Organic acids can be separated using either a reversed phase column (usually C8) or a ion exclusion column. For simple separation, where few organic acids have to be separated, the reversed phase option is the least expensive. But if a large number of organic acids need to be resolved and identified, the ion exclusion method is the gold standard!

For ion-exclusion, Shodex offer the RSpak KC-811 column. This uses a combination of ion exclusion and reversed phase modes. Organic acids are usually only partially ionised. They are ionised at pHs above their pKa , so the lower their pKa, the more ionised they are. When in the ionised form, they are excluded by the negatively charged sulfonate groups on the column, and so in ion exclusion mode, the organic acids elute in order of decreasing acidity, or of increasing pKa. When in the unionised form, ion exclusion does not occur, and the organic acids are retained by hydrophobic interaction with the resin substrate (as in reversed phase mode). Hence in this mode they elute in order of decreasing polarity, as we would expect for any reversed phase column.

The separations on KC-811 columns take place in a perchloric acid eluent, and are dependent upon both the buffer concentration and the temperature. For maximum resolution it is normal to use two of these columns in series, and the retention times of more than 40 organic acids are listed in the Shodex Technical Notebook 3 (available from us as a paper copy or pdf on request) at various temperatures and buffer concentrations.

For low polarity organic acids it is recommended to use RSpak NN-814, which has a higher polarity packing material. This is particularly useful when analysing aromatic organic acids.

For maximum sensitivity, organic acids can be detected using post-column derivatisation. This is simply the addition of a pH indicator to the eluent after the column, and dedtecting the coloured product that forms in the visible region. Normally Bromothymol Blue is used, detecting at 430nm. Bromocresol purple gives a higher sensitivity but a lower linearity of response. We can supply a  pdf copy of a paper outlining this procedure on request.

In spite of the above, the most commonly used separation mode for organic acids remains reversed phase. Using silica columns, the C8 column is most commonly used. However since Shodex specialises in polymer-based reversed phase columns, they offer one of these for this analysis. The recommended column is RSpak DE413. Attraction to the column occurs when the organic acid is not dissociated, so it is important to work at low enough pH to ensure that this is the case for all organic acids being analysed. As a guide, this means working at least 1.5 pH units below the lowest pKa of the organic acids being analysed.

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Reversed phase column for separation of organic acids in order of decreasing polarity.

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