11. I want to use reverse-phase chromatography. Which columns and mobile phases can I use?
The polysaccharide type CSPs seem to be most frequently used with organic eluents (alkane mixtures or the polar solvents discussed above). However, resolution of enantiomers on these columns by aqueous eluents has a history almost as long as their applications with organic mobile phases [K. Tachibana et al., J. Chromatogr. A, 906 (2001) 127]. The choice of RP mode has often been related to the need for direct injection of the samples of biological extracts; to address the low retentivity of certain compounds under normal phase conditions, the low solubility of polar and ionic samples in organic mobile phases or the lack of appropriate enantioselectivity in organic solvents. An additional and increasingly important reason to favorite RP methods is their suitability for LC/MS applications.
RP chromatography is often complementary to normal phase (NP) mobile phases. The presence of water in the mobile phase undoubtedly has an impact on the enantiorecognition and offers the possibility of enhancing the chances of resolution success.
The nature of the molecules to be analysed should be taken into account for mobile phase selection. Under this primary guideline, a “decision tree” can be defined to start the method development. To learn more, click here.
The selected mobile phases do not really differ from the proposals for achiral RP screening.
The selection of the mobile phase for neutral compounds is straightforward: plain water is suitable to be used as the aqueous component. As for the additives, the most common and effective pH value of the aqueous solution for the resolution of acidic molecules is in the range 2.0-2.5 in which the ionisation of most organic acids will be suppressed. Formic acid is a good choice where LC/MS compatibility is required while other acids (TFA, acetic acid, H3PO4) may be used for other applications. Where RP methods suitable for MS detection for basic compounds are being developed, ammonium bicarbonate adjusted to pH 9 is an excellent option, although some other buffers or solutions at basic pH (phosphates, borates, ammonium acetate or KPF6 aqueous solutions) may also be useful in terms of enantiorecognition.
The organic modifier plays an important role in regulating retention and modulating enantioselectivity. The first choices of modifiers to be combined with water are acetonitrile (ACN) and methanol (MeOH). The use of other organic modifiers, such as EtOH, 1-PrOH and 2-PrOH can also be considered. In our practice, however, these modifiers would be tried only if no successful hit is achieved with ACN and MeOH.
This is the typical pattern for method development in RP-mode with any of our polysaccharide-derived columns. On the immobilised supports, other solvents, such as THF, can be employed. Moreover, sample injection can also be performed in DMSO, DMF or acetone, for example, in case of need. THF may sometimes be a good alternative to ACN and MeOH, but in a general way, it seems to be a less versatile organic modifier if compared with ACN and MeOH. The relative eluting strength of these three organic modifiers can be ranked as follows: THF > ACN > MeOH.
An interesting feature of immobilised polysaccharide-derived supports is that it would be possible to use the same column in organic and water containing solvents without compromising the reproducibility of results on both modes. This possibility does exist due to the fact that a washing protocol between RP and organic mode in strong solvents (such as THF and DMF) can be applied, in order to eliminate water traces adsorbed on the polymer. This protocol is described in detail on the instruction manual of the immobilised columns, but will not be compatible with the previously existing coated CSPs.
To learn more about the preparation of buffers, including also the MS-compatible ones, click here.
References for RP-method development:
Reversed-phase screening strategies for liquid chromatography on polysaccharide-derived chiral stationary phases, T. Zhang, D. Nguyen, P. Franco, J. Chromatogr. A, 1217 (2010) 1048–1055
P. Franco, T. Zhang, Common screening approaches for efficient analytical method development in LC and SFC on columns packed with immobilised polysaccharide stationary phases, in Chiral Separations, Methods and Protocols, second ed., G. Scriba (Editor), Humana Press, 2013, 113-126
Reversed-phase liquid chromatographic separation of enantiomers on polysaccharide type chiral stationary phases, K. Tachibana, A. Ohnishi, J. Chromatogr. A, 906 (2001) 127-154
