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HPLC columns have a finite lifespan.
Although the exterior is made of stainless steel, the interior is
packed with silica or polymer particles. Over time, the packing
inside the column undergoes gradual changes due to the stress caused
by passing samples and mobile phases. Additionally, the surface of
the stationary phase may undergo chemical changes, leading to
degradation that manifests as changes in analytical results.
The following explains how to determine whether an HPLC column
has degraded.
1. Track Changes in the Measured
Values of Analytes
The best way to determine column
degradation is by evaluating the peak shape of the analytes.
Tracking changes in peak height and theoretical plate number is
one of the most practical methods. Record the initial values of
these metrics and use them as a benchmark to define the column's
usage limit. If the values fall below acceptable levels and the
analysis is no longer feasible, the column can be considered to have
reached the end of its life.
Depending on the analytical method, it may also be necessary to
establish limits for other parameters, such as changes in retention
time or increases in column pressure, in addition to peak shape.
2. Measure the Resolution (Rs)
Between the Target Analyte and Adjacent Impurity PeaksTrack Changes
in the Measured Values of Analytes
This method involves monitoring the
separation between the target analyte and nearby impurity peaks
using the resolution (Rs) metric. It is particularly effective for
applications where HPLC separation is critical.
As the column degrades, peak broadening occurs, leading to a
decrease in resolution. By setting an acceptable limit for
resolution, the column can be considered to have reached the end of
its usable life if the resolution drops below this threshold.
In cases of reversible degradation, column performance may be
recoverable through proper cleaning. However, if the degradation is
due to chemical changes in the stationary phase or alterations in
the packing material, the damage is irreversible, and column
performance cannot be restored.
3. Regular Measurements Using an
In-House Standard Substance
While the methods described in points 1 and
2 are practical for managing column degradation using actual
samples, there are cases where practical monitoring is difficult.
This includes situations where the analyte is present at very low
concentrations, is structurally unstable, or produces poor peak
shapes. In such cases, it is recommended to establish an analytical
method using a stable and readily available compound as a substitute
standard, which can then be used to monitor column degradation.
To implement this approach effectively, it is necessary to
develop a robust analytical method that considers the stationary
phase properties, sample structure, and mobile phase composition.
For long-term operational goals, creating and managing such a
standardized method provides an objective means of monitoring
changes in column performance over time.
4. Perform Measurements Under the
Manufacturer's Inspection Conditions
HPLC columns come with inspection data
(Certificate of Analysis, COA) issued at the time of shipment. This
document serves as the manufacturer's record to confirm that the
column meets the specified packing quality and performance
standards.
At Imtakt, the COA includes details
about the mobile phase composition and the concentration of the
analyte, among other analytical conditions. Additionally, by
entering the Prod# and Ser#, users
can reissue the COA through the following system:
[COA
Reissuance Program]
When purchasing a column, performing an analysis based on the COA
conditions and setting acceptable limits for peak shape degradation
can provide a guideline for determining when to replace the column.
However, since HPLC instruments may vary, the manufacturer's COA
data and the user's analytical results may not always match.
Therefore, it is essential to use the initial performance values
obtained by the user as a reference.
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When assessing column degradation, if the column's lifespan seems
unusually short, it may be necessary to review the analytical method
before questioning the performance of the column itself.
[Reference]
Causes of HPLC Column Degradation
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