A Hadamard transform (HT) detection method for microchip capillary electrophoresis with laser-induced fluorescence and a charge-coupled device (CCD) is described and compared to signal-averaged detection. A low-noise CCD camera is used to image a section of a separation channel where each camera pixel can be thought of as a unique detector. For signal averaging, electropherograms corresponding to individual pixels can be averaged for improved S/N. HT detection is performed on each pixel electropherogram to generate a contour plot electropherogram. The multiple injections required for HT provides an enhancement at the cost of longer times for the pseudorandom injection sequences. A short sample injection length of 0.25 s is used to reduce the overall analysis time and improve sensitivity compared to previously published results. An injection sequence is performed on the microchip that is based on a cyclic S-matrix of 513 elements that generates an 8-fold improvement in S/N compared to a single injection. This spatially resolved HT detection method is also capable of performing a multicomponent separation. Signal-averaged HT and single-injection data are compared to experimental HT and single-injection results. The unique capabilities of each method are described.
This paper describes an improved format for Shah convolution Fourier transform (SCOFT) detection that utilizes the spatial resolution of a charge-coupled device (CCD) rather than a fixed optical mask to perform a Shah or sine convolution over a fluorescence signal. The laser-induced fluorescence from a 9-mm section of microfabricated channel is collected with a CCD at 28 Hz. Each image frame is multiplied by a convolution function to modulate the collected signal through space. Each frame is then summed to generate an intensity-versus-time data set for Fourier analysis. The fluorescence signal oscillates at a frequency dependent upon both the convolution function multiplied across each data frame and the velocity of fluorescent microspheres or a plug of fluorescent dye flowing through the channel. This SCOFT technique affords more flexibility over formats that employ a physical mask and provides data that can be optimized for signal-to-noise (S/N) or resolution information. A 1,000-fold improvement in S/N is demonstrated for a plug of fluorescein dye. Detection of fluorescent beads exhibited frequency signals that were dependent upon the bead size distribution, the electric field, and the electrophoresis buffer concentration. Data are presented demonstrating the quantitation of fluorescent microspheres.
A Hadamard transform-capillary electrophoresis-UV (HT-CE-UV) detection technique is described for the analysis of biological samples. Pseudorandom injections of sample and buffer according to a simplex matrix obtained from the corresponding Hadamard matrix is performed with conventional capillaries. Alternating injections are achieved with a novel capillary "T" connector created by drilling conventional capillary dimensions through a 1-cm diameter polycarbonate disc. This connector design coupled with a switching system allows for rapid, electrokinetic injections of solution into alternating sample and buffer capillary arms for UV detection. The standard mixtures of nitric oxide (NO) metabolites, nitrite and nitrate, dissolved in physiological saline solution are injected into the separation capillary according to an 83-element injection sequence to obtain a signal-to-noise ratio (S/N) enhancement of ca. 4.5 over a single injection. Nitrite, being the less concentrated metabolite in NO detection and thereby more difficult to detect, was calibrated with the HT-CE-UV method and a limit of detection (LOD) of 0.56 microM was obtained. Rat blood plasma was analyzed with this detection system and demonstrated to be comparable with NO metabolite concentrations of previously published results. This HT-CE-UV method is described where a unique reservoir tube design that contains 8-microL standard nitrite sample volumes is placed over the end of the capillary arm to explore low volume limits for biological samples.
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