TSP interface

The most important area for packed column use involves modified mobile phases (MPs). Consequently, pSFC needs detection systems in which the MP modifier and possible additive(s) do not interfere, and in which detection of low or non-UV-absorbing molecules is possible in combination with pressure/modifier gradients. The disadvantage of adding even small amounts of modifier is that FID can no longer be used as a detector. In the presence of polar modifiers in pSFC the detection systems are restricted basically to spectroscopic detection, namely UVD, LSD, MSD (using PB and TSP interfaces as in LC). ELSD can substitute FID and covers the quasi-universal detection mode, while NPD and ECD cover the specific detection mode in pSFC on a routine basis. As ELSD detects non-UV absorbing molecules dual detection with UV is an attractive option. 

Different options are available for LC-MS instruments. The vacuum system of a mass spectrometer typically will accept liquid flows in the range of 10-20 p,L min-1. For higher flow-rates it is necessary to modify the vacuum system (TSP interface), to remove the solvent before entry into the ion source (MB interface) or to split the effluent of the column (DLI interface). In the latter case only a small fraction (10-20 iLrnin ) of the total effluent is introduced into the ion source, where the mobile phase provides for chemical ionisation of the sample. The currently available commercial LC-MS systems (Table 7.48) differ widely in characteristics mass spectrometer (QMS, QQQ, QITMS, ToF-MS, B, B-QITMS, QToF-MS), mass range m/z 25000), resolution (up to 5000), mass accuracy (at best <5ppm), scan speed (up to 13000Das-1), interface (usually ESP/ISP and APCI, nanospray, PB, CF-FAB). There is no single LC-MS interface and ionisation mode that is readily suitable for all compounds... 

Capillary HPLC-MS has been reported as a confirmatory tool for the analysis of synthetic dyes [585], but has not been considered as a general means for structural information (degradant identification, structural elucidation or unequivocal confirmation) positive identification of minor components (trace component MW, degradation products and by-products, structural information, thermolabile components) or identification of degradation components (MW even at 0.01 % level, simultaneous mass and retention time data, more specific and much higher resolution than PDA). Successful application of LC-MS for additive verification purposes relies heavily and depends greatly on the quality of a MS library. Meanwhile, MB, DLI, CF-FAB, and TSP interfaces belong to history [440]. 

The TSP interface was very popular and attractive to chromatogra-phers in the 1980s, as a result of its ease of operation and dependable performance. Commercial TSP LC/MS systems are equipped with an electron emitter filament to enhance the Cl process. 

At present, the most powerful and promising interfaces for drug residue analysis are die particle-beam (PB) interface that provides online EI mass spectra, the thermospray (TSP) interface diat works well with substances of medium polarity, and more recently the atmospheric pressure ionization (API) interfaces that have opened up important application areas of LC to LC-MS for ionizable compounds. Among die API interfaces, ESP and ISP appear to be the most versatile since diey are suitable for substances ranging from polar to ionic and from low to high molecular mass. ISP, in particular, is compatible with the flow rates used with conventional LC columns (70). In addition, both ESP and ISP appear to be valuable in terms of analyte detectability. These interfaces can further be supplemented by preanalyzer collision-induced dissociation (CID) or tandem MS as realized with the use of triple quadrupole systems. Complementary to ESP and ISP interfaces with respect to the analyte polarity is APCI with a heated nebulizer interface. This is a powerful interface for both structural confirmation and quantitative analysis. 

The TSP interface is widely used for tlie determination of drug residues in foods (86). TSP is typically used with reversed-phase columns and volatile buffers. 

The TSP interface is typically combined with quadrupole MS, but coupling with ion-trap (91) or magnetic sector MS (92) has been also reported. Drawbacks of LC-TSP-MS are the requirements for volatile modifiers and the control of temperature, particularly for thermolabile compounds. Lack of structural information from LC-TSP-MS applications can be overcome by the use of LC-TSP-MS-MS. Use of this tandem MS approach provides enhanced selectivity, generally at the cost of a loss of sensitivity as a consequence of a decreased ion transmission. 

In contrast to TSP interface, no extensive temperature optimization is needed with APCI. For systems providing a countercurrent drying gas, it is claimed that volatile as well as nonvolatile buffers can be used. Uncharged volatile material is swept away from the nozzle by the countercurrent drying gas, whereas nonvolatile contamination deposited in the source chamber can readily be wiped away without the need to switch off tire vacuum system. 

A series of highly complex interfaces were described between 1978 and 1980 by Blakley et al. [62-64] in an attempt to develop a system capable of the introduction of up to 1 ml/min aqueous mobile phase into an El mass spectrometer. The systems contained extensive multistage vacuum systems and ingenious heating devices, e.g., laser evaporation or hydrogen-flame heaters, to achieve rapid solvent evaporation. These systems subsequently developed towards the TSP interface (Ch. 4.7). 

While in the TSP interface the heated solvent is nebulized into a medium-pressure ion source region, other systems have been described in which nebulization into an atmospheric-pressure system is performed. In an atmospheric-pressure spray system, as described by Sakairi and Kambara [94-95], a TSP nebulizer is used for the efficient introduction and evaporation of a mobile phase into an APCI source. In other systems, a heated nebulizer is used to achieve sample introduction in an APCI source. 

The TSP interface was developed in the laboratories of Vestal at the University of Houston. It was the result of a long-term research project which started in the mid-70 s, aiming at the development of an LC-MS interface which is compatible with 1 ml/min of aqueous mobile phase and capable to provide both El and solvent-independent Cl [62]. The initial interface was a highly complex system, which subsequently was greatly simplified with respect to vaporizer design and vacuum system [58, 62-65]. Developments in vaporizer design are summarized in Table 4.1. Finally, direct electrically-heated vaporizers were applied [64-65]. 

The performance of the TSP interface is determined by many interrelated experimental parameters, such as solvent composition, flow-rate, vaporizer temperature, repeller potential, and ion source temperature. These parameters have to be optimized with the solvent composition nsed in the analysis. This optimization procedure is often performed by column-bypass injections, in order to save valuable analysis time. However, for several compounds the spectral appearance may differ between column-bypass and on-column injection, owing to the influence of subtle differences in solvent composition or matrix effects. 

With the development of the TSP interface for LC-MS (Ch. 4.7), Vestal et al. [4, 16-18] also introduced a new ioiuzation technique. While the analyte ionization in their first experiments was initiated by electrons from a filament, they subsequently demonstrated that collision of the vapour-droplet beam from the TSP nebulizer with a nickel-plated copper plate leads to soft ioiuzation of analytes. Next, the collision was found not to be a vital step in the process [18]. The presence of a volatile buffer or acid in the mobile phase appeared more important in TSP, i.e., in charging the droplets generated by TSP, and in generation of preformed ions in solution. The ionization phenomena were explained in terms of the ion evaporation (lEV) model [4]. 

The thermospray (TSP) interface is widely used for the determination of drug residues in foods. Thermospray is typically used with reversed-phase columns and volatile buffers. Aqueous mobile phases containing an electrolyte, such as ammonium acetate, are passed through a heated capillary prior to entering a heated ion source. As the end of the capillary lies opposite a vacuum line, nebulization takes place and a jet of vapor containing a mist of electrically charged droplets is formed. As the droplets move through the hot source area, they continue to vaporize, and ions present in the eluent are ejected from the droplet and... 

Complementary to ESP and ISP interfaces is the APCI interface equipped with a heated nebulizer. The nebulized liquid effluent is swept through the heated tube by an additional gas flow, which circumvents the nebulizer. The heated mixture of solvent and vapor is then introduced into the ionization source where a corona discharge electrode initiates APCI. The spectra and chromatograms from APCI are somewhat similar to those from TSP, but the technique is more robust, especially with gradient LC, and it is often more sensitive. Atmospheric pressure chemical ionization is particularly useful for heat labile compounds and for low-mass, as well as high-mass, compounds. In contrast to the TSP interface, no extensive temperature optimization is needed with APCI. 

The PB interface accommodates common reverse and normal phase chromatographic mobile phases at flow rates up to 0.5 ml/min, and is mechanically simple, rugged, and easy to operate. Unlike the TSP interface, buffer ions are not required to effect ionization, which is instead accomplished by El or Cl in a conventional MS source (3,8). Thermospray LC-MS allows higher flow rates, but requires a special source, and generally requires a volatile buffer to ionize neutral molecules. Thermospray LC-MS provides mostly molecular weight information, whereas PB-LC-MS provides El as well as Cl spectra. 

Three LC-MS interfacing techniques were compared. When using the thermospray (TSP) interface, [M — H] or [M - - CHsCOO]" were obtained as the main ions. APCI and ion spray (ISP) interfaces gave [M — H] at 20-30 V as the main ion. Calibration graphs were linear from 1 to 100 ng for each compound with repeatability values of 15-20%. Instrumental LOD for APCI were 3-180 ng in full scan and from 0.001-0.085 ng in SIM mode. Instrumental LOD for ISP and TSP were larger by approximately one order of magnitude . 

Because of its unsatisfactory sensitivity, a result of the low flow rate and the dogging of the diaphragms separating the eluting analytes from the high vacuum of the ion source, the appHcation of the DLI interface technique was successively reduced. Because of all the disadvantages observed with its application, the DLI approach was soon replaced by the appHcation of the more robust TSP interface. 

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. 

During the second half of the 1980s, the TSP interface, which had first been introduced in 1983 [197], became the most widely-used technique for coupling LC and MS. The main disadvantage of DLI, its low sensitivity, had soon led to its replacement by the TSP interface, before, in the mid-1990s, the commercial breakthrough of API technology took place. 

A standard commercial TSP interface was modified to increase the sensitivity to sulfonated azo dyes and to permit their analysis. The sensitivity could be increased in order to determine sub-pg amounts of the dyes. A by-product of AZO 4 (2,2 -di-hydroxy-4-sulfonyl-6-nitro-l,T-azobisnaphthalene) which could not previously be identified by LC-MS, was then confirmed to be a structural isomer of AZO 4 [202]. 

TSP-LC-MS with explosives was one of the topics Arpino [198] presented when he reviewed the applications of TSP interfacing. General operating principles, optimization strategies and possible ionisation mechanisms were presented. 

LC-MS methodologies applying TSP interfacing for unequivocal identification of isomers, oligomers and homologues of surfactants and their biodegradation intermediates in environmental samples at trace levels have been reviewed [28] very extensively [23, 40]. 

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