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Study Notes: The Mobile Phase

The selection/preparation of the sample is crucial to the effectiveness of the HPLC technique, as the sample must interact with both the stationary and mobile phases. Additionally, selection of a correct mobile phase is important.

The sample itself must satisfy a number of criteria before it can be analysed. The main points to remember are the following.

  • The mobile and stationary phases should be selected to provide optimum separation of the analytes in the sample.
  • The sample should be dissolved in a buffer that is compatible with the mobile and stationary phases.
  • The sample should not contain any particulate matter.
  • The sample should not contain any material that will ‘poison’ (that is, irreversibly bind) the column.
  • The sample should be concentrated in a small volume (injection volume is small).

The mobile phase itself is of equal importance to the stationary phase and is chosen to elicit separation of the analytes of interest during the HPLC run. A good mobile phase has the following characteristics.

  • Compatible with the column packing/stationary phase and the sample.
  • Gives good separation of the analytes of interest.
  • Is of the highest purity.
  • Must not contain particulate matter.
  • Must be de-gassed immediately prior to use.

In reversed-phase HPLC (often used for protein separations) the mobile phase commonly used is the polar hydrocarbon acetonitrile (CH3CN, also called methyl cyanide). This is often run in a gradient elution against water. A common elution would be:

0% to 60% acetonitrile in 0.05% w/v trifluoroacetic acid (TFA) in water. TFA helps to solubilise the protein.

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