Investigation of Protein Recovery and Memory Effects in Reversed-Phase and Ion-Exchange Chromatography

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Amy E. Daly;Martin Gilar;John C. Gebler [Waters]
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Carbonic anhydrase, conalbumin, ovalbumin, BSA. Ribonuclease A, Bovine serum albumin, Beta-lactoglobulin A
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We investigated the separation of intact proteins by reversed-phase high performance liquid chromatography (RP-HPLC) and ion exchange chromatography using polymeric and silica-based sorbents. Ribonuclease A, bovine serum albumin, beta-lactoglobulin A, and ovalbumin were chosen as a model sample for RP-HPLC. 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 RP-HPLC columns tested. Carbonic anhydrase, conalbumin, ovalbumin, BSA, and soybean trypsin inhibitor were chosen as a model sample for anion exchange while myoglobin, alpha-chymotrypsinogen, cytochrome C, and lysozyme were chosen for cation exchange chromatography. Protein recovery was determined using an external calibration; recovery varied between 90-100 %. There was only a negligible memory effect observed for the ion exchangers. The goal of the study was to identify stationary phases with high recovery and low memory effect that would be suitable for protein analysis, purification, and characterization by liquid chromatography/mass spectrometry (LC-MS). Recommendations for column selection are also presented.

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