Reference: 

Gokhale SB, Practical book of  Pharmacognosy and Phytochemistry II , Nirali prakashan, Pune.

 

Requirement:

Stationary phase (Paper): Whatman filter paper No. 1; developing chamber, glass rod, lid, etc

 

Principle

Chromatography is separation technique, in which separation of mixture of components in to the individuals using stationary phase and mobile phase. The sugars are separated using paper chromatography. The paper chromatography is partition chromatography, in which the mobile phase and stationary phase are liquid. The mixture of compounds is separated based on their partition coefficient. The components have more soluble in stationary phase travels slower and components, which are more soluble in  mobile phase, travels  faster.  Thus, compounds are separated because of their differences in partition coefficient. No two components have the same partition coefficient for the combination of stationary phase, mobile phase and other conditions. In paper chromatography, the cellulose layer on the filter paper contains moisture, which acts as stationary phase. The organic solvents or buffers are utilized as mobile phases.

 

Preparation of solution

i)  Standard solutions:  Prepare the aqueous solution of any three  of the  following  by dissolving in water at concentration of 20-50 µg/ml of each sugar. The sugars are: D-glucose, D-fructose, D-xylose, L-rhamnose, D-galactose, Lactose, maltose, sucrose, D-mannose.

ii) Preparation of mixture solution of sugars: Add few drops of each sample sugar solution in a dry test tube.

iii) Sample solution:  The plant material or fruit juice  were grinded and extracted with suitable solvent to concentrate the sugars. These sugars are diluted or concentrated or applied directly.




Procedure

1.     Prepare the mobile phase selected for  the analysis and transfer in  the developing chamber, cover the led and allow the chamber to be saturated with mobile phase.

2.     Cut the sheet of whattman 1 chromatography paper in proper size, remark the solute application line about 1.5cm from the lower edge of the paper with the help of pencil.  

3.     Mark the line at equal distance on the solute application line based on the number of standard and sample.

4.     Label the paper at the top with the name of each of the sugars and label the last unknown.

5.     Use a fine capillary to place the drops of the solutions of the sugars, glucose, fructose, maltose, lactose and the mixture.

6.     After spotting, dry the paper with hot air dryer for one minute, repeat this step again.

7.     Place the  spotted paper in  the chromatographic tank  and make the development by using the ascending technique.

8.     Close the tank with lid, allow the solvent to flow for about 30-45 minutes.

9.     Remove the paper and immediately mark the position of the solvent front with a pencil.

10.  After the chromatogram has dried, spray the paper with the detecting reagent.

11.  Circle the position of each spot with pencil.

12.  Calculate the Rf value for each spot and also for the spots the mixture contained.

Chromatographic conditions

Mobile phase Solvents:  

a.    n-butanol-acetic acid-water (4:1:5 v/v)

b.   Isopropanol-pyridine-water-acetic acid (8:8:4:1 v/v)

Spray reagent:

Resorcinol reagent:   1% ethanolic solution of resorcinol and 0.2N HCl (1:1 v/v) visualize spots by heating at 90OC

Formulae

Rf Value = Distance travelled by solute / Distance travelled by solvent

Sugar

Rf Values

Mobile phase a

Mobile phase b

Glucose

0.64

0.18

Galactose

0.62

0.16

Fructose

0.68

0.25

Ribose

0.76

0.31

Lactose

0.46

0.09

Maltose

0.50

0.11

Sucrose

0.62

0.14