BCMB 203: Principles of Biochemical Techniques

Chromatography: Partition coefficient and chromatographic systems.
Basis of separation: adsorption and partition (polarity); ion-exchange (ionic nature), exclusion/gel (molecular size and shape). Principles and applications (HPLC, FPLC, GLC, TLC, Paper, Chromatofocusing and two-dimensional electrophoresis). Analytical aspects: retention time and volume, capacity ratio, peak resolution theoretical plates/plate height, peak capacity, internal and external standardization and analyte quantitation.
Centrifugation: Basic principles of sedimentation, RCF value, relationship between v, s and G. Centrifuges and rotors (types and uses). Preparative centrifugation: differential and density gradient; preparation of gradients, recovery and monitoring of fractionates. Analytical centrifugation: determination of relative molar mass (sedimentation velocity and equilibrium methods), purity and shape of macromolecules.
Electrophoresis: General principles. Low voltage thin sheets (paper, cellulose acetate, thin layer) and high voltage gels (agarose, polyacrylamide - native, gradient and SDS-PAGE). Applications; purity and molecular weight determination of proteins and nucleic acids, DNA sequencing. Iso-electric focusing and isotachophoresis.