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Separation of proteins by cation-exchange sequential injection chromatography using a polymeric monolithic column

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Abstract

Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24 % GMA, 16 % EDMA, 20 % cyclohexanol, and 40 % 1-dodecanol (all % as w/w) containing 1 % of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na2CO3, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH2PO4/K2HPO4) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 μL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 μL s−1) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

Sequential injection chromatograph for cation-exchange separation of proteins in a iminodiacetatemodified polyglycidyl methacrylate-co-ethylene dimethacrylate, IDA-poly(GMA-co-EDMA), monolithic column, where MP1 = 20 mM KH2PO4/K2HPO4 (pH 7.0), HC = Holding coil, SP = Syringe Pump, RV = Relef Valve and D = UV-Detector

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Acknowledgments

This work was funded by grants 2013/18507-4 from the São Paulo Research Foundation (FAPESP) and 306075/2013-0 from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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  1. Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970, São Paulo, SP, Brazil

    Jorge Cesar Masini

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Masini, J.C. Separation of proteins by cation-exchange sequential injection chromatography using a polymeric monolithic column. Anal Bioanal Chem 408, 1445–1452 (2016). https://doi.org/10.1007/s00216-015-9242-9

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