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To separate and identify glucose and fructose from a mixture by descending paper chromatography and to calculate the Rf values of the compounds.

Aim

To separate and identify glucose and fructose from a mixture by descending paper chromatography and to calculate the Rf values of the compounds.



 *Principle* 

Paper chromatography separates components of a mixture by differential partitioning between a mobile phase (solvent) and a stationary phase (paper cellulose). In descending chromatography the solvent rises up the paper due to capillary action while the paper strip slowly descends (or solvent moves downward along the strip, depending on the setup). Components travel different distances; the ratio of distance traveled by a compound to the distance traveled by the solvent front is the Rf value (retention factor).

 *R_f* = Distance from origin to centre of spot}\Distance from origin to solvent front


 *Apparatus & Reagents* 

Whatman filter paper (or chromatography paper) strips, ~20–25 cm long

Chromatography tank / tall beaker + watch glass or lid

Solvent (mobile phase) — common solvent system for sugars: 
n-butanol–acetic acid–water (4:1:1) 

Capillary tubes or micropipette for spotting
Pencil, ruler (cm scale)
Spatula / scalpel to cut paper, clips or paper support

Sample: glucose + fructose mixture (aqueous) and standards of glucose and fructose .

Spray reagent or detector for sugars often periodic acid–Schiff or aniline/phloroglucinol methods can be used or sugar-specific color reagent; or heat spray with sulfuric acid.

Gloves, goggles, lab coat

 *Preparation of Paper* 

1. A strip of chromatography paper ~20–25 cm long and 2–3 cm wide is cut.
2. About 2 cm from the top of the strip, two small holes are punched and a short paper clip is attached to hang it in the tank (so the top is fixed and bottom free).
3. With a pencil, a horizontal origin line is drawn (baseline) 5 cm from the bottom (this is where the sample is spotted). three small spots are marked on the line: 
4. left = glucose standard 
       Middle = mixture,   
       right = fructose   
       standard 

 *Procedure (Descending paper chromatography)* 

1. The solvent is poured into the chromatography tank to a depth of about 1.0–1.5 cm (below the origin line). The tank is covered and let solvent vapor equilibrate for 5–10 minutes.
2. Using a capillary tube, A very small concentrated spot of the sample solution is applied on the origin line. It is allowed to dry and if needed, a second tiny spot in the same place is applied to concentrate the band.
3. The paper strip is hanged such that the origin line is above and just touching the solvent surface (for descending technique the strip is placed in solvent so solvent climbs and paper gradually descend .
4. It is ensured that paper does not touch the tank walls; clamp or hang centrally.
4. The tank is closed and solvent is allowed to ascend. The chromatography is started running until the solvent front reaches about 2–3 cm from the top of the strip (or predetermined distance).
5. Remove the strip, immediately mark the solvent front with a pencil, and allow the strip to dry.
6. Visualize spots by spraying with the detection reagent or by heating as required. Mark the centre of each visible spot with a pencil.


Diagram 

A) Chromatography tank setup 



Labels: paper strip, origin line (above solvent), solvent pool, watch glass/lid to saturate vapour.

B) CHROMATOGRAPH


Calculation of Rf 

Formula:

R_f = Distance from origin to centre of spot \Distance from origin to solvent front

OBSERVATION TABLE:

Substance

Distance from Origin to Center of Spot  (cm)

Distance from Origin to Solvent Front (cm)

Retention Factor Value

Glucose Standard

3.60

9.00

0.400

Fructose Standard

5.10

9.00

0.567

Glucose + Fructose Mixture (Spot 1)

3.60

9.00

0.400

Glucose + Fructose Mixture (Spot 2)

5.10

9.00

0.567


Substance
Distance from Origin to Center of Spot (cm)
Distance from Origin to Solvent Front (cm)
Retention Factor Value
Glucose Standard
3.60
9.00
0.400
Fructose Standard
5.10
9.00
0.567
Glucose + Fructose Mixture (Spot 1)
3.60
9.00
0.400
Glucose + Fructose Mixture (Spot 2)
5.10
9.00
0.567

Using the sample values in the table:

1. Glucose

Distance (origin to glucose spot) = 3.60 cm

Distance (origin to solvent front) = 9.00 cm
Compute digit-by-digit:
( 3.60 ÷ 9 ) = 0.4

2. Fructose

Distance
 (origin to fructose spot) = 5.10 cm

Distance 
(origin to solvent front) = 9.00 cm

Compute: ( 5.10 ÷9 )
≈ 0.567 

 *Interpretation* :

 Different Rf values indicate successful separation. Spots are compared with standards to identify which is glucose and which is fructose.

 *Result** 

The mixture was separated by descending paper chromatography. The measured Rf values (sample data) are:

Glucose: Rf = 0.400

Fructose: Rf ≈ 0.567

 **Precaution* 

Ink is not used to mark the paper.Pencil is used only.

Very small concentrated spots are applied; large spots reduce resolution.

Solvent depth is kept below the origin line.

The tank is closed to ensure vapour saturation (improves band shapes).

Solvent is allowing front to reach the same distance for samples and standards to compare Rf values reliably.

The paper is dried and handled gently to avoid smearing spots.

 *Conclusion* 

Descending paper chromatography allowed separation of glucose and fructose, and Rf values were determined. The difference in Rf values confirms distinct mobility of the two sugars in the chosen solvent system.

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