Performing Thin-Layer Chromatography to Assess Bacterial Exopolysaccharide Degradation

Start with fermented supernatants containing degraded and undegraded exopolysaccharides, or EPS.

These EPS are obtained during fermentation, where distinct fecal bacterial populations in each supernatant act on EPS differently.

Spot the supernatants near the base of a thin-layer chromatography plate pre-coated with a polar stationary phase.

Air-dry the plate, then place it in a chamber containing a mobile phase with lower polarity than the stationary phase.

As the mobile phase rises, EPS migrate based on their polarity and size.

Large, undegraded EPS interact strongly with the stationary phase and remain near the origin.

Smaller degraded fragments bind weakly and migrate upward with the mobile phase.

Remove the plate and air-dry it.

Immerse the plate in a carbohydrate-staining reagent that reacts with EPS.

Air-dry the plate, then heat it to dehydrate the EPS and form colored bands.

A single band near the origin confirms undegraded EPS, while multiple bands confirm EPS degradation by bacteria.

Load 0.2 microliters of the fermented supernatants onto each pre-coated silica gel 60 TLC aluminum plate and swirl to spread. Then, use a hair dryer with hot air to dry.

Immerse one end of the sampled pla