Multidimensional separations of biological samples

The separation of complex biological mixtures requires a separation technique that has a high peak capacity. The use of multidimensional separations can solve this requirement as the overall peak capacity of a multidimensional separation is equal to the product of the peak capacities of each individual dimension. In this work combinations of reversed phase liquid chromatography (RPLC), capillary zone electrophoresis (CZE), and electrospray ionization-time of flight mass spectrometry (ESI-TOF MS) are employed to separate various biological samples.; Chapter 1 introduces the combination of RPLC and CZE with laser induced fluorescence detection. This two dimensional (2D) system has an estimated peak capacity of 2600. The system was used to successfully separate a tryptic digest of bovine serum albumin (BSA) and several targeted biomolecules from the adrenal medulla. However, the 2D RPLC-CZE system has difficulties identifying peaks in the separation, while the derivitization of the sample to render the analytes fluorescence is demonstrated to cause degradation of some of the samples.; Chapters 2 and 3 focus on replacing the laser induced fluorescence detection with a third dimension of ESI-TOF MS. These chapters examine the use of the two major types of interfaces, sheath-flow and sheathless, between the CZE and ESI-TOF MS, and various combinations of running conditions. The peak capacity of this system was estimated to be 1.3 million. The 3D RPLC-CZE-ESI-TOF MS system is used to separate a mixture of peptides. Although the system is successful in separating a portion of the mixture, there are two issues that make this system unsuitable for further study. First, combining CZE and ESI-TOF MS requires the use of two power supplies and many electrical pathways. This electrical system creates a situation where it is difficult to maintain stable electrospray. More importantly, the 3D RPLC-CZE-ESI-TOF MS system can only detect components that have a concentration of 50 μM or greater. Many peptide concentrations in biological media are in the low nM range and would not be detectable by this system.; The final chapter describes a system modification of the 3D RPLC-CZE-ESI-TOF MS to address the concerns brought up in the previous chapters. The decoupling of the RPLC from the CZE-ESI-TOF MS removes the interface between the RPLC and the CZE. This reduces the number of electrical pathways, decreases the complexity of electrical system and makes it easier to achieve stable electrospray. Furthermore, the decoupling allows for sample concentration techniques that extend the lower limit of detection to the low nM. (Abstract shortened by UMI.)...