Preparative SFC

Examples with other Pirkle-type CSPs have also been described [139, 140]. In relation to polysaccharides coated onto silica gel, they have shown long-term stability in this operation mode [141, 142], and thus are also potentially good chiral selectors for preparative SFC [21]. In that context, the separation of racemic gliben-clamide analogues (7, Fig. 1-3) on cellulose- and amylose-derived CSPs was described [143]. 

Packed column SFC has also been applied to preparative-scale separations [42], In comparison to preparative LC, SFC offers reduced solvent consumption and easier product recovery [43]. Whatley [44] described the preparative-scale resolution of potassium channel blockers. Increased resolution in SFC improved peak symmetry and allowed higher sample throughput when compared to LC. The enhanced resolution obtained in SFC also increases the enantiomeric purity of the fractions collected. Currently, the major obstacle to widespread use of preparative SFC has been the cost and complexity of the instrumentation. 

On-line SFE-pSFC-FTD, using formic or acetic acid modified CO2 as an extraction solvent, was used to analyse a dialkyltin mercaptide stabiliser in rigid PVC (Geon 87444) [114]. Hunt et al. [115] reported off-line SFE-pSFC-UV analysis of PVC/(DIOP, chlorinated PE wax, Topanol CA), using methanol as a modifier. Individual additives are unevenly extracted at lower pressures and temperatures, where extraction is incomplete. Topanol CA, the most polar of the three PVC additives studied, could not be fully extracted in the time-scale required (15-20min), even at the highest CO2 temperature and pressure obtainable. However, methanol-modified CO2 enhances extraction of Topanol CA. PVC film additives (DEHP, fatty acids, saturated and aromatic hydrocarbons) were also separated by off-line SFE-preparative SFC, and analysed by PDA and IR [116]. 

Chiral SFC can be performed in open tubular [41,42], and packed column [43,44] modes. Packed column SFC can be further categorized into analytical, semipreparative, and preparative SFC [7, 8], Packed column SFC is more suitable for fast separations than open tubular column SFC, since a packed column generally provides low mass transfer resistance and high selectivity [45, 46], Packed column SFC also provides high sample loading capacity [27,47], which can increase sensitivity. Only packed column SFC is suitable for preparative-scale enantioseparation. This chapter will focus on chiral separation using packed column SFC in the analytical scale. 

Figure 7.5 Separation of a cis/trans isomer mixture by SFC using 6% methanol, isocratic elution, (a) Analytical SFC separation. Conditions column 250 X 4.6 (i.d.) mm Berger NH2 flow rate 2.5 mbmin oven temperature 35°C nozzle temperature 40°C outlet pressure 120 bar sample concentration 5 mg/ml in methanol injection volume 5 pi UV 220 nm. (b) Preparative SFC separation. Conditions column 150 X 21.2 (i.d.) mm Berger NH2 flow rate 50 mPmin oven temperature 35°C nozzle temperature 60°C outlet pressure 100 bar sample concentration 50 mg/ml in methanol injection volume 1 ml UV 220 nm.

Except for analytical and small preparative instruments, CO2 recychng after solute separation is common practice. If this were not the case, CO2 consumption would easily exceed 10 or even 20kg of liquefied gas per hour for a preparative SFC system equipped with a 50-nim id column. Gas leaving the separators should be brought back into the same physical state and be at the same pressure as a fresh fluid delivered from the supply unit. Since liquid pumps are most often used in SFC equipment, gaseous eluent must be liquefied prior to recycling. 

Sample Preparation. SFC grade C02 (< 5 ppm O2) was purchased from Scott and pyrene (99 %) was obtained from Aldrich. The pyrene purity was checked by reversed phase HPLC (C18) and all reagents were used as received. Stock solutions of pyrene were prepared in absolute ethanol. 

Experiments have been performed on a preparative SFC system using pure CO2 as the mobile phase under significant pressure drop. The retention times, pressure drop characteristics and the mass transfer behaviour were studied. The trends observed differ from the behaviour of HPLC systems. These trends also emphasize the complexity involved in analyzing the data for SFC measurements, which imply in turn greater complexity of the SFC model as compared to standard liquid chromatography model. 

Semi-Preparative SFC/MS," in Proceedings ofthe 50th ASMS Conference on... 

Farrell, W. Ventura, M. Aurigemma, C. Tran, P Fiori, K. Xiong, X. Lopez, R. Osbonubi, M. Analytical and Semi-Preparative SFC for Combinatorial Chemistry, presented at The International Symposium on Supercritical Fluid Chromatography, Extraction, and Processing, Myrtle Beach, SC, August, 2001. 

Nicoud, R.M. Clavier, J.Y Perrut, M. Preparative SFC Basics and Applications, pp. 397-429 in Caude, M. Tlriebaut, D., eds.. Practical Supercritical Fluid Chromatography and Extraction, Harwood, Amsterdam, The Netherlands (1999). 

Nicoud R-M, Clavier J-Y, Perrut M. Preparative SFC basics and applications. In Caude M, Thiebaut D, eds. Practical Supercritical Fluid Chromatography and Extraction. Amsterdam Flarwood Academic, 1999, Chap 10. 

Xu R, Cai Z, Fogelman K, Wikfors R, Worle V, Stublen N, Kassel DB. Mass-directed purification of compound libraries by automated semi-preparative SFC/MS. Oral presentation and poster. American Society for Mass Spectrometry. Orlando, FL, 2002. 

The SFC systems are grouped into two categories analytical SFC for chemical analysis and preparative SFC for scale-up chemical synthesis and purification. On a fundamental level, the instrumentation for SFC consists of the following (1) a fluid delivery system with high-pressure pumps to transport the sample in a mobile phase and to control the pressure (2) the column in a thermostat-controlled oven where the separation process occurs (3) a restrictor to maintain the high pressure in the column (4) a detection system and (5) a computer to control the system as well as to record the results (see Figure 9.5 as an example). In SFC the mobile phase... 

Searle, P.A., Glass, K.A., and Hochlowski, J.E., Comparison of preparative HPLC/ MS and preparative SFC techniques for the high-throughput purification of compound libraries, J. Comb. Chem., 6(2), 175, 2004. 

In spite of this critical note, the potential of SFC in analytical and preparative-scale enan-tioseparations has been already illustrated. Technical development in this field may open even more challenges for this technique. The advantage of SFC for preparative separations is that the high-pressure liquid carbon dioxide used as mobile phase can easily be removed from the product. In addition, carbon dioxide is non-hazardous and relatively inexpensive. On the other hand, this mobile phase creates the following problems the solubility of polar compounds is limited, and alcohols or other polar modifiers have to be used. Although this makes the technical advantage of SFC questionable, the method may offer some advantages for chiral compounds that may dissolve in SFC mobile phases. Selected examples of preparative SFC enantioseparations are summarized in Table 10 [168-171]. 

Preparative SFC systems with different sizes are available ... 

Because of its increased efficiency, preparative SFC is being used for separations that are difficult to effect by HPLC. But, to take advantage of the narrow peaks obtained in SFC, very little overloading can be done for these difficult separations. As a result the maximum amount of material obtained in a run is on the order of 100 mg in SFC compared with the 1-g amounts obtainable sometimes in HPLC. 

Wang and co-workers have reported that a preparative SFC system can be interfaced with a single quadrupole mass spectrometer for mass-directed fraction collection. Samples with no chromophore (Ginsenoside Rb, Ginsenoside Rc, and Ginsenoside Re) were isolated near homogeneity. A more sophisticated preparative SFC system was patented by Maiefski et al. There are four parallel ehannels in this system, and there is a UV detector for each channel. Since the eluent can be also splitted into a mass spectrometer, this system is capable of both UV and MS directed purification. 

SFE is a very promising method for sample preparation and it has already been employed extensively in connection with lipid analysis, where it can be a substitute for traditional Soxhlet extraction. In addition, there is considerable potential for further development of preparative SFC. 

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