Separation of multiple compounds coexisting with acid and base, during method development, it often occurs that when the acid retention peak shape is good, the peak shape retention of the basic component is not ideal, and when the basic component retention peak shape is satisfactory, the peak shape and retention of the acidic component Not ideal. In this case, the ion-pairing reagent can be selected to increase the retention of acid or base, and then the appropriate mobile phase pH and organic phase ratio can be selected so that all substances can be separated and have good peak shape.
For example
Separation of azelaic acid and matrine:
Column: Ultisil® Polar RP, 4.6×250mm, 5μm.
| Time/min | 0.1% trichloroacetic acid (%) | Acetonitrile (%) | |
| 0 | 95 | 5 | |
| Mobile Phase | 10 | 85 | 10 |
| 20 | 40 | 60 | |
| 21 | 95 | 5 | |
| 35 | 95 | 5 |
Detection wavelength: 220nm;
Column temperature: 40℃;
Flow rate: 1.0mL/min;
Injection volume: 20 μL.
In liquid chromatography, pH value has a great influence on the retention and peak shape of acid-base components. When pH decreases, acid retention increases and alkali retention decreases. On the contrary, as pH increases, acid retention decreases and alkali retention increases. . Therefore, for acid-base samples, it is very important to choose the appropriate pH value.
In this case, azelaic acid is an acidic component, matrine is an alkaline component, and trifluoroacetic acid has a weak ion pair effect, which can appropriately increase the retention of matrine. However, the pH of 0.1% trifluoroacetic acid is between 2.0 and 2.5, and azelaic acid has strong retention under acidic conditions. The gradient elution method is used to make the peak shape and retention of the two components reach a satisfactory peak shape. with reservation.
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