Reports of the Academy of Sciences of the USSR

1. Vol. 149, No. 6

CHEMISTRY

L. D. BERGELSON, E. V. DYATLOVITSKAYA, V. V. VORONKOVA

DESCENDING THIN-LAYER CHROMATOGRAPHY OF POLYHYDROXY COMPOUNDS

(Presented by Academician M. M. Shemyakin, 13 XI 1962)

In connection with work in the field of macrolide antibiotics, we developed a method of ascending chromatography of polyhydroxy compounds on plates with a fixed layer of cellulose, which has a number of advantages

Fig. 1. Plate holder

over chromatography on paper (a considerable reduction in development time, the possibility of chromatographing smaller amounts of substances, smaller spot size) (¹). However, this method did not make it possible to separate certain mixtures of substances with very close \(R_f\) values (glucose—galactose, lyxose—ribose, arabinose—mannose, etc.). For the separation of such “critical pairs” we have now developed a method of descending flow chromatography.

Chromatography was carried out on plates coated with a fixed layer of cellulose (¹), in a glass chamber with a support made of glass or stainless steel, from which was suspended the trough customarily used in paper chromatography. A duralumin vertical holder for the plates was placed beside it (Fig. 1). The eluent was fed onto the plate through a strip of filter paper (previously wetted with the same eluent), one edge of which was placed in the trough, while the other was in close contact with the adsorbent layer. The plates were coated with a paste of cellulose powder, gypsum, and water, as described earlier (¹). After drying to an air-dry state (10–12 hr), the side edges of the plates were cleaned of adsorbent (5 mm on each side) so that the adsorbent layer did not come into contact with the metal surface of the holder.

The shape of the adsorption layer has a great influence on the resolving power of the plate. In the chromatography of carbohydrates, optimum results were obtained when the adsorption layer was given the form shown in Fig. 2. The substance was applied at a distance of 15 mm from the upper edge of the plate and at a distance of 10 mm from the edge of the strip of filter paper transferring the eluent to the plate*.

Fig. 2. Separation of carbohydrate mixtures. 1 — system: n-butanol—25% \(N\) \( \mathrm{H_4OH} \)—water (16:1:2); time 7 h, arabinose—xylose—ribose—rhamnose; 2 — system: n-butanol—pyridine—water (10:3:3); time 10 h; galactose—glucose—mannose—xylose—ribose—rhamnose. 3 — system: phenol—n-butanol—acetic acid—water (5:5:2:10) (upper layer, amount of phenol in grams) time 10 h, a — mannose—arabinose; b — lyxose—ribose. The separated substances are listed in the order of their distance from the starting line.

Optimum separation of carbohydrate mixtures with close \(R_f\) values was achieved in systems containing bases: 1) n-butanol—pyridine—water (10:3:3) (I) and n-butanol—25% \( \mathrm{NH_4OH} \)—water (16:1:2) (II). Only in the case of the mixtures lyxose—ribose and arabinose—mannose was separation carried out in the acidic system phenol—butanol—acetic acid—water (5:5:2:10) (upper layer; the amount of phenol is given in grams) (IV). The time for developing the chromatograms took (depending

* To obtain reproducible data it is necessary to observe all the indicated dimensions.

from the system) from 7 to 18 hours (in paper chromatography, development in the same systems takes from 3 to 9 days ($^2$)). For the detection of carbohydrates, as in the preceding work ($^1$), the reagent aniline—phthalic acid was used. The described method for separating carbohydrates proved to be approximately 10–20 times more sensitive than paper chromatography in a stream ($^2$), and makes it possible to detect 0.5–1 $\gamma$ of substance when the mixture is applied as a spot and 5–10 $\gamma$ when the mixture is applied as a band.

Using the indicated method, we separated a number of carbohydrate mixtures that do not separate in ascending chromatography on cellulose plates (Fig. 2). The values of $R_g$ and $R_a$ are presented in Table 1.

Table 1

Values of $R_g$ and $R_a$ for polyoxy compounds*

* Roman numerals denote the developer systems;
$a$ — application as a spot, $b$ — as a band.

The method of descending thin-layer chromatography on cellulose also proved very effective for the separation of stereoisomeric polyoxy acids. The most suitable system in this case was a mixture of n-butanol — 8% NH$_4$OH — water (saturated with borax) (8 : 1 : 2) (III); detection was carried out with a solution of periodate—potassium cuprate ($^3$). Using this method, threo- and erythro-2,3-dioxy-2-methylpentanoic acids ($^4$) were separated (see Table 1), which cannot be achieved by any of the previously used thin-layer chromatography methods ($^{1,5}$).

Thus, the method of descending chromatography in a thin layer of cellulose can be recommended for the separation of complex mixtures of polyoxy compounds (including carbohydrates) with very close $R_f$ values.

Institute of Chemistry of Natural Compounds
Academy of Sciences of the USSR

Received
12 X 1962

CITED LITERATURE

1. E. V. Dyatlovitskaya, V. V. Voronkova, L. D. Bergelson, DAN, 145, 325 (1962).
2. G. N. Zaitseva, T. P. Afanas’eva, Biokhimiya, 22, 1035 (1957).
3. T. G. Bonner, Chem. and Ind., 1960, 345.
4. L. D. Bergelson, E. V. Dyatlovitskaya et al., Izv. AN SSSR, OKhN, 1962, 1612.
5. L. D. Bergelson, E. V. Dyatlovitskaya, V. V. Voronkova, DAN, 141, 84 (1961).