Amino Acid Chromatography

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In this experiment paper chromatography was used in order to identify two unknown amino acids using eight known amino ones. The two unknown ones were identified by comparing the distance they travelled up the chromatography paper and their Rf values to the corresponding values of the other eight known amino acids. The unknown amino acids identified were Glycine and Methionine.

Proteins in cells are important in many ways. There are different types of proteins such as contractile proteins, enzymes, hormonal proteins, structural proteins and transport proteins. They are vital to regular cell functioning. Proteins are made up of amino acids that are joined together by peptide bonds. When fewer than 50 amino acids are joined together, a polypeptide is formed. All proteins have two groups in common. They have a carboxylic group and an amino group. There are 20 types of amino acids that bond together in different combinations to perform different functions. The primary structure of proteins is the order and number of amino acids. Secondary, tertiary and quarternary structures are formed from chains of peptides that are folded into sheets, ribbons and coils so that they form a 3D shape and are more stable. Different weights of amino acid make them differ in polarity. This characteristic enables the separation of proteins by polarity using chromatography. Paper chromatography is an example of a chromatography technique called absorption chromatography. The paper is the adsorbent, which will bind the components of the mixture. The substance will be “spotted” onto the chromatography paper and put into a beaker filled with solvent. The solvent will then flow through the paper. The solvent chosen depends highly on its polarity as this will be the characteristic that will separate the different substances. Petroleum, ether, hexanes, cyclohexanes and toluene are some examples of solvents with different polarities as well as increasing polarities. In some cases, mixtures of solvents are made to reach a certain polarity. If substances that are needed to be separated are polar, then the solvent must be slightly less polar. Non-polar substances need a polar solvent to be separated. The solvent travels faster than the samples. The Rf value is the ratio of the distance traveled by the sample and the distance travelled by the sample.

Rf = distance travelled by amino acid sample from the origin in mm distance travelled by the solvent from the origin in mm

Factors affecting how far the amino acids travel depend on how high the solvent is allowed to rise on the paper, the type of absorbent, the type of concentration of the solvent, temperature and the distance of the origin from the solvent. One type of test to detect proteins is the Ninhydrin test. This test makes the amino acids spots visible. Ninhydrin is a pale yellow solid and it reacts with the amino group in the amino acids and proteins and produces a purple product. Heat must be used in order to speed up the reaction.


The objective of this experiment was to spot various amino acids and an unknown mixture on chromatography paper and run it with a chromatography solvent. The lab period following included treating the samples with Ninhydrin solution and heating it so that the amino acids could be visible. The distance of the samples were then measured in mm from the origin. The measurements were then used to calculate the Rf values for each sample and thus the unknown sample could be identified.


Alanine, 1% Solution
Arginine, 1% Solution
Asparagine, 1% Solution
Aspartic acid, 1% Solution
Glycine, 1% Solution
Lysine, 1% Solution
Methionine, 1% Solution
Tyrosine, 1% Solution
Unknown, 1% Solution
Chromatography Solvent, 20mL
Ninhydrin solution, 2%, 10mL
Beaker, 600mL
Chromatography paper, 20X10 cm
Graduated Cylinder, 25-mL
Heat source, drying over or hot plate
Microtip pipets, 9
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