Application of biological samples treated by wet-digested mineralization to cation-exchange high-performance liquid chromatography with post-column reaction by 4-(2-pyridylazo)-resorcinol.
To quantify metals in biological samples, we tried to find good conditions for wet-digested mineralization of the samples and for separation of metal ions in chromatography. A 500 microliters volume of aliquot was transferred to a glass tube, and was evapolated at 100 degrees C for 2 hours. A 5.5 ml volume of a mixture of concentrated nitric acid-70% perchloric acid (10: 1, v/v) was added and heated, consecutively, at 80 degrees C for 12 hours, at 140 degrees C for 2 hours, at 180 degrees C for 2 hours, and finally at 190 degrees C for 1 hour to evapolate the residual acids. After addition of 500 microliters of 10 mM nitric acid, the metals were extracted by a suspension mixer (32 r.p.m., 1 hour). One hundred microliters of the extracted solution was applied to the chromatographic system: cation-exchange column, TSKgel IC-Cation SW (Tosoh Co.); eluent, 0.35 M lactic acid-0.35 M sodium lactate (pH 3.0); flow rate, 0.7 ml/minute; column temperature, 30 degrees C. After adding a color-forming reagent (100 mg/l 4-(2-pyridylazo)-resorcinol in 40 g/l Na2CO3; flow rate, 0.7 ml/minute) to the effluent, five different metal ions of Cd2+, Co2+, Cu2+, Ni2+ and Zn2+ were detected at 520 nm. The peaks were separated in approximately 25 minutes, and were quantified even at the 1-10 ppb levels. The present procedures were considered to provide simultaneous detection and accurate quantitation of the above five metals in the biological samples.