Separation Techniques Coupled with Mass Spectrometry

2 Separation Techniques Coupled with Mass Spectrometry 

Modern chromatographic and electrophoretic techniques (e.g., UHPLC, GC, and CE) are characterized with high apparent numbers of theoretical plates (typically, 10 -10 ). While chromatograms and electropherograms obtained with these separation techniques can 

Please note that analytes introduced to GC columns need to be volatile and amenable to detection using the detector at the column outlet. To render less volatile compounds amenable to analysis by GC-MS, derivatization is often conducted by reacting sample components with a generic or specific reagent. While derivatization is normally conducted off-line, in principle, it can be done on-line. In one smdy, a system was developed in which analytes were first separated in an LC column followed by on-line derivatization, and introduction of the resulting mixture to a GC column for further separation and detection by MS using an electron ion source and a quadrupole ion trap analyzer [11]. 

An important issue to address is matching the flow rates of separation systems and ion sources. Conventional LC systems operate at high flow rates (milliliters per minute) 

Modern chromatographic systems are equipped with autosamplers. Samples can be injected directly into the separation column, or they can be passed through enrichment (trapping) columns. The concentrated analytes can subsequently be directed to the separation columns. This step extends analysis time however, it may be indispensable in the case of analysis of low abundance components of matrix-rich samples. 

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Erny, G. L., and Cifuentes, A. (2006). Liquid separation techniques coupled with mass spectrometry for chiral analysis of pharmaceuticals compounds and their metabolites in biological fluids. ]. Pharm. Biomed. Anal. 40, 509—515. 

Chromatographic techniques coupled with mass spectrometry (GC-MS, GC-MS/MS, LC-MS and LC-MS/MS) have also been applied in many combinations. A library of reference mass spectra is an integral part of each type of GC-MS apparatus, enabling typing of spectra that are potentially similar to the identified compound. For the GC-MS-EI technique, a combination of two previously separate libraries, Wiley and NIST 2008 (W8/N08), should be mentioned because of its extensiveness. Library W8/N08 contains 562,000 El spectra, 5308 spectra of parent ions (precursors) subjected to fragmentation by the MS/MS technique, over 2 million names of chemical compounds and their synonyms, 35,000 structural formulae and 43,000 GC retention indices. The Automated Mass Spectrometry... 

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. 

Either gas chromatography (GC) or liquid chromatography (LC) can be used as a separation technique coupled with a variety of detection methods. Mass spectrometry (MS) is one of the most popular means of detection. When using GC-MS, a capillary column should be used, while any suitable LC column can be used for LC-MS. It is advisable to obtain a print-out of the chromatogram so that the shapes of individual peaks can be assessed. Electronically produced data using integrators should be treated with some suspicion and always examined visually to check the selected baseline, start- and end-points of peak integration, etc. 

Halket and Lisboa (25) examined several Vitamin D derivatives by capillary gas chromatography coupled with mass spectrometry. This technique offered the advantages of great sensitivity and separating power. Retention times and fragmentation patterns for ergocalciferol, cholecalciferol and calcitriol were reported. 

Whilst these methods are informative for the characterisation of synthetic mixtures, the information gained and the nature of these techniques precludes their use in routine quantitative analysis of environmental samples, which requires methods amenable to the direct introduction of aqueous samples and in particular selective and sensitive detection. Conventionally, online separation techniques coupled to mass spectrometric detection are used for this, namely gas (GC) and liquid chromatography (LC). As a technique for agrochemical and environmental analyses, high performance liquid chromatography (HPLC) coupled to atmospheric pressure ionisation-mass spectrometry (API-MS) is extremely attractive, with the ability to analyse relatively polar compounds and provide detection to very low levels. 

Very complex mixtures can be separated by this technique. When coupled with mass spectrometry as a detection system, virtually positive identification of the eluted compounds is possible at very high sensitivity, creating a very powerful analytical system. 

GC has been widely used for amine analyses because of its simplicity, high resolving power, high sensitivity, and low cost. Coupled with mass spectrometry (GC-MS), it is a technique most commonly employed for the analysis of volatile organic pollutants in environmental samples. In this combination, the GC separation usually provides isomer selectivity, while the MS shows compound class homologue specificity. The MS fragmentation pattern can provide unambiguous component identification by comparison with library spectra. 

Gas chromatography is often coupled with mass spectrometry in a technique called GC/MS analysis. The gas chromatograph separates components of a mixture, while the mass spectrometer then gives structural information about each one (Fig. 9.45). 

Apart from the mentioned technological advances, the separation power and selectivity of HPLC have been drastically improved over the last years. In this context, ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) represents a relatively new category of analytical coupled techniques that respects the principles of the classic HPLC-MS while bringing some improvements in chromatographic resolution, speed, and sensitivity. 

However, full characterization of functionalized siloxanes, particularly those containing high degrees of polymerization, can be difficult. Therefore, separation techniques coupled with various detection devices are typically employed. Supercritical fluid chromatography is effective for the separation of silicone oligomers that have molecular weights less than 3000 Da (688,689). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is applicable up to 10,000 Da (660). 

The use of selective detectors is the most promising route to the identification of unknown components in a complex mixture. Chromatography can separate complex mixtures into individual components, but it cannot positively identify them. Most spectrometric methods, on the other hand, supply sufficient information to identify complex compounds, but only if they are so pure that all the spectral clues can be ascribed safely to the same compound. Among the possible combinations, coupling with mass spectrometry, and to a lesser degree IR spectrometry, have been the most fruitful, because the amount of sample easily handled in classical chromatography is well suited to these techniques. Less sophisticated detectors have also been used to advantage. 

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