Many liquid phase methods usebuffer salts. It has been found that many people do not understand the application of buffer salts in liquid phase methods, so I would like to share some of my views and stimulate discussion.
If the sample or the key impurities to be studied in the sample have ionization tendencies, other specific cases will be discussed separately. Among the components with strong ionization tendencies, some are in molecular state and combine better with the reverse phase chromatographic stationary phase; others are in ionic state and have a stronger affinity for the mobile phase. If the ionization state of the molecule state and the ionic state is not controlled by using buffer salts, it is easy to have peak tailing and bifurcation. In this case, water and organic systems should not be used as the mobile phase, and buffer salts and organic systems should be selected.
When using buffered electrolyte salts in the mobile phase, it should have a certain buffering capacity. If reducing the amount of sample injection can improve the peak shape, it may indicate that the buffering capacity of the buffer salt solution is insufficient. The buffering capacity of the buffer salt solution is related to the concentration of the buffer salt and the pH value of the buffer salt solution. The higher the concentration of the buffer salt, the stronger the buffering capacity; the closer the pH value of the buffer salt solution is to its pKa, the stronger the buffering capacity. Generally, the pH value of the buffer salt solution should be within the range of pKa ± 1. In order to ensure that the retention time of the main component does not deviate greatly due to slight changes in pH value, the pH value of the buffer salt solution should be outside the range of sample pKa ± 2.
Each buffer salt has a corresponding cutoff wavelength, and the detection wavelength of the method should not be less than the cutoff wavelength of the buffer salt solution. The closer the detection wavelength is to the cutoff wavelength of the mobile phase, the higher the baseline noise, the greater the response value error, and the smaller the component response value and sensitivity.
1. The concentration ofbuffer salt is generally 10-50 mmol/L. The concentration of the buffer salt should be selected according to the properties of the sample and the amount of injection to meet the required buffering capacity.
2. The maximum allowable proportion of the organic phase in gradient elution should be determined by mixing experiments ofbuffer salts and organic phases. Different buffer salt-organic phase proportions should be taken to determine the maximum allowable proportion of the organic phase, such as 10:90, 20:80, 30:70, etc. Buffer salts should not be set at a high proportion on the instrument to prevent the precipitation of buffer salts from damaging the instrument.
3. Choose buffer salts according to multiple constraints such as cutoff wavelength, pH value, and buffering capacity, etc. For example, if the detection wavelength is at the end absorption, phosphate should generally be selected.
4. Commonbuffer salt solutions include: 0.1% phosphoric acid/trifluoroacetic acid/acetic acid/formic acid solution, 0.02-0.05mol/L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid), etc.
5. For alkaline compounds, potassium salts are preferred, which can better suppress the tailing effect.