Atmospheric Pressure Ionization API Methods

In the sources so far described, ionization takes place in the vacuum region of the mass spectrometer, thus requiring removal, either through additional pumping or by a reduction in the flow rate of the mobile phase. The production of ions prior to entry to the MS high vacuum regions, i.e. at atmospheric pressure, would obviate these requirements. Development of atmospheric pressure ionization techniques has led to a rapid and exciting development in LC-MS instrumentation. Although API methods have been available for a number of years, it was not until the pioneering work of Fenn et al. that their potential was realized. The two variants normally employed in conjunction with HPLC are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

Electrospray ionization ESI produces charged particles directly from solution at atmospheric pressure. Since its introduction in the mid-1980s it has developed into one of the most popular ionization techniques, especially for biomolecules. The source design is relatively simple and extraction of ions into the mass spectrometer is readily achieved. Although a variety of source designs have been developed and commercial instruments differ in this respect, the basic processes of ion information and extraction are similar.

In its simplest form, ESI is realized from a sample solution (flow rate 2-10 ^Lmin-1) introduced through a capillary into the ion source, which is at atmospheric pressure. The emerging liquid is formed into a fine spray of charged droplets by the presence of a potential difference of + 3-5 kV applied between the capillary and a counter-electrode. The formation of gaseous ions from the sample solution occurs as a result of this droplet formation and subsequent desolvation. Formation of the charged droplets is reasonably well understood, but the process of ion formation from them is the subject of debate.

A typical source is shown in Figure 4.

The capillary delivering the liquid flow is contained, in a co-axial arrangement, within an outer stainless-steel capillary. A flow of gas through this outer capillary aids droplet formation and extends the usable flow rate up to & 1.5 mL min~\ Nitrogen is the usual nebulizing gas employed in this modification, sometimes referred to as ion spray. Beyond the counterelectrode is a sampling cone (or in some instruments this may be a short glass or steel capillary) which may be maintained at a low voltage (& 30-250 V). Between this cone and the counterelec-trode, a countercurrent flow of gas (usually nitrogen) is introduced. This gas, known as the drying or curtain gas, aids the desolvation process. Additionally, the source may be held at elevated temperatures (&60°C) as a further means of helping desolvation. Entry into the analyser region of the mass spectrometer proceeds via a skimmer held at ground potential. Stages of differential pumping (or cryopumping) reduce the source pressure (atmospheric) to that of the analyser (&10~5mmHg).

Electrospray is an extremely soft ionization technique and results in the formation of ions representa-

Figure 4 Ion source for electrospray ionization. For operation in APCI mode, a discharge needle would be placed between the inlet capillary and the counter-electrode.

tive of the intact molecule with virtually no fragmentation. For small molecules the mass spectra have a very simple appearance, generally showing just the protonated molecule ion and/or adduct ions, e.g. (M + Na) +. The mass spectra of larger molecules, however, become more complicated because of the production of multiply charged ions. A series of molecule ions of the form (M + nH +)n+ is produced, where n varies according to the number of sites on the molecule which are able to accept a proton. The molecular mass of a compound is calculated from the ion series by a deconvolution algorithm contained in the instrument's software (though it can be done manually!). An example of a typical ESI spectrum and the result of deconvolution is shown in Figure 5.

Formation of negative ions occurs in electrospray, with both singly and multiply charged species being formed. The choice of ionization mode depends on the proton affinities of the analytes.

In addition to molecular weight information, ESI mass spectra may contain ions representative of both specific and nonspecific interactions that are noncovalent in nature. Fragmentation of intact molecule ions may be induced within the source region by manipulation of the cone voltage. The application of a voltage (up to 250 V) between the counter-electrode and the first sampling cone will lead to low energy collisions between sample ions and the curtain gas. Depending on the energy of these collisions, declus-tering and then fragmentation may occur.

Figure 5 (A) ESI mass spectrum of cytochrome c; (B) deconvoluted mass spectrum of cytochrome c.

Since its introduction, ESI has undergone rapid development and has seen widespread application, especially in the biochemical field. Commercial instruments with dedicated ESI sources are readily available, ranging from simple benchtops to sophisticated tandem mass spectrometers.

The development of orthogonal sources has allowed the use of non-volatile buffers for LC-MS whereas 'in-line' sources are restricted to volatile buffers.

Atmospheric pressure chemical ionization An APCI

source relies on the formation of reactant ions and their subsequent reaction with sample molecules. These reactant ions are formed at atmospheric pressure by a corona discharge achieved by maintaining a stainless-steel needle at a voltage of 3-6 kV. The source design for APCI, for LC-MS, is very similar to that of ESI, the major difference being the addition of the discharge needle in the region between inlet capillary and the counterelectrode. The LC eluent is converted into a fine droplet spray by a nebulizing gas and this is followed by vaporization in a heated region (up to 500°C, depending on the instrument) of the capillary. This rapid desolvation and vaporization minimizes any thermal decomposition. Chemical ion-ization of the sample is effected via ion molecule reactions: the reactant ions are formed from the LC mobile phase. Operation in either positive ion or negative ion mode is possible depending on the nature of the analyte. The use of a curtain gas aids decluster-ing in a manner similar to electrospray. Molecular weight information is readily provided, but to obtain structural information the use of collision induced decomposition (CID) experiments is required.

Mobile phase flows from 0.1 to 2.0 mL min"1 can be accommodated, eliminating the need for splitting. Both volatile and, with the advent of orthogonal sources, nonvolatile buffers are tolerated and mobilephase compositions of up to 100% water are permitted.

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