Use a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture, and the Eluant
specified in the monograph. Apply equal volumes (20 µL) of the Test Solution
and Standard Solutions
to the plate, using a stream of nitrogen to dry the spots.
Allow the chromatogram to develop in a pre-equilibrated chamber until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and air-dry. View the plate using the visualization technique(s) specified. Locate any spots other than the principal spot, in the chromatogram of the Test Solution, and determine their relative intensities by comparison with the chromatograms of the appropriate Standard Solutions. The total of any ordinary impurities observed does not exceed 2.0%, unless otherwise specified in the individual monograph.
KEY FOR VISUALIZATION TECHNIQUES
(1) Use UV light at 254 nm and at about 366 nm.
(2) Use Iodoplatinate TS.
(3) Solution AMix 850 mg of bismuth subnitrate with 40 mL of water and 10 mL of glacial acetic acid.
Solution BDissolve 8 g of potassium iodide in 20 mL of water. Mix A and B together to obtain a Stock Solution which can be stored for several months in a dark bottle. Mix 10 mL of the Stock Solution with 20 mL of glacial acetic acid, and dilute with water to make 100 mL, to prepare the spray reagent.
(4) Ninhydrin SprayDissolve 200 mg of ninhydrin in 100 mL of alcohol. Heat the plate after spraying.
(5) Acid SprayIn an ice bath, add slowly and cautiously, with stirring, 10 mL of sulfuric acid to 90 mL of alcohol. Spray the plate, and heat until charred.
(6) Acid-Dichromate SprayAdd sufficient potassium dichromate to 100 mL of sulfuric acid to make a saturated solution. Spray the plate, and heat until charred.
(7) VanillinDissolve 1 g of vanillin in 100 mL of sulfuric acid.
(8) Chloramine T-Trichloroacetic AcidMix 10 mL of a 3% aqueous solution of chloramine T with 40 mL of a 25% alcoholic solution of trichloroacetic acid. Prepare immediately before use.
(9) Folin-CAdd 10 g of sodium tungstate and 2.5 g of sodium molybdate to 70 mL of water, add 5 mL of 85% phosphoric acid and 10 mL of 36% hydrochloric acid, and reflux this solution for 10 hours.
(10) KMnO4Dissolve 100 mg of Potassium Permanganate in 100 mL of water.
(11) DABMix 1 g of p-dimethylaminobenzaldehyde in 100 mL of 0.6 N hydrochloric acid.
(12) DACMix 100 mg of p-dimethylaminocinnamaldehyde in 100 mL of 1 N hydrochloric acid.
(13) FerricyanideMix equal volumes of a 1% ferric chloride solution and a 1% potassium ferricyanide solution. Use immediately.
(14) Fast Blue BReagent ADissolve 500 mg of Fast Blue B Salt in 100 mL of water.
Reagent B0.1 N sodium hydroxide.
Spray first with A, then with B.
(15) Alkaline Ferric CyanideDilute 1.5 mL of a 1% potassium ferricyanide solution with water to 20 mL, and add 10 mL of 15% sodium hydroxide solution.
(16) Iodine SprayPrepare a 0.5% solution of iodine in chloroform.
(17) Expose the plate for 10 minutes to iodine vapors in a pre-equilibrated closed chamber, on the bottom of which there are iodine crystals.
(18) Solution ADissolve 0.5 g of potassium iodide in 50 mL of water.
Solution BPrepare a solution of 0.5 g of soluble starch in 50 mL of hot water.
Just prior to use, mix equal volumes of Solution A and Solution B.
Dissolve 20 g of p
-toluenesulfonic acid in 100 mL of alcohol, spray the plate, dry for 15 minutes at 110
, and view under UV light at 366 nm.
(20) o-Tolidine SprayDissolve 160 mg of o-tolidine in 30 mL of glacial acetic acid, dilute with water to make 500 mL, add 1 g of potassium iodide, and mix until the potassium iodide has dissolved.
(21) Mix 3 mL of chloroplatinic acid solution (1 in 10) with 97 mL of water, followed by the addition of 100 mL of potassium iodide solution (6 in 100) to prepare the spray reagent.
(22) IodineMethanol Spray
Prepare a mixture of iodine TS
and methanol (1:1).