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The success of combinatorial chemistry, and the increasedemphasis on single well-characterised compounds of highpurity, has had a significant impact on analytical andpurification technologies. The requirement for ever-increasingthroughput has led to the automation and parallelisation ofthese techniques. Advances have also been made indeveloping faster methods to augment throughput further.

AddressesGlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow,Essex CM19 5AW, UK*e-mail: ian_hughes-1@gsk.com†e-mail: david_hunter-1@gsk.com

Current Opinion in Chemical Biology 2001, 5:243–247

1367-5931/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsAPCI atmospheric pressure chemical ionisationCLND chemiluminescent nitrogen detectionDI direct injectionELSD evaporative light scattering detectionESI electrospray ionisationFIA flow injection analysisFT Fourier transformICR ion cyclotron resonanceMALDI matrix-assisted laser desorption/ionisationSFC supercritical fluid chromatography

IntroductionThe field of combinatorial chemistry, once dominated bylarge libraries of mixtures or beads, has matured through thedevelopment of automated and parallel techniques [1] to apoint where high-throughput synthesis of single, well-char-acterised products in milligram quantities is an establishedpractice. The deconvolution of mixture-based combinatorial

libraries and the structural determination (decoding) of com-pounds on single synthesis beads necessitated imaginativeand sophisticated analytical techniques [2]. In contrast, thesynthesis of many single compounds in milligram quantitiesintroduces a new set of challenges. In principle, there is suf-ficient material to apply any of the modern analyticalmethods, such as HPLC, MS and NMR. The challenge nowis to achieve sufficient thoughput of the chosen analyticaltechniques and, importantly, the interpretation of the largequantity of data generated. Additionally, when dealing withsingle compounds, there is the possibility of purifying any orall of the synthetic products, to provide material of high puri-ty and hence ensure more meaningful screening results. Therecent development of high-thoughput purification tech-niques makes this a realistic goal, and also provides theopportunity to reduce the often resource-intensive reactionoptimisation stages of library synthesis. This review focuseson the development and implementation of processes foranalysis and purification of libraries of discrete, single com-pounds on the milligram scale. As with synthesis, automationand/or parallelisation are the foundation stones of high-throughput analysis and purification. A recent publicationsummarises the approaches in use at three companies [3].

High-throughput analysisHigh-throughput analytical techniques set out to answerthree questions about a sample: what is it, how pure is it,and how much is there? A summary of techniques in use isgiven in Table 1. Often a combination of techniques areemployed to provide all three answers.

Structural analysisThe most generally applied method for structure confirma-tion is undoubtedly MS [4]. Not only is the method rapid,

Techniques for analysis and purification in high-throughputchemistryIan Hughes* and David Hunter†

Table 1

Summary of high-throughput analytical techniques.

Analytical technique Relative Structural Purity Quantitative Commentsthroughput* analysis* analysis* analysis*

FIA-MS +++ + + Mass onlyMALDI-FT + ++ Accurate massESI-FT-ICR-MS + ++ Accurate massDI-NMR + +++ ++ ++ Data interpretation issueHPLC–UV ++ ++ Extinction coefficient variationsHPLC–UV/MS ++ + ++ Parallel methods increase throughputGravimetric +++ ++ Solvent entrapment possibleHPLC–ELSD ++ + ++ Volatiles, low melting point compounds

not detectedFIA-CLND +++ + Total nitrogen in sampleHPLC–CLND ++ + +++ Nitrogen-free solvent systems required

*An indication of relative throughput and applicability for structural, purity and quantitative analysis (+++ = highest) of the techniques reviewed inthis paper. See text for abbreviations.

but data interpretation can be as simple as comparing thefound molecular ion with the calculated mass and reportingthe result as a red/green colour-coded plate. A group atNovartis [5,6•] have reported the use of flow injectionanalysis (FIA) MS to perform 35,000 analyses per year on asingle instrument and additionally report estimated puritiesbased on the reconstructed ion current. Electrospray ioni-sation (ESI) is generally favoured over atmosphericpressure chemical ionisation (APCI) because the formergives less unanticipated fragmentation. In contrast, Duleryet al. [7] prefer APCI because it gives a higher detectionpercentage, but leaves the choice of method to the syn-thetic chemist, who has knowledge of the behaviour of thecompounds. Sorting the sampling sequence such that mol-ecular weight differences are maximised and the similarityof building blocks are minimised between consecutivesamples has been used by the Novartis group [8,9] to sim-plify detection of carry-over and hence reduce cycle timesfrom 168 to 44 seconds. A back-flush elution procedure hasbeen described by Marshall [10] to provide in-line removalof components responsible for ion-suppression.

Colour-rendered visualisation for automated carry-oversurveillance was reported [11] to contribute to a through-put of more than 70,000 samples in two years. By using amultiprobe autosampler with FIA-MS, Wang et al. [12]have reported the analysis of 80 samples in 10 min.Kyranos and Hogan [13] have discussed the advantagesand limitations of FIA-MS in high-throughput analysis,and its implementation, in conjunction with HPLC toanalyse more than 20,000 samples per month. Software toautomate the interpretation of high-throughput MS datahas been reported by Tong et al. [14••].

Tutko et al. [15] have described the application of matrix-assisted laser desorption/ionisation (MALDI) Fouriertransform (FT) MS to the analysis of 20 compounds perhour with root mean square errors of less than 5 ppm forparent and fragment ions. Walk et al. [16] have reported theuse of ESI-FT ion cyclotron resonance (ICR) MS for highaccuracy mass measurements on 300 samples per night.The mass accuracy is >500 times that of ESI quadrupole,using 10-fold less sample (4 pmol) and hence allows reli-able structure confirmation. An interesting development[17•] is the application of a multichannel device, with anarray of 96 electrospray tips, to the ESI-MS analysis of 96samples in 480 seconds.

More recently, the use of direct injection (DI) NMR hasbeen demonstrated [18••] as a valuable high-throughputanalytical technique suited to samples presented inmicrotitre plate format; 350 µl of 25 mM solutions areinjected into the probe and after approximately 60 secondsof data acquisition, the sample is returned to its well.Additionally, quantitation is made possible by the use of aninternal standard. Data interpretation is more time con-suming than for MS, but a variety of views have beendeveloped to faciltate this, including a matrix of small

spectral plots in microtitre plate format for pattern-recognition analysis.

Purity analysisHPLC is the ‘universal’ means of delivering high-throughput purity estimates and, although there remainsdebate about the most appropriate detection method, UVis most commonly employed. Key to achieving highthroughput has been the reduction in cycle times and thedevelopment of generic methods suitable for the range ofstructural types analysed.

Goetzinger and Kyranos [19] have investigated the varia-tion of chromatographic performance as gradient times andcolumn sizes are decreased and found that run times of lessthan 60 seconds are possible, whilst retaining sufficientpeak separation to be of value in purity assessment. C-8column packings were preferred to the more commonlyused C-18 because of easier flushing of very lipophilicmaterials. The use of HPLC retention times predictedfrom the relative retention times of substituents has beenadvocated [20] as an inexpensive means of characterisingarrays of compounds.

The use of MS detection in combination with UV hasadvantages of simultaneously returning structural andpurity information. Greig [21] describes an HPLC–UV/MSsystem running a single method over a 90 second gradient(4.25 min cycle time), in which UV (260, 305 nm) and MSprovide complementary detection methods. Dulery’sgroup [7] also makes use of HPLC–UV/MS, running 7 minto 10 min gradients.

De Biasi et al. [22•] have reported an alternative strategyfor increasing HPLC–MS throughput, whereby fourHPLC columns are run in parallel, and the output isanalysed by ESI time of flight spectrometry using a four-channel multiplexed electrospray source. Using a 2 mingradient, 96 samples were analysed in 1 hour, comparedwith 4.5 hours using a serial approach with the same gradi-ent. Extension to an eight-way system, recently installed atGlaxoSmithKline (V de Biasi, A Organ, personal commu-nication), has increased throughput further. The group atCombiChem [23] have described the use of a two-columnsystem with a dual electrospray ion source to achieve parallel HPLC–MS of 384 samples per day. They haverecently reported [24•] extension of the concept to four-and eight-column multi-parallel systems.

A recent development [25,26] is the application of gradient-packed column supercritcal fluid chromatography (SFC), inwhich analysis speed and recovery is up to 10 times fasterthan HPLC. Additionally, being a normal-phase method,the results are complementary to those obtained by con-ventional reversed-phase HPLC. Similarly, mixed-modeanion–cation exchange/hydrophilic interaction liquid chro-matography–ESI-MS has been reported [27] as analternative, orthogonal separation system.

244 Combinatorial chemistry

Although, ideally, all samples should undergo a completeanalysis, some groups have adopted strategies of statisti-cal sampling (of around 25% samples) under certaincircumstances [7,19].

Quantitative analysisThe use of automation renders gravimetric analysis viableas a high-thoughput quantitation technique [3,28,29].Although weighing thirty racks of 96 samples in 24 hoursto 0.1 mg accuracy is possible [3], inaccuracies fromentrapped solvents is a concern, especially when productsare obtained as oils or gums.

In principle, quantitation of any (resolved) component in thesample is possible by using HPLC followed by an appropriatedetector. All detection methods known to the reviewers sufferlimitations — the lack of a truly universal detector accounts forthe variety of approaches reported in the literature.Additionally, most compounds are being prepared for the firsttime, so the availability of standards for accurate calibration israre. UV is the ubiquitous HPLC detection method, but suf-fers from significant (wavelength dependant) variations of theextinction coefficient. UV is thus more often used as a purityestimation rather than a quantitation method [30•,31].

HPLC evaporative light scattering detection (ELSD) is anattractive quantitation tool [30•], because detection dependson the mass of material remaining after evaporation of sol-vent. However, the inability to detect low melting andvolatile materials (e.g. reagents) make it less well-suited topurity estimation. A detailed comparison [32••] of the quan-titation errors arising from UV and ELSD detection of 90well-characterised standards from 15 libraries demonstratedan average error of 18.5% for ELSD, which in most caseswas more accurate than UV. Of the UV wavelengths studied,214 nm generally performed better than 220 and 254 nm,although variation from library to library would be expected.

The development of chemiluminescent nitrogen detec-tion (CLND) is of considerable interest because it canprovide accurate quantitation when standards are not avail-able [33]. The response is related to the number ofnitrogen atoms in the sample, and it can detect impuritiesinvisible to other methods (e.g. triethylammonium trifluo-roacetate). Taylor et al. [31] have compared the applicationof CLND with FIA or HPLC analysis. Although FIA givesgood linearity, the presence of nitrogen-containing impuri-ties and solvent contributes to the ‘total’ nitrogenrecorded. Hence, HPLC–CLND may be of more value,but critically requires nitrogen-free solvent systems, suchas water/methanol/isopropanol. However, because of thesesolvent limitations, Shah’s group [34] use simultaneousFIA-MS and DI-CLND for the routine structure confir-mation and quantitation of 1000 compounds per day.

High-throughput purificationApproaches to the purification of combinatorial libraries havebeen reviewed [35]. The application of non-chromatographic

methods, such as phase separation, reagent or by-productsequestration and other polymer-supported purification tech-niques is an important development. Because these methodshave been the subject of a number of recent, comprehensivereviews [36–39,40••,41–43], they will not be discussed furtherhere. However, in many instances, these tools prove inade-quate or are inappropriate, and in such circumstances a trulychromatographic technique is required.

In 1997 workers at Parke Davis [44] reported the purifica-tion of 50 to 100 samples per day on a 10 to 50 mg scaleusing an automated preparative HPLC system. Data fromanalytical HPLC–MS acquired prior to the preparative runenabled prediction of retention-time windows for the col-lection of desired products. During the run, only peaksabove a preset UV threshold and within the predictedretention-time window were collected. UV-triggered frac-tionation was also employed at MDS Panlabs [28], butthroughput was enhanced by operating four chromatogra-phy columns in parallel. Up to 288 samples (75 to 100 mgscale) were purified per day on a single instrument.Fractions were collected throughout the run whenever UVthresholds were exceeded (intelligent fraction collection).All fractions were analysed by FIA MS (and ELSD forquantitation), followed by recombination and evaporation.In our own laboratories [45], we use the same four-columnsystem, and apply similar parameters for the UV-triggeredcollection of fractions. However, analytical HPLC–MSdata acquired before the preparative run is used to guidethe selection of fractions for onward processing throughconfirmatory HPLC–MS and recombination. Any errors inthe selection process can be corrected because allUV-active fractions have been retained. An integrated synthesis/purification system has been reported byTommasi et al. [46], in which syntheses are performed onthe bed of an autosampler, which allows direct UV-trig-gered HPLC fractionation of 16 reactions on a 50 to250 µmol scale. The integration of parallel preparativeHPLC into a high-throughput organic chemistry systemhas been described by Coffey et al. [47].

Several groups have reported the use of mass-triggered frac-tionation as an alternative to UV-triggered methods, withthe attraction that only material of the desired molecularweight is collected. The group at CombiChem [48,49] havereported the use of mass-triggered fractionation to collect upto four products of predefined mass per sample.Additionally, they demonstrated the use of high flow rates(>70 ml/min) to give cycle times of around 5 to 8 min persample. By operating two columns in parallel and collectinga single product per tube, throughput was doubled [23].Recently, extension to a system capable of running fourpreparative or analytical columns in parallel, with dual wave-length UV and MS detection, has been reported [3].Kiplinger et al. [29] also describe the implementation of anautomated system using both UV and MS to make real-timedecisions. Workers at Bristol-Myers Squibb [3] described asystem with variable-wavelength UV detectors and the

Techniques for analysis and purification in high-throughput chemistry Hughes and Hunter 245

option of mass-direction of fraction collection on a 2 to100 mg scale. Detailed descriptions of their development ofsuitable chromatographic methods have been reported [50].GlaxoSmithKline are reported [3] to employ a mass-trig-gered system for sample sizes of 1 to 10 mg. Larger samplesuse a preparative HPLC–UV system in which the tediousnature of fraction management is highlighted.

Apart from a brief mention of failures attributed to poorionisation or false mass triggering [23] and a discussion onthe effects of peak-trailing on cross-contamination [29], weare unaware of reports comparing the reliability and gener-ality of mass-triggered versus UV-triggered methods inhigh-throughput systems.

More recently, SFC has begun to emerge as an alternative toconventional HPLC for high-throughput purification.Coleman [51] has described an HPLC system modified tomake use of SFC using a fast gradient (7 min cycle) with UVdetection and, optionally, ELSD for quantitation. The use ofcarbon dioxide as the major eluent offers advantages of simplified evaporation, reduction of waste disposal costs andassociated environmental impact, as well as the true poten-tial to collect only one fraction per product. Many of themajor issues involved in developing SFC for high through-put have been described by Berger et al. [52•], a keydevelopment being a novel separator that avoids the aerosolsnormally generated by the rapid evaporation of carbon dioxide as the pressure is released on exiting the system.

ConclusionsThe ability to analyse and purify large numbers of com-pounds is an important component of the high-throughputchemistry process. For compound characterisation, MS iscurrently the method of choice. However, it is expectedthat developments in NMR will render it a key high-throughput technology, especially as software becomesavailable to automate data interrogation. The use of inter-nal standards will enable concurrent characterisation andquantitation [18••,53]. Photoionisation (PI) MS is emerg-ing as a near universal ionisation technique for manyclasses of compounds [54], with minimal fragmentation.Hence, PI-MS is likely to find additional applications inpurity assessment and mass-triggered fractionation. Arange of purification techniques are now available to thechemist, and parallelisation of these techniques has greatlyincreased their throughput. Further developments in SFCchromatography offer the potential of significant advancesover conventional HPLC.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

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3. Giger R: High-throughput analysis, purification and quantificationof combinatorial libraries of single compounds. Chimia 2000,54:37-40.

4. Suessmuth RD, Jung G: Impact of mass spectrometry oncombinatorial chemistry. J Chromatogr B 1999, 725:49-65.

5. Gorlach E, Richmond R, Lewis I: High-throughput flow injectionanalysis mass spectroscopy with networked delivery of color-rendered results. 2. Three-dimensional spectral mapping of 96-wellcombinatorial chemistry racks. Anal Chem 1998, 70:3227-3234.

6. Richmond R, Gorlach E, Seifert J-M: High-throughput flow injection• analysis-mass spectrometry with networked delivery of colour

rendered results: the characterisation of liquid chromatographyfractions. J Chromatogr A 1999, 835:29-39.

This paper describes tools for facilitating the visualisation of the many datapoints generated by high-throughput purification and analytical techniques.

7. Dulery BD, Verne-Mismer J, Wolf E, Kugel C, Van Hijfte L: Analyses ofcompound libraries obtained by high-throughput parallelsynthesis: strategy of quality control by high-performance liquidchromatography, mass spectrometry and nuclear magneticresonance techniques. J Chromatogr B 1999, 725:39-47.

8. Richmond R, Gorlach E: Sorting measurement queues to speed upthe flow injection analysis mass spectrometry of combinatorialchemistry syntheses. Anal Chim Acta 1999, 394:33-42.

9. Richmond R: The analytical characterisation of sub-minutemeasurement duty cycles in flow injection analysis massspectrometry, by their carry-over. Anal Chim Acta 2000, 403:287-294.

10. Marshall PS: Development and applications of a rapid back-flushmicroseparation system coupled to a mass spectrometer for thequality control of combinatorial libraries. Rapid Comm Mass Spec1999, 13:778-781.

11. Richmond R, Gorlach E: The automatic visualisation of carry-overin high-throughput flow injection analysis mass spectrometry.Anal Chim Acta 1999, 390:175-183.

12. Wang T, Zeng L, Strader T, Burton L, Kassel DB: A new ultra-highthroughput method for characterizing combinatorial librariesincorporating a multiple probe autosampler coupled with flowinjection mass spectrometry analysis. Rapid Comm Mass Spec1998, 12:1123-1129.

13. Kyranos JN, Hogan JC: High-throughput characterization ofcombinatorial libraries generated by parallel synthesis. AnalChem 1998, 70:389A-395A.

14. Tong H, Bell D, Tabei K, Siegel MM: Automated data massaging,•• interpretation, and e-mailing modules for high throughput open

access mass spectrometry. J Am Soc Mass Spectrom 1999,10:1174-1187.

This paper presents interesting software for interpretation of MS data. Itincludes a number of valuable tabulations of peak types commonly observedin mass spectra.

15. Tutko DC, Henry KD, Winger BE, Stout H, Hemling M: Sequentialmass spectrometry and MSn analyses of combinatorial librariesby using automated matrix-assisted laser desorption/ionisationFourier transform mass spectrometry. Rapid Comm Mass Spec1998, 12:335-338.

16. Walk TB, Trautwein AW, Richter H, Jung G: ESI Fourier transform ioncyclotron resonance mass spectrometry (ESI-FT-ICR-MS): a rapidhigh-resolution analytical method for combinatorial compoundlibraries. Angew Chem Int Ed Engl 1999, 38:1763-1765.

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electrospray tips for high-throughput mass spectrometry. AnalChem 2000, 72:3303-3310.

This paper describes devices with the potential to increase MS throughputby an order of magnitude.

18. Keifer PA, Smallcombe SH, Williams EH, Salomon KE, Mendez G,•• Belletire JL, Moore CD: Direct-injection NMR (DI-NMR): a flow

NMR technique for the analysis of combinatorial chemistrylibraries. J Comb Chem 2000, 2:151-171.

This paper describes high-throughput NMR, a valuable addition to MS forthe analysis of libraries. Newly developed tools for the display and analysisof the NMR data produced are presented.

19. Goetzinger WK, Kyranos JN: Fast gradient RP-HPLC for high-throughput quality control analysis of spatially addressablecombinatorial libraries. Am Lab 1998, 30(8):27-37.

246 Combinatorial chemistry

20. von Roedern EG: A new method for the characterization ofchemical libraries — solely by HPLC retention times. Mol Diversity1998, 3:253-256.

21. Greig M: Use of automated HPLC-MS analysis for monitoring andimproving the purity of combinatorial libraries. Am Lab 1999,31(24):28-32.

22. de Biasi V, Haskins N, Organ A, Bateman R, Giles K, Jarvis S: High• throughput liquid chromatography/mass spectrometric analyses

using a novel multiplexed electrospray interface. Rapid CommMass Spec 1999, 13:1165-1168.

This paper describe the significant gains in throughput made possible byanalysing the flow streams from four HPLC columns with a single multi-plexed MS interface.

23. Zeng L, Kassel DB: Developments of a fully automated parallelHPLC mass spectrometry system for the analyticalcharacterization and preparative purification of combinatoriallibraries. Anal Chem 1998, 70:4380-4388.

24. Wang T, Zeng L, Cohen J, Kassel DB: A multiple electrospray• interface for parallel mass spectrometric analyses of compound

libraries. Comb Chem High Throughput Screen 1999, 2:327-334.This paper describes the sequential sampling of four or eight HPLC flowstreams multiple times per second, hence enhancing throughput whilstretaining good sensitivity and peak shape.

25. Ventura MC, Farrell WP, Aurigemma CM, Greig M: Packed columnsupercritical fluid chromatography/mass spectrometry for high-throughput analysis. Anal Chem 1999, 71:2410-2416.

26. Berger TA, Wilson WH: High-speed screening of combinatoriallibraries by gradient packed-column supercritical fluidchromatography. J Biochem Biophys Methods 2000, 43:77-85.

27. Strege MA, Stevenson S, Lawrence SM: Mixed-mode anion-cationexchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometry as an alternative to reversedphase for small molecule drug discovery. Anal Chem 2000,72:4629-4633.

28. Schultz L, Garr CD, Cameron LM, Bukowski J: High throughputpurification of combinatorial libraries. Biorg Med Chem Lett 1998,8:2409-2414.

29. Kiplinger JP, Cole RO, Robinson S, Roskamp EJ, Ware RS,O’Connell HJ, Brailsford A, Batt J: Structure-controlled automatedpurification of parallel synthesis products in drug discovery. RapidComm Mass Spec 1998, 12:658-664.

30. Hsu BH, Orton E, Tang SY, Carlton RA: Application of evaporative• light scattering detection to the characterization of combinatorial

and parallel synthesis libraries for pharmaceutical drug discovery.J Chromatogr B 1999, 725:103-112.

This paper gives a good introduction to the theory of ELSD, as well as dis-cussing its role in quantitation of small-molecule libraries.

31. Taylor EW, Qian MG, Dollinger GD: Simultaneous on-linecharacterization of small organic molecules derived fromcombinatorial libraries for identity, quantity, and purity byreversed-phase HPLC with chemiluminescent nitrogen, UV, andmass spectrometric detection. Anal Chem 1998, 70:3339-3347.

32. Fang L, Wan M, Pennacchio M, Pan J: Evaluation of evaporative•• light-scattering detector for combinatorial library quantitation by

reversed phase HPLC. J Comb Chem 2000, 2:254-257.This paper provides a detailed comparison of the quantitation errors inher-ent in UV and ELSD detection methods.

33. Fitch WL, Szardenings AK: Chemiluminescent nitrogen detectionfor HPLC: an important new tool in organic analytical chemistry.Tetrahedron Lett 1997, 38:1689-1692.

34. Shah N, Gao M, Tsutsui K, Lu A, Davis J, Scheuerman R, Fitch WL,Wilgus RL: A novel approach to high-throughput quality control ofparallel synthesis libraries. J Comb Chem 2000, 2:453-460.

35. Weller HN: Purification of combinatorial libraries. Mol Diversity1999, 4:47-52.

36. Curran DP: Strategy-level separations in organic-synthesis: fromplanning to practice. Angew Chem Int Ed Engl 1998,37:1175-1196.

37. Flynn DL: Phase-trafficking reagents and phase-switchingstrategies for parallel synthesis. Med Chem Rev 1999, 19:408-431.

38. Parlow JJ, Devraj RV, South MS: Solution-phase chemical librarysynthesis using polymer-assisted purification techniques. CurrOpin Chem Biol 1999, 3:320-336.

39. Nilsson UL: Solid-phase extraction for combinatorial libraries.J Chromatogr A 2000, 885:305-319.

40. Ley SV, Baxendale IR, Bream RN, Jackson PS, Leach AG,•• Longbottom DA, Nesi M, Scott JS, Storer RI, Taylor SJ: Multi-step

organic synthesis using solid-supported reagents andscavengers: a new paradigm in chemical library generation.J Chem Soc Perkin Trans 1 2000:3815-4195.

This paper provides a very comprehensive review of the literature.

41. Thompson LA: Recent applications of polymer-supported reagentsand scavengers in combinatorial, parallel, or multistep synthesis.Curr Opin Chem Biol 2000, 4:324-337.

42. Peng SX, Henson C, Strojnowski MJ, Golebiowski A, Klopfenstein SR:Automated high-throughput liquid–liquid extraction for initialpurification of combinatorial libraries. Anal Chem 2000, 72:261-266.

43. Booth RJ, Hodges JC: Solid-supported reagent strategies for rapidpurification of combinatorial synthesis products. Acc Chem Res1999, 32:18-26.

44. Kibbey CE: An automated system for the purification ofcombinatorial libraries by preparative LC/MS. Lab Robot Autom1997, 9:309-321.

45. Hughes I: Separating the wheat from the chaff: high throughputpurification of chemical libraries. J Assoc Lab Autom 2000,5:69-71.

46. Tommasi RA, Whaley LW, Marepalli HR: AutoChem: automatedsolution-phase parallel synthesis and purification via HPLC.J Comb Chem 2000, 2:447-449.

47. Coffey P, Ramieri J, Garr C, Schultz L: An open, multivendorspecification for high-throughput organic chemistry (HTOC). AmLab 1999, 31(4):57-71.

48. Zeng L, Wang X, Wang T, Kassel DB: New developments inautomated prepLCMS extends the robustness and utility of themethod for compound library analysis and purification. CombChem High Throughput Screen 1998, 1:101-111.

49. Zeng L, Burton L, Yung K, Shushan B, Kassel DB: Automatedanalytical/preparative high-performance liquid chromatography-mass spectrometry system for the rapid characterization andpurification of compound libraries. J Chromatogr A 1998,794:3-13.

50. Weller HN, Young MG, Michalczyk SJ, Reitnauer GH, Cooley RS,Rahn PC, Loyd DJ, Fiore D, Fischman SJ: High-throughput analysisand purification in support of automated parallel synthesis. MolDiversity 1997, 3:61-70.

51. Coleman K: High-throughput preparative separations fromcombinatorial libraries. Analysis 1999, 27:719-723.

52. Berger TA, Fogleman K, Staats T, Bente P, Crocket I, Farrell W,• Osonubi M: The development of a semi-preparatory scale

supercritical-fluid chromatograph for high-throughput purificationof ‘combi-chem’ libraries. J Biochem Biophys Methods 2000,43:87-111.

This paper discusses the merits of SFC and issues surrounding its suc-cessful implementation.

53. Gerritz SW, Sefler AM: 2,5-Dimethylfuran (DMFu): an internalstandard for the "traceless" quantitation of unknown samples via1NMR. J Comb Chem 2000, 2:39-41.

54. Syage JA, Evans MD, Hanold KA: Photoionisation massspectrometry. Am Lab 2000, 32(24):24-29.

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