Separating Amino Acids by Thin Layer Chromatography

In this separating amino acids by Thin Layer Chromatography post we have briefly explained principle, Thin Layer Chromatography technique requirements, and thin layer chromatography technique.

Separating Amino Acids by Thin Layer Chromatography

Thin Layer Chromatography technique is an analytical tool commonly used in chemistry laboratories to study the purity of organic compounds or to separate and analyses the components of complex mixtures. Thin Layer Chromatography technique is a solid-liquid chromatography method in which the stationary phase is solid and the mobile phase is liquid. The stationary phase is created by coating a rectangular solid support with a very thin layer of a polar adsorbent.


Thin-layer chromatography is performed on a glass, plastic, or aluminium foil sheet coated with a thin layer of adsorbent material, most commonly silica gel, aluminium oxide (alumina), or cellulose. The adsorbent layer is referred to as the stationary phase.

After the sample is placed on the plate, a solvent or solvent combination (known as the mobile phase) is drawn up the plate via capillary action. Different analytes ascend the Thin Layer Chromatography technique plate at different rates, resulting in separation.

Depending on the adsorbent, treatment, and solvents used, it is thus based on the principles of adsorption chromatography, partition chromatography, or a combination of the two. Components with a higher affinity for the stationary phase move more slowly.

Once separated, the individual components are depicted on the plate as spots with varying degrees of movement, with components with a lower affinity for the stationary phase moving faster. To determine their nature or character, detection techniques are used.



1. Chemical fume hood

2. Hot air oven

3. Weighing balance


1. Silica

2. Sodium acetate

3. 1-Butanol

4. Glacial acetic acid

5. Ninhydrin

6. Serine–Phenylalanine–Lysine mixture


1. A pair of gloves

2. Development chamber

3. Filer paper

4. Glass capillaries

5. Glass microscopic slide

6. Glass pipette

7. Glass rods

8. Pencil

9. Ruler

10. Tweezers

Reagent Preparations

Solvent Preparation

Eluent: Butanol : Glacial acetic acid : Water (4 : 1 : 1) Pour 40 ml of 1-butanol, 10 ml of glacial acetic acid, and 10 ml of water in a 100 ml flask.

0.2% Ninhydrin solution

0.2% Ninhydrin solution: Weigh 0.2 g of ninhydrin and dissolve it in 97 ml of 1-butanol. Add 3 ml of glacial acetic acid.

Thin Layer Chromatography Technique

Plate Preparation

1. Make a 20 mL solution of sodium acetate at 20 mM. Weigh 10 g of silica and dissolve it in 20 ml of sodium acetate solution. To make a slurry, thoroughly combine all of the ingredients.

2. Pour the silica slurry on one edge of the slide and roll the glass spreader (glass rod) in a single stroke towards the other edge.

3. Allow the slides to air dry until they are white and smooth. Bake the slides for 30 minutes at 120 °C in a hot air oven. Remove the coated slides and set them aside to cool to room temperature. The Thin Layer Chromatography technique plate is now ready to be used.

Sample Spotting

Separating Amino Acids by Thin Layer Chromatography

1. Draw a light line parallel to and 1 cm away from one of the small edges with a  pencil. Make sure the silica layer does not get scraped off.

2. Prepare standard solutions of serine, phenylalanine, and lysine in water at 1% (w/v). Load the serine standard solution, phenylalanine standard solution, lysine standard solution, and the given amino acid mixture as four small spots on the spotting reference line in glass capillaries.

3. Load each of these solutions 5 times with intermittent drying in the air or with a hot-air blower. When the sample loading is finished, air-dry the Thin Layer Chromatography technique plates for 10 minutes.


Separating Amino Acids by Thin Layer Chromatography

1. Fill the development chamber with eluant to get an eluant column of ~ 0.5–0.8 cm. Dip a piece of filter paper in the eluant and stick it inside the development chamber’s wall. Close the development chamber and set it aside for two hours to allow the solvent vapours to saturate the chamber.

2. Place the dried Thin Layer Chromatography technique plate in the development chamber, making sure it does not come into contact with the filter paper kept inside the development chamber.

3. Allow the eluant to rise until the eluant front reaches the desired height (about 1 cm from the plate’s top edge). Take out the plate and mark the solvent front using a pencil. Air-dry the plate for 20 minutes followed by 2 minutes drying in a hot air oven at 70⁰C.

Visualization and analysis

1. Evenly spray the developed and dried Thin Layer Chromatography technique plate with the 0.2% ninhydrin solution. Heat the plate at ~120 ºC for 3-5 minutes for drying the plate; purple-blue spots should appear under white light.

2. Outline the spots using a pencil and measure the distance travelled by the spots relative to the spotting reference line. Calculate the Rf values of the spots and compare them with the standard spots.

Rf = (distance covered by the sample) / (distance covered by the solvent)

Further Readings