Thin Layer Chromatography        

Cat# BB-C030 (10 reactions) 

Aim: To separate a mixture of amino acids by Thin Layer Chromatography (TLC) and identifying the unknown amino acids in comparison with known amino acids by measuring their R_f values.

Principle: Thin Layer Chromatography (TLC) is a sensitive, fast, simple and in-expensive analytical technique. It is a solid-liquid form of chromatographic technique in which one phase is solid (or stationary phase) and another one is liquid (or moving phase). It involves spotting of the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an adsorbent. The adsorbents most commonly used in chromatography are silica gel (SiO₂ x H₂O) and alumina (Al₂O₃ x H₂O). Both of them are polar, but alumina is more. Silica is acidic whereas alumina is available in neutral, basic, or acidic forms.

The sheet is placed on end in a covered jar containing a layer of solvent (generally 1-2 cm layer from the bottom). As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent and the stationary adsorbent phase. The more strongly a given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the plate.

Kit Description: Cat# BB-C030: This kit is designed to perform 10 experiments.

Bio Bharati Thin Layer Chromatography Teaching Kit contains 5 different standard amino acids stock solutions and different mixture of test solutions, TLC plates, TLC solvent, Ninhydrin solution are provided in this kit. The reagents are more than enough to carry out 10 experiments. Bio Bharati Thin Layer Chromatography Teaching Kit enables separation of mixture of different amino acids and their identification by measuring their respective R_f value.

Students will carry on the experiment as stated in “Kit procedure” and identify the amino acids by determining the R_f values.

Duration of Experiment: 2 hours.

Materials required but not provided:

Glass wares: TLC jar with lid, Glass capillary for loading the sample on plate.

Other requirements: Hot oven, Sprayer, Gloves, Whatman paper, Vaseline to seal the TLC jar lid, Forceps.

Storage: Bio Bharati Thin Layer Chromatography Teaching kit is stable for 6 months from the date of receipts without showing any change in performance if stored properly as recommended. Once receipt, store all the amino acid solutions

into 4°C refrigerator and rest of the item in room temperature. Remember to store TLC plates and developing solution at dark.

Important Instructions:

• Read the procedure carefully before starting the
• Never touch the TLC plate with bare Always wear gloves when handle the TLC plates.
• Standard amino acid solutions and test mixtures are provided Don’t mix it.
• Store TLC plate and developing solutions in dark.

Procedure:

• Place Whatman paper into three sides of the jar.
• Pour the TLC solvent in the TLC jar until the level reach 1 cm from the
• Seal the jar with lid and keep the jar at least for 1 hour before starting the experiment.
• Take out the TLC plate in a clean work area from the pack provided in the
• Draw two straight lines 1 cm above the bottom of the plate and 1 cm below from the upper side of the
• Put 1µl of each amino acid provided in the kit on the lower straight Make sure there will be even gap between each sample of amino acids.
• Wait until the samples become dry in the Carefully remove the lid of the jar, place the plate as early as possible into the jar with a help of a forceps and seal the lid again.
• While placing the plate within the jar make sure that the solvent level must remain below the lower line.               
• Keep the plate into the jar, until the solvent front reach to the upper line of the plate.

• Carefully remove the plate from the jar and air-dry the plates for 10-15 minutes.
• Spray the ninhydrin solution throughout the plate and keep the plate at hot air oven (~70°C) until the spot develop into the plate. Normally it will take around 5-7 minutes to visualize the color spot into the

Calculate the Rf value of the sample by using following formula. Standard amino acid

• Identify the amino acid present in the test sample by comparing with the Rf values of known Standard amino acid.

 Observation and Result: In the above case we have loaded Alanine, Methionine, Serine, Leucine and Lysine.

The distance travelled by these amino acids and the solvent front are listed below in the table:

The R_f Value of the amino acids are as follows:

Interpretation: In Thin Layer Chromatography amino acid samples are first spotted on the TLC plate which subsequently is placed in a chamber containing appropriate solvent (mobile phase) for separation. After the solvent front reaches certain level (generally 1 cm below from the top of the plate), it dries and the developing reagent is applied for the visualization of the individual amino acids along with the test sample. By calculating the R_f values of amino acid samples the constituents of the test sample is determined.

Troubleshooting Guide:

Technical Assistance: At Bio Bharati LifeScience Pvt. Ltd., we always believe to deliver the best products for quality research at our customers end. All the reagents are made at our in house R&D facility with utmost care to produce the best optimum results. We are always happy to stand beside our researcher friend at their need. For any assistance please write to us at [email protected] Or Contact at our telephone no. (033)-40077640, Cell 09836508080.

Adapted from internet