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Journal of Chromatography A, 1216 (2009) 685–699

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .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 con-

cerning 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686

2. Electrospray ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686

2.1. Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687

2.2. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688

2.2.1. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688

2.2.2. Concentration of additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689

2.3. Adduct formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689

2.4. Ion-pairing and ion exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689

3. Atmospheric pressure chemical ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690

3.1. Positive ion APCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691

3.1.1. Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691

3.1.2. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692

3.2. Negative ion APCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692

4. Atmospheric pressure photoionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694

4.1. Positive ion APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694

4.1.1. Dopants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694

4.1.2. Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695

4.1.3. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696

4.2. Negative ion APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696

4.2.1. Solvents and additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696

4.3. Effect of solvent flow rate in APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697

5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697

∗ Corresponding author. Tel.: +358 9 191 59134; fax: +358 9 191 59556.

E-mail address: [email protected] (R. Kostiainen).

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.chroma.2008.08.095

686 R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699

1. Introduction

During the last 10 years, liquid chromatography–mass spec-

trometry (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], atmo-

spheric pressure chemical ionization (APCI) [9,10] and atmospheric

pressure photoionization (APPI) [11,12]. All three provide high sen-

sitivity, 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 par-

ticle 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 revolu-

tionalized 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 tech-

nique 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 inter-

nal diameter of 3–4.6 mm. Shorter columns with the same internal

diameter provide faster analysis but at cost of resolution. Capil-

lary columns, with internal diameter of 0.05–0.3 mm, offer high

separation power and sensitivity, but analysis times are long. Mono-

lithic columns, as well as the recently introduced ultra performance

liquid chromatography (UPLC), provide good chromatographic res-

olution 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 success-

ful 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 parame-

ters, 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 trifluo-

roacetic acid (TFA), may significantly suppress the ESI signal. Thus,

the selection of eluent composition for LC–MS is usually compro-

mise 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 tech-

niques 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 analyt-

ical 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 capil-

lary which is set to high voltage (typically 3–5 kV). Owing to the

high electrostatic field at the tip of the capillary, negative counte-

rions (when positive ions are analyzed) drift away from the liquid

surface towards the wall of the capillary, where they are neutral-

ized, and the positive ions drift downfield towards the liquid front

(Fig. 1A). As a result, the liquid forms a cone jet, the so-called Tay-

lor cone [16], in which the positive ions drift towards the surface of

the liquid jet (Fig. 1B). When the electrostatic repulsion at the sur-

face 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 (counterelec-

trode) of the API source, and during the flight the surface area of the

R. Kostiainen, T.J. Kauppila / J. Chromatogr. A 1216 (2009) 685–699 687

droplet starts to decrease due to evaporation of the solvent from the

droplet, and the charge density at the surface increases. At a cer-

tain 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 gas-

phase ions. Two models have been proposed for the generation of

gas-phase ions: the charge residue model [6,19] and the ion evapo-

ration model [20,21]. The theory of electrospray ionization process

is presented in more detail in earlier reviews [22–27]. The require-

ment 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 neu-

tral 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 depro-

tonated 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 com-

pounds with PAs higher than that of ammonia (853.6 kJ/mol) can be

ionized efficiently via gas-phase proton transfer reaction in ESI. Elu-

ent 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 charac-

teristics of the solvents and additives, such as volatility, surface

tension, viscosity, conductivity, ionic strength, dielectric constant,

electrolyte concentration, pH and gas-phase ion–molecule reac-

tions, 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 sol-

vation 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 ion-

ization 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 emit-

ter 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 pro-

cess 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 ana-

lyte 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-

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 poten-

tial 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 ten-

sion 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 sur-

face 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 sol-

vent. For these reasons, the disintegration of the charged droplets is

less efficient with water than with organic solvents, and the num-

ber of droplets capable of emitting gas-phase ions is decreased.

Hence, the ESI response is lower when water alone or highly aque-

ous 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 distri-

bution 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 distribu-

tion towards higher m/z values. Similarly, the addition of organic

solvent, often methanol or acetonitrile, to water may lead to con-

formational 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 reversed-

phase 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 com-

pounds 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 modi-

fiers 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


better ESI efficiency than acetonitrile [51] and gives better peak

shape for basic compounds [52], which include most pharmaceuti-

cally 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 percent-

age 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 reversed-

phase 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 com-

pounds [1,46,54,55–57]. The stationary phases in HILIC are polar –

aminopropyl- (NH2), cyanopropyl- (CN) and 2,3-dihydroxypropyl-

modified (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 sol-

vent 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 reversed-

phase 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 sig-

nal 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 analy-

sis of basic drugs (Fig. 2). The power of HILIC–ESI/MS has been

demonstrated in many applications, including the analysis of neu-

rotransmitters [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 concentra-

tion 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 contamina-

tion of the ion source resulting in decreased sensitivity and stability.

Also the strong volatile acids, such as TFA, commonly used as ion-

pairing reagents in the LC analysis of peptides and proteins, may

cause significant signal suppression in ESI [48,62,63]. Although var-

ious 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 car-

boxylic 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 ana-

lytes 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 sen-

sitivity 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 proto-

nated 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 gas-

phase proton transfer reaction or via reactions at the liquid–gas

interface of the droplet [29,65]. The gas-phase proton transfer reac-

tion is dependent on the amount of reagent ions in the gas phase,

and it has been shown that increase in the concentration of ammo-

nium 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, result-

ing in decreased ESI response (see below). It has also been shown

that acidic compounds may be efficiently ionized under acidic con-

ditions. 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


gas-phase proton transfer reaction. The significance of gas-phase

ion–molecule reactions in ESI has also been demonstrated in anal-

ysis 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 ion-

ization”, 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 ioniza-

tion and reduced sensitivity. Enke and co-workers [77] presented

an equilibrium partitioning model, which predicts that analyte

response is proportional to concentration at electrolyte concentra-

tions 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 concen-

trations 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 activ-

ity 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 con-

centration 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 stud-

ied 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 concen-

tration levels of 0.1% or even below caused clear suppression. It has

also been reported that ammonium formate, ammonium biphos-

phonate 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 result-

ing 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 sur-

face 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

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 sensitiv-

ity 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, carbo-

hydrates, 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+CH3COO]−). 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], tetra-

cyclines [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 sam-

ple 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 for-

mation of sodium clusters may be deleterious in the analysis of

polyprotic organic acids such as oligonucleotides [92] and bispho-

sphonates [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 repeatabil-

ity 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+CH3COO]−) 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 explo-

sives [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 multi-

ply 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 CCl3COO− >

CF3COO− > CH3COO− = 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-


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 ion-

pairing 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 addi-

tion 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 concen-

trated in the charged droplet due to the stronger evaporation of

TFA resulting in decreased suppression (Fig. 3). Although the “TFA-

fix” 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 pep-

tides 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 com-

pounds, 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,N-

dimethyl-n-butylamine, and tri-n-butylamine) for aromatic sul-

fonates [84]. The ion-pairing reagents used in the analysis of acids

may also cause suppression. Storm et al. [84] showed that alky-

lamines at concentrations higher than 2.5 mM resulted in a strongly

reduced signal in the analysis of aromatic sulfonates. As a con-

clusion, 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 elu-

tion 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 ace-

tonitrile at pH 6 (mobile phase A) and pH 10.5 (mobile phase B)

as solvents [122]. Multidimensional LC combined to ESI/MS utiliz-

ing IEC and reversed-phase LC has been widely used in proteomics

instead of two-dimensional electrophoresis [123–127]. The pep-

tides 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 alterna-

tive ionization method to ESI. In APCI, analytes eluting from LC are

vaporized at high temperature (300–500 ◦C) and ionized via gas-

phase 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 63Ni source, but this was soon replaced by a corona discharge nee-

dle, 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 com-

pounds are vaporized to the gas phase by thermal energy, the

method is not suitable for labile and large biomolecules such as


Table 1Ionization reactions in positive ion APCI [130]

N2 + e− → N2+• + 2e− (1)

N2+• + 2N2 → N4

+• + N2 (2)

H2O+• + H2O → H3O+ + HO• (3)

H3O+ + H2O + N2 → H+(H2O)2 + N2 (4)

H+(H2O)n − 1 + H2O + N2 → H+(H2O)n + N2 (5)

A + B+• → A+• + B (6)

A + BH+ → AH+ + B (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 concen-

trations 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 ioniza-

tion (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 diox-

ide, 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, Reac-

tion (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 2Ionization energies (IE) and proton affinities (PA) of atmospheric gases and selected

LC solvents [28]

IE (eV) PA (kJ/mol)

Nitrogen 15.581 493.8

Oxygen 12.1 421.0

Carbon dioxide 13.777 540.5

Water 12.6 691.0

Methanol 10.84 754.3

Ethanol 10.48 776.4

Acetonitrile 12.2 779.2

Ammonia 10.07 853.6

n-Hexane 10.13 –

Chloroform 11.37 –

2-Propanol 10.17 793

Isooctane 9.89 –

Benzene 9.243 750.4

Toluene 8.83 784.0

Acetone 9.703 812.0

Anisole 8.20 839.6

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 addi-

tion of 6% of methanol to water results in 50% of protonated

methanol molecules and 50% of protonated water molecules. Sim-

ilarly, 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 sup-

pression 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 mobile-

phase 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 liq-

uid 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, tri-

ethylamine, 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 reversed-

phase solvents when the aim is to ionize the analytes through

charge exchange [143,144]. For example, the post-column addi-

tion of benzene has been reported to enhance the formation of

M+• ions and the overall signal of non-polar environmental tox-

ics [145]. Kolakowski et al. [144] have studied the effect of common

reversed- and normal-phase solvents (water/acetonitrile, acetoni-

trile, 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 cyclo-

hexane and nonane promoted the formation of M+• ions. Of the

hydrocarbon solvents, isooctane and n-hexane gave the best over-

all sensitivity for both M+• and MH+, and pentane and cyclohexane


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 aque-

ous 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, ammo-

nium 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 ion-

ization 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 ana-

lytes having high PA [45,73,136,147]. A significant increase in the

Table 3Ionization reactions in negative ion APCI [148]

M + e− → M−•, ifEA(M) > 0 (1)

O2 + e− → O2−•, sinceEA(O2) = 0.48 eV (2)

O2−• + M → M−• + O2, ifEA(M) > EA(O2) = 0.48 eV (3)

HA + O2−• → A− + HO2

•, if�Gacid(HA) < �Gacid(HO2•) (4)

A + [B−H]− → [A−H]− + B, if�Gacid(A) < �Gacid(B) (5)

M + O2−• → [M−X + O]− + OX•, whereX = halogen, NO2orH (6)

M−• + O2 → [M−X + O]− + OX• (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 ana-

lytes 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 ammonium-

containing buffers has also been reported to produce ammonium

adducts and thus improve the overall sensitivity [73]. The forma-

tion 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 suppres-

sion [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 elec-

trons produced at the tip of the corona discharge needle. The

thermal electrons can be captured by compounds that possess pos-

itive 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 super-

oxide 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 com-

pounds. 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 deter-

mined 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 acidi-

ties 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, tri-

fluoroacetate) 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-


Table 4Gas-phase acidities and electron affinities of selected gases and solvents [28]

Compound EA (eV) �Gacid (kJ/mol)

Oxygen 0.451 –

HO2• – 1451

Water – 1607

Methanol – 1565

Acetonitrile 0.01 1528

Chloroform 0.622 1464

Acetic acid – 1429

Formic acid – 1415

Trifluoroacetic acid – 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 com-

pounds 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 reac-

tion.

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, N-

methylmorpholine, gave better ionization efficiency for all analytes

than the acidic buffers or solvent without buffer (Fig. 5).

Compounds that do not possess high gas-phase acidities or

positive electron affinities and cannot be ionized by deproto-

nation, charge exchange or electron capture can sometimes be

ionized by adduct ion formation through the addition, for exam-

ple, of chloroform or chloride salts ([M+Cl]−) or certain acids

([M+HCOO]−, [M+CH3COO]−) 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 Numa-

jiri [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 adja-

cent 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]).


Table 5Ionization reactions in positive ion APPI [167,168]

M + h� → M+• + e− (1)

M+• + S → MH+ + [S−H]• (2)

D(dopant) + h� → D+• + e− (3)

D+• + M → M+• + D, ifIE(M) < IE(D) (4)

D+• + S(solvent molecule) → [D−H]• + SH+, ifPA(S) > PA([D−H]•) (5)

SH+ + M → MH+ + S, ifPA(M) > PA(S) (6)

S + h� → S+• + e− (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 ioniza-

tion process is initiated by using a vacuum ultraviolet (VUV) lamp

instead of a corona discharge needle. Most often, a krypton dis-

charge lamp, which emits 10.0 and 10.6 eV photons, is used. The

compounds that can be directly ionized by the photons must pos-

sess 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 back-

ground and improved analytical selectivity are obtained as a result.

Two excellent reviews have been published on the theory and appli-

cations 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 hydro-

gen 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), tetrahydrofu-

ran (9.40 eV), and toluene (8.83 eV) [28]. The reactant ions that are

formed depend on the solvent. For example, isooctane tends to pro-

duce 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 molecu-

lar 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 C7H7• (884.0 kJ/mol [28]). This

applies to high PA additives such as ammonia, but also to the com-

mon reversed-phase LC solvents such as methanol and acetonitrile.

Although the PAs of methanol and acetonitrile are below the PA of

C7H7•, 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 trans-

fer 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 forma-

tion 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


Fig. 6. The absolute abundances of the total ion currents for the studied com-

pounds 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, ben-

zene, 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 pre-

ferred 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 com-

pounds. 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 gener-

ally 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 ioniza-

tion 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, ethylac-

etate 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]).


Table 6Ionization reactions in negative ion APPI [185]

D + h� → D+• + e− (1)

O2 + e− → O2−• (2)

M + e− → M−•, ifEA(M) > 0 eV (3)

M + O2−• → M−• + O2, ifEA(M) > EA(O2) = 0.451 eV (4)

M + O2−• → [M−H]− + HO2

•, if�Gacid(M) < �Gacid(HO2•) (5)

S + O2−• → [S−H]− + HO2

•, if�Gacid(S) < �Gacid(HO2•) (6)

M + [S−H]− → [M−H]− + S, if�Gacid(M) < �Gacid(S) (7)

M + O2−• → [M−H + O]− + OH• (8)

M−• + O2 → [M−H + O]− + OH• (9)

on the solvent system: best peak areas were obtained with

hexane/CHCl3 (1:1), isooctane, n-hexane/2-propanol (1:1), CH2Cl2,

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 ion-

ize 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 recom-

bination 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, phospho-

ric 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 contamina-

tion 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 concentra-

tion 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 elec-

trons 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 ambi-

ent 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 sol-

vent 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 elec-

tron 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 effi-

cient 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 depen-

dent 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 affin-

ity (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]−, [CH3COO]− and [CF3COO]− can sup-

press 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 sup-

press 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 sol-

vent resulting in less efficient charge exchange or electron capture

reactions. However, Song et al. [193] report that while the addi-

tion of halogenated solvents suppressed the ionization of analytes

through electron capture, dissociative electron capture or proton

transfer, the halide attachment promoted the ionization of ana-

lytes. 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-1,3,5,7-

tetrazocane (HMX). The best sensitivity for these compounds was

achieved with 1% methylene chloride addition in toluene and mon-

itoring 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 cataly-

sis 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.


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 dopant-

assisted 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 con-

ditions, 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 temper-

ature [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 acetoni-

trile as the solvent, since it has a high photoabsorption cross-section

at 10 eV energy [164]. The loss of photons would affect the forma-

tion 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 elu-

ent 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 separa-

tion, 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. Unfortu-

nately, many of the solvents that are optimal for ESI cannot be

used in LC and compromises between ionization and chromato-

graphic separation efficiencies must be made. This is especially

true with reversed-phase LC–ESI/MS analysis, where the best chro-

matographic 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 popu-

lar 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, ammo-

nium acetate and ammonium formate, which may have a significant

effect on the ionization process. In ESI the concentration of addi-

tive 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: non-

polar and neutral compounds can be ionized more efficiently with

APCI and especially with APPI than with ESI; both polar and non-

polar 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

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 gas-

phase 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 photoab-

sorption cross-section, that result in decreased number of photons

available for ionization reactions and thereby in decreased sensi-

tivity. 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 sol-

vents without any additives. However, if additives are used, their

ion energetics must be favourable for the efficient ionization of ana-

lytes. 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 com-

plex and many ionization pathways and gas-phase ion–molecule

reactions may take place simultaneously. Therefore, it is impos-

sible 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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