On-the-fly frequency-domain fluorescence lifetime detection in capillary electrophoresis

On-the-fly fluorescence lifetime detection in capillary electrophoresis (CE) is demonstrated. Virtually continuous detection is achieved by interfacing a commercial CE instrument with a commercial, multiharmonic Fourier transform lifetime fluorometer (MHF). The CE capillary cartridge was modified to allow the capillary to pass through a specially constructed capillary column mount capable of micropositioning in the MHF sample chamber. Optimization of the CE/MHF interface was achieved through the incorporation of a focusing lens, appropriate alignment of the laser beam on the capillary, and use of appropriate optical filters in the emission beam. Both fluorescence intensity and lifetime are recovered from the dynamic MHF data, which is analyzed using either conventional nonlinear least-squares or the maximum entropy method, which allows for lifetime recovery without a priori knowledge of the system. Continuous, on-the-fly fluorescence lifetime detection using the MHF technology adds the dimension of fluorescence lifetime without sacrificing the resolution and speed of CE. Its application to the CE separation of a mixture of the fluorescent dyes NBD-hexanoic acid and fluorescein is demonstrated.