Selection of Modern HPLC Columns

Stefan Lamotte, Stavros Kromfdas, and Frank Steiner 

From the beginning, a wide range of different materials have been used as stationary phases in liquid chromatography. Their selection depends primarily on three key properties chemical stability, mechanical stabihty, and specific surface area. The chemical stability is important for achieving the greatest possible compatibility with the mobile phase used, and on this depends also the possibihty of bringing the analytes to be determined into solution. 

The second equally important point is the mechanical stabihty. Since HPLC in the form UHPLC can take place at pressures up to and above about 130-150 MPa (1300-1500 bar, bar is used throughout the chapter), the materials of the stationary phase are potentially subject to high mechanical loads. As a consequence, only certain supports come into question. 

Finally, for liquid chromatography, it is important to use base materials having a high specific surface area. This is necessary to guarantee a certain phase relation between the mobile and stationary phases and thus ensure an appropriate loadability of the stationary phase. Such a high specific surface area can only be achieved with a porous support. The porosity in turn has an influence on the mechanical stability of the stationary phase. 

a variety of materials can be ruled out, and the selection is limited to metal oxides such as Si02, AI2O3, Ti02, and Zr02, as well as cross-linked polymers based on polystyrene-divinylbenzene and acrylates, and also carbon. 

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Improvement of the resolution of poorly resolved analytes then could be pursued in two different ways either by increasing the efficiency or by improving the selectivity. The resolution value equal to 1.5 is usually regarded as sufficient for the baseline separation of closely eluted peaks and if we consider that typical average efficiency of modern HPLC column is equal to 10,000 theoretical plates, then we can calculate the selectivity necessary for this separation to get a resolution of 1.5. It will be also useful to compare what would be required in terms of efficiency and selectivity to improve the resolution from 1 to 1.5. Corresponding calculations are shown in the Table 1-1. 

If the separation factor is unity, the peaks coincide, and no separation has occurred. If the separation factor is 1.3, the column selectively retards one component 30% more than the other. The larger the a value, the easier the HPLC separation is to achieve however, an a value of 1.1-1.4 is typically desired. As we show later in this chapter, because resolution is influenced by three factors, separations can be attained for a values smaller than 1.1. In some modes of modern HPLC, meaningful resolution can be achieved with a values as low as 1.05, which means that the column retards the second component only 5% more than it retards the first component. 

This chapter provides an overview of modern HPLC equipment, including the operating principles and trends of pumps, injectors, detectors, data systems, and specialized applications systems. System dwell volume and instrumental bandwidth are discussed, with their impacts on shorter and smaller diameter column applications. The most important performance characteristics are flow precision and compositional accuracy for the pump, sampling precision and carryover for the autosampler, and sensitivity for the detector. Manufacturers and selection criteria for HPLC equipment are reviewed. 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

It is also important to understand, that retention in RP-LC is never solely based on a pure hydrophobic or dispersion interaction, but always influenced by secondary retention mechanisms. These secondary mechanisms can be both of wanted or of an unwanted nature. A good understanding of these relationships is extremely helpful for selectivity optimization and the fundamentals are discussed in more detail in Chapter 4 on modern HPLC columns. 

Whenever a measured value exceeds a certain threshold (an internally defined limit or a legal restriction criterion) then a confirmation procedure is recommended or even necessary. The purpose of confirmation analysis is to prove or disapprove the measurement result obtained by the usual analytical method. Generally, the difference from the confirmation procedure compared to the usual test method should be due to only either the use of a completely different separation column (with completely different retention behaviour) in the same detection system or the use of an alternative detection method with sufficient sensivity. For the latter case and especially for GC methods, the prefered procedure should be to apply analyte selective mass spectroscopy (MS) detection. In some cases, derivatisation of the analyte followed by MS detection can also be the method of choice. In the case of HPLC methods, different polarity of another column in connection with full exploitation of modern UV diode array detection systems may be useful to selectively allow confirmation of the analyte. It is extremely important to make sure that the confirmation procedure works at the restriction criterion level or other self-defined concentration limit ... 

Supercritical fluid chromatography has some of the same characteristics of both HPLC and gas chromatography (GC). Packed column SFC uses the same column technology as HPLC, and when used with binary or tertiary solvents, has a broad range of applicability [1]. This range is much broader than GC, because compounds need not be volatile or thermally stable. As in GC, SFC can be coupled to most modern chromatographic detectors, such as element-specific detectors. These detectors are often very selective for... 

If no rational starting point for the separation of a racemate can be found, a random screening procedure has to be applied. In most cases 250 X 4 or 4.6 mm columns are used for this purpose. Modern HPLC systems often include a column switching device with up to 12 columns. Most systems have programmable software options for the set of different mobile phase compositions. Combination of UV and polarimetric detection allows even very small selectivities to be found and used as a starting point for further optimization. Commercial systems with deconvolution and optimization options are available, together with a given set of 12 different chiral stationary phases (www.pdr-chiral.com). After an initial detection of selectivity on a CSP, the mobile phase composition and additives as well as temperature have to be varied either manually or by means of an automated system. 

Modern HPLC has been developed to a very high level of performance by the introduction of selective stationary phases of small particle sizes, resulting in efficient columns with large plate numbers per litre. 

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