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Monitor and record the volume of acid or base used to set the pH for any particular buffer, and as a quality control step make sure that a similar volume is always added. , 10–20% (v/v) acetonitrile) to limit hydrophobic interactions with the exchanger, and the solvent should be added after the pH of the buffer has been set. 5. Batch vs Column Purification IEX separations can be carried out using either a batch technique, or with the exchanger packed into a chromatographic column (or with prepacked columns supplied by the manufacturer).
6), and 3) column stripping buffer (1 M NaCl), before actual use. Re-equilibrate the column with 5–10 column volumes of starting buffer and ensure that both the pH and conductivity of the eluent are the same as the starting buffer. 2. Pilot Study to Choose Optimal pH 1. A pilot test to determine the optimal pH to use for ion-exchange chromatography relies on binding of the molecule(s) of interest to an aliquot of the ion-exchanger, and its subsequent disappearance from solution (4). 5 to 1 pH unit.
Welling, G. , and Welling-Wester, S. (1987) Column liquid chromatography of integral membrane proteins. J. Chromatogr. 418, 223–243. 26. , Braat, A. and Duine, J. A. (1987) Assessment of protein purity by chromatography and multiwavelength detection. Anal. Biochem. 162, 65–73. 27. , and Eriksson, R. B. (1986) High performance liquid chromatography and diode array detection for the identification of peptides containing aromatic amino acids in studies of endorphin-degrading activity in human cerebrospinal fluid.