The technique is well-suited for the capture or intermediate steps in a purification protocol. HIC media separate proteins with differences in hydrophobicity. Hydrophobic Interaction Chromatography (HIC) To reduce separation times and buffer consumption, transfer to a step elution after method optimization as shown in Figure A11.4. Check recommended flow rates for the specific medium and column. Select the highest flow rate that maintains resolution and minimizes separation time.Usually start with a 10 to 20 column volume linear gradient. Select the steepest gradient to give acceptable resolution at the selected pH.This optimization step can be combined with optimizing the ionic strength of the sample and binding buffer. Begin 0.5 to 1 pH unit away from the isoelectric point of the target protein if known. Scout for optimal pH to maximize capacity and resolution.(HiTrap ® columns have a 2.5 cm bed height, and HiScreen columns have a 10 cm bed height). If a longer packed bed is required use prepacked HiScreen IEX columns. Select optimal ion exchanger using small 1 mL columns as in the HiTrap ® IEX Selection Kit or HiTrap ® Capto IEX Selection Kit to save time and sample.
![principle app no gradient principle app no gradient](https://principleformac.com/static/video/flyoutposter.8a2c87d964cb8e60c2bef448e68b1d9c.png)
AC is also used to remove specific contaminants for example, Benzamidine Sepharose 4 Fast Flow (high sub) removes serine proteases. The key stages in an AC separation are shown in Figure A11.1.
![principle app no gradient principle app no gradient](https://mir-s3-cdn-cf.behance.net/project_modules/max_1200/9729bb62566489.5a94a0f48922d.png)
Samples are concentrated during binding, and the target protein is collected in purified and concentrated form. Elution is performed specifically, using a competitive ligand, or nonspecifically, by changing the pH, ionic strength, or polarity. Unbound material is washed away, and bound target protein is recovered by changing conditions to those favoring elution. The sample is applied under conditions that favor specific binding to the ligand. The target protein(s) is/are specifically and reversibly bound by a complementary binding substance (ligand). It is frequently used as the first step (capture step) of a two-step purification protocol, followed by a second chromatographic step (polishing step) to remove remaining impurities. AC offers high selectivity and usually high capacity. The technique is well-suited for a capture or as an intermediate purification step and can be used whenever a suitable ligand is available for the protein(s) of interest.
![principle app no gradient principle app no gradient](https://titan.as/wp-content/uploads/2019/05/principle-app-for-mac-icon-1.jpg)
AC media separate proteins on the basis of a reversible interaction between a protein (or a group of proteins) and a specific ligand attached to a chromatographic matrix.