Practical Considerations for HIC Separation

This section covers detailed aspects of each step in a HIC separation, together with practical hints and tips to improve resolution and overall performance. In practice the steps in a separation can be summarized as follows:

  1. Equilibrate column with 5–10 column volumes of start buffer or until UV baseline and conductivity are stable.
  2. Adjust the sample to the chosen salt concentration (and pH if necessary). Filter and apply to the column.
  3. Wash with 5–10 column volumes of start buffer or until the UV baseline and conductivity are stable, that is, when all unbound material has washed through the column.
  4. Begin elution using a gradient volume of 10–20 column volumes, increasing the proportion of elution buffer until the salt concentration reaches a minimum, that is, salt-free buffer (100% elution buffer).
  5. Alternatively, if gradient-making equipment is not available, elute bound proteins with up to 5 column volumes of elution buffer at a salt concentration lower than that in the start buffer. Repeat, lowering the salt content at each step until the target protein(s) has been eluted.
  6. Wash with 2–5 column volumes of salt-free elution buffer to elute any remaining hydrophobically bound material.
  7. Re-equilibrate with 5–10 column volumes of start buffer or until conductivity reaches the required value.

These steps are highlighted together with more detailed hints and advice throughout this section.

Buffer volumes are expressed in column volumes, for example, 3 CV=3 ml for a column with a 1 ml bed volume. Using column volumes to describe a separation profile facilitates method development and transfer of methods to columns of different dimensions. The number of column volumes used at each stage of the separation can often be reduced by optimization. For example, the gradient volume can be reduced if resolution can be maintained, and less buffer may be required for washing when separating reasonably clean samples.

  • Maintain sample, start and elution buffers, columns and chromatographic equipment at the same, constant temperature throughout a separation to ensure consistent, reproducible results.

Fig 18. A typical purification strategy has three phases: Capture, Intermediate Purification and Polishing (Cipp). Each phase has a specific objective, dependent largely on the properties of the starting material. Select the appropriate HIC medium according to the objective of the purification step and the condition of the starting material.

Note: STREAMLINE™ products, based on expanded bed adsorption technology, enable proteins to be purified from crude, particulate feedstock without the need for separate clarification, concentration or initial purification. STREAMLINE™ products are designed for use in industrial-scale processes and for producing gram quantities of product. STREAMLINE™ Phenyl is available only as a custom-designed product.

Materials