Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Ion-exchange chromatography in a fixed bed of ion exchanger

Ion-exchange Thin-Layer Chromatography in a fixed bed of ion-exchange resin is a novel technique in biochemistry. Undoubtedly, Ion-exchange chromatography remains the premier fractionation method known today, a fact corroborated by numerous experiments over the past two decades.

Ion-exchange chromatography has enabled the automation of Amino acid analysis, heralding a new era in Protein Biochemistry and various fields of molecular biology. However, when employing classical Column ion-exchange chromatography, the time factor plays an exceptionally critical role. The primary challenges in column chromatography Methods arise during the evaluation of fractionation and the execution of parallel experiments.

By the very Nature of the method, evaluation requires structurally complex and expensive semi-automatic or automatic instrumentation. In its absence, non-automatic fractionation—where each fraction from the collector must be analyzed individually—is extremely time-consuming.

The throughput of an automatic amino acid analyzer is determined by the number of samples analyzed within a given timeframe; it is quite obvious that this significantly exceeds the throughput of non-automatic fractionation.

Clearly, conducting parallel experiments necessitates either multiple automated units—which, financially speaking, is feasible for only a small number of laboratories—or a large staff of researchers. Yet, many instances call for a high volume of analyses. For instance, determining the complete Amino Acid Sequence of an average molecular weight protein in Cell/13.html">Protein Structure research requires performing approximately 3,000 to 4,000 analyses.

¹ This chapter was contributed specially for the Russian edition by T. Dévényi.— Ed. note.

Approximately the same number of tests must be conducted in plant breeding to select a single new variety with an elevated protein content or higher levels of Lys and Met. In clinical practice, there is also a domain where amino acid analysis is utilized in mass screening to detect Amino acid METABOLISM disorders in newborns. In many countries, such screening has already been made mandatory, albeit utilizing microbiological methods, since investigating a massive number of samples via other amino acid analysis methods is practically unfeasible.

Evidently, column chromatography is inapplicable to such studies, whereas thin-layer chromatography is a very simple, rapid, and convenient Procedure in which separated substances are detected quite easily; it does not require expensive equipment and is suitable for mass testing. Admittedly, its resolving power is considerably inferior to that of column ion-exchange chromatography. Despite this, this relatively young technique is spreading rapidly across all areas of chemistry, and not by chance.

For separating, for example, a mixture of 16 Amino Acids, thin-layer chromatography, much like paper chromatography, must be performed in two dimensions. This diminishes the value of the method, as two-dimensional chromatograms are rather difficult to evaluate, particularly when a sample contains more than 16 amino acids. Moreover, two-dimensional chromatography requires a separate plate for each sample, generating additional complications related to workspace, chromatogram Processing, etc.

The high resolving power of ion-exchange chromatography and the simplicity of thin-layer chromatography sparked a desire to combine these two techniques and develop a technology for thin-layer chromatography in a fixed ion-exchanger bed.

At first, accomplishing this seemed quite straightforward. Various sorbents (silica gel, aluminum oxide, polyamide, various Cellulose derivatives, etc.) and various binding agents (gypsum, starch, dextran, polyvinyl alcohol, etc.) are utilized in thin-layer chromatography. It was assumed that these agents and the conditions under which they are applied would be entirely suitable for fixing resins.

Unfortunately, initial experiments demonstrated that the matter was not so simple; to perform chromatography in an ion-exchanger bed, the following problems had to be resolved:

1. The layer must be robust. Its mechanical strength should be comparable to that of silica gel layers, meaning it must be sturdy enough to be handled by hand, written on with a soft pencil lead, and stored stacked.

2. Ion-exchange chromatography is viable only In aqueous solutions; consequently, the layer must be moisture-resistant.

3. The binding agent must not diminish the Swelling capacity of the fixed ion-exchanger (as determined by its capacity) or its resolving power.

4. The layer must retain its integrity in organic Solvents, because the visualization of fractionated components (e.g., via the ninhydrin reaction) typically involves their use (acetone, butanol, etc.).

We successfully developed a method for preparing an ion-exchange resin layer with a binding agent that meets all the necessary requirements, and we found the conditions for performing chromatography within it. Our method was adopted by the Chinoin-Nagyteteny plant (Hungary) and Machery-Nagel (Germany).

Currently, “Fixion” plates of Hungarian manufacture and “Ionex” plates of German manufacture are commercially available.