Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Chromatography
Thin-Layer Chromatography Method

In Thin-Layer Chromatography (TLC), a solid stationary phase is applied (and frequently immobilized) on a backing plate made of Glass, plastic, or aluminum. The solid phase typically consists of silica gels, aluminum oxides, ion-exchange resins (most commonly modified celluloses), or Gel filtration media (such as a porous polyacrylamide gel). Methodologically, TLC is performed in much the same way as paper chromatography (PC).

Both paper chromatography and TLC are suitable for analyzing virtually any mixture. Their key advantages include simplicity, high resolution and reproducibility, rapid analysis time (ranging from 20 minutes to 3 hours), and high sensitivity (requiring very small amounts of the sample).

Methods for Amino Acid Detection

The methods for detecting and quantitatively determining Amino Acids following PC and TLC Separation are identical. The chromatograms are sprayed with a 0.5% solution of ninhydrin in acetone, air-dried, and heated for 30 minutes at T=75°C. Amino acids appear as blue spots. These spots are excised, and the colored amino acid-ninhydrin complex is extracted with an alcoholic copper sulfate solution. The optical density of the resulting solution is then measured at 575 nm. The quantities of the respective Amino acids are determined using standard calibration curves.

Certain amino acids can be analyzed (identified) post-chromatography by employing specific color reactions (see sections 1.1.7 and 1.1.8). For instance, Histidine and Tyrosine can be visualized by treating the chromatogram with diazotized sulfanilic acid in an alkaline environment, yielding red and brown spots, respectively. Proline and hydroxyproline produce an intense blue coloration when the chromatograms—already treated with ninhydrin—are further subjected to hydrogen peroxide. Under these conditions, the spots for all Other Amino Acids are bleached.



Last update: 06/08/2026

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