How does the aqueous phase ratio affect the separation in HILIC mode?

Elution adjustment direction in HILIC mode

The principle of elution is mainly similar polarity compatibility and hydrophilic separation and distribution mode. The more polar solvents during elution, the shorter the retention time; the order of peaks is weak polarity first, and strong polarity last, in contrast to reversed phase, so it is suitable for retaining substances with strong polarity that cannot be retained on reversed phase.

Instance

• Separation of CTP, ATP, GTP, UTP:
• ATP: Adenosine Triphosphate
• GTP: Guanosine triphosphate
• CTP: Cytosine Triphosphate
• UTP: Uridine Triphosphate

Ultisil® AQ-C18 4.6*250mm 5μm Phosphate buffer (neutral pH)-acetonitrile system, the gradient run is shown in the table below:

Effect drawing

Analysis: The peaks are well separated, but the peaks are all in the pure salt phase, and the retention is weak. The peak shape of some target peaks is not good, the method repeatability is not good, and it is easy to overlap with the solvent peak.

According to the working principle of HILIC, this mode is suitable for the separation of such polar substances, so Topsil® Silica 4.6*250mm 5μm:

Phosphate-acetonitrile (25/75)

Analysis: The four substances were completely separated with moderate retention, but there was a risk of interference from impurity peaks. There was room for optimization of peak shape, reducing aqueous phase and extending retention.

Phosphate-acetonitrile (20/80)

Analysis: good separation, improved peak shape, no interference of impurities, much enhanced retention of each target peak, long running time for one needle, and the proportion of water in the mobile phase has a greater impact on retention and separation, which is in line with the separation principle of HILIC. For the strong elution ability, the change of the small proportion has a great influence on the chromatographic effect, and the proportion of the water phase decreases.

Phosphate-acetonitrile (22/78)

Analysis: As expected, the retention time is shortened, and the impurities can be separated better. Gradients can be tried. In HILIC mode, small changes in the water phase have a great impact on the chromatogram. Therefore, the equilibration time between the two injections is recommended to be 10 times the column. Above the volume, and the equilibration time is the same, the system can also achieve a dynamic equilibrium to ensure the reproduction of the separation effect.

In conclusion

When running HILIC mode:

1. Consider the polarity and solubility of the sample: First, the target is generally a strongly polar substance, and it is best to have a certain solubility in a high proportion of the organic phase. If the sample is insoluble or has a low solubility in the organic phase, or is prone to other If it is reactive, it does not apply;
2. Consider the mutual solubility of the buffer salt and the organic phase: When adjusting the mobile phase ratio, if the mobile phase contains salt, the solubility of the salt in the organic phase should be considered to avoid the phenomenon of salting out;
3. Pay attention to the real balance state of the system: System balance is not only concerned with the problem of baseline noise and drift, but also the continuous injection of samples to obtain stable retention time and peak area. During the test, the system will encounter the problem of slow balance, which needs to be attached solutions or improvements;
4. Pay attention to the stability of the sample: ensure that the sample solution used is the closest to the initial state, pay attention to the stable state of the solution during the test, and use a new one if necessary; determine the final preparation method according to the main peak of the sample during the whole method. preservation method.