Separation of Ink Pigments Using Paper Chromatography

In majority forms of counter-current extraction column the very small drop required for the rapid achievement of equilibrium, and thus for high productivity, cannot be using due to the problem of preventing it from moving in the wrong direction. In the case of the solid, however, for any rational size of particle a filter will stop it from moving in any unwanted direction. Consideration of such facts led us to try absorbing water in silica gel etc., and then using the water-saturated solid as one phase of a chromatogram, the other being some fluid immiscible with water, the silica acting merely as mechanical support.

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This type of chromatography division hence rely upon differences in the partition between two liquid phases of the substances to be divided, and not, as in all previously described chromatogram, on differences in absorption between liquid and solid phases.

The difficulties of using chromatogram have been lowered when the substances to be separated are made visible by colouring. Different techniques have been used for this (cf. Zechmeister & Cholnoky, 1936;Cook, 1941), though none of these was suitable for our problems. As the substances which we want to separate were acids, and one of our phases is water, we were able to get visual evidences of the presence of any of these acids by adding a suitable indicator to the water with which the gel was saturated.

In the current paper we introduce a rough theory of chromatographic separation, and described an application of the new chromatogram to the micro-determination of the higher monoamino-acid in protein hydrolysates. This technique is based on the partition of acetamino-acids between chloroform and water phases, and supersedes the macro-method described by us( Martin & Sygne,1941,1), being rapid and economical both of materials and of apparatus.

Work is in progress, using ethyl acetate as the less polar phase in the chromatogram, on the separation of the acetyl derivatives of most of the other naturally occurring amino-acids, and the technique promises also to be of use in analogous separations of simple peptides. We wish to emphasize, however, that the possible fields of usefulness of the new chromatogram is by no means restricted to protein chemistry. By using suitable phase pairs, many other substances should be separable. Where water is suitable as one of the phases, an indicator may be used to make visible the separation of organic acids or bases. Even where this is not possible, as with neutral substances, the theory given below will enable ‘cuts’ of known partition coefficient to be taken. In the usual adsorption chromatogram optically active adsorbents have been used for optical resolutions (cf. Henderson & Rule, 1937; Karagunis & Coumoulus, 1938), and resolution of racemic substances may be expected in the new chromatogram when either phase is optically active (cf. Bailey & Hass, 1941).

The mobile phase may not be a liquid but might be a vapour. We show below that the efficiency of contact between the phases (theoretical plates per unit length of column) is far better in the chromatogram than in normal distillation or extraction columns. Very specific separations of volatile substances should therefore be possible in a column in which permanent gas is made to flow over gel impregnated with a non-volatile solvent in which the substances to be separated approximately obey Roult’s law, as in azeotropic distillation.

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The method might also be useful in the separation of isotopes, e.g. of nitrogen isotopes, by passing ammonia gas over gel impregnated with ammonium sulphate solution (cf. Urey et al, 1937).