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Switching
between Normal Phase to Reversed Phase
Protocols
Normal phase solvents
order of polarity:
hexane > dichloromethane >
chloroform
> isopropanol>ethanol, etc.
Reversed phase
solvents order of hydrophobicity:
buffers > water > methanol
> acetonitril
> ethanol > isopropanol
REVERSED
PHASE to NORMAL PHASE
waters -> methanol -> isopropanol
-> hexane
- 1. Remeber: H2O,
ACN, MeOH do not mix with hexane!
- Do not keep any normal
phase solvent in the
reservoirs when working with reversed phase solvents!
2. Isopropanol (IPA)
mixes with hexane or with H2O, ACN, MeOH
- When finishing with
reversed phase solvents
always wash the entire system with H2O:
pump-autosampler-detector-sealwash
and needlewash with H2O
to remove residues of buffers. Use a zero-dead-volume-union
instead of the column.
- Wash with MeOH:
minimum of 10 ml should pass through each line A, B, C, D, and through
the injector (Purge injector) and through
the detector (just use the flow).
- Wash with IPA:
minimum of 10 ml should pass through each line A, B, C, D, and through
the injector (Purge injector) and through
the detector (just use the flow).
- Only after the entire
system is flushed with IPA
you can introduce the normal phase solvents.
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NORMAL
PHASE to REVERSED PHASE
hexane -> isopropanol -> methanol -> waters
1. Remeber: H2O,
ACN, MeOH do not mix with hexane!
- Do not keep any of those
solvents in the reservoirs
when working with normal phase solvents!
2. Isopropanol
(IPA) mixes with hexane or with H2O, ACN, MeOH
- Always keep isopropanol as a
reservoir when
working with normal phase solvents, it will be used as the final wash
solvents
to the entire HPLC system.
- When finishing with normal
phase solvents
always wash the entire system: pump-autosampler-detector-sealwash
and
needlewash with Isopropanol. Use
a zero-dead-volume-union instead of the column.
- Wash with IPA:
minimum of 10 ml should pass through each line A, B, C, D, and through
the injector (Purge injector) and through
the detector (just use the flow).
- Wash with MeOH:
minimum of 10 ml should pass through each line A, B, C, D, and through
the injector (Purge injector) and through
the detector (just use the flow).
- Wash with H2O:
minimum of 10 ml should pass through each line A, B, C, D, and through
the injector (Purge injector) and through
the detector (just use the flow).
- Only after the system was
washed with H2O
it
is ready for reversed phase.
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