Preparative-scale HPLC separations

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... 

Preparative-Scale HPLC Separations. Results of the analytical-scale HPLC separations are used to develop an approach in the scale-up of the HPLC separations for the preparation of subfractions of the residue organics in quantities suitable for mutagenesis testing and compound isolation. If the analytical-scale separation results indicate the... 

Isolation of Mutagens. Applying the general preparative-scale HPLC separation procedure outlined in the previous section (Step I),... 

Fritz and co-workers (F5) described the use of reversed-phase HPLC for the isolation of protected oligonucleotide intermediates in the stepwise synthesis of deoxyribonucleotides. They report that the use of reversed-phase preparative scale HPLC reduces the time required for synthesis of oligonucleotides by 30%. In addition, they report on the RPLC separation of unprotected S -hydroxylated oligonucleotides. 

The MaNP acid method has been successfully applied to various racemic alcohols listed in Table 9.3 for preparation of enantiopure secondary alcohols and the simultaneous determination of their absolute configurations. If the separation factor a is as large as in the case of l-octyn-3-ol 56 (entry 2 in Table 9.3, a. = 1.88), a large-scale HPLC separation of diastereomeric MaNP esters is feasible. For example, in the case of esters 64a and 64b derived from alcohol 56, ca. 0.85-1.0 g of the mixture was separable in one run by the HPLC (hexane/EtOAc = 20 1) using a silica gel glass column (22 x 300 mm) (Figs. 9.19 and 9.20). 

An example of the application of HPLC is the separation of fullerenes (see Section 14.4). Columns packed with stationary phases designed specifically for preparative-scale fullerene separation are commercially available (e.g. CosmosiF Buckyprep columns). 

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

In this context, the enantiomeric pair containing the eutomer of cyclothiazide can be resolved by HPLC on cellulose-derived coated CSPs. Nevertheless, the poor solubility of this compound in solvents compatible with this type of support makes this separation difficult at preparative scale. This operation was achieved with a cellulose carbamate fixed on allylsilica gel using a mixture of toluene/acetone as a mobile phase [59]. 

In addition to the development of the powerful chiral additive, this study also demonstrated that the often tedious deconvolution process can be accelerated using HPLC separation. As a result, only 15 libraries had to be synthesized instead of 64 libraries that would be required for the full-scale deconvolution. A somewhat similar approach also involving HPLC fractionations has recently been demonstrated by Griffey for the deconvolution of libraries screened for biological activity [76]. Although demonstrated only for CE, the cyclic hexapeptides might also be useful selectors for the preparation of chiral stationary phases for HPLC. However, this would require the development of non-trivial additional chemistry to appropriately link the peptide to a porous solid support. 

Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), was used for the separation and fractionation of macromolecules on an analytical and preparative scale [17]. The separation occurs predominantly by the hydrodynamic volume of the macromolecules in solution, however, in some cases the polarity of the molecules can also influence the retention times. Like HPLC, the SEC technique is generally very reproducible with regard to its elution times (typically < 1 h) and hence can be used for automated synthesis. But because the cost for an automated SEC system is high, it must be considered as a serial separation technique. In addition, larger scale separations > 100 mg, usually require repetitive injection of small aliquots. 

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