The Handbook of Analysis and Purification of Peptides and Proteins by Reversed-Phase HPLC

 

This handbook presents the basic principles of reversed-phase HPLC for the
analysis and purification of polypeptides. For further details regarding
reversed-phase HPLC separations of polypeptides please refer to the technical
references at the back of the Handbook or contact the Grace Vydac Technical
Support Group.
Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Mechanism of Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
The Role of the Column in Polypeptide Separations . . . .  . . .8
Analytical Conditions: The Role of the Mobile Phase and Temperature . . . .17
Reversed-Phase HPLC/Mass Spectrometry . . . . . . . . . . . . . . .26
The Role of Reversed-Phase HPLC in Proteomic Analysis . . . . . . . .30
Examples of the Use of Reversed-Phase HPLC
in the Analysis of Polypeptides . . . . . . . . . .  . . . . . . . .32
HPLC as a Tool to Purify and Isolate Polypeptides . . . . . . .. . . . . .40
Viral Inactivation During Reversed-Phase HPLC Purification . . . . . . . . .50
Appendices
Appendix A: Column Characteristics . . . . . . . . . . . . . .52
Appendix B: The Care and Maintenance of Reversed-Phase Columns . . . . .53
Appendix C: The Effect of Surfactants on Reversed-Phase Separations . . . .56
Appendix D: Ion Exchange Chromatography:
Orthogonal Analytical Techniques . . . . . . . . . .  . . . . . . . . .58
Appendix E: The Effect of System Hardware
on Reversed-Phase HPLC Polypeptide Separations . . . .. . . . .60
Technical References . . . . . . . . . . . . . . . . . . . . . . . . . .62

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