Plate count chromatograph

All packing materials produced at PSS are tested for all relevant properties. This includes physical tests (e.g., pressure stability, temperature stability, permeability, particle size distribution, porosity) as well as chromatographic tests using packed columns (plate count, resolution, peak symmetry, calibration curves). PSS uses inverse SEC methodology (26,27) to determine chromatographic-active sorbent properties such as surface area, pore volume, average pore size, and pore size distribution. Table 9.10 shows details on inverse SEC tests on PSS SDV sorbent as an example. Pig. 9.10 shows the dependence... 

Each SynChropak column is tested chromatographically to assure that it has been packed according to specifications. For SynChropak GPC columns, a mixture of a high molecular weight DNA and glycyltyrosine, a dipeptide, is used to evaluate internal volume and efficiency. The mobile phase used for the test is 0.1 M potassium phosphate, pH 7, and the flow rate is 0.5 ml/min for 4.6-mm i.d. columns. Minimum plate count values and operational flow rates are listed in Table 10.4 for 4.6-mm i.d. columns of all supports and the various diameters of the SynChropak GPC 100 columns. 

Biological characteristics include microscopic and chromatographic examinations, plate counts, total heterotrophs, petroleum degrading, total hydrogen degrading, bacteria microscopic examination, and TOC. 

In chromatographic and related separation techniques the basic requirements for the resolution (Rs) of two peaks are a column with a high number of plate counts and a factor to induce some selectivity for the separation. Basically resolution is the product of separation efficiency and selectivity ... 

Comment on the resolution of the chromatograph in relation to (a) the plate count and (b) to separation between the members of a pair of narrow distribution samples. 

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. 

Eqn.(7.15) is the key equation for the optimization of chromatographic sensitivity. Naturally, the peak height is proportional to the concentration of the solute in the sample and to the volume of the injected sample. However, this proportionality holds over a limited range and we cannot increase these two quantities indefinitely without having to sacrifice another vital characteristic of the system, the linearity of detection. The proportionality between cmax and the product cgV-in ends when N may no longer be considered as a constant. Consequently, the aforementioned product may be increased until the plate count starts to be affected. 

The most common performance indicator of a column is a dimensionless, theoretical plate count number, N. This number is also referred to as an efficiency value for the column. There is a tendency to equate the column efficiency value with the quality of a column. However, it is important to remember that the column efficiency is only part of the quality of a column. The calculation of theoretical plates is commonly based on a Gaussian model for peak shape because the chromatographic peak is assumed to result from the spreading of a population of sample molecules resulting in a Gaussian distribution of sample concentrations in the mobile and stationary phases. The general formula for calculating column efficiency is... 

The efficiency at which the column or plate count is packed initially in chromatography is important because (1) it determines the shape of the individual band profiles, and (2) it can effect the void fraction and thus the operating pressure. For chromatographic processes where the plate count is important (see Section 7.8.1 on flow rate), developing methods to measure the plate count of the column after it is packed or of a prepacked column is important. There are several methods for measuring the plate count of a packed column, as given below. 

Two related terms are widely used as quantitative measures of chromatographic column efficiency (1) plate height H and (2) plate count or number of theoretical plates N. The two are related by the equation... 

Cut and Count. Chromatographic plates may be analyzed using a Nal(Tl) scintillation counter. The plates are cut in segments (up to ten) and transferred into tubes to be counted. The radioactivity on the plate can be plotted as a histogram. The sensitivity is much higher compared with the ionization chamber (even dilution may be necessary), and the resolution is dependent on the number of segments. It is important to keep the... 

The plate count required for a resolution of 1.0 using different separation conditions is summarized in Table 1.10 [146]. Practically all chromatographic separations have to be made in the efficiency range of 10 -10 theoretical plates. The importance of optimizing the separation factor and retention factor to obtain an easy separation is obvious from the data in Table 1.10. Easy separations require chromatographic systems that maximize the separation factor and provide at least a minimum value for the retention factor. A common optimization strategy for difficult separations with a limited number of components is to fix the value of the retention factor between 1 and 3 for the two components most difficult to separate in the mixture. 

Approximate plate heights can also be determined for a given type of packing by thin-layer chromatographic measurements. Thus, for sample 2 in Figure 28-,3()a, the plate count is given by the equation... 

Figure 16.17 Effective plate count versus flow rate. Chromatographic conditions Gradient from 59% methanol to 95% methanol in 40 min. Column HiBar Cn Lichrosorb Cig, 5/tm, 4.6 mm x 250 mm. The sample is pentachlorophenol.)...

This chromatographic mode represents a simple on-off technique. The compound of interest is fished-out from a more complex mixture via a very selective interaction with a ligand immobilized on solid support. Once the entire sample is processed through the column and followed by washing, the captured compound is released using a strong eluent. Thanks to the simple binary separation mechanism, plate counts are not important and the development of devices mostly focuses on binding capacity and selectivity. 

Column effects. In order to establish optimal operating conditions, it is useful to consider the effects of system parameters on the resolution characteristics of an HDC system. HDC has been described as a chromatographic method with very low capacity but very high efiBciency. For example, the calibration curves show that the spectrum of sizes from less than 100 nm to greater than 300 nm is encompassed in less than about 5% of the column void volume. On the other hand, the theoretical plate count corresponding to the marker peak is typically in the range of several thousand per foot. Comparisons in terms of the specific resolution factor, enable a more precise analysis, since both the separation factor and peak dispersion are included in its definition. A simple form for the specific resolution between two particle populations of diameter Dpi and Dp2 is [11]. 

The positional criteria can lead to reliable resolution optima using the retention data from a few mobile phases. However, as the shape and width of the chromatographic peaks are not considered, for largely overlapped asymmetric peaks, an unacceptable optimum can be obtained. If this is the case, only an increase in plate count will provide the desired separation, using for instance two identical columns in series [8]. Alternatively, a response surface related to a different criterion must be examined. 

The major drawback of practical separations applying MLC is still the low chromatographic efficiency, caused by the resistance to mass transfer in the processes involving micelles and a surfactant-modified stationaiy phase as exposed in Chapter 6. This is especially important since the increase in micelle concentration causes a decrease in plate count, resulting in a varying efficiency over the variable space. It is thus worthwhile to examine the inclusion of the expected peak shape in the expression of the chromatographic quality. 

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