Post-column electrochemical reduction reactor for fluorescence detection of aldehydes using high performance liquid

Abstract

Some commonly encountered difficulties in High Performance Liquid Chromatographic (HPLC) analyses are the inadequate detectability of analytes in the chromatographic eluent, and overlapping bands resulting in poor resolution of peaks on the chromatogram. These problems make separation, identification and quantization of aldehydes difficult. One common mode of detection frequently used in HPLC, ultraviolet (UV) absorption, is most problematic in this regard. To remedy this problem, a post-column on-line electrochemical reactor was employed, followed by an attempt at on-line fluorogenic derivatization. It was reasoned that using an on-line electrochemical reactor would ensure a controlled, complete, and reproducible reduction reaction, as well as eliminate the need for reagents. Also, concerns of eluent pH were eliminated, since the reaction occurred after the pH-sensitive column. In this study, a mixture of aldehydes was derivatized with an acidic solution of 2,4-dinitrophenylhydrazine prior to injection onto the chromatographic column. The mixture was injected onto the reverse-phase column using a solution of 0.10 M sodium perchlorate in 55% acetonitrile -45% water as the mobile phase. Immediately after elution from the column, the analytes were electrochemically reduced to 2, 4 -diaminophenylhydrazone derivatives, followed by an attempt on-line chemical derivatization using fluorescamine. Detection via a fluorimeter was attempted but was unsuccessful. A three electrode system employing a Ag/AgCl reference electrode was attempted but was unsuccessful due to the high pressure of the eluent forcing the reference electrode solution out of the reference electrode housing. Even though fluorimetry and the three-electrode reduction were unsuccessful, use of the two electrode reduction system proved significant. Significant differences between the absorption spectrum of the 2, 4 - DNPH-aldehyde derivatives and their reduced counterparts were observed. This led to the ability to absolutely identify 2, 4-DNPH-aldehyde components in a chromatogram by simply looking at 212 nm/345 nm absorbance ratios.