On-column coaxial flow chemiluminescence detection for underivatized amino acids by pressurized capillary electrochromatography using a monolithic column.

A new method for pressurized capillary electrochromatography (pCEC) coupling with chemiluminescence (CL) detection using a modified on-column coaxial flow detection interface was developed. To evaluate the feasibility and reliability of the experimental setup, the typical CL compounds luminol and isoluminol were separated and detected by using this pCEC-CL system. A detailed investigation of CL detection interface and postcolumn CL reagent flow rate parameters was described. The excellent resolution and detection sensitivity was achieved by using 3-microm ODS-C18 packed column with 30% ACN (v/v), 5 mmol/L phosphate buffer (pH 8.0). Moreover, with the presence of Co(II) (1.0 x 10(-4) mol/L) in the mobile phase, the linear range of the concentration for luminol was 2.0 x 10(-9)-2.0 x 10(-6) mol/L with a detection limit (S/N = 3) of 2.0 x 10(-10) mol/L, and 2.5 x 10(4) theoretical plates was achieved. In addition, separation and detection of the underivatized amino acids (l-threonine and l-tyrosine) were accomplished by using a polymerized monolithic column based on the principle of the luminol-H2O2-Cu(II)-amino acid CL system. Under the optimum conditions, the mixture of amino acids was efficiently separated with satisfactory results.