Chromatographic separation with combinatorial libraries

Figure 13.5. Mass-directed purification of a combinatorial library. Chromatographic separation was carried out using gradient elution of 10-90%acetonitrile in water for 7 min after an initial hold at 10%acetonitrilefor 1 min. (a)Total ion chromatogram showingdesired product and impurities, (b) Computer-reconstructed ion chromatogram (RIC) corresponding to the expected product, (c) Postpurification analysis of the isolated component with a purity >90%. (Reproduced from Ref 15 by permission of Elsevier Science.)...

A UV-triggered purification system was described by Kibby [44] in support of the purification of combinatorial libraries generated at Parke-Davis. This system is operated in either reverse-phase or normal-phase mode, and is employed as well for chiral separations. Multiple column sizes allow the system to accommodate the purification of samples in weight up to 50 mg. The operational protocol involves an initial scouting run by analytical HPLC with APCI-MS detector. The conditions that are selected are based on structural information. Fraction collection is controlled by customized software, and sample identity, UV, MS data along with chromatographic data are imported from the analytical LC-MS. Peaks are collected only when the UV threshold is met within an appropriate collection window thus, the number of fractions obtained is limited. Postpurification loop injection mass spectra are collected on these fractions to determine the desired component from each sample. 

Further improvement of throughput in LC-MS analysis may be achieved by step-gradient elution. This elution format is essentially an on-line solid-phase extraction (SPE) process, where the samples are loaded onto the column, washed with aqueous mobile phase to remove water soluble impurities, and compounds are eluted with a mobile phase of high organic content. The technique combines the simplicity of FIA with the benefit of the removal of impurities and buffer components before mass spectrometry detection. In this case, selectivity is achieved by mass spectrometry alone without chromatographic separation. The technique has been used for compound purity assessment and quantitation. An on-line back-flush SPE-MS technique has been used by Marshall for quality assessment of the combinatorial libraries [112]. This back-flush elution procedure provides a very effective in-line removal method... 

The miniaturisation of liquid chromatographic (LC) separation techniques has become increasingly important over the last ten years. The introduction of new synthetic chemistry techniques, such as combinatorial chemistry, has produced libraries of small-volume samples that require fast throughput, but also efficient and sensitive analysis. The use of miniaturised liquid chromatographic techniques offers the possibility of analysing these submicrolitre samples with high efficiency and short analysis times. 

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