Preparative-scale chromatography injection

Menet et al. [6] have compared performances of CCC and preparative HPLC owing to a separation of two antibiotics X and Y. The CCC apparatus used was a centrifugal partition chromatograph (CPC, Sanki LLN) of 250 mL internal volume. For this purpose, classical parameters of preparative-scale chromatography were calculated experimental duration, including the sample preparation and the separation time, solvent consumption, including the volume of the mobile phase, the stationary phase and the injection solvent, and purity of the purest fraction in Y. The parameter purity in Y was chosen because Y is the solute most difficult to purify because of its physical properties (particularly hydrophobicity) which are close to those of the main impurities. The hourly yield (g/h) is defined as the ratio of the recovered quantity to the experimental duration. The volumic yield (g/L) is defined as the ratio of the recovered quantity to the solvent consumption. Table 1 summarizes the results of... 

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. 

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). 

Figure 1.2 Preparative scale liquid chromatography unit with 6 ft (1.8 m) long column (in center) having an inner diameter of 6 in (15 cm). Up to 2 L of sample solution can be injected and processed in a single 90-min run. (Courtesy Thomas J. Filipi and Whatman Chemical Separation Division.)...

In addition to requiring significant bulk material, the timeframe to complete the isolation is considerable. If the maximum analytical load for a 4.6 mm x 150 mm column has been determined to be 5 mg, assuming the isolation will be performed using semi-preparative chromatography (20 mm x 300 mm column), approximately 190 mg of sample can be loaded onto the preparative column. For a 0.1% level unknown, this translates to 190 pg of unknown injected onto the preparative column. Therefore, a total of 27 injections are required. If the assay time were estimated to be 1 hr, it would take at least 27 hr to perform the injections needed to obtain 5 mg (once again assuming 100% recovery). This timeframe does not include the time needed for method scale-up development, concentration and... 

The technique of boxcar injections (not to be confused with boxcar chromatography) can be extremely productive for iso-cratic elution in any mode of chromatography and should always be considered when scaling up a separation. The preparative HPLC of an enantiomeric mixture utilising a chiral stationary phase is described here to demonstrate the approach for separation of a binary mixture. 

The method of analysis for the final determination of the seven CB congeners used by each participating laboratory was based on capillary gas chromatography with electron capture detection. In addition, mass spectrometry was used for compound identification and confirmation, but not for quantification. Each laboratory used their own proven procedures for the sample preparation, clean-up, method of injection, choice of carrier gas and chromatographic condition. The mineral oils were disssolved in an appropriate solvent and analysed without any preliminary extraction from the matrix. The clean-up and the final determination were completed within the normal time scale for these analyses. 

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