Preparative scale separations

Application of rotating coiled columns has become attractive for preparative-scale separations of various substances from different samples (natural products, food and environmental samples) due to advantages over traditional liquid-liquid extraction methods and other chromatographic techniques. The studies mainly made during the last fifteen years have shown that using rotating coiled columns is also promising for analytical chemistry, particularly for the extraction, separation and pre-concentration of substances to be determined (analytes) before their on-line or off-line analysis by different determination techniques. 

Although some applications for preparative-scale separations have already been reported [132] and the first commercial systems are being developed [137, 138], examples in the field of the resolution of enantiomers are still rare. The first preparative chiral separation published was performed with a CSP derived from (S -N-(3,5-dinitrobenzoyl)tyrosine covalently bonded to y-mercaptopropyl silica gel [21]. A productivity of 510 mg/h with an enantiomeric excess higher than 95% was achieved for 6 (Fig. 1-3). 

Liquid-liquid extraction is a basic process already applied as a large-scale method. Usually, it does not require highly sophisticated devices, being very attractive for the preparative-scale separation of enantiomers. In this case, a chiral selector must be added to one of the liquid phases. This principle is common to some of the separation techniques described previously, such as CCC, CPC or supported-liquid membranes. In all of these, partition of the enantiomers of a mixture takes place thanks to their different affinity for the chiral additive in a given system of solvents. 

CE is generally more suited to analytical separations than to preparative-scale separations. However, given the success of CE methods for chiral separations, it seems reasonable to explore the utility of preparative electrophoretic methods to chiral separations. Thus, the purpose of this work is to highlight some of the developments in the application of preparative electrophoresis to chiral separations. Both batch and continuous processes will be examined. 

Ultimately, however, it should be noted that these examples of classical gel electrophoretic separations are batch processes and therefore limited in sample throughput. To achieve true preparative-scale separations by electrophoresis, it becomes necessary to convert to continuous processes. 

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. 

A new brush-type CSP, the Whelk-0 1, was used by Blum et al. for the analytical and preparative-scale separations of racemic pharmaceutical compounds, including verapamil and ketoprofen. A comparison of LC and SFC revealed the superiority of SFC in terms of efficiency and speed of method development [50]. The Whelk-0 1 selector and its homologues have also been incorporated into polysiloxanes. The resulting polymers were coated on silica and thermally immobilized. Higher efficiencies were observed when these CSPs were used with sub- and supercritical fluids as eluents, and a greater number of compounds were resolved in SFC compared to LC. Compounds such as flurbiprofen, warfarin, and benzoin were enantioresolved with a modified CO, eluent [37]. 

GENERAL CONDITIONS FOR PREPARATIVE-SCALE SEPARATIONS BY FLASH CHROMATOGRAPHY... 

Figure 4.37 Preparative-scale separation of bilirubin laomarm by high pressure liquid chronatography. The analytical separation was optimized to maximize the separation factor at the smallest practical value for the capacity factor and then the saiq>le size scaled-up to that allowed by the larger amount of packing in the preparative column. (Reproduced with permission from Perkin-Elmer Corporation).

There is a wide variety of commercially available chiral stationary phases and mobile phase additives.32 34 Preparative scale separations have been performed on the gram scale.32 Many stationary phases are based on chiral polymers such as cellulose or methacrylate, proteins such as human serum albumin or acid glycoprotein, Pirkle-type phases (often based on amino acids), or cyclodextrins. A typical application of a Pirkle phase column was the use of a N-(3,5-dinitrobenzyl)-a-amino phosphonate to synthesize several functionalized chiral stationary phases to separate enantiomers of... 

White recently illustrated the use of fast supercritical fluid and EFLC for drug discovery and purification [46]. The optimized isocratic separations used to scale up to preparative-scale separations were often EFL mixtures. For example, Figure 9.13 shows the optimized conditions for the separation of a drug candidate included 30% methanol (with 0.2% isopropyl amine)/C02 on a Chiralcel OJ-H column at 5 mL/min [46]. His work also illustrates by using gradients that start in supercritical conditions and then move into EFL mixture conditions provides efficient and fast separations. 

Geiser et al. [50,51] illustrated the screening of different chiral stationary phases and the separation of highly polar amine hydrochlorides using EEL methanol/C02 mixtures and the columns, Chiralpak-AD-H, Chiralpak-AS. This method is advantageous because no acid or base additive was required to achieve base line separation of the racemates and conversion to free base form for enantiomer separation was not required. Preparative-scale separations of the amine-hydrochloride were accomplished using similar mobile phase conditions [51], Furthermore, this is believed to be the first chiral separation of highly polar solutes without the addition of acid or base additive to effect the separation. 

High-performance liquid chromatography (HPLC) is one of the most widespread analytical and preparative scale separation techniques used for both scientific investigations and industrial and biomedical analysis. Now in its second edition, this revised and updated version of the Handbook of HPLC examines the new advances made in this field since the publication of the benchmark first edition twelve years ago. It reports detailed information on fundamental and practical aspects of HPLC related to conventional format and sophisticated novel approaches that have been developed to address a variety of separation problems in different fields. 

For preparative scale separations one needs the highest possible chromatographic selectivity (a-values) to optimize throughput and yield per time unit. The selection of CDA may, therefore, become an important issue for calculating cost-benefit factors. 

Additional Preparative-Scale HPLC Separations. After mutagenesis assessment of the HPLC fractions from the initial preparative-scale separation just discussed, those fractions containing mutagenic constituents are further separated on HPLC by employing the following strategy For example, if the mutagenic constituents were found to be in Fraction D from an initial reverse-phase HPLC preparative-scale separation, that is, a mobile-phase composition of 25 water 75 acetonitrile, a... 

Gas chromatography can be utilized for preparative-scale separations as well as for analysis. For the qualitative and quantitative analyses of a small sample (see Chapter 4), microliter or microgram sample sizes are used. Once having worked out the conditions for such an analytical separation, one may wish to isolate larger amounts of one or more components of a mixture. Use of GC for this purpose is referred to as preparative gas chromatography. 

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