Analytical HPLC temperature column operation

Since the separation process in CEC has a number of attributes similar to those of HPLC, the most important variables affecting the separation are the same for both of these techniques. However, in HPLC mobile phase, flow and separation are independent variables. Therefore, the most important operational variables are the analyte-sorbent interactions that can be modulated by the chemistry of the packing, composition of the mobile phase, and temperature. In contrast, the CEC column has a dual role as it serves as both (i) a flow driving device and (ii) separation unit at the same time. Although the set of variables typical of HPLC is also effective in CEC, their changes may affect in one way or another both column functions. Therefore, optimization of the separation process in CEC is more complex than in HPLC. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Qualification Approaches If an analytical instrument is comprised of different functionally discrete modules, a modular approach to qualification testing that focuses on the specific operations of the individual module can be suitable for certain aspects of some operational testing (such as the flow rate precision and accuracy testing of a HPLC pump and the temperature accuracy column compartment). 

The separation and purification of ethyl esters of EPA, DHA, and the heretofore-minor unreported polyunsaturate octadecatetraenoic acid (C18 4 3, OTA) on a preparative scale by modification of an analytical RP-HPLC procedure has been described by Beebe et al. (48). They used a liquid chromatograph equipped with a differential refractometer as detector operated at room temperature and an ST Macrobore column (350 X 4.6-mm ID) of C18 reverse-phase material, 25-yum particle size. 

Figure 7.16A depicts a flexible SFE-HPLC coupled assembly developed by Ischi and Haerdi [106] that consists of three main parts [viz. the SFE system (Al), the interface (A2) and the HPLC system (A3)] each furnished with appropriate valves operating as shown in Fig. 7.16B. Thus, valve 5 in Fig. 7.16A is used to provide extraction with or without a modifier, via a tee connector on the other hand, valve 10 allows switching between static and dynamic extraction. The former is done by having the valve close the outlet of the extraction cell after the desired temperature is reached. By switching the valve back, the dynamic state is restored. Valve 13 enables trapping of the extracted analytes, either on a C, silica column placed in an oven for on-line preconcentration and insertion of non-polar or low-polar analytes into the chromatograph after elution or into a liquid phase to implement an off-line operation. When polar ionic analytes are to be preconcentrated, the eluent from the extractor is diverted to valve 18 and retained on the ion-exchange material packed in the column. Preconcentration of both non-polar, low-polar and polar ionic analytes can be accomplished by using both valves (13 and 18) [106],...

Reproducibility, as defined by ICH, represents the precision obtained between laboratories with the objective of verifying if the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified, parameters of the method (interlaboratory tests). Various parameters affect reproducibility. These include differences in room environment (temperature and humidity), operators with different experience, equipment with different characteristics (e.g., delay volume of an HPLC system), variations in material and instrument conditions (e.g., in HPLC), mobile phases composition, pH, flow rate of mobile phase, columns from different suppliers or different batches, solvents, reagents, and other material with different quality. 

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