A: Infrared Absorption 197K—

B: In the test for Chromatographic purity, the principal spot in the chromatogram obtained from Test solution 2, observed after spraying with the ninhydrin solutions, corresponds in RF value, color, and size to that in the chromatogram obtained from Standard solution 1.

C: It meets the requirements of the test for Phosphate 191.

Solvent— Prepare a mixture of 13.5 N ammonium hydroxide and dehydrated alcohol (3:2).

Test solution 1— Prepare a solution of Piperazine Phosphate in Solvent containing 100 mg per mL.

Test solution 2— Mix 1 mL of Test solution 1 and 9 mL of Solvent.

Standard solution 1— Prepare a solution of USP Piperazine Phosphate RS in Solvent containing 10 mg per mL.

Standard solution 2— Prepare a solution of ethylenediamine in Solvent containing 0.25 mg per mL.

Standard solution 3— Prepare a solution of triethylenediamine in Solvent containing 0.25 mg per mL.

Resolution solution— Prepare a solution in Solvent containing 0.25 mg of triethylenediamine and 10 mg of Piperazine Phosphate per mL.

Procedure— Separately apply 5-µL portions of Test solution 1, Test solution 2, Standard solution 1, Standard solution 2, Standard solution 3, and the Resolution solution to a suitable thin-layer chromatographic plate (see Chromatography 621), coated with a 0.25-mm layer of chromatographic silica gel. Allow the spots to dry, and develop the chromatograms in a solvent system consisting of a freshly prepared mixture of acetone and 13.5 N ammonium hydroxide (80:20) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and dry the plate at 105. Spray the plate with a 0.3% solution of ninhydrin in a mixture of butyl alcohol and glacial acetic acid (100:3). Spray the plate again with a 0.15% solution of ninhydrin in dehydrated alcohol, dry the plate at 105 for 10 minutes, and examine the plate: any secondary spot in the chromatogram obtained from Test solution 1 is not more intense than the principal spot in the chromatogram obtained from Standard solution 2 (0.25%). Spray the plate with 0.1 N iodine TS, allow to stand for 10 minutes, and examine the plate: any spot corresponding to triethylenediamine in the chromatogram obtained from Test solution 1 is not more intense than the principal spot in the chromatogram obtained from Standard solution 3 (0.25%). In a valid test, the chromatogram obtained from the Resolution solution shows a spot due to triethylenediamine clearly separated from the principal spot. Disregard any spot at the origin of any chromatogram.