Abstract
A new technique of on-line electrochemical derivatization using glassy carbon column electrode as a reactor is established. Materials eluting from a separation column are converted in the reactor to form fluorescent or intensely UV-absorbing products, the products being monitored by a suitable detector. An example of the use of this technique is the detection of homovanillic acid (HVA) in urine. HVA is applied to the column (Hitachi #3011-N, 250×4mm i.d.) of HPLC and derivatized to the fluorescent dimer (2, 2-dihydroxy-3, 3-dimethoxybiphenyl-5, 5-diacetic acid) by electrochemical oxidation in the reactor. The intensity of the fluorophore is measured with a spectrofluorometer (λex325nm, λem420 nm) and recorded graphically. Linearity of the relationship between peak hight and amount of HVA is obtained between 5 ng and 120 ng. The reproducibility, expressed in terms of coefficient of variation, is 1.6% (20ng, n=10). As none of the derivatizing reagent need to be added to eluent stream, this technique has a little or no effect on the dilution of eluate, back ground noise based on the derivatizing reagent and peak broadening. This technique will extend to many substances.
