Preparative-scale LC separation

Preparative Scale LC. Separation of JP-5 and DFM on the Waters radially compressed silica column gave many fractions. 

Figure 2. Preparative-scale LC separation of Shale-11 DFM 254-nm UV detector (----------) 313-...

The problem with organoleptic experiments is the extreme difficulty of avoiding contamination of the sample by compounds which should not be present. Consider for example the result of a preparative scale LC separation on an octadecylated silica gel column. 

The only field in which CE has no chance of application is of course in the preparative scale LC separation of the compounds. This will play an essential role in the organoleptic evaluation of all the compounds involved. For this reason alone it is best not to abandon LC completely since the preparative separations will necessarily be based on systems developed on an analytical scale first. 

As can be seen from this experiment, the spectrophotometric detector is excellent for analytical scale detection of the phthalates which have a good molar absorptivity at 254 nm. However, as was observed in this experiment, the spectrophotometer is much less useful for preparative scale chromatographic separations because it tends to become optically saturated when relatively large amounts of materials are injected, that is, all light entering the detector cell is absorbed and no separation can be seen because of the off-scale readout. Also, spectrophotometric detectors will be blind to molecules that have no chromophore in their structure. The differential refractometer is the preferred detector for preparative LC because generally... 

S. Andersson, Semi-preparative and LC Separation of Enantiomers in the Development of Pharmaceutical Compounds, in Proceedings of EuroTech 98, Preparative Process Scale Separations, G. Subramanian (ed.), Cambridge (UK), 1998. 

As in analytical chiral LC, Daicel derivatised polysaccharide CSPs are the most frequently used materials in preparative scale chiral separations. Recently CSPs have been prepared in which derivatised polysaccharides have been covalently bonded to the solid support rather than coated on as in the Diacel materials. The rationale for this is that it is advisable to reduce the chance of the chiral selector leeching off the column in trace amounts to contaminate samples of chiral dmgs isolated by production scale LC. However, the extent to which the Daicel coated CSPs are now used in production scale chiral LC would tend to suggest that such a problem, if it exists, is not a very significant risk. 

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]. 

Other online HPLC techniques (such as LC-CD or LC-IR) are likely to be exploited. For example, a mixture of diastereoisomeric biflavonoids from the African plant Gnidia involucrata (Thymelaeaceae) could not be separated on a preparative scale by HPLC or crystallization. However, their analytical separation on a Cig column was sufficient to run an online LC-CD investigation and provide stereochemical information about the individual isomers. 

Packed-column SFC also is suitable for preparative-scale enatioseparations. Compared with preparative LC, sub- or supercritical fluid chromatography results in easier product and solvent recovery, reduced solvent waste and cost, and higher output per unit time. Because of its reduced sample capacity, SFC usually allows the separation of 10-100 mg samples per run. Chromatographers can compensate for these sample amounts by using shorter analysis times and repetitive injections (Wolf and Pirkle, 1997). 

The chromatographic methods use gas or liquid separately as the mobile phase, hence the terms gas chromatography (GC) and liquid chromatography (LC). Gas chromatography could not be accepted as the method of choice for the chiral resolution of racemic compounds mainly because of its requirement for the conversion by derivatization of the racemic compound into a volatile species. Besides, the separated enantiomers cannot be collected for further pharmacological and other studies. Moreover, GC cannot be used at the preparative scale. 

The preparative-scale separation of enantiomers on chiral stationary phases (CSPs) by GC cannot match the overwhelming success achieved in the realm of liquid chromatography (LC) (Francotte, 1994, 1996 and 2001). Modern commercial instrumentation for preparative-scale GC is not readily available. In contrast to LC, separation factors a in enantioselective GC are usually small (a = 1.01 - 1.20). This is beneficial for fast analytical separations but detrimental to preparative-scale separations. Only in rare instances are large chiral separation factors (a > 1.5) observed in enantioselective GC. Only in one instance, a separation factor as high as a = 10 was detected in enantioselective GC for a chiral fluorinated diether and a modified 7-cyclodextrin (Schurig and Schmidt, 2003) (vide supra). 

The efficiencies obtained with pressure supported CEC whereat pressurization takes place at one end of the capillary are always higher than those observed with LC. Another advantage is the shorter elution time resulting from the combination of pressure-driven and electro-driven flow. An advantage of the LC method is the availability of a diversity of commercially available instruments which are simple and robust. Enantiomer separation on a preparative scale is easier accomplished with LC than with CEC. 

After the sample is prepared by any or all of the techniques just discussed, the last two steps on the flow chart (Fig. 6-33) involve developing the analytical HPLC separation and scaling it up to the final column upon which the actual preparative separation is to take place. Once the preparative LC separation is accomplished, the purity of the fractions can be determined by analysis using the original HPLC separation. In developing the separation,... 

Dehydroabietyl isocyanate, [39], was used by Falck et al. to determine the absolute configuration of hydroxyeicosatetraenoic acid methyl esters in a study of the enzymatic epoxidahon of arachidonic acid (175). In another study of similar metabolites, the stereochemical identity of 12-hydroxy-5,8,10,14-eicosatetraenoic acid derived from the lesional scale of patients with psoriasis was studied via separation of the enantiomers after derivatization with [39] (176). In these studies (175,176), LC separation of the derivatives was used. CDA [39] can be prepared from commercially available resolved dehydroabietylamine. It would be worthwhile to examine the applicability of this CDA to the resolution of other compounds. 

For analytical LC, standard packed columns (4-8 mm I.D.), capillary packed colunms (50-100 pm I.D.), and microbore packed colunms (0.5-1.5 mm I.D.) are used widely. Colunm sizes depend on the application, e.g., analytical, preparative, or commercial separations. Special configurations also exist, including membrane chromatography modules (stacks and hollow fibers) that offer lower pressure drops and easier scale-up than packed beds. 

LC Separation. Preparative scale liquid chromatography was performed with Waters PrepPak radially compressed silica columns. Fuel charges of 6 to 10 ml were carried through the column with n-pentane at a flow of 200 ml/minute. Refractive index, 254 nm ultraviolet, and 313 nm ultraviolet detectors monitored the LC effluent and keyed the collection of fractions. 

There are also reports of HPLC-NMR-MS in which the separation system is coupled to both NMR and MS (39). The power and potential of LC-NMR and related hyphenated techniques are likely to be enormous, extending the scope of analytical separations and obviating the need for much time-consuming preparative scale work and reducing the risk of chemical degradation of the compounds. This will allow extraction work to concentrate on natural products that... 

In addition to the miniaturization of HPLC enantioseparations, another current trend occurs in the opposite direction, namely the scaling-up of separations. The techniques of preparative-scale enantioseparations using liquid chromatography (LC) are described below. 

However, these pumps tend to be expensive. With such pumps, direct pumping recycle chromatography is impossible. Another disadvantage is the limited volume of solvent held in the cylinder (although sometimes two pumps can be used in tandem). This volume is 250-500 ml for most of these pumps, whereas the flow--rate through LC columns is usually 0,2 to a few ml/min, so that this disadvantage is not serious in analytical separations, but remains important in preparative--scale chromatography. 

Diastereomeric amides from camphor and camphor-related compounds have been used for the chiral resolution of amino acids by both gas and liquid chromatographic techniques. Nambara et al. [146] recommended the use of L-teresantalinyl chloride (56) to form the amides from alkylated amino acids for GC analyses. Although camphorsulphonamide p-nitrobenzyl esters have been used for LC separations [147,148], Aberhart el al. [149] found that these derivatives were difficult to prepare on a small scale, e.g. for enzyme assays. 

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