Recovery and Memory Effects in Reversed-Phase Chromatography of Proteins

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Amy E. Daly;Martin Gilar;Peter J. Lee;John C. Gebler [Waters]
FAll ACS 2002; 224th ACS National Meeting; Boston August 18-22
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Protein; BSA; Bovine Serum Albumin; beta-Lactaglobulin A; Ovalbumin
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We investigated the separation of intact proteins by reversed-phase high performance liquid chromatography (RP-HPLC) using polymeric and silica-based sorbents. Ribonuclease A, bovine serum albumin, beta-lactoglobulin A, and ovalbumin were chosen as a model sample. Protein recovery was determined using both an internal and external calibration; the recovery varied between 30-100 %. Residual amounts of protein eluted in up to three successive blank gradients following the protein mixture injection. This column memory effect was observed to a varying degree for all columns tested. The goal of the study was to identify stationary phases with high recovery and low memory that would be suitable for quantitative chromatography as well as liquid chromatography/mass spectrometry (LC-MS) for protein analysis.

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