Electrospray ionisation source

MS measurements were performed on a Sciex API I Plus single quadruple mass spectrometer equipped with an electrospray ionisation source. The mass spectrometer was operated in negative or positive-ion mode. Ion spray voltage/orifice voltages were selected at - 4 KV/- 70 V and + 5 KV/+ 60 V, respectively. 

Figure 2,33 Schematic diagram of an electrospray ionisation source (Figure used by kind...

Commercially, Agilent Technologies produces chips for both direct infusion into a mass spectrometer and for HPLC-MS applications. The chips accommodate nanoflow rates with an electrospray ionisation source, are about the size of a credit card and are rensable. The infusion chip is for collecting direct MS or tandem MS data. The protein HPLC chip has both a sample enrichment and CIS separation column on the chip, as well as the connections and spray nozzles for electrospray. There are also a small molecule chip and a glycan chip. The chip being used is placed in a Chip Cube MS interface which positions the sprayer tip perpendicular to the MS inlet (Figure 10.7). 

Fig. 1.31 a Plan of a typical electrospray ionisation source operating in positive ion mode. Adapted from [99]. b Electrospray ionisation plume photographed from a Thenno LTQ Ion Trap... 

ES ionisation can be pneumatically assisted by a nebulising gas a variant called ionspray (IS) [129]. ESI is conducted at near ambient temperature too high a temperature will cause the solvent to start evaporating before it reaches the tip of the capillary, causing decomposition of the analyte during ionisation and too low a temperature will allow excess solvent to accumulate in the sources. Table 6.20 indicates the electrospray ionisation efficiency for various solvents. 

ESI and APCI are soft ionisation techniques which usually result in quasi-molecular ions such as [M + H]+ with little or no fragmentation molecular weight information can easily be obtained. However, experimental conditions can also be chosen in such a way that a sufficiently characteristic pattern is obtained, allowing verification [540]. ESI is amenable to thermally labile and nonvolatile molecules. Both ESI and APCI are much more sensitive than PB and very well suited for quantitative analysis, but less so for unknown samples. The choice among the two is usually determined by the application. Recently, nanoscale LC-ESI-MS has been developed [541]. The nano-electrospray ion source offers the highest sensitivity available for LC-MS (atto-to femtomole range) and can also be used as an off-line ion source. 

Undoubtedly, mass spectrometric detection has a substantial role to play in condensed-phase chromatographic analyses of toxic impurities. As in GC/MS, it can be highly sensitive, although this is probably more analyte-specific than in GC/MS. Selectivity can be gained by SIM on single quadrupoles or, if necessary, SRM on MS/MS instruments. What must be considered is the appropriate ionisation mode to be used in LC/MS. Most modern instruments use atmospheric pressure ionisation sources, including electrospray ionisation (ESI), atmospheric pressure chemical ionisation (APCI) and more recently atmospheric pressure photoionisation (APPI). 

Triple-quadrupole mass spectrometer coupled with an electrospray ionisation (ESI) source. 

Modem mass spectrometers within the pharmaceutical industry are more usually fitted with atmospheric pressure ionisation sources that are ideally suited to be connected to HPLC equipment. They are very robust which enables them to be used unattended for many weeks without the need for source cleaning or routine maintenance. There are two types of atmospheric ionisation sources, namely Electrospray Ionisation (ESP) or Atmospheric Pressure Chemical Ionisation (APCl) [15]. Both ionisation modes provide soft ionisation which favours quasi-molecular ion production with little or no fragmentation. Most typically MH ions are observed but MNa, MNHj and MK may also be produced. 

Electrospray ionisation (ESI) is a technique that takes place at atmospheric pressure and is considered to be a soft ionisation process. It is very useful for liquids. Unlike hard processes, the molecule is not normally fragmented and so the resulting mass spectrum is much simpler, the principal peak of which will be the pseudo-molecular ion, i.e. a pro-tonated or sodiated peak. It is therefore much easier to decipher the molecular weight of a compound from an ESI source but there is less structural information given about the molecule, if any. 

Two new independently developed techniques called Dart ° (direct analysis in real time) and Desi (desorption electrospray ionisation) are making a huge impact on mass spectrometry. Together they remove the need for sample preparation and vacuum, speed up analysis time and can work in the open air. The sample is held in a gas or liquid stream at room temperature and the impact induces the surface desorption of ions. The ions then continue into the vacuum interface of the MS for analysis. Samples can be hard, soft or even liquid in nature. Ifa et al. have used Desi to image biological samples in two dimensions, recording images of tissue sections and the relative concentrations of molecules therein. Jeol have launched a commercial Dart ion source for non-contact analysis of materials in open air under ambient conditions. 

Similarly to LC (section 4.3.4), also CE instruments can be coupled to a mass spectrometer, providing a powerful system for analysis of complex samples. The output of the electrophoresis capillary is connected to an electrospray ionisation (ESI) source, CE-ESI-MS. Usually a make up flow is necessary to increase the flow rate for a stable spray. To avoid contamination of the ion source, it is essential to utilise only volatile buffer components such as ammonium acetate and volatile additives such as methanol, acetonitrile and acetic acid. 

Electrospray ionisation (ESI) Electrospray ionisation is a method in which the analyte is sprayed at atmospheric pressure into an interface to the vacuum of the mass spectrometric ion source [28]. The sample solution is sprayed across a high potential difference (1 4 kV) from a needle tip into an orifice of the mass spectrometer. 

Electrospray ionisation generates analyte ions this is accomplished by spraying the eluent (mobile phase solvent + any analytes eluting from the EC system) into a chamber at atmospheric pressure. This is done in the source in the presence of a heated drying gas (usually Nj) and a strong electrostatic field. The pressure of the electrostatic field causes further dissociation of the analyte molecules and the drying gas causes the solvent to evaporate (see Figure 5.16). 

The electrospray ionisation mass spectrometric analyses were performed using a Finnigan LCQ ion trap mass spectrometer. The samples were dissolved in methanol or chloroform methanol system (10 1 v/v) and such solutions were introduced to the ESI source by continuous infosion by means of the instrument syringe pmnp with the rate of 3 iL/min. The ESI source was operated at 4.25 kV and the capillary heater was set to 200°C. For ESI-MS" experiments mass selected mono-isotopic parent ions were isolated in the trap and collisionally activated with 33% ejection RF-amplitude at standard He pressure. The experiments were performed in the positive and negative-ion mode. 

The mass spectra of mixtures are often too complex to be interpreted unambiguously, thus favouring the separation of the components of mixtures before examination by mass spectrometry. Nevertheless, direct polymer/additive mixture analysis has been reported [22,23], which is greatly aided by tandem MS. Coupling of mass spectrometry and a flowing liquid stream involves vaporisation and solvent stripping before introduction of the solute into an ion source for gas-phase ionisation (Section 1.33.2). Widespread LC-MS interfaces are thermospray (TSP), continuous-flow fast atom bombardment (CF-FAB), electrospray (ESP), etc. Also, supercritical fluids have been linked to mass spectrometry (SFE-MS, SFC-MS). A mass spectrometer may have more than one inlet (total inlet systems). 

In specific cases, when long chain compounds such as esters and TAGs have survived and have not yet been hydrolysed or oxidised, it may be useful to carry out soft ionisation techniques in order to fully characterise the structure of these biomarkers by direct infusion into an electrospray source after adapted purification treatments. 

To summarise, a fractionation step allows the isolation of the compounds of interest from the other molecular constituents, particularly from the fatty acids that are well-ionised. To compensate for the low ionisation yield of some compounds, such as TAGs, the solutions may be doped with a cation. Samples are then directly infused into the ion electrospray source of the mass spectrometer. A first spectrum provides an overview of the main molecular compounds present in the solution based on the peaks related to molecular cations. The MS/MS experiment is then performed to elucidate the structure of each high molecular compound. Table 4.2 shows the different methods of sample preparation and analysis of nonvolatile compounds as esters and TAGs from reference beeswax, animal fats and archaeological samples. 

Non-volatile buffers such as phosphates, borates, perchlorates and phosphoric acid should be avoided at all costs because of high background ion current, source contamination and blockages, and in the case of perchlorates, explosions. Figure 6.4 shows the mass spectrum of typical background when using phosphoric acid in the eluent. If the solvent system for a particular analysis does not assist the electrospray process, it is possible to enhance ionisation by postcolumn addition of a suitable volatile buffer. 

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