Dionex MSQ User Manual
MSQ
Document No. 031871
Hardware
Revision 02
October 2003
™
©2003 by Dionex Corporation
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Revision 01, July 2002
Revision 02, October 2003
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Published by Technical Publications, Dionex Corporation, Sunnyvale, CA 94086.
Chapter 1
Introducing the MSQ 1.
Introducing the MSQ.................................................................................................................1-i
Introduction ....................................................................................................................................1-1
System Overview............................................................................................................................1-2
What Is Mass Detection?...................................................................................................1-4
Exterior Features of the MSQ............................................................................................1-5
The Source–An Introduction to API Techniques ...........................................................................1-8
Electrospray.......................................................................................................................1-9
Atmospheric Pressure Chemical Ionization.....................................................................1-13
Source Fragmentation......................................................................................................1-16
Cone Voltage Ramping ...................................................................................................1-18
Polarity Switching ...........................................................................................................1-19
Application of API Techniques .......................................................................................1-20
The Self-Cleaning Source: Cone Wash ........................................................................................1-23
Introduction .....................................................................................................................1-23
Functional Description ....................................................................................................1-24
The Reference Inlet System..........................................................................................................1-25
Introduction .....................................................................................................................1-25
Functional Description ....................................................................................................1-25
The Mass Analyzer and Detector .................................................................................................1-26
The Vacuum System.....................................................................................................................1-27
The Data System...........................................................................................................................1-28
Software...........................................................................................................................1-28
Raw Data .........................................................................................................................1-30
Raw Data Types...............................................................................................................1-31
____________________________MSQ Hardware Manual _____________________________ 1-i
Introducing the MSQ
Introduction ____________________________________________________________________________
1-ii____________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
___________________________________________________________________________ Introduction
Introduction
The MSQ™ MS detector has been specifically designed and engineered for
liquid chromatographic detection using Atmospheric Pressure Ionization
(API) and Mass Spectrometry (MS) technology. These technologies can
provide sensitive and selective detection of organic molecules.
Interfacing High Performance Liquid Chromatography (HPLC or LC) and
MS provides the separation scientist with one of the most powerful
analytical tools available. Both LC and MS have developed to a point
whereby they represent two of the most important techniques in
characterizing and detecting organic compounds. Although the potential
benefits of interfacing LC to MS have been clearly recognized for many
years, producing a truly automated “connect-and-use” interface has proven
to be a challenging task.
Atmospheric Pressure Ionization (API) techniques now provide highly
sensitive detection using conventional to capillary LC flow rates on benchtop MS detector systems. LC/MS works with typical solvent compositions,
whether the separation is achieved by isocratic or gradient elution.
Historically, LC/MS has been compatible only with volatile buffer systems
using modifiers such as trifluoroacetic acid, formic acid, and acetic acid.
Phosphate buffers, although extensively used in LC separations, were not
suited to LC/MS due to rapid blocking of the ion sampling region caused by
the deposition of involatile phosphate salts. The self-cleaning API source
allows for extended periods of operation in LC/MS with chromatographic
buffers such as phosphates or ion-pairing agents and samples in dirty
matrices.
API using Electrospray (ESI) or Atmospheric Pressure Chemical Ionization
(APCI) interfaces has proved to be invaluable in meeting sensitivity
requirements in quantitative methods. It can also provide structural
information, which is complementary to techniques such as NMR and infra
red spectroscopy.
This introduction focuses on the principal components of the system.
___________________________MSQ Hardware Manual ____________________________ 1-1
Introducing the MSQ
System Overview ________________________________________________________________________
System Overview
The MSQ MS detector is an integral part of the LC detection system. Key
points of the system are:
The sample is introduced into the ion source using an LC system,
•
possibly through a column.
•
In an API MS detector, the part of the source where ionization takes
place is held at atmospheric pressure, giving rise to the term
Atmospheric Pressure Ionization (API).
•
In ESI, the sample is ionized in the liquid phase, while in APCI,
ionization occurs in the gas phase. In both cases, efficient desolvation is
needed to remove the solvents from the sample.
•
Ions, now in the gas phase, are passed through the mass analyzer and are
collected at the detector.
•
The detected signal is sent to the data system and stored ready for
processing.
LC System
Sample
introduction
LC Column
Separation
Figure 1-1. The key components of the MSQ API LC detection system
Ion Source
Ionization &
transmission
Mass Analyzer
Sorting of ions
Turbomolecular
& rotary pumps
Molecular weight information
Structural information
Positive identification
Quantitative information
Detector
Detection
of ions
Data System
Windows NT
1-2 ___________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________________________________System Overview
The main features of the MSQ MS detector are:
Dual ESI/APCI orthogonal probe •
• • Self-cleaning API-LC/MS interface
M-Path™ triple orthogonal source
Quadrupole
mass analyzer
Split flow
turbomolecular
pump
Square
quadrupole
RF lens
Rotary
pump
Exit cone
Entrance cone
From HPLC
Cone wash
Orthogonal sample
introduction probe
Figure 1-2. Schematic diagram of the MSQ API inlet, analyzer, and
detector system
The LC eluent is ionized at the API probe and the resulting ions are focused
into a square quadrupole RF lens. The quadrupole mass analyzer filters the
ions before detection.
___________________________MSQ Hardware Manual ____________________________ 1-3
Introducing the MSQ
System Overview ________________________________________________________________________
What Is Mass Detection?
Mass detection is a very powerful analytical technique used in a number of
fields, including:
Identification of unknown compounds •
• • Quantitation of known compounds
Determination of chemical structure
The basic function of an MS detector is to measure the mass-to-charge ratio
of ions.
The unit of mass used is the Dalton (Da). One Dalton is equal to 1/12 of the
mass of a single atom of carbon-12. This follows the accepted convention
that an atom of carbon-12 has exactly 12 atomic mass units (amu). The MS
detector does not directly measure molecular mass, but the mass-to-charge
ratio of the ions. Electrical charge is a quantized property and so can exist
only as an integer; that is, 1, 2, 3, and so on. The unit of charge used here (z)
is that which is on an electron (negative) or a proton (positive). Therefore,
the mass-to-charge ratio measured can be denoted by m/z. Most ions
encountered in mass detection have just one charge. In this case, the massto-charge ratio is often spoken of as the “mass” of the ion.
1-4 ___________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________________________________System Overview
Exterior Features of the MSQ
This section highlights the exterior features of the MSQ. The parts labeled
here may be referred to in later chapters of this manual or other manuals
supplied with the MSQ.
Status light
Figure 1-3. Front view of the MSQ
Figure 1-3 shows the front view of the MSQ. The main feature is the status
light.
___________________________MSQ Hardware Manual ____________________________ 1-5
Introducing the MSQ
System Overview ________________________________________________________________________
Table 1-1. Instrument status light
Instrument Status Light
Vented Red
Venting Red
Pumping down Flashing yellow
Under vacuum (above
vacuum trip)
Under vacuum (ready
for use)
Operate on (MSQ in
use)
Source enclosure open Red
Red
Yellow
Green
The vacuum trip is the pressure below which it is safe to switch on the
voltages in the source. When the instrument is functioning normally, the
status light will go from flashing yellow to solid yellow and Operate can be
switched On. If the pressure in the instrument rises above the operating
pressure, the status light turns red to indicate that the pressure is above a
safe level. See the chapter Shutting Down and Restarting the System for
information on pumping down the MSQ.
Figure 1-4 shows the MSQ with the doors open. The source enclosure and
reference inlet are now visible.
Source
enclosure
Figure 1-4. The MSQ with the doors open
1-6 ___________________________ MSQ Hardware Manual ____________________________
Reference
inlet
Introducing the MSQ
_______________________________________________________________________System Overview
Figure 1-5 is a schematic of the rear view of the MSQ.
PUMP RELAY
Rotary pump power
To P C
Contact closure and
analog inputs
To rotary pump
USB
SOURCE
USER I/O
EXHAUST
Exhaust from API
source
BACKING
To rotary pump
Figure 1-5. Rear view of the MSQ
RESET
MODEL:
RATING: 220-240v
GAS IN
6 BAR MAX
50/60 Hz
1000 VA
MAINS ON/OFF
Gas inlet for nebulizer
and sheath gas
Reset communications
Power switch
MAINS IN
Power supply
___________________________MSQ Hardware Manual ____________________________ 1-7
Introducing the MSQ
The Source–An Introduction to API Techniques ________________________________________________
The Source–An Introduction to API Techniques
The source, or interface, performs four main functions:
Separates the analytes from the solvent and buffer systems used in LC •
• • Ionizes the analyte molecules
Allows efficient transfer of ions into the mass analyzer for detection
LC eluent enters the source through the orthogonal sample introduction
probe. The primary objective of an orthogonal probe is to direct any
involatile components present in the LC eluent, such as those from buffers,
ion-pairing agents, or matrices, away from the entrance orifice. Under
operating conditions, however, both the sample ions and the charged liquid
droplets (containing any involatile components, if they are present) are
deflected by the electric field towards the entrance orifice. This leads to a
gradual buildup of involatiles and a concomitant loss in sensitivity with
time. The self-cleaning source delivers a constant, low flow of solvent (the
cone wash™) to the edge of the inlet orifice, helping to prevent a buildup of
involatiles during an LC/MS run.
1-8 ___________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________ The Source–An Introduction to API Techniques
Rotary
pump
Exit cone
To the mass
analyzer
Entrance cone
From HPLC
Figure 1-7. Schematic of the MSQ source showing the cone wash
Cone wash
Orthogonal sample
introduction probe
Two types of API are commonly encountered. These are Electrospray
Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI).
The following sections discuss the mechanism of ion generation in each.
Electrospray
Electrospray Ionization (ESI) is regarded as a soft ionization technique
providing a sensitive means of analyzing a wide range of polar molecules.
Since the first combined ESI LC/MS results were announced in 1984, and
its first application to protein analysis four years later, the technique has
become an established analytical tool in separation science.
When applied to smaller molecules up to 1000 Daltons in molecular mass,
electrospray ionization results in either a protonated, [M+H]
1-8) or deprotonated, [M-H]
-
, molecule. Choice of ionization mode is
governed by the functional chemistry of the molecule under investigation. In
ESI, fragmentation is generally not apparent; however, increased source
voltages can induce fragmentation to provide structural information.
___________________________MSQ Hardware Manual ____________________________ 1-9
+
(see Figure
Introducing the MSQ
z
The Source–An Introduction to API Techniques ________________________________________________
100
240
OH
NH
tBu
HO
%
HO
Chemical structure of salbutamol,
(molecular weight 239)
0
60 80 100 120 140 160 180 200 220 240 260 280 300
241
m/
Figure 1-8. Electrospray mass spectrum of salbutamol in positive ion
mode
The base peak at m/z 240 (see Figure 1-8) corresponds to the protonated
salbutamol molecule. It is notable that ESI results in a prominent base peak
with minimal fragmentation, quite dissimilar from the results often achieved
with GC/MS.
Mechanism of Ion Generation
Electrospray ionization operates by the process of emission of ions from a
droplet into the gas phase, a process termed Ion Evaporation. A solvent is
pumped through a stainless steel insert capillary that carries a high potential,
typically 3 to 5 kV (see Figure 1-9). The strong electric field generated by
this potential causes the solvent to be sprayed from the end of the insert
capillary (hence, electrospray), producing highly charged droplets. As the
solvent is removed by the desolvation process, the charge density on the
surface of the droplets increases until the Rayleigh limit is exceeded; after
this, a multitude of smaller droplets are formed by coulombic explosion.
This process is repeated until charged sample ions remain. These ions are
then available for sampling by the ion source.
-
+
Insert capillary +3-5 kV
+
+
+
+
+
Droplet
containing
ions
+
+
+
+
-
+
+
+
+
-
+
+
+
+
+
+
+
As the droplet
evaporates, the
electric field
increases and ions
move towards
the surface
Figure 1-9. Positive ion electrospray mechanism
-
+
+
+
-
+
+
+
+
+
-
+
-
+
+
+
-
+
+
++
+
+
+
Ions evaporate
from the surface
1-10 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________ The Source–An Introduction to API Techniques
Electrospray Ionization Using the MSQ Source
The sample, in solution, enters the source via a stainless steel insert capillary
held at a voltage of 3 to 5 kV. The insert capillary is surrounded by a tube
that directs a concentric flow of nitrogen nebulizing gas past the droplets of
liquid forming at the probe tip. The action of the nebulizing gas, high
voltage, and heated probe produces an aerosol of liquid droplets containing
ions of the sample and solvent. The ion evaporation process is assisted by a
second concentric flow of heated nitrogen gas. This is the sheath gas. This
highly efficient evaporation process close to the entrance cone enables the
routine use of high LC flow rates (up to 2.0 mL/min) in ESI mode.
The newly formed ions then enter the focusing region through the entrance
cone. This is due to the following:
The high electric field. The insert capillary is at 3 to 5 kV with respect
•
to the rest of the source, which is typically at 20 to 30 V.
• The gas flow into the focusing region.
Ions then exit the focusing region and pass into the RF lens. The RF lens
(square quadrupole) helps to focus the ions before they enter the mass
analyzer region.
Region
Intermediate
Pressure
Region
Exit cone
Entrance cone
Probe
Insert capillary
LC eluent
Nebulizing gas, N
Sheath gas, N
2
2
Insert
Cone Wash
Atmospheric
Pressure
Region
Figure 1-10. Schematic of the ESI source on the MSQ, showing the
principal components and pressure regions
Rotary
pump
___________________________MSQ Hardware Manual ___________________________ 1-11
Introducing the MSQ
The Source–An Introduction to API Techniques ________________________________________________
Spectral Characteristics
Polar compounds of low molecular weight (<1000 amu) typically form
singly charged ions by the loss or gain of a proton. Basic compounds (for
example, amines) can form a protonated molecule [M+H]
analyzed in positive ion mode to give a peak at m/z M+1. Acidic
compounds (for example, sulphonic acids) can form a deprotonated
molecule [M-H]
-
, which can be analyzed in negative ion mode to give a
peak at m/z M-1. As electrospray is a very soft ionization technique, there is
usually little or no fragmentation and the spectrum contains only the
protonated or deprotonated molecule.
Some compounds are susceptible to adduct formation if ionization takes
place in the presence of contamination or additives such as ammonium or
sodium ions. The spectra will show other ions in addition to, or instead of,
the quasi-molecular ion. Common adducts are ammonium ions NH
[M+18]
100
+
, sodium ions Na+ [M+23]+, and potassium ions K+ [M+39]+.
+
, which can be
+
4
[M+H]
322
+
%
+
100
103
80
99
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360
141
145
181
187
241
244
261
279
282
[M+Na]
344
363
m/z
Figure 1-11. Electrospray spectrum showing a sodium adduct
The singly charged ions arising from samples of relatively low molecular
masses can be interpreted directly, as they represent the protonated or
deprotonated molecule. Electrospray, however, can produce multiply
charged ions for analytes that contain multiple basic or acidic sites, such as
proteins and peptides. As an MS detector measures mass-to-charge ratio
(m/z), these ions appear at a m/z value given by the mass of their protonated
molecule divided by the number of charges:
n
+
nHM
+
n
zm
=
Where, M = actual mass, n = number of charges, and H = mass of a proton.
Electrospray allows molecules with molecular weights greater than the mass
range of the MS detector to be analyzed. This is a unique feature of
electrospray.
1-12 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________ The Source–An Introduction to API Techniques
Flow Rate
The electrospray source can be used with flow rates from 5.0 µL/min to
2.0 mL/min.
Atmospheric Pressure Chemical Ionization
Atmospheric Pressure Chemical Ionization (APCI) is also a very soft
ionization technique and has many similarities to electrospray ionization.
Ionization takes place at atmospheric pressure and the ions are extracted into
the MS detector in the same way as in electrospray.
Similarly, as observed in ESI, [M+H]
providing molecular weight information. Fragmentation can be induced in
the source by increasing the source voltage to give structural information.
Mechanism of Ion Generation
+
and [M-H]- ions are usually formed
In APCI, the liquid elutes from an insert capillary, surrounded by a coaxial
flow of nitrogen nebulizing gas into a heated region. The combination of
nebulizing gas and heat form an aerosol that evaporates quickly to yield
desolvated neutral molecules (see Figure 1-12).
At the end of the probe is a corona pin held at a high potential (typically 2.0
to 3.5 kV). This produces a high-field corona discharge that causes solvent
molecules eluting into the source to be ionized. In the atmospheric pressure
region surrounding the corona pin, a series of reactions occur that give rise
to charged reagent ions. Any sample molecules, which elute and pass
through this region of reagent ions, can be ionized by the transfer of a proton
to form [M+H]
+
or [M-H]-. This is a form of chemical ionization; hence the
name of the technique, Atmospheric Pressure Chemical Ionization.
Heated nebuliser
N
2
Liquid
N
2
Solvent molecules
An aerosol
is formed
Sample molecules
Solvent and
sample molecules
are desolvated
Corona pin
Collisions and
+
+
+
proton transfer
+
+
+
+
Solvent molecules
are ionized
+
+
Sample
+
[M+H]
ions formed
Figure 1-12. Positive ion APCI mechanism
___________________________MSQ Hardware Manual ___________________________ 1-13
Introducing the MSQ
The Source–An Introduction to API Techniques ________________________________________________
APCI Using the MSQ Source
The sample is carried to a spray region via a stainless steel insert capillary.
The action of both the nebulizing gas and the heated probe leads to the
formation of an aerosol. The desolvation process is assisted by a second
concentric flow of nitrogen gas, the sheath gas.
In contrast to electrospray, APCI is a gas phase ionization technique.
Ionization occurs as the aerosol leaves the heated nebulizer region. A corona
pin, mounted between the heated region and the entrance cone, ionizes the
sample molecules with a discharge voltage of approximately 3.0 to 3.5 kV
in positive ion mode and 2.0 to 3.0 kV in negative ion mode.
Region
Intermediate
Pressure
Region
Exit cone
Rotary
pump
Entrance cone
Pressure
Region
Cone Wash
LC eluent
Nebulizing gas, N
Sheath gas, N
Probe
2
2
Atmospheric
Corona pin
Figure 1-13. Schematic of the APCI source on the MSQ, showing the
principal components and pressure regions
The newly formed ions then enter the focusing region through the entrance
orifice and pass into the RF lens region. The RF lens (square quadrupole)
helps to focus the ions before they enter the mass analyzer region.
1-14 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________ The Source–An Introduction to API Techniques
Spectral Characteristics
Like electrospray, APCI is a soft ionization technique and forms singly
charged ions–either the protonated, [M+H]
+
, or deprotonated, [M-H]-,
molecule–depending on the selected ionization mode. Unlike electrospray,
however, APCI does not produce multiply charged ions and so is unsuitable
for the analysis of high molecular weight compounds such as proteins or
peptides.
Although a high temperature is applied to the probe, most of the heat is used
in evaporating the solvent, so the thermal effect on the sample is minimal. In
certain circumstances (for example, with very thermally labile (unstable)
compounds), the heated probe may cause some thermal fragmentation.
Flow Rate
Flow rates of 0.2 to 2.0 mL/min can be used with APCI.
___________________________MSQ Hardware Manual ___________________________ 1-15
Introducing the MSQ
The Source–An Introduction to API Techniques ________________________________________________
Source Fragmentation
Both electrospray and APCI are regarded as soft ionization techniques.
Ionization generally results in spectra dominated by either the protonated
molecule [M+H]
(negative ion mode), depending on whether positive or negative ionization
mode has been selected. Choice of ionization mode is governed by the
functional chemistry of the molecule under investigation.
Source fragmentation can be induced to give additional information on a
compound, such as diagnostic fragment ions for structural determination or
an increased response on a particular confirmatory ion for peak targeting.
Formation of Diagnostic Fragment Ions
The MSQ allows the simultaneous acquisition of MS data at a number of
different source voltages. For example, the MSQ can be programmed to
acquire data at source voltages of 20, 40, and 60 V on an alternating scan
basis within a single acquisition. The benefits of setting up acquisitions in
this way are:
+
(positive ion mode) or deprotonated molecule [M-H]-
The optimum source voltage for a particular ion can be determined in
•
one acquisition for compounds where sample volume is at a premium.
• The intensity of fragment ions can be maximized to gain structural
information.
Fragmentation at increased source voltages is useful for most compounds.
For example, using source fragmentation of salbutamol in electrospray
ionization, a number of confirmatory fragment ions can be generated and
their intensity maximized (see Figure 1-14).
The mechanism for the formation of the fragment ions is characteristic for
not only salbutamol, but also for related β-agonists such as clenbuterol,
terbutaline, and metaproterenol. It involves loss of water (-18 amu; resulting
in the fragment ion at m/z 222 (middle trace)) and an additional loss of the
tert-butyl group (-56 amu; resulting in the fragment at m/z 166 (lower
trace)).
Note. The MSQ uses the term “cone voltage” to represent source voltage.
1-16 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
+
+
+
u
_______________________________________________ The Source–An Introduction to API Techniques
100
Source voltage 10V
%
0
50 75 100 125 150 175 200 225 250 275 300
100
Source voltage 25V
%
0
50 75 100 125 150 175 200 225 250 275 300
100
Source voltage 35V
%
0
50 75 100 125 150 175 200 225 250 275 300
148
166
240
241
240
222
241
240
222
241
m/z
m/z
m/z
Protonated molecule at m/z 240
OH
+
tBu
NH
HO
HO
2
Fragment ion at m/z 222
NH
tBu
HO
HO
2
Fragment ion at m/z 166
NH
HO
HO
3
[M+H]
[M+H]+-H2O
[M+H]+-H2O-tB
Figure 1-14. Source fragmentation of salbutamol in electrospray
ionization
Optimum Response for Confirmatory Ions
When acquiring fragment ions for confirmation purposes, the applied source
voltage per compound would require some optimization to maximize the
intensity of these ions. It is generally observed that small changes to the
source voltage result in only small intensity changes; thus, fine-tuning of
this voltage is usually not critical (typically +/- 5 V is adequate).
___________________________MSQ Hardware Manual ___________________________ 1-17
Introducing the MSQ
The Source–An Introduction to API Techniques ________________________________________________
Cone Voltage Ramping
Source voltage ramping can be used in Full Scan operation (see page 1-32)
in electrospray when the compound of interest forms multiply charged ions.
A spectrum similar to that in Figure 1-15 will be produced, showing the
multiply charged envelope.
771
808
848
893
942
944
998
999
1060
1131
1212
1305
m/z
100
%
738
707
694
0
700 800 900 1000 1100 1200 1300
Figure 1-15. Electrospray spectrum of horse heart myoglobin
This envelope is represented in diagrammatic form in Figure 1-16. The first
diagram shows the envelope at a source voltage of 30 V. Next, the envelope
is shown at 60 V. There is no ramping applied here. The envelope has the
same shape but has moved to a higher m/z value. This is due to the charge
stripping that occurs at high source voltages, which lowers the charge state
and produces an apparent higher mass distribution. If the source voltage is
ramped between 30 and 60 V, the charge distribution envelope is extended
and it resembles the lower diagram.
1-18 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
f
_______________________________________________ The Source–An Introduction to API Techniques
Intensity
Without s ource voltage ramping
Source voltage 30V
Increasing
number of
charges
Intensity
Decreasing
number o
charges
With source voltage ramping 30-60V
m/z
Intensity
Without s ource voltage ramping
Source voltage 60V
m/z
Figure 1-16. Cone voltage ramping
Cone voltage ramping is often used for proteins and peptides during:
Calibration (to extend the calibration range) •
• Analysis (to increase the number of charge states leading to greater
accuracy of molecular weight determination)
m/z
Note. Cone voltage ramping will not perform source fragmentation because
the same cone voltage corresponds to the same m/z value each time.
Polarity Switching
Switching between positive and negative ionization modes in a single
analytical run is supported by the MSQ. Rapid polarity switching is a
technique that is applied to several important areas of MS analysis; for
example:
Quantitation of different chemistries within the same run. In drug
•
metabolism studies, certain compounds preferentially ionize in positive
ion mode because they may contain a primary amino group. Other
metabolites, such as glucuronide metabolites, are likely to lose a proton
and respond in the negative ion mode.
• Rapid screening of unknown analytes; for example, in combinatorial
chemistry. If the compound has a carboxylic acid group that is sterically
unhindered, it is likely that the compound will lose a proton in negative
ion mode and not respond in positive ion mode.
___________________________MSQ Hardware Manual ___________________________ 1-19
Introducing the MSQ
A
The Source–An Introduction to API Techniques ________________________________________________
Application of API Techniques
Both electrospray and APCI are ideal for online liquid chromatography
detection, providing an additional dimension of information. With many
compounds, it is possible to analyze them by both APCI and electrospray. It
may be difficult to decide which is the more appropriate technique,
especially when the compounds of interest lack polar functionalities.
Points to note:
Electrospray is one of the softest ionization methods available, whereas
•
APCI, although also a soft ionization technique, may not be suitable for
some very thermally labile compounds as there may be thermal
fragmentation (see Figure 1-17).
214
231
231
PCI mass spectrum,
source voltage 25V.
Electrospray mass
spectrum, source
voltage 25V.
100
%
0
130 140 150 160 170 180 190 200 210 220 230 240
100
%
0
130 140 150 160 170 180 190 200 210 220 230 240
156
173
Figure 1-17. Thermal fragmentation of the herbicide asulam in APCI
APCI does not yield multiply charged ions like electrospray, and so is
•
unsuitable for the analysis of high molecular weight compounds such as
proteins.
m/z
m/z
• Both APCI and electrospray generally provide data from which it is
simple to infer molecular weight values.
In many cases, with the correct conditions, only one major peak is
observed in the spectrum: either the protonated molecule [M+H]
ion) or deprotonated molecule [M-H]
-
(-ve ion). However, some
+
(+ve
compounds are more susceptible to fragmentation than others, so
different degrees of fragmentation may be seen from compound to
compound. When determining molecular weights, always take account
of possible adduct ions. Common adducts are [M+18]
+
NH
+
4
(ammonium adducts seen in the presence of buffers such as ammonium
acetate), [M+23]+ Na+ (sodium adducts), and [M+39]+ K+ (potassium
adducts).
1-20 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
_______________________________________________ The Source–An Introduction to API Techniques
Source fragmentation is used in both APCI and electrospray to give
•
structural information.
In general, increasing the voltage applied to the source block (the cone
voltage) yields increasing amounts of fragmentation, depending on the
nature of the compound. The optimum source voltage required to give
the maximum intensity of the protonated or deprotonated molecule is
compound-dependent, as is the source voltage required for
fragmentation. The energies involved in source fragmentation are low,
so usually only weaker bonds such as C-N and C-O are broken.
Since there are many similarities between electrospray and APCI, there are
many applications common to both.
Compounds suitable for analysis by electrospray are polar and of molecular
weight less than 100,000 amu. The higher molecular weight compounds,
such as proteins, can produce multiply charged ions. As it is the mass-tocharge ratio (m/z) that is measured by the MS detector, these can often be
seen at lower masses. For example, if the molecular weight is 10,000, a
doubly charged ion (2+ in +ve ion) would be seen at m/z 5001, 10+ at m/z
1001, etc.
Typical electrospray applications are: peptides, proteins, oligonucleotides,
sugars, drugs, steroids, and pesticides.
Compounds suitable for analysis by APCI are generally polar (although less
polar than electrospray) and of molecular weight <1000 amu.
Typical APCI applications are: pesticides, drugs, azo dyes, and steroids.
A summary comparing electrospray and APCI is shown in Table 1-2.
___________________________MSQ Hardware Manual ___________________________ 1-21
Introducing the MSQ
The Source–An Introduction to API Techniques ________________________________________________
Table 1-2. Comparison of ESI and APCI
LC/MS technique Electrospray (ESI) Atmospheric Pressure Chemical
Ionization (APCI)
Compound polarity Polar Polar, some non-polar
Examples Drugs, proteins, biopolymers,
oligonucleotides, steroids, and
pesticides
Pesticides, azo dyes, drugs,
metabolites, agrochemicals, and
steroids
Sensitivity fg to pg (compound-dependent) fg to pg (compound-dependent)
Type of spectra [M+H]+ for +ve ion mode, [M-H]- for
-ve ion mode, fragmentation via
source voltage
+
[M+H]
for +ve ion mode, [M-H]- for
-ve ion mode, fragmentation via
source voltage
Flow rates 2.0 µL/min to 2.0 mL/min 0.2 to 2.0 mL/min
LC columns Capillary to 4.6-mm ID columns 2.1- to 4.6-mm ID columns
Mobile phases H2O, CH3CN, CH3OH are most
frequently used.
H2O, CH3CN, CH3OH are most
frequently used. Non-polar
solvents can be used.
Typical mass range <100,000 <1000
1-22 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
______________________________________________________The Self-Cleaning Source: Cone Wash
The Self-Cleaning Source: Cone Wash
This section introduces the cone wash. Information on how and when to use
it is provided in the chapter LC/MS and the Cone Wash.
Introduction
The API source on the MSQ includes a self-cleaning solvent delivery
system (the cone wash). This makes the source extremely robust and
productive and greatly increases the number of samples that can be analyzed
before maintenance is required.
The orthogonal API probe serves to direct the LC eluent away from the inlet
orifice. However, under typical LC/MS conditions, both the ions and the
charged liquid droplets (containing involatile components) are deflected by
the electric field towards the inlet orifice. This effect leads to a gradual
buildup of involatile components and an associated loss in sensitivity with
time.
The self-cleaning API source delivers a constant, low flow of solvent to the
edge of the inlet orifice (see Figure 1-18). This prevents the buildup of
involatile components during LC/MS analysis with typical chromatographic
buffers (for example, phosphates and ion-pairing agents). This greatly
improves the quantitation precision of analysis without the need to
compromise the LC method, and more importantly, dramatically extends the
length of time possible for analysis.
Figure 1-18. Dispersion of involatile components from the inlet orifice
___________________________MSQ Hardware Manual ___________________________ 1-23
Introducing the MSQ
The Self-Cleaning Source: Cone Wash _______________________________________________________
Functional Description
As shown in Figure 1-18, the cone wash nozzle consists of a stainless steel
capillary, which is fed to the edge of the inlet orifice of the entrance cone.
The capillary is attached to PEEK tubing, internal to the instrument, which
when connected to an LC pump delivers the solvent (typically HPLC-grade
water or organic solvents such as methanol) at a controlled flow rate.
Note. It is necessary to use the cone wash only for compounds in dirty
matrices or when involatile buffers are used. Choose the cone wash solvent
to give the most effective solubility for the expected contaminants.
1-24 __________________________ MSQ Hardware Manual ____________________________
Introducing the MSQ
______________________________________________________________ The Reference Inlet System
The Reference Inlet System
This section introduces the reference inlet system. Information on how to set
up the system is provided in the chapter Reference Inlet System.
Introduction
The recommended way to introduce a sample for tuning and mass
calibration in electrospray is to use the reference inlet system.
Functional Description
As shown in Figure 1-20, the reference inlet system consists of a pressurized
reservoir containing the reference sample and a PEEK delivery tube. One
end of the PEEK delivery tube is inserted into the reference inlet reservoir,
while the other end is attached to the switching valve. When the pressure in
the reservoir is increased, the sample is forced through the PEEK tube and
infused directly into the 100 µL sample loop.
Figure 1-20. The reference inlet system
___________________________MSQ Hardware Manual ___________________________ 1-25
Introducing the MSQ
The Mass Analyzer and Detector____________________________________________________________
The Mass Analyzer and Detector
The mass analysis and detection system comprises two main components:
A quadrupole mass analyzer •
• A detector whose main component is a channeltron electron multiplier
The square quadrupole RF lens helps to focus the ions before they are
filtered, according to their mass-to-charge ratio in the mass analyzer.
The analyzer in the MSQ is a quadrupole. This is one of the most widely
used types of analyzer and can be easily interfaced to various inlet systems.
By applying carefully controlled voltages to the four rods in the quadrupole,
only ions of a specific mass-to-charge ratio are allowed to pass through at
any one time.
The ions then reach the detector, whose main component is a channeltron
electron multiplier. In positive ion mode, the ions exit the analyzer and
strike a conversion dynode, which results in the emission of electrons. The
electrons are accelerated towards the channeltron electron multiplier, which
then creates an electron cascade. The current is then converted and amplified
into a voltage signal that is analyzed and processed by the MSQ’s on-board
data acquisition system. The resultant peak information is sent to the data
system.
Ion successfully transmitted
by the mass analyzer
Quadrupole
mass analyzer
Square
quadrupole
RF lens
Source exit cone
Figure 1-21. Schematic of the MSQ analyzer and detector
Detector
Ion trajectory incorrect
for transmission trough
the mass analyzer
Split flow
turbomolecular
pump
1-26 __________________________ MSQ Hardware Manual ____________________________
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