate improvement in the separation of the higher polymers.

(c ) For separating non-linear from linear isomers of the lower polyglycevols-Coat the plates with a slurry of 15 g of kieselguhr G (E. Merck, 8129) plus 15 g of silica gel (Whatman SG41) in 60 ml of 0.045 M calcium chloride, then dry them and apply the polyglycerol samples as described in (a) above. Develop the chromatogram in ethyl acetate - isopropyl alcohol - water (55 + 30.5 + 14.5). A single development over 15 cm has normally taken 75 to 100 minutes. Three developments in the same direction are worthwhile in this instance (compare plates B and C in Fig. l), the solvent being allowed to evaporate at room temperature after each development.

(d) Detection-Spray the plate (from either procedure (b ) or (c) above) fairly heavily with a fresh 0.5 per cent. solution of thymol in ethanol - sulphuric acid (95 + 5), then heat in an oven at 120" to 125" C for 1 hour. Polyglycerols (0-5 pg or more) show up initially as purple - black spots on a mauve background that fades on standing.

The above method, which was applied to sugars by A d a ~ h i , ~ ~ has been found the best on the basis of sensitivity, reproducibility and permanence. Certain other methods, among many tested, also gave satisfactory results. The spots could also be detected on the calcium chloride impregnated plates at room temperature with either a 0.16 per cent. solution of potassium permanganate in acetone (immediate yellow spots on a mauve background) or with a 1 per cent. solution of lead tetra-acetate in dry benzene (pale orange-yellow spots on a brownish orange background up to 24 hours after with 1,2-diols). On sodium hydrogen sulphite impregnated plates, a modification of the method described by Akita and Ikekawa26 gave clear permanent spots (slate-blue spots on a dull green background after spraying with a solution of 0-5 per cent. potassium permanganate in acetone, followed, after 15 minutes, by a 0.2 per cent. solution of bromophenol blue in ethanol).

RESULTS AND DISCUSSION

Typical separations obtained with the above procedures are illustrated in Fig. 1. In general, temperature, humidity and the ratio of kieselguhr to silica gel did not appear very critical, but it was found necessary to make up the solvent accurately with analytical-grade

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1 2 3 4 5 6 7 8 9 1 0 I I 12 13 14 15

A B C

Fig. 1 . Separation of polyglycerol by thin-layer chromatography on a 1 -k 1 mixture of kieselguhr and silica gel. lYate A, by procedure (b ) ; plate B, by procedure (c) and with single development; plate C, also by procedure (c), but with three developments in the same direction and with the same solvent. Strips 1, 6, antl 11, 1 , 2’-diglycerol, I Ia , (5 pg) +- cyclic diglycerol, I I Ia , (10 p g ) ; strips 2, 7, and 12, sample (20 pg) of polyglycerol prcparcd in the laboratory; strips 3, 8, antl 13, glycerol + synthetic linear di, tri and tetraglycerol (5 pg of each); strips 4, 9, and 14, sample (20 pg) of typical commercial polyglyccrol; and strips 5 , l O and 15, pure glycerol (6 pg).

Cyclic diglycerol, I I Ia , comes well above glycerol on each plate and cyclic diglycerol, I I Ib , not illus- trated, separates just below cyclic diglycerol, I I Ia , on thc calcium chloride impregnated plates

[To face p. 636

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October, 19671 CHROMATOGRAPHY O F POLYGLYCEROLS 637 reagents and, where relevant, to incorporate the correct amount of calcium chloride into the adsorbent layer. It may be added that there is some indication that the layer thickness is fairly critical for a given load per spot. As with most thin-layer chromatographic separations, care had to be taken to ensure good liquid - vapour equilibrium in the developing tank.

Strips 1 to 5 illustrate separations on a sodium hydrogen sulphite impregnated layer by using procedure ( b ) ; in the linear polyglycerol series it may be seen that glycerol itself (see strip 5) has the highest R, value. By using such a layer, separation is mainly according to the number of glycerol units in the molecule, the separation of the acyclic isomers from the linear molecules being much less than on a calcium chloride impregnated layer. The resolution of a typical commercial polyglycerol (strip 4) becomes rapidly worse above what is inferred to be heptaglycerol and is probably caused by the increasing abundance and variety of non-linear isomers as the molecular weight increases ; with 30-cm development (about 5 hours) in tall tanks it was just possible to distinguish a series of ten spots, starting from glycerol. Procedure ( b ) , therefore, gives a good indication of the molecular-weight range in a sample of polyglycerol. As, however, the physical and physiological properties of a polyglycerol mixture are likely to depend also on the relative amounts of non-linear isomers present, procedure (c), too, must be considered necessary for the analysis of a polyglycerol sample.

Separations with procedure (c) are illustrated by strips 6 to 15 in Fig. 1. The advantage of the multiple development technique described by Starka and Hamp12’ can be seen by comparing plates B and C. Strips 7 and 12 show a sample of polyglycerol prepared in this laboratory; when the latter strips are compared with strips 9 and 14 (typical commercial polyglycerol), the difference in relative abundance of certain isomers is clearly seen. The identity of the spot visible just above glycerol on strip 12 is still being sought; the spot also on strip 12 and just below the position of cyclic diglycerol, IIIa (strip l l ) , has the same RF value as cyclic diglycerol, IIIb. Of the two spots lying below glycerol, but above linear diglycerol, the lower one has the same RF value as 1,2’-diglycerol, IIa.

The rBle of the sodium hydrogen sulphite is not certain; its ability to form addition com- pounds with carbonyls is believed to be the reason for its effectiveness in the chromatography of sugars.25 Nor is the r61e played by the calcium chloride known; the fact that sodium acetate also improves the resolution of the isomers suggests that pH is not responsible, a conclusion supported by results on pH-gradient plates prepared with a gradient spreader. Incorporation of boric acid into the adsorbent has not been found to have any advantage.

A single procedure giving good resolution of the isomers, as well as of the higher polymers, has not been found possible. Further research here is now being directed towards the development of a quantitative thin-layer chromatographic procedure and studies will be undertaken to see how the quantitative gas - liquid chromatographic results compare with those obtained by such a procedure.

The authors are indebted to Miss M. McMullin, Miss G. Good and Mr. D. Knights for their assistance with the experimental work, and to Dr. I. P. Freeman for providing a sample of the cyclic diglycerol, IIIb.

1. 2. 3.

4.

5. 6.

7. 8. 9.

10. 11. 12. 13.

REFERENCES

Hodansky, M., Herrmann, C. L., and Campbell, K., Biochem. J. , 1938, 32, 1938. Babayan, V. K., Kaunitz, H., and Slanetz, C. A., J . Amer. Oil Chem. SOC., 1964, 41, 434. “Emulsifiers and Stabilisers in Food Regulations,” Statutory Instrument No. 720, H.M. Stationery

Federal Register, July 2, 1963, p. 6783; March 19, 1963, pp. 2675 and 2833; FdENgng, 1963,35,

Nash, N. H., and Babayan, V. K., Bakers’ Dig., 1963, 37, 72. Rabayan, V. K., Kaufman, T. G., Lehman, H., and Tkaczuk, R. J., J . SOC. Cosmet. Chem., 1964,

Wurziger, J., and Gebauer, W., Fette Seifen Anstr-Mittel, 1961, 63, 523. Zajic, J., Sb. Vys. s k . Chew.-Technol. Praze, 1962, 6 (3), 179. Wurziger, J., and Gebauer, W., Brot Geback, 1962, 16, 209. Tustanowski, S., Nowicki, R., Nowicka, I., and Zielinski, A. Z., Chemia Analit., 1964, 9, 623. Hartman, L., J . Chrounat., 1964, 16, 223. Barrett, C. B., Sen, N., and Keating, M., J . Gas Chromat., 1967, 5, 269. Seher, A., Fette Seifen Anstr-Mittel, 1964, 66, 371.

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638 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

DALLAS AND STEWART

Rangier, M., C.R. Hebd. Se'anc. Acad. Sci., Paris, 1928, 187, 345. Instin, M., A n n l s Fac. Sci. Marseille, 1940, 13, 5 ; Chem. Abstr., 1947, 41, 2392. Wright, H. J., and Du Puis, R. N., J . Amer. Chem. SOC., 1946, 68, 446. Wittcoff, H., Roach, J. R., and Miller, S. E., Ibid., 1947, 69, 2655. Siegel, H., Bullock, A. B., and Carter, G. H . , A%aZyt. Chem., 1964, 36, 502. Wittcoff, H., Roach, J. R., and Miller, S. E., J . Amer. Chem. SOC., 1949,71, 2666. Roach, J. R., and Wittcoff, H., Ibid., 1949, 71, 3944. Woodward, R. B., and Brutcher, F. V., Ibid., 1958, 80, 209. Gibson, G. P., J . SOC. Chem. Ind. , Lond., 1931, 50, 949. Battegay, M., Buser, H., and Schlager, E., C.R. Hebd. Siaanc. Acad. Sci., Paris, 1929, 188, 796. Summerbell, R. K., and Stephens, J. R., J . Amer. Chem. SOC., 1954, 76, 6401. Adachi, S., J . Chromat., 1965, 17, 295. Akita, E., and Ikekawa, T., Ibid., 1963, 12, 250. Starka, L., and Hampl, R., Ibid., 1963, 12, 347.

Received February 24th, 1967

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