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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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In order to be successful, a proper method, like any other type of assay, run on the appropriate instrument, must be employed. In addition, software, both for systems operation/integration and data analysis, also plays a central role. This chapter will address how chemists, using chromatography, have adapted in answering the challenges presented by a combinatorial chemistry program of drug discovery. While not intended to be an exhaustive review, this chapter focuses predominantly on liquid chromatography (LC), discussing various applications and techniques that highlight its use in the drug discovery process.
Since application of fast, generic LC methods with mass spectrometry (LC/MS) is emerging as the technique of choice for assessing the progress and final quality of large combinatorial arrays in drug discovery, it will be discussed in some detail, along with other detection techniques. Mass-directed purification and characterization on the preparative scale will also be addressed.
II. BASIC HIGH-THROUGHPUT ANALYSIS AND CHARACTERIZATION
A. Chromatographic Optimization and Injection Overhead Reduction
The first step in drug discovery is lead identification before high-throughput screening. This step is characterized by use of combinatorial libraries varying in size from a few hundred to tens of thousands (or more) compounds. Assays should provide simple, basic information such as compound identification on a molecular weight basis, and the synthesis yield (purity). In order to answer these questions, methods must satisfy certain requirements. Gradient reverse phase liquid chromatography (RP-LC) with various detection modes can satisfy these requirements and is currently the method of choice for the analysis of combinatorial libraries and synthesis-related products (1-5).
The sheer number of samples, their diversity, and a lack of suitable standards for quantitation can at first seem an insurmountable challenge. Since method development for individual samples is not feasible due to time and economic factors, a broadly applicable ‘‘generic’’ method must be developed, without sacrificing information content. Also, an inject-to-inject cycle time of
Liquid Chromatography
115
2-5 min (or less!) is desired to accomplish the required sample throughput. Methods and instruments also need to be compatible with various detection methods in addition to MS, such as photodiode array (PDA), evaporative light scattering, and nitrogen- and sulfur-specific chemiluminescence detection. Finally, it is important to keep in mind that once a lead has been identified, it may be necessary to scale up the separation (with instrument and chemistry implications) to isolate larger amounts of pure material for additional studies.
Chromatographic instruments used in support of combinatorial chemistry require features somewhat different from those of traditional systems (58). The basic components of a system are the same: a solvent manager (pump), a sample manager (autosampler or injector), a detector(s), a data system, and, in some instances, a fraction collector. However, some important differences exist. The most significant difference is the sample manager. Samples can be presented in standard vials, tubes, or microtiter plates of various sizes and capacities, either singly or in various combinations. Therefore some type of ‘‘XYZ’’ sample management device is dictated. Other important differences include software instrument access and control, and data reduction and reporting.
Let’s examine a typical hypothetical situation. Assume an analyst has just received a combinatorial library on six high-density 384-well microtiter plates, for a total of 2304 samples of which all are unknown, all are different, and all differ in the degree of purity, for analysis in the lab. The traditional approach would be to use a gradient RP-LC method similar to that presented in Fig. 1. Using a long column and a shallow gradient is typically the first step in developing and optimizing a method. However, given the number of compounds involved, individual method development is highly impractical. Under the conditions used in Fig. 1, including 20 min of postrun reequilibration time, analysis of the 2304 samples would take more than 96 days! Although multiple systems could be used to analyze the samples, additional steps must be taken to improve sample throughput and to maintain final method detector compatibility in a full-time combinatorial chemistry support laboratory.
Several options, either alone or in combination, can be employed to improve sample throughput. Obvious options include using shorter columns, higher flow rates and temperatures, and/or sacrificing resolution. Figure 2A shows a separation of the same sample illustrated in Fig. 1, but with a smaller column at higher flow rates. The chromatographic test sample used spans a wide elution range; however, the peak capacity of the method remains high. Under these conditions, the total time for the analysis of the 2304 samples was decreased by an order of magnitude to less than 8 days. The column used
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