 Dear students, chromatography is an analytical technique that can be used to identify and separate biomolecules based on their chemical properties. A mixture of amino acids or biomolecules can be separated and identified by means of paper chromatography. The presence of amino acids on the chromatogram can be detected by spraying it with NIN Hydrin which gives a highly colored product upon reaction with amino acids. In paper chromatography, the biomolecule or a solution of biomolecules is spotted on a piece of filter paper which is then placed in an organic solvent. The organic solvent rises up the filter paper by means of capillary reaction. As it moves along the filter paper, it takes the biomolecule along with it. The distance travelled by the biomolecule depends on its relative affinity for the hydrophobic organic solvent which is the mobile phase and the hydrophilic filter paper which acts as the stationary phase. The distance travelled by the biomolecule is specific for each biomolecule and can be calculated to detect which biomolecule is present in the sample. Today we will be performing the paper chromatography of amino acids. To begin with, we will take the mobile phase or the organic solvent which contains butanol, acidic acid and water in the ratio 60, 15 and 25. Take a chromatography tank and add the solvent to it ensuring that it covers the base of the tank forming a layer half to one inch in thickness. Cover the tank and allow the atmosphere to get saturated with vapours of the organic solvent or mobile phase. Next, take a clean piece of filter paper. Using a lead pencil, draw a line at least 2.5 cm from the base of the filter paper. Do not use any ink pen or ballpoint to draw this line since they also contain compounds that will then interfere with the results of the chromatography. In the next step, carefully place a drop of the amino acid solution on the filter paper. Allow the spot to dry. If more than one sample has to be analysed, the distance between the spots of the two samples should be at least 2 cm. Using any point of this experiment, do not touch the filter paper with bare hands since skin secretions also contain compounds that will react with ninhydrin and give us false positive results. Allow the sample to air dry completely. Once the sample has dried completely, carefully place this filter paper in the chromatography tank. While the base of this filter paper should be in contact with the chromatography solvent, the line where we have placed our biomolecule or amino acid solution should not be immersed in the solvent. Leave the filter paper in this chromatography tank till the solvent has risen to at least two thirds of the length of this entire filter paper. This usually takes about 40 to 60 minutes. At any point during chromatography, the filter paper should not come in contact with the walls of the tank. After about 60 minutes, you will observe that the solvent has risen to approximately two thirds the length of the filter paper. At this stage, carefully remove the filter paper from the chromatography tank. Allow the chromatogram to dry completely in the air before moving on to the next step. Once the chromatogram has dried completely in the air, mark the distance travelled by the solvent with a lead pencil. This is known as the solvent front. Next, we will be spraying this chromatogram with NIN hydrogen so that we are able to visualize if the amino acids are present in this sample. Now place the chromatogram in an oven at 95 degree centigrade for approximately 4-5 minutes. After 5 minutes, we will remove this chromatogram from the oven. We will be able to clearly see the spot of the amino acid on the chromatogram. We will now proceed towards the calculation of the RF value or the retardation factor also known as relative mobility or retention factor. For this, we will first measure the distance travelled by the solvent on the filter paper. We have already marked the solvent front. This comes out to be 15 cm for our sample. We will then calculate the distance travelled by the spot or sample on the filter paper. We will measure the distance from the center of the spot. This comes out to be 2.1 cm for our sample. Hence, the RF value comes out to be 0.14 which is the RF value for the amino acid lysine. Therefore, we can identify that our sample contained the amino acid lysine. Paper chromatography is a valuable tool for the identification of amino acids. Each amino acid has a different RF value based upon the presence of the R group in that amino acid. The RF values can be used to identify the amino acid present in the solution. This indicates that our sample contains the amino acid lysine. Paper chromatography is a valuable tool for the identification of amino acids. Each amino acid has a different RF value based upon the presence of the type of R group in that amino acid. Therefore, using RF values, we can identify which amino acids are present in our sample.