Phase Collapse

It always can be heard in the hydrophobic interaction chromatograph/reverse chromatography, comes as no reservation to the matter, peak time ahead making no reservation between peaks.


Chromatography column: Ultimate® XB-CB 21.2×250mm, 10μm
Flowing phase: approximate pure water

The 1st day → the normal activation feeding chromatogram

No feeding in the night, because of no additive like acid and alkali in the flowing phase, so shutting down after cleaning auto-feeder.

The 2nd day → directly using the flowing phase to wash 2 times column volume feeding chromatogram
(already feeding more than 30 times)

So far so good, make the column free and the sample is clean enough. In order to experiment fast on Monday, we still clean the feeder and shut it down.

Monday → balance 2 times column volume feeding:

Peaking abnormal and no peak, perhaps balancing the column unwell, after all it came for a weekend, so let’s wash it for a long while, after that, peaking is normal, but peaking came 2 minutes ahead and some impurity peaks are separated.

Checking the cases:

  1. If the flowing phase is wrong?
  2. If instrument parameters are set normally?
  3. If the instrument flowing speed is normal?

If everything goes normally, we doubt it’s the phase collapse. Although C8’s polarity is strong and it can bear the high proportion water even if the pure water, it’s in approximate pure water for 48 hours. Let’s wash 5 times column volume by 80% acetonitrile, then feeds normally.

Knowledge: the reverse chromatography stationary phase.

For example: C18, C8, PHEYNL and etc. bear high proportion water even if the pure water, actually it means the phase collapse doesn’t come out in this flowing phase, the reservation time peak square is stable and it won’t influence the daily test.

In this case, the phase collapse was come out from C8 bonding phase which was in approximate pure water for a long while. So it also needs to be washed by the high proportion organic solvent water after without using for a long while.

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