Fractionation of Amino Acid
Result:
Part A:
Table 1: Fractionation of amino acid Fraction No | Response to Ninhydrin test(level of blue colour change) | Response to Sakaguchi test (colour change) | 1 | X | yellow | 2 | Dark blue | yellow | 3 | Dark blue | Pale orange | 4 | Blue | Pale orange | 5 | Pale blue | Pale orange | 6 | Blue | Pale orange | 7 | Pale blue | Pale orange |
Discussion
Part A
In the separation and purification of a single protein or amino acids, a solution containing the desired analyte is passed through a column of ion exchange media, and the eluent checked to ensure that the desired component of mixture has bound to the column. The column is then washed with 2 - 5 column volumes of low ionic strength buffer to remove any unbound material which has simply adhered to the column. There are four steps of mechanism, firstly is the selective adsorption of the molecules to be separated by the ion exchange resins followed by release of the exchangeable group from the matrix. Next is the elution of the absorbed molecule by specific eluants and regeneration of the matrix by recharging with the original exchangeable groups (James, 1972). Since amino acids do not absorb light in the visible region therefore are not coloured and cannot be seen with the "naked" human eye. In this case, the visualization reagents must be used to identify this compound. Amino acids react with a dye called ninhydrin to form a highly conjugated aromatic derivative which absorbs light in the visible portion of the spectrum. Furthermore, ninhydrin amino acid derivative became blue-violet color and easily visible. For example, determination of tryptophan in proteins and feed stuff has been carried out by ion exchange chromatography with the use of ninhydrin in amino acid estimation (Khan, 2008). Besides, Ninhydrin reacted to decarboxylate the amino acids, and yield an intensively blue-purple coloured product with having absorption maximum at 570nm and the mechanism shown below:
Part A:
Table 1: Fractionation of amino acid Fraction No | Response to Ninhydrin test(level of blue colour change) | Response to Sakaguchi test (colour change) | 1 | X | yellow | 2 | Dark blue | yellow | 3 | Dark blue | Pale orange | 4 | Blue | Pale orange | 5 | Pale blue | Pale orange | 6 | Blue | Pale orange | 7 | Pale blue | Pale orange |
Discussion
Part A
In the separation and purification of a single protein or amino acids, a solution containing the desired analyte is passed through a column of ion exchange media, and the eluent checked to ensure that the desired component of mixture has bound to the column. The column is then washed with 2 - 5 column volumes of low ionic strength buffer to remove any unbound material which has simply adhered to the column. There are four steps of mechanism, firstly is the selective adsorption of the molecules to be separated by the ion exchange resins followed by release of the exchangeable group from the matrix. Next is the elution of the absorbed molecule by specific eluants and regeneration of the matrix by recharging with the original exchangeable groups (James, 1972). Since amino acids do not absorb light in the visible region therefore are not coloured and cannot be seen with the "naked" human eye. In this case, the visualization reagents must be used to identify this compound. Amino acids react with a dye called ninhydrin to form a highly conjugated aromatic derivative which absorbs light in the visible portion of the spectrum. Furthermore, ninhydrin amino acid derivative became blue-violet color and easily visible. For example, determination of tryptophan in proteins and feed stuff has been carried out by ion exchange chromatography with the use of ninhydrin in amino acid estimation (Khan, 2008). Besides, Ninhydrin reacted to decarboxylate the amino acids, and yield an intensively blue-purple coloured product with having absorption maximum at 570nm and the mechanism shown below:
References: 1. James, V., Benson, J.R., 1972. Multipurpose Resins for Analysis of Amino Acids and Ninhydrin-Positive Compounds in Hydrolyzates and Physiological Fluids. Journal Of Science Direct, 50, 477-493. 2. Khan, A.S., Faiz, F., 2008. Amino Acids Analysis Using Ion Exchange Rennins. Journal of Biology Chemistry, 48, 1-16. 3. Li, H., Liang, X.H., Feng, L.D., Liu. Y.L., Wang, H., 2008. A Simple and Fast Method for Arginine Determination in Grape Juice. Journal of Food and Drug Analysis, 16, 53-58. 4.