Some Applications of hplc

In this section we are going to look at some case studies to see how hplc experimental methods are developed. 1 am not going to give a long list of applications, because these are easy to find elsewhere, and sometimes do not make very interesting reading. Most textbooks on hplc have lists of applications, eg the book by Hamilton and Sewell (2nd Edn, Chapter 8), and applications can also be obtained from a number of journals (eg Analytical Chemistry annual reviews). 

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Table 10.16 summarizes some applications of HPLC in Pharmaceutical Chemistry which encompass drug stability studies, the determination of trace impurities or decomposition products in bulk drug samples, and the assay of drugs and metabolites in body fluids. 

Mondello et al. (54) have developed some applications of on-line HPLC-HRGC and HPLC-HRGC/MS in the analysis of citrus essential oils. In particular, they used LC-GC to determine the enantiomeric ratios of monoterpene alcohols in lemon, mandarin, bitter orange and sweet orange oils. LC-GC/MS was used to study the composition of the most common citrus peel, citrus leaf (petitgrain) and flower (neroli) oils. The oils were separated into two fractions, i.e. mono- and sesquiterpene... 

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

HPLC methods have been widely used for the analysis of OTC in different samples. As described above in the Section 2.3, the HPLC method is described in most of compendia [1,2,4,7] for determination of OTC in bulk drug substances and in some pharmaceutical preparations. The application of HPLC methods for the analysis of antibiotics including oxytetracycline has been recently reviewed by Diaz-Cruz et al. [37] and Lunn [38], A summary of HPLC method for the analysis of OTC is presented in Table 3. 

HPLC and the extensive complexity of ESI and MALDI spectra for multicomponent polymers with molar mass over about 10 g mol. Some applications of MALDI MS in polymer HPLC can be found for example in [300-303],... 

A number of criteria could be apphed to organize this chapter, depending on the point of view by which foods are considered. In this chapter, application of HPLC to food analysis will be described considering homogeneous classes of food components lipids, carbohydrates and related substances, proteins, peptides, amino acids, biogenic amines, phenolics, vitamins, and some selected contaminants. 

Table 19.4 reports some applications concerning HPLC analysis of carbohydrates in food samples. 

The aim of this chapter is not a detailed description of the technique and instrumentation but to show applications of HPLC in the preparative separation of flavonoids. Some representative examples are given in Table 1.1. In a 1982 review of isolation techniques for flavonoids, preparative HPLC had at that time not been fully exploited. However, the situation is now very different and 80% of all flavonoid isolations contain a HPLC step. Approximately 95% of reported HPLC applications are on octadecylsilyl phases. Both isocratic and gradient conditions are employed. 

Gas chromatography of dimer methyl esters at high temperature on a short column also has been reported (73). The separation of trimer from dimer was incomplete in this method, and it is likely that some sample degradation occurs during the analysis because of the high temperatures involved. More promising approaches have been developed with the application of HPLC to this problem (74,75). 

This present chapter will summarise the application of HPLC-NMR and HPLC-NMR-MS to studies of biomedical and pharmaceutical interest and include some limited examples of further hyphenation (hypernation) of UV, NMR, MS and IR spectroscopy that illustrate the potential of these combinations in this field [3,4]. This chapter excludes examples in the drug metabolism field involving the separation, identification, and in some cases, quantification of metabolites in biofluids and their extracts as these are considered elsewhere in this volume. However, because of their general relevance, HPLC-NMR-MS studies of the chemical reactivity of such molecules will be discussed. 

Applications of HPLC for Hg speciation studies have been reviewed by Harrington.83 Practically all HPLC methods for Hg speciation reported in the literature are based on reversed-phase separations, involving the use of a silica-bonded phase column and a mobile phase containing an organic modifier, a chelating or an ion pair reagent, and in some cases, a pH buffer. 

In most analytical applications of HPLC, all these discrepancies are quietly and conveniently forgotten, and selection of some so-called nonretained component as a void volume marker is a common way for void volume measurement. In the majority of recent analytical publications, either thiourea or uracil were used as the void volume markers. As a disclaimer, we have to say here that for the purposes of analytical method development, qualitative or quantitative separation of complex mixtures which involves the use of a nonretained component as a void volume marker is acceptable insofar as there are no physicochemical generalization, thermodynamic development, or futher theoretical development performed upon the basis of these pseudo void volume determinations. 

Among some recent reviews of the applications of HPLC are a general one of the application to a wide range of products [261], the application to the resolution of complex biological mixtures [262], its application in pharmaceutical analysis [263], and its use within the clinical laboratory [264]. 

These will now be considered briefly in relation to protein separation the reader is referred to some of the numerous books and reviews on HPLC for further details of the theory and applications of HPLC (B22, C8, D9, H5, KIO). 

The utility of ion-exchange HPLC for the separation of biomolecules is specially noteworthy and some applications of the technique in this field are listed in Table 10.11. 

Some examples of the application of HPLC in the chemical industry have been given earlier in this chapter during the course of discussion of working of the individual modes of HPLC. Table 10.21 lists some more examples of the application of HPLC in the chemical industry. 

The following sections discuss the application of HPLC to the various methods used in the isolation and analysis of proteins, peptides and amino acids. The development of new stationary phases coupled with instrumentation which allows unattended gradient development has transformed the task of purification. Some of these aspects will also be discussed in this section. 

Quantitative analysis could be improved by the introduction of the joint application of HPLC and NMR spectroscopy in drug impurity profiling. This is demonstrated by GSrog et al. for some examples, such as the 17a and 17 3 isomers of an ethinylestradiol and impurities of enalapril maleate [40]. The details of this technique and further applications will be discussed in the section NMR in body fluids. 

Forensic science encompasses a number of different fields of science. In this book, we are explaining the theories associated with high performance liquid chromatography, the different forms that it may take, and its use in some forensic applications. In this chapter, we will examine applications of HPLC in drug analysis, toxicology, analysis of explosives, analysis of coloured materials, and environmental science. 

Chapter 11 looks at some of the applications of HPLC within the field of forensic science in greater detail. We have covered the most common areas, but we acknowledge that both HPLC and forensic science are far-reaching fields of study. 

The majority of applications of HPLC in clinical laboratories are based on the use of blood, plasma/ serum, or urine samples, although feces and tissue samples are also used. The high protein content of blood samples ( 70gl in plasma or serum) necessitates some form of sample pretreatment prior to injection onto the column to reduce background interference and to avoid column clogging. The specific preparation steps found most appropriate for the applications listed above will be described in the... 

Analysis of the components of other fractions of simple lipids like free sterols, sterol esters, and wax esters by means of HPLC has some drawbacks, and it is usually performed using high-temperature GC. However, the most remarkable field of application of HPLC is the analysis of oxidized sterol and their esters, of biological interest, that used to be performed earlier using RP-LC with postcolumn fluorometric detection and is nowadays be carried out using LC coupled to MS, providing structural information on these complex derivatives. 

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