Plate count test

In order to maintain or improve over laboratory performance in manufacturing it is important to obtain the same number of plates as that obtained in the laboratory. That means that a plate count test needs to be developed. One of the simplest tests would be to use the product and load a small volume and at a small concentration to perform an analytical injection on manufacturing column and compare the results with the laboratory column. In this way, an additional compound is not introduced into the manufacturing process. The plate count for the product can be correlated with standard test solutions on laboratory columns to determine how close the reduced plate count, / , compares with industrial standards. A reduced plate count of 2 or less describes a... 

After packing and testing the column, one may find that the plate count or the peak symmetry is unsatisfactory. In this case some adjustments need to... 

PSS uses the following formulae (22,23) to calculate plate count, asymmetry, and resolution. PSS uses test conditions that conform to the ISO/EN 13885 and DIN 55672 requirements for SEC sample testing. 

There are different ways to calculate the theoretical plate count of a column. PSS SEC column plate counts are measured using the so-called half-height method. Figure 9.9 shows a graphic representation of this test. 

Plate count should always be tested with a monodisperse sample of low molecular weight. Polymers can also be used, but they show much lower plate counts because their diffusion coefficients are much smaller than those of low molecular weight compounds. 

The reader should note that the value of the theoretical plate count depends on the sample chosen for testing. PSS always specifies on the PSS column quality certificate which sample has been used for measuring plate count and the exact test conditions. 

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... 

After column packing, each column is tested for theoretical plate count, peak symmetry, resolution, pore volume, and back pressure. If one of these tests fails the column is removed from the production cycle. If a PSS SEC column is kept in storage for a longer time, it is retested for theoretical plate count, peak symmetry, resolution, pore volume, and back pressure prior to shipping to the customer to prove up-to-date column performance. 

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. 

Most GPC columns are provided with vendor estimates of the plate count of the column and a chromatogram of a series of test peaks. These plate count estimates are usually obtained using small molecule analytes that elute at the total permeation volume (Vp) of the column. The Gaussian peak shape model... 

Because physiological deterioration is generally accompanied by an increase in bacterial population, as pointed out by Nielsen, Wolford, and Campbell (33), estimation of bacterial numbers might serve as the basis of a test for condition. Obviously, the plate count method is not adaptable because of the time limitations imposed. Direct microscopic count would be much more appropriate, especially if a positive field count were substituted for cell count as suggested by Wolford (39). 

Having chosen the test mixture and mobile diase composition, the chromatogram is run, usually at a fairly fast chart speed to reduce errors associated with the measurement of peak widths, etc.. Figure 4.10. The parameters calculated from the chromatogram are the retention volume and capacity factor of each component, the plate count for the unretained peak and at least one of the retained peaks, the peak asymmetry factor for each component, and the separation factor for at least one pair of solutes. The pressure drop for the column at the optimum test flow rate should also be noted. This data is then used to determine two types of performance criteria. These are kinetic parameters, which indicate how well the column is physically packed, and thermodynamic parameters, which indicate whether the column packing material meets the manufacturer s specifications. Examples of such thermodynamic parameters are whether the percentage oi bonded... 

If the test solutes show reasonable peak asymmetry with lower than average plate count values for well retained solutes (k > 2) the column is most probably poorly packed. The number of theoretical plates, normalized to one meter column length by... 

The efficiency, or plate count of a column N is often calculated as 5.54 (tr/a)2, where tr is the retention time of a standard and a is the peak width in time units at half-height.1 2 5 This approach assumes that peaks are Gaussian a number of other methods of plate calculation are in common use. Values measured for column efficiency depend on the standard used for measurement, the method of calculation, and the sources of extra-column band broadening in the test instrument. Therefore, efficiency measurements are used principally to compare the performance of a column over time or to compare the performance of different columns mounted on the same HPLC system. 

The test for mutagenic activity of the detergent (Tween) extract did not induce any revertants all plate counts were in the range of spontaneous mutation rate, with and without S-9 mix. From the ethanol distillate we got a positive response demonstrated in Table II. In the case of milk-crumb the numbers of induced revertants were about a hundredfold those of the spontaneously reverted colonies with the most sensitive strains (TA 98, 100) only without metabolic activation. In the same experiment the test strain TA 100 responded to the soya-crumb sample, also only without metabolic activation. 

It should be noted that the tests and specifications listed in Table 7 are provided for guidance and should be set for each individual method based on its application and performance. For example, there may be no resolution requirement for an HPLC dissolution assay. For the same method, it may also be impractical to set the injector repeatability requirement at an RSD of < 1% if the peak response for the API is very small. Again, in the same example it is also impractical to require a check standard to be within 1% of the working standard if the RSD requirement for replicate analyses of the working standard is <2 %. Some ion exchange columns may not provide plate counts of > 2000 or tailing factors of < 2. 

The most commonly used criterion for judging column performance is efficiency as measured by the number of theoretical plates or column plate count (N) exhibited by the column during the separation of a test mixture. The larger the number of theoretical plates, the more likely it is that the column will produce the desired separations. However, while popular, N is not a complete performance parameter for making comparisons. For example, N does not take into account particle size as does the reduced plate height, h. Another measurement, hmin, accounts for all of these factors as well as the mobile phase linear velocity and sample diffusion. However, N is the term most commonly recognized as being related to resolution (2), as shown in Equation 1 ... 

Test for Yeasts and Molds The plate method is used for testing molds and yeast in semisolids. The procedure is similar to that of the total count test. Instead of SBCDA medium, Sabouraud dextrose agar (SDA) medium or potato dextrose agar (PDA) medium is used. Samples are incubated for five to seven days at 20-25 °C to identify the presence of yeasts and molds. 

Usually, total aerobic bacteria, molds, and yeasts are counted by using a standard plate count in order to test the microbial limits. The microbial limit test may be customized by performing a screening for the occurrence of Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepacia, Escherichia coli, and Salmonella sp. [56],... 

Agar plate test Soil burial test Saturated atmosphere test Count test... 

For the enumeration procedure, each laboratory received 60 capsules and a maximum number of 50 enumerations was requested. The extra capsules were supplied to replace the capsules that were not acceptably dissolved. The capsules were dissolved in 2 rounds each of 30 capsules. For each series also a control was tested, consisting of a Petri-dish containing peptone saline to which no capsule was added. The procedure can be summarised as follows (1) dissolution of a capsule in 5 mL peptone saline solution in a Petri-dish for (45 5) min in an incubator at (38.0 0.5)°C while shaking at ca. 100 rpm (2) repair of Salmonella in Plate Count Agar (incubation time (4 1) h at (37 ... 

There are many pasteurized milk products that can be stored at room temperature. These products are occasionally contaminated by a small number of bacteria, so it is necessary to do sterility testing using agar plates. This sterility test typically takes a long time. For example, pasteurized products are incubated at 30 °C or 37 °C for 2-3 days. Incubation followed by plate counting is time-consuming, yielding results after... 

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