Automated purification methods

Solution-phase combinatorial synthesis provides a homogeneous reaction medium and overcomes the drawbacks of a solid-phase strategy. An easy and reliable purification method is required in solution-phase combinatorial (parallel) synthesis to facihtate automation. The throughput in solution-phase automated synthesis is directly related to the facility of performing a purification process (work-up), compound separation, etc.15... 

Purification A variety of creative and innovative open-access LC/MS formats were developed to address throughput needs within the industrial laboratory. As the preparation of large libraries for lead discovery became routine, the burden placed on analytical techniques focused mainly on throughput and quality (Kyranos and Hogan, 1998 Van Hijfte et al., 1999). Biological assay requirements, however, normally required pure compounds. Thus, the focus shifted toward the use of automated high-throughput purification methods applied to libraries of discrete compounds (Weller, 1998-99). 

The problems associated with high-throughput analysis of SP reaction products, automation of purification techniques, and automation of methods for stmcture determination in SPS are typically encountered in combinatorial chemistry and will be dealt with more thoroughly in Chapters 6-8. 

Work up/purification procedures have also largely benefited from the commercial availability of semiautomated or automated instrumentation. The panel of tools spans from small, 96-well based devices to increase the throughput of manual operations (67, 68) to semiautomated systems able to purify in parallel combinatorial samples (69) or to concentrate in parallel large, discrete solution libraries (70, 71). Several systems developed in-house have also been recently rejxirted (72, 73), and a recent review covered the most recent trends in automated high-throughput purification methods for solution discrete libraries (74). 

The analysis of phytochemicals is a tedious process involving several steps in which care must be taken to avoid degradation and contamination. Recent advancements in extraction, concentration, purification and analytical procedures of phytochemicals have been made, but additional developments are needed to assist in the identification and quantification of the diverse array of phytochemicals present in plants and foods, as well as metabolites in biological samples. Specifically there is a need to automate sample extraction, clean-up, and concentration steps to facilitate the screening of phytochemicals develop analytical methods with improved sensitivity, resolution and throughput that utilize less organic solvents and develop concentration and purification methods to produce analytical standards that are not available commercially. Continued advancements in sample preparation and analytical techniques will assist researchers in their quest to identify and quantify the vast array of phytochemicals present in plants... 

Of course, to consider using any purification method on such potentially large numbers of samples meant that the system must be compatible with automation. This was not the only requirement that any system developed would need to fulfil other criteria included the ability to separate/remove closely related structural contaminants, the ability to scale the purification method up funher to multi-gram scale if needed and the ability to handle all sorts of compound classes/polarities using a single or at least a small number of generic methods. 

With many sensitive liquid chromatographs, little or no pretreatment is necessary, but with GLC partial purification and isolation are required. Since most biological substances have low volatility or poor thermal stability, it is usually necessary to prepare suitable volatile and stable derivatives before analysis. At present, pretreatment methods involve little or no specialized instrumentation, but this will undoubtedly be developed for use with faster automated chromatographs. Methods of sample injection for GLC are relatively crude and an internal standard is necessary to compensate for errors in the volume injected. If large numbers of samples are to be analyzed repeatedly, some form of automatic injection, linked to a timing device, will need to be developed (J6, Zl). 

SH DeWitt. Automated parallel purification methods. Solid and Solution Phase Combinatorial Synthesis, New Orleans, 1997. 

Solid-phase parallel synthesis mimics the previously described solution phase strategy. This approach easily lends itself to both semi- and full automation. In contrast to the solution phase method, purification is easily achieved by simply washing the resin beads, and the reactions can be driven to completion by excess reagents, multiple cycles, and microwave techniques. The initial building block or scaffold is attached to the resin bead by a detachable linker. At the end of the synthesis, the final construct is released under the appropriate cleavage conditions for automated purification, usually by high-pressure liquid chromatography (HPLC). This allows bioanalysis of the final product in aqueous solution under standard assay conditions. 

Today, multiple microreactor designs have been reported for the aerobic oxidation of organic compounds. Most of the reactors are built out of simple polymer or stainless steel tubing, assembled together as prototype reactor concepts. But also multigram to kilogram-scale reactor setups have been reported, with automated control and inhne purification methods. 

This enables the samples to be introduced as narrow bands, higher and rapid resolution of the mixtures and an incorporation of automated analytical method to faciUtate purification processes. An improvement in the speed of separation for biomacromolecules can be achieved by the use of superficially porous support packed into microbore columns... 

There are many methods used to purify polypeptides and proteins. The specific methods one chooses depend on the source of the protein (isolation from a natural source or chemical synthesis), its physical properties, including isoelectric point (p7), and the quantity of the protein on hand. Initial purification methods may involve precipitation, various forms of column chromatography, and electrophoresis. Perhaps the most important final method for peptide purification, HPLC, is used to purify both peptides generated by automated synthesis and peptides and proteins isolated from nature. 

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

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