The acidity or basicity of a sample is usually defined by the pH value. Choosing a suitable pH for ionizing molecules often creates sharp and symmetric peaks in HPLC. Symmetrical peak shape is essential in quantitative analysis to achieve lower detection limits. For weak acids pKa values are usually in the range of 4 - 6. So, at a pH value of 3 or less, the acid must exist as a neutral molecule. At a pH value of 7 and above, the acid has a negative charge. The charge state of the analyte is extremely significant in the separation of high-performance liquid chromatography; this is why some form of pH modification is almost constantly needed.A charged analyte typically has a higher solubility in water compared to a neutral compound and therefore has much lower retention on a reversed-phase column.
If we wish to use a reversed-phase column, the acid must be in its neutral form, where retention should be high. If compounds have some or no polar groups, and it contains a considerable number of carbons, our work is normally quite easy. We prefer to use 0.1% formic acid or 0.1% phosphoric acid, all of which must be kept in their neutral form. Subsequently, we set the amount of organic solvent as required, a less amount of organic solvent means more retention time of a molecule. If the analyte is more polar, then the retention time may decrease at any of these phases.
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