Preparative chromatography sample valves

The dimensions of the exit tube from the detector are not critical for analytical separations but they can be for preparative chromatography if fractions are to be collected for subsequent tests or examination. The dispersion that occurs in the detector exit tube is more difficult to measure. Another sample valve can be connected to the detector exit and the mobile phase passed backwards through the detecting system. The same experiment is performed, the same measurements made and the same calculations carried out. The dispersion that occurs in the exit tube is normally considerably greater than that between the column and the detector. However, providing the dispersion is known, the preparative separation can be adjusted to accommodate the exit tube dispersion and allow an accurate collection of each solute band. 

Procedure - Equilibrate the Standard Preparation and the Assay Preparation in the dark at 80° for 2.5 hours, accurately timed. Cool, and introduce equal volumes (5 to 10 1) of the heat-equilibrated Standard Preparation and Assay Preparation into the high-pressure liquid chromatograph (See chromatography <621>) by means of a suitable sampling valve. Measure the peak responses obtained for the Assay Preparation and the Standard Preparation, and calculate the quantity, in mg, of C27H44O in the portion of Cholecalciferol taken by the formula 0.25C(Ay/Ae)> in which C is the concentration, in g per ml, of USP Cholecalciferol RS in the Standard Preparation, and Ay and As are the peak responses for cholecalciferol obtained for the Assay Preparation and the Standard Preparation, respectively. 

Injection Process transferring the desired amount of feed or sample into the mobile phase stream, just upstream of the column inlet. In analytical chromatography, it is carried out using syringes. In preparative chromatography, it is done by valve switching or by replacing the mobile phase by the feed at the pump. 

Multidimensional liquid chromatography encompasses a variety of techniques used for seunple separation, cleanup and trace enrichment [12,279-289]. A characteristic feature of these methods is the use of two or more columns for the separation with either manual or automatic switching by a valve interface of fractions between columns. These techniques require only minor modification to existing equipment, and of equal importance, enable the sample preparation and separation procedures to be completely automated. 

Although SFE and SFC share several common features, including the use of a supercritical fluid as the solvent and similar instrumentation, their goals are quite distinct. While SFE is used mainly for the sample preparation step (extraction), SFC is employed to isolate (chromatography) individual compounds present in complex samples (11 -15). Both techniques can be used in two different approaches off-line, in which the analytes and the solvent are either vented after analysis (SFC) or collected (SFE), or on-line coupled with a second technique, thus providing a multidimensional approach. Off-line methods are slow and susceptible to solute losses and contamination the on-line coupled system makes possible a decrease in the detection limits, with an improvement in quantification, while the use of valves for automation results in faster and more reproducible analyses (16). The off-line... 

The calibration method most often used in ion chromatography is direct comparison of the peak area in an unknown sample with that of a solution with a known content of the same substance. This method requires the injection of constant volumes under constant chromatographic conditions. Errors in the sample delivery, however, are almost excluded upon application of a sample loop valve. A prerequisite is the existence of reference compounds for all sample components to be analyzed. In practice, several different standard solutions in the investigated concentration range are prepared and chromatographed [8], When the resulting peak area is plotted versus the concentration of the standards, one obtains a substance-specific calibration function. 

The first of the separation techniques to be used in process measurement was gas chromatography (GC) in 1954. The GC has always been a robust instrument and this aided its transfer to the process environment. The differences between laboratory GC and process GC instruments are important. With process GC, the sample is transferred directly from the process stream to the instrument. Instead of an inlet septum, process GC has a valve, which is critical for repetitively and reproducibly transferring a precise volume of sample into the volatiliser and thence into the carrier gas. This valve is also used to intermittently introduce a reference sample for calibration purposes. Instead of one column and a temperature ramp, the set up involves many columns under isothermal conditions. The more usual column types are open tubular, as these are efficient and analysis is more rapid than with packed columns. A pre-column is often used to trap unwanted contaminants, e.g. water, and it is backflushed while the rest of the sample is sent on to the analysis column. The universal detector - thermal conductivity detector (TCD)-is most often used in process GC but also popular are the FID, PID, ECD, FPD and of course MS. Process GC is used extensively in the petroleum industry, in environmental analysis of air and water samples" and in the chemical industry with the incorporation of sample extraction or preparation on-line. It is also applied for on-line monitoring of volatile products during fermentation processes" ... 

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