Journal of Chromatography A, 1216 (2009) 685–699
Contents lists available at ScienceDirect
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
Review
Effect of eluent on the ionization process in liquid
chromatography–mass spectrometry
Risto Kostiainen ∗ , Tiina J. Kauppila
Faculty of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, P.O. Box 56, FIN-00014, Helsinki, Finland
a r t i c l e
i n f o
Article history:
Available online 2 September 2008
Keywords:
Electrospray ionization
Atmospheric pressure chemical ionization
Atmospheric pressure photoionization
Liquid chromatography–mass spectrometry
Solvent effect
a b s t r a c t
The most widely used ionization techniques in liquid chromatography–mass spectrometry (LC–MS) are
electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure
photoionization (APPI). All three provide user friendly coupling of LC to MS. Achieving optimal LC–MS
conditions is not always easy, however, owing to the complexity of ionization processes and the many
parameters affecting mass spectrometric sensitivity and chromatographic performance. The selection of
eluent composition requires particular attention since a solvent that is optimal for analyte ionization often
does not provide acceptable retention and resolution in LC. Compromises must then be made between
ionization and chromatographic separation efficiencies. The review presents an overview of studies concerning the effect of eluent composition on the ionization efficiency of ESI, APCI and APPI in LC–MS. Solvent
characteristics are discussed in the light of ionization theories, and selected analytical applications are
described. The aim is to provide practical background information for the development and optimization
of LC–MS methods.
© 2008 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electrospray ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1.
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2.
Concentration of additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Adduct formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Ion-pairing and ion exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Atmospheric pressure chemical ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Positive ion APCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Negative ion APCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Atmospheric pressure photoionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Positive ion APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1.
Dopants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2.
Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3.
Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Negative ion APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.
Solvents and additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Effect of solvent flow rate in APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +358 9 191 59134; fax: +358 9 191 59556.
E-mail address: risto.kostiainen@helsinki.fi (R. Kostiainen).
0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.08.095
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1. Introduction
During the last 10 years, liquid chromatography–mass spectrometry (LC–MS) has become a major technique in analytical
laboratories, especially in the pharmaceutical and biotechnology
industries [1–5]. This is the result of extensive basic research
on atmospheric pressure ionization (API) techniques, which
today offer a user friendly way to couple LC to MS. The main
API techniques are electrospray ionization (ESI) [6–8], atmospheric pressure chemical ionization (APCI) [9,10] and atmospheric
pressure photoionization (APPI) [11,12]. All three provide high sensitivity, stable performance and good repeatability and, as a result,
have almost totally replaced the earlier interfacing techniques such
as continuous flow fast atom bombardment, thermospray and particle beam. The triumphal march of LC–MS started in a big way in
early 1990s when the API techniques became widely available.
ESI, APCI and APPI differ in their applicability [13]. ESI revolutionalized biochemical research by offering a highly sensitive and
specific method for the analysis of large biomolecules [14,15]. ESI
has been widely used also for smaller polar organic molecules, and
it is the most widely used atmospheric pressure ionization technique today. The ionization efficiency tends to be poor for more
non-polar compounds, however. For these, APCI and especially APPI
are more suitable. These three ionization techniques are able to
ionize a wide variety of organic molecules from small molecules to
biological macromolecules.
The selectivity and sensitivity of LC–MS analysis not only
depends on the ionization technique and mass spectrometer but
also on the LC technique. Reversed-phase LC is most commonly
used, but also many other LC techniques are applied in LC–MS.
These include ion exchange, ion-pair, affinity and size exclusion
chromatography. The separation efficiency and analysis times in LC
are dependent on the column diameter and solid-phase material.
The most popular columns are 100–200 mm long and have an internal diameter of 3–4.6 mm. Shorter columns with the same internal
diameter provide faster analysis but at cost of resolution. Capillary columns, with internal diameter of 0.05–0.3 mm, offer high
separation power and sensitivity, but analysis times are long. Monolithic columns, as well as the recently introduced ultra performance
liquid chromatography (UPLC), provide good chromatographic resolution with shorter analysis time.
Combinations of the latest technologies in LC–MS offer powerful
and convenient approaches to the analysis of organic compounds
present in minimal amounts in complex matrices. The successful operation of LC–MS nevertheless requires educated personnel
and a clear understanding of the operational parameters. The
signal response is directly dependent on the instrumental parameters, analyte characteristics and eluent composition. MS detection
is not compatible with all solvents and eluent additives that
are commonly used in LC separation. For example, non-volatile
mobile-phase additives cannot be used in practice since they cause
excessive background noise and rapid contamination of the ion
source. Some additives, for example strong acids such as trifluoroacetic acid (TFA), may significantly suppress the ESI signal. Thus,
the selection of eluent composition for LC–MS is usually compromise between LC separation and ionization efficiency.
This review presents an overview of studies dealing with the
effect of eluent composition on the ionization efficiency of ESI,
APCI and APPI, which are the most widely used ionization techniques in LC–MS. The relationship between eluent characteristics
and ionization efficiency is discussed in the light of ionization
theories, with the aim of providing practical information for the
development and optimization of LC–MS methods. Selected analytical applications are described including the most important LC–MS
methods.
Fig. 1. (A) Ionization process in ESI, (B) Taylor cone, (C) droplet evolution scheme
due to solvent evaporation at constant charge and coulomb fissions at the Rayleigh
limit (reproduced with permission from refs. [30,34]).
2. Electrospray ionization
The basis of electrospray ionization as an ionization technique
in analytical devices was presented about 40 years ago, when Dole
et al. [6] studied the ionization mechanism of ESI by ion mobility
spectrometry. Later Yamashita and Fenn [7] successfully combined
ESI and MS, but the real breakthrough took place in 1988 when
Fenn and co-workers [14,15] recognized that ESI is highly suitable
for large biomolecules. This work was honoured by the Noble Prize
in 2002 in chemistry.
In ESI, the sample solution is directed through a narrow capillary which is set to high voltage (typically 3–5 kV). Owing to the
high electrostatic field at the tip of the capillary, negative counterions (when positive ions are analyzed) drift away from the liquid
surface towards the wall of the capillary, where they are neutralized, and the positive ions drift downfield towards the liquid front
(Fig. 1A). As a result, the liquid forms a cone jet, the so-called Taylor cone [16], in which the positive ions drift towards the surface of
the liquid jet (Fig. 1B). When the electrostatic repulsion at the surface overcomes the surface tension of the liquid at the cone tip, the
jet breaks up and small electrically charged droplets are formed.
The initial droplets travel towards the interface plate (counterelectrode) of the API source, and during the flight the surface area of the
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
droplet starts to decrease due to evaporation of the solvent from the
droplet, and the charge density at the surface increases. At a certain radius, called the Rayleigh limit [17], the charge density at the
surface becomes so high that the repulsion forces on the surface
exceed the surface tension of the droplet [18]. As a consequence,
a set of charged smaller droplets is formed (Fig. 1C). This process
is repeated until the size of droplet is small enough to emit gasphase ions. Two models have been proposed for the generation of
gas-phase ions: the charge residue model [6,19] and the ion evaporation model [20,21]. The theory of electrospray ionization process
is presented in more detail in earlier reviews [22–27]. The requirement for both the processes is that the analyte is ionized already in
liquid phase.
ESI not only produces charged analytes to the gas phase but also
large quantities of charged eluent species derived from solvents
and additives, which act as reagent ions in gas-phase ion–molecule
reactions. The charged eluent molecules are typically protonated or
deprotonated solvent or additive molecules, which may ionize neutral analytes present in the gas phase by proton transfer reactions if
the gas-phase proton affinity (PA) of the analyte is higher than that
of the eluent molecule (positive ion mode) or if the PA of the deprotonated eluent molecule is higher than that of the analyte (negative
ion mode). It is worth noting that gas-phase PAs are not necessarily
related to dissociation constants determined in liquid phase. PAs
for numerous organic compounds are listed in the NIST Chemistry
WebBook [28]. The rule of thumb seems to be that neutral compounds with PAs higher than that of ammonia (853.6 kJ/mol) can be
ionized efficiently via gas-phase proton transfer reaction in ESI. Eluent components with high PA, in turn may suppress the ionization
of compounds with lower PA [29].
The overall ESI process is highly complex and several characteristics of the solvents and additives, such as volatility, surface
tension, viscosity, conductivity, ionic strength, dielectric constant,
electrolyte concentration, pH and gas-phase ion–molecule reactions, influence ionization process and thereby the signal response.
Sensitivity is also influenced by chemical and physical properties of
the analyte, including pKa , hydrophobicity, surface activity, ion solvation energy, and proton affinity, and by operational parameters
such as flow rate, temperature and ESI voltage. A free selection of
mobile-phase composition in LC–ESI/MS is not possible since only
polar solvents and volatile additives can be used in practice, and the
selection of the mobile phase often must be balanced between ESI
response and LC separation efficiency. The optimal mobile-phase
composition must be determined separately for each case, which
means that a through understanding of solvent effects on the ionization efficiency and chromatographic behaviour is required in the
development of an LC–ESI/MS method.
2.1. Solvents
The first step in the ESI process is charge separation (cations from
anions), which is achieved with a strong electric field at the ES emitter tip [30,31]. The efficiency of charge separation can be measured
as a spray current, which is dependent on solvent conductivity and
thus on electrolyte concentration [32]. The charge separation process is dominated by the electrophoretic migration of ionic species
in a solvent [33,34], and results in the formation of charged droplets
[30,35]. It has been shown with various solvents that at low analyte concentrations there is a strong correlation between the ESI
response and the spray current [32]. The conductivity of the solvent
must be sufficient for efficient charge separation if high sensitivity
and good stability are to be achieved. Solvents suitable for ESI vary
from polar to medium polar, the most widely being water, methanol
and acetonitrile. Non-polar solvents with low conductivity can be
used in LC–ESI/MS only with a post-column addition of a polar sol-
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vent compatible with ESI [36,37]. Water alone is a poorer solvent
for ESI than are organic solvents such as methanol, acetonitrile and
dichloromethane [32]. This is partly because the viscosity of water
is higher, and therefore the electrophoretic mobility of ions is lower,
leading to inefficient charge separation and difficulties in producing
a stable spray. Spray formation can be facilitated by increasing the
electric field strength at the sprayer tip, but too high fields may lead
to electric discharge, which is deleterious for the ESI process since
it leads to unstable ion currents and decreased sensitivity. Stable
electrospray is more difficult to obtain in negative than in positive
ion mode [38] because electric discharge occurs at lower electric
fields in negative ion mode. This is because electric discharge is
facilitated in the negative mode due to the strong negative potential at the needle, which favours emission of electrons from the
needle surface. The onset voltage of electric discharge (the lowest
voltage, at which electric discharge takes place) is dependent on
the solvent. Straub and Voyksner [39] showed, for example, that
methanol and isopropanol provide a more stable spray and better
sensitivity in negative ion mode than do acetonitrile and ethanol.
The risk of electric discharge in negative ion mode can be reduced
through use of a scavenger gas such as oxygen [7,40], SF6 [41] or the
vapor of a chlorinated solvent [42] which is capable of capturing
electrons.
Once the initial charged droplet has been formed, the efficiency
of a droplet to emit gas-phase ions is dependent on the surface tension and volatility of the solvent [30,43,44]. In discussions about
solvent effects in ESI/MS, the relatively poor sensitivity for analytes
dissolved in water has been attributed to high surface tension, low
volatility and the efficient solvation of ions in water. The relatively
high sensitivity for analytes dissolved in organic solvents is in turn
attributed to low surface tension, higher volatility and less efficient
solvation of ions in organic solvents [32]. Water, having higher surface tension than organic solvents (e.g. methanol and acetonitrile),
produces larger initial droplets. Also, the evaporation of water from
the charged droplet is slower than the evaporation of an organic solvent. For these reasons, the disintegration of the charged droplets is
less efficient with water than with organic solvents, and the number of droplets capable of emitting gas-phase ions is decreased.
Hence, the ESI response is lower when water alone or highly aqueous solvent is used [39,45,46]. In applications where highly aqueous
solvents are mandatory, the performance of the electrospray can
be improved by using sheath flow of organic solvent [47,48] or a
pneumatically assisted electrospray called ionspray [49].
The polarity of the solvent may affect the charge state distribution of multiple charged ions in ESI spectra. Cole and Harrata
[42,44] showed that more polar solvents, which can better stabilize
multiple charged ions in solution, shift the charge state distribution towards higher m/z values. Similarly, the addition of organic
solvent, often methanol or acetonitrile, to water may lead to conformational changes or denaturation of proteins, which will shift
the charge state distribution towards lower m/z values [50].
Most LC–ESI/MS analyses have been carried out by reversedphase LC with non-polar C18 - or C8 -bonded silica stationary phases
[13]. The mobile phase often has consisted of water and organic
modifier and always is a compromise between chromatographic
performance and ESI sensitivity. The retention of non-polar compounds is stronger than that of polar compounds, and therefore the
compounds are eluted with decreasing polarity order in gradient
runs with an increasing amount of organic solvent. Normally the
sensitivity is better for compounds that are eluted at higher organic
solvent content [39,45,46]. The most widely used organic modifiers are methanol and acetonitrile, in LC–ESI/MS. Although a clear
conclusion as to which provides better performance in LC–ESI/MS
analysis cannot be made, methanol has been preferred as an organic
modifier in most LC–ESI/MS applications. Methanol offers slightly
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R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
better ESI efficiency than acetonitrile [51] and gives better peak
shape for basic compounds [52], which include most pharmaceutically active compounds. Methanol is also preferred over acetonitrile
in the analysis of pesticides by gradient LC–ESI/MS because its lower
eluotropic strength causes compounds to elute at a higher percentage of organic solvent and thereby offers increased sensitivity [53].
On the other hand, acetonitrile is reported to give larger gain than
methanol in the ESI/MS signal for morphine [45].
Since the signal response in ESI tends to increase with the
amount of organic solvent, for the reasons presented above, it
is advantageous if the retention of an analyte to the reversedphase stationary phase is increased so that the analyte is eluted
with higher content of organic solvent. Polar compounds may
have low retention on reversed-phase columns and are eluted
with low organic solvent content, and their ionization efficiency in
LC–ESI/MS can be low. The retention of highly polar compounds
can be improved with use of ion-pair reagents. However, these
may suppress ionization of the analyte (see below). Coupling of
hydrophilic interaction liquid chromatography (HILIC) with MS has
proven to be an effective alternative in the analysis of polar compounds [1,46,54,55–57]. The stationary phases in HILIC are polar –
aminopropyl- (NH2 ), cyanopropyl- (CN) and 2,3-dihydroxypropylmodified (OH) and unmodified silica – on which polar compounds
have higher affinity than do non-polar compounds. The compounds
are eluted in order of increasing polarity with water–organic solvent mixtures as eluent and a gradient with decreasing organic
solvent content. It follows that polar analytes are typically eluted
with higher organic modifier content in HILIC than in reversedphase LC and the ESI response is improved. For example, in a
study of the effects of pentafluorophenyl (PFP), OH and CN phases
and of C8 - and C18 -phases on retention, peak shape and ESI signal response, Needham et al. [46,58] showed that significantly
increased sensitivity and better peak shape are achieved with
HILIC–ESI/MS than with reversed-phase LC–ESI/MS in the analysis of basic drugs (Fig. 2). The power of HILIC–ESI/MS has been
demonstrated in many applications, including the analysis of neurotransmitters [57,59], metabolites [56] and peptides [60,61].
2.2. Additives
Additives and buffers are used in LC mobile phases to improve
resolution and reproducibility. Chemical properties and concentration of the additive, as well as pH, have a significant effect on analyte
response in ESI. Unfortunately, many of the additives and buffers
commonly used in LC are not compatible with ESI/MS. In general,
non-volatile buffers such as phosphate and borate tend to cause
increased background, signal suppression, and rapid contamination of the ion source resulting in decreased sensitivity and stability.
Also the strong volatile acids, such as TFA, commonly used as ionpairing reagents in the LC analysis of peptides and proteins, may
cause significant signal suppression in ESI [48,62,63]. Although various volatile additives have been employed in LC–ESI/MS, the most
widely used are acetic acid, formic acid, ammonium hydroxide,
ammonium acetate and ammonium formate [13].
2.2.1. pH
Often the best sensitivity in ESI is achieved when the analyte
is ionized already in a liquid phase by using acidic mobile phase
for basic analytes, such as amines, (pH two units below pKa of
the analyte) and basic conditions for acidic analytes, such as carboxylic acids and phenols (pH two units above pKa of the analyte)
[64]. On the other hand, the best chromatographic performance
in reversed-phase LC, with good retention factors and resolution,
is achieved by adjusting the pH so that the acidic or basic analytes are non-ionized in the mobile phase. In many cases, however,
Fig. 2. Overlaid chromatograms to show similar retention yet increased signal on a
CN phase compared to a C18 phase for the HPLC–ESI–MS analysis of (A) nortriptyline
and (B) pindolol (about 25 ng) (reproduced with permission from ref. [46]).
adequate chromatographic performance can be achieved although
the analyte exists as a preformed ion in the mobile phase if the
interaction between the hydrophobic moiety of the analyte and
reversed-phase material is sufficient. On the other hand, high sensitivity in LC–ESI/MS can be achieved in some cases although the pH
is adjusted so that the analyte is non-ionized in the mobile phase.
It has been shown that, contrary to expectation, abundant protonated molecules can be produced in basic conditions and abundant
deprotonated molecules in acidic conditions. This phenomenon is
referred to as “wrong-way-round ionization” [65]. For example,
high sensitivity in positive ion mode has been achieved for basic
compounds by using a basic mobile phase containing ammonium
hydroxide [66–68]. This is not in agreement with ion evaporation
theory, and it is likely that the ionization takes place either via gasphase proton transfer reaction or via reactions at the liquid–gas
interface of the droplet [29,65]. The gas-phase proton transfer reaction is dependent on the amount of reagent ions in the gas phase,
and it has been shown that increase in the concentration of ammonium hydroxide from 0.05% to 1% enhances the ESI response of basic
analytes in positive ion mode [69]. Note that in cases where the ions
are formed by ion evaporation mechanism, increased concentration
of an additive in the mobile phase may suppress ionization, resulting in decreased ESI response (see below). It has also been shown
that acidic compounds may be efficiently ionized under acidic conditions. For example, in their study of the effect of various acidic and
basic solvents on the responses of acidic flavonoids, Rauha et al. [70]
achieved the highest sensitivity in positive ion mode with acidic
solvents containing formic acid (pH 2.3). The flavonoids were in
neutral form at pH 2.3, and most likely the protonation occurred via
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
gas-phase proton transfer reaction. The significance of gas-phase
ion–molecule reactions in ESI has also been demonstrated in analysis of nucleosides [71], proteins [72] and steroids [73]. As noted
above, the unexpected behaviour of ESI could also be due to redox
reactions taking place at the tip of the ESI sprayer, which result in
significant difference in pH (as much as 1–4 pH units) between the
charged ESI droplet and the bulk sample solution [65,74,75,76]. In
positive ion mode the pH of the charged droplet is lower than the
pH of the sample solution, and in negative ion mode it is higher. This
means that a non-ionized analyte in the mobile phase may become
ionized in the ESI droplet. These examples show the complexity of
ionization process in ESI and alternative ionization mechanisms to
the ion evaporation mechanism, such as “wrong-way-round ionization”, can well be exploited in the development of LC–ESI/MS
methods.
2.2.2. Concentration of additives
The concentrations of additives required in LC are often at the
level of 100 mM that is too high for ESI, which can tolerate only low
additive concentrations. In practice, the additive concentrations
should not exceed 10 mM in order to avoid suppression of ionization and reduced sensitivity. Enke and co-workers [77] presented
an equilibrium partitioning model, which predicts that analyte
response is proportional to concentration at electrolyte concentrations below 10−3 M. At higher concentrations the analyte response
may decrease. The decrease may be attributed to the repulsive
forces caused by the increased charge density at high buffer concentrations and these repulsive forces cause spreading of the spray. The
density of ions at the centre of the spray is then reduced, and fewer
ions are collected by the API source for mass analysis. Spreading of
the spray at higher salt concentrations has been visually observed
[77]. The decreased sensitivity at high buffer concentrations may
also be due to the competition of ions for a site at the surface of
the ESI droplet or due to the formation of a solid residue [78]. The
suppression effect may also depend on the surface activity of an
additive [22,24,79,80] so that electrolytes with higher surface activity can be expected to suppress ionization of an analyte more than
those with lower surface activity.
Mallet et al. [69] studied the influence of several additives and
their concentrations on the ESI responses of acidic and basic drugs.
The results showed a clear decrease in the response when the concentration of the additive (formic acid, acetic acid, trifluoroacetic
acid, ammonium formate, ammonium biphosphonate, ammonium
bicarbonate) was increased from 0.05% to 1%. The suppression effect
was also reported by de Leenheer and co-workers [45], who studied the effect of eluent composition on the ionization efficiency for
morphine. They showed that volatile acids and buffers (formic acid,
acetic acid, ammonium acetate and ammonium formate) at concentration levels of 0.1% or even below caused clear suppression. It has
also been reported that ammonium formate, ammonium biphosphonate and ammonium bicarbonate have a stronger supression
effect than do acidic (formic acid, acetic acid) and basic buffers
(ammonium hydroxide) on the ESI response of selected acidic
and basic drugs [69]. The suppression of ionization by TFA resulting in unstable spray has been demonstrated in several studies
[62,63,69,81,82]. The spray instability and signal reduction have
been presented to be due to either the high conductivity and surface tension of the aqueous eluent including TFA [48,83] or strong
ion-pairing between the TFA-anion and the protonated molecule.
The ion-pairing process is described as masking the protonated
molecules and thereby decreasing the efficiency of the ESI droplet
to emit protonated molecules to the gas phase [63]. Ion-pairing
may also lead to reduced charge separation at the tip of the ESI
sprayer and thereby to decreased ionization efficiency [45,84]. The
results noted above show that too high concentrations of additives
689
lead to decreased sensitivity in ESI. In many cases, however, the
ESI response can be enhanced through the addition of a sufficient
amount of buffer. For example, Kamel et al. showed for a series of
tetracyclines [85] and nucleosides [71] that the addition of 1% acetic
acid resulted in good HPLC separation and the greatest sensitivity in positive ion mode, while the addition of 50 mM ammonium
hydroxide resulted in the greatest sensitivity in negative ion mode.
2.3. Adduct formation
Polar neutral compounds that cannot be ionized by protonation
or deprotonation in liquid phase (amides, esters, ethers, carbohydrates, many lipids etc.) can sometimes be ionized by adduct
ion formation, for example with ammonium, sodium or lithium
ions in positive ion mode ([M+NH4 ]+ , [M+Na]+ , [M+Li]+ ) and with
chloride, formate or acetate ions in negative ion mode ([M+Cl]− ,
([M+HCOO]− , ([M+CH3 COO]− ). The use of ammonia-based buffers
(ammonium formate, ammonium acetate, ammonium hydroxide)
may result in the formation of ammonium adducts, [M+NH4 ]+ ,
instead of the protonated molecule. Ammonium adduct formation
is common for compounds having a proton affinity close to that
of ammonia (853.6 kJ/mol), for example for steroids [86,87], tetracyclines [71], saccharides [88], certain wax esters [89] and lipids
[90,91]. Sodium adducts [M+Na]+ are often formed in addition to
[M+H]+ ions since sodium is always present in the mobile phase at
concentrations of 0.01–0.1 mM due to impurities derived from sample vials, LC-lines or solvents (even from HPLC grade solvents). The
concentration of sodium depends on experimental conditions and
the origin of the sample, and the relative abundance of [M+Na]+
may vary, decreasing the repeatability of the analysis. The formation of sodium clusters may be deleterious in the analysis of
polyprotic organic acids such as oligonucleotides [92] and bisphosphonates [93]. The formation of sodium adducts and clusters can
be decreased by adding formic acid to the eluent after the column,
for example as sheath liquid.
The addition of a controlled amount of sodium or lithium salts
has been used to enhance ionization and improve repeatability in the analysis of trichothecenes [94], carbohydrates [95–98],
and lipids [99,100]. In negative ion mode, chloride, formate
and acetate anions are effective in promoting the formation of
adducts ([M+Cl]− , [M+HCOO]− , [M+CH3 COO]− ) for analytes that
do not readily undergo deprotonation [101]. Chlorinated solvents
(dichloromethane, chloroform, carbon tetrachloride) and chlorine
salts have been used as a source of chloride anions for example
in the analysis of carbohydrates [102,103], lipids [104] and explosives [105]. Formate and acetate adducts have been utilized in the
analysis of glycosides [106] and explosives [105]. In practice, only
low concentrations of salts (likely below 0.1 mM) can be added to
facilitate ionization in ESI via adduct ion formation since higher
concentrations may lead to strong background interference and
rapid contamination of the ion source.
Salts may also have effect on the charge distribution of multiply charged peptides and proteins. Mirza and Chait [107] showed
that the charge states of peptides and proteins were shifted
to lower values due to neutralization of the positive charge by
a counterion present in solution. The nature of the counterion
influenced the magnitude of the shift in the order CCl3 COO− >
CF3 COO− > CH3 COO− = Cl− . The influence of counterions on the ESI
spectra of peptides and proteins has been summarized by Wang
and Cole [108].
2.4. Ion-pairing and ion exchange
Ion-pairing can be used in reversed-phase LC–ESI/MS to improve
the retention and resolution of polar ionic compounds. Volatile ion-
690
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
Fig. 3. Effect of “TFA-fix” on signal intensity (A) 100% water + 0.2% TFA (B) 100% water + 0.2% TFA + “TFA-fix” (IPA 25 ␮L/min and 75 ␮L/min propanoic acid) (reproduced with
permission from ref. [62]).
pairing agents, such as TFA, pentafluoropropanoic acid (PFPA) and
heptafluorobutanoic acid (HFBA), have commonly been used in the
analysis of polar basic compounds [62,109–113]. These ion-pairing
agents form relatively stable ion-pairs with basic compounds,
decreasing the secondary interactions between free silanol groups
of the stationary phase, and resulting in decreased peak tailing,
improved resolution and better retention. However, acidic ionpairing agents may suppress ionization. For example, Gustavsson
et al. [109] showed that the use of fluorinated carboxylic acids as
ion-pairing agents at useful concentrations (a few mM) decreased
the ESI signal of certain amines by about 30–80% relative to the
signal intensity with formic acid–ammonium formate buffer. The
suppression effect of TFA, which is often used as an ion-pairing
agent in the analysis of peptides and proteins in LC, is well known
in LC–ESI/MS. [48,63,107]. The deleterious effect of TFA can be
eliminated by the “TFA-fix” method, i.e., by post-column addition of propionic acid in 2-propanol (75:25, v/v). Improvement in
the signal-to-noise ratio is 10–100-fold [62]. A weak acid, in this
case propionic acid, added at high concentration, becomes concentrated in the charged droplet due to the stronger evaporation of
TFA resulting in decreased suppression (Fig. 3). Although the “TFAfix” method is feasible, it complicates the analysis, and formic acid,
which provides adequate chromatographic performance without
suppression, has routinely been preferred in the analysis of peptides by LC–ESI/MS. The effect of ion-pairing agents and buffers in
the analysis of peptides and proteins by LC–ESI/MS is summarized
by Carcia [114].
Alkylamines are commonly used as ion-pairing reagents in
reversed-phase LC-negative ion ESI/MS analysis of acidic compounds, such as nucleoside mono-, di- and triphosphates [115,116]
sulfonates, sulfates, sulfonated dyes, and halogenated acids [117].
For example, 50 mM aqueous triethylammonium bicarbonate
has been used for nucleic acids [118], N,N-dimethylhexylamine
for nucleosides [115], and trialkyl amines (triethylamine, N,Ndimethyl-n-butylamine, and tri-n-butylamine) for aromatic sulfonates [84]. The ion-pairing reagents used in the analysis of acids
may also cause suppression. Storm et al. [84] showed that alkylamines at concentrations higher than 2.5 mM resulted in a strongly
reduced signal in the analysis of aromatic sulfonates. As a conclusion, although ion-pair reversed-phase LC–ESI/MS may offer a
useful method for strong acids and bases, the ion-pairing agent may
cause signal suppression and increased background disturbance.
Ion-exchange chromatography (IEC)–ESI/MS is an alternative to
the ion-pair reversed-phase LC–ESI/MS for the analysis of ionic
compounds. There are four types of ion exchangers: weak anion,
weak cation, strong anion and strong cation exchangers. The mobile
phase normally consists of water and organic modifier, and the
ionic compounds are eluted by increasing the salt concentration
or changing the pH in the mobile phase. High salt concentrations,
commonly used in the elution of ionic compounds in IEC, suppress
ionization and rapidly contaminate the ion source in IEC–ESI/MS.
Salts must therefore be removed after the column by an on-line
desalting method, such as on-line dialysis [119], use of membrane
suppressor [120] or use of solid-phase chemical suppressor [121].
The use of a pH gradient instead of salt gradient for the elution of ionic analytes is more compatible with IEC–ESI/MS. For
example, nucleoside triphosphates were successfully analyzed by
IEC–ESI/MS using a pH gradient with ammonium acetate in acetonitrile at pH 6 (mobile phase A) and pH 10.5 (mobile phase B)
as solvents [122]. Multidimensional LC combined to ESI/MS utilizing IEC and reversed-phase LC has been widely used in proteomics
instead of two-dimensional electrophoresis [123–127]. The peptides are fractionated by IEC, and the fractions are transferred for
desalting and separation by using reversed-phase LC and eluents
compatible with ESI/MS.
3. Atmospheric pressure chemical ionization
Atmospheric pressure chemical ionization provides an alternative ionization method to ESI. In APCI, analytes eluting from LC are
vaporized at high temperature (300–500 ◦ C) and ionized via gasphase ion–molecule reactions initiated by corona discharge needle.
APCI was introduced and combined with MS analysis in the early
1970s [9,10]. In the first APCI sources, the ionization was initiated by
a 63 Ni source, but this was soon replaced by a corona discharge needle, which provides significantly higher signal intensity [10,128].
APCI is best suited for relatively stable and small molecules of
molecular weights less than about 1000–2000 Da. Since the compounds are vaporized to the gas phase by thermal energy, the
method is not suitable for labile and large biomolecules such as
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
Table 1
Ionization reactions in positive ion APCI [130]
N2 + e− → N2 + • + 2e−
N2 + • + 2N2 → N4 + • + N2
H2 O+ • + H2 O → H3 O+ + HO•
H3 O+ + H2 O + N2 → H+ (H2 O)2 + N2
H+ (H2 O)n − 1 + H2 O + N2 → H+ (H2 O)n + N2
A + B+ • → A + • + B
A + BH+ → AH+ + B
(1)
(2)
(3)
(4)
(5)
(6)
(7)
proteins, larger peptides or oligonucleotides. The main advantages
of APCI over ESI are that neutral and less polar compounds can
be ionized with good sensitivity, polar and non-polar solvents can
be used, and the system tolerates higher salt and additive concentrations than does ESI. The most important gas-phase reactions in
APCI are proton transfer, charge exchange and adduct formation,
the reactions being the same as those in classical chemical ionization (CI) [129]. Because the ionization in APCI takes place in gas
phase, the ionization mechanism is less complicated than that in
ESI. However, understanding of the gas-phase reactions that lead to
the formation of reactant and analyte ions in APCI in light of solvent
properties is essential. Because the ionization process for positive
and negative ions in APCI is different, positive ion and negative ion
APCI are discussed separately below.
3.1. Positive ion APCI
In the absence of solvent the primary reacting molecules in APCI
originate from atmospheric species, such as nitrogen, carbon dioxide, oxygen and water. The primary reactant ions of the gases are
formed by the corona discharge (Table 1, Reactions (1)–(5)). When
solvent is introduced to the APCI source, further reactions take place
via proton transfer or charge exchange reactions (Table 1, Reactions
(6) and (7)). The formation of reactant and analyte ions in APCI
has previously been reviewed by Carroll et al. [130]. The ionization
reactions in the gas-phase are governed by the ion energetics of the
reacting species, i.e., ionization energies (IEs) and proton affinities
(PAs) in positive ion mode [28]. In proton transfer (Table 1, Reaction (7)) the proton is transferred to the species of highest proton
affinity. The charge exchange reaction (Table 1, Reaction (6)) can
take place with the compounds having low ionization energy.
Table 2 shows the PAs and IEs of some atmospheric gases and
solvents commonly used in LC. Water, methanol and acetonitrile,
the most widely used solvents, have higher PAs and lower IEs than
atmospheric gases, and protonated solvent molecules acting as
reagent ions are efficiently formed in APCI. The analytes are then
Table 2
Ionization energies (IE) and proton affinities (PA) of atmospheric gases and selected
LC solvents [28]
Nitrogen
Oxygen
Carbon dioxide
Water
Methanol
Ethanol
Acetonitrile
Ammonia
n-Hexane
Chloroform
2-Propanol
Isooctane
Benzene
Toluene
Acetone
Anisole
IE (eV)
PA (kJ/mol)
15.581
12.1
13.777
12.6
10.84
10.48
12.2
10.07
10.13
11.37
10.17
9.89
9.243
8.83
9.703
8.20
493.8
421.0
540.5
691.0
754.3
776.4
779.2
853.6
–
–
793
–
750.4
784.0
812.0
839.6
691
ionized via proton transfer between protonated solvent molecule
and an analyte, if PA of the analyte is higher than that of the solvent
molecule. Note that the solvents, especially at lower temperatures,
can form solvent clusters, which have higher PAs and IEs than the
individual solvent monomers [131,132]. Solvents that possess low
PAs and IEs (e.g., benzene) can form molecular ions (M+• ), which
can react further through charge exchange (Table 1, Reaction (6)).
It is important to note that the reagent ion composition changes
when the concentration of the eluent species changes during LC
gradient runs. Even small changes in eluent composition may
lead to significant changes in reagent ion composition, which
is highly dependent on the differences in PAs between solvent
components. Enke and co-workers [29] showed that the addition of 6% of methanol to water results in 50% of protonated
methanol molecules and 50% of protonated water molecules. Similarly, when the concentration of ethanol in methanol exceeds
10%, protonated ethanol molecules become dominant, and when
the concentration of propanol in ethanol exceeds 15%, protonated
propanol molecules become dominant. The PAs of water, methanol,
ethanol and propanol are 691, 754, 776, and 793 kJ/mol, respectively
(Table 2). The results show that the larger the difference between
the PA of the solvent species, the lower concentration of solvent
with higher PA is needed to produce a reagent ion composition
dominated by protonated molecules of the higher PA solvent.
3.1.1. Solvents
The most popular polar mobile phase in reversed-phase
LC–APCI/MS applications consists of a mixture of methanol or
acetonitrile and water. Several groups have reported signal suppression for low PA analytes when acetonitrile is used in place of
methanol as the organic modifier [45,133,134]. Most likely this is
because PA of acetonitrile is higher than that of methanol. Fig. 4
shows the reversed-phase LC–APCI/MS analysis of steroids by Ma
and Kim [133] with acetonitrile and methanol as organic mobilephase modifiers. For all steroids except testosterone, which have the
highest proton affinity, a significantly stronger signal was obtained
when methanol was used as the LC solvent. Hence, methanol may
be a better choice than acetonitrile for LC separations, especially
when, the PAs of the analytes are relatively low.
Both polar and non-polar solvents can be used in APCI, whereas
only polar or medium polar solvents can be used in ESI. In view
of this, several groups have chosen APCI for normal-phase liquid chromatography (NP-LC) applications [135–142]. NP solvents
commonly used in APCI include n-hexane, 2-propanol, methanol,
ethanol, isohexane, isooctane, tetrahydrofurane, chloroform and
ethoxynonafluorobutane, with additives such as diethylamine, triethylamine, dimethylethylamine, formic acid, acetic acid, ammonia
and trifluoroacetic acid. The possible suppression effect of strongly
acidic or basic additives depends on the analytes and must be taken
into consideration (see Section 3.1.2 below).
Non-polar solvents may work better than high PA reversedphase solvents when the aim is to ionize the analytes through
charge exchange [143,144]. For example, the post-column addition of benzene has been reported to enhance the formation of
M+• ions and the overall signal of non-polar environmental toxics [145]. Kolakowski et al. [144] have studied the effect of common
reversed- and normal-phase solvents (water/acetonitrile, acetonitrile, dichloromethane, cyclohexane, isooctane, pentane, hexane,
heptane, octane and nonane) in the ionization of polyaromatic
hydrocarbons (PAHs). The PAHs were observed to form both M+•
and MH+ ions, depending on the solvent used. Isooctane provided
the best overall sensitivity for M+• and MH+ ions, whereas cyclohexane and nonane promoted the formation of M+• ions. Of the
hydrocarbon solvents, isooctane and n-hexane gave the best overall sensitivity for both M+• and MH+ , and pentane and cyclohexane
692
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
Table 3
Ionization reactions in negative ion APCI [148]
M + e− → M− • , ifEA(M) > 0
O2 + e− → O2 − • , sinceEA(O2 ) = 0.48 eV
O2 − • + M → M− • + O2 , ifEA(M) > EA(O2 ) = 0.48 eV
HA + O2 − • → A− + HO2 • , if Gacid (HA) < Gacid (HO2 • )
A + [B−H]− → [A−H]− + B, if Gacid (A) < Gacid (B)
M + O2 − • → [M−X + O]− + OX• , whereX = halogen, NO2 orH
M− • + O2 → [M−X + O]− + OX•
Fig. 4. Total ion current (TIC) obtained from APCI LC/MS analysis of a mixture
containing 11 steroids. The gradient systems used were methanol/1% acetic acid
(a) in water and acetonitrile/1% acetic acid in water (b). The 11 standards used
were: A, testosterone; B, DHEA; C, epitestosterone; D, 5␣-DHT; E, progesterone;
F, allo-THDOC; G, androsterone; H, pregnenolone; I, 5␣-DHP; J, pregnanolone; K,
allopregnanolone (reproduced with permission from ref. [133]).
the lowest. The proportion of MH+ ions increased when the solvent
was doped with water.
Some groups have been concerned about the explosion hazard
associated with the use of flammable solvents, such as hexane, at
high flow rates and high temperature in the presence of corona
discharge [146]. This risk may be overcome by adding polar or aqueous modifiers to the LC solvent or post-column, or by using N2 as
auxiliary and nebulizer gases [136,137,139].
3.1.2. Additives
The ammonium-containing additives commonly used in
LC–ESI/MS (ammonium hydroxide, ammonium acetate, ammonium formate) can be used in LC–APCI/MS only for analytes having
high PA. This is because ammonium-containing additives have high
PA, and even low concentrations of these additives in solvent will
neutralize the other solvent molecules in gas phase by proton
transfer, and the reagent composition will be dominated by the
ammonium ions. The proton transfer reaction can occur only with
analytes having PA higher than that of ammonia while the ionization of analytes having lower PA will be suppressed since the
proton transfer from the reactant ion to the analytes is energetically
unfavourable [70,135]. More basic additives, such as diethylamine
or triethylamine are generally more detrimental than ammonia due
to their even higher PAs. However, through careful choice of the
additives, the selectivity of the ionization can be improved to allow
only the ionization of high PA analytes, without ionization of matrix
components with lower PAs.
In some cases the addition of relatively high concentrations of
basic additives in APCI can improve the ionization efficiency of analytes having high PA [45,73,136,147]. A significant increase in the
(1)
(2)
(3)
(4)
(5)
(6)
(7)
ionization efficiency in APCI has been reported when large amounts
(even 100 mM) of buffer (e.g., ammonium acetate, formic acetate)
are added to the solvent [147]. The increase has been observed in
both positive and negative ion modes, but only for highly basic analytes in positive ion mode and highly acidic analytes in negative ion
mode. This result could be due to the increased amount of reactant
ions that become available for the proton transfer reaction. Another
possibility, as suggested by Schaefer and Dixon [147], is that the
primary ionization product is in fact a buffer adduct rather than
a protonated or deprotonated molecule. The use of ammoniumcontaining buffers has also been reported to produce ammonium
adducts and thus improve the overall sensitivity [73]. The formation of ammonium adducts is favoured with compounds having
somewhat lower PA than that of ammonia.
The amount of protonated reactant ions in the gas-phase, and
thus the efficiency of the proton transfer reaction can sometimes
be enhanced by adding acidic additives to the solvent [70,45]. The
most widely used acidic additives are formic and acetic acids, but
trifluoroacetic acid (TFA) has also been experimented by some
groups. For the most part, formic and acetic acids have given good
results, whereas TFA tends to cause significant signal suppression [70,136]. Rauha et al. [70] have suggested that this is due
to gas-phase neutralization of positively charged ions by the TFA
ions.
3.2. Negative ion APCI
In negative ion APCI the ionization is initiated by thermal electrons produced at the tip of the corona discharge needle. The
thermal electrons can be captured by compounds that possess positive electron affinities, i.e., gases, solvents or analytes (Table 3,
Reaction (1)). Oxygen is an important reactant gas in negative ion
APCI since it possesses positive electron affinity (EA = 0.48 eV) [28]
and it is always present in the atmospheric pressure ion source. In
the electron capture reaction, oxygen forms a highly reactive superoxide ion O2 −• (Table 3, Reaction (2)), which can react further with
other gas-phase species through charge exchange or proton transfer
[148]. Charge exchange takes place when the electron affinity of the
reacting species is greater that of O2 (Table 3, Reaction (3)). This is
true for many quinones, diketones and halogenated and nitro compounds. O2 −• is also a relatively strong gas-phase base and thus
it can accept protons from species of higher gas-phase acidities
(Table 3, Reaction (4)).
The reactant ion composition in negative ion APCI is determined by the gas-phase acidities of the solvents, so that solvent
species possessing the highest gas-phase acidities are deprotonated
(Table 3, Reaction (5)), and solvent species with lower gas-phase
acidities are neutralized. Electron affinities and gas-phase acidities of typical APCI gases and solvents are listed in Table 4. Those
analytes having higher gas-phase acidity than the additive (e.g.,
formic acid, acetic acid, trifluoroacetic acid, acetate, formate, trifluoroacetate) can be ionized by proton transfer, but the ionization
of analytes with lower gas-phase acidity may be suppressed [147].
Furthermore, the response for analytes having higher gas-phase
acidities may be improved by high buffer concentrations. Schae-
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
Table 4
Gas-phase acidities and electron affinities of selected gases and solvents [28]
Compound
EA (eV)
Gacid (kJ/mol)
Oxygen
HO2 •
Water
Methanol
Acetonitrile
Chloroform
Acetic acid
Formic acid
Trifluoroacetic acid
0.451
–
–
–
0.01
0.622
–
–
–
–
1451
1607
1565
1528
1464
1429
1415
1328
fer and Dixon [147] compared the signals of carboxylic acids and
phenols with 10 and 100 mM ammonium acetate added to the
LC solvent. They observed that the signal of weakly acidic compounds was weaker with 100 mM than 10 mM ammonium acetate,
whereas the signal of the strongest acids improved with the
100 mM concentration. The suggested explanation of the stronger
signal at higher buffer concentrations was the increased number
of acetate ions resulting in more efficient proton transfer reaction.
The basic buffers that can accept protons may enhance the
deprotonation reaction and thus the ionization efficiency. This
was shown by Schaefer and Dixon [147], who studied the effect
of different buffers to the ionization efficiency of carboxylic
acids and phenols in negative ion APCI. The basic buffer, Nmethylmorpholine, gave better ionization efficiency for all analytes
than the acidic buffers or solvent without buffer (Fig. 5).
693
Compounds that do not possess high gas-phase acidities or
positive electron affinities and cannot be ionized by deprotonation, charge exchange or electron capture can sometimes be
ionized by adduct ion formation through the addition, for example, of chloroform or chloride salts ([M+Cl]− ) or certain acids
([M+HCOO]− , [M+CH3 COO]− ) directly to the LC mobile phase or
post column [70,142,147,149,150]. Optimal adduct ion formation
usually requires low vaporizer temperatures [150]. Kato and Numajiri [149] achieved efficient ionization of carbohydrates by APCI
when 0.5% of chloroform was introduced to the LC mobile phase
solvent flow. Chloroform was found to provide better sensitivity
than either dichloromethane or carbon tetrachloride. According to
Kato and Numajiri [149], formation of the chloride adduct ion is
efficient for compounds such as carbohydrates that possess adjacent OH-groups. Carbon tetrachloride has been added to achieve
ionization of nitroglycerin via chloride adduct ion formation [150].
The addition of chlorinated solvents can also prevent in-source
fragmentation in APCI, as was reported by Zencak and Oehme for
polychlorinated n-alkanes [142].
In addition to charge exchange and proton transfer reactions,
substitution reactions between a neutral analyte and superoxide
ion (Table 3, Reaction (6)) or between the negative molecular ion
of an analyte and oxygen (Table 3, Reaction (7)) may produce ions
of the form [M−X+O]− . These types of ion are commonly formed
with aromatic compounds containing a halogen or a nitro-group
[151–154]. For certain chlorine-substituted aromatic compounds,
the formation of phenoxide ions has been reported to compete with
the formation of negative molecular ions, so that in low pressure
conditions, where it is possible to remove oxygen from the ion-
Fig. 5. Reconstructed ion traces for the [M−H]− at m/z 153 for Z-norbomaneacetic acid obtained using HPLC mobile phases composed of a 1:1 mixture of acetonitrile with
the following aqueous buffers: (A) 10 mM N-methylmorpholine, (B) 10 mM ammonium acetate, (C) 100 mM ammonium acetate, or (D) 10 mM formic acid. The value in the
upper right corner of each trace represents signal height (reproduced with permission from ref. [147]).
694
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
Table 5
Ionization reactions in positive ion APPI [167,168]
M + h → M+ • + e−
M+ • + S → MH+ + [S−H]•
D(dopant) + h → D+ • + e−
D+ • + M → M+ • + D, ifIE(M) < IE(D)
D+ • + S(solvent molecule) → [D−H]• + SH+ ,
SH+ + M → MH+ + S, ifPA(M) > PA(S)
S + h → S+ • + e−
ifPA(S) > PA([D−H]• )
(1)
(2)
(3)
(4)
(5)
(6)
(7)
ization area, the formation of negative molecular ions takes place,
but when the amount of oxygen exceeds 0.5 ppm, phenoxide ions
dominate the spectrum [152].
4. Atmospheric pressure photoionization
Atmospheric pressure photoionization was developed with the
aim of widening the group of analytes that can be analyzed by
LC–MS towards less polar compounds [11,12]. Similarly to APCI,
the solvent is vaporized with a heated nebulizer, but the ionization process is initiated by using a vacuum ultraviolet (VUV) lamp
instead of a corona discharge needle. Most often, a krypton discharge lamp, which emits 10.0 and 10.6 eV photons, is used. The
compounds that can be directly ionized by the photons must possess ionization energies below 10 eV (or 10.6 eV) (Table 5, Reaction
(1)). This includes most analytes, whereas commonly used gases
and LC solvents such as methanol and acetonitrile have higher
ionization energies and are not ionized (Table 2). Low solvent background and improved analytical selectivity are obtained as a result.
Two excellent reviews have been published on the theory and applications of APPI [155,156]. As for APCI, positive and negative ion APPI
are discussed separately.
4.1. Positive ion APPI
The ionization reactions in positive ion APPI are divided into
those in direct APPI, solvent-mediated reactions that take place
without dopant addition and dopant-mediated reactions that take
place when a dopant is added to the system as an extra solvent
with the purpose of enhancing or initiating the ionization. In direct
APPI the initial reaction is the formation of a molecular ion (M+• )
by photoionization of the analyte, which must possess ionization
energy below the energy of the photons (Table 5, Reaction (1)). In
the presence of a protic solvent (e.g., methanol, water, 2-propanol,
cyclohexane), the molecular ion of the analyte abstracts a hydrogen atom from the solvent to form a protonated molecule (Table 5,
Reaction (2)) [157].
In solvent-mediated APPI the solvent is directly ionized by the
photons [158–163]. Many normal-phase LC solvents possess IEs
below 10.0 or 10.6 eV and can be ionized by the photons emitted
by krypton discharge lamp. Among these solvents are 2-propanol
(10.17 eV), n-hexane (10.13 eV), isooctane (9.89 eV), tetrahydrofuran (9.40 eV), and toluene (8.83 eV) [28]. The reactant ions that are
formed depend on the solvent. For example, isooctane tends to produce protonated molecules via self-protonation, which can react
further with the analytes through proton transfer, whereas toluene
forms molecular ions, which can react further by charge exchange
reaction [162]. Ionization of the solvents possessing higher IEs than
10.6 eV can also be achieved by replacing the krypton discharge
lamp with an argon lamp, which emits photons with 11.7 eV energy
[158,164]. With the argon lamp methanol (10.84 eV) can be ionized
directly by the photons, which results in large amount of protonated
reactant molecules in the ion source and more efficient proton
transfer reactions. However, at the same time the charge exchange
reaction producing M+• ions becomes unfavourable.
In dopant-assisted APPI the ionization efficiency is improved
by the addition of a dopant, an additional solvent with ionization
energy below the energy of the photons [11]. A usual flow rate
of dopant is about 1/10 of the eluent flow rate, which in turn is
typically 100–300 ␮L/min. The dopant is directly ionized by the
photons (Table 5, Reaction (3)), after which the dopant molecular ions react with the analyte or solvent molecules through charge
exchange (Table 5, Reaction (4)) or proton transfer (Table 5, Reaction
(5)). The addition of dopant is believed to enhance the efficiency of
ionization reactions because of the longer lifetime of the dopant
molecular ions than of the photons.
4.1.1. Dopants
The most commonly used dopants in APPI are acetone, toluene
and anisole. Ionization energies of all three are below 10.0 eV
(Table 2) and thus they can be ionized directly by the 10.0 and
10.6 eV photons emitted by the krypton discharge lamp. However,
the different proton affinities of the dopants create differences in
their behaviour.
Acetone is reported to be best suited for the analysis of polar
compounds that can be ionized through proton transfer [165,166].
Acetone favours formation of protonated acetone instead of acetone
molecular ion. Thus, charge transfer reaction (Table 5, Reaction (4))
is often not favoured, and ionization through proton transfer is the
main ionization route (Table 5, Reaction (6)). Since the PA of acetone
is relatively high, only compounds that possess a higher PA than
that of acetone can be efficiently ionized, and the use of acetone in
the analysis of low PA analytes leads to poor sensitivity [165].
Use of toluene as a dopant provides ionization of a wider range of
compounds than acetone, either through proton transfer or charge
exchange [165,166]. Toluene favours formation of a molecular ion,
which can react with analytes through charge exchange, if the IE
of the analyte is lower than that of toluene (Table 5, Reaction (4)).
However, the molecular ion of toluene can also react through proton
transfer with the solvent to produce a protonated solvent molecule
(Table 5, Reaction (5)), if the PA of the solvent is above that of the
deprotonated toluene molecular ion C7 H7 • (884.0 kJ/mol [28]). This
applies to high PA additives such as ammonia, but also to the common reversed-phase LC solvents such as methanol and acetonitrile.
Although the PAs of methanol and acetonitrile are below the PA of
C7 H7 • , the PAs of their clusters are above it [131]. Neutralization
of the toluene radical cation prevents the ionization of analytes
through charge exchange, but the ionization through proton transfer is still possible through a reaction with the protonated solvent
molecules (Table 5, Reaction (6)) [167]. Toluene has been reported
to give higher background noise than acetone, possibly due to trace
impurities [158,160,168].
Since the proton transfer reaction is not efficient for very low PA
compounds, ionization through charge exchange and the formation of molecular ions of the analytes is often desired. One way to
achieve this is to reduce the flow rate of eluent to 10–50 ␮L/min, so
that the ratio of the flow rate of dopant and eluent is increased, and
the toluene molecular ions are not completely consumed by the
eluent [169]. The charge exchange reaction can also be achieved if
a low PA solvent such as hexane, chloroform or pure water is used
with the toluene dopant [167]. This is impractical, however, if RP-LC
is the preferred separation method. Ionization of analytes through
charge exchange in the presence of RP solvents (water, methanol,
acetonitrile) is achieved by choosing a dopant with high PA, such
as anisole, which efficiently produces radical cation (molecular ion)
[170]. Since anisole molecular ion has higher PA than do RP solvents
it is not neutralized by proton transfer reaction with the solvents,
and the anisole molecular ion stays in the system and can react with
the analytes through charge exchange. Ionization through proton
transfer with anisole can be less efficient. Fig. 6 shows the ionization
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
695
Fig. 6. The absolute abundances of the total ion currents for the studied compounds in acetonitrile by using anisole and toluene as dopants. The numbers indicate
the studied compounds: (1) naphthalene, (2) ethylnaphthalene, (3) 2-naphthol, (4)
anthracene, (5) diphenylsulfide, (6) luteolin, (7) catechin, (8) carbamazepine, (9)
verapamil (10) propranolol, (11) 1-naphthalenemethylamine, (12) testosterone, (13)
acridine and (14) midazolam (reproduced with permission from ref. [170]).
of a group of compounds with different IEs and PAs with toluene and
anisole used as dopants and acetonitrile as the solvent. A significant
increase was observed in the signals of several low PA compounds
(2-naphthol, anthracene and diphenylsulfide, compounds 3, 4 and
5, respectively) that were ionized through charge exchange.
Other low IE solvents, such as tetrahydrofuran, pyridine, benzene, heptane, isooctane and hexafluorobenzene have also been
tested as APPI dopants [168,171–173]. For some applications the
best results have been obtained with a mixture of dopants [174].
4.1.2. Solvents
For reversed-phase LC–APPI/MS water and methanol are preferred to acetonitrile, which has repeatedly been reported to give a
lower ionization efficiency than methanol or even to suppress the
signal for some analytes [159,160,169,175–177]. This can partially
be explained by the higher PA of acetonitrile than of methanol, and
therefore acetonitrile may suppress the ionization of low PA compounds. Another explanation is the absorption of photons emitted
by the VUV lamp by acetonitrile due to its high photoabsorption
cross-section that results in decreased number of photons available
for ionization reactions [159,160]. It has also been suggested that
acetonitrile is isomerized producing ions with low IE, which can be
directly ionized by the 10 eV photons [178]. These ions can in turn
react with the analytes through unexpected gas-phase reactions.
As in APCI, highly volatile normal-phase LC solvents are generally well suited to APPI, since the solvent phase has to be vaporized
before the ionization. Lower vaporizer temperatures can be used
with easily vaporizable solvents, and this may be useful when
analyzing thermolabile compounds [159]. Many normal-phase
solvents possess ionization energies below the 10.6 eV photons
emitted by the krypton discharge lamp (e.g., 2-propanol 10.17 eV,
n-hexane 10.13 eV, isooctane 9.89 eV, tetrahydrofuran 9.40 eV [28]),
and can be directly ionized without dopant addition. On the other
hand, the use of low proton affinity normal-phase solvents (hexane,
chloroform) with toluene as a dopant can enhance the ionization through charge exchange and thereby improve the ionization
efficiency for non-polar compounds [167]. Normal-phase solvents
successfully applied to APPI analysis include ethanol, 2-propanol,
hexane, heptane, cyclohexane, isooctane, tetrahydrofuran, ethylacetate and chloroform [158–163,168,179].
Fig. 7 shows the effect of mobile phase on the signal of the
protonated molecule of the methyl ester of eicosapentaoic acid
(EPA methyl ester) and the baseline [159]. The tested mobile
phases include solvents commonly used in normal-phase LC and
dopants commonly used in APPI. The signals of the EPA methyl
ester and the background were found to be highly dependent
Fig. 7. Effect of the LC mobile phase on (a) peak area, (b) baseline intensity, and
(c) S/N ratio of EPA methyl ester (m/z 317) in APPI. Injection amount = 100 ng
(10 ng/␮L × 10 ␮L), mobile-phase flow rate = 100 ␮L/min, cone voltage = 25 V, probe
temperature = 450 ◦ C (reproduced with permission from ref. [159]).
696
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
Table 6
Ionization reactions in negative ion APPI [185]
D + h → D+ • + e−
O 2 + e− → O 2 − •
M + e− → M− • , ifEA(M) > 0 eV
M + O2 − • → M− • + O2 , ifEA(M) > EA(O2 ) = 0.451 eV
M + O2 − • → [M−H]− + HO2 • , if Gacid (M) < Gacid (HO2 • )
S + O2 − • → [S−H]− + HO2 • , if Gacid (S) < Gacid (HO2 • )
M + [S−H]− → [M−H]− + S, if Gacid (M) < Gacid (S)
M + O2 − • → [M−H + O]− + OH•
M− • + O2 → [M−H + O]− + OH•
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
on the solvent system: best peak areas were obtained with
hexane/CHCl3 (1:1), isooctane, n-hexane/2-propanol (1:1), CH2 Cl2 ,
hexane and 2-propanol, and highest backgrounds with toluene and
hexane/CHCl3 . The best signal-to-noise ratios were obtained with
isooctane and hexane. The solvent properties with greatest effect
on the ionization of the analytes were listed as solvent volatility,
polarity, proton affinity, ionization energy and photon absorption
cross-section. Photon absorption cross-section was considered to
be the most important.
4.1.3. Additives
Several groups have reported that the ionization efficiency in
positive ion APPI decreases when basic or acidic buffers are added
to the APPI solvent [70,165,167,180,181]. High PA additives, such
as ammonium hydroxide, ammonium formate and ammonium
acetate produce efficiently ammonium ions, which are able to ionize only high PA compounds and may suppress ionization of lower
PA compounds. The addition of acids such as TFA and formic acid
in positive ion APPI may weaken the signal response due to recombination of the negatively and positively charged species resulting
in fewer reactant ions available for ionizing of the analytes [70].
The use of non-volatile capillary electrophoresis (CE) buffers
and surfactants (e.g., potassium and sodium phosphate, phosphoric acid, sodium borate, sodium dodecyl sulfate) in APPI without
signal suppression has been widely reported [165,166,182–184]. In
all the applications, the flow rate of the sheath liquid (15 ␮L/min)
was higher than that of the CE (0.5 ␮L/min) [165,166]. Hence,
the final concentrations of the non-volatile additives in the
APPI source were low. Non-volatile buffers cause contamination of the ion source, however, and they should be avoided.
Although APPI tolerates higher buffer concentrations than ESI,
the best sensitivity is reportedly achieved when the concentration of the buffer (if necessary for chromatography) is minimized
[70,180].
4.2. Negative ion APPI
In negative ion APPI, ionization is initiated by the thermal electrons released in the photoionization reaction (Table 6, Reaction (1))
or by photoelectron emission from the metallic surfaces of the ion
source [185,186]. These electrons are captured by analytes, solvents
or gases with positive electron affinity (Table 6, Reactions (2) and
(3)). Since oxygen is always present in APPI operating under ambient conditions it is highly probable that oxygen, due to its positive
electron affinity, will capture electrons. The resulting superoxide
ion (O2 −• ) can react with other species through charge exchange
(Table 6, Reaction (4)), proton transfer (Table 6, Reactions (5) and
(6)) or substitution reactions (Table 6, Reaction (8)) [185]. Charge
exchange takes place in case the electron affinity of the reacting
species is above that of O2 (0.451 eV). O2 −• is also a relatively
strong gas-phase base and thus it can accept protons from solvent species of higher gas-phase acidities (Table 3, Reaction (4))
producing deprotonated solvent molecules.
The analytes are ionized via proton transfer, charge exchange
or electron capture reaction. The proton transfer reaction takes
place with compounds having higher gas-phase acidity than of
the solvent (Table 6, Reaction (7)) or protonated superoxide ion
(HO2 • ) (Table 6, Reaction (5), Table 4). The charge exchange reaction
between superoxide ion and analyte, producing negatively charged
molecular ion, can take place if the analyte possesses higher electron affinity than oxygen (Table 6, Reaction (4)). An analyte with
positive electron affinity may also be ionized by electron capture
reaction (Table 6, Reaction (3)). However, it has been suggested
that the number of thermal electrons is not sufficient for efficient electron capture reactions [156,185]. In some cases, especially
with aromatic compounds, substitution reactions may take place
between the negative molecular ion of an analyte and oxygen or
between a neutral analyte and superoxide ion, producing ions of
the form [M−X+O]− (Table 6, Reactions (8) and (9)).
4.2.1. Solvents and additives
Similarly to negative ion APCI, negative ion APPI is highly dependent on the solvent composition. Acetonitrile has been reported to
provide lower ionization efficiency than methanol in negative ion
APPI [187–190], perhaps due to its slightly positive electron affinity (Table 4) [190]. The lower ionization efficiency might also be
explained by the high photoabsorption cross-section of acetonitrile
and subsequent consumption of photons [159,160].
In analyses, where ionization takes place via proton transfer
reactions, strong organic acids or buffers (e.g. formic acid, acetic
acid, TFA, ammonium formate, ammonium acetate) producing low
PA anions such as [HCOO]− , [CH3 COO]− and [CF3 COO]− can suppress the ionization of less acidic analytes [167,187,191,192]. For
highly acidic analytes, however, the use of weaker gas-phase acids
may improve the selectivity and sensitivity [167]. Strong acids can
also protonate and neutralize the superoxide ion, which may suppress ionization of high EA analytes through charge exchange [167].
Solvents or additives that possess positive electron affinities (e.g.
halogenated solvents such as chloroform) are reported to suppress
the ionization in negative ion APPI [185,193]. The suppression has
been explained in terms of the consumption of electrons from the
ion source or the neutralization of superoxide ions by the solvent resulting in less efficient charge exchange or electron capture
reactions. However, Song et al. [193] report that while the addition of halogenated solvents suppressed the ionization of analytes
through electron capture, dissociative electron capture or proton
transfer, the halide attachment promoted the ionization of analytes. This was especially advantageous for analytes that could
not be ionized by any other mechanism, such as the explosives
cyclotrimethylenetrinitramine (RDX) and 1,3,5,7-tetranitro-1,3,5,7tetrazocane (HMX). The best sensitivity for these compounds was
achieved with 1% methylene chloride addition in toluene and monitoring of [M+Cl]− ions.
As in negative ion APCI, quinones and halogenated and nitro
compounds can react with oxygen in negative ion APPI to form
[M−X+O]− ions (Table 6, Reactions (8) and (9)) [185]. In some cases,
the formation of [M−X+O]− ions is reported to be more intense
in negative ion APPI than in APCI [185], perhaps due to catalysis by the metal surfaces in the APPI source. This explanation is
supported by the observation that the proportion of [M−X+O]−
ions is much lower with an open micro-APPI source than with
the conventional APPI source [194]. Kauppila et al. [167] observed
for 1,4-naphthoquinone that the proportion of [M−X+O]− ions
increases and the proportion of negative molecular ions decreases
when acetic acid, ammonium acetate or ammonium hydroxide is
added to the solvent. Acetonitrile and hexane-containing solvents
have the opposite effect.
R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699
4.3. Effect of solvent flow rate in APPI
The optimum flow rate of the solvent in APPI is lower than that
in APCI. The ionization efficiency in both direct as well as dopantassisted APPI is reported to decrease with solvent flow rates higher
than about 100–200 ␮L/min [195–197]. The effect seems to be less
pronounced in direct APPI and for analytes that are ionized through
proton transfer [159,169,196–198]. In aqueous reversed-phase conditions, when the mobile phase contains a high percentage of
water, the decrease in efficiency at higher flow rates can partly be
explained by insufficient vaporization of the solvent. This can be
compensated to a certain point by increasing the vaporizer temperature [159]. This cannot be the only explanation, however, since a
similar effect has not been reported for APCI sources, which employ
the same geometry and temperatures as APPI. Another reason for
the signal loss at high flow rates could be the loss of photons
through photon absorption by the larger amount of solvent vapor in
the ion source [64]. The effect would be emphasized with acetonitrile as the solvent, since it has a high photoabsorption cross-section
at 10 eV energy [164]. The loss of photons would affect the formation of ions by direct photoionization, as well as by charge exchange
and proton transfer, since the total amount of reactant ions available
for ion–molecule reactions would be reduced. In dopant-assisted
APPI the signal loss at high flow rates could also be due to the
larger amounts of high PA impurities and solvent clusters that may
neutralize the dopant molecular ions. Neutralization would have a
direct effect on the charge exchange reaction and thus the signal of
molecular ions of the analytes [196–198].
5. Summary
Many studies have demonstrated the significant effect of eluent composition on the ionization efficiency in ESI, APCI and APPI.
In ESI the ionization takes place via an ion evaporation process,
and the solvent species have a significant effect on charge separation, the formation of charged droplets and ion emission. Solvents
with sufficient conductivity, low surface tension, low vaporizing
point and low solvation energy are favourable in ESI. Unfortunately, many of the solvents that are optimal for ESI cannot be
used in LC and compromises between ionization and chromatographic separation efficiencies must be made. This is especially
true with reversed-phase LC–ESI/MS analysis, where the best chromatographic performance is achieved when the analyte is in neutral
form, but often the sensitivity is highest when the compound is in
ionic form. During recent years the use of hydrophilic interaction
chromatography (HILIC) coupled to ESI/MS has become more popular in the analysis of polar compounds. Significantly larger amounts
of organic modifier are required for the elution of polar compounds
in HILIC than for their elution in reversed-phase LC and ESI signals
are enhanced as a result. A buffer must be used to achieve good
chromatographic performance in the analysis of acidic and basic
compounds by LC–MS. The most widely used buffers or additives
in ESI are acetic acid, formic acid, ammonium hydroxide, ammonium acetate and ammonium formate, which may have a significant
effect on the ionization process. In ESI the concentration of additive should be low, likely below 10 mM, to avoid suppression of the
ionization of analytes.
APCI and APPI provide alternative ionization techniques to ESI.
The advantages of APCI and APPI over ESI are the following: nonpolar and neutral compounds can be ionized more efficiently with
APCI and especially with APPI than with ESI; both polar and nonpolar solvents can be used whereas only polar and medium polar
solvents can be used in ESI; and higher buffer concentrations are
tolerated in APCI and APPI than in ESI. Furthermore, APCI and
APPI are more compatible with reversed-phase LC eluents than
697
ESI, since the analyte can be in neutral form in the mobile phase
resulting in enhanced retention towards reversed-phase materials.
However, APCI and APPI are suitable only for relatively stable and
small compounds, whereas ESI can also be used in the analysis of
large biomolecules. The ionization process in APCI and APPI takes
place in gas phase via ion–molecule reactions between ionized
eluent species and analyte molecules. The most important gasphase reactions are proton transfer, charge exchange and adduct
ion formation. The effect of the eluent depends therefore on ion
energetics (e.g., proton affinity, electron affinity, ionization energy)
of the eluent species and analytes. In reversed-phase LC with APCI
or APPI, methanol may be preferable to acetonitrile due to higher
proton affinity of acetonitrile. In APPI, acetonitrile absorbs photons
more efficiently than methanol does owing to its high photoabsorption cross-section, that result in decreased number of photons
available for ionization reactions and thereby in decreased sensitivity. Non-polar solvents such as n-hexane, isooctane, 2-propanol,
tetrahydrofurane, and chloroform can be used in APCI and APPI,
and this allows the use of normal-phase chromatography, which
may be advantageous in the analysis of non-polar compounds. The
use of buffers or additives is not necessary in the LC–MS analysis of
compounds that do not show acid–base behaviour. Often the best
sensitivity with APCI and APPI is achieved with the use of pure solvents without any additives. However, if additives are used, their
ion energetics must be favourable for the efficient ionization of analytes. If not, the ionization is suppressed and sensitivity decreased.
The choice of dopant strongly affects the sensitivity and selectivity
in APPI.
The effect of the mobile-phase composition has been shown to
have a significant influence on the ionization efficiency in ESI, APCI
and APPI in many studies. The ionization processes are highly complex and many ionization pathways and gas-phase ion–molecule
reactions may take place simultaneously. Therefore, it is impossible to give universally applicable rules for the optimization of
the mobile-phase composition for LC–MS analysis. Although many
detailed and excellent studies have been carried out dealing with
fundamentals of ESI, APCI and APPI, the full understanding of the
ionization processes is still a challenge. Therefore every study on
this topic is highly welcome.
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