Decreased HPLC Column Pressure and Prolonged Retention Time

In HPLC analysis, there are instances where pressure suddenly drops, leading to prolonged retention times for solutes. Before attributing such drops in analytical pressure to the column, it's advisable to consider the 'HPLC pump'.

The prevalent HPLC pumps today are of the 'double plunger' type. This design features two 'plungers' within two pump heads operating 180 degrees out of phase, allowing for the aspiration and expulsion of the mobile phase and enabling low pulsation fluid delivery. To draw in and expel the mobile phase, 'check valves'—pressure-resistant valves with directional flow—are essential at both ends of each pump head. A typical parallel pump has a total of four check valves.

A common issue when restarting HPLC after a weekend shutdown is 'flow rate reduction'. If the mobile phase does not flow for a period, it may indicate a malfunction in one of the check valves, preventing the plungers from properly aspirating and expelling the mobile phase. This does not necessarily mean that the column is damaged.
The check valves, which control the passage of the mobile phase by opening and closing, are vital for high-pressure stable delivery. However, problems may arise if debris gets lodged in the valve, causing gaps that allow backflow, or if the mobile phase components adhere like glue, preventing the valve from opening and closing.

In the worst-case scenario, where both pump head check valves fail, the mobile phase ceases to flow entirely, which is immediately apparent. The real challenge arises when the flow rate drops by half, reducing pressure and significantly extending retention times. Experienced HPLC users first suspect the instrument, while novices might assume column issues because there's no visible change to the pump head, and the mobile phase appears to be flowing. Because analysis is ongoing, users tend to doubt the column.

If one pump head's check valve malfunctions while the other operates normally, only half the set flow rate reaches the column, halving the pressure while doubling the retention time. Astute users quickly associate pressure halving with a clogged check valve, but identifying which valve is clogged can be a daunting task. With four check valves in a parallel-type double plunger pump, identifying and cleaning the malfunctioning valve is a significant endeavor, as is verifying normal flow rates post-maintenance, potentially taking half a day.

An even more challenging issue is when bubbles entangle in the check valve, leading to a flow rate drop. If left alone, the bubbles might resolve themselves, but if this occurs repeatedly during analysis, it results in non-reproducible retention times on the chromatogram. This can be particularly problematic since the flow rate might appear normal, typically leading to suspicions about the column. Non-reproducible retention times usually indicate either bubble entanglement or a lack of robustness in the method. In such cases, it's crucial to use a pump pressure monitor to record and verify pressure fluctuations during analysis.

HPLC (High-Performance Liquid Chromatography) involves operating machinery (a high-performance liquid chromatograph). Proficiency in machine operation is necessary to perform analyses.

WK07 / YAZAWA Itaru,