Optimization of gradient chromatofocusing for the protein separation and LC-MS/MS determination of NGP1-01 in mouse serum, brain, and retina

Gradient chromatofocusing (GCF) is a chromatographic technique developed by our laboratory that generates linear pH gradients using weak anionexchange HPLC with inexpensive low-molecular buffer components. This method shows significant advantages over conventional chromatofocusing and salt gradient ionexchange chromatography in protein separation on DEAE column. In GCF an elution buffer (consisting of multiple acidic buffers with evenly spaced pKa values) is mixed in successively greater proportions with application buffer (composed of multiple basic buffers with evenly spaced pkas), to generate a smooth linear pH gradient. GCF has the advantage of giving an estimate of the protein pI and being able to be directly interfaced to a mass spectrometer when volatile buffers are used. Although GCF yields considerable success in protein separation, extensive study has not been done yet. Further characterization and method optimization studies in GCF are done in the present work. Chapter 1 describes the characteristics of the conventional ion-exchange chromatography, chromatofocusing, and gradient chromatofocusing for protein separation.