Abstract
The micro-determination of the free amino acids in presence of albumin in human embryos' culture medium using different analytical techniques is the main target of this manuscript. This test involved using ninhydrin reagent in micro-determination of both glycine and tryptophan present in the tested medium in pure form. The molar absorptivity of the product was found to increase gradually with the period of heating at 60 ̶ 70oC until 8 ̶ 10 minutes. The absorbance attained maximum values at 8.0 min. The effect of pH was also studied and showed that pH= 5 is the preferred value for reaction between glycine and ninhydrin while pH = 8 is preferred for tryptophan determination. It was shown that ninhydrin reacts with glycine or tryptophan with the ratio 1:1 under optimum conditions (λmax = 560 nm and pH = 5). Calibration graphs were constructed for micro-determination of both glycine and tryptophan in pure forms with recovery values ranging from 98.93 ̶ 101.9 % and 98.06 ̶ 100.7 % respectively. Studying the interfering possibility of some main components of the tested culture medium showed that there is no interfering possibility. The calibration curve for determination of the total glycine and tryptophan content in a binary mixture was constructed and it is linear in the concentration range 8.356 ̶ 66.64 µg mL ̶ 1. Therefore; this proposed method can be successfully used to determine the total amino acids in the mentioned concentration range (8.356 ̶ 66.64 µg mL ̶ 1) with a high accuracy, precision and sensitivity. The determined amino acids in actual medium by the proposed spectral method gives concentration of the total amino acids = 510.98 μg mL̶ 1. This value is compared with that obtained by analysis of the same medium using ninhydrin method at Agriculture Research Center (ARC); which is found to be 481 µg mL ̶ 1. The identification and individual determination of amino acids present in actual culture medium were performed by using ion chromatographic method and the summation of concentrations of these free amino acids is found to be 301.41 µg mL ̶ 1. This result is correlated in a confidence level of 95% with that found in literature on the same medium which is 327.54 µg mL ̶ 1.