Aliquots of 0.25 - 2.0 mL of the standard solution
(100 μg/mL) were transferred to 10-mL calibrated flasks.
One milliliter of potassium hydrogen phthalate buffer
(pH 4.0) was added, followed by 0.5 mL of reagent solution
(0.2% w/v). After 2 min vortexing the tubes were
allowed to separate the two layers. The complex was
extracted three times with 3 mL of chloroform. After
the phases had been separated by centrifugation, the
combined extracts were adjusted to 10 mL with the same
solvent. The absorbance of the yellow-colored complex
was measured at 424.5 nm, against a corresponding
reagent blank similarly prepared. All measurements were
made at room temperature (25 ± 2°C).