Separation in HPLC

A. M. Krstulovic (Ed.), "Chiral Separations in HPLC , Ellis Norwood, Chichester, OK, 1989. 

Discussed below are various modes of separations in HPLC. Included here is brief coverage of mobile-phase selection for various modes of chromatography and elementary information on mechanism, choice of solvents and columns, and other practical considerations. It should come as no surprise that reversed-phase HPLC is discussed at greater length in this section because it is the most commonly used technique in HPLC (more detailed discussion is provided in Section 15.8). Clearly,... 

It is clearly not possible to cover all these applications within the scope of this chapter. Detailed applications can be found in the literature [1-3,5,7]. Reproduced below is a case study to show how one may select a mode of separation in HPLC and at the same time develop a better understanding of separation mechanism. 

Because the HPLC mobile phase is a liquid, there are some very obvious differences between HPLC and GC. First, the mechanism of separation in HPLC involves the specific interaction of the mixture components with a specific mobile phase composition, while in GC the vapor pressure of the components,... 

Although the overwhelming majority of separations in HPLC are carried out with the supports described above, separations using other basic principles have also found application in HPLC. 

Chiral separation of drng molecules and of their precursors, in the case of synthesis of enantiomerically pure drugs, is one of the important application areas of HPLC in pharmaceutical analysis. Besides HPLC, capillary electrophoresis (CE) is another technique of choice for chiral separations. Chapter 18 provides an overview of the different modes (e.g., direct and indirect ones) of obtaining a chiral separation in HPLC and CE. The direct approaches, i.e., those where the compound of interest is not derivatized prior to separation, are discussed in more detail since they are cnrrently the most frequently used techniques. These approaches require the use of the so-called chiral selectors to enable enantioselective recognition and enantiomeric separation. Many different molecnles have been nsed as chiral selectors, both in HPLC and CE. They can be classified into three different groups, based on their... 

For the majority of the direct chiral separations in HPLC, different kinds of CSPs are used. The CSP can consist of small chiral molecules or polymers [22] and is often immobilized on agarose [23], silica gel [23,24], or polymer particles [23]. During the two last decades, a number of new phases have been introduced. Most commonly used CSPs are listed in Table 17.1. 

Since increased temperature is known to improve separation in HPLC, Sandra et al. performed CEC separations of triglycerides at temperatures of up to 50°C and did not observe any bubble formation or breakdown of the current [18]. However, Knox and McCormack [19] demonstrated that high temperature might also affect the sample injection. If the rate of thermal expansion of the liquid within the capillary is faster than the rate of electromigration of the slowest component, some compounds present in the injected sample may not enter the capillary. 

Example of a set of columns (I-1II) that may be used to realize separations in HPLC requiring up to 10,000 theoretical plates. Column IV may be used to extend the set to 20,000 plates. Conditions are given in figure 7.2. 

A rapid method for making acetonides, coupled with liquid chromatography-MS (LC-MS) has been used to study the structure of ecdysteroid metabolites. Information about the structure can be deduced from chromatographic retention times. Mono-, di-, and tri-acetonides are well separated in HPLC.55... 

Other Examples of the Use of Principal Properties Characterization by principal properties has been reported for classes of compounds in applications other than organic synthesis Aminoacids, where principal properties have been used for quantitative structure-activity relations (QSAR) of peptides [64], Environmentally hazardous chemicals, for toxicity studies on homogeneous subgroups [65]. Eluents for chromatography, where principal properties of solvent mixtures have been used for optimization of chromatographic separations in HPLC and TLC [66],... 

Cela, R. et al. PREOPT-W off-line optimization of binary gradient separation in HPLC by simulation IV phase 3. Comput. Chem. 1996, 20, 315-330. 

High-performance liquid chromatography (or less common, high-pressure liquid chromatography, HPLC) is a preferred method of analysis for many compounds because it does not require the high temperatures used in gas chromatography. Separations in HPLC can be based on either a size exclusion or on an adsorption principle. The size exclusion mode is useful for separating fatty acids from... 

Gradient high-performance liquid chromatography (HPLC) has been useful for the characterization of copolymers (14-19). In such experiments, careful choice of separation conditions is a conditio sine qua non. Otherwise, low resolution for the polymeric sample will obstruct the separation. However, the separation in HPLC, dominated by enthalpic interactions, perfectly complements the entropic nature of the SEC retention mechanism in the characterization of complex polymer formulations. 

Edkins, T. J. and D. C. Shelly, Measurement concepts and laser-based detection in high-performance micro separation, in HPLC Detection Newer Methods (G. Patonay, ed.), VCH, New York, 1992, pp. 1-15. 

The mobile phase controls the separation Whereas the stationary phase provides a media for analyte interaction, the mobile phase controls the overall separation. In HPLC method development, efforts focus on finding a set of mobile phase conditions for separating the analyte (s) from other components. Exceptions to this rule are size exclusion, chiral, and affinity chromatography. 

Several books are devoted to the chiral separations in HPLC [69,93,104,108,110]. Among several hundreds of CSPs described, ligand-exchange [105], Pirkle-type [113], protein and peptide [114], polysaccharide [115], macrocyclic [93,116], and synthetic polymeric [117-119] CSPs are most widely used. 

Separation in HPLC can be achieved by exploiting a Variety of sorption processes, of which the more important are shown in Table 6.1 together with the dominant sorption mechanisms. 

The value represents the sum of M6 and M7 radioactivity since the two metabolites were not separated in HPLC-LSC analysis. 

CyDs have been used as major chiral mobile phase additives (CMPAs) for enantio-separations in HPLC. The first application of 8-CyD as a CMPA in combination with an achiral reversed-phase material for HPLC enantioseparations was reported by Sybilska and co-workers in 1982 [27]. These authors could achieve partial resolution of the enantiomers of mandelic acid and derivatives. The CMPA method played an important role in HPLC enantioseparations before the development of effective chiral stationary phases (CSPs) but is now rarely used. The major disadvantage of this technique, together with difficulties associated with the isolation of resolved enantiomers, is the rather large consumption of chiral selector. 

This relatively simple system demonstfates the potential of a microfluidic chip for separations in HPLC mode. It was certainly not ideally suited for the separations of more complex mixtures since it was only used in isocratic conditions and the several minutes long retention times are too long for... 

Today, the majority of separations in HPLC are conducted on reversed-phase materials. These materials can be balanced more rapidly, require a solvent with aqueous organic components and are more versatile with regard to their areas of application. 

There are many parameters which control the enantiomeric resolution by HPLC. The most important of them include parameters of the stationary phase, such as particle size of CSP, pore size of column, and kind of chiral selector, composition, and pH of the mobile phase, flow rate of mobile phase, and temperature. Systematic variation of column temperature should be considered as one way to improve chiral separations in HPLC. From the practical point of view, it is easier to vary column temperatures than mobile phase composition. In addition, variable temperature runs can provide useful information concerning the thermodynamic parameters for the CSP-analyte interactions. The effect of temperature on the resolution... 

Edkins, T. J. and D. C. Shelly, Measurement concepts and laser-based detection in high-performance micro separation, in HPLC Detection Newer Methods (G. Patonay, ed.), VCH, New York, 1992, pp. 1-15. Goodall, D. M. and D. K. Lloyd, A note on an optical rotation detector for high-performance liquid chromatography, in Chiral Separations (D. Stevenson and D. Wilson, eds.). Plenum Press, New York, 1988, pp.131-133. 

---