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Analitical techniques in combinatorial chemistry - Swarth M.E.

Swarth M.E. Analitical techniques in combinatorial chemistry - Marcel Dekker, 2000. - 311 p.
ISBN 0-8247-1939-5
Download (direct link): analyticaltechniquesincombinatorialchemistry2000.pdf
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Figure 10 Injection pooling schematic.
chemiluminescent nitrogen detection (CLND) and its cousin, chemilumines-cent sulfur detection (CLSD). Used alone or in combination with UV and MS, use of these auxiliary techniques allows a more accurate and complete assessment of purity to be obtained.
The principles of ELSD date back a number of years (13-15). More recently, ELSD has been applied specifically to pharmaceutical analyses and combinatorial uses (2,16-18). Detection by light scattering is based on the available mass and not absorptivity (UV) or ionization efficiency (MS), making it more accurate in some applications. In ELSD, nebulized column effluent enters a heated drift tube where rapid evaporation of the LC mobile phase takes place. A stream of nitrogen gas sweeps any nonvolatile solutes toward a detection region. Detection is accomplished by a laser and photodiode at an angle of 90°, perpendicular to the central axis of the drift tube. As the solute particles pass through the laser beam, the source is scattered. The intensity of the scattered light measured by the photodiode is proportional to the amount of solute in the column effluent.
Liquid Chromatography
Although most compounds respond well to ELSD, the volatility of some low molecular weight compounds may cause problems during the evaporation process. Varying response in different stages of the gradient can also cause problems. Use of ELSD in combination with UV detection minimizes these potential limitations.
CLND and, more recently, CLSD play a role similar to that of ELSD in quantitative assays of true unknowns. The CLND and CLSD detectors respond to the nitrogen and sulfur content of a compound, respectively. Both detectors operate under relatively the same principles and have been used with considerable success in drug discovery (19-21). In CLND, the analyte is oxidized at 1050°C, converting nitrogen-containing compounds to nitric oxide. The nitric oxide reacts with ozone to produce nitrogen dioxide in the excited state, which releases a photon when decaying to the ground state. The photons are measured by a photomultiplier tube and converted to an analog signal dependent only on the total mass injected.
Since a vast majority of drug compounds contain nitrogen, CLND is very useful for pharmaceutical analyses. The CLND response has been shown to be independent of gradient composition and, again, is often used in combination with other (UV and MS) detection systems (18). However, the presence of nitrogen-containing impurities in the sample or solvent will bias results. Mobile phases must of course be nitrogen-free, dictating the use of methanol or another alcohol rather than acetonitrile as mobile phase modifier.
A. Open Access Instrument Operation
Traditionally, MS analyses have been performed in a centralized facility, often on highly specialized instruments that required constant operator intervention and maintenance. This situation is highly impractical when supporting a combinatorial program because it inhibits high throughput and general access to instrumentation and data. In response, instruments are now often operated in an ‘‘open access’’ environment. In such an environment, people not trained in LC or MS can submit samples on a continuous basis and get rapid turnaround. The use of MS and its wealth of information is promoted, and spec-trometrists are freed from the mundane, tedious task of repetitive sample analysis of perhaps thousands of samples.
An open access setup usually consists of a workstation at the point of need. The walkup user has access to the workstation as well as to the sample
Figure 11 Interplate pooling with internal standards. Separation conditions are identical to those reported in Figure 2B. Peaks A and B are acetylfuran and valerophenone. The internal standards for peaks A and B are acetanilide and benzophenone, respectively. Data system run time was extended to 15 minutes to capture LC instrument timing. Cycle to cycle injection time is 4.7 minutes including interplate pooling.
manager or autosampler used for injection. A system administrator is responsible for setting up the system and has access to and control of all components. The walkup user, perhaps a medicinal or synthetic organic chemist, logs in sample(s) (or an entire plate) at the workstation, places them into the directed location, and, depending on the queue and system setup, gets a postanalysis report at the point of use or e-mailed to his desk. Results are typically displayed in an integrated browser format, similar to that illustrated in Fig. 13. The browser provides a graphical display for review of the results, a confirmation of molecular weight, and displays corresponding chromatograms and spectra.
B. Mass-Directed Autopurification
As mentioned previously, during lead optimization and testing leading to candidate drug selection, compounds are often required in larger quantities. Analytical techniques used in lead discovery eventually give way to semipreparative or preparative mass-directed autopurification techniques, capable of isolating and purifying 10-20 mg or more of the compound of interest during a single chromatographic analysis. Short, wide-diameter columns operated at
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