Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase.

The mobile phases employed in current supercritical fluid chromatography (SFC) are usually composed of a mixture of pressurized carbon dioxide and a co-solvent. The co-solvent is most often an alcohol and may contain a third component in small proportions, called an additive (acid, base or salt). The polarity of such mobile phase compositions is here re-evaluated with a solvatochromic dye (Nile Red), particularly to assess the contribution of additives. It appears that additives, when employed in usual concentration range (0.1% or 20mM) do not modify the polarity in the immediate environment of the probe. In addition, the combination of carbon dioxide and an alcohol is known to form alkoxylcarbonic acid, supposedly conferring some acidic character to SFC mobile phases. Direct measurements of the apparent pH are impossible, but colour indicators of pH can be used to define the range of apparent pH provided by carbon dioxide-alcohol mixtures, with or without additives. Five colour indicators (Thymol Blue, Bromocresol Green, Methyl Red, Bromocresol Purple, and Bromothymol Blue) were selected to provide a wide range of aqueous pKa values (from 1.7 to 8.9). UV-vis absorption spectra measured in liquid phases of controlled pH were compared to those measured with a diode-array detector employed in SFC, with the help of chemometric methods. Based on these observations, it is concluded that the apparent pH range in carbon dioxide-methanol mobile phases is close to 5. Increasing the proportion of methanol (in the course of a gradient elution for instance) causes decreasing apparent pH. Strong acids can further decrease the apparent pH below 1.7; strong bases have little influence on the apparent pH, probably because, in this range of concentrations, they are titrated by alkoxylcarbonic acid or form ion pairs with alkoxycarbonate. However, bases and salts could stabilize the acidity in the course of gradient runs.