APPLICATION NOTES

HPTLC Identification of Bearberry leaf (Arctostaphylos uva-ursi)

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Sample: In a flask 0.5 g powdered drug are heated under reflux with 5 mL a methanol water mixture 1:1 for 10 min. The hot mixture is filtered. Flask and filter are washed with methanol water 1:1 and the filtrate is diluted to a final volume of 5 mL. This is the test solution. Standards (optional): 1 mg each of quercetin-3-O-arabinofuranoside, quercetin-3O-arabinopyranoside, hyperoside, and rutin are dissolved in 1 mL methanol. This is standard mixture A. 1 mg each of arbutin, hydroquinone, and gallic acid are dissolved in 1 mL methanol. This is standard mixture B. Derivatization reagents (method requires one plate for each reagent): Dichloroquinone chlorimide reagent (DQC): Solution a) 1 g dichloroquinonechlorimide is dissolved in 100 mL methanol. Solution b) 10 g anhydrous sodium carbonate are dissolved in 100 mL water. Sulfuric acid reagent: 20 mL sulfuric acid are carefully added to 180 mL ice-cold methanol. Chromatographic conditions: Stationary phase: Mobile Phase: Sample application: Development: HPTLC plates silica gel 60 F254 (Merck), 10x10 cm or 20x10 cm Ethyl acetate, formic acid, water (88:6:6) 10 L test solution and standard are applied as 8 mm bands, min. 2 mm apart, 8 mm from lower edge of plate. 10x10 cm or 20x10 cm Twin Trough Chamber, saturated for 20 min (filter paper), 5 mL (respectively 10 mL) developing solvent per trough, developing distance 60 mm from lower edge of plate. The plate is then dried with a hair dryer (cold air) for 5 min.

06/03

Please contact CAMAG for more application notes and products for analysis of herbals!

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APPLICATION NOTES

Detection:

a) UV 254 nm b) DQC reagent: the plate is first sprayed with solution a, dried, then sprayed with solution b, and dried. Examination in white light. c) Sulfuric acid reagent: a second plate is immersed in reagent for 1 s, then heated at 100C for 5 min. Examination in white light. d) Examination of derivatized plate c) under UV 366 nm.

Results: Compare to the chromatograms below: 1 2 3 1 2 3

a) UV 254 nm Track assignment:

b) DQC reagent

1: Arbutin, gallic acid, hydroquinone (with increasing Rf) 2: Rutin, hyperoside, quercetin-3-O-arabinopyranoside, quercetin-3-O-arabinofuranoside (with increasing Rf) 3: Acrtostaphylos uva ursi

06/03

Please contact CAMAG for more application notes and products for analysis of herbals!

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APPLICATION NOTES

a) UV 254 nm Track assignment:

c) White light, sulfuric acid

d) UV 366 nm, sulfuric acid

1: Rutin, hyperoside, quercetin-3-O-arabinopyranoside, quercetin-3-O-arabinofuranoside (with increasing Rf) 2: Acrtostaphylos uva ursi 3: Arbutin, gallic acid, hydroquinone (with increasing Rf)

06/03

Please contact CAMAG for more application notes and products for analysis of herbals!

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