Harper and Davis present a two-dimensional thin-layer chromatography (TLC) method for analyzing amino acids in bacterial cell walls, a chemotaxonomic approach used in bacterial systematics. Using cellulose-coated aluminum sheets and two sequential solvents—isopropanol-acetic acid-water followed by methanol-pyridine-hydrochloric acid-water—they achieved excellent separation of 15 amino acids, including the critical distinction between LL-diaminopimelic acid (DAP) and meso/DD-DAP isomers. This method improves upon traditional paper chromatography by reducing analysis time and eliminating the need for extra work to identify DAP forms. The authors validated their approach on gram-positive bacteria including Corynebacterium, Actinomyces, Propionibacterium, Brevibacterium, and Erysipelothrix species. The technique requires minimal sample volume (2-4 microliters of hydrolysate from ~10 mg dry cell weight), simple equipment, and produces discrete, easily identifiable spots. The method successfully detects significant cell wall amino acids while distinguishing them from trace contaminants through spot density differences. The authors conclude this approach offers practical advantages for taxonomic classification work, being faster and more reliable than existing chromatographic methods.

Key findings

• Two-dimensional TLC successfully separates 15 bacterial cell wall amino acids with clear distinction between LL-DAP and meso/DD-DAP isomers using specific solvent combinations
• The method requires minimal sample volume (2-4 µL) and simple equipment, making it more practical than paper chromatography for routine bacterial taxonomy
• Application to selected gram-positive bacteria demonstrates the method's reliability for chemotaxonomic classification based on cell wall amino acid composition
• Thin-layer chromatography offers greater sensitivity than paper chromatography, allowing detection of trace contaminants but enabling ready recognition of significant amino acids through spot density differences