Column evaluation plate number

The Waters company recommends a system check of the chromatographic equipment that is used for plate number determination and analyses (2) the columns in the GPC unit used are replaced by a zero dead volume union. Then the test sample is injected under the same conditions such as a plate number determination. The 5a peak width measured on a suitable recorded peak is evaluated this 5or width of a 20-/a1 injection should be lower than 150 /a1. 

Two linear columns from Showa Denko, Shodex SB-806M and Shodex SB-806MHQ, and two linear columns from TosoHaas, TSK GM-PWxl and TSK GM-PW, were evaluated. Prior to the evaluation, the number of theoretical plates for Shodex SB-806MHQ, SB-806M, PWxl, and PW was determined to be 15,100, 15,700, 11,390, and 4710, respectively, as per manufacturer inspection. The lower plate count of the TSK PW column is due to the larger particle size of this column. Two mobile phases, water with 0.1 M LiNOi and 50 50 methanol/water (v/v) with 0.1 M LiNOi, were used for each of the four columns. These four columns were new and only PEO and PVP were analyzed with these columns in this study. Waters Ultrahydrogel columns have also been used in this laboratory. However, Ultrahydrogel columns are exactly the same as the TSK GM-PWxl columns based on the calibrations curves supplied by the manufacturers and by the pyrolysis GC data discussed later. 

Note that with this procedure, the effect of the number of theoretical plates arailable can be determined. In an existing column where the number of trays are fixed, the theoretical trays can be obtained by evaluating an efficiency for the system. 

In order to evaluate the efficacy of the expanded bed technique the plate height (HETP), plate number (N), resolution (Rs), Bodenstein number (Bo), particle Peclet number (Pep) and axial dispersion coefficient (DJ have been calculated and compared with the corresponding values of a traditional HPLC column. N can be expressed by... 

The performance of columns is determined by using a simple procedure for both new and used columns. The test method and the results are usually printed on a sheet provided by the column manufacturer. Each manufacturer uses the most suitable method for their column to obtain the best theoretical plate number. The column evaluation provides important information for all users of liquid chromatography. 

There are several parameters which can be used to evaluate the operation of a column and to obtain infomation about a specific system. If we plot the concentration of solute (in %) versus volume of mobile phase or number of plate volumes for the tenth, twentieth, and fiftieth plate in the column, we will obtain a plot as shown in Figure 2.12. Improved separation of component peaks is possible for columns that have a larger plate number,. Similar information is obtained if we plot concentration of solute (% of total) versus plate number. Figure 2.13 shows the band positions after 50, 100, and 200 equilibrations with the mobile phase. 

With the introduction of capillary open tube columns (Chapter 4) it became possible to obtain chromatograms corresponding to hundreds of thousands (even >10 ) of theoretical plates as evaluated via Equation [3.20] or [3.21]. However, as a consequence of the issues discussed in Section 3.4.6, such enormous efficiencies could be obtained only for compounds with very low k values, i.e., those that eluted very close to the column dead volume. To provide a more realistic measure of column efficiency in such cases, the effective plate number (Ng) was defined by replacing t, by (L—tg) in Equations [3.20-3.21], where tg is the elution time after injection for unretained solutes ... 

Even though the plate number is a very useful column specification, it is certainly not sufficient to evaluate the suitability of a tested column for a given separation problem. Esfiecially for RP columns, a variety of column performance tests have been developed [77]. These tests give important information about the quality of the column bed (well packed or not), the kinetic and thermodynamic performance, basic and acidic behavior, and... 

Column efficiency (number of theoretical plates) As in batch chromatography, one needs to determine the efficiency of the column in order to evaluate the dispersion of the fronts due to hydrodynamics dispersion or kinetics limitations. The relationship of N proportional to L can be expressed in terms of the equation for height equivalent to a theoretical plate (HETP) as ... 

The performance of the HPLC system should be evaluated from the column efficiency and the symmetry of the peak. The column efficiency is determined as the number of theoretical plates, N, which should be greater than 5000. It is calculated as follows ... 

This measure was based upon the ratio of the minimum necessary number of plates, A min (averaged over the reboiler composition) in a column to the actual number of plates in the given column, Nj. Christensen and Jorgensen assumed that the mixture has a constant relative volatility a and the column operates at total reflux using constant distillate composition (x o) strategy (section 3.3.2) and evaluated Nmin using the Fenske equation ... 

It is important to note that the use of Logsdon et al. s objective function will not change the number of plates and operating policies if the mixtures were to share the column by a certain fraction of time (the profit function of Logsdon et al. does not include the allocation time). However, for different allocation time to each mixture, the total number of batches for each separation, individual and total yearly profit, etc. can be evaluated by using the results presented in Table 7.7. These are summarised in Table 7.8. Comparison of the results in Table 7.4 and 7.8 clearly... 

The reduced parameters are also helpful in evaluating column performance. The best columns have a reduced plate height of 2 to 5—a number that can be thought of as representing the number of particles between sorptions—and 2 is a practical minimum. The reduced velocity represents the ratio between the flow velocity and the diffusion rate over one particle diameter typical values should be in the range of 3 to 20. 

An example is shown in Figure 8-71, where the column stability was evaluated with two different buffers, phosphate buffer ("pFI 11) and 1-methyl piperidine (wpH 11) [43]. Plots of H (plate height) or N versus number of column volumes can be generated. Remember that H is inversely proportional to the efficiency ... 

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