Introduction:
Reversed-phase chromatography is by far the most
widely used technique in high performance liquid chromatography (HPLC). It is
popular because it is applicable to most non-polar analytes and to many
ionizable and ionic compounds. Most of the stationary phases used in reversed phase
chromatography are hydrophobic in nature. Therefore, analytes are separated by
their degree of hydrophobic interaction with the stationary phase and matrices
with hydrophobic components can also be retained in a similar manner.
In HPLC analysis, Stationary phase is very important
and need to be maintained with more care. Column life will be decided by the maintenance
and washing. If the column washed properly after every analysis and stored with
end capped, then column life will be increased and the problem raises due to
column will get reduce.
What is Reversed-Phase HPLC Columns?
A reverse phase column, or reversed-phase HPLC columns, are chromatography columns that contain a non-polar stationary phase.
What Causes Contaminant Build up in Reversed-Phase
Columns and How to solve?
Sample matrix:
Injecting the sample without filtration.
Usually, sample matrix contains compounds that are of
no interest to analysts. If sample matrix components are retained strongly on
the column and if the mobile-phase solvent composition itself never becomes
strong enough to elute them, these adsorbed or absorbed compounds will
accumulate, usually at the head of the column, after many injections.
Make sure that the sample must be filtered before injecting
into the HPLC. Flush the column with appropriate washing solvent at end of every
analysis.
pH of the Mobile phase:
In general, HPLC columns are stable within a pH range
of 2 to 8. If you are measuring a pH value, the measurement must be done in the
aqueous media before mixing the eluent with organic solvents. Now a days HPLC
columns can be used out side of the pH range. Check with the vendor certificate
before using the silica-based column outside the pH range of 2 to 8. However,
best lifetimes are obtained between pH 2.0 and pH 8.
Mechanical Vibration:
Basically, in general Stationary phases based on
silica are mechanically very stable. The packed columns show no pressure limit
and can be used at more than 40 MPa (6000 psi) without any problem. However,
avoid pressure shocks on the column. Pressure shocks lead to channeling in the
column, which results in peak splitting in the corresponding chromatogram.
Mobile Phase & Solvent:
Silica based stationary phases are compatible with all
organic solvents in the above mentioned pH range. Always use correct grade of
solvents and salts during the mobile phase preparation.
It is a very good practice to filter the mobile phase
by 0.45µ membrane filter (0.22µ if UPLC) before using in HPLC instrument.
The use of non pure solvents in HPLC causes
irreversible adsorption of impurities on the column head. These impurities
block adsorption sites, change the selectivity of the column and lead to peak
splitting in the chromatogram. In gradient elution, impurities cause so called
“Ghost Peaks”. Ghost peaks are peaks that always appear in the same position on
the chromatogram. Their origin is not the sample, but the impurities from the
solvents or solvent additives. Therefore, it is highly recommended to run a
gradient without injection in the beginning of each method to determine the
ghost peaks.
Column Washing & Storage:
1. A recommended column washing system for a typical
bonded-silica column and a mobile phase without buffer salts is to use
2. After completion of the analysis, flush your column
without buffer and store in the proper storage solvent.
3. For short term storage, like overnight, columns can be
stored in the eluent used in last analysis.
4. For middle term storage, like 2 days or over the
weekend, columns should be flushed with pure water to prevent microbial growth.
5. For long term storage, silica-based columns should be
stored in an aprotic solvent. The water content should not be higher than 50%.
The best storing solvent is Acetonitrile for most of the solvent. (Storage
solvents depends product sometimes)
6. After the analysis, column should not flush with 100%
organic solvent, if buffer is used as mobile. Make sure that all buffers are
washed out of the column before flushing with Acetonitrile. Buffer salts are
mainly not soluble in Acetonitrile and can block the capillaries and the column.
7. Do not allow the column to dry out. Keep it tightly
capped when not in use.
8. Use the column in the direction indicated on the
label. An unusually high operating pressure may indicate a plugged inlet frit
and may be cleared by reversing flow through the column for 10-20 mL.
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