Phase Collapse

Hydrophobic interaction chromatography is a term often heard in reversed-phase chromatography. In the process of experiment, the substance is not retained, and the peak time is ahead, so that there is no retention between peaks.

Chromatographic Conditions

Column: Ultisil® XB-C8 21.2×250mm, 10μm
Mobile phase: Approximate pure water

The 1st day → Chromatogram of normal activation injection:

No injection was performed at night. Since there were no additives such as acid and alkali in the mobile phase, the autosampler was shut down after cleaning.

The 2nd day → Chromatogram of direct mobile phase washing 2 times column volume injection:
(already injecting more than 30 times)

Everything is normal. The chromatographic column is free and the sample is clean enough. In order to do the experiment faster next Monday, the automatic sampler is still cleaned and shut down.

Monday → Equilibrate 2 column volume injections::

The peak is abnormal and there is no peak. It is suspected that the chromatographic column is not well balanced. After all, after the weekend, washing for a period of time, the peak is normal, but the peak is 2 minutes ahead of schedule. Several impurity peaks are separated


  • Whether the mobile phase is wrong;
  • Whether the instrument parameter setting is normal;
  • Whether the actual flow rate of the instrument is normal.

After they were all normal, it was suspected that the chromatographic column collapsed. Although C8 had strong polarity and tolerated a high proportion of water or even pure water, it was soaked in near pure water for 48 hours, washed with 80% acetonitrile water for 5 times the column volume, and then injected normally.

Knowledge point: Reversed phase chromatography stationary phase.

For example, C18, C8, Pheynl, etc. are resistant to high proportion of water or even pure water. In fact, it means that there is no phase collapse in such mobile phase system, and the retention time and peak area are stable, which does not affect the daily detection.

In this case, the C8 bonded phase still collapsed in the system immersed in near pure water for a long time. Therefore, when it is not used for a long time, it still needs to be washed and maintained with a high proportion of organic solvent water

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