Direct Hadamard Transform Capillary Zone Electrophoresis without Instrumental Modifications

We report the first successful implementation of a multiplexing method on a standard capillary electrophoresis system with UV detection that is independent of additional hardware. This was achieved using the Hadamard transform approach and employing vial exchange and voltage suspensions for translation of pseudorandom binary sequence elements into sample and background electrolyte injections of a capillary zone electrophoresis separation. Sequences exceeding peak capacity of the capillary were subdivided into shorter subsequences measured successively and realigned afterward based on EOF marker or analyte peaks. This way, we realized and deconvoluted modulation sequences as long as 8-bit (255 injections) for two systems containing either AMP or a mixture of the nucleotides (A,C,G,U)MP resulting in electropherograms of considerably improved signal-to-noise ratio. We achieved factors of intensity enhancement of around 6.9 and 5.2, respectively (theoretical maximum 8.0). This contribution, further, presents experimental and simulation studies on the effects on zones during injection and separation when experiencing voltage suspensions. Besides analysis of EOF behavior and influence of diffusion dispersion, we also provide data on the significance of specific electrophoretic errors such as peak position shift, inconsistent sample injection, and peak broadening on the quality of the inverse Hadamard transform. Moreover, the application of our approach to the practical analysis of a milk sample is described. The results demonstrate the applicability of multiplexing on unmodified standard CE instrumentation and establish a new suitable methodology to enhance the low sensitivity of on-column UV detection in capillary electrophoresis.