Considerations for the establishment of reverse phase ion pair chromatography

After reading the previous article, you have a clear understanding of reversed ion pair chromatography. Today, I will share with you a practical case:

Formamide and other 6 substances separation

Initial condition

Column: Ultisil® LP-C18 (4.6×250mm, 5μm)
Mobile phase: 0.1% sevofluoro-butyric acid solution/acetonitrile
Detection wavelength: 210nm
Column temperature: 30℃
Flow rate: 1mL/min
Sample size: 10μL

Chromatographic diagram

Discuss:

Formylhydrazine combined with 4-amino-triazole, 3-amino-triazole peak before the inverted peak interference, triazole peak type is not good, formamide and diformylhydrazine separation is not baseline separation, increase ion pair reagent concentration.

Mobile phase: 0.3% sevofluoro-butyric acid solution/acetonitrile

Chromatographic diagram

Discuss:

The separation effect and baseline were improved by increasing the concentration of ion pair reagent, but there was an inverted peak at about 9 minutes. The separation degree of formamide and diformylhydrazine was not separated at baseline, and the number of carbon atoms in the ion pair reagent was increased.

Mobile phase: 10mmol/L KH₂PO₄(containing 1g/L sodium octane sulfonate, pH1.6)/ acetonitrile =85/15

Chromatographic diagram

Discuss:

There was no improvement after switching to sodium octane sulfonate, and the type of ion pair reagent was changed.

Mobile phase: 10mmol/L KH₂PO₄(containing 2g/L sodium hexane sulfonate, pH1.5)/ acetonitrile =90/10

Chromatographic diagram

Discuss:

First, the separation of the two peaks is good, other substances need to be improved, adjust the proportion of organic phase.

Mobile phase: 10mmol/L KH2PO4(containing 2g/L sodium hexane sulfonate, pH1.5)/ acetonitrile =95/5

Test results:

The retention and separation were good, the separation was complete, and the peak shape of triazole could meet the quantitative requirements of peak area.

Discuss:

In the separation of formamide and other 6 substances, these are large polarity, the reverse phase chromatography retention is weak, to achieve the separation of six components, first need to increase its retention, and then according to the situation, adjust the amount of ion-pair reagent, pH value, organic phase type and concentration to achieve separation, Ultisil® LP-C8 (pH tolerance range 0.5-8) column was selected to withstand strong acid conditions in the experiment. By adjusting the type, concentration of ion-pair reagents, and proportion of organic phase, each substance was well separated to meet the needs of daily detection.