24
May/June 2013
be in their cationic form. In this case, addition of an acidic additive (as acetic acid, trifluoroacetic acid, formic acid or citric acid) with a pKa
below that of the analyte can
Figure 3: Chromatograms of basic drugs of high pKa values: pethidine (a), buprenorphine (b), dextromethorphan (c), codeine (d), pholcodine (e), and morphine (f). Acquity UPC2 BEH 2-EP, gradient elution with methanol (top), or methanol with 20 mM NH4OH (bottom); 40°C, 150 bar, 2.5 mL/min. Reprinted from [15], with permission from Elsevier.
polar compounds elution
The purpose of increasing the polarity of the mobile phase by adding co-solvents and additives to carbon dioxide is not only to increase the solubility of analytes, but also possibly to mask ‘active sites’ of the stationary phase that might cause irreversible adsorption.
Initially, a distinction should be made between polar neutral compounds and ionic compounds. Polar neutral compounds can be easily eluted in packed-column SFC, provided the mobile phase composition is adjusted to ensure sufficient solubility of the analytes, as proven with many examples from sugars to polar lipids (Table 1).
To elute ionic and ionisable species, additives are usually considered as an absolute necessity. The truth is that most SFC chromatographers introduce additives in the mobile phase in a quasi-systematic fashion in the course of method development. However, several examples prove that charged species can be eluted without any additive. For instance, Geiser et al. achieved preparative isolation of hydrochloride salts using simple carbon dioxide – methanol mobile phases [23]
.
Similarly, Bhoir et al. achieved elution of anionic phenytoin and phethenylate sodium salts from an octadecyl-bonded silica phase [24]
.
Shown in Figure 2 an example separation of basic compounds, analogues of metoprolol, eluted without additives from an aminopropyl- bonded silica phase.
Ionisable species (acidic or basic) make up a majority of the compounds analysed in the pharmaceutical industry. Moreover, basic active pharmaceutical ingredients are often present as cationic amine salts. As mentioned above, the estimated pH of carbon dioxide –alcohol mobile phases might be close to 4-5. In such acidic conditions, it is likely that the most acidic compounds would be in their anionic form, while basic compounds would
restore the neutral state of acidic species, and protonate basic compounds. Basic additives (diethylamine, triethylamine or isopropylamine) would restore the neutral state of basic compounds but deprotonate acidic compounds. Influence of the acidic and basic additives on the charge state of the stationary phase would be similar. In many cases, an acidic and a basic additive were used in conjunction. Current practice of packed column SFC often favours ammonium acetate or even ammonia, not only to promote solubility but also for their compatibility to mass spectrometric detection[16, 25-26]
. Shown in
Figure 3 example chromatograms to illustrate the effect of adding ammonia to the mobile phase when analysing basic compounds with high pKa values.
There is also the possible problem of analyte reaction with mobile phase
components. Indeed, amines are known to react with carbon dioxide to form carbamic acids. However, it appears that any reaction is probably reversible and that the original amines are retrieved after the back- pressure regulator when carbon dioxide is depressurised[27]
. There are
also alcohol-sensitive compounds which might react with the alcohol solvent (esterification reactions, for instance). Byrne et al. have shown that 2,2,2- trifluoroethanol was an interesting substitute to avoid such reactions [28]
.
Tables 2 and 3 provide examples of acidic and basic species that should be ionic in the usual carbon dioxide – methanol mobile phases and that were successfully analysed in SFC.
Permanently charged analytes are not affected by alterations in the pH, but additives may be useful in achieving their elution. Table
4 presents some analytes with permanent charges that were successfully eluted in SFC.
. An additive of the same nature as the analyte (acidic additives for acids, basic additives for bases) should therefore be selected. However, Blackwell observed that resolution of neutral chiral analyte enantiomers was also greatly affected by the choice of mobile phase additive, while the competitor theory is difficult to fit to this observation [30]
Whatever the charge state of the analyte, the mechanism through which additives participate in the chromatographic process is still unclear. They were often believed to act as competitors for ‘active sites’ of the stationary phase [29]
.
Also difficult to understand is the improvement in the enantioselectivity of metoprolol (a basic amino-alcohol) when a
Table 2: Acidic compounds successfully analysed with packed column SFC
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
