The volume of tryptophan 0.016 M solution was varied; 0.1, 0.2, 0.25, 0.3, 0.4, 0.6, 0.8, 1.0 mL, while other mixture constituents and test procedure remained unchanged, see paragraph 2.1. As can be seen in Figure , there is a proportional increase in absorbance with increased added volume of tryptophan solution until reaching 0.3 mL. At this point, tryptophan amount (0.48 x 10 -5 moles) should have reacted with the complete amount of glyoxylic acid present in the sample (amounting to 0.7 x 10 -5 moles). Afterwards, absorbance increased sluggishly nearing a plateau at 0.6 mL (or 0.6*0.016=0.96*10 -5 mol). The later increase in absorbance with larger tryptophan solution volume can be ascribed to the interaction between tryptophan and iron II ion produced during reaction. We decided to keep the amount of tryptophan in this excess in all after coming tests to compensate for its loss in reaction with iron II ion. produced differing colors from crimson-violet and lacked reproducibility. This is not astonishing, because tryptophan-glyoxylic acid reds are known to be greatest unstable pigments. They readily lose CO 2 and soon oxidize on little warming .