Thermo Electron introduces the Biomolecules Guide, a reference for the separation of biological molecules for liquid chromatography and mass spectrometry
The new guide describes techniques for the separation and purification of proteins, peptides, oligosaccharides, and various other biological molecules from a liquid chromatographic standpoint.
Various modes of HPLC, including reversed phase, ion exchange and size exclusion are described in detail for the separation of biomolecules.
Guidance is given to help choose the appropriate column and separation mode for the different biomolecule applications.
Also included in the guide are comparisons between various Thermo Electron phases.
These comparisons between BioBasic, BetaBasic, and Hypercarb outline the importance of pore size and phase characteristics when performing HPLC on biomolecules using both ultraviolet and mass spectrometric detection.
The fundamentals of gradient time and slope in relation to pore size are discussed when analysng digests with outcomes reported in sequence coverage.
The guide describes the chromatographic modes associated with proteins and peptides in meticulous detail and contains several quick reference tables.
The mechanisms of retention are explained for reversed phase (RP), ion exchange chromatography (IEC), and size exclusion chromatography (SEC) as well as the fundamental differences between these three common modes for separating and purifying proteins.
The tables outline molecular weights of common proteins, buffer capacities and appropriate working pH ranges, optimum flow rates and injection volumes for columns of capillary dimension (75u ID) up to preparative sizes (100mm ID), and properties associated with common HPLC solvents.
Proteomics discussions include both top-down and bottom-up approaches and the inherent difficulties and advantages associated with each methodology.
Two dimensional LC/MS are discussed for all three modes (RP, IEC, and SEC) along with mobile phase considerations for analyzing biomolecules in a mass spectrometer.
