Preconditioning of chromatography chamber 1. Prepare chromatography solvent by mixing isobutanol, acetone, formic acid, and water in a 160:160:1:39 proportion (vol. by vol.). 2. Pour the solvent into a chromatography chamber, the liquid level should be around 5 mm from the bottom. Seal the solvent-containing chamber for at least 1 h to saturate the chamber with solvent vapors. Preparation of chromatography paper 1. Scissor out a strip of chromatography paper with the appropriate dimensions. The width of the strip depends on the number of samples to be analyzed (the distance between sample spots and between spots and paper edges should be at least 20 mm). The height depends on the difference in Rf values of separated compounds and usually is 100-400 mm. 2. Draw a line 20 mm from the bottom of the strip with a graphite pencil (starting line). Mark dots on this line for future sample spots. 3. Load spots of the test samples and standards on the starting line using a capillary tube. Microliter quantities are used to prepare spots. The diameter of the spots should be only up to a few mm. In order to get a concentrated spot, the spotting can be repeated several times. If it is necessary, wait till the spot is dried and then repeat the spotting. In the case of urocanic acid and deaminohistidine, each spot should contain about 10 nmol of one of these compounds. Chromatography 1. To develop the chromatogram, place the prepared strip into the saturated chromatography chamber and seal the chamber. 2. Remove the strip from the chamber when the solvent front has traveled up to about 20 mm from the top of the paper. Mark the position of the solvent front on the strip. Detection 1. Air dry the strip. 2. Using a spraying nozzle, spray the dried strip first with the Pauly diazo reagent until the paper becomes humid, followed by spraying the humid strip with a 10% Na2CO3 solution until spots of imidazolyl derivatives become colored (urocanic acid – orange, deaminohistidine – red). The color is stable within several weeks. 3. Calculate Rf values [(distance traveled by a component)/(distance traveled by the solvent)].