3.3: Atmospheric Pressure Chemical Ionisation (APcI)

OPDAC 3.3: Atmospheric Pressure Chemical Ionisation (APcI) 3.3.1 Introduction The atmospheric pressure chemical ionisation (APcI) interface nebulises the LC column effluent in a heated vaporiser tube placed concentrically around the capillary. Once vaporised the plasma of eluent and sample components enters an API source where it encounters a cloud of electrons emitted from the tip of a corona electrode pin (Figure 3.3 (1)). The eluent molecules, which are in vast excess compared to the analyte, are ionised and act as the reagent gas in a chemical ionisation process that closely resembles that of medium pressure chemical ionisation (CI). Analyte molecules are ionised by the reagent gas plasma and are subsequently sampled into the nozzle-skimmer region of the mass spectrometer in a similar fashion as described for electrospray ionisation. Figure 3.3 (1): Schematic representation of an APcI source APcI interfacing for LC-MS is amongst the eldest approaches, initial work being carried out by Horning in 1974. However, due to the lack of commercially available instrumentation, the first wide-scale implementation of the technique took place in the early 1990 s.

Today, APcI approaches to analyte ionisation are amongst the most widely applied interface strategies. Due to the essential similarity between the electrospray and APcI interfaces most modern LC-API-MS systems are equipped for both ionisation modes with a minimum of operator interaction or equipment modification. Although APcI is not as widely implemented as electrospray ionisation, a growing number of applications is reported within the literature. The region of applicability for the APcI technique is shown in Figure 3.3 (2)) [1]. Figure 3.3 (2): Applicability of various modes of ionization in LC and GC -MS APcI is the method of choice for many drugs and metabolites and is probably the most widely used technique for high throughput target applications (such as BAPK and combinatorial techniques), particularly in the pharmaceutical industry. Figure 3.3 (4): Typical application of APcI in quantitative bio-analysis 3.3.2 Suitable samples for APcI The sensitivity, ruggedness and reliability of APcI puts it ahead of electrospray ionisation techniques for many pharmaceutical applications. APcI appears to be much less sensitive to chemical interferences than electrospray-ms and the ionisation process associated with APcI is one of the most efficient, approaching 100% under ideal conditions (Figure 3.3 (5)). LC-MS for the Chromatographer Crawford Scientific, 2001 2

Figure 3.3 (5): APcI interfaces are less prone to ion suppression than the ESI interface The only major limitation with APcI applies to all API techniques in that the ability to collect and detect all of the ions generated in the interface is limited, with only approximately 1% of ions formed being detected. Examples of analytes suitable for analysis via APcI include PAHs, PCBs, fatty acids, phthalates, and triglycerides. Samples that contain heteroatoms such as benzodiazepines and carbamates also work well. Figure 3.3 (6): Suitable sample types for APcI analysis LC-MS for the Chromatographer Crawford Scientific, 2001 3

Avoid samples that are thermally labile as these may decompose or react in the heated nebuliser of the source. Samples that are typically charged in solution such as proteins, peptides, amino acids, or oligonucleotides should also be avoided, as sensitivity will be much reduced. These species also tend to be thermally labile and due to their high masses will be less volatile. Figure 3.3 (7): Unsuitable sample types for APcI analysis A summary of analyte types suitable for APcI-MS analysis may include: Small molecules that are moderately polar to non-polar Samples that contain heteroatoms Avoid samples that typically are charged in solution Avoid samples that are very thermally unstable or photosensitive 3.3.3 APcI Interfacing Details Figure 3.3 (8): Schematic diagram of a typical APcI interface APcI interfaces usually consist of the following components (Figure 3.3 (8)); LC-MS for the Chromatographer Crawford Scientific, 2001 4

An HPLC system delivering column eluent at flow rates between 0.5 and 2.0 ml/min. (i.e the majority of current conventional HPLC solvent delivery systems) A device for nebulising the column effluent and vaporisation of the droplets produced. An atmospheric-pressure ion source, containing a corona discharge electrode, and is capable of delivering the ions produced from the atmospheric pressure region into the high vacuum region of the mass spectrometer. In this respect, APcI sources differ from ESI sources ion two major respects; The inclusion of a heated region that extends beyond the nebulising capillary tip through which a gas is passed to rapidly vaporise the eluent and sample components The inclusion of a corona discharge pin to produce a cloud of electrons that are used to initiate the chemical ionisation process in the vapour phase. This section will therefore concentrate on the major differences between the APcI and the electrospray source which has already been discussed in detail. 3.3.3.1 Nebuliser Types In contrast to electrospray ionisation, APcI ionisation occurs in the gas phase as a result of strong heating of the nebulised eluent. Several nebulising and vaporising devices are available from a variety of manufacturers these are briefly described below; Figure 3.3 (9): APcI nebuliser design after Covet et. Al. The nebuliser first introduced by Covey et. al. [2] is used extensively in modern instrument designs and consists of a concentric pneumatic nebuliser and a quartz vaporiser tube extending beyond the tip of the nebuliser capillary (Figure 3.3. (9)). In this system flows of up to 1.5 ml/min. column eluent are nebulised with a inert gas (usually nitrogen) at pressures of approximately 0.8Mpa. LC-MS for the Chromatographer Crawford Scientific, 2001 5

The aerosol created is swept through the heated nebuliser tube (usually 300-500 o C), assisted by a make-up gas (nitrogen at 1-3 L/min., 0.5 MPa). This results in a vapour temperature of approximately 100 o C, which achieves almost complete nebulisation of the aerosol components. The corona discharge electrode is positioned, off axis, near the exit of the quartz vaporiser tube and this source type is available from Applied Biosystems (Foster City, CA, US. Pneumatic nebulisers, comprising three concentric tubes (i.e. a liquid carrying capillary in the center, a nebuliser gas tube and an auxiliary gas tube), are used extensively [3]. The nebuliser gas passes through a heater built into the tube and the pre-heated aerosol passes into a heated vaporiser region directly in-front of the nebuliser to complete the vaporisation process. The corona electrode is placed axially and near to or within the heated vaporiser region of the source. This source design has been used by manufacturers such as Micromass (Manchester, UK) and Thermo Finnigan (Manchester, UK). In general the flow rates of gas used as nebulising, auxiliary, make-up and counter current flows in the APcI interface vary widely between instrument manufacturers. The flow rates of gas used will also very depending upon chemical nature of the eluent system. However, it should be noted that the required nitrogen supply could be as high as 600 L/h in some systems with a line pressure of 0.7MPa this will almost always require the Nitrogen supply to be directly pumped from a liquid nitrogen pressurised vessel or from a nitrogen generating unit supplied with high-pressure compressed air supply. 3.3.3.2 Ion sampling and transfer in APcI interfaces Ultimately the products of the APcI interface are gas phase ion products of eluent and analyte components, very similar to the products of the electrospray interface with the addition of eluent reagent ions such as large water clusters and heavily hydrated gas phase acids and bases (Figure 3.3. (10)). Figure 3.3 (10): Formation of hydrated cluster ions in the APcI source Therefore, the hardware employed to de-cluster and transfer these gas phase ions to the high vacuum region of the mass spectrometer is essentially similar to that previously described for electrospray instruments. LC-MS for the Chromatographer Crawford Scientific, 2001 6

The important aspects of the interface and ion transfer hardware are highlighted below by way of a summary and reminder of the material covered in previous sections; Figure 3.3 (11): Ion optics of a typical APcI source The atmospheric pressure region terminates in an ion sampling opening (sampling orifice) or a transfer capillary (heated), which acts as a nozzle at the low-pressure face. A region between the nozzle and a skimmer plate acts as a momentum jet separator and represents the first vacuum region of the instrument (Figure 3.3 (11)). Lenses may be employed in this region to promote the preconcentration of analyte ions relative to neutral and low molecular weight ionic species arising from the eluent. Figure 3.3 (12): Ion optics of a typical APcI source Some systems employ a second skimmer to allow a second differentially pumped region of the instrument. An ion focusing device such as an rf only, quadrupole, hexapole or octapole ion bridge used to pre-concentrate analyte species and direct the ion beam onto an axial path prior to the mass analysing device (Figure 3.3 (12)). This region is usually pumped by an oil diffusion or turbo-molecular pump. LC-MS for the Chromatographer Crawford Scientific, 2001 7

3.3.3.3 Analyte ion declustering The effects of declustering analyte species require more attention with APcI, as many of the gas phase analyte ions produced are clustered with water molecules and reagent ions derived from the eluent system (Figure 3.3 (13)). Figure 3.3 (13): Cluster ion formation at the corona electrode in APcI Declustering of analyte ions may be achieved using one, or a combination, of the following approaches; Using a counter current gas flow at the nozzle plate, also known as a curtain gas. Figure 3.3 (14): Declustering using a curtain gas LC-MS for the Chromatographer Crawford Scientific, 2001 8

Using a heated transfer capillary between the API region and the nozzleskimmer region. Figure 3.3 (15): Declustering using a heated transfer capillary into the nozzle-skimmer region Using a drift voltage between the nozzle and skimmer plates to promote intermolecular collisions between the analyte clusters and the background gas molecules known as collision induced dissociation. Figure 3.3 (16): Declustering using collision induced dissociation Large solvent clusters may also form that do not contain an analyte ion and it is important that these clusters are removed to prevent noisy baselines and obscuring of the analyte signal. Increasing the drying gas flow and temperature in the main interface can reduce the formation of the solvent clusters. However, too high a drying gas flow will help to eliminate solvent clusters but will also attenuate the signal from the analyte [4]. LC-MS for the Chromatographer Crawford Scientific, 2001 9

Figure 3.3 (17): Drying gas flow and nebuliser temperatures should be optimized to reduce cluster ion formation 3.3.4 APcI Ionisation Mechanisms 3.3.4.1 Overview (1) APcI is based on chemical ionisation by ion-molecule or electron capture reactions carried out on gas phase products of the vaporised LC eluent at atmospheric pressure. APcI can occur in a number of ways; Proton transfer or charge exchange in the positive ion mode LC-MS for the Chromatographer Crawford Scientific, 2001 10

Figure 3.3 (18): Ionisation mechanisms in APcI (+ve mode) Proton absraction or electron capture in the negative ion mode. Figure 3.3 (19): Ionisation mechanisms in APcI (-ve mode) Charge transfer with positive ions takes place between a reactant ion generated from a molecule possessing a high ionisation potential, and a gas phase molecule with a lower ionisation potential. With a careful choice of the reactant ion, charge transfer can be a selective ionisation method. Benzene molecular ions have been used in positive ion mode as the reactant for the selective ionisation of polychlorinated biphenyls and the O 2 - ion may be perfused into the API source to promote charge transfer reaction in negative ion mode. Electron capture occurs if thermalised electrons are present in the API source, where suitable sample molecules are ionised by resonance electron capture, producing molecular anions, or by dissociative electron capture, producing negative fragment ions. Suitable analyte molecules posses electronegative functional groups, such as conjugated carbonyl species, nitro groups or halogen atoms. 3.3.4.1 Overview (2) The majority of ion-molecule reactions in APcI are gas phase acid-base type reactions [5]. For positive ion operation the reactant ions are acids - protonated water, methanol and acetonitrile are all mild acids and the ammonium ion is a weak selective acid (Figure 3.3 (20)) [6]. For negative ion operation, the reactant ions are bases hydroxide ions are stong gas phase bases, CH 3 O - and NCCH 2 - are mild bases and CH 3 COO - and Cl - are weak bases. If reactant gases are mixed, protons are transferred to form the weakest possible reactant ion (Figure 3.3 (21)). If acetonitrile is added to water, H 3 O + transfers its proton to the acetonitrile gas phase ion. Similarly if ammonia is added to LC-MS for the Chromatographer Crawford Scientific, 2001 11

methanol vapour, the initially formed protonated methanol transfers its proton to ammonia, giving NH 4 +. It is not possible to convert a weak reagent into a stronger one. Figure 3.3 (20): Gas phase acidity and basicity of various reactant species in APcI Figure 3.3 (21): Formation of weakest reagent ion in APcI Which ionization mechanism dominates depends upon the chemistry of the mobile phase and analyte as well as the ion polarity (either positive or negative). In general however, negative APcI does not yield the same dramatic increase in sensitivity found when operating in negative CI in the gas phase in gas chromatography-mass spectrometry (GC-MS). 3.3.4.2 Reagent Gas Formation The chemical ionisation reactions are initiated by means of electrons emitted from a corona discharge electrode kept at 5-10kV with a discharge current of 1-5µA. The energetic electrons released from the tip of the corona electrode (or corona pin as it is more usually known), begin a series of reactions; N 2 + e - N 2 +. + 2e - N 2 + 2N 2 N 4 +. + N 2 LC-MS for the Chromatographer Crawford Scientific, 2001 12

The major benefit to ionisation at atmospheric pressure is that all gas phase ions undergo significant collisions with surrounding gas phase molecules. This results in interactions of all surrounding gases (N 2, H 2, H 2 O and air) and a cascade reaction induced is shown (Figure 3.3 (22)) [7]. Figure 3.3 (22): Cascade reaction resulting in water cluster reagent ions The major resulting ions are N 2 +, O 2 +, H 2 O + and NO + and in the presence of only trace amounts of water, cascade reactions are initiated that ultimately result in production of water cluster ions, which predominate the reagent gas mass spectrum. Since these water cluster ions are the major source of reagent ions, the proton affinity of these clusters relative to the gas phase proton affinity of the analyte and eluent ions has a great affect on the sensitivity [8-9]. Figure 3.3 (23): Predicting reactivity in APcI based on proton affinity LC-MS for the Chromatographer Crawford Scientific, 2001 13

relative to the main reagent ions In order to make some rudimentary predictions about the relative sensitivities of each analyte ion and any possible suppression effects attributable to eluent components such as volatile buffers, a consideration of relative proton affinity can be useful (Figure 3.3 (23)). The optimum response region represented on Figure 3.3 (23) above is for analytes with greater proton affinity than water but one must consider than proton affinity for ALL eluent components as the buffers and additives used may compete for protons with the analyte, potentially reducing sensitivity [10]. Figure 3.3 (24): Proton affinity values for selected reagent ions and organic species in APcI 3.3.4.3 Effect of eluent additives on APcI Ionisation (1) Gas phase acid-base chemistry is of overriding importance in LC-APcI-MS where reactant gas mixtures are the rule rather than the exception. Additives in the LC eluent used to improve separation may entirely change the chemical ionisation process. When eluents are used with additives and volatile buffers the APcI reactant ion spectrum is dominated by solventderived reactant ions and their clusters with water, methanol etc. [11]. If ammonium acetate is added to the eluent, the reactant ions are ammonium and acetate ions, clustered with water, ammonia, acetic acid and other polar components in the eluent system. LC-MS for the Chromatographer Crawford Scientific, 2001 14

Figure 3.3 (25): Eluent additive cluster ion spectrum in the APcI interface Where water is the main component in the eluent system, the most abundant positive reactant ion series is H 3 O + clustered with water molecules, as has been previously stated. It can be demonstrated that the gas-phase acidity of H 3 O + (H 2 O)n decreases with increasing n. The ionisation of weakly basic samples is less effective or even not possible if n is increased. A decrease of n with increase of proton transfer efficiency is effected at higher temperatures in the interface region around the corona discharge electrode [11]. Figure 3.3 (26): With increased source temperature water cluster ion gas-phase acidity increases ensuring efficient ionization of the analyte. 3.3.4.3 Effect of eluent additives on APcI Ionisation (2) Gas phase acid-base chemistry is of overriding importance in LC-APcI-MS where reactant gas mixtures are the rule rather than the exception. Figure 3.3 (27): Effect of solvents / eluent additives on instrument response in APcI LC-MS for the Chromatographer Crawford Scientific, 2001 15

In general, because of the possibility of charge exchange reactions, methanol is a better choice in APcI-MS than acetonitrile. Using acetonitrile will tend to reduce the sensitivity of the analysis (Figure 3.3 (27)) [12]. Samples with lower proton affinities than ammonia or triethylamine will often loose a proton, become neutralised or form adducts in the gas phase when these eluent additive species are present and therefore their use should be carefully controlled (Figure 3.3 (27)). One very common problem with gas-phase acid base chemistry is signal quenching by acidic mobile-phase constituents in negative ion APcI. Addition of 10mmol/L formic acid or 10 to 100 mmol/l ammonium acetate to the LC eluent significantly decreased the response of selected model compounds in a study by Schafer and Dixon [13]. The extent of the quenching (or signal suppression) was directly correlated to gas phase acidities and basicities. The use of N-methyl morpholine in the eluent appears to be a better alternative in terms of signal quenching. In general positive ion APcI is preferred over negative ion mode whenever this is possible. 3.3.4.4 Other ionisation mechanisms in APcI A less well documented mechanism for APcI is derived via the triboelectric effect (Figure 3.3 (28)). As the mobile phase and analyte species exit the nebuliser, the sheer forces generated by the nebulising gas tear the liquid stream into droplets. The friction generated by this process generates an electric charge (the triboelectric effect) at the liquid gas interface. This charge can cause ion formation in some non-volatile analytes. Figure 3.3 (28): Other ionization processes possible in the APcI interface LC-MS for the Chromatographer Crawford Scientific, 2001 16

Triboelectric APcI does not depend upon the electrons generated by the corona dicharge electrode and ions may be generated (with their associated signals in the mass spectrum), even when the corona discharge is very low or even turned off. This effect will also occur when the corona electrode is switched on. Triboelectric APcI is most common for analyte molecules that are moderately polar and/or non-volatile. Ion ejection is the mechanism that is responsible for most of the ion production in ESI-MS, but may also occur in APcI-MS (Figure 3.3 (28)). Some analyte molecules in the LC eluent will exist as ions in the solution before they reach the APcI ion source. After nebulisation, the droplets of eluent pass through the vaporiser tube or heated region and as they shrink due to solvent vaporisation the pre-formed analyte ion concentrate at the droplet surface. From this point the mechanism of gas phase ion production closely match that described in the previous sections dealing with ESI-MS. This mechanism is common with analytes that are highly polar or ionic in solution and may be promoted or suppressed by adjusting the ph of the eluent solution 3.3.5 APcI Source Parameter Optimisation 3.3.5.1 APcI Source Parameter Optimisation (1) In general APcI-MS interfaces are considered as extremely simple to operate. This is bourn out by the fact that hardly any optimisation of the interface parameters is reported in many of the APcI application papers. Choosing the optimum settings for the more important interface parameters such as the eluent flow rate, the solvent composition, the nebuliser and auxiliary gas flows and the vaporiser temperature, which are highly interrelated, is far less critical than for electrospray interfaces. Figure 3.3 (29): Effect of vapourising temperature LC-MS for the Chromatographer Crawford Scientific, 2001 17

on instrument response in APcI Of all the parameters that may be optimised within the APcI, the vaporising probe temperature has perhaps the most profound effect on signal intensity (Figure 3.3 (29)). The case study shows Vitamin D3 and furosemide showing little or no signal at a probe temperature of 200 o C [14]. As the temperature is raised to 400 o C the signal due to these analytes has improved appreciably this may be explained due to the increasing efficiency of volatilisation of these two analytes, ensuring full conversion to the gas phase in the region around the corona electrode pin. It should be noted that this approach may not be suitable for thermo-labile analytes that may decompose in the heated zone that can reach temperatures in excess of 200 o C. A further interesting point of note is the APcI signal intensity of compounds such as penicillin, methylene blue, basic yellow 2, and basic violet 10, which are normally charged in solution. These compounds will be less volatile in typical reverse phase eluent systems and so volatilisation will be less effective due to the high intermolecular attractions between the charged analyte species and the polar eluent constituents. Where ph may not be used to ensure that the analyte is neutral in solution, the interface of choice will be ESI. Similarly, higher molecular weight species such as gentamicin and streptomycin will be less volatile and therefore will not be efficiently ionised in the gas phase at the corona electrode. Again the ionisation method of choice will be ESI. In general, higher molecular weight species such as peptides and proteins are not suitable for APcI analysis due to the extremely high temperatures required to vaporise these species in the region immediately after the nebuliser. 3.3.5.1 APcI Source Parameter Optimisation (2) As has already been discussed, any non-volatile buffer species within the LC eluent can act to trap the analyte species either through intermolecular attractions between the less volatile species and the analyte or by a physical trapping effect as the non-volatile particles agglomerate within the evaporating eluent droplets (Figure 3.3 (30)). To demonstrate typical operating conditions for the APcI interface and highlight the generic approach to optimisation an experiment performed by Garcia et.al [15] is outlined. LC-MS for the Chromatographer Crawford Scientific, 2001 18

Figure 3.3 (30): Trapping of analyte species by non-volatile eluent additives leading to reduced instrument sensitivity.. Ten parameters were evaluated for their effect on the performance of the Finnigan MAT APcI system: Mobile phase (5 95% water) Flow rate (0.1 1 ml/min.) Acetic acid concentration (0.1% in water) Nebuliser gas pressure (0.14 0.42 Mpa) Vaporiser temperature (300-500 o C) Corona discharge current (2.8 6.8 ma) Transfer capillary temperature (150 300 o C) Capillary voltage (20-200V) Tube lens voltage (20-200V) Figure 3.3 (31): APcI source parameter optimization after Garcia et. Al. LC-MS for the Chromatographer Crawford Scientific, 2001 19

Ibuprofen was chosen as the model compound with optimum performance found at a flow rate of 0.1 ml/min., nebuliser gas pressure of 0.42 Mpa, transfer capillary temperature of 225 o C, tube lens voltage 55V. These settings proved to be the optimum setting not only for ibuprofen but also for 40 other chemical and pharmaceutical compounds. A caveat to the above findings however must be that all analytes will need a small amount of optimisation for each new experimental set up although, in general, the optimisation required for APcI interfaces will be much less than for the electrospray interface. LC-MS for the Chromatographer Crawford Scientific, 2001 20

3.3.6 References [1] W.M.A Niessen and A.P. Tinke, J. Chromatogr. A., 703, (1995), 37-57. [2] T.R. Covey, E.D. Lee and J.D. Henion, Anal. Chem., 58, (1986), 2453. [3] D.R. Doerge, S. Bajic and S. Lowes, Rapid Commun. Mass Spectrom., 7, (1993), 1126. [4] E.C. Horning, D.I. Carroll, I. Dzidic, S.-N. Lin, R.N. Stillwell and J.-P. Thenot, J. Chromatogr., 142, (1977), 481-495. [5] A.P. Bruins, Adv. Mass Spectrom., 10, (1986), 119 131. [6] A.P. Bruins, Mass Spectrom. Reviews, 10, (1991), 53-77. [7] M.L. Huertas, J. Fontan, Evolution Times of the Tropospheric Positive Ions, Atmospheric Environ., 9, (1875), 1018 as shown in The API book, PE Sciex, Toronto, Canada. [8] J. Sunner, G. Nichol and P. Kebarle, Anal. Chem., 60, (1988), 1300. [9] J. Sunner, M.G. Ikonomou, G. Nichol and P. Kebarle, Anal. Chem., 60, (1988), 1308. [10] Data reproduced from Agilent Technologies Publication number G1999-90001, (1998), Palo Alto, CA, USA. [11] F. Hunt, Reagent gases for CI. Adv. Mass Spectrom., 6, (1974), 6334-6341. [12] Data reproduced from Agilent Technologies 1100 LCMSD Solution Chemistry Handbook, Agilent Technologies, Palo Alto, Ca, USA, 1998. [13] W.H. Schaefer and F. Dixon, Jr, J. Am. Soc. Mass Spectrom., 7 (1996), 1059. [14] Data reproduced from Agilent Technologies 1100 LCMSD Solution Chemistry Handbook, Agilent Technologies, Palo Alto, Ca, USA, 1998. [15] D.M. Garcia, S. K. Huang and W.F. Stansbury, J. Am. Soc. Mass Spectrom., 7, (1996), 59. LC-MS for the Chromatographer Crawford Scientific, 2001 21