25


Figure 4 reproduces some of these chromatograms.


Consequently, there is still a poor understanding of why in some cases, additives provide a significant improvement in resolution, while in other cases they simply have no effect or even deteriorate the quality of the separation.


Is water the solution?


Figure 4: SFC/MS of large polypeptides (angiotensin I, II and III, urotensin and sauvagine). Princeton Ethylpyridine, gradient elution with 13 mM TFA/methanol; 40°C, 120 bar, 2 mL/min. Reprinted with permission from [39]. Copyright 2006 American Chemical Society.


strongly acidic additive (trifluoroacetic acid) was used on a stationary phase made of polysaccharide coated onto silica[31]


.


In some cases, ion-pairing was shown to occur. For instance, Steuer et al. used ion-pairing agents to elute a number of ionic drug substances[32]


. Suto et al. used an ion-pairing


agent to achieve extraction and separation of cationic alkaloids, berberine and palmatine [33]


. Patel et .


Gyllenhaal et al. separated metoprolol tartrate and metoprolol succinate, and related amino- alcohols with an ion-pairing agent[34]


al. showed that ion-pairing facilitated the elution of peptides[35]


, while Zheng et al.


demonstrated an ion-pairing mechanism to elute a number of cationic compounds from a cyanopropyl-bonded silica column with the help of sulfonate salts [18]


the ion-pairing agent in the mobile phase might also be a concern if used to elute ionic solutes.


In other cases, the additive is simply believed to suppress ionisation of the analyte [36-37]


.


Blackwell showed that retention of acidic phenylalanine analogues correlated well with the pKa


of the acidic additive [38] . Taylor et al.


succeeded in eluting polypeptides and polypeptide salts with up to 40 residues [39-40]


.


Some of these compounds did not dissolve in methanol, unless acidic or trifluoroacetic acid (TFA) was introduced. This was an interesting indication as to the best mobile phase composition because those polypeptides required larger concentrations of TFA in the methanol co-solvent (13mM) to elute as sharp peaks. It was believed that TFA acted in protonating the acid and amino functions.


Water is possibly the most interesting additive (or co- solvent?) to use in SFC. It is of little use as a single co- solvent because the miscibility of water in carbon dioxide is very limited, but it can be included in a ternary composition comprising carbon dioxide and an alcohol co-solvent. Some old studies paved the way for future SFC practice. Water was often found in mobile phase compositions employed in the early years of packed column SFC, in proportions usually ranging from 0.1 to 10%, with or without any other solvent. An advantage of very small proportions of water without any other solvent was to possibly retain compatibility to flame ionisation detection, and even improve the detector response. Geiser et al. used it to improve the


. However, solubility of


separation of free fatty acids [41]


. Pyo employed it to achieve SFC separation of vitamins [42]


and sulphonamide antibacterials [43]


, free fatty acids . Thiébaut et


al. achieved elution of underivatised amino acids [44] and imidazole derivatives [45]


.


Salvador et al. used it to analyse carbohydrates[46]


.


Strangely, water then seems to have been completely forgotten in the following decennia. It is now appearing again in several papers that have promoted its


advantages. While most of them simply advocate small proportions (0.5 to 5%) to enhance peak shape [26,47,48], others introduce it in much larger proportions (5


to 30%) allowing the elution of very polar compounds[49] and Thurbide[50]


. Figure 5 illustrates this point. Li demonstrated that


isopropanol was the most advantageous solvent, allowing larger proportions of water to


Table 3: Basic compounds successfully analysed with packed column SFC


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