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E. Other Aspects
Successful planar chromatographic separation is dependent on several key factors. One relates to the quality of sample application.
The reproducibility of sample amount and spot size is obviously quite important, but to achieve good chromatographic resolution and sensitivity of detection, the shape of the spots of the applied sample is of great importance. Techniques and apparatus for sample introduction are reviewed by Fennimore (28,29), Janchen (30), and Kaiser (31).
In summarizing the practical problems associated with various sampling procedures, the following principles should be considered:
1. There is a narrow working range for the sample volume load if no focusing step is included in sample application.
2. Sampling in the presence of solvents with a measurable elution strength can start chromatography, resulting in significant loss in separation efficiency.
3. If a solvent used for sample preparation remains in the layer in or around the sampling area, the selectivity and the relative or absolute spot position in the chromatogram may be markedly altered.
4. If part or all of the sample is solidified or adsorbed onto the stationary phase surface, a slow dissolution effect can cause significant tailing of the spots.
The application of the sample should be made in the form of spots or streaks. The streak type of sample application which forms very narrow lines of starting shape, results in sharper spots and
Pharmaceuticals and Drugs
increased resolution allowing for optimum separation efficiency. However, streak-type sample application is less precise compared to spot-type sample application. Among the other aspects, phase system optimization, detection, quantitation, and validation will be treated in detail in the following sections.
II. TYPES OF ANALYTICAL AIMS IN PHARMACEUTICAL ANALYSIS
The most important tasks of capillary action planar chromatography in pharmaceutical analysis are summarized in Table 2. In general, it can be stated that although HPLC has superseded TLC in many application areas, TLC both in instrumentalized or in noninstrumentalized forms have remained as standard methods for solving many difficult analytical problems.
With respect to applicability and significance of TLC in pharmaceutical analysis, the methods used in Pharmacopoeias can be separated from those used in industrial pharmaceutical analysis.
Due to the useful role of TLC when cost effectiveness is essential, TLC is widely used as a standard technique for rapid and accurate identification of the raw materials or finished products as well as for purity testing of raw materials and formulations in various pharmacopoeial prescriptions.
The importance ofTLC methods as assay methods considering the several difficulties experienced during its use, has decreased considerably, and only few official methods can be found in the pharmacopoeias usually with a spot elution technique for quantitation.
In the field of industrial pharmaceutical analysis the situation is different, because TLC instrumentation has reached a relatively high level. In some special application areas, such as the analysis of the extracts of medicinal plants, fermentation mixtures, etc., modem TLC (precoated or HPTLC layers, densitometric evaluation) has a distinct role, because the interference of so-called unknown background materials can be more easily eliminated than with other chromatographic techniques. Many chromatographers working in the pharmaceutical industry prefer to use reversed phase HPLC in conjunction with normal phase TLC or HPTLC to analyze raw materials for purity and impurities as well as for stability testing.
III. METHOD DEVELOPMENT
Method development, which resulted in sufficient separation, is one of the most critical steps of qualitative and quantitative analyses. In Figure 1 the main steps are summarized.
A. Stationary Phase Selection
Separations for pharmaceuticals and drugs can be performed using modified, nonmodified, and impregnated stationary phases due to a difference in chemical properties between the sorbent material and the compounds of the sample to be separated (32). Different types of chromatographic process can be distinguished (straight phase-, reversed phase-, partition-, and ion exchange chromatography) based on the type of determining interactions involved. In TLC, (>90%) silica is used mainly for the separation of pharmaceuticals and drugs. However, in the last period chemically bonded phases are used more and are more popular to solve special separation problems (33).
In straight phase chromatography, it is the hydroxyl groups on the surface of the silica which are the polar, active centers where most of the interactions take place in the retention of the compounds to be separated. These interactions are mainly hydrogen-bonding and induced dipole-dipole. Generally, the phase can be characterized with the following three characteristic values: specific surface area, specific pore volume, mean pore diameter.