Hyphenated Liquid Chromatography Techniques

Keywords Crystallization analysis fractionation Field Flow Fractionation High performance liquid chromatography Hyphenated techniques Liquid chromatography Polyolefin analysis SEC-NMR coupling Size exclusion chromatography Temperature rising elution fractionation Two-dimensional liquid chromatography... 

Mass spectrometry, on its own, does not find much, if any, use in forensic science due to the nature of the samples that are analysed (sample matrices tend to very complex, e.g., blood). In this section, we shall look at the aspects of mass spectrometry as it relates to hyphenation with liquid chromatography. Hyphenation is the combining of the two techniques whereby HPLC is used to separate a mixture of components each analyte is introduced into the mass spectrometer as a single compound (i.e., no longer part of the matrix of the original sample). 

The spectrum of new analytical techniques includes superior separation techniques and sophisticated detection methods. Most of the novel instruments are hyphenated, where the separation and detection elements are combined, allowing efficient use of materials sometimes available only in minute quantities. The hyphenated techniques also significantly increase the information content of the analysis. Recent developments in separation sciences are directed towards micro-analytical techniques, including capillary gas chromatography, microbore high performance liquid chromatography, and capillary electrophoresis. 

Gas and liquid chromatography directly coupled with atomic spectrometry have been reviewed [178,179], as well as the determination of trace elements by chromatographic methods employing atomic plasma emission spectrometric detection [180]. Sutton et al. [181] have reviewed the use and applications of ICP-MS as a chromatographic and capillary electrophoretic detector, whereas Niessen [182] has briefly reviewed the applications of mass spectrometry to hyphenated techniques. 

Because carotenoids are light- and oxygen-sensitive, a closed-loop hyphenated technique such as the on-line coupling of high performance liquid chromatography (HPLC) together with nuclear magnetic resonance (NMR) spectroscopy can be used for the artifact-free structural determination of the different isomers. 

Pasch, H. Analysis of Complex Polymers by Interaction Chromatography. Vol. 128, pp. 1 -46. Pasch, if. Hyphenated Techniques in Liquid Chromatography of Polymers. Vol. 150,... 

As liquid chromatography plays a dominant role in chemical separations, advancements in the field of LC-NMR and the availability of commercial LC-NMR instrumentation in several formats has contributed to the widespread acceptance of hyphenated NMR techniques. The different methods for sampling and data acquisition, as well as selected applications will be discussed in this section. LC-NMR has found a wide range of applications including structure elucidation of natural products, studies of drug metabolism, transformation of environmental contaminants, structure determination of pharmaceutical impurities, and analysis of biofiuids such as urine and blood plasma. Readers interested in an in-depth treatment of this topic are referred to the recent book on this subject [25]. 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The... 

The concurrent identification and quantification of organic impurities is a principal use of liquid chromatography in the pharmaceutical industry. However, the application of liquid chromatography to this task highlights a weakness of this technique when compared to gas chromatography specifically, the lack of a universal detector. Great strides have been made to create detectors and hyphenated techniques to address these problems. However, multiple detectors and analytical procedures may be necessary to accurately and specifically identify and quantify the impurities in complex systems. 

Mass spectrometry (MS) is probably a famhiar tool to chemistry and biology students as a technique commonly used to measure the molecular mass of a sample. Often, MS is used in tandem with other techniques for chromatic separation of the sample before mass measurement. Some common hyphenated techniques include HPLC-MS, high-pressure liquid chromatography coupled to MS GC-MS, gas chromatography coupled to MS or CE-MS, capillary electrophoresis coupled to MS. 

Pasch, H. Hyphenated Techniques in Liquid Chromatography of Polymers. Vol. 150,... 

Modern drug discovery approaches involve HTS, where, applying full automation and robotics, hundreds of molecules can be screened using several assays within a short time, and with very little amounts of compounds. In order to incorporate natural products in the modern HTS programmes, a natural product library (a collection of dereplicated natural products) needs to be built. Dereplication is the process by which one can eliminate recurrence or re-isolation of same or similar compounds from various extracts. A number of hyphenated techniques are used for dereplication, e.g. LC-PDA (liquid chromatography-photo-diode-array detector). 

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