12. Can I use my columns in SFC? How should I approach the method development in SFC?
SFC applications in the separation of enantiomers are a growing field and a number of applications have been already described. Experiments to date with polysaccharide-derived CSPs show that excellent success rates can be achieved by using alcohol co-solvents. In particular, MeOH, EtOH, and 2-PrOH are very good initial co-solvent choices. ACN seems to be less successful, but it may be an option for a secondary screening. If the elution of the analytes is not sufficient when ACN is used, an alcohol can be added to the CO2/ACN mixture. To learn more about method development strategies, click here.
To resolve basic compounds, add a small amount (normally 0.5-1% in the co-solvent) of a basic additive (often DEA or TEA). For acidic compounds, an acidic additive (i.e. TFA, formic or acetic acid) may be helpful for better resolution, although the acidity of CO2 could in certain cases be sufficient for good enantiomer resolution acidic compounds.
Moreover, immobilised polysaccharide-derived CSPs are compatible with a much wider range of solvents than are the coated phases. The use of such “extended range” solvents was investigated for SFC screening in a similar manner as for LC separations. Initially these solvents were used to enhance sample solubility, to solve difficult separations or to process compounds being unstable in alcohols. Subsequently we have found that these solvents can also offer exceptional enantioselectivity profiles. THF, MtBE, DCM or ethyl acetate, for example, can be used as the CO2 modifier. In some cases, pure DCM, MtBE and ethyl acetate may not be strong enough to elute certain compounds. In these cases, the addition of small percentages of an alcohol mixed with the co-solvent is advised. To learn more about method development strategies, click here.
As already mentioned, success rate is already very high with the alcohol modifiers. Therefore, it has become established practice to use either the immobilised columns or alternatively the coated ones – in the column set for screening with the alcohol modifiers as first strategy. Then the solvent range can be broadened on the immobilised columns if no satisfactory separation is achieved or sample solubility/stability becomes an issue. SFC and LC are complementary techniques and comparable success rates are achieved with the same set of columns.
Analytes are preferably dissolved in the solvent mixture that is used as the co-solvent in SFC. If this is not possible, choose a better sample diluent (e.g., ethanol) and monitor any potential perturbance of the baseline due to the injection solvent. For samples insoluble in alcohols or ACN, it is possible to dissolve these in DCM or DCM mixtures for injection when using immobilised phases. In such cases, caution should be taken to avoid on-line precipitation of the compounds. This step cannot be done with coated phases without compromising the life-time of the column. Please note that coated CSPs are only compatible with alcohols and acetonitrile mixtures in SFC.
A common concern is the effects of high pressures used in SFC on column stability. The pressure drop across the column is the important factor in column stability. This pressure drop is lower in SFC than HPLC and Daicel columns have proved very stable to SFC conditions. We recommend that when the column is not in use, it be removed from the SFC, flushed briefly with isopropanol to displace CO2 (that would evaporate leaving a dry column), and capped. When using a Daicel column in SFC that had been used in HPLC, it is necessary to first flush the hexane with isopropanol, as CO2 will not efficiently flush hexane, and a noisy baseline will result. Chiral Technologies has recently extended its line of Daicel SFC columns to new dimensions: 3-mm internal diameter and different column lengths. These new columns, packed with 3-µm immobilised or coated CSPs, are designed for ultra-fast SFC analysis. To learn more about high-speed and efficient chiral separations, in less than one minute, using the 3-mm chiral SFC columns, click here.
SFC can offer several advantages in preparative applications. Separations are faster and isolation of the product from the mobile phase is also faster as the bulk of the mobile phase evaporates as part of the collection process. With the lower pressure drop experienced in SFC, the use of higher efficiency 5-micron columns for preparative application is feasible. Chiral Technologies offers 1-, 2-, 3- and 5-cm i.d. columns packed with 5 µm materials in SFC column hardware. For analytical screening 3-micron columns are also available. Our technical teams will help you chose the best column dimensions commensurate with SFC systems available in your lab. Depending on your region, please contact us: Chiral Technologies Europe (CTE), support@cte.daicel.com, Chiral Technologies, Inc. (CTI), questions@cti.daicel.com, or Daicel Chiral Technologies India (DCTI), chiral@chiral.daicel.com
References for SFC-method development:
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
Enantiomer resolution screening strategy using multiple immobilised polysaccharide-based chiral stationary phases, T. Zhang, D. Nguyen, P. Franco, J. Chromatogr. A, 1191 (2008) 214-222
Separation of enantiomers and conformers of Tofisopam using Daicel immobilised polysaccharide-derived chiral columns using the Agilent 1260 Infinity Analytical SFC System, T. Zhang, N. Nguyen, P. Franco, M. Vollmer, Application Note (Agilent , 2011)
Enantiomer separation of non-steroidal anti-inflammatory drugs, T. Zhang, N. Nguyen, P. Franco, M. Vollmer, Application Note (Agilent, 2011)
