Since ionizable compound retention in HPLC assay is highly influenced by the pH of the mobile phase, it is necessary to have control over the pH. Studies can achieve this control by adding a buffer in the mobile phase. As most compounds contain an acidic or basic functional group, HPLC labs use buffers to control the pH of the mobile phase. If the study does not adequately monitor the pH, it may become a source for several technical problems.
Buffers are generally weak acid or weak base conjugated to its acid or base in the solution. This conjugated acid or base is at least 50% aqueous. The conjugated pair exists in equilibrium, in such a way that the equilibrium shifts upon the addition of an acid or base to compensate and maintain the same pH.
Why is pH regulation so important?
A change in the pH of the mobile phase may influence the ionization of the analytes, altering its relative hydrophobicity and thus the retention time. It is recommended to use a buffer with a pH that is at least two units apart from the pKa value of the analyte of interest, or else it is common to observe split peaks or shoulders. For an acidic analyte, pH above the pKa value results in ionized form, whereas pH below the pKa value results in non-ionized species. It is the other way round with a basic analyte. For several analytes, it is better to adjust the pH in a way that all analytes exist in either an ionized or non-ionized form.
Common buffers for HPLC assay
Phosphate and acetate buffers are the most common HPLC assay buffers for UV detectors. Both the buffers are effectively used as they can detect at wavelengths less than 220 nm. But with the increasing use of the LC-MS method and lc ms ms analysis, the volatile nature of the mobile phase becomes critical. As the LC-MS interface vaporizes the mobile phase, using phosphate buffers is not feasible. TFA (0.1%), acetate, ammonium formate, formic acid, and ammonium bicarbonate are some buffers that are volatile enough to be used for LC-MS methods and LC-MS/MS analysis.
What is an ideal buffer concentration?
The lowest concentration buffer that is reproducible for the intended use is ideal for an HPLC assay. Even a slight temperature fluctuation or increase in the organic content may lead to precipitation in higher concentration buffers. Generally, 2-50 mM concentration is adequate for reverse phase-HPLC. When it comes to reverse phase-HPLC, it is crucial to keep in mind that an increase in ionic strength decreases the ionic species retention, while a decrease in ionic strength facilitates increased retention.
It is highly recommended to never leave and shut off the HPLC system buffer for an extended time, or it may give rise to microbial growth and buffer precipitation. Before flushing a strong solvent for hardly retained materials, it could be beneficial to switch to a water/organic mobile phase. As a golden rule, contaminants should be avoided, buffers must be filtered after preparation, and should never be left alone when the system is not in use.