Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

• Published in: Journal of Chromatography A Vol. 1217; no. 42; pp. 6484 - 6495
• Main Authors: Morrison, Christopher J., Gagnon, Pete, Cramer, Steven M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.10.2010; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Analytical chemistry; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Ceramics - chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid - methods; Displacement; Durapatite - chemistry; Eluents; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; High-throughput screening; High-Throughput Screening Assays - methods; Hydroxyapatite; Models, Chemical; Molecular Weight; Other chromatographic methods; Proteins; Proteins - isolation & purification; Resins; Robotics; Selective displacement; Selectivity; Separation; Sodium Chloride; High-throughput screening; Hydroxyapatite; Selective displacement; High throughput screening; Purification; Phase composition; Selectivity; Mobile phase; Displacement; Protein; Ion exchange chromatography; Separation method; Ion exchanger; Stationary phase
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2010.08.038

A detailed study was carried out to combine the unique selectivity of ceramic hydroxyapatite (CHA) with the separation power of selective displacement chromatography. A robotic liquid handling system was employed to carry out a parallel batch screen on a displacer library made up of analogous compounds. By incorporating positively charged, metal chelating and/or hydrogen bonding groups into the design of the displacer, specific interaction sites on CHA were targeted, thus augmenting the selectivity of the separation. The effect of different mobile phase modifiers, such as phosphate, sulfate, lactate and borate, were also investigated. Important functional group moieties and trends for the design of CHA displacers were established. Selective batch separations were achieved between multiple protein pairs which were unable to be resolved using linear gradient techniques, demonstrating the applicability of this technique to multiple protein systems. The specific interaction moieties used on the selective displacer were found to dictate which protein was selectively displaced in the separation, a degree of control not possible using a mono-interaction type resin in displacement chromatography. Mobile phase modifiers were also shown to play a crucial role, augmenting the selectivity of a displacer in a synergistic fashion. Column separations were carried out using selective displacers and mobile phase modifiers identified in the batch experiments, and baseline separation of the previously unresolved protein pairs was achieved. Further, the elution order in these systems was able to be reversed while still maintaining baseline separations. This work establishes a new class of separations which combine the selectivities of multi-modal resins, displacers/eluents, and mobile phase modifiers to create unique selectivity windows unattainable using traditional modes of operation.