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Automated Liquid-Liquid Extraction and Ion-Exchange Solid-Phase Extraction for Initial Purification


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Automated Liquid–Liquid Extraction and Ion-Exchange Solid-Phase Extraction for Initial Purification
Sean X. Peng and Charles Henson 1. Introduction The development of combinatorial chemistry has made a profound impact on the pharmaceutical industry by producing a large number of structurally diverse compounds in a very short amount of time. Combined with highthroughput screening, bioinformatics, and laboratory automation, the combinatorial chemistry approach has led to a significantly accelerated drug discovery process compared to a traditional one-compound-at-a-time approach (1). With a potential explosion of the number of biological targets available for various diseases from genomics and proteomics, combinatorial chemistry and parallel synthesis will be further embraced by the pharmaceutical industry, leading the way to rapidly generate a large number of chemical libraries to screen for different disease targets. Currently, thousands of potentially bioactive compounds are made every week by combinatorial chemistry synthesis. Subsequently, these compounds go through various high-throughput biological screens in different therapeutic areas to find biologically active compounds or hits. However, in order to ensure true hits from these various screening assays, initial sample purification to remove assay-interfering components is required to prevent false positive or negative results. This initial sample cleanup step poses a great challenge to synthetic and analytical chemists, because purification in a high-throughput and fully automated fashion is needed to keep up with the fast pace of combinatorial synthesis and high-throughput biological screening. In general, combinatorial chemistry employs either solid-phase or solutionphase synthesis (2). The solid-phase synthesis has the advantage of generating cleaner samples, because the solid support material, i.e., resin beads, can be
From: Methods in Molecular Biology, Combinatorial Library Methods and Protocols Edited by: L. B. English ? Humana Press Inc., Totowa, NJ

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filtered and washed after the reaction is complete and purer reaction product is obtained upon cleavage of the linker on the resin. However, excess cleaving reagents are often needed to achieve complete cleavage in a short amount of time because many cleavage reactions are slow with stoichiometrical amounts. For solution-phase synthesis, excess starting reagents and by-products often remain in the intermediate and final product and are difficult to remove. When these excess reagents and by-products interfere with biological screening assays of interest, the screening results become unusable. To address these problems, tremendous ongoing efforts have been made in the purification of combinatorial library products. Several different approaches have been developed and employed for initial purification of crude library samples. These include liquid–liquid extraction (LLE) (3–5), various modes of solid-phase extraction (SPE) (6–8), solid-phase scavenging (SPS) (9–12), and parallelcolumn preparative high-performance liquid chromatography (HPLC) (13). LLE, SPE, and SPS are generally employed for initial or primary purification to remove unspent starting reagents, by-products, and impurities from reaction mixtures. Because these methods can be fully automated in a high-throughput fashion such as in a 96-well format, they are generally employed as the first purification step immediately after crude library samples are made. After this initial purification, the purified samples pass through high-throughput biological screens. When a hit sample is found, a parallel-column preparative HPLC method is typically employed to isolate the hit compound for an additional confirmatory biological assay. Preparative HPLC can also be used for initial purification to provide high purity samples for biological screening. Because of its limited throughput, this HPLC method may be best suited for purification of hit samples for hit identification and confirmation as the number of samples to be purified is significantly reduced after initial biological screening. For the initial biological screening, however, the assay-interfering components are typically removed using LLE, SPE, and SPS as these approaches offer higher sample throughput, faster speed, lower cost, and easier adaptation to automation. While the LLE method exploits the differences in partitioning between the desired product and unwanted components in two immiscible organic and aqueous phases, the SPE and SPS methods take advantage of the differences in interactions (reversible and non-covalent interaction in SPE versus irreversible and covalent bonding in SPS) between the solid-phase resins or scavengers and the desired products or unwanted components. Currently, automated LLE and ion-exchange SPE are the two commonly used methodologies for initial purification of crude combinatorial library samples. LLE is a well established and widely utilized extraction technique in organic synthesis. It is simple, rapid, and convenient, producing extremely clean extracts with high product recovery. Recent development of a fully automated

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96-well LLE methodology has made LLE a very attractive and practical method for post-reaction purification of combinatorial libraries for high-throughput screening (3). The automated LLE method selectively removes unreacted starting materials, by-products, and impurities from reaction mixtures. Automated ion-exchange SPE has also been commonly employed to separate ionizable by-products and impurities from neutral target compounds, or to remove unwanted neutral components from ionizable target compounds, for purification of small molecule libraries (7,8). In ion-exchange SPE, two strategies are commonly employed: either the desired final products or the unwanted by-products and excess reagents are extracted onto the ion-exchange resins. Typically, cationic ion-exchange SPE is employed to remove cationic by-products and excess reagents from neutral target molecules by extracting them onto the cationic resins or to selectively extract cationic target compounds and then elute them from the resins. Conversely, anionic ion-exchange SPE selectively removes unwanted anionic components in the reaction mixture by extracting them onto the anionic resins or selectively attaches anionic target compounds onto the resins and then washes them off the resins. Because most of the starting reagents for combinatorial library synthesis are acids such as acid chlorides or bases such as amines, the resulting unwanted by-products or unreacted excess reagents are typically ionizable components, such as carboxylic acids and primary and secondary amines, which can be readily removed by either cationic (e.g., for amine removal) or anionic (e.g., for carboxylic acid removal) ion-exchange resins. Here, we describe the protocols for post-reaction purification by automated 96-well LLE and ion-exchange SPE, the two most commonly used techniques for removal of unwanted or assay-interfering components from crude reaction mixtures in combinatorial library synthesis. In these protocols, a robotic liquid handler and various types of 96-well plates are used to facilitate automation, increase sample throughput, and reduce solvent consumption. A schematic representation of each purification technique is shown in Figs. 1 and 2. 2. Materials 2.1. Chemicals for LLE
1. 2. 3. 4. 5. Butylacetate (Aldrich, Milwaukee, WI). Hydrochloric acid (Aldrich). Sodium hydroxide (Aldrich). Dimethylsulfoxide (Aldrich). Hydromatrix diatomaceous earth material (Varian, Harbor City, CA, USA).

2.2. Apparatus for LLE
1. 96-well (2-mL capacity) hydrophobic GF/C glass fiber filter plate (Whatman, Clifton, NJ).

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Fig. 1. Schematic representation of automated 96-well liquid–liquid extraction for the removal of basic and water soluble components such as amines from crude library samples.

2. 3. 4. 5. 6.

96-well (1- and 2-mL capacity) collection plate (Varian). Polypropylene reagent reservoir with 2 baffles (Tomtec, Hamden, CT). Manifold disposable waste tray (Waters, Milford, MA). Quadra 96 model 320 96-channel liquid handler (Tomtec). SPE Dry-96 96-well plate sample concentrator (Jones Chromatography, Lakewood, CO).

2.3. Chemicals for Ion-Exchange SPE
Methanol (J. T. Baker, Phillipsburg, NJ). Acetonitrile (J. T. Baker). Ammonia, 2 M in methanol (Aldrich). Sodium hydroxide (Aldrich). Trifluoroacetic acid (J. T. Baker). Dimethylsulfoxide (Aldrich). Dowex strong cation exchanger 50WX8-100 ( see Note 1 ), with sulfonic acid as the functional group on polystyrene-based resins (Supelco, Bellefonte, PA). 8. Dowex strong anion exchanger 1X8-100 (see Note 1), with trimethylbenzyl ammonium as the functional group on polystyrene-based resins (Supelco). 1. 2. 3. 4. 5. 6. 7.

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Fig. 2. Schematic representation of automated 96-well ion-exchange solid-phase extraction for the removal of anionic components such as carboxylic acids from crude library samples.

2.4. Apparatus for Ion-Exchange SPE
1. 2. 3. 4. 5. 6. Empty 96-well plate (2-mL capacity) with bottom frits (Varian). 96-well (2-mL capacity) collection plate (Varian). Polypropylene reagent reservoir with two baffles (Tomtec). Manifold disposable waste tray (Waters). Quadra 96 model 320 96-channel liquid handler (Tomtec). SPE Dry-96 96-well plate sample concentrator (Jones Chromatography, Lakewood, CO).

2.5. Chemicals for Purity Determination
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. HPLC-grade methanol (J. T. Baker). HPLC-grade acetonitrile (J. T. Baker). Formic acid (J. T. Baker). Triethylamine (Aldrich). Heptafluorobutyric acid (Aldrich). Tetrabutylammonium dihydrogenphosphate (Aldrich). Monobasic sodium phosphate (Aldrich). Dibasic sodium phosphate (Aldrich). Phenyl isothiocyanate (Aldrich) (see Note 2). Phenyl isocyanate (Aldrich) (see Note 2). p-Bromophenacyl bromide (Aldrich) (see Note 3).

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2.6. HPLC Instrumentation for Purity Determination (see Note 4).
1. 2. 3. 4. A Waters 2690 separation module (Waters). A Waters Symmetry C18 column (150 × 3.9 mm, 5 mm particle size) (Waters). A Waters 996 photodiode array (PDA) detector (Waters). An Alltech evaporative light scattering detector (ELSD) ELSD2000 (Alltech Associates, Deerfield, IL), connected to the outlet of a PDA detector.

3. Methods 3.1. LLE Plate Preparation
1. Load diatomaceous earth particles into a 96-well collection plate (1-mL capacity) fully; slowly shake the plate to remove extra particles so that the particles are evenly distributed (about 200 mg per well) in each well (see Note 5). 2. On the top of the particle-filled collection plate, place an empty 96-well hydrophobic GF/C glass fiber filter plate (2-mL capacity) upside down. 3. Firmly hold the two plates together and invert both plates quickly to unload the particles from the collection plate to the filter plate; tap the bottom of the collection plate lightly to ensure that all particles in the collection plate are transferred. 4. Remove the collection plate and cover the LLE plate (i.e., the particle-filled filter plate) with a sheet of aluminum foil. 5. Repeat steps 1–3 to prepare additional LLE plates as necessary.

3.2. LLE Extraction
1. Place an LLE plate in the vacuum manifold and a collection plate inside of the manifold. 2. Program the Quadra 96 liquid handler to execute the following sequentially (see Note 6): a. Aspirate 800 ?L of butylacetate and dispense it into the sample plate containing combinatorial samples. b. Perform six aspirate–dispense mixing cycles to dissolve the combinatorial samples in butylacetate. c. Aspirate 800 ?L of 2 N hydrochloric acid (for removal of basic components) or 2 N sodium hydroxide (for removal of acidic components) and dispense it into the LLE plate. d. Pull 800 ?L of combinatorial samples from the sample plate and place it into the LLE plate. e. Wait for 3–5 min and then apply gentle vacuum (< 3 in. Hg) to the vacuum manifold. f. Load 800 ?L of butylacetate into the LLE plate to elute the sample from the LLE plate to the collection plate. 3. Take the collection plate and place it in an SPE Dry-96 96-well plate sample concentrator; evaporate the solvent completely under a stream of heated nitrogen (45°C).

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4. Store the collection plate containing the dry sample or redissolve the sample in dimethylsulfoxide (DMSO) using the Quadra 96 for storage or for purity determination by HPLC/UV/ELSD.

3.3. Ion-Exchange SPE Plate Preparation
1. Load ion-exchange resins, either cationic or anionic ion exchangers, into an empty 96-well plate with frits (about 600 mg per well), shake the plate until the particles are evenly distributed in each well (see Note 7). 2. Cover the ion-exchange SPE plate with a sheet of aluminum foil. 3. Repeat steps 1–3 to prepare additional ion-exchange SPE plates as necessary.

3.4. Cationic Ion-Exchange SPE Extraction
1. Place an ion-exchange SPE plate filled with cationic exchange resins in the vacuum manifold and a waste tray inside of the manifold. 2. Program the Quadra 96 liquid handler to execute the following sequentially (see Note 8): a. Aspirate 800 ?L of acetonitrile and dispense it into the sample plate containing combinatorial samples. b. Perform six aspirate–dispense mixing cycles to dissolve the combinatorial samples in acetonitrile. c. Aspirate 600 ?L of water and dispense it into the ion-exchange SPE plate. d. Aspirate 600 ?L of acetonitrile and dispense it into the ion-exchange SPE plate. e. Remove the waste tray from the vacuum manifold, discard the collected eluent, and place a 96-well collection plate inside the vacuum manifold. f. Pull 800 ?L of combinatorial samples from the sample plate and place it into the ion-exchange SPE plate. g. Wait for 1–2 min and then apply gentle vacuum (< 3 in. Hg) to the vacuum manifold to elute the sample from the resins (see Note 9). 3. Take the collection plate and place it in an SPE Dry-96 96-well plate sample concentrator; evaporate the solvent completely under a stream of heated nitrogen (45°C). 4. Store the collection plate containing the dry sample or redissolve the sample in DMSO using the Quadra 96 for storage or for purity determination by HPLC/ UV/ELSD.

3.5. Anionic Ion-Exchange SPE Extraction
1. Place an ion-exchange SPE plate filled with anionic exchange resins in the vacuum manifold. 2. Program the Quadra 96 liquid handler to execute the following sequentially (see Note 10): a. Aspirate 800 ?L of acetonitrile and dispense it into the sample plate containing combinatorial samples. b. Perform six aspirate–dispense mixing cycles to dissolve the combinatorial samples in acetonitrile.

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c. Aspirate 800 ?L of water and dispense it into the ion-exchange SPE plate, drain it with low vacuum applied. d. Add 5 mL of 4 N sodium hydroxide to the ion-exchange plate and drain it with low vacuum applied (see Note 11). e. Add 4 mL of water to the ion-exchange plate and drain it with low vacuum applied. f. Aspirate 800 ?L of acetonitrile and dispense it into the ion-exchange SPE plate. g. Remove the waste tray from the vacuum manifold, discard the collected eluent, and place a 96-well collection plate inside the vacuum manifold. h. Pull 800 ?L of combinatorial sample from the sample plate and load it into the ion-exchange SPE plate. i. Wait for 1–2 min and then apply gentle vacuum (< 3 in. Hg) to the vacuum manifold to elute the sample from the resins (see Note 9). 3. Take the collection plate and place it in an SPE Dry-96 96-well plate sample concentrator; evaporate the solvent completely under a stream of heated nitrogen (45°C). 4. Store the collection plate containing the dry sample or redissolve the sample in DMSO using the Quadra 96 for storage or for purity determination by HPLC/ UV/ELSD.

3.6. Purity Determination
Purity of the samples extracted by the automated LLE or ion-exchange SPE can be determined by HPLC/UV/ELSD. The UV wavelength in a UV or PDA detector is typically set at 210 nm to observe both strong and weak UV-absorbing components. The ELSD detector is used to monitor non-volatile components that do not contain strong UV-chromophores.
1. Sample preparation: An appropriate amount of the dry sample is dissolved in DMSO to give a concentration of about 100 mg/mL. The sample can then be directly injected into the HPLC system for the determination of product purity and recovery. However, when extraction efficiency (e.g., removal of certain components such as amines and carboxylic acids) needs to be evaluated and higher detection sensitivity is required for small amounts of impurities that lack strong UV-chromophores, chemical derivatization can be employed to improve UV detection sensitivity and retention. Amines can be derivatized using phenyl isothiocyanate (PITC) or phenyl isocyanate (PIC) as follows (see Note 2): 5 ?L of PITC or PIC reagent is added to 100 ?L of the extracted sample dissolved in 1 mL acetonitrile to form UV-absorbing phenylthiocarbamoyl or phenylcarbamoyl derivatives (reaction at room temperature for 10 min). Subsequently, 10-?L aliquots of the resulting solutions are injected onto the HPLC system. For carboxylic acids, they can be derivatized using p-bromophenacyl bromide as follows (see Note 3): 10 ?L of p-bromophenacyl bromide reagent is added to 100 ?L of the extracted sample dissolved in 1 mL acetonitrile, followed by addition of 10-?L aliquots of triethylamine (as a catalyst), to form UV-absorbing ester deriv-

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2.

3. 4.

5.

atives (reaction at 50°C for 20 min). Subsequently, 10-?L aliquots of the resulting solutions are injected onto the HPLC system. HPLC conditions: A generic 15-min linear gradient method is used where 20 ?L of sample is injected into a Waters Symmetry C18 column (150 × 3.9 mm ID, 5 mm) and eluted from mobile phase A (acetonitrile:water:formic acid; 5:95:0.1; v/v/v) to mobile phase B (acetonitrile:water:formic acid; 95:5:0.1; v/v/v) with a flow rate of 1.0 mL/min (see Note 12). A PDA detector is connected with an ELSD detector in series to monitor both UV and light scattering signals. PDA UV detector settings: wavelength 200–400 nm with the monitoring wavelength at 210 nm and resolution at 1.2 nm. ELSD detector settings: in the “Impactor On” mode—drift tube temperature at 40°C and a nitrogen flow rate at 1.5 L/min; in the “Impactor Off” mode—drift tube temperature at 95°C and a nitrogen flow rate at 2.2 L/min. HPLC assay: The purity of the final combinatorial products is determined by the aforementioned gradient HPLC method. The library samples before and after extraction are injected onto the HPLC system. The product and impurity peak areas are then compared to assess the product purity and recovery. The product purity is determined by the percent peak area of the product of interest in the post-extraction sample solution. The product recovery and the removal of the excess reagents, by-products, or impurities are evaluated by the ratio of the peak areas of the respective component before and after extraction.

4. Notes
1. Any other equivalent (cation or anion) ion-exchange resins from different manufacturers may be used. Other types of products may be better in some situations. 2. Both phenyl isocyanate (PIC) and phenyl isothiocyanate (PITC) are used as derivatization reagents for aliphatic amines that lack strong UV-chromaphores to improve detectability and retention on a reversed-phase HPLC column. PIC is for both primary and secondary amines, while PITC is mainly for primary amines (14). They are used to determine extraction efficiencies of individual amines. 3. It is used as a derivatization reagent for aliphatic carboxylic acids that lack strong UV-chromaphores to improve detectability and retention on a reversed-phase HPLC column (14). It is used to determine extraction efficiencies of individual carboxylic acids. 4. Any equivalent analytical HPLC system can be used. If no ELSD detector is available, a UV detector may be sufficient enough for purity determination. An ELSD detector is preferred over a UV detector because it provides more uniform responses independent of the optical properties of compounds. 5. A 1-mL 96-well collection plate is used only as a template to facilitate the packing of an LLE plate so that an equal amount of particles can be easily transferred to each well of a 96-well filter plate. 6. The Tomtec Quadra 96 liquid handler can be replaced with another robotic liquid handler with 96-, 12-, or 8-channels. Each aspiration step by the Quadra 96 is generally proceeded by 50 ?L of air gap to aid in complete and accurate dispensing.

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7. When packing dry ion-exchange resins, use a 1-mL 96-well collection plate as a template and load the resins into the collection plate; then place the filter plate on the top of the collection plate upside down and invert both plates together to transfer the resins from the collection plate to the filter plate. 8. Each aspiration step by the Quadra 96 is generally proceeded by 50 ?L of air gap to aid in complete and accurate dispensing. This extraction procedure is used to remove excess amines or other basic by-products and impurities in the final combinatorial products. It takes advantage of the difference in basicity between the impurities such as amines (pKa 9.5–10.5 for most of their conjugate acids or amonium ions) and the desired product (pKa < 0 for most of their conjugate acids). Therefore, amines are protonated cations at a neutral pH and retained by the cation exchange SPE resins, while the desired product is not. If basic ionizable final products (e.g., amines) are to be purified and nonionizable impurities are to be removed, then an additional final elution step with a basic solvent (e.g., 1 mL of 2 M ammonia in methanol) is needed to obtain the desired final product that is retained on the resins after step g. 9. To further improve product recovery, an additional 200 ?L of acetonitrile may be added into the ion-exchange SPE plate to elute the residual product remained in the SPE plate. 10. Each aspiration step by the Quadra 96 is generally proceeded by 50 ?L of air gap to aid in complete and accurate dispensing. This extraction procedure is used to remove carboxylic acids or other acidic by-products and impurities in the final combinatorial products. It takes advantage of the difference in acidity between the impurities, such as acids (pKa < 5, deprotonated anions at neutral pH) and the desired product (unionized form at neutral pH). Therefore, acids are retained by the anion exchange SPE resins, while the desired product is not. If acidic ionizable final products (e.g., acids) are to be purified and nonionizable impurities are to be removed, then an additional final elution step with an elution solvent (e.g., 1 mL of 1–2 M trifluoroacetic acid in methanol) is needed to obtain the desired final product that is retained on the resins after step g. 11. A 4 N sodium hydroxide solution is used to change the counter-ion on the resin from chloride (higher affinity anion) to hydroxide (lower affinity anion) for better retention of carboxylic acids. 12. For the library samples that contain very polar by-products, excess reagents, or impurities, an ion-pair reagent may be added to the mobile phase to improve retention of the polar components on the reversed-phase HPLC column. For small polar basic components such as amines, 0.1–1% of heptafluorobutyric acid can be added into the mobile phase containing acetonitrile or methanol in water. For small polar acidic components such as carboxylic acids, 5–20 mM of tetrabutylammonium dihydrogenphosphate can be added into the mobile phase containing acetonitrile or methanol in 50 mM phosphate buffer (pH 7.4).

References
1. Dolle, R. E. (2000) Comprehensive survey of combinatorial library synthesis: 1999. J. Comb. Chem. 2, 383–433.

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2. Czarnik, A. W. (1998) Combinatorial chemistry. Anal. Chem. 70, 378A-386A. 3. Peng, S. X., Henson, C., Strojnowski, M. J., Golebiowski, A., and Klopfenstein, S. R. (2000) Automated high-throughput liquid-liquid extraction for initial purification of combinatorial libraries. Anal. Chem. 72, 261–266. 4. Johnson, C. R., Zhang, B., Fantauzzi, P., Hocker, M., and Yager, K. M. (1998) Libraries of N-alkylaminoheterocycles from nucleophilic aromatic substitution with purification by solid supported liquid extraction. Tetrahedron 54, 4097–4106. 5. Cheng, S., Comer, D. D., Williams, J. P., Myers, P. L., and Boger, D. L. (1996) Novel solution phase strategy for the synthesis of chemical libraries containing small organic molecules. J. Amer. Chem. Soc. 118, 2567–2573. 6. Nilsson, U. J. (2000) Solid-phase extraction for combinatorial libraries. J. Chromatogr. A 885, 305–319. 7. Gayo, L. M. and Suto, M. J. (1997) Ion-exchange resins for solution phase parallel synthesis of chemical libraries. Tetrahedron Lett. 38, 513–516. 8. Siegel, M. G., Hahn, P. J., Dressman, B. A., Fritz, J. E., Grunwell, J. R., and Kaldor, S. W. (1997) Rapid purification of small molecule libraries by ion exchange chromatography. Tetrahedron Lett. 38, 3357–3360. 9. Coates, S. W., Kirkland, J. J., Langlois, T., Majors, R. E., Szafranski, C. A., Thompson, L. A., and Wang, Q. (2000) New solid-phase scavengers improve recovery and speed throughput in parallel and related synthesis. LC-GC 18, S30–S34. 10. Booth, R. J. and Hodges, J. C. (1997) Polymer-supported quenching reagents for parallel purification. J. Am. Chem. Soc. 119, 4882–4886. 11. Kaldor, S. W., Siegel, M. G., Fritz, J. E., Dressman, B. A., and Hahn, P. J. (1996) Use of solid supported nucleophiles and electrophiles for the purification of nonpeptide small molecule libraries. Tetrahedron Lett. 37, 7193–7196. 12. Parlow, J. J., Devraj, R. V., and South, M. S. (1999) Solution-phase chemical library synthesis using polymer-assisted purification techniques. Curr. Opin. Chem. Biol. 3, 320–336. 13. Zeng, L. and Kassel, D. B. (1998) Development of a fully automated parallel HPLC/mass spectrometry system for the analytical characterization and preparative purification of combinatorial libraries. Anal. Chem. 70, 4380–4388. 14. Li, F. and Lim, C. K. (1993) Colored and UV-absorbing derivatives, in Handbook of Derivatives for Chromatography, second edition (Blau, K. and Halket, J., eds.), John Wiley & Sons, pp. 157–174.


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