Application

Note: AN04-01

Mobile Phase Selection in LC/API-MS

Luisa Pereira, Thermo Electron Corporation, Runcorn, Cheshire UK

Key Words
Mobile phase
Electrospray
APCI
Buffer pH
Solvent
composition
Additives

Introduction

API Techniques

In LC/UV, selectivity is generally altered by changing

mobile phase composition or stationary phase
functionality. The most successful and convenient option
in the past, particularly in the analysis of ionizable
compounds, has been to optimize the mobile phase
composition by changing solvent type and strength,
buffer type and concentration, and pH. In LC/MS with
atmospheric pressure ionization (API) techniques, the
mobile phase composition needs to balance the
chromatographic requirements with the ionization
efficiency. Solution and gas phase chemistries need to
be optimized in order to maximize ionization. Also,
components which may cause ionization suppression,
may cause background ions or induce the formation of
several molecular weight adducts, need to be removed.
This application note discusses the selection of mobile
phase when developing a method in RP-LC/API-MS.
Parameters discussed are solvent nature, buffer and
additive type and concentration, mobile phase pH, and
the effects these have not only on the chromatographic
resolution but also on the detector response.

The two most widely used API techniques are Electrospray

Ionization (ESI) and Atmospheric Pressure Chemical
Ionization (APCI). These MS inlets or interfaces must
transfer the analyte from the liquid phase into the gas
phase, remove the mobile phase, and generate sample
ions. In electrospray (Figure 1), the LC eluent containing
the analytes is sprayed into a chamber at atmospheric
pressure by the action of a high potential difference and
a concentric flow of nebulizing gas. The resulting spray
contains highly charged droplets. The ejection of the ions
from the charged droplets occurs by the action of heated
drying gas.
Essentially, electrospray is a desorption technique as
it transfers ions from the liquid phase into the gas phase.
Thus, an understanding of solution chemistry is essential
as ions are formed in solution. Acid-base equilibrium,
redox reactions, and interaction with electrolytes in the
mobile phase will all affect the formation of analyte ions
and thus detector response. In contrast to electrospray, ion
formation in APCI occurs in the gas phase (Figure 2). The
LC eluent containing the analyte is sprayed into a heated
chamber at atmospheric pressure. The heat evaporates
solvents and analytes; once in the gas phase the neutral
analyte molecules are ionized by collision with reagent
ions produced by interaction of the source gases with a
corona discharge. Ionization can occur by proton transfer

To MS

Figure 1: Schematic of the electrospray process

To MS

Figure 2: Schematic of the Atmospheric Pressure Chemical Ionization (APCI) process.

or charge exchange, and thus the proton affinity of all

sample and mobile phase components is an important
parameter to consider. Also, since neutral molecules are
more volatile than ions, selection of mobile phase
conditions which will maintain analytes in a neutral form
in the liquid phase (acid-base equilibrium) will aid
ionization in the gas phase and maximize MS sensitivity.
The complimentary nature of ESI and APCI make both
valuable as LC interfaces to MS.

Although solution chemistry is not as critical in APCI

as in electrospray, the solvent properties still need to be
considered for best performance. Protic solvents such as
methanol improve positive ionization, whereas in negative
APCI solvents which can capture electrons are more
beneficial. Low polarity solvents generally used in normal
phase chromatography can also be used in APCI, since
the solvent evaporation and ionization processes are not
simultaneous like in ESI.

Solvents

Buffers and Additives

The most common solvents used in LC/API-MS are water,

methanol, acetonitrile and mixtures of the above.
The solvent composition (aqueous-to-organic ratio) is
particularly important in the electrospray nebulization
and ionization process. The efficiency of the electrospray
process depends on the conductivity and surface tension
of the liquid being nebulized. When the conductivity
and/or the surface tension are too high (i.e. highly
aqueous) it is difficult to produce a stable spray and to
vaporize the droplets formed by the action of the high
voltage and nebulizing gas. Because the surface tension
of water is much higher than the surface tension of
methanol or acetonitrile, the sensitivity is reduced when
using more than 70-80% of aqueous mobile phase. If
the chromatographic separation requires high aqueous
content, then one alternative is to add a sheath liquid
prior to ionization. The sheath liquid is highly organic
(example isopropanol) to help the spray and vaporization,
but of course this involves a more complex setup
(Figure 3). The aqueous-to-organic ratio is more
significant when working at high flow rates since there is
more solvent to be nebulized and vaporized. A very high
organic content may also lower the sensitivity, especially
if no additive is used, because the conductivity of pure
organic solvent is too low. A small percentage of water
in the mobile phase aids the droplet formation.

Additives are often used in the mobile phase, either to

enhance the LC separation and resolution, or to enhance
ionization of the sample by post-column addition.
The preferred additives are formic and acetic acids (0.01
to 1% v/v) because they improve protonation of basic
analytes in positive ionization. The most compatible
buffers are ammonium formate, ammonium acetate,
and ammonium hydroxide at concentrations of 10 to
20mM in ESI and up to 50mM in APCI (Table 1).
Other additives occasionally used include
trifluoroacetic acid (TFA), triethylamine (TEA) and
diethylamine (DEA) but these need to be used at low
concentrations (<0.1% v/v) since they may cause
ionization suppression. TFA is the most commonly used
ion-pairing agent in RP-LC of polypeptides. The signal
suppression by TFA is caused by ion-pairing with the
positive charged analyte and surface tension. The TFA
anion masks the positive charge on the analyte molecule
and thus prevents ion evaporation of that ion1. Postcolumn addition of propionic acid (10%) in isopropanol
(75:25 v/v) has been used to counteract the signal
suppression (TFA fix)2. Also, TFA has a high surface
tension, thus it enriches the droplet surface during droplet
formation, preventing the analytes from migrating to the
surface and being evaporated.

Buffer

pK

Buffer range

Concentration

Trifluoracetic acid

<1

1.5 2.5

< 0.1% (v/v)

Formic acid / ammonium formate

3.8

2.8 4.8

10 20 mM

Acetic acid / ammonium acetate

4.8

3.8 5.8

10 20 mM

Ammonium hydroxide / ammonia

9.2

8.2 10.2

10 20 mM

Table 1: Buffers for use in LC/MS

DEA and TEA have high proton affinities and thus

will compete with analyte molecules for protons,
preventing positive ionization of the sample molecules.
Traditionally, TEA has been used in the analysis of basic
compounds at intermediate pH to ensure deactivation
of the acidic silanols on the column. Newer silica-based
stationary phases have fewer acidic silanols and thus
symmetrical peaks are obtained for basic compounds
at neutral pHs, without the need for ion-pairing agents.
If the chromatographic method requires an ion-pairing
agent then, heptafluorobutyric acid (HFBA) and
tetraethylammonium hydroxide (TEAH) can be used in
LC/API-MS as anionic and cationic reagents respectively.
Involatile buffers are very popular in RP-LC because
of their good buffering capacity and wide buffer range.
However, the use of high purity silica-based stationary
phases with volatile buffers allows for good
chromatographic performance to be obtained. Involatile
buffers such as phosphates, citrates and borates, ion
pairing agents and inorganic acids are generally avoided
in LC/API-MS method development, although modern
orthogonal (off-axis) sources are more robust, and have
been designed to operate with low concentrations
(<10 mM) of involatile buffers or additives with minimal
sample clean-up. Since blockage of the ion sampling cone
can still occur with involatile buffers, a more robust
orthogonal source is the M-Path triple orthogonal
sampling system on the Finnigan Surveyor MSQ. This
interface includes a self-cleaning feature with patented
cone wash that can operate effectively over the range of
buffer concentrations commonly used in classical RP-LC.
Post-column modification of the mobile phase is the
approach taken when the conditions necessary to promote
ionization interfere with chromatographic performance.
Generally, an additive is introduced into the LC eluent
either via a T-piece (Figure 3) or coaxially if the source
design allows it.

Mobile Phase pH
The pH of the mobile phase determines the ionization
state of the analytes, when working with acids, bases or
amphoteric species, and therefore affects the response in
LC/API-MS. The mobile phase pH also determines
chromatographic selectivity for ionizable compounds:
at pH values where the analyte is in its non-ionic form
then the hydrophobic interactions with the stationary
phase are maximized and so is retention; at pH values
where analytes are fully ionized then retention decreases
in RP-LC; at pH values within 2 units of the pK the
biggest changes in retention occur (Figure 4). For good
pH control of the mobile phase it should be within 1 pH
unit of the buffer pK value (Table 1).
Experimental conditions:
Column: HyPURITY C18, 5m, 50x2.1mm;
Mobile phase:
Aqueous (50%)
0.1% Formic acid
pH 3 - Ammonium formate 20 mM
pH 5 - Ammonium acetate 20 mM
pH 8.2 - Ammonium acetate 20 mM
pH 9 - Ammonium acetate 20 mM

Organic (50%)
MeOH
MeOH
MeOH
MeOH
MeOH

Flow rate: 0.2 mL/min;

Temperature: 25C;
Detection: +ESI, 450C, 4.5kV, 20V (-ESI, 450C, 3.5kV, 20V); Scan:
120 480u. Analytes: Nortriptyline, Propranolol, Tetracycline,
Caffeine, Paracetamol, Tryptophan, Salicylic acid, Nicotinic acid

Figure 4: Effect of mobile phase pH on the retention factor of several

compounds in RP-LC

Figure 3: Sheath liquid addition

Information on the pK of each proton on the analyte

will allow prediction of the ionization state of the analyte
as a function of pH. With APCI, which requires the
sample to be vaporized before ionization, pH should be
adjusted to a value where analytes exist in the neutral
form: for acids, 2 pH units below pK, for bases 2 pH
units above pK. Conversely, for ESI the analyte should
be ionized in solution, thus pH needs to be adjusted to
2 pH units below pK for bases and 2 pH units above pK
for acids (Figure 5).
However, these are general guidelines, often the pK of
the analytes is not known, or may be difficult to predict
based on the pK values for each proton, particularly if the
analytes are complex molecules containing diverse
functionalities. In this situation, several regions of pH
need to be evaluated for best response both in positive and
negative polarity modes. Figure 6 and 7 illustrate the
variation of response in positive and negative ESI-MS with
pH of the mobile phase for 8 compounds with basic,
acidic or both functionalities. The best response in positive
ESI (Figure 6) is obtained with 0.1% formic acid, for
nortriptyline, propranolol, tryptophan and nicotinic acid.
The response of the most basic compounds (nortriptyline
and propranolol) decreases as the pH increases, other
compounds such as tetracaine and tryptophan show a
different trend because of the presence of functional
groups with diverse acidity or basicity. Salicylic acid
cannot be detected under positive ionization. In negative
ESI (Figure 7), the response of salicylic acid and nicotinic
acid increases as the pH increases, as the carboxylic group
becomes deprotonated.
The response for compounds such as tetracycline and
tryptophan show a similar trend in positive and negative
ESI as the basic and acidic group become protonated or
deprotonated respectively.

In addition to these
offices, Thermo Electron
Corporation maintains
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organizations
throughout the world.

Figure 5: Representation of the favored pH regions for APCI and Electrospray:

relative abundance of an acid (AH) and its conjugated base
(A), and a base (B) and its conjugated acid (BH+) at various pH values

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Figure 6: Effect of mobile phase pH on the peak area obtained in positive

ESI-MS for several compounds. For experimental conditions refer to Figure 4.

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Conclusion

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The mobile phase composition is an important parameter

to consider when developing a method in RP-LC/API-MS.
The nature of the buffer and its concentration, the buffer
pH, the organic solvent nature and the aqueous/organic
ratio all affect chromatographic selectivity, resolution and
also the detector response.

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References
1. W. Niessen, Liquid Chromatography-Mass Spectrometry,
Chromatographic Science Series, Volume 79, page 320.
2. A. Apffel, S. Fisher, G. Goldberg, P.C. Goodley, F.E. Kuhlman,
J. Chromatogr. A, 712 (1995) 177.

Figure 7: Effect of mobile phase pH on the peak area obtained in negative

ESI-MS for several compounds. For experimental conditions refer to Figure 4.

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