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Atmospheric Pressure Ionization

In atmospheric pressure ionization sources (API) the ions are first formed at atmospheric pressure and then transferred into the vacuum. In addition, some API sources are capable of ionizing neutral molecules in solution or in the gas phase prior to ion transfer to the mass spectrometer. Because no liquid is introduced into the mass spectrometer these sources are particularly attractive for the coupling of liquid chromatography with mass spectrometry. Pneumatically assisted electrospray (ESI), atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) are the most widely used techniques.

API offers unique opportunities for the implementation of new sources or to develop new applications. Atmospheric pressure matrix assisted laser desorption (AP-MALDI) [21] can be mounted on instruments such as ion traps which were originally designed only for electrospray and LC-MS. New API desorption techniques such as desorption electrospray (DESI) [22] or direct analysis in real time (DART) [23] have been described and offer unique opportunities for the analysis of surfaces or of solid samples.

The sampling of ions from atmospheric pressure into to the high vacuum region of the mass analyzer region requires significant pressure reduction. A gas stream introduced into a vacuum system expands and cools down. When this gas stream contains ions and solvent vapors the formation of ion-solvent clusters is observed. To obtain good sensitivities and high quality spectra one of the key roles of the interface is to prevent cluster formation. Different instrument designs have been proposed, including single stage pumping or differential stage pumping. Figure 1.7 depicts a typical single stage interface with curtain gas. The space between the orifice and the curtain plate is flushed with heated pure nitrogen. Ions are moved through the curtain gas into the mass analyzer with the help of an electric field formed between the curtain plate and the orifice. In this way, neutral solvent molecules cannot penetrate into the high vacuum region, which prevents

plate

Curtain gas

Fig. 1.7 Single stage pumping atmospheric pressure ionization interface with curtain gas. The size of the orifice is ca. 100 mm, q0 acts as a focusing quadrupole and the nitrogen curtain gas prevents neutral molecules being introduced into the mass spectrometer. T = Temperature of the cryoshells (in Kelvin); p = pressure.

the formation of cluster ions. In a single-stage pumping interface, as described in Fig. 1.7, the size of the orifice is ca. 100 mm and to maintain a high vacuum cryogenic pumps are mandatory. Declustering can also be performed by applying a potential difference between the orifice and quadrupole q0 [24]. If the value of

Fig. 1.8 Differential pumping design with heated capillary. This configuration requires a dual stage pumping system before the ions are introduced into the quadrupole mass analyzer which needs to be operated at high vacuum. The role of the lenses is to focus ions. In some systems the lenses are replaced by hexapoles or octapoles.

Fig. 1.8 Differential pumping design with heated capillary. This configuration requires a dual stage pumping system before the ions are introduced into the quadrupole mass analyzer which needs to be operated at high vacuum. The role of the lenses is to focus ions. In some systems the lenses are replaced by hexapoles or octapoles.

the declustering potential is set too high ''in source'' or ''up front'' collision-induced dissociation can be observed. Cryogenic pumps have high pumping capacity (10000 L s-1 and more) but they need to be recycled every 48 h, which jeopardizes automated use of the instrument. Turbomolecular or diffusion pumps have much lower pumping capacities (50-800 L s-1). To achieve the desired vacuum in the mass analyzer, differential pumping designs were developed. An instrument design using differential pumping with a heated capillary interface is illustrated in Fig. 1.8. In a first step ions flow through a heated capillary (T = 150-300 °C) which helps desolvatation. The internal diameter of the capillary is typically 0.5 mm. A reduced vacuum is achieved in the first pumping region with the help of a rotary pump. Ions are then pushed through a skimmer or an orifice into a second vacuum chamber where the vacuum is produced by a turbo molecular pump and then analyzed in the mass analyzer. Most modern instruments use differential pumping either with capillary skimmer or with an orifice skimmer setup with or without curtain gas.

1.3.2.1 Electrospray

A spray of small droplets at atmospheric pressure can be generated by: (i) a nebulizing gas, (ii) the application of heat, (iii) the application of ultrasounds iv) the application of an electric field. Electrospray ionization (ESI) is a process were charged droplets result from the nebulization of a solution in an electric field. The liquid flows through a stainless steel or a fused silica capillary while the potential (typically 3-6 kV in positive mode) is applied directly on the capillary or on a counter electrode. In negative mode to avoid discharge, the range is somewhat lower (typically 3-4 kV). After nebulization the charged droplets reduce their size and subdivide, up to a point where gas phase ions escape from the droplets. A stable spray can be obtained at flow rates of 1-10 ml min-1. When performing LC-MS with standard bore LC columns (4.6 mm i.d.) the LC effluent must be split. To overcome this limitation, the spray process can be assisted by a nebulizing gas such as nitrogen or air [25] (Fig. 1.9). This way of operation was originally named ionspray but the term is less and less used. With liquid chromatography most sources use air or nitrogen to assist the electrospray process (pneumatically assisted electrospray). Stable sprays can be observed with flow rates above 1 ml min-1, allowing direct interfacing of LC with MS. Most modern commercial instruments operate with pneumatically assisted electrospray placed orthogonally to the entrance of the MS. The nebulizing process can be further assisted with the use of heat, where either the sprayer is heated or a hot stream of nitrogen is directed orthogonally towards the formed droplets.

Very low flow electrospray is called nanoelectrospray [26] where the samples are infused into the mass spectrometer at the nanoliter flow rate range. The infusion of a few microliters will result in a stable signal for more then 30 min, using pulled capillaries or chip-based emitters [27]. With infusion, signal averaging allows to improve the limit of detection in tandem mass spectrometry. Nanoelec-trospray is particularly important in combination with nanoflow liquid chroma-tography or chip-based infusion for the analysis of peptides and proteins.

Fig. 1.9 Pneumatically assisted electrospray. The coaxial nitrogen gas assists the electrospray process allowing to operate at flow rates of several hundred microliters.

ESI is a condensed phase ionization process and the ions have to be already present in solution. To generate ions, the pH has to be adjusted in such a way that ionizable groups are either protonated or deprotonated. In some cases neutral molecules can be analyzed by the formation of adducts with ions such as ammonium, sodium, potassium, acetate or silver.

Peptides and proteins have several ionizable sites, resulting in the formation of multiply charged ions [14]. Figure 1.10 shows the ESI spectrum of human gamma interferon (Mr = 16908.50). The mass spectrum of the protein corresponds to a distribution of multiply charged ions obtained through protonation ([M+zH]z+). The ion at (m/z)1 846.4 corresponds to human gamma interferon protonated 20 times [z1- (m/z)1 = Mr + z1 ■ mp], Mr being the relative molecular mass of the protein, z1 the number of charges and mp the mass of the proton. Because each pair of ions differs by one proton [(m/z)2 806.1 bears 21 protons] the charge state (zi) of any ion and therefore the relative molecular mass of an unknown protein can be determined with the following equations:

Z2 ^ (m/z) - mp 2 (m/z)1 - (m/z)2 Mr = Z2 ■ [(m/z)2 - mp] (2)

where z is charge, m is mass and mp is proton mass.

The relative molecular mass determination of an unknown protein is generally performed automatically using various deconvolution algorithms, but the procedure is limited to relatively simple mixtures.

Electrospray ionization can be considered as an electrolysis cell (Fig. 1.11) where, in the positive mode, cations are enriched at the surface of the solution and negative ions move inside the capillary. Oxidation of the analyte has been observed at certain occasions, in particular at very low flow rates. Also in the case of

Fig. 1.10 (A) Positive mode electrospray spectrum of human gamma interferon on a quadrupole mass analyzer. (B) Deconvoluted spectrum of human gamma interferon. The molecular mass was measured at 16 908 ± 2 Da.

stainless steel sprayers nickel or iron ions can be released and form positively charged complexes with certain types of analytes.

The mechanisms for the formation of gas phase ions from droplets are not fully understood and two therories have been proposed: the ion evaporation model (IEV) and the charge residue model (CR) [28]. The IEV model proposes that the ions are directly emitted into the gas phase when, after evaporation and

Fig. 1.11 Electrospray as an electrophoretic cell. Adapted with permission from reference [28].

coulomb droplet fission, the droplets reach a certain radius. In the case of the CR model it is assumed that gas phase ions are produced when no further solvent evaporation is possible. In the case of small molecules it is believed that the IEV model predominates while for the proteins the CR model is assumed to occur.

A very interesting characteristic of electrospray MS is that it behaves, under controlled settings, like a concentration-sensitive detector [29]. This means that the MS response is directly proportional to the concentration of the analyte. A direct consequence is that LC post-column splitting does not affect the intensity of the MS signal. Another important point is that the reduction of the internal diameter of the column results in an increase in the MS response proportional to the squared ratio between the internal diameters of the greater i.d. column to the smaller i.d. column. Assuming that the same amount of analyte is injected onto a 0.3 mm i.d. column instead of a 2.0 mm i.d. column, a 44-fold increase in response is observed. Or the same response is obtained using a 44 times smaller sample volume. The use of smaller sample volumes is attractive for qualitative analysis where sample consumption can be critical. Because the injection volumes have also to be much lower with smaller i.d. columns, column-switching approaches become mandatory to really benefit from the gain of sensitivity in quantitative analysis [30]. Generally the trapping column is of a larger i.d. than the analytical column, allowing the rapid injection of 50-100 mL of sample.

1.3.2.2 Atmospheric Pressure Chemical Ionization

Atmospheric pressure chemical ionization (APCI) is a gas phase ionization process based on ion-molecule reactions between a neutral molecule and reactant ions [31]. The method is very similar to chemical ionization with the difference that ionization occurs at atmospheric pressure. APCI requires that the liquid sample is completely evaporated (Fig. 1.12). Typical flow rates are in the range 200-1000 mL min-1, but low flow APCI has also been described. First, an aerosol is formed with the help of a pneumatic nebulizer using nitrogen. The aerosol is directly formed in a heated quartz or ceramic tube (typical temperatures 200-500 °C) where the mobile phase and the analytes are evaporated. The temperature of the nebulized mobile phase itself remains in the range 120-150 ° C due to evapo-

Fig. 1.12 Atmospheric pressure chemical ionization source. A Analyte.

ration enthalpy. In a second step, the evaporated liquid is bombarded with electrons formed by corona discharge. In positive mode primary ions such as N2+' are formed by electron impact. These ions react further with water in several steps by charge transfer to form H3O+. Ionization of the analyte A occurs then by proton transfer. In negative mode ions are formed either by: (i) resonance capture (AB ! AB—), (ii) dissociative capture (AB ! B—) or (iii) ion-molecule reaction (BH ! B—). Generally APCI is limited to compounds with Mr < 2000 which do not undergo thermal decomposition. Singly charged ions [M+H]+ or [M—H] — are predominantly observed. While electrospray is a condensed phase ionization process, APCI is a gas phase ionization process where the analyte ionization efficiency depends on its gas phase proton affinity. APCI ionization has become very popular for liquid chromatography coupled with mass spectrometry because it can handle very easily liquid flow rates from 200 mL min—1 to 1 mL min—1. In contrast to electrospray, the application of heat may generate thermal decomposition of the analyte. At atmospheric pressure, ionization occurs with the high collision frequency of the ambient gas and rapid desolvation and vaporization limits the thermal decomposition of the analyte. Figure 1.13A shows the electrospray full-scan spectrum of the sulfuric acid monoester of 3-hydroxy retinoic acid, which is a phase II metabolite of 3-hydroxy retinoic acid without any degradation. In the APCI spectrum of the same analyte (Fig. 1.13B) several intense ions at m/z 315 and m/z 297 can be observed. These ions are not generated by collision-

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