European Pharmacopoeia Method for Sucrose Identification Test B using silica gel G TLC plates

By: Dr Sanjay Poman, Merck KGaA, Darmstadt, Germany, Life Science Application Centre (GAC), Mumbai, India

Introduction

Sucrose is a common naturally occurring carbohydrate found in many plants and plant parts. The molecule is composed of two monosaccharides; glucose and fructose with the formula C12H22O11.

Current European pharmacopoeia (version 9.5) indicates that a TLC silica gel G plate should be used for the Sucrose identification test B. TLC silica gel G plate complies with the requirements for a TLC silica gel plate R but with the following modification, it contains calcium sulfate hemihydrate as binder. In this application note we elaborate how Silica Gel G plates can be used for this test.

Sucrose

Experimental Conditions
TLC Plate Silica Gel 60G F254  20x20 cm (preheated at 105 °C for one hour) Injection volume 2 µL
Detection Spray with a solution of 0.5 g thymol in a in a mixture of 5 ml of sulfuric acid and 95 ml of ethanol. Heat the plate at 130˚C for 10 minutes Diluent Water:Methanol (2:3)
Mobile Phase Cold saturated solution of boric acid, 60% v/v solution of glacial acetic acid, anhydrous ethanol, acetone, ethyl acetate (10:15:20:60:60 (v/v/v/v/v)
Plate development Place the plate in an unsaturated tank containing the mobile phase as mentioned above. Spot 2µl of each spot of reference solution and sample solution. Dry the plate at room temperature and spray with thymol reagent and observe the spots after drying.  
Development   Over 2/3rd of the plate
Drying In current of warm air
Standard Dissolve 10 mg of Sucrose, Fructose, Glucose and Lactose each in diluent and make up the volume to 20 ml
Sample Dissolve 10 mg substance to be examined in diluent and dilute to 20 ml with the same diluent.

 

TLC plate after development

TLC plate after development

Chromatograms

Chromatograms

Chromatographic Data (Standard Solution)

Chromatographic Data (Standard Solution)

 

  Compound Rf
1 Sucrose 0.31

Chromatographic Data (Sample Solution)

Chromatographic Data (Sample Solution)

 

  Compound Rf
1 Sucrose 0.31

Conclusion

  1. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution.
  2. The Silica Gel G TLC plate shows clear separation of 4 clear spots of sugars.
  3. Silica gel G plates can be used for the Sucrose identification test B and they comply to all requirements as per the European Pharmacopoeia.

Materials