Separation Techniques with Mass Spectrometry

2 Coupling of Separation Techniques with Mass Spectrometry 

An additional dimension to library analysis is introduced when mass spectrometry is coupled with common separation techniques, for example liquid chromatography (LC) and capillary electrophoresis (CE). While these couplings are compatible with spray ionization techniques such as ESI or APCI, they more or less exclude the use of the MALDI technique. Several contributions deal with LC-ES-MS [36, 37] and CE-ES-MS-coupling [38], For molecules with isobaric nominal masses, MS/MS-experiments are performed to confirm the identity of a library component. Separation and analysis of compound mixtures may also be performed by GC-MS [39, 40], As a supplement to the more common- 

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Interfacing of solution-based separation techniques with mass spectrometry has historically been a challenge because of the incompatibility of the used solvent with the vacuum system. Standard electron impact (El) ionization with techniques such as particle beam require samples to be vaporized under high vacuum for ion formation to occur. 

The first approaches to the coupling of liquid-phase separation techniques with mass spectrometry were designed for HPLC needs, starting in the 1970s with since-forgotten techniques such as direct liquid introduction (DLI) and moving belt. In the 1980s, techniques such as thermospray, continuous-flow-fast atom bombardment (CF-FAB), and particle beam arose. 

In order to analyse a complex mixture, for example natural products, a separation technique - gas chromatography (GC), liquid chromatography (LC) or capillary electrophoresis (CE) - is coupled with the mass spectrometer. The separated products must be introduced one after the other into the spectrometer, either in the gaseous state for GC/MS or in solution for LC/MS and CE/MS. This can occur in two ways the eluting compound is collected and analysed off-line or the chromatograph is connected directly to the mass spectrometer and the mass spectra are acquired while the compounds of the mixture are eluted. The latter method operates on-line. Reviews on the coupling of separation techniques with mass spectrometry have been published in the last few years [1-4]. 

The most obvious advantage drawn from coupling a separation technique with mass spectrometry consists of obtaining a spectrum used for identifying the isolated product. However, this is not the only goal that can be reached. The ideal detector should ... 

The most frequently chosen method for compound characterization in the pharmaceutical industry is LC/MS [6]. Replacing flow injection by a chromatographic separation prior to MS analysis offers three major advantages i) impurities or by-products are separated in time from the product of interest, rendering a purity assessment of the sample possible ii) ionization suppression of the compound of interest by salts, detergents, or by-products is avoided iii) the interpretation of mass spectra of pure compounds is much easier than the MS analysis of mixtures. Combinations of separation techniques with mass spectrometry have been reviewed recently by Tomer [39]. 

Edwards, E. and Thomas-Oates, J., Hyphenating liqnid phase separation techniques with mass spectrometry on-line or off-line. AnaZy f, 130, 13, 2005. 

Why do we need separation techniques As will be discussed in Sections 5 and 6, state-of-the-art mass analyzers and tandem mass spectrometry allow mass spectrometry to be a powerful tool for the analysis of complex mixtures. The coupling of classical separation techniques with mass spectrometry further improves the utility of these combined techniques for mixture analysis. Mass spectrometers are the most sensitive and structure-specific detectors for separation techniques that, in general, provide more detailed and reliable structural information on components of complex mixtures than other conventional detectors (such as flame ionization, UV, reflective index detectors, etc.). 

High-temperature high-resolution gas chromatography (HTGC) is an established technique for the separation of complex mixtures of high molecular weight compounds (HMW) which do not elute when analyzed on conventional GC columns [530]. The combination of this technique with mass spectrometry (i.e., HTGC-MS) is not so common, however, Elias et al. [530] used this novel application to evaluate and identify the occurrence of HMW tracers (> C40) from smoke aerosols. 

However, mass spectrometry itself offers two additional degrees of freedom . One can either resolve the complexity of a sample by going to high or even ultra-high mass resolution or one can employ tandem MS techniques to separate the fragmentation pattern of a single component from that of others in a mixture. [2,3] In practice, the coupling of separation techniques to mass spectrometry is often combined with advanced MS techniques to achieve the desired level of accuracy and reliability of analytical information. [1,7,24-27]... 

The online coupling of a separation device with mass spectrometry is an analytical approach that can help in the analysis of real-world samples such as environmental samples and biological tissues and fluids. This online marriage between two stand-alone analytical techniques can provide an unequivocal characterization of individual components of such complex samples and greatly increases the information content of those components. Several issues that must be addressed to achieve an ideal combination. A major concern is the pressure mismatch. The solvent incompatibility also becomes an issue in the coupling of LC and CE with mass spectrometry. Thus, online coupling requires an interface that can transport the separated components into the ion source without affecting their resolution or the performance of a mass spectrometer. Not all types of mass spectrometers... 

Analytical Approaches. Different analytical techniques have been appHed to each fraction to determine its molecular composition. As the molecular weight increases, complexity increasingly shifts the level of analytical detail from quantification of most individual species in the naphtha to average molecular descriptions in the vacuum residuum. For the naphtha, classical techniques allow the isolation and identification of individual compounds by physical properties. Gas chromatographic (gc) resolution allows almost every compound having less than eight carbon atoms to be measured separately. The combination of gc with mass spectrometry (gc/ms) can be used for quantitation purposes when compounds are not well-resolved by gc. 

The recent development and comparative application of modern separation techniques with regard to determination of alkylphosphonic acids and lewisite derivatives have been demonstrated. This report highlights advantages and shortcomings of GC equipped with mass spectrometry detector and HPLC as well as CE with UV-Vis detector. The comparison was made from the sampling point of view and separation/detection ability. The derivatization procedure for GC of main degradation products of nerve agents to determine in water samples was applied. Direct determination of lewisite derivatives by HPLC-UV was shown. Also optimization of indirect determination of alkylphosphonic acids in CE-UV was developed. Finally, the new instrumental development and future trends will be discussed. 

The combination of chromatography and mass spectrometry (MS) is a subject that has attracted much interest over the last forty years or so. The combination of gas chromatography (GC) with mass spectrometry (GC-MS) was first reported in 1958 and made available commercially in 1967. Since then, it has become increasingly utilized and is probably the most widely used hyphenated or tandem technique, as such combinations are often known. The acceptance of GC-MS as a routine technique has in no small part been due to the fact that interfaces have been available for both packed and capillary columns which allow the vast majority of compounds amenable to separation by gas chromatography to be transferred efficiently to the mass spectrometer. Compounds amenable to analysis by GC need to be both volatile, at the temperatures used to achieve separation, and thermally stable, i.e. the same requirements needed to produce mass spectra from an analyte using either electron (El) or chemical ionization (Cl) (see Chapter 3). In simple terms, therefore, virtually all compounds that pass through a GC column can be ionized and the full analytical capabilities of the mass spectrometer utilized. 

If we consider only a few of the general requirements for the ideal polymer/additive analysis techniques (e.g. no matrix interferences, quantitative), then it is obvious that the choice is much restricted. Elements of the ideal method might include LD and MS, with reference to CRMs. Laser desorption and REMPI-MS are moving closest to direct selective sampling tandem mass spectrometry is supreme in identification. Direct-probe MS may yield accurate masses and concentrations of the components contained in the polymeric material. Selective sample preparation, efficient separation, selective detection, mass spectrometry and chemometric deconvolution techniques are complementary rather than competitive techniques. For elemental analysis, LA-ICP-ToFMS scores high. 

Hyphenated techniques like combination of optical detection methods based on reflectometry or refractometry and separation techniques are of future interest. The same is valid for the intention to couple SPR or RIfS with mass spectrometry like MALDI33. 

Over the past two decades, capillary electrophoresis (CE) and related techniques have rapidly developed for the separation of a wide range of analytes, ranging from large protein molecules to small inorganic ions. Gas chromatography has been considered as a powerful tool due to its sensitivity and selectivity, especially when coupled with mass spectrometry. Nevertheless, liquid chromatography is the most used method to separate and analyze phenolic compounds in plant and tissue samples. 

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