Purification of combinatorial libraries

I High-Throughput Sample Preparation Techniques and Their Application to Bioanalytical Protocols and Purification of Combinatorial Libraries... 

Scavenger-Based Purification of Combinatorial Libraries Generated... 

T. Karancsi, L. Godorhazt, D. Szalay, and F. Darvas, UV-triggered main-component fraction collection method and its application for high throughput chromatographic purification of combinatorial libraries, / Comb. Chem. 7 (2005), 58-62. 

L. Zeng, D.B. Kassel, Developments of a fully automated parallel IC-MS system for the analytical characterization and preparative purification of combinatorial libraries. Anal, chem., 70 (1998) 4380. 

DB Kassel. A fully automated mass spectrometry based system for the rapid analysis and purification of combinatorial libraries. Solid and Solution Phase Combinatorial Synthesis, New Orleans, 1997. 

Zeng, L. Kassel, D.B. Developments of a Fully Automated Parallel HPLC/Mass Spectrometry System for the Analytical Characterization and Preparative Purification of Combinatorial Libraries, Anal. Chem., 70(20), 4380 388 (1998). 

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. 

All of the aforementioned HPLC purification systems employ detectors other than mass spectrometers as the selection criteria by which peaks are collected. With these systems, however, mass spectrometry is generally employed as a primary structural validation tool. More recently, systems have been described whereby a mass spectrometer has been added to preparative HPLC format to detect compounds of interest for collection. A recent review by Kassel [9] discusses the relative merits of employing MW-triggered versus UV-triggered fraction collection in various environments for the purification of combinatorial libraries. 

FIA is the simplest form of sample introduction into the mass spectrometer, and this injection format has been widely used in the analysis of combinatorial library samples. This technique offers the highest throughput combined with ease of use and facile automation. Richmond et al. [67-69] reported methods to minimize sample carryover for the FIA-MS analysis of combinatorial libraries. Samples were sorted before the analysis to maximize the molecular-weight difference between samples in the analysis queue and to minimize the conditions where consecutively measured wells contain samples similar to building blocks. Cycle times of less than a minute were reported with a carryover of 0.01%. A software appUcation was developed to automatically report the sample purity and calculate sample carryover by an automatic spectrum comparison method [70,71]. A quasi-molecular ion discovery feature was also implemented [72] in the automated data-processing program. Automated FIA-MS analysis and reporting were also used in the analysis of fractions from the purification of combinatorial libraries [73]. Whalen et al. developed software to allow automated FIA-MS analysis from 96-well plates [74]. The system optimizes the interface for mass spectrometry and MS/MS conditions, and reports the results in an unattended fashion. 

The MS instrumentation is the most expensive part of the LC-MS system, hence efforts to improve the throughput of the LC-MS analysis often involve the use of parallel multiple columns that feed into a single mass spectrometer. Zeng and Kassel [99] developed an automated parallel analytical/preparative LC-MS workstation to increase the throughput for the characterization and purification of combinatorial libraries. The system incorporates two columns operated in parallel for both LC-MS analytical and preparative LC-MS purifications. A multiple-sprayer ESI interface was designed to support flows from multiple columns. The system is under complete software control and delivers the crude samples to the two HPLC columns from a single autosampler. The authors demonstrated characterization of more than 200 compounds per instrument per day, and purification of more than 200 compounds per instrument per night. De Biasi et al. [100] described a four-channel multiplexed... 

Searle, P. An Automated Preparative HPLC-MS System for the Rapid Purification of Combinatorial Libraries, presented at The Strategic Institute Conference High-Throughput Compound Characterization, Dallas, TX, March, 1998. 

Schultz, L. Garr, C.D. Cameron, L.M. Bukowski, J. High Throughput Purification of Combinatorial Libraries, Bioorg. Med. Chem. Lett. 8, 2409-2414(1998). 

Barker, G.E. Romano, S.J. Short, K.M. Slee, D. Park, W. Purification of Combinatorial Libraries by OntoCHROMTM A Novel High-throughput SFC Prep Technique, in ACS Proceedings, San Diego, CA, USA, (2001) 221-ORGN-046. 

Peng, S.X. Henson, C. Strojnowski, M.J. Golebiowski, A. Klopfenstein, S.R. "Automated High-Throughput Liquid-Liquid Extraction for Initial Purification of Combinatorial Libraries, Anal. Chem. 72,261-266 (2000). 

Figure 6.2. Schematic diagram of an LC/MS system for high-throughput purification of combinatorial library compounds using the MS signal as trigger for fraction collection. Reprinted with permission from reference 12.

Nilsson, U.J. (2000) Solid-phase extraction for combinatorial libraries. J. Chromatogr. A 885 305-319. Takahashi, T. (2001) New tools for isolation and analysis in combinatorial chemistiy. Chromatography 22 45 -48. Schultz, L., Garr, C.D., Cameron, L.M. and Bukowski, J. (1998) High throughput purification of combinatorial libraries. Bioorg. Med. Chem. Lett. 8 2409-2414. 

Yan, B. et al. High-throughput purification of combinatorial libraries I A high-throughput purification system using an accelerated retention window approach. J. Comb. Chem. 2004, 6, 255-261. 

In this chapter we review various purification strategies, factors that impact on the purification efficiency, and recent progresses in high-throughput purification of combinatorial libraries. 

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