High pressure liquid

chromatography (HPLC)
High pressure liquid chromatography (HPLC) enables the
dissection of complex mixtures into individual constituents.
Mass spectrometry (MS) is an analytical technique that
allows sensitive and accurate determination of the
molecular weight and additional characterization of ionised
compounds in the gas phase.
The combination of HPLC and MS combines the
power of both techniques, allowing the dissection and
characterization of complex mixtures of a variety of
compounds in a single analysis.
separation of complex
mixtures
positive and negative ions
proteins, peptides and
DNA
The principle of HPLC
High pressure liquid chromatography (HPLC)
enables the dissection of complex mixtures
into their individual components. This can
be achieved by making use of different
interactions of compounds in solution, with
a stationary phase. By selecting a particular
combination of a mobile and stationary
phase, the mode of separation can be chosen
and optimised.
A commonly used form of HPLC is so-called
reversed phase HPLC. Here, a hydrophobic
stationary phase is used and compounds
are loaded under aqueous conditions.
Consequently, hydrophobic compounds
preferably interact with the stationary
phase, rather than remaining dissolved in the
aqueous phase. After loading, the conditions
in the liquid phase are slowly changed from
aqueous to organic. This results in the elution
of compounds from the stationary phase
in order of hydrophobicity. Detection of
eluting compounds yields an output such as
shown in Figure 1, called a chromatogram. If
needed, quantifcation of compounds can be
performed by calculating the area under the
curve of the peaks in the chromatogram.
Depending on the types of stationary phase,
the interaction strength in the reversed
phase can be varied. Next to reversed phase
HPLC, several other types of columns are
available differing in interaction type and/or
strength. Some examples are given in Table
1. Moreover, chromatographic modes can be
used in sequence, enabling the separation of
complex mixtures into individual compounds
based on a variety of (orthogonal)
characteristics. Finally, depending on sample
availability, the chromatographic scale can be
varied (see Characteristics).
Detection
Even though UV is a sensitive detection
method, not every compound exhibits UV
absorption. This can cause problems in the
detection and quantifcation of particular
analytes. Therefore, a mass spectrometer can
be used as (a second) detector. In this way,
the range of detectable compounds can be
improved (see Figure 1).
The principle of mass spectrometry
A mass spectrometer generally consists of
three modules: an ion source, a mass analyser
and a detector. This is depicted in Figure 2.
When using an electrospray ion (ESI) source,
compounds in an acidifed liquid phase are
introduced into the mass spectrometer
through a heated capillary. Consequently, liquid
0 5 10 15 20 25 30 Time [min]
UV Chromatogram,
254 nm
0 5 10 15 20 25 30 Time [min]
BPC 150 - 1500 +All MS
Fig. 1: UV chromatogram (light blue) and MS positive ion chromatogram (dark blue) of the same
HPLC separation of a crude mixture obtained after organic synthesis. The MS signal provides a
different view of the sample compared to the UV signal.
HPLC outlet
3D ion trap
Time of flight
Ion formation
ESI
source
Ion optics Mass analyser/detector
Mass spectrum
Table 1: Examples of some column types available for a wide range of separations.
Fig. 2: Schematic setup of a mass spectrometer: the outlet of the LC is coupled directly to the
electrospray ionisation source. Ions formed are introduced into the mass spectrometer and ion
optics are subsequently used to focus the ions. The mass analyser, which can for example be a
3D ion trap or Time of Flight, separates by mass the ions that fnally reach the detector yielding
a mass spectrum.
Type of column Mode of separation
Reversed / normal phase Differences in hydrophobicity (polarity)
Ion exchange Differences in charge at a certain pH
Gel filtration Differences in size (molecular weight)
(Bio-)affinity Differences in interactions with a ligand
vaporizes and ions are formed. The resulting
ions enter the mass analyser, which can, for
example, be an ion trap (IT) or a time of fight
(ToF) tube. The mass analyser separates ions
based on their mass and fnally a detector (the
red squares in Figure 2) is used for detection of
the ions. This can be done either in the positive
or the negative ion mode.
The mass spectrum
A mass spectrometer determines the
mass-over-charge (m/z) ratio of ions, which
can be used to calculate the mass of the
corresponding molecules.
Due to the occurrence of natural (stable)
isotopes, the vast majority of compounds is
represented by multiple peaks in the mass
spectrum. Figure 3 shows a typical mass
spectrum, in which several ions of average
organic compounds are present. Some of
these ions are singly charged, others are
doubly (or multiply) charged as can be seen in
the inset. The difference between such charge
states can be determined by the distance
between the isotope peaks.
Ion trap mass spectrometry: compound
fragmentation
An ion trap mass spectrometer cannot
only determine the m/z ratio, but is also
capable of performing so-called tandem mass
spectrometry (MS/MS or MS
2
), in which a
certain ion is selected for fragmentation. This
can help to identify particular elements in
a molecule. An example of a fragmentation
study is depicted in Figure 4. During
fragmentation, (labile) bonds in the molecule
break, thereby producing fragment ions
that are characteristic for certain chemical
moieties. In some cases, highly labile bonds
are present, and fragmentation will yield
only little compositional information. In that
case it is possible to perform sequential
fragmentation (MS
n
), which enables isolation
and subsequent fragmentation of fragment
ions. This, in theory, makes it possible to
break a molecule down to very small pieces,
yielding more detailed information on the
molecular structure.
Time of fight mass spectrometry: high
mass accuracy
In contrast to an ion trap, a time of fight
(ToF) is not capable of performing tandem
mass spectrometry. On the other hand, it
can determine molecular masses of ionised
compounds with much higher accuracy
(< 5 ppm) than the ion trap. Accurate mass
determination can also aid in resolving
the elemental composition of an unknown
compound. Moreover, determining a relative
mass shift on a particular compound resulting
from a chemical modifcation can help in
determining the reaction effciency. In Figure
5 a magnifed version of a mass spectrum
generated by a ToF is given, which shows
baseline resolution for individual isotopes of
a singly charged ion.
200 250 300 350 400 450 500 550
169.0
208.0
223.0
257.1
266.1
291.0
308.1
330.2
353.2
373.2
396.2
416.2
479.2
498.3
577.5
600 m/z
225.1
281.2
307.2
356.3
391.2
410.3
449.3
480.4
528.3
561.5
597.4
613.4
764.5
819.7
597.4
598.5
599.4
597.4
MS survey scan
Precursor selection
MS/MS: fragmentation
0
1
2
3
4
5
x10
7
Intens
200 300 400 500 600 700 800 900 m/z
0
1
2
3
4
5
x10
7
Intens
200 300 400 500 600 700 800 900 m/z
0.0
0.2
0.4
0.6
0.8
1.0
x10
8
Intens
2.5
x10
7
Intens
379.2
406.2
429.0
497.8
510.4
557.2
577.3
604.4
642.9
673.3
696.4
735.2
780.5
811.5
834.5 872.4
894.2
922.3
994.5
1019.6
0.0
0.5
1.0
1.5
2.0
400 500 600 700 800 900 1000 m/z
811.5
812.4
813.4
814.4
696.9
696.4
697.3
697.8
698.2
1+ ion
2+ ion
Fig. 4: The process of MS/MS fragmentation in a (schematically drawn) 3D ion trap, illustrated for
an ion with an m/z of 597.4. After detection of the ion in an MS survey scan (top panel), the ion
of interest (precursor) is selected (middle panel) and subsequently fragmented (bottom panel),
yielding information about the composition of the ionised molecule.
Fig. 3: Typical mass spectrum, containing a large number of ions. In the inset an example of
both a singly and doubly charged ion are given.
2008 Koninklijke Philips Electronics N.V.
All rights reserved.
Characteristics
Information obtained
Sample composition, molecular
weight and structural information on
individual components
Qualitative and (semi-)quantitative
information
Sample type
Solid or liquid (dissolved in aqueous
buffers or solvents like DMF or DCM)
Analytical range
Percentages using normal HPLC
dimensions (2.1x100mm columns, 0.3
mL/min fow rates)
Femtomoles using capillary HPLC
(0.3x200mm columns, 1L/min fow
rates)
Nano LC lowers column dimensions
and fow rates even further, thereby
increasing sensitivity
Typical sample size
0.5-10 L
Accuracy
< 5ppm (for LC-ESI-ToF analyses)
Destructive
Yes
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offers a full range of analytical methods
and expertise to support both research
and manufacturing, serving customers
by taking an integral, solution-oriented
approach.
World-class expertise
working for you
For more information:
Tel./fax: +31 -40-2748044/42944
E-mail: materials@miplaza.com
www.miplaza.com/materials
Technical Note 23
July 2008
Applications
Characterization of reaction mixtures
resulting from organic synthesis
- purity of the starting material
- the presence of remaining starting
compounds and/or unexpected side
products
- identifcation based on fragmentation
patterns
Quantitation of (a) specifc compound(s)
within a mixture
Identifcation and characterization of
biomolecules
- protein sequencing
- molecular weight determination of
proteins or DNA
Fig. 5: Example of an accurate mass determination, using an
ESI-ToF mass spectrometer.
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
554 555 556 557 558 559 560 561 m/z
I
n
t
e
n
s
i
t
y

(
c
o
u
n
t
s
)
557.17814
558.18152
559.18576

Antibodies are proteins that are
increasingly used for diagnostic purposes.
To visualize them in cells or the
human body, fuorescent or radioactive
reporter groups have to be attached
to the antibody. The resulting modifed
antibodies can be studied using LC-MS,
giving insight into the labelling effciency.
By measuring the intact protein both
before and after labelling, the (average)
number of modifcations present per
protein molecule can be determined
using the mass difference before and after
labelling.

Subsequent proteolytic digestion of antibodies
into peptides and LC-MS/MS analysis of
the resulting peptide mixtures allows for
comparison of unmodifed and chemically
modifed proteins. This enables the localization
of chemical modifcations in the primary
sequence of the protein. Large numbers of
protein 3D structures are given in public
databases. This information can be used
to determine the site of modifcation on a
protein. In this way, information is obtained
about possible activity loss as a result of the
chemical modifcation of amino acids that are
essential for protein function.
A typical application: Characterization of chemical modifcations on proteins