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Analitical techniques in combinatorial chemistry - Swarth M.E.

Swarth M.E. Analitical techniques in combinatorial chemistry - Marcel Dekker, 2000. - 311 p.
ISBN 0-8247-1939-5
Download (direct link): analyticaltechniquesincombinatorialchemistry2000.pdf
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Liquid Chromatography
10-50 mL/min are the norm. Isolation and purification on the preparative scale involves fraction collection and reanalysis. Fractions are reanalyzed on the analytical scale to see how efficient the purification was. The analytical scale separation is also run on the system to check to develop the preparative method prior to actual use. Generic methods exactly like those outlined previously and scalable column chemistries are used.
Automated purification has evolved over the years from ‘‘collect everything’’ on a time basis to UV-based collection. In both instances, time-consuming secondary analyses must be carried out to correctly identify the correct or desired fraction. More recently, techniques have evolved using mass-directed and intelligent automated purification. Mass-directed fraction collection is defined as collecting a fraction of a certain specified mass only. Intelligent automated fraction collection takes fraction collection one step further by allowing fraction collection based on masses, substructures/fragments, multiple masses, adducts, etc. This results in higher quality data, single fractions, less time, fewer steps, and less chance for errors. A typical high-throughput LC/MS mass-directed autopurification system is highlighted in Fig. 14.
The system as diagrammed in Fig. 14 used two-column regeneration to improve throughput. The system has both preparative and analytical capability so that samples can be run on the same system for rapid screening of original samples, or an initial purity assessment, as well as fraction collection capability. In addition to the two 6-port column-switching valves, two flow splitters
LC Pump
Optional UV Detectors
Figure 12 Schematic of an automated multichannel LC/MS system.
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Figure 13 MassLynx™ (Micromass UK Limited, Manchester, UK) OpenLynx™ open access browser data report. Plate configuration is shown in upper left. MS and UV data is displayed for the selected well position. If the requested mass is found, the well position is highlighted in green. If it is not, it is highlighted in red. Browser’s of this type are also used to track fractions in mass directed auto-purification systems.
are also used in this configuration. The upstream splitter divides the preparative flow and is an integral part of the system. The second splitter splits the analytical flow for parallel MS and PDA detection.
The use of an upstream splitter as outlined in Fig. 14 has several distinct advantages. Besides being easy to use and reproducible, this type of splitter provides constant delay times across a wide flow range, without the need for plumbing different tubing lengths or diameters. The use of a makeup flow allows high column loading without saturating the detector (by dilution) and allows flow to be split to multiple detectors without back streaming (explained below). The splitter essentially has two sides: a high-pressure side (column to collector) and a low-pressure side (makeup to detectors). As long as this balance is maintained the system works correctly. In operation, however, as the gradient composition changes from aqueous to organic, the back pressure
Liquid Chromatography
Figure 14 High-throughput mass-directed auto purification system schematic. System as shown is configured for preparative two-column regeneration and an analytical column for method development or to reanalyze fractions.
in the system decreases. Eventually the gradient back pressure can fall below the constant pressure of the makeup pump, resulting in splitter back streaming which prevents sample from getting to the MS probe and prevents late eluting peaks from being seen or collected. By using a lower viscosity organic solvent (e.g., 100% MeOH) as the makeup solvent, the back pressure is reduced to below that of the lowest point in the gradient, and back streaming is prevented. Subtle variations in split ratio delay times and band broadening are nominal across all gradient compositions.
Mass-directed autopurification systems are designed so that a peak is detected at the MS prior to reaching the fraction collector. Different flow rate-dependent delay times must be taken into account, and the software controls the synchronization between detection and collector trigger times. Figures 15 and 16 highlight the results of a mass-directed autopurification experiment (22). In this experiment, a three-component synthetic mixture was fractionated on the preparative scale and the fractions reanalyzed on the same system. Figure 15 shows the total ion chromatogram, UV signal, and fraction collection signal for the 20 mg/mL preparative run. As can be seen from the fraction collection signal, as the chromatography changes, the intelligent fraction collection compensates. The MS response directed the fraction collection based on the molecular weight of the three components in each instance. Figure 16 depicts the reanalysis of the second fraction on the analytical scale, on the same system. In spite of the two closely eluting peaks before and after, the data show a clean spectrum free of contamination.
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