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- Efficient Phase Conversion Of HPLC Instruments
Posted by : kaushik zala
Sunday, March 30, 2014
While reversed-phase (RP) HPLC is still by far the most common
mode, normal-phase (NP) HPLC is increasing in popularity
with the introduction of new highly polar columns with
excellent retention, selectivity and stability. It is easy to interconvert
between RP and HILIC (a type of NP) because both
modes employ polar aqueous mobile phases; however, many
compounds are not polar enough to be retained under aqueous
HILIC conditions. When aqueous solvents must be replaced
with non-aqueous conditions to study non-polar samples,
immiscibility situations can arise during changeover.
The first best practice is to dedicate instruments to a specific
mode. Significant seal wear-and-tear can be caused by
expansion, contraction and extra friction of changing solvents.
If possible, columns should also be dedicated to one mode for
trouble-free operation. If dedicating the instrument or column is
not possible, one should use the following procedure.
Our regular practice is to replace the column with tubing or
a union and flush extensively with isopropanol (IPA) before
going over to water or hydrocarbon. Before beginning the
changeover process, remove the HPLC column, cap and store
in the appropriate storage solvent unless the same column
is to be used in the new mode. Columns such as Cyano and
Fluorophenyl (F5) can work in either RP or NP mode and can
remain installed if desired. After IPA flush, the column can be
removed and capped to avoid excessive wear on the valuable
component. In the flushing steps, be sure to include the entire
fluid path (pump, autosampler, valves, detector, etc.). Also,
include the sample loop and any other fluid paths that are
encountered for the normal operation of making injections. This
can vary considerably depending on whether the autosampler
is an external loop design, or an internal loop design. As part of
all the washes, make certain the injection needle gets washed as
well. It is best to do several full loop injections of a solvent such
as IPA that is miscible with both high aqueous and high organic
mobile phases. The total volume of IPA needed will vary with
instrument design, but the waste volume should be monitored
(record the volume as a guide for future changeovers) and
observed for uniform appearance. UV detectors may remain on
(ca. 250 nm) during this step to indicate when the system has
returned to a stable baseline.
The second wash step after the IPA should be with methanol
(or ethanol). Follow the previous procedure that was used
for the IPA wash before going to water. Methanol will help
flush the IPA out faster than going directly from IPA to water. If
excessive baseline noise or drift is observed with a UV detector,
repeat the procedures and allow more time to flush out any
poorly swept flow regions.
Some Dos and Don’ts for Solvent Changeover:
- Do remove all additives and start with 100% isopropanol in all reservoirs.
- Isopropanol is fully miscible with all common solvents and is the safest changeover solvent for either direction.
- Do use low flow - about half of normal to avoid excessive seal wear and damage due to over-pressuring.
- Don’t use acetonitrile routinely as the changeover solvent- it is better than methanol, but is not fully miscible with pure hydrocarbons.
- Don’t use methanol routinely as the organic- it is not fully miscible with many normal phase conditions.
- Either acetonitrile or methanol may be used to routinely change from reversed-phase and back (remember to remove additives).
- Do check miscibility (use small external vessel) with target mobile phase before starting, especially if IPA is not selected.
- Do use organic (such as IPA) in all lines of a gradient instrument to make certain that water or hydrocarbon is removed from all fluid areas.
- Do operate the injector valve and any other selector valves while doing the IPA flush procedure.
- Do monitor pressure and detector signals during changeover as these are excellent methods to confirm full system equilibration evaporative detectors such as MS and ELSD cannot be used for this purpose.
- Incomplete mixing shows up as severe detector baseline noise or pressure fluctuations (globules of immiscible solvent can resemble bubbles or particles).
- Do flush detectors and all other components even if baseline is not monitored.
- Total time for changeover can vary but should take about an hour. Do not rush; this may actually slow down the process.
- Do record the volumes of solvent used during changeover for use as a future guide; if changeover is unsuccessful, use more solvent the next time.
- Don’t expect fast changeover and baseline equilibration with refractive index detectors- they are extremely slow to equilibrate after changeover.
- Do check gradient blank runs for excessive baseline noise and drift that might indicate pockets of immiscible solvent.
- Good chromatography in the target mode is the most sensitive !nal test- start with simple binary mobile phases and standard test mixes and work toward real samples with mobile phase additives.