Method parameters typical variations

Parameters that should be tested in HPLC method development are flow rate, column temperature, batch and supplier of the column, injection volume, mobile phase composition and buffer pH, and detection wavelength [2], For GC/GLC methods, one should investigate the effects of column temperature, mobile phase flow rate, and column lots or suppliers [38], For capillary electrophoresis, changes in temperature, buffer pH, ionic strength, buffer concentrations, detector wavelength, rinse times, and capillaries lots and supplier should be studied [35, 36], Typical variation such as extraction time, and stability of the analytical solution should be also evaluated [37],... 

The evaluation of robustness should be considered in the development of the assay and will depend on the type of procedure under development. It must show the reliability of a method with respect to deliberate variations in method parameters. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement might be included in the procedure. One consequence of the evaluation of robustness may be that a series of system suitability parameters is established to ensure that the validity of the analytical procedure is maintained whenever used. Typical parameters to be tested would be the following sample concentration, sample stability, labeling variability (if applicable), injection variability, reagent lot-to-lot variability, and capillary vendor. 

Robustness is the capacity to remain unaffected by small, but deliberate, variations in method parameters. In a typical HPLC validation this exercise would include minor variations in flow rate or pH of the buffer. When considering cleaning verification, this parameter may be extended to extraction time of the swabs, pH of extraction solvent, etc. However, it is fairly uncommon to evaluate such parameters due to the above-mentioned variability that may be found in swab methods. 

Relatively small fractionating volumes are associated with HDC systems. Therefore, the Rf market method (27) typically is used to compensate for possible variations in the operating parameters during the separation. Figure 13 shows a HDC calibration plot that was obtained by the marker method. The arbitrary log sol diameter versus Rf plot produced a linear relationship for this series of 40-600-nm SdFFF-characterized sols as standards. With this particular HDC system, silica sols can be routinely measured with precisions of about 15% (relative) with the peak-position calibration method. This precision level is a direct result of the relatively poor resolution of HDC separations. 

Kinetic methods call for no measurements of absolute values of the parameter typically used to monitor reactions (absorbance, fluorescence intensity, potential), but rather for their temporal variations as a result, kinetic measurements are free from the effects of factors that introduce errors in absolute values (e.g., turbidity, the liquid-liquid junction potential, and the presence of other absorbing or fluorescent substances provided they do not take part in the reaction of interest or alter the parameter response). However, strict timing and temperature control (to within 0.01-0.1°C) are essential to kinetic methods, which thus require modern, powerful instrumentation. 

The above material focuses on the conventional electrochemical methods, which typically means studies employing conventional-sized electrodes, DC waveforms, and solution phase redox chemistry in organic solvents containing electrolyte to provide adequate conductivity. With modern forms of electrochemistry, each of these parameters may be altered to facilitate studies in non-conventional media with respect to usual conditions employed in studies of coordination compounds. Examples of variations of methodology that broaden the scope of redox studies of coordination compounds include ... 

In the low conversion regime, considerable effort has been made to study the dependence of the free-radical termination rate coefficient (or the total polymerization rate) on the solution viscosity [e.g. 40, 61-85]. To study this effect, two basic approaches have been carried out. The viscosity can either be varied using different solvents with inherently different viscosities or, alternatively, various amounts of polymer can be dissolved as viscosity modifier. Although the effects of both methods upon the variation of kt can turn out to be quite different, the former method (variation of the type of solvent) is hard to separate from the latter one (variation of the amount of dissolved polymer) in experimental studies, as some conversion must always be tolerated to obtain kinetic parameters. The number of studies on this topic is rather large and therefore only a limited number with typical examples will be discussed. 

It can be used under very high pressure and temperatures. Oil reservoirs are found typically at 100°C and 300 atm pressure. The surface tension of such systems can be conveniently studied by using high pressure and temperature cells with optical clear windows (sapphire windows 1 cm thick up to 2000 atm). For example, yof inorganic salts at high temperatures (ca. 1000°C) can be measured using this method. The variation in surface tension can be studied as a function of various parameters (temperature and pressure additives [gas, etc.]). 

Robustness. Examples of typical possible sources of variation in automated methods are homogenization speed, homogenization time, age of sample, accuracy of solvent dispense, and temperature variation. If all studies described in the method development have been performed, the robustness of the sample preparation has been demonstrated and does not require additional testing. Parameters in relation to the measurement technique may need to be considered and are covered in the relevant chapter. 

The tubers of Jerusalem artichoke typically comprise about 80% water, 15% carbohydrate, and 1 to 2% protein. Data on the composition of Jerusalem artichoke are relatively sparse in comparison to other vegetables, however, and significant variation has been recorded for certain parameters. Differences in cultivar, time of harvest, production conditions, postharvest treatment, and preparation methods most likely account for this variation (Table 5.1). 

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