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Pressure increase is a common problem in HPLC. The solution to pressure increase in reversed phase separation is discussed herein.
If the system pressure increases, you should disconnect the column, run the system without a column, and determine the line pressure. If the line pressure is high, piping or tubing may be clogged. If there is no excessive line pressure, then the column pressure may be high. The column needs washing. Washing by reversed direction flow will be very effective. Although generally the relative proportion of the organic solvent of mobile phase is increased when washing, the key consideration is to choose, in accordance with the characteristics of the sample, an appropriate solvent that easily dissolve the adsorbed material. Reversed phase separation often causes protein to be adsorbed by the packing material, resulting in high pressure. This problem can be fixed effectively by gradient washing with acetonitrile/ water containing 0.1% TFA, rather than washing with an organic solvent. If the cause is believed to be insoluble material in samples or precipitation of a sample in separation, washing or replacing a frit (filter) might be successful.
However, in reality a column will become difficult to restore despite washing once it has undergone pressure increase. It is desirable that column pressure should be prevented from increasing by conducting sample preparation such as protein removal and using a guard column, as well as conducting sample filtration.
