Amino acids adsorption analysis of mixtures

Adsorption Analysis of Amino Acid Mixtures Arne Tiselius... 

The primary process of lEC involves adsorption and desorption of ionic species from ionogenic groups located in the packing. lEC was the first of the traditional column techniques to be exploited for modern LC, due principally to the need for the fast routine analysis of amino acids and protein mixtures. 

The technique and instrumentation for liquid-liquid chromatography are much the same as for adsorption chromatography the main distinction lies in the nature of the stationary phase. In the original development of the method, Martin and Synge effected a separation of acetylated amino acids by using silica gel as support for an aqueous stationary phase. The mobile phase was a mixture of chloroform, butanol, and water. The method was used for analysis of the amino acids of various proteins. ... 

This isotherm model has been used successfully to accoimt for the adsorption behavior of numerous compounds, particularly (but not only) pairs of enantiomers on different chiral stationary phases. For example, Zhou et ah [28] foimd that the competitive isotherms of two homologous peptides, kallidin and bradyki-nine are well described by the bi-Langmuir model (see Figure 4.3). However, most examples of applications of the bi-Langmuir isotherm are found with enantiomers. lire N-benzoyl derivatives of several amino acids were separated on bovine serum albumin immobilized on silica [26]. Figure 4.25c compares the competitive isotherms measured by frontal analysis with the racemic (1 1) mixture of N-benzoyl-D and L-alanine, and with the single-component isotherms of these compounds determined by ECP [29]. Charton et al. foimd that the competitive adsorption isotherms of the enantiomers of ketoprofen on cellulose tris-(4-methyl benzoate) are well accounted for by a bi-Langmuir isotherm [30]. Fornstedt et al. obtained the same results for several jS-blockers (amino-alcohols) on immobilized Cel-7A, a protein [31,32]. 

The advantage of this type of adsorption analysis evidently lies in the stability of the zones theoretically at.least one should be able to use as large columns as desired and therefore to carry out also very difficult separations. The main drawback of the elution method—the tailing— is eliminated. The fact that the different zones are in close contact is a difficulty which,- however, may be eliminated by interposing substances of intermediate adsorption and of a different chemical nature so that they may easily be removed afterwards. Examples of the application of tills procedure may be found in a paper by Hahn and the author (Tiselius and Hahn, 1943), in which various saccharides were separated, and some experiments on amino acids and peptides already mentioned. In case of strong adsorption, the affinity of the adsorbent has to be reduced by pre-treatment with suitable substances as described in the last mentioned paper. Nevertheless, losses are sometimes serious in this method. The experience so far seems to show that with the adsorbents now available the displacement method is applicable chiefly for mixtures of closely related substances, which are able to displace each other completely. However, for fatty acids in organic solvents on carbon the method has failed, but in the separation of gaseous hydrocarbons it seems quite satisfactory (Claesson, 1946). 

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