The use of aqueous mobile phase is a key step in the analysis and detection of ionic compounds by reversed-phase chromatography: including the effect of pH on analyte retention, the type and concentration of buffer salts, the solubility of buffer salts in organic solvents and their effects on analyte retention. detection effects, etc. Improper selection of buffer salt types, ionic strength, and pH values can result in poor or non-reproducible retention and tailing in reversed-phase separations of polar and ionizable compounds.
The analyte was partially ionized, strong interaction between the analyte and the free silanol groups and other active sites of the stationary phase, etc., can be solved by selecting the appropriate buffer range, the correct type of buffer salt and its concentration (ionic strength) to improve. Sensitive LC-MS analysis with greater emphasis on the choice of acids, bases, buffer salt types and other additives: Buffers do not inhibit ionization of analytes.
In reversed-phase mode, the retention of ionic compounds is primarily determined by the pH of the mobile phase. The dissociation properties of ionic functional groups also affect compound retention. The retention of non-ionic compounds is hardly affected by the pH of the mobile phase. For compounds containing acidic groups (mostly carboxylates), a pH value lower than the compound pKa can enhance retention, and for compounds containing basic groups (mostly amines), a pH value higher than the compound pKa can enhance retention.
Smaller pH changes have little effect on the ratio of aproton-protic species at pH values farther from the pka. Therefore, moderate pH changes do not have a significant effect on retention. Small changes in pH around pKa can have a noticeable effect on the ratio of the two species. Therefore, changing the pH close to the pKa can significantly affect compound retention. When pH is used as a means to increase compound retention, the consideration should be to minimize ionization of the analyte. A buffer is a solution of a weak acid and its conjugate base, or a weak base and its conjugate acid.
Buffers reduce the influence of hydrogen ions, hydronium ions, and hydroxide ions, resulting in less pH fluctuations including dilution. In reversed-phase mode, the pH tolerance range of classic silica-based packing materials is 2-8. The choice of buffer is usually determined by the desired pH. The pKa of the buffer must be close to the desired pH, as pH is easiest to control at the pKa. The rule of thumb is to choose a buffer with a pKa value <2 units of the desired mobile phase pH.
pHa value and effective buffer range of different buffers
|Buffer solution||pKa（25℃）||Effective pH Range|
General idea of buffer salt application
- The concentration of buffer salt is generally 10-50mmol/L. Select the buffer salt concentration according to the nature of the sample and the injection volume to make it meet the due buffering capacity.
- The maximum allowable ratio of organic phase in gradient elution should be determined according to the miscibility experiment of buffer salt and organic phase. Take different buffer salt-organic phase ratios to determine the maximum allowable value of the organic phase ratio, such as buffer salt: organic phase in different ratios such as 10:90, 20:80, 30:70. Do not arbitrarily set a high proportion of organic phase on the instrument to prevent the instrument from being damaged by the precipitation of buffer salts.
- Select the buffer salt according to multiple constraints such as cut-off wavelength, pH value and buffer capacity. If the detection wavelength absorbs at the end, phosphate should generally be selected.
- Commonly used buffer salt solutions include: 0.1% phosphoric acid/trifluoroacetic acid/acetic acid/formic acid solution, 0.02-0.05mol/L potassium dihydrogen phosphate solution (adjust pH to 3.0 with phosphoric acid), etc.
- The basic compound is preferably potassium salt, and the effect of suppressing tailing is better.