Plate count analysis

Fig. 1. Plot of plate count analysis time for high-powered isocratic separations.

It is difficult to decide what should serve as adequate column quality parameters for describing the performance of a set of GPC columns. The two most common measures are plate count and resolution. While both of these can be useful for monitoring the performance of a column set over time, it is not generally possible to a priori specify the performance needed for a specific analysis. This will depend on the nature of the polymer itself, as well as the other matrix components. 

Columns - yBondagel 2000A, E-linear, 125 A Detection - RI 16X, SP4050 Attenuation 10 Chart speed - 4 cm/min Analysis time - 10 min Total plate count 10 plates... 

In more demanding separations that require higher plate counts, specially designed rapid analysis columns packed with very high efficiency 2 to 3 /.an porous particles are available from several manufacturers. In addition, monolithic columns with improved flow-through characteristics are also commercially available. Figure 13.4 depicts a comparison of inlet pressure and flow rate for 4.6 mm inner diameter x 50, 100, and 150 mm columns packed with 5 /an particles. 

Laboratory analysis of site soils should be made to determine the presence and population density of naturally occurring microbes that are capable of degrading the contaminant. At a minimum, these analyses should include plate counts to determine the relative number of microbes of several types, the substrate (food) type that reflects the type of chemicals they are likely to consume, and if toxic substances are present. 

HPLC systems have recently become commercially available, which allow the use of pressures up to 1000 bar. Columns of particle size around 1.7 p,m and up to approximately 10-cm long can be operated at their optimum flow rate within the pressure capability of the system. The main advantage of these columns is that they can generate the same plate count as longer columns of larger particles but in a shorter analysis... 

First, we look at isocratic separations. Let us assume that the analysis can be accomplished within a retention factor of 10. We also suppose that the analysis is carried out with a typical reversed-phase solvent and a sample with a typical molecular weight of a pharmaceutical entity. In order to manipulate the analysis time, we will keep the mobile phase composition the same and vary the flow rate. The maximum backpressure that we will be able to apply is 25MPa (250 bar, 4000psi). In Figure 1, we have plotted the plate count as a function of the analysis time for a 5 J,m 15-cm column. We see that the column plate count is low at short analysis times and reaches a maximum at an analysis time of about 1 h. A further increase in analysis time is not useful, since the column plate count declines again. This is the point where longitudinal diffusion limits the column performance. The graph also stops at an analysis time of just under 5 min. This is the point when the maximum allowable pressure drop has been reached. 

FIGURE I Plate count vs. analysis time for a 5-gm 15-cm column. 

Studied responses were resolution, " " analysis time, migration time, plate count, tailing factor, tablet content, peak area, peak height, peak width, and peak area/migration time ratio. ... 

Third, the efficiency (N) may be adapted to meet the requirements set by the values of k and a. The value of N is determined by the column characteristics and the flow rate. While increasing the plate count may require great sacrifices in terms of analysis time, the reverse is also true. If the values of k and a allow the use of a column with a low N value to achieve the separation, then the analysis time may be reduced dramatically. 

Another factor that contributes to Rs is the plate count N. However, we have seen in section 1.5 that optimization through an increase in N is expensive, not only in terms of equipment and columns, but also in terms of analysis time. Therefore, as long as the shape of the peaks and the plate height (length of the column divided by N) are satisfactory, we should not rely on the number of plates for optimization, unless as a last resort. Methods which may be used to optimize the chromatographic system with respect to the required number of plates will be described in chapter 7. 

In GC we have a real choice between packed columns (dp = 100-200 pm 150-65 mesh) and open columns (dc= 50-500 pm). Capillary columns have the advantage of enhanced speed of analysis (eqn.7.6). In order to exploit this advantage, narrow-bore capillaries (dp< 100 pm) should ideally be used. However, such columns require relatively high inlet pressures (especially for high plate counts) and considerable experimental modifications and have a very low sample capacity [702],... 

Until 1990, when Giovannoni and Ward applied 16S rRNA sequence analysis by extracting community DNA, microbiologists had to rely on plate counts of bacteria which could be cultured and isolated to obtain an idea of the number of bacteria in the sample.43,44 This new approach allowed the study of the remaining 99% of bacteria which could not be isolated by nutrient media and liquid enrichment. 

Since the goal is to reduce analysis time by minimizing Hlu while holding N constant (at the minimum required plate count), the approximation can be made that H dp, and therefore N LIdp. This means as the particle diameter is reduced, the column length must also be reduced proportionally. 

The use of modeling to collect information on the importance of various parameters is seen in the discussion of each variable. In the analysis of the importance of plate count, modeling can reduce significantly the number of experiments and the time or cost of collecting range finding data. 

Plots of Po/w or vs. plate counts for benzene and 2-ethylanthraquinone eluted with micellar SDS, in the presence of several alkanols and alkane diols, show an initial steep increase in efficiency, after which an approximately constant value is reached (Fig. 3). Among the alcohols, maximal efficiency for benzene and 2-ethylanthraquinone is obtained with the butanols and the pen-tanols, with enhancement factors of 2.5 and 25 (compared to pure SDS), respectively. However, final efficiencies for the latter compound are much lower compared to that for benzene. Dipolar aprotic modifiers (acetonitrile or dimethylsulfoxide) appear to be somewhat more effective in enhancing efficiencies than alcohols with comparable Pq/w- Some recent work has shown the advantage of using acetonitrile as additive in MLC for the analysis of sulfonamides, tetracyclines, and the most polar steroids. 

Fluorescence lifetimes were measured by time-correlated single photon counting using a mode-locked, synchronously pumped, cavity-dumped pyridine I dye laser (343 nm) or Rhodamine 6G dye laser (290 nm). Emissive photons were collected at 90° with respect to the excitation beam and passed through a monochromator to a Hamamatsu Model R2809U microchannel plate. Data analysis was made after deconvolution (18) of the instrument response function (FWHM 80 ps). 

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