External, preparative chromatography

Statistical process control methods are applied to preparative chromatography for the case where cut points for the effluent fractions are determined by on-line species-specific detection (e.g., analytical chromatography). A simple, practical method is developed to maximize the yield of a desired component while maintaining a required level of product purity in the presence of measurement error and external disturbances. Relations are developed for determining tuning parameters such as the regulatory system gain. 

We foimd that the ratiometric method is superior because it is not dependent on pyranine concentration and therefore free of error in pipeting (18,22,54). Calibration curves were constructed by preparing liposomes in which the hydration of the lipids to form MLV was done using solutions of high concentration at the desired pH in the range of 3.0 to 10.0. Gel-exclusion chromatography on a Sephadex column, as mentioned above, yielded a series of liposome preparations with a fixed external pH (pH 7.5), but different internal pH values determined by the buffer used for lipid hydration. Neither KI nor DPX, which quench the fluorescence of aqueous solutions of pyranine, has much effect on the fluorescence intensity of pyranine in the void volume after gel-exclusion chromatography, which indicates the complete removal of the pyranine from the extraliposomal medium. 

To a solution of 20.2 g diisopropylamine in 200 mL anhydrous THF that had been cooled to -10 °C under a He atmosphere with an external ice/MeOH bath, there was added 125 mL of a 1.6 M solution of butyllithium in hexane. There was then added, in sequence, 5.1 mL of dry CH,CN followed by the dropwise addition of 15.3 g 4-bromo-1,3-diethoxy-2-ethylthiobenzene diluted with a little anhydrous THF. There was only a modest color development. Analysis by thin-layer chromatography showed that the reaction components were largely starting bromide and only alittle product nitrile. An additional 2.5 mL dry CH3CN was added, followed immediately by a solution of lithium diisopropylainide prepared separately from 14... 

Calibrations were carried out for the GC/PID or the GC/MS daily. Calibration standards were prepared based on standard reference materials obtained from Supelco Chromatography products. A check standard was analyzed every ten samples to assure calibration and accuracy. A reagent blank was included in each analytic batch of samples. Blanks were made from reagent or make-up water and matrix similar to the sample. A spiked sample was analyzed every twenty samples. This was done by splitting an appropriate sample into two subsamples and adding a known quantity of TCE to one of the split samples. The purpose of a spiked sample is to determine the extent of matrix bias or interference on TCE recovery and sample to sample precision. Accuracy was assessed by analysis of external reference standards (separate from calibration standards) and by percent recoveries of spiked samples. Precision was assessed by means of replicate sample analysis. It is expressed as relative percent difference (RPD) in the case of duplicates or relative standard deviation (RSD) for triplicate (or more) analyses. Recovery was 96% or more for all spiked samples, and RPD/RSD are less than 7% for all samples. 

K. Nakajima and T. Yasuda, Simultaneous determination of 1-menthol, dl-camphor and methyl salicylate in pharmaceutical preparations for external application using headspace gas chromatography, Chiba-Ken Eisei Kenkyusho Kenkyu Haboku, 5 14-18 (1984). 

John R. C., Kimberly C. J. and Ranmali W., External recycle Chromato- graphy A practical method for preparative purifications. Journal of Chromatography A, 462 (1989), 85-94. 

GC has system requirements similar to those of HPLC. The sample is volatilized and an inert gas is used as the mobile phase to carry the sample through the chromatography column. External standard solutions of the solvent in question are prepared and will be compared to sample solutions. The amount of residual solvent can then be calculated using the peak responses. 

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