Source: Tropicos.org. Missouri Botanical Garden. 27 Feb 2014 <http://www.tropicos.org/Image/100253567> ^[1]

G. glabra reference standard

Licorice (root) (Glycyrrhiza glabra)

Sample Preparations: Extract 1.0 g of coarsely powdered Glycyrrhiza root in 50 mL of water by boiling for about 5 minutes, and filter. Repeat for 4-5 times or until the extract is colorless. Combine the extracts, concentrate to about 100 mL, and cool to room temperature. Before injection, filter through a membrane filter of 0.45-um or finer pore size, discarding the first 5 mL of the filtrate.

Column: C18, 25-cm x 4.6 mm, 5-um

Mobile Phase: 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL phosphoric acid, mix, complete to volume with water, and mix (Solution A); and acetonitrile (Solution B)

Elution: Gradient program, see Table below

Flow rate: 1.5 mL/min

Detection: UV, 254 nm

Injection volume: 20 uL

• 

  G. glabra reference standard

• 

  G. inflata reference standard

• 

  G. uralensis reference standard

Source: Natural Remedies Pvt Ltd ^[17]

Table: Gradient program

Glycyrrhiza glabra HPTLC ID - Developed, UV 366 nm

Licorice (root) (Glycyrrhiza glabra)

Lane Assignments Lanes, from left to right (Track, Volume, Sample):

1. 2 μL Ammonium glyccyrhizate
2. 2 μL Glycyrrhizic acid
3. 2 μL Licorice root 1 (Glycyrrhiza glabra)
4. 2 μL Licorice root 2 (Glycyrrhiza glabra)
5. 2 μL Licorice root 3 (Glycyrrhiza glabra)
6. 2 μL Licorice root (Glycyrrhiza glabra) 

Other Notes
Reference Standard Solution: 0.1 mg/mL ammonium glycyrrhizate in ethanol and water (7:3). Or 0.1 mg/mL glycyrrhizic acid in ethanol and water (7:3).

Reference Sample Preparations: Mix 0.5 g of powdered sample with 10 mL of ethanol and water (7:3, v/v), sonicate for 10 minutes, centrifuge or filter the solution, and use the supernatant / filtrate.

Stationary Phase: HPTLC, Silica gel 60 F254

Mobile Phase: Ethyl acetate, acetic acid, formic acid, water (15:1:1:2)

Development: Saturate chamber for 20 minutes; developing distance 70 mm from lower edge of the plate; relative humidity 33%, temperature 25°.

Derivatization reagent: Methanol sulfuric acid reagent- 180 mL of ice-cooled methanol are mixed with 20 mL of sulfuric acid.

Detection: a. Examine under UV light at 254 nm b. Dip (time 0, speed 5) in Derivatization reagent, heat at 100°C for 10 min, leave to cool, and examine under visible light.

Procedure:

Reference Standard Solutions, Stationary Phase, Mobile Phase, Development, Derivatization reagent, and Detection, as described above.

Test Sample Preparation: Prepare test sample as described under Reference Sample Preparations and apply 2 uL.

Identification: Compare Test Sample Preparation chromatogram with chromatograms of Reference Sample Preparations. The Test Sample Preparation chromatogram is similar to that of the Reference Sample Preparations chromatograms. Additional weak zones may be present.

Under UV light, the Test Sample Preparation chromatogram exhibits a quenching zone in the lower-third section of the chromatogram corresponding to the zone due to ammonium glycyrrhizate in the Reference Standard Solution chromatogram. Above it there are four quenching zones corresponding to those marked with red arrows in the Reference Sample Preparations chromatograms.

After derivatization and under visible light, the Test Sample Preparation chromatogram exhibits a brown zone in the lower-third section of the chromatogram corresponding to the zone due to ammonium glycyrrhizate in the Reference Standard Solution chromatogram. Above it there are four yellow zones corresponding to those marked with black arrows in the Reference Sample Preparations chromatograms.

Note: Images presented in this entry are examples and are not intended to be used as a bases for setting specifications for quality control purposes.

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  Glycyrrhiza glabra HPTLC ID - Developed, UV 366 nm

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  Glycyrrhiza glabra HPTLC ID - Developed, White RT

Source: HPTLC Association ^[18]