Reasons for Increasing Pressure in HPLC

1. Column issues: Column clogging (sample retention, particulate accumulation): Failure to filter samples through 0.22 μm membranes, removal of impurities in the mobile phase, or lack of derivatization for large-molecule samples containing proteins or polysaccharides can lead to impurity accumulation within the column, resulting in progressive increase in column pressure. Chronic clogging may cause a decline in column efficiency, potentially leading to irreversible loss of performance.

Column bed collapse or packing material degradation: Reversed chromatographic column usage or abrupt pressure changes (e.g., sudden pump shutdown or rapid switching of mobile phase) may cause column bed collapse; inappropriate selection of mobile phase (e.g., using solvents incompatible with the packing material) or improper storage conditions (e.g., humidity or high temperature) may lead to packing material degradation. Both scenarios can result in increased column pressure.

Low column temperature increases mobile phase viscosity: When the column temperature is below the set value or the temperature fluctuates due to column temperature chamber malfunction, the viscosity of the mobile phase rises, leading to increased resistance and pressure in the chromatographic column. This also affects separation efficiency, resulting in peak tailing and retention time drift.

2. Online filter clogging in the filtration system: The online filter is a critical component for protecting the chromatographic column. Prolonged use leads to retention of impurities, which can clog the filter membrane, resulting in sustained pressure elevation in the system. Regular disassembly, cleaning, or replacement is required.

Saturation of the guard column or pre-column: If the guard column/pre-column is not replaced for an extended period, the retained impurities reach saturation, rendering it incapable of further filtration. This not only leads to increased pressure but also results in the loss of its protective function, allowing impurities to enter the analytical column.

Sample retention and crystallization of mobile phase may block the sealing gasket of the injector rotor, leading to increased pressure at the injection port and affecting the accuracy of sample injection, which may result in ghost peaks and poor reproducibility of peak areas.

3. Operational conditions: Excessive flow rate settings: When the flow rate exceeds the maximum tolerance range of the chromatographic column or surpasses the rated output flow rate of the instrument pump, it may cause a sharp increase in system pressure. Prolonged operation at high flow rates can also damage the chromatographic column and pump components.

Changes in solvent viscosity during gradient elution: During gradient elution, variations in the mixing ratio of different solvents can cause abrupt changes in the viscosity of the mobile phase (e.g., the methanol-water system reaches peak viscosity at a certain ratio). Failure to anticipate and adjust the gradient program in advance may lead to a sudden pressure surge.

Sample overload or complex sample matrix: Excessive sample loading or the presence of high levels of interfering impurities in the sample matrix can increase the burden on the chromatographic column, leading to elevated column pressure, as well as issues such as peak broadening and splitting.