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Direct methods can be further divided into two major groups:
1. Visual comparison, when the spot intensities are established by visually comparing them with the intensities of simultaneously developed reference spots.
2. In situ densitometry quantifying the chromatogram directly on the plate by measurement of optical density, native or induced fluorescence, etc., of the separated spots.
The semiquantitative estimation of impurities by visual comparison is generally used in different pharmacopoeias for the purity testing of active raw materials and formulated products, as well. Four purity criteria can be defined:
1. The criterion does not allow the appearance of any other spots than the principal one on the chromatogram (single spot criterion).
In this case a known quantity of the sample is transferred onto the plate. This procedure is simple, does not require the simultaneous application of any reference materials; however, the result does not reflect the impurity profile of the material, and could be dependent on experimental conditions (e.g., light intensity of the UV lamp, plate type and efficiency, etc.).
Szepesi and Nyiredy
2. The criterion specifies the maximum allowable number of impurities and limits the quantity of each individual impurity.
Only the presence of those impurities are allowed that show identical Ł values with the simultaneously developed reference compounds. Although this procedure is more accurate compared with the previous one, it requires close conformity of the materials (the impurity profile should be the same, which is the function of the manufacturing process).
3. The criterion limits the total intensity of the separated impurity spots.
The criterion does not limit the number and quantity of the individual impurities, but limits the total intensity of the separated impurity spots in comparison with the intensity of spot(s) of simultaneously developed reference standards (principal component) applied in known quantity.
Although this procedure is generally used for the evaluation of pharmacopoeial purity tests, it suffers from two major limitations:
1. Spot intensity may be the function of the chemical structure and Rf value of the impurities.
2. The spot intensities must be summed visually.
4. The criterion limits the quantity of each individual impurity and the total amount of impurities, as well.
Standard dilution of reference materials are simultaneously applied to the plate and the purity of the material to be tested is evaluated by comparison of the spot intensities of known impurities with those of standard dilutions of the same compounds; the quantity of other spots different from the reference spots is expressed in term of the principal component.
This evaluation procedure is the most accurate one among the methods based on visual comparison.
Advantageous and disadvantageous properties of conventional and instrumental detection modes are discussed in Section IV. A brief summary of the most important advantages and limitations are shown in Table 3.
As with any quantitative method based on the interpretation of detector responses, several methods exist for evaluation of the results obtained. In the case of quantitative TLC, a nonlinear relationship between the detector signal and the amount of substance to be tested exists. This nonlinearity is valid for both peak height and peak area measurements. A detailed interpretation of the problem can be found in the papers of Ebel (95-101). Derivative recording using smoothed numerically generated derivatives have been designed to overcome this problem (95).
The next section deals with method validation of quantitative TLC methods. Two questions should, however, be answered prior to discussing the validation experiments: namely, whether the statistical evaluation of data elements, such as precision, accuracy, and reproducibility should be calculated on the basis of measured peak heights or peak areas, and whether the internal or external standard methods, or area normalization should be used to yield quantitative results for the assay. Without going into detail, the most important advantages and limitations of peak height and peak area measurements, and those of the different methods of quantification are summarized in Table 4.
As is apparent from the table, it is not easy to select the most appropriate method for quantification. The plot of peak height (peak area) against concentration is linear only within a narrow range of concentrations and, therefore, the application of a 5-point calibration covering the total range of concentrations is highly recommended. Similarly, the usefulness of the internal standard method relates mainly to those analytical tasks requiring complex sample preparation procedures. Area normalization can not be used for the evaluation of the TLC results.
Pharmaceuticals and Drugs
Table 3 Advantageous and Disadvantageous Properties on Spot Elution Technique and In Situ Densitometry