Step 1: Prevent the Three Most Common Causes of
Column Failure
Most people would like to extend column lifetime. This can be done by minimizing the
causes of column failure. The three most likely reasons for reversed-phase column failure
are:
Loss of Bonded Phase
As bonded phase strips off the silica support, retention times and resolution will
change, and peaks may tail more.This is the most common reason that columns "wear
out."
Cause:
- Hydrolysis of the siloxane bond of the bonded phase or endcapping reagents. It is most
severe with a mobile phase pH of less than 3.
Prevention:
- Use stabilized bonded phases (i.e. StableBondŽ) when you need to operate below pH 3
(e.g. phosphate buffered mobile phases pH 1-3). StableBond column materials are more
resistant to acid hydrolysis. Operate other types of columns within a pH range of 3 to 7.
- Operate at a mobile phase temperature of less than 60°C. A StableBond SB-C18 column can
be used up to a temperature of 90°C at low pH.
Monitor:
Capacity factor, selectivity and tailing
factor. (See Step 4)
Column Voiding
When a void develops at the head of an HPLC column, efficiency drops (low N);
resolution between peaks decreases, and peaks broaden, tail, or split into doublets.
Causes:
- Poorly packed columns that settle during use.
- Dissolving the silica column packing by operating at high pH.
- Excessive system back pressure or pressure surges due to poorly operating pumps or
sample injection valves.
Prevention:
- Keep the operating pressure under 5000 psi and eliminate any large pressure surges.
- Purchase quality columns from suppliers with the expertise to pack columns well.
- When using a mobile phase pH greater than or equal to 7, operate columns at less than
40°C to reduce silica solubility in the mobile phase.
- Operate with a mobile phase pH of less than 7. If you need to operate at or above pH 7
use a silica saturator column between the pump and the injector. The silica in this column
will dissolve first, saturating the mobile phase with dissolved silica, extending column
life. Use columns with dense, hydrophobic bonded phases, such as the Eclipse™ XDB-C8,
to protect silica from dissolution.
Monitor:
Theoretical plates and tailing factor. (See Step 4)
Column Contamination
Column fouling results from a buildup of retained material on the stationary phase.
Buildup can lead to shifts in peak retention and loss of resolution. Peak shape may also
be adversely affected by column fouling. Particulate matter or precipitated samples and
buffers will also foul a column by plugging column frits and column packing.
Causes:
- Samples containing compounds that do not elute from the column with the mobile phase
being used. This is common when using low organic mobile phases.
- Impurities in the mobile phase that adsorb to the column's stationary phase. This is
common when using ion-pair chromatography and other mobile phase additives with low
organic isocratic mobile phases.
- Particulate matter in the samples or mobile phase plugging the column inlet frit.
Prevention:
- Clean up samples before injection. This may include filtering the samples to remove
particulates and solid phase extraction techniques to remove highly retained sample or
matrix components.
- Use HPLC grade solvents for the mobile phase and filter buffer solutions.
- Routinely flush the column with a strong solvent (i.e. 100% acetonitrile) to elute
retained material off the stationary phase.
- Place a guard column in-line between the sample injection valve and the analytical
column to collect non-eluting; compounds and protect the analytical column. Change the
guard column frequently.
Monitor:
Theoretical plates, capacity factor,
selectivity, and back pressure. (See Step 4)
Table of Contents | Section 2 | Step 2 |
Step 3 | Step 4
MAC-MOD Analytical, Inc. -- info@mac-mod.com -- 1-800-441-7508
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