Method adjustment
Organic phase, pH value, injection volume, column temperature, etc.
Injection volume
Mobile phase: methanol: 10mmol/L sodium hexanesulfonate (pH2.8 buffer salt) = 35:65
| Molar concentration ratio of aniline to ion-pairing reagent | Number of theoretical plates | Symmetry factor | Peak shape |
| 1:8 | 16689 | 1.05 | Normal |
| 1:4 | 13786 | 1.13 | Normal |
| 1:2 | 10167 | 1.50 | Slightly cutting edge |
| 1:1 | 6527 | 2.01 | Serious frontier |
| 2:1 | 5931 | 2.21 | Twin peaks |
Conclusion: When designing the method, it is recommended that the ion-pairing reagent concentration is at least 4 times greater than the sample, and the limit ratio is 1:1. If the sample concentration is too much greater than the ion-pairing reagent concentration, double peaks will appear.
Column temperature
Changes in temperature can cause significant changes in relative retention.
Since the column temperature will change the amount of ion-pair reagents adsorbed on the chromatographic column, the selectivity will change due to the difference in the concentration of the ion-pair reagents.
Column type
There will be some variance, but since in ion-pair chromatography the column packing materials are partially covered by the ion-pair reagent, switching column types is not the preferred approach.
Buffer salt type
There will be a certain effect, with little impact on selectivity.
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