Pressure increase is a common problem in HPLC. Possible methods to reduce pressure increase in reversed phase separation are discussed below.
If the system pressure increases, you should disconnect the column and run the system without a column to determine the line pressure. If the line pressure is high, hardware such as injectors or connection tubing may be clogged. If there is no excessive line pressure, then the high pressure may be due to the column. The column needs washing. Washing by reversed direction flow is very effective. Although generally the relative proportion of the organic solvent in the mobile phase is increased when washing, the key consideration is to choose, in accordance with the characteristics of the sample, an appropriate solvent that will dissolve easily the adsorbed material. Reversed phase separation often causes protein to be adsorbed by the packing material, resulting in high pressure. This problem can be overcome by gradient washing in the opposite flow direction with acetonitrile/ water containing 0.1% TFA, rather than washing with an organic solvent. If the high back pressre is thought to be the result of insoluble material in samples or precipitation of a sample during the separation, washing or replacing a frit (filter) might be successful.