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The lifespan of an HPLC column is not
fixed, as the rate of column degradation varies depending on usage
and analytical conditions.
There are extreme cases where, in continuous 24-hour operations
for drug discovery works, one
column might last about a week, while in less frequent quality
control analyses, one column could last up to
10 years.
Generally, a single column is expected to support
several hundred analyses. If a column degrades after only a few to
several dozen analyses, it should be considered that the method may
not be optimized. This could indicate that the molecular
interactions have not been adequately considered, leading to poor
reproducibility, or that the analytical conditions are too harsh for
the column.
The following are factors that can significantly impact column
degradation.
1. Impact of Flow Rate
In high-speed analysis with high flow
rates, which increase the linear velocity across the column
cross-section, the pressure differential along the axial direction
of the column becomes significant. If this large pressure
differential is sustained, it places a burden on the packing
material particles, causing them to shift within the column. This
can result in the formation of voids (gaps) at the column inlet,
leading to deteriorated peak shapes. When conducting high-throughput
(high-flow rate) analysis, it's important to be aware that the
column's lifespan may be shortened.
2. Impact of Mobile Phase Composition
Mobile phase compositions that are unfavorable for the column can
significantly shorten its lifespan. For conventional silica-based
columns, pH is the most critical factor influencing column
degradation.
Low pH (<2): Mobile phases with a pH below 2 can
cause acid hydrolysis, leading to the detachment of ligands from the
stationary phase and resulting in decreased retention.
High pH (>7): Mobile phases with a pH above 7
can hydrolyze the silica substrate itself, causing the silica
particles to dissolve and leading to peak splitting due to the loss
of packing material.
Even if the mobile phase pH is within the appropriate range, the
presence of high concentrations of inorganic salts, such as
phosphates that have poor solubility in organic solvents, can lead
to salt precipitation within the column, damaging the packing
material. It is preferable to use organic salts that do not
precipitate in organic solvents, such as ammonium acetate or
ammonium formate.
When the viscosity of the mobile phase is high, the column
pressure increases, placing stress on the packing material. In such
cases, it is advisable to reduce the flow rate or increase the
column temperature, for example to 50°C, to lower the pressure.
For ODS stationary phases, when the water content of the mobile
phase is high (for example, 100% water), the ODS ligands can be
excluded due to the hydrophobic effect of water, leading to a
reduction in effective separation sites, a sudden drop in retention,
and the formation of voids due to the contraction and movement of
packing material particles. The use of a 100% water mobile phase
with ODS columns can negatively impact column lifespan. In such
cases, it is recommended to switch to a normal phase mode using a
high concentration of organic solvents.
3. Impact of Sample Injection
When insoluble particles are present in the sample solution, they
can accumulate at the column inlet, increasing column pressure and
leading to degradation. Although connecting a guard column can help
prevent this, it also causes an increase in column pressure. The
real issue arises when the frequency of guard column replacement is
not properly monitored; repeated injections can eventually result in
insoluble particles contaminating the analytical column, potentially
leading to its failure as well. It’s not enough to rely on a guard
column alone; pre-treatment of the sample solution to remove
insoluble particles should also be considered.
Sample concentration and injection volume also impact column
lifespan. Injecting large volumes of highly concentrated samples can
place significant stress on the column inlet due to the increased
viscosity, potentially leading to void formation. To prolong column
life, attention should be paid to sample concentration and, in
particular, the viscosity of the sample solution, including the
solvent.
Moreover, even with clean samples, each injection adds stress to
the column. In the case of loop injectors, when a sample is injected
into the sample loop at atmospheric pressure and then the flow path
is switched, a sudden increase in pressure occurs. This pressure
fluctuation is transmitted to the column inlet. Repeated pressure
fluctuations from repeated injections can gradually alter the
packing arrangement at the column inlet, eventually leading to
changes in peak shape. The magnitude of these pressure fluctuations
during injection can be influenced by the sample solvent, flow rate,
mobile phase composition, and even the design of the injection
system.
4. Impact of Column Temperature
Organic stationary phases bonded to the silica surface are
susceptible to hydrolysis, a process that accelerates at elevated
temperatures. For instance, performing reverse-phase separations of
proteins at 80°C can increase the likelihood of ligand hydrolysis.
While temperature is a critical factor for improving peak shape, it
is essential to balance the benefits of high-temperature analysis
with the potential trade-off in column lifespan.
5. Impact of Elution Conditions
In HPLC, "isocratic elution" is often treated as the standard,
but just as "temperature programming" is the norm in GC, "gradient
elution" should be considered the fundamental approach in HPLC.
Isocratic analysis is only feasible when the interaction between the
solute and stationary phase, such as with ODS, is
very weak. However, it is difficult to achieve stable
isocratic analysis for ionic substances that require careful
consideration of pH and ionic strength. Repeated injections can lead
to the accumulation of impurities on the stationary phase or changes
in ligand properties due to hydrolysis, often resulting in the
conclusion that the "column has deteriorated." This method lacks
"robustness," and rather than relying on the durability of the
column, it is advisable to consider a more robust analytical method.
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One thing that can be said with certainty about column
degradation is that the performance of an HPLC column is highest
when it is first shipped from the manufacturer. Over time, not only
does the column degrade through analysis and cleaning operations,
but, much like pharmaceuticals, it can also deteriorate even when
left unused after purchase. Therefore, it is advisable to use the
column as soon as possible after purchase.
[Reference]
How to Determine HPLC Column
Degradation
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