Chromatography baseline determination

Figure 3.10 exemplifies the application of packed-column SFC for the rapid determination of the enantiomeric purity of pharmaceutical materials. The method employed a CHIRALCEL OD CSP with a carbon dioxide/methanol/ isopropylamine mobile phase under isocratic conditions. A flow rate of 4 mFmin was used to generate a high linear velocity whilst maintaining an outlet pressure of 200 bar by use of a back-pressure regulator to ensure reproducible chromatography. Baseline resolution of the enantiomers of propanolol was achieved in approximately 3 min with an overall cycle time of 4 min. Detection was by UV... 

The right chromatography column should separate the sample sufficiently to enable identification or quantitative measurement of the components within a reasonable period of time. The resolution factor (Rs) for two sample components is determined by the width of the two peaks and the distance between the peak maxima. In general, Rs values of 1.0 are required for good qualitative or quantitative work, whereas Rs values >1.5 indicate baseline resolution for two components (3). 

Gas chromatography was used to determine n-paraffin distribution in the oil and wax samples. An F and M Instrument Company Model 500 chromatograph was used with an uncompensated single column, a helium carrier gas flow rate of 25 ml/min., and a thermal conductivity detector. The column was 4.8 mm in diameter and 3.3 m in length, and was packed with 3% Dexil 300 on Chromo-sorb P. The block and injection port temperatures were maintained at 673 K. The column was temperature-programmed from 348 K to 673 K at a rate of 5.7 K per minute. Peak identification was aided by the use of internal standards of decane, dodecane, and hexadecane. The baseline was determined by heating without sample injection. Response values were not available for the various areas on the traces, so the analyses were reported as % by area. 

Preparative enrichment of enantiomers should be followed by determination of their chemical and enantiomeric purities, e.g., by enantioselective liquid chromatography.25,26 In addition to the sorbents mentioned above, others may be used which are available in only smaller amounts. In our laboratory, this is true for (+)-poly(trityl methacrylate) on silica,35 which can be used for analytical purposes. Preparative separations on a column 0.5 cm in diameter, however, would require many injections of small amounts of racemate. In the case of a baseline chromatogram like that in Figure 2, the determination of enantiomeric purity by measurement of the two peak areas of the enantiomers is straightforward. An analytical chromatogram showing some separation by photometric detection in spite of peak overlap (Figure 4) can still be used for optical purity determina-tion. The simultaneous use of both photometric and chiroptical detection as... 

Is the method specific and stability indicating as shown by analysis of samples subjected to stressed stability studies (pH, light, heat, oxidation) Normally, specificity is determined through peak purity using ultraviolet (UV) diode array or liquid chromatography (LC)/mass spectrometry (MS) analysis. In methods for analysis of drug products, placebo formulations (and stressed placebos) must yield blank chromatographic baselines. The development... 

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