You are given a mixture of the following amino acids: as[artate, histidine, vali

Question

You are given a mixture of the following amino acids: as[artate, histidine, valine, and argininie. Design an ion-exchange chromatography experiment that will allow you to effectively seperate these amino acids. The design must include the following:

a. column matrix used (cation or anion exchanger) and why

b. buffers and appropriate pH used to equilibrate the column and elute the amino acids and the reason behind choice of buffer/pH

c. expected order of elution under your experimental conditions

Explanation / Answer

Adsorption and separation of the amino acids on the ion exchanger depends upon their dissociation constants. Thus Anion exangers are used as Anion exchangers in acid form will adsorb all amino acids and any non-electrolytes such as sugar will be separated from them. Weakly acidic resins are more suitable for separation of all types of amino acids. Four columns are used in the work. The columns are: i) charcoal, ii) wolfatit C (a carboxylic acid resin), iii) wolfatit KS (a sulphonic acid resin) and iv) Amberlite IR 4 (Cl)-. The first column adsorbed the aromatic amino acids and let the others pass. The second removed the basic amino acids (arginine, histidine), and third adsorbed the remaining neutral and acidic amino acids. The eluate from the third column was passed through the fourth column containing Amberlite IR 4 (Cl)-, where neutral acid (Valine) and those containing two carboxylic groups (Aspartate) were separated by elution with water and 1M HCl, respectively.

Citrate-bicarbonate buffers of progressively increasing pH from 3.4 to 11.0. Small amounts of detergent and thiodiglycol were added to the solution of acids to increase the rate of flow and reduce losses due to the oxidation of methionine, respectively. Such kind of buffers are chosen where at first acidic pH is maintained to elute out acidic amino acid and then the neutral and then basic amino acids.

Order of Elution: Aspartate, Valine, Hstidine, Arginine

Hope my answer helps. Thank You.

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