Particle beam HPLC

Particle Beam HPLC/MS. The particle beam HPI.C/MS analysis was performed on a Hewlett-Packard 5988A (Palo Alto, CA) mass spectrometer. The mass spectrometer was operated at 70 eV electron energy with a source temperature ranging from 150-350°C for acquisition of the El spectra. The Cl spectra was obtained using methane reagent gas, at an electron energy of 100 eV, and with a source temperature between 100-300°C. The instrument was scanned from m/z 50-550 in 1 second. The mass spectrometer was tuned and calibrated daily with FC--43. The particle beam (Hewlett-Packard, Palo Alto, CA) was operated at the conditions determined optimal from a previously reported study for the analysis of the various drugs and antibiotics [13]. 

The diversity of compounds that can be analyzed by HPLC currently preclude the use of any one HPLC/MS technique to specifically detect trace quantities. Thermospray is one of the most popular HPLC/MS techniques due to its ability to ionize nonvolatile and thermally labile compounds with minimal compromises on a HPLC separation or MS operation. Ion evaporation ionization (no filament or discharge) is suitable for numerous compounds, but often results in spectra with insufficient structural information (i.e. one ion spectra), with sensitivities varying drastically between compound classes. In cases where thermospray specificity or sensitivity is not sufficient, complementary HPLC/MS approaches need to be employed. The use of thermospray HPLC/MS and complementary techniques (i.e. immobilized enzyme bioreactors, chemical degradation and particle beam HPLC/MS) have been evaluated for the specific analysis of three major classes of compounds--peptides, pharmaceuticals, and pesticides. 

Pesticides. There are numerous references to the use of HPLC/MS for the analysis of pesticides and herbicides [14, 16-20]. Some major classes of pesticides and herbicides including carbamate, triazines, organophosphorus, and phenolic acid have been analyzed by HPLC/MS using Cl or ion evaporation ionization. While these ionization techniques often resulted in excellent sensitivity (thermospray/MS full scan detection limits of 1-10 ng), usually only [M+H] and/or [M+NH4] ions were formed. This limitation can be overcome using tandem4MS [20], moving belt [17], and most recently through the use of particle beam HPLC/MS. 

Figure 7.12 Particle beam HPLC-MS interface and data system reports (a) VG particle beam line interface (b) total ion chromatogram for a series of pesticides obtained using the LINC Particle Beam interface, in the El mode. Peaks A and B are identified below as Rotenone and Atrazine by the library search.

The nebulization and evaporation processes used for the particle-beam interface have closely similar parallels with atmospheric-pressure ionization (API), thermospray (TS), plasmaspray (PS), and electrospray (ES) combined inlet/ionization systems (see Chapters 8, 9, and 11). In all of these systems, a stream of liquid, usually but not necessarily from an HPLC column, is first nebulized... 

The development of methods of analysis of tria2ines and thek hydroxy metabohtes in humic soil samples with combined chromatographic and ms techniques has been described (78). A two-way approach was used for separating interfering humic substances and for performing stmctural elucidation of the herbicide traces. Humic samples were extracted by supercritical fluid extraction and analy2ed by both hplc/particle beam ms and a new ms/ms method. The new ms /ms unit was of the tandem sector field-time-of-flight/ms type. 

The particle-beam interface provides El spectra from HPLC eluates and this is of great advantage over other interfaces which provide only molecular weight information. Why then, is it of advantage to be able to generate Cl spectra from the particle-beam interface ... 

The particle-beam interface has been developed primarily to provide El spectra from HPLC eluates but may be combined with other ionization techniques such as CL If quantitative studies are being undertaken, a detailed study of experimental conditions should be undertaken. Isotope-dilution methodology is advocated for the most accurate results. 

The performance of the particle-beam interface deteriorates as the percentage of water in the HPLC mobile phase increases. 

The obvious alternative for the in-line flow-through cell in HPLC-FTIR is mobile-phase elimination ( transport interfacing), first reported in 1977 [495], and now the usual way of carrying out LC-FTIR, in particular for the identification of (minor) constituents of complex mixtures. Various spray-type LC-FTIR interfaces have been developed, namely, thermospray (TSP) [496], particle-beam (PB) [497,498], electrospray (ESP) [499] and pneumatic nebulisers [486], as compared by Som-sen et al. [500]. The main advantage of the TSP-based... 

LC-MS inlet probes support all conventional HPLC column diameters from mobile phase must be eliminated, either before entering or from inside the mass spectrometer, so that the production of ions is not adversely affected. The problem of removing the solvent is usually overcome by direct-liquid-introduction (DLI), mechanical transport devices, or particle beam (PB) interfaces. The main disadvantages of transport devices are that column... 

Only the particle-beam interface produces El spectra for direct comparisons with computerized library spectra of fragmentation patterns. The other systems enable the relative molecular mass (RMM) of analytes up to 105 and above to be established. An example of an HPLC-APCI separation and identification of some benzodiazepine tranquillizers is shown in Figure 4.39. The most appropriate choice of LC-MS interface for a particular... 

The particle beam interface [55] borrowed and built upon some of the key elements and concepts of its predecessors. Eluent from the HPLC was nebulized into a spray of small droplets by a flow of helium. The spray of droplets entered a heated chamber where evaporative processes further reduced the droplet size creating an aerosol. The next step of the process involved the spraying of the aerosol (i.e., the... 

In recent decades the hyphenated technique, HPLC-MS, also become the method of preference in HPLC practice. Various techniques have been developed and applied for MS detection such as thermo-spray interface, atmospheric ionization (API), electrospray or ionspray ionization (ESI or ISI), and particle beam ionization. 

A Solvent Extractable Nonvolatile Compounds by HPLC/Thermospray/MS or UV Detection 8325 Solvent Extractable Nonvolatile Compounds by HPLC/Particle Beam/MS... 

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. 

Figure 16.14—HPLC/MS interface using a particle beam interface. The angular diffusion of heavy molecules under vacuum is narrower that that of smaller molecules of solvent, thus they have a higher probability of being transported to the mass spectrometer along the central axis of the system.

The particle beam interface is an example of an HPLC/MS interface (see Fig. 16.14). In this interface, the mobile phase is vaporised in the form of a spray into a desolvation chamber before entering the area where the sample is concentrated by evaporation. Because solvent molecules are lighter, their angular dispersion is wider than that of the analyte, which can be transferred into the transfer capillary. This type of interface is slowly being abandoned in favour of others that have a greater sensitivity. 

The APCI interface uses a heated nebulizer to form a fine spray of the HPLC eluate, which is much finer than the particle beam system but similar to that formed during thermospray. A cross-flow of heated nitrogen gas is used to facilitate the evaporation of solvent from the droplets. The resulting gas-phase sample molecules are ionized by collisions with solvent ions, which are formed by a corona discharge in the atmospheric pressure chamber. Molecular ions, M+ or M , and/or protonated or de-protonated molecules can be formed. The relative abundance of each type of ion depends upon the sample itself, the HPLC solvent, and the ion source parameters. Next, ions are drawn into the mass spectrometer analyzer for measurement through a narrow opening or skimmer, which helps the vacuum pumps to maintain very low pressure inside the analyzer while the APCI source remains at atmospheric pressure. 

R Andreoli, M Careri, P Manini, G Mori, M Musci. HPLC Analysis of fat-soluble vitamins on standard and narrow bore columns with UV, electrochemical and particle beam MS detection. Chro-matographia 44 605-612, 1997. 

The availability of commercial bench-top mass spectrometry detectors for HPLC is facilitating the development of HPLC-MS methods for many analytes. This is more common in pharmaceutical than food applications. As is generally the case, mass spectrometry is first being applied to standard solutions and relatively simple samples before being applied to more complex food matrices. A standard mixture of ten vitamers, AA, DHAA, PN, PL, PM, thiamine, nicotinic acid, nicotinamide, pantothenic acid and biotin, were recently determined by HPLC-particle beam... 

Yinon et al. (228) used an HPLC interfaced with a triple-quadrupole mass spectrometer by means of a particle beam for the identification of several azo dyes. Characterization of the dyes was achieved by observing typical fragment ions formed by cleavage of the N-C and C-N bond on either side of the azo linkage and/or cleavage of the N=N double bond with the transfer of two hydrogen atoms to form an amine. Sensitivity was observed to be two to three orders of magnitude worse than with thermospray ionization. 

The use of liquid chromatography-mass spectrometry (LC-MS) is becoming more popular because of the increasing number of LC-MS interfaces commercially available thermospray (TSP), particle beam (PB), and atmospheric pressure ionization (API). Coupled with mass spectroscopy, HPLC provides the analyst with a powerful tool for residue determination. 

Recent advances in electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), thermospray, and particle beam LC-MS have advanced the analyst toward the universal HPLC detector, but price and complexity are still the primary stumbling blocks. Thus, HPLC-MS remains expensive and the technology has only recently been described. Early commercial LC-MS uses particle beam and thermospray sources, but ESI and APCI interfaces now dominate. Liquid chromatography MS can represent a fast and reliable method for structural analyses of nonvolatile compounds such as phenolic compounds (36,37), especially for low-molecular-weight plant phenolics (38), but the limited resolving power of LC hinders the widespread use of its application for phenolics as compared to GC-MS. 

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