Plate number maximizing

The theoretical plate number for the separation of these model proteins has a maximal value at pH 7. However, the peak capacity increases with pH because... 

Obviously, the aim is for a chromatographic separation in which peak width is narrow relative to the time of elution (wi/Vr is minimized), i.e., the number of theoretical plates is maximized. There are three main factors that give rise to band broadening (1) multiple path effect (eddy diffusion), (2) axial (longitudinal) diffusion, and (3) mass transfer—slow transfer/equilibration between mobile and stationary zones. 

If you do not do preparative work, the goal should be not the maximal resolution, but the optimal or sufficient resolution. If you have a selective chromatographic system, you only need a small theoretical plate number for your separation. You can even afford to give away plates by increasing the temperature if you gain on time (see Tip No. 16). 

Since plate height is inversely proportional to plate number, a small value indicates a narrow peak—the desirable condition. Thus, each of the three constants. A, B, and C should be minimized in order to maximize column efficiency. 

Fig. 25 Simulation results for the Baker-Williams column for different values of the maximal plate number in the column using (15) (a) and (16) (b) for the feed distribution of the copolymer. The numbers are the considered volume element...

The plate height, and thus the total number of theoretical or effective plates, depends on the average linear carrier gas velocity (van Deemter relationship) and, for a particular carrier gas, the efficiency will maximize at a particular flow rate. Only at the optimum carrier gas flow rate are n, N, and HETP Independent of the column length. The efficiency will also depend on the column diameter (see section 1.7.1) where typical values for n, N, and HETP for different column types can also be found. Values for n, N, and HETP are reasonably independent of temperature but may vary with the substance used for their determination, particularly if the test substance and statioKary phase are not compatible. 

Several statistical approaches have been applied to the results of plate incorporation assays (Wahrendorf et al., 1985 and Mahon et al., 1989). These authors make a number of important suggestions to maximize the power of statistical analyses those that relate to the method of analysis are reproduced below. 

Incubate at least four series, cells with three or more different concentrations of the preparation to be examined and the reference preparation in a microtitre plate and include in each series appropriate controls of untreated cells. Choose the concentrations of the preparations such that the lowest concentration produces some protection and the largest concentration produces less than maximal protection against the viral cytopathic effect. At a suitable time add the cytopathic virus to the wells with the exception of a sulScient number of wells in ah series, which are left with uninfected control cells. Determine the cytopathic effect of virus quantitatively with a suitable method. Calculate the potency of the preparation to be examined by the usual statistical methods for a parallel line assay. 

System suitability parameters [6,17] are defined as an interval in which a response (e.g. retention time, resolution, number of theoretical plates) are allowed to vary for a robust method. They can be derived from the minimal and maximal result for the considered response as seen with a design in which the quantitative results of the method were found to be rugged. 

E. Extracolumn Band Broadening or Variance To maximize the effective number of theoretical plates, the contribution of the entire chromatographic system to band broadening (system variance, o-2ys) must be minimized. The system variance may be broken down into contributions from the column variance, a 01, as described above, and extracolumn diffusion and mixing processes, cr2x. As with the case of the column variance, extracolumn variance is an additive property and may be broken down into the major components ... 

The wet chemical methods are summarized in Table IV. Basically this approach centers on the water-soluble extract obtained from Alter substrates, impactor plates, or other collection surfaces. The extraction process has to be done with some care to ensure that all of the water-soluble material is removed. Standard extraction methods now involve the use of ultrasonic devices to maximize extraction efficiency. Once the extract is obtained, it can be subjected to a number of the methods listed in Table IV, such that a detailed elemental breakdown by inorganic and (water-soluble) organic carbon is accomplished. 

Maximization of the minimum resolution value observed in the chromatogram (eqn.4.25) corresponds to aiming at the minimum number of plates required to effectuate the separation. 

In some cases the speed of separation becomes crucial, so we change our focus, as in Section 8.9, to that of maximizing the number of plates per unit time Nit. From Eq. 9.17, N = XIH is... 

To ensure the integrity and stability of compounds used in HTS assays and to maximize the number of HTS screens, which can be performed with microgram quantities of compounds, all research sites at Roche use prealiquoted compound samples that are delivered to the HTS labs in sealed, ready-to-use sample plates . The sample plates contain aliquots of frozen millimolar compound solutions in water-free DMSO, 1 aL per well, corresponding to a small liquid droplet of approximately 1 mm diameter. The sample plate is a standardized, low-cost 384-well plate made from polypropylene with a tapered or parabolic well bottom forming a cavity for the small aliquot. This sample distribution concept for the HTS compound library is based on the following rationale ... 

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