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

Ion-exchange chromatography in a fixed bed of ion exchanger
Sample application

In classical Thin-Layer Chromatography, the test material must be desalted prior to application onto the plate. Desalting is labor-intensive and time-consuming, and when dealing with small quantities of substance, it leads to significant sample loss. In ion-exchange thin-layer chromatography, the salt applied alongside the sample does not interfere with the Separation process, since the concentration of the eluting buffer solution generally exceeds the salt concentration in the sample. To achieve complete fraction separation, the following requirements must be met:

1. The pH of the test sample should be below 3; it is advisable to adjust it to 2. If the sample is in dry form (e.g., a dried hydrolysate or extract), it is recommended to dissolve it in 0.01 N HCl.

2. If the test material is applied as a spot, its diameter should not exceed 2–3 mm; if applied as a band, its width should not exceed 1 cm.

3. The sample is applied to the plate using a fine capillary. After each droplet is applied, the application site must be dried to prevent damaging the layer.

4. The higher the concentration of the applied sample, the faster and more convenient the analysis. A maximum of 1–2 µg of The amino acid mixture should be applied to the plate. When separating a mixture of Amino Acids with close R_f values, a 4-to-5-fold difference in their concentrations is permissible to obtain reproducible results. When the R_f values of the Amino acids differ significantly, this, of course, does not matter.

5. Hydrolysates, if necessary (e.g., for Met determination), can be applied directly to the plate. In a warm stream of air, the acid evaporates instantly, leaving no time for Met to decompose.

6. Alkaline hydrolysates, such as those used in Trp determination, are applied after neutralization or acidification; the salt formed in the process does not interfere with chromatography.

7. If the test material dissolves in ethanol in the presence of acid, it is suspended in a solution of 0.1 N HCl in 95% ethanol, which precipitates trace Proteins that would otherwise hinder separation. For physiological fluids (Blood, plasma, etc.), such pretreatment of the applied material is of paramount importance.

8. All ion-exchange thin-layer chromatography Methods developed to date are limited to one-dimensional separation. This is one of the advantages of this method over classical thin-layer chromatography techniques. To obtain a clear and easily evaluated pattern, appropriate control mixtures should be applied at two locations on the plate (as a spot or band). This greatly facilitates identification, and if for any reason the chromatographic pattern differs from expectations, the control mixture can help determine the cause of incomplete separation and ascertain the COMPOSITION OF THE sample;