high-throughput puri cation: triage and optimization in .NET

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high-throughput puri cation: triage and optimization
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Agilent (Wilmington, DE) 1100 Series HPLC and PDA detector, and a Sedere (Alfortville, France) Sedex 75 detector. The columns used on the analytical instruments are Phenomenex LunaR C8 columns in a 5-m particle size and 2.1 50 mm format. Standard gradient elution systems used are 10 100% acetonitrile : 0.1% aqueous TFA, at 1.5 mL per minute ow rate with positive ion APCI mode detection or 10 100% acetonitrile : 10-mM ammonium acetate, at 1.5 mL per minute ow rate with negative ion APCI. These systems are mostly employed by medicinal chemists. Reports generated on this system are sent directly to the desktop of the submitting chemist via a custom program integrated to the company s e-mail system. Data are archived to the central server and are available to the synthetic chemist for further processing or future retrieval. Analytical LC/MS/UV/ELSD: Microtiter Format A microtiter injection format analytical HPLC/MS/UV/ELSD is available to the high-throughput organic synthesis (HTOS) group at Abbott so as to be compatible with their automated library syntheses format. This system is comprised of a Waters (Milford, MA) ZMD/Alliance LC/MS with APCI probe operated in the positive ion mode and a Sedere (Alfortville, France) Sedex 75 detector. Columns used on the analytical instruments are Phenomenex LunaR C8 columns in a 5-m particle size and 2.1 50 mm format. A standard gradient of 10 100% acetonitrile : 0.1% aqueous TFA, at 1.5 mL per minute ow rate is used exclusively on this instrument. A software package Masslynx Diversity BrowserR, commercially available from Waters allows the HTOS chemist a global view of the success of library prior to submission to puri cation. 11.2.3. Puri cation Work ow
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Samples are submitted for puri cation on a continual basis to the HTP service by a large number of chemists. These samples are generated in diverse formats, varying in weight of individual sample, library set size, yield, polarity and functionality, and in chromatographic behavior. For each sample or set submitted, however, the same general work ow is followed. This work ow begins with an Intranet-based sample log-in system, available to client chemists from their desktop. Data entered into the sample log-in system begins the automated process of sample tracking and provides information used in the selection of a puri cation method, such as sample weight and structure. Data are imported in different formats into the various puri cation systems available within HTP, but the system is designed such that output format and sample labeling are transparent to
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puri cation systems and analytical support
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the submitting chemists. As described in the previous instrumentation section, samples are puri ed either by SFC or HPLC and by UV-, ELSD-, or MW-triggered fraction collection. In each case mass spectral validation data are returned to the synthetic chemist generated during either puri cation, as in the case of MW-triggered HPLC, or postpuri cation by loop injection MS, in the case of UV- or ELSD-triggered collection. After the acquisition of mass spectra, samples to be returned to the chemist are selected within interactive sample processing software packages, as described in the previous section. Labels are generated upon peak selection, and the appropriate fractions pulled, labeled, and dried-down on a series of centrifugal evaporation systems. These dried and labeled samples are returned to the submitting chemist along with a puri cation report and MS data, and an e-mail is sent to the chemist informing them that samples are ready for pickup. The process is accomplished within 72 hours of sample receipt. In the event that the submitting chemist subsequently requests the return of additional samples from the crude reaction mixture after evaluation of the puri cation report, all fractions collected from each run are retained for approximately one week. 11.2.4. Puri cation Triage
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Of the systems just described, each may be more amenable to the puri cation of speci c sample sets based on considerations such as sample sets size, sample weights, and structural characteristics. Additionally consideration is given to the availability of each instrument at the time at which samples are submitted and how the puri cation can be more ef ciently and rapidly returned to the submitting chemist. UV-triggered HPLC represents the highest puri cation capacity in HTP with four instruments available. This technique is therefore employed as the rst of choice for all routine samples. These instruments are always used when chromatographic and spectroscopically amenable for the many small sample sets submitted in a given day. Further, when more than one component is requested for return, or particularly when the MW of some requested components are unknown, UV-triggered HPLC is employed. All samples in weight above 70 mg are puri ed on these systems as they are the only instruments presently set up in an automated column switchable format that accommodates larger sized columns. The only requisite for use of the systems is that a good UV chromophore, else a good ELSD response, be observable based on the analytical LC/MS data. In order to most ef ciently use the instruments available, several small sample sets from different submissions may be combined into a single puri cation set if the optimal gradient method and trigger wavelength are compatible.
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