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