SELECTION OF CHROMATOGRAPHIC METHODS

Before we can inject a sample into a chromatograph, we should be able to decide upon which of the above chromatographic techniques is suitable for the separation problem at hand. Clearly, this requires some information about the sample. Completely unknown samples may require a combination of different techniques. For example, an unknown liquid may contain a volatile fraction that can be analysed by GC and a non-volatile fraction, for which LC is required. In many cases, we know something about the sample, enough to decide which of the many possible analytical techniques can be applied. A scheme to decide on the appropriate chromatographic technique based on the nature of the sample is shown in figure 2.1. 

The first question to be answered is whether or not the sample is volatile enough to be analyzed by GC. GC columns are currently available with upper temperature limits of around 350 °C. Hence, compounds should be sufficiently volatile at this temperature to be analyzed by GC. A second requirement for the sample, which becomes the more relevant the higher the temperatures used, is the thermal stability of the sample, both in the column, as well as in the injector, which in conventional GC is operated at a temperature slightly above that of the column. Because of the limited stability of organic substances at higher temperatures, extremely high temperatures do not seem to be very 

For the volatile samples we then have a choice between GSC and GLC. For all but the permanent gases, which possess a very high intrinsic volatility and need strong specific adsorption sites to be sufficiently retained, GLC is usually the preferred method. 

For those samples that are not compatible with GC, the first question to ask involves the size (molecular weight) of the solute molecules. Their size should be compared to the pores of the packing materials that can be used in LC. If the size of the molecules is not negligible relative to the (average) pore size, then part of the pores and hence part of the stationary phase present in the column will not be accessible to the solute molecules. Hence, the simple relationship between chromatographic retention and thermodynamic distribution (eqn.l. 6) loses its significance. To avoid that, wide pore materials can be used for the separation of large molecules (e.g., proteins) based on their distribution over the two phases [202]. 

The effect of limited penetration of the pores by the largest molecules may also be applied beneficially for the separation of very large molecules. Depending on the size of the molecules (in solution), they will be more ore less excluded from the pores, and hence the retention times will be affected. This effect is used in size exclusion chromatography (SEC) or gel permeation chromatography (GPC). In this technique, any interactions between the solute molecules and the stationary phase are purposefully avoided. The solute molecules remain exclusively in the mobile phase, but the accessible mobile phase volume, and hence the retention volume, may vary between the total volume of the mobile phase and the so-called exclusion volume, which is the total volume of mobile phase outside the pores. The latter elution volume applies to very large solute molecules (excluded solutes), 

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Chart 1. Selection of chromatographic method based on solubility. 

Section 2.2.1 briefly addresses the possibility to incorporate a scheme for the selection of chromatographic methods in a computer program, a so-called expert system. This is a relatively recent proposition, and progress may be expected in this area. 

Liquid chromatography, a seemingly simpler procedure than GC in the speciation analysis of arsenic because it does not require the derivatization stage, also generates a number of potential errors. The selection of chromatographic method involves preliminary evaluation of the solubility of compounds to be separated. The... 

T. Hanai, Selection of chromatographic methods for biological materials, in Advanced Chromatographic and Electromigration Methods in BioScience, ed. Z. Deyl, I. Miksik, F. Tagliaro and E. Tesarova, Elsevier, Amsterdam, 1998. 

Hanai, T. Selection of chromatographic method for biological materials. In Advanced Chromatographic and Electromigration Methods in Biosciences Deyl, Z., Ed. J. Chromatogr. Library 60, Elsevier Amsterdam, 1998 1-51. 

In the post-World War II years, synthesis attained a different level of sophistication partly as a result of the confluence of five stimuli (1) the formulation of detailed electronic mechanisms for the fundamental organic reactions, (2) the introduction of conformational analysis of organic structures and transition states based on stereochemical principles, (3) the development of spectroscopic and other physical methods for structural analysis, (4) the use of chromatographic methods of analysis and separation, and (5) the discovery and application of new selective chemical reagents. As a result, the period 1945 to 1960 encompassed the synthesis of such complex molecules as vitamin A (O. Isler, 1949), cortisone (R. Woodward, R. Robinson, 1951), strychnine (R. Woodward, 1954), cedrol (G. Stork, 1955), morphine (M. Gates, 1956), reserpine (R. Woodward, 1956), penicillin V (J. Sheehan, 1957), colchicine (A. Eschenmoser, 1959), and chlorophyll (R. Woodward, 1960) (page 5). ... 

Vander Heyden, Y., Questier, R, and Massart, D. L. (1998). Ruggedness testing of chromatographic methods selection of factors and levels. /. Pharm. Biomed. Anal. 18, 43-56. 

The selectivity of a method is a measure of how capable it is of measuring the analyte alone in the presence of other compounds contained in the sample. The most selective analytical methods involve a chromatographic separation. Detection methods can be ranked according to their selectivity. A simple comparison is... 

Chromatographic separation of amino acids in the test protein is required for chemical scoring methods. The selectivity and sensitivity of chromatographic methods vary and comparing results of more than one method may be required, particularly when proteins with unusual amino acid profiles are being tested. 

Because of their importance in pharmaceutical analyses, much attention has been focused on harmonizing the parameters necessary for the validation of chromatographic methods. While some of these parameters are applicable to other analytical techniques, it is the responsibility of the analytical chemist to select and tailor the appropriate parameters and acceptance criteria for the particular method to be validated. Since most analytical chemists are not experts on regulatory matters, it is essential for the regulatory affairs professional to understand the requirements of method validation and work closely with the analytical chemist to select appropriate validation parameters and meaningful acceptance criteria. 

CONTENTS 1. Chemometrics and the Analytical Process. 2. Precision and Accuracy. 3. Evaluation of Precision and Accuracy. Comparison of Two Procedures. 4. Evaluation of Sources of Variation in Data. Analysis of Variance. 5. Calibration. 6. Reliability and Drift. 7. Sensitivity and Limit of Detection. 8. Selectivity and Specificity. 9. Information. 10. Costs. 11. The Time Constant. 12. Signals and Data. 13. Regression Methods. 14. Correlation Methods. 15. Signal Processing. 16. Response Surfaces and Models. 17. Exploration of Response Surfaces. 18. Optimization of Analytical Chemical Methods. 19. Optimization of Chromatographic Methods. 20. The Multivariate Approach. 21. Principal Components and Factor Analysis. 22. Clustering Techniques. 23. Supervised Pattern Recognition. 24. Decisions in the Analytical Laboratory. 

Nonvolatile Samples. Figure 12.2 shows one flow sheet for selecting a chromatographic method. Normally nonvolatile samples are run by LC, but consideration should be given to the possibility of derivatizing the sample to get volatility adequate for GC. (Consult Chapter 11 for possible deri-vatizations.) Other variables considered in Figure 12.2 are the number of samples and the importance of speed. 

The use of UV-vis and FT-IR spectroscopy in determining drug impurities without previous chromatographic separation is limited because of the low selectivity of both methods. In some cases, when the impurity has spectral properties that are very different from those of the active substance, the direct measurement of absorbance can give some useful data about the structure of the impurity. But, in many cases the impurity has a similar structure to the active substance and, therefore, their UV and IR absorption spectra are similar, and overlap with each other additively. This means that direct use of spectroscopic methods in drug purity analysis is limited [49]. 

An especially challenging task is maintaining the selectivity of the method for separation of compounds whose elution time is very short, close to the dead time. In such cases, it is necessary to perform a preliminary review of the planned chromatographic conditions, including the composition of the analyzed material. For example, a typical eluent employed in anion-exchange chromatography (with pH of 8.5) is intended to facilitate the dissociation of separated compounds. Neglecting the time necessary to achieve acid/base equilibrium of substances loaded into the column in a neutral solution can result in their elution in the dead volume. The phenomenon is observed, for example, for MMA(V), whose consecutive dissociation constants are p/sTi 3.6 and p/sT2 8.22 [164]. 

In the lifetime of a chromatographic method different stages can be considered. In a first instance the analyst selects a method or a technique method selection) which could serve for the purpose he has in mind, i.e. to determine a given substance in a given matrix. The selection of the method depends on the properties of the analyte(s) to be determined and on the availability of analytical techniques in a given laboratory. For instance, one might decide to determine the substance(s) of interest by R(eversed) P(hase) HPLC. Expert systems (Section 6.8) may help in this step. 

One cannot fail to note the vast expansion of the collection in the last few decades. Surely this was not fueled by additional biologic assays. Underlying the initial growth phase was the widespread utilization of spectrophotometry for identification and assay. Separation science was the second phase in pharmaceutical industry control laboratories. As a corollary, USP and NF method selection moved in the same direction. Spectrophotometric identity tests and assays are more reliable, especially for compliance testing, when performed in the relative mode, which uses a reference standard, rather than the absolute mode, which is the norm in titrimetry. There is some residual difference of opinion in other countries on this point, but that is rendered moot by the widespread adoption of separation science by the pharmaceutical industry and, thus, by the compendia. It is a characteristic of chromatographic methods that a reference standard be required, sometimes more than one for a procedure. The accumulation of modern tests and assays results in 5 to 10 uses for many reference standards. 

Air analysis is very difficult to do because of the complexity of the matrix. To obtain reliable results one must choose the best sampling process. The selectivity of the method in the case of air analysis cannot assure the best reliability without any separation method. The development of chromatographic techniques have made them suitable for most separation processes utilized for air analysis. 

Determination of solubility by headspace analysis offers several advantages over spectrophotometric techniques. First, because of the selectivity of chromatographic analysis, compound purity is not a critical factor second, absolute calibration of the gas chromatographic detector is not necessary if the response is linearly related with concentration over the range necessary for the measurements and finally, this method does not require the preparation of saturated solutions, since a partition coefficient, not a solubility, is actually measured. However, headspace methodology would probably not be applicable for determining PAH solubilities for three reasons. First, there is little data in the literature on the vapor pressures of PAHs. Second, the aqueous solubilities of most PAHs are too low to be measured by this procedure. Finally, adsorptive losses of PAHs to glass surfaces from the vapor phase would cause errors. 

The successful application of the CFD method in combination with subsequent gas chromatographic separation and the use of an BCD has resulted in the extensive development of this technique. However, other CFD methods aimed at obtaining derivatives that can be selectively detected by other selective detectors (e.g., sodium thermionic, flame photometric) have not been developed adequately, despite their obvious promise. It seems that the high selectivity of the method should be used for the elaboration of selective methods of functional group analysis in order to identify compounds at the picogram level. This is especially pertinent to the analysis of microsamples on capillary columns. 

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