In chromatographic separation, there are many factors that affect selectivity and separation, among which the type of organic solvent in the mobile phase and different solvent strengths are one of the influencing factors. For reversed-phase chromatography, commonly used organic solvents are methanol, acetonitrile, tetrahydrofuran, etc. We can change the selectivity and resolution by changing the type of organic phase in the mobile phase or adjusting its ratio to show different separation effects.
Application
Separation of 4-(chloromethyl)benzoic acid & 3-(chloromethyl)benzoic acid
The structure is shown in the figure:
The two substances are meta and para isomers, which cannot be completely separated in the acetonitrile system. After changing the type of organic phase, better separation can be achieved.
Chromatographic Condition 1
Column: Ultisil® XS-C18 4.6*250mm, 5μm
Mobile phase: 0.1% phosphoric acid water and acetonitrile, gradient elution
Analysis: The two target peaks have a tendency to separate, but the separation is not complete. Adjust the gradient to isocratic conditions.
Chromatographic Condition 2
Column: Ultisil® XS-C18 4.6*250mm, 5μm
Mobile phase: 0.1% phosphoric acid water/acetonitrile=70/30
Analysis: The separation has been improved, but the separation is still not complete, and the separation cannot be improved by adjusting the strength of the solvent.
Chromatographic Condition 3
Method optimization was performed on this column, replacing acetonitrile with methanol and testing again
Column: Ultisil® XS-C18 4.6*250mm, 5μm
Mobile phase: 0.1% phosphoric acid water/methanol=60/40
Result: The peak shapes of the two targets were good, and the complete separation could be achieved.
0 Comments