Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Detection of compounds in paper chromatography and thin-layer chromatography
Fluorescent detection methods

After drying, the chromatogram is examined under UV light to locate fluorescent spots. The solvent front is typically visible as well; its shape can be used to monitor any flow disruptions within the chromatographic system.

The Procedures outlined below are used for chromatogram visualization by immersion in a reagent solution or by spraying under appropriate conditions.

1. Immerse the paper sheet in a solution of fluorescamine in acetone (25 mg per 1 L containing 0.5% triethylamine). Examine under UV light; the detection limit is 1 nmol of peptide. A less concentrated fluorescamine solution (4 mg/L) is used for preparative work. After washing with acetone, other staining Methods can be applied.

2. Required: fluorescamine solution (10 mg/100 mL of acetone) and triethylamine solution (1% in acetone). Dry the chromatogram, wash with acetone to remove the solvent, and dry again. Spray with the triethylamine solution and, after drying in a fume hood for 15 min, with the fluorescamine solution. Dry, spray with acetone, and dry again. Detect fluorescent spots under a UV lamp (336 nm). To visualize faint peptide spots, repeat the Treatment Procedure. Heating the chromatogram in an oven at 110 °C for 3 h allows the detection of Peptides with an N-terminal Proline residue [131].

3. Required: o-phthalaldehyde solution (30 mg/100 mL of acetone) and triethylamine solution (1% in acetone, containing 0.05% 2-mercaptoethanol). The procedure for treating the chromatogram with this reagent is similar to that described in step 2. OPA is comparable in sensitivity to fluorescamine while being more cost-effective.