4. Demonstration of paper chromatography of amino acids.

Separation of amino acids using paper chromatography

 

Materials and Equipment

Materials:

Chromatography paper, amino acids (1% solutions): tyrosine, serine, glutamine,

glycine, arginine, glutamic acid and unknown, eluting solution (isopropyl alcohol,

0.5 M NH4OH; 0.2% ninhydrin spray).

 

Equipment:

600-mL beaker, pencil, ruler, evaporating dish, hair dryer, stapler and blotting

paper.

 

Preparation of Chromatography Paper

The procedure for ascending paper chromatography method is quite simple as compared to other methods of chromatography. The chromatography paper is cut into rectangular strips and marks a line on the paper with pencil at about 2 cm from the bottom. With the help of capillary tube, the samples are applied at different points on the starting line Now, place the chromatography paper in the developing chamber, which contains the mobile phase. 

While placing the paper, it is important that the solvent level should not reach the starting line or the sample spots and paper shouldn’t touch the walls of the developing chamber. After sometime the solvent rises up the paper or the stationary phase by capillary action and dissolves the sample. The components of the sample move along with the solvent in upward direction. 

Check if the solvent has reached near the top level of chromatography paper. Then the paper is removed when it reaches the top and marked the level with pencil. 

This level (or) height is called the “solvent front”.  Each spot represents a specific component of the sample.

 Acquisition of Chromatogram

1. Take a 500-mL beaker and pour 10-mL of 0.5 M NH4OH and 20 mL of isopropyl alcohol (eluting solution) into the beaker. Obtain your evaporating dish and use it to cover the beaker.

2. Gently place the paper cylinder into the beaker and cover the top with plastic wrap. Remember that the spots must be above the liquid level for the experiment to work. Watch the eluent creep up the paper until it is about 2 cm from the top. It will take about 45-60 minutes for the solvent front to reach the finish line.

3. When the solvent front reaches the finish line, remove the paper from the beaker, being careful to touch only the top. Let excess eluent drip into the beaker. Gently remove the staple and lay the chromatogram on a piece of paper towel. Use a hair dryer to dry the chromatogram completely. Pour the eluting solution in the organic waste container under the fume hood.

4. Working in the fume hood, spray the chromatogram lightly with the ninhydrin solution. Dry the sprayed chromatogram with a hair dryer, distinct colored spots will appear as a result of the ninhydrin reacting with the amino acids.

Interpretation of Chromatogram

1. Circle around each color spot.

2. Use a ruler and draw a plus sign in the center of each spot. Measure the distance from the starting line to each plus sign. Record this distance for each spot on your lab report. These are the DD values, in cm.

3. Measure the distance between the starting line and the finish line or, the farthest up that the solvent front reached. Record this distance. This is the FF value, in cm.

4. Calculate the retention factor (Rf) for each spot and record the values in your lab report.

5. You and your lab partner will hand in your lab reports at the same time, with the paper chromatogram stapled to one of the lab reports.

 

Formula –

 

Results

RF1 = -----------------

 

RF2 = -----------------

R3 = -----------------