Atmospheric pressure chemical separation techniques

Samples containing mixtures of peptides can be analyzed directly by electrospray. Alternatively, the peptides can be separated and analyzed by LC/MS coupling techniques such as electrospray or atmospheric pressure chemical ionization (APCI). 

A number of analytical techniques such as FTIR spectroscopy,65-66 13C NMR,67,68 solid-state 13 C NMR,69 GPC or size exclusion chromatography (SEC),67-72 HPLC,73 mass spectrometric analysis,74 differential scanning calorimetry (DSC),67 75 76 and dynamic mechanical analysis (DMA)77 78 have been utilized to characterize resole syntheses and crosslinking reactions. Packed-column supercritical fluid chromatography with a negative-ion atmospheric pressure chemical ionization mass spectrometric detector has also been used to separate and characterize resoles resins.79 This section provides some examples of how these techniques are used in practical applications. 

Rosenberg, E. (2003). The potential of organic (electrospray- and atmospheric pressure chemical ionization) mass spectrometric techniques coupled to liquid-phase separation for speciation analysis. /. Chromatogr. A 1000, 841 — 889. 

A striking feature of the ILs is their low vapor pressure. This, on the other hand, is a factor hampering their investigation by MS. For example, a technique like electron impact (El) MS, based on thermal evaporation of the sample prior to ionization of the vaporized analyte by collision with an electron beam, has only rarely been applied for the analysis of this class of compounds. In contrast, nonthermal ionization methods, like fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), atmospheric pressure chemical ionization (APCI), ESI, and MALDI suit better for this purpose. Measurement on the atomic level after burning the sample in a hot plasma (up to 8000°C), as realized in inductively coupled plasma (ICP) MS, has up to now only rarely been applied in the field of IE (characterization of gold particles dissolved in IE [1]). This method will potentially attract more interest in the future, especially, when the coupling of this method with chromatographic separations becomes a routine method. 

The number of detectors that are sensitive and selective enough to be applied online with LC is limited because the solvents used are not compatible, as in the case of immunochemical detection after reversed- or normal-phase LC. The technology of coupling is still under development and not yet available in a large number of laboratories not specialized in techniques such as LC-MS. Therefore, LC separations are frequently followed by offline detection. Confirmatory analysis of suspected liquid chromatographic peaks can be made possible by coupling liquid chromatography with mass spectrometry. Atmospheric-pressure chemical ionization LC-MS has been employed for the identification of six steroid hormones in bovine tissues (448). 

In recent years the rapid development of high-sensitivity analytical techniques such as mass spectrometry (MS) and liquid chromatography (LC) supported the investigation of the endocannabinoids as part of a complex lipid network. The identification of lipid components of the endocannabinoid system can be achieved in a single analytical step by state-of-the-art platforms such as tandem mass spectrometry (MS/MS), which provides the detailed structural information necessary for characterization of lipids and increases specificity in complex biological matrices. Furthermore, the implementation of ionization techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) allow the coupling of LC to MS, and permits the separation and analysis of endocannabinoids with greater speed and accuracy. 

Application of LC-MS/MS techniques to the analysis of phthalate ester metabolites in urine have also been developed. For example, Blount et al. (2000b) have developed an assay to quantify the monoester metabolites (including MEHP) of eight phthalate diesters in urine, utilizing HPLC coupled with atmospheric pressure chemical ionization and tandem mass spectrometric (APCI-MS/MS) detection techniques. Urine samples were treated with -glucuronidase to release the free phthalate monoesters followed by a two-step solid phase extraction procedure. After evaporative concentration of the eluant, the analytes in the purified samples are further separated on a phenyl reverse phase HPLC column and quantified by APCI-MS/MS, following careful optizimation of the APCI-MS/MS instrument. The limits of detection for MEHP were determined to be 1.2 ng/ml urine with recovery efficiencies of between 78 and 91%. 

The most recent products of this evolution are electrospray ionization (ESI)18 and atmospheric pressure chemical ionization (APCI).19 These techniques enable the routine on-line analysis of a wide variety of compounds using a variety of separation conditions (i.e., solvents, additives or buffers, flow rates, etc.). 

It has become painfully obvious that most of the excellent approaches and techniques that have been developed for use in liquid chromatography are not applicable to liquid chromatography/mass spectrometry (LC/MS) with atmospheric pressure ionization. Chapter 5 described the reagents and the range of mobile-phase compositions that are compatible with electrospray and atmospheric pressure chemical ionization (APCI), and these are limited to volatile components that do not cause significant ion suppression. Certain problems that are not significant with standard LC separations become difficult to deal with because of the limitations placed on the mobile phase by atmospheric pressure ionization (API) LC/MS. 

Suitable analytes exhibit high electron capture capacity or high electron affinity. Cl is the technique of choice for the analysis of isomers in environmental samples. In APCI (Atmospheric Pressure Chemical Ionization) solvent evaporation and analyte ionization are two separate processes. 

Probably the most common separation systems used in the laboratory today require the sample to be in solution (e.g. HPLC, CE). The solvent may be aqueous or solvent based. However, onemL of such solution yields far too much vapour (1-2L) to be accommodated by a mass spectrometer s vacuum system. Thus the aim of a sample introduction system for such solutions would require the sample to be ionised and the solvent to be separated from these sanple ions. In addition the interface must maintain the integrity of the chromatography. The chromatographic separation must be maintained as well as allowing sufficient analyte through to generate a mass spectmm. A number of methods have been developed to do this, but the two main techniques used today are electrospray and atmospheric pressure chemical ionisation (APCI for short). These are described below under ionisation techniques. 

Separation is more usually carried out in the liquid, rather than gas phase. Neither El nor Cl can deal very effectively with samples in solution and a number of techniques to overcome this have arisen over the last few years. The first widely used method was thermospray [16], However, this has been largely superseded by atmospheric pressure chemical ionisation (APCl). The other commonly applied technique is electrospray. These techniques are described in detail below. 

Right from the outset of the 1990s, a selection of those interfaces that could be adapted to a routine LC-MS analysis was observable. This trend had been initiated by pharmacological and pharmaceutical research, although it had the TSP interface at its disposal, which was a well-adapted and reliable type of interface that had shown its fiiU capacity in manifold appliances. The sample material, being available only in very limited quantities for such research, and improved separation techniques, as, for example, capillary electrophoresis (CE) or capillary zone electrophoresis (CZE) necessitated different types of interfaces that could be operated with considerably smaller amounts of sample than the TSP interface, which reached its optimized sensitivity with flow rates of about 2 mL min. Such a desirably lower sample demand is guaranteed by atmospheric pressure ionisation (API) interfaces, atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI) interface. 

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