Test solution: 110 mg per mL, in 6 N hydrochloric acid.

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Dissolve an accurately weighed quantity of Histidine in water to obtain a solution having a concentration of 10 mg per mL. Apply 5 µL.

Standard solution— Dissolve an accurately weighed quantity of USP l-Histidine RS in water to obtain a solution having a known concentration of about 0.05 mg per mL. Apply 5 µL. [note—This solution has a concentration equivalent to about 0.5% of that of the Test solution.]

System suitability solution— Prepare a solution in water containing 0.4 mg each of USP l-Histidine RS and USP l-Proline RS per mL. Apply 5 µL.

Spray reagent— Dissolve 0.2 g of ninhydrin in 100 mL of a mixture of butyl alcohol and 2 N acetic acid (95:5).

Developing solvent system— Prepare a mixture of butyl alcohol, glacial acetic acid, and water (60:20:20).

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. After air-drying the plate, spray with Spray reagent, and heat between 100 and 105 for about 15 minutes. Examine the plate under white light. The chromatogram obtained from the System suitability solution exhibits two clearly separated spots. Any secondary spot in the chromatogram obtained from the Test solution is not larger or more intense than the principal spot in the chromatogram obtained from the Standard solution: not more than 0.5% of any individual impurity is found; and not more than 2.0% of total impurities is found.