|
The
HPLC Column Pressure is High!
There are several possible causes that can explain this issue:
1. Pressure of a New Column
If you notice
that the pressure is high when starting analysis with a brand-new
column, it may be because the new column differs from the one you
previously used.
Under the same
analytical conditions, the smaller the particle size of the packing
material, the higher the column pressure.
For example, if you previously used a column with 5 µm particles and
switched to one with 3 µm particles (with the same column
dimensions), the column pressure will be more than
twice as high under
the same conditions.
Column pressure is
inversely proportional to
the square of the particle size. A 1.5 µm column, for
instance, will generate four
times the pressure of a 3 µm column. However, resolution
only improves in proportion to the
square root of the
particle size, meaning the resolution gain is only about
1.4 times.
Choosing a
small-particle column without proper consideration may increase the
risk of pressure-related issues such as solvent leaks in the HPLC
system. Considering potential connection problems and maintenance
due to leaks, a column with a particle size of around
3 µm is generally
the most appropriate choice for most current HPLC systems.
(Reference)
The
Practical Advantages of 3 µm Particle Columns
2. Insoluble Components in the
Injected Sample
The sample
solution injected into the column must be completely clear, even if
it is colored.
If the solution is
even slightly turbid, particulate matter can be introduced into the
column, causing a rapid increase in backpressure.
To remove turbidity, it
is common to filter the sample using a
0.45 µm or 0.2 µm membrane
filter, but this does not guarantee complete removal.
Submicron particles,
typically in the range of a few hundred nanometers, can still pass
through and accumulate at the column inlet with each injection,
gradually raising the column pressure.
In most cases, once
pressure increases, it cannot be fully restored. However, in rare
situations where clogging occurs at the
inlet frit,
reversing the column connection may allow the accumulated particles
to be flushed out, resulting in a drop in pressure.
Note that not all columns are designed for reverse flow—always
check the manufacturer’s guidelines before attempting
reverse flushing.
3. Macromolecular impurities
Biological samples often
contain a variety of high-molecular-weight compounds, such as
proteins, DNA, and polysaccharides. These substances are generally
hydrophilic and may dissolve completely in the injection solvent,
resulting in a clear solution. However, once injected into the
column, they can be trapped by the packing material and remain
inside the column.
This is especially
problematic when using organic solvents like acetonitrile in the
mobile phase, which tend to promote aggregation.
High-molecular-weight compounds that form aggregates inside the
column can lead to increased backpressure and are typically very
difficult—or even impossible—to remove through standard washing
procedures.
Therefore, if such
macromolecules are present in the sample but are not the target of
analysis, they must be removed through sample pretreatment. Since
they are dissolved in solution,
membrane filtration is not
effective for their removal.
One method for removing
serum proteins involves mixing the sample with an equal volume of
0.4 M perchloric acid
or 5% sulfosalicylic acid
to denature and precipitate the proteins, followed by
centrifugation to
separate them from the solution.
(Reference)
Deproteinization
for LC-MS Analysis of Amino Acids in Serum
4. Column Degradation
When the packing material
undergoes hydrolysis due to the mobile phase, the silica particles
may degrade and break down into fine particles, leading to clogging
inside the column. This can result in a rise in column pressure. In
such cases, voids or
channeling often occur, causing
severely distorted peak
shapes.
Column regeneration is
not possible in this situation, and the only solution is to
replace the column with a
new one.
For silica-based
columns, prolonged analysis under
alkaline conditions (pH > 7)
increases the likelihood of silica hydrolysis. If alkaline analysis
is not required, it is advisable to perform the analysis at
neutral or acidic pH
to preserve column integrity.
As a column is used over time, various
factors can gradually cause an increase in pressure.
If the column pressure is high but there are no issues with peak
shape or compound separation, it is often more efficient to
continue using the column
as is—by raising the pump’s
pressure limit and carefully monitoring for any
potential leaks in the flow
path.
|
