Short Specialist Review

Quadrupole mass analyzers:

theoretical and practical
considerations

Frank A. Kero and Richard A. Yost

University of Florida, Gainesville, FL, USA

Randall E. Pedder
Ardara Technologies LP, Monroeville, PA, USA

1. Introduction

Quadrupole mass analyzers are dynamic mass filters that play a central role in
mass spectrometric proteomic investigations. Quadrupoles are used both alone and
in tandem for the isolation (by mass-to-charge) of ions; they may also be used
(in RF-only mode) to contain ions during collision-induced dissociation (CID)
(Yost and Enke, 1979). Gas-phase ions are generated by a source external to the
quadrupole mass analyzer, often at atmospheric pressure. It should be noted that
ion production at one atmosphere is an important practical advantage, in that it
allows for the easy exchange of ion sources without disturbing the vacuum of the
quadrupole region (Cole, 1997). Peptides and proteins are typically introduced into
the ion source in the liquid phase either from an HPLC column (for LC/MS),
by flow injection analysis (with no HPLC column), or by continuous direct
infusion through a fused silica capillary. For heterogeneous samples, a sample
cleanup or separation technique such as HPLC or solid-phase extraction is typically
incorporated into the method development scheme to allow for the sequential
introduction of multiple analytes into the ion source. Simultaneous ionization
of multiple analytes, particularly at widely varying concentrations, may result in
suppression and prove deleterious in quantitative investigations (Cole, 1997; King
et al ., 2000; Constantopoulos et al ., 1999).
The most common ionization source coupled with quadrupole mass analyzers for
proteomics studies is electrospray ionization (ESI), since it is ideal for compounds
such as peptides and proteins that can be ionized in solution. The fundamentals
of ESI mechanisms have been described elsewhere (Cole, 1997; Kebarle, 2000;
Cech and Enke, 2001). A key advantage of ESI is the ability to impose multiple
2 Core Methodologies

charges on an analyte upon ionization. Recall that ions are separated in mass
analyzers as a function of their respective mass-to-charge ratios (m/z ) (Throck
Watson, 1997). It follows that multiple charging reduces the mass-to-charge ratio
of ions, and thereby extends the mass range of the instrument. Atmospheric pressure
chemical ionization (APCI) is a highly efficient alternative to ESI for less polar
compounds. ESI and APCI are complementary techniques, as reflected in the phase
in which charge transfer processes occur (King et al ., 2000). Ionization for ESI
occurs in the liquid phase, whereas ionization for APCI occurs in the gas phase.
Both APCI and ESI are suitable ionization sources for a quadrupole mass analyzer
because the ions are generated and transmitted as a continuous beam of ions.
The other primary ionization method for proteomics studies is matrix-assisted
laser desorption/ionization (MALDI). Since MALDI produces narrow pulses of
ions, it is a far better marriage with pulsed mass analyzers (such as time-of-flight
mass analyzers; see Article 11, Nano-MALDI and Nano-ESI MS, Volume 5
and Article 14, Sample preparation for MALDI and electrospray, Volume 5)
than with the continuous mass analyzers such as quadrupoles. Furthermore,
MALDI yields almost exclusively singly charged ions, and thus benefits from the
essentially unlimited mass range of the time-of-flight mass analyzer. Quadrupole
mass analyzers are also commonly used in mass spectrometers employing electron
ionization (EI) and chemical ionization (CI), such as those coupled with gas
chromatographs (GC/MS). These ionization techniques do not play a significant
role in modern proteomic investigations, however.
The purpose of this communication is to provide the reader with a balanced
treatment of practical and theoretical considerations for quadrupole mass analyzers,
and to present the advantages of interfacing quadrupoles in tandem.

2. Theory and practice of quadrupole mass filters

A quadrupole mass analyzer consists of four electrically isolated hyperbolic or
cylindrical rods linked to RF (radio frequency) and DC (direct current) voltages,
as described by the schematic in Figure 1 (Pedder, 2001). The combination of RF
and DC voltages creates a region of strong focusing and selectivity known as a
hyperbolic field. In simplest terms, the ratio of RF/DC allows for the selective
transmission of ions of a narrow range of mass-to-charge from the total population
of ions introduced from the ionization source.
The idealized hyperbolic field can be described in terms of Cartesian coordinates
(x and y directions toward the rods, and z direction along the rods’ axis). Ions of
a selected m/z follow a stable trajectory around the center of the field, and are
transmitted in the z direction through the device (Dawson and Douglas, 1999).
Motion of these “stable ions” in the x and y directions are small in amplitude,
as the lowest energy pathway lies toward the center of the hyperbolic field. Ions
of other m/z will have unstable trajectories, with increasing displacement in the x
and y directions away from the center of the hyperbolic field, and thus strike the
quadrupole rods, where they neutralize upon contact. These ions will not transmit
efficiently through the quadrupole to the detector. The application of RF and DC
voltages creates a region of stability for transmission of ions of a limited m/z
 Short Specialist Review 3

−
+ +
−

RF RF
secondary secondary

RF drive
Resolving circuit Resolving
−DC + DC

DC pole
bias offset

Figure 1 The quadrupole mass filter and its power supply

range. In this regard, the quadrupole analyzer is operating as a mass filter. A mass
spectrum is produced by ramping the RF and DC voltages at a nearly constant
ratio. Ions of increasing m/z will sequentially achieve stable trajectories and reach
the detector in order of increasing m/z .
To understand the behavior of ions in the quadrupole, a brief introduction to the
mathematics associated with ion motion is worthwhile. For a complete discussion,
the reader is referred to the work of March and Hughes (1988).
The motion of ions through a quadrupole is described by a second-order linear
differential equation called the Mathieu equation, which can be derived starting
from the familiar equation relating force to mass and acceleration, F = ma, yielding
the final parameterized form, with the following substitutions for the parameters a
and q (Dawson, 1976):

d 2u 8eU 4eV
+ (au − 2qu cos 2ξ )u = 0 au = qu = (1)
dξ 2 mr0 2 2 mr0 2 2

The u in the above equations represents position along the coordinate axes
(x or y), ξ is a parameter representing t/2, t is time, e is the charge on an
electron, U is the applied DC voltage, V is the applied zero-to-peak RF voltage,
m is the mass of the ion, r 0 is the effective radius between electrodes, and  is
the applied RF frequency in radians s−1 . The parameters a and q are proportional
to the DC voltage U and the RF voltage V , respectively.
The analytical solution to this second-order linear differential equation is:
∞

∞


u(ξ ) =  C2n exp(2n + β)iξ +   c2n exp −(2n + β)iξ (2)
n=−∞ n=−∞
4 Core Methodologies

80
70
60
50
40
30
20
10
U, DC voltage 0
−10 0 50 100 150 200 250 300 350 400 450 500 550
V, RF voltage (Vo-p)
−20
−30
−40
−50
−60
−70

Figure 2 Mathieu stability diagram for an ion of m/z 219 in a quadrupole mass filter with
9.5-mm diameter round rods and an RF frequency of 1.2 MHz (Pedder, 2001)

which reduces to a similar infinite sum of sine and cosine functions:

∞

∞


u(ξ ) = An sin ωn + An cos ωn (3)
n=−∞ n=−∞

The solutions to the Mathieu equation can be presented graphically, as shown in

Figure 2 in a so-called stability diagram. Points in (U , V ) space (DC, RF voltage
space) within the lines lead to stable trajectories for the m/z 219 ion; points outside
the lines will lead to an unstable trajectory. The dotted line is called the scan line,
and shows the ramp of RF and DC voltage at a nearly constant ratio (the slope of
the line). If the line passes just under the apex of the stability region, as shown
here, the m/z 219 ions will have a stable trajectory at those voltages, and will be
transmitted to the detector.
Figure 3 shows the stability diagram for three different ions, of m/z 28, 69, and
219. Note that ions of lower m/z have stable trajectories at lower RF and DC volt-
ages (i.e., they are stable at points in (U , V) space closer to the origin). The dotted
scan line again shows the ramp of RF and DC voltage at a constant ratio (Pedder,
2001). The scan line passes through the apices of the stability regions for all three
ions. As the RF and DC voltages ramp up in amplitude, ions of increasing m/z have
stable trajectories and are transmitted, as shown in the dotted mass spectrum below
the stability diagram. If the scan line has a lower slope (the solid line), it passes
through a larger portion of each stability region, resulting in a mass spectrum with
lower resolution, as shown as well.
The selection of RF and DC voltages, V and U , the RF frequency, , and the
inscribed radius r 0 between the rods determines the performance of the quadrupole
 Short Specialist Review 5

80
m/z 219
70

60
DC voltage (volts)

50

40

30
m/z 69
20
m/z 28
10

0
0 50 100 150 200 250 300 350 400 450 500 550
(a) RF voltage (Vo-p)
Intensity

(b) m/z ->

Figure 3 (a) Mathieu stability diagram for ions of m/z 28, 69, and 219. Two scan lines are
shown, one dotted and a second solid. (b) Simulated mass spectra showing these ions as the RF
and DC voltages are scanned along the two scan lines (dotted and solid) shown

mass filter. Typical values are 4 kV RF(0-p), 700 V DC, 1 MHz RF frequency,
and 1 cm radius, and yield a mass-to-charge range of ∼3000. As can be noted
from equation (4), the Mathieu q parameter is proportional to the RF voltage and
inversely proportional to (Constantopoulos et al ., 1999; Pedder, 2002)
4eV
qu = (4)
mr0 2 2
the square of the inscribed radius and RF frequency; thus, the mass range of the
mass filter can be increased by increasing the RF and DC voltages, or by decreasing
the inscribed radius or RF frequency. The mass resolution is a function of the ratio
of the RF and DC voltages (the slope of the scan line in Figures 2 and 3); increasing
the slope to move the scan line closer to the apex of the stability region (moving
from the solid line to the dotted line, for instance) increases the mass resolution, as
shown in the spectra in Figure 3(b). Unfortunately, the increase in mass resolution
is accompanied by a loss of sensitivity (decrease in transmission of ions through
the mass filter). The ultimate resolution that can be achieved is determined not only
by how close the scan line approaches the stability region apexes but also by the
precision and accuracy of the quadrupole power supplies and the quadrupole rod
dimensions.
An important operational mode of the quadrupole is achieved when U , the DC
potential, is equal to zero (a = 0). This corresponds to a scan line along the x -axis
in Figures 2 and 3. This RF-only mode results in transmission of ions of a very
6 Core Methodologies

wide range of m/z values, and is thus often termed “total ion mode” (Dawson
and Douglas, 1999). Note that in this mode, there is a limit to the lowest m/z
ion that can achieve a stable trajectory. This can be seen as the right-hand edges
of the stability regions in Figure 3. This total ion mode is used for the collision
cell in tandem quadrupole mass spectrometers, as discussed below; it is also often
employed in quadrupoles used to help transmit ions from the ion source region to
the quadrupole or other mass analyzer.

3. Tandem mass spectrometry

Most applications of mass spectrometry in proteomics employ mass spectrometry
in tandem with one or more other analytical stages (Busch et al ., 1998; Niessen,
1999). One of those stages is often a separation stage, typically liquid chromatog-
raphy or capillary electrophoresis (Cole, 1997), leading to LC/MS and CE/MS, as
discussed elsewhere in this volume (see Article 13, Multidimensional liquid chro-
matography tandem mass spectrometry for biological discovery, Volume 5).
Another stage may be a second stage of mass spectrometry (the combination of
two or more stages of mass spectrometric analysis in series is termed tandem mass
spectrometry (McLafferty 1983; Fetterolf and Yost, 1983) or MS/MS, or even MSn ,
where n can be ≥2). Indeed, a common approach in proteomics investigations is
combining a stage of separation with tandem mass spectrometry, that is LC/MS/MS
and LC/MSn . These tandem (sometimes termed hyphenated) methods offer dramat-
ically improved selectivity and information to solve tough analytical problems, as
often encountered in proteomics.
Informing power is a figure of merit that provides a means of quantifying the
amount of information available in such an analytical procedure. Fetterolf and
Yost (1984) demonstrated the use of informing power to illustrate the advantages
of hyphenated methods such as LC/MS/MS over single-stage analytical methods.
Note that MS/MS is particularly important in LC/MS (compared to combined gas
chromatography/mass spectrometry – GC/MS) for two reasons. First, LC typically
provides much poorer chromatographic resolution than GC, meaning that LC sep-
arations are often incomplete, with coeluting peaks that MS/MS can help resolve.
Second, LC/MS employs ionization methods such as ESI and APCI that provide
only molecular-type ions and little or no structural information (GC/MS, in contrast,
typically employs EI, which provides significant fragmentation and, therefore, struc-
tural information with only a single stage of mass spectrometry). Thus, LC/MS/MS
is invaluable in providing structural information for the identification of components
eluting from the LC column and for detecting those compound with high selectivity.
MS/MS is readily performed “tandem-in-space” (i.e., with two mass analyzers,
typically quadrupole mass filters, in sequence). Note that MS/MS can also be
performed “tandem-in-time”, with two or more stages of mass analysis performed
sequentially in time in a single ion trap such as a quadrupole ion trap (see Article 9,
Quadrupole ion traps and a new era of evolution, Volume 5).
The triple quadrupole mass spectrometer (TQMS, see Figure 4) is the most com-
mon MS/MS instrument in use (Yost and Enke, 1979). The operational principles
of the triple quadrupole tandem mass spectrometer can be described in a similar
 Short Specialist Review 7

Ion Quad Quad collision Quad Particle

source mass filter chamber mass multiplier
filter
Sample

Ionization Component ion Ion fragmentation Fragment ion Ion

selection selection detection

Figure 4 Schematic of a triple quadrupole mass spectrometer (TQMS)

fashion to the single quadrupole. Ions from the ion source enter the first quadrupole
mass filter (Q1), which selects ions of a selected m/z and transmits them to the
second quadrupole. The second quadrupole (Q2) functions as a collision cell in RF-
only mode; CID is accomplished by adding a collision gas, typically nitrogen or
argon, at a pressure of around 1–10 millitorr (1–10 microbar). The third quadrupole
(Q3), another mass filter, provides a means of mass-analyzing the products of CID
from the collision cell (Fetterolf and Yost, 1983).
Note that the RF-only quadrupole collision cell in triple quadrupole instruments
is often replaced with a higher-order multipole (a hexapole or an octopole, with 6
or 8 rods, respectively) (McCloskey, 1990). The use of an RF-only higher-order
multipole provides effective transmission of a wider mass range than an RF-only
quadrupole. The stability diagram for a hexapole or octopole (as in Figure 2 for
the quadrupole) has less well-defined stability boundaries, so the low-mass cut-off
characteristic of an RF-only quadrupole is not an issue with higher-order multipole
collision cells. A practical advantage that should be noted is that these multipoles
require less demanding tuning procedures for the optimization of voltages and
frequencies.
A fundamental understanding of the scan modes associated with the TQMS
is essential for understanding the MS and MS/MS capabilities of the instrument.
These are summarized in Table 1 (McLafferty, 1983; Fetterolf and Yost, 1983;
Busch et al ., 1998; Dawson and Douglas, 1999).
First, note that the triple quadrupole tandem mass spectrometer can readily be
used to perform a single stage of mass spectrometry (MS) by setting two of the
three quadrupoles into RF-only (total ion) mode. Either Q1 or Q3 can be used as a
mass filter, scanned to obtain a full spectrum, or set to pass a single fixed m/z (or a
few fixed m/z values), a mode termed selected ion monitoring (SIM). When Q1 and
Q3 are both used as mass analyzers, there are four possible MS/MS scan modes. In
the daughter ion scan mode (also called product ion scan), Q3 is scanned to obtain a
spectrum of all the daughter ions generated by CID in q2 of the parent or precursor
ion mass-selected with Q1. In the parent ion scan mode (also called precursor ion
scan), Q1 is scanned to obtain a spectrum of all the parent ions that upon CID in
q2 produce the daughter or product ion mass-selected with Q3. In a neutral loss
scan, both Q1 and Q3 are scanned at the same rate but with a fixed difference in
mass to detect only those ions that undergo a specific neutral loss in q2 such as a
loss of mass 18 (H2 O). An important MS/MS scan mode for quantitating targeted
compounds with maximum sensitivity is the selected reaction monitoring mode
(SRM) in which one (or a few) selected parent-ion-to-daughter-ion transitions are
8 Core Methodologies

Table 1 MS and MS/MS operational modes of a triple quadrupole mass spectrometer, showing
the modes for the two mass filters (Quadrupole 1 and Quadrupole 3) and the quadrupole collision
cell (q2)

Scan mode Q1 q2 Q3
MS – full scana Scan m/z RF-only RF-only
MS – selected ion monitoring Fixed m/z RF-only RF-only
(SIM)a
MS/MS – daughter ion scan Fixed m/z RF-only collision gas Scan m/z
MS/MS – parent ion scan Scan m/z RF-only collision gas Fixed m/z
MS/MS - neutral loss scan Scan m/z RF-only collision gas Scan m/z b
MS/MS – selected reaction Fixed m/z RF-only collision gas Fixed m/z
monitoring (SRM)
a Note that the MS scan modes can be performed by mass selection with either Q1 or Q3, with

the other one in RF-only (total ion) mode.

b Q3 scanned at a difference in mass corresponding to the mass of the selected neutral.

monitored by setting Q1 and Q3 to pass specific m/z values. SRM is the MS/MS
scan mode analogous to the MS selected ion monitoring scan mode (SIM).
The relationship between the MS/MS scan modes can be explored by examining
the plot of the three-dimensional MS/MS data – intensity (on a logarithmic scale)
versus parent ion m/z versus daughter ion m/z – for a single compound, as shown
in Figure 5 (Yost and Enke, 1979). Such a three-dimensional data set could be
obtained by a series of daughter ion scans of each parent ion formed in the ion
source, or by a series of parent ion scans of each daughter ion. It could even be
obtained by a series of neutral loss scans for every possible neutral loss (i.e., by
interrogating the surface in a series of lines parallel to the front line of the plot).
Finally, a series of SRM experiments could measure the intensity of every possible
parent ion–daughter ion pair (i.e., every point on the surface).

1
Relative intensity

−1
10
−2
10
−3
10
84 −4
10
15
69
30
55
44
40
27 57

69

27 29 39 4142 43
28 40 51 53 55 57
Spectrum 54 56 EI spectrum 69 83 85
84
of parent ions Spectrum +
yielding 51+
M
of fragment
ions of 69+

Figure 5 Plot of the three-dimensional dataset generated by electron ionization/MS/MS of cyclohexane. A similar
plot of 1-propanol could be found in Fetterolf and Yost (1983)
 Short Specialist Review 9

Finally, note that all of these MS and MS/MS scan modes are readily achieved
on a TQMS, and indeed, on most tandem-in-space MS/MS instruments. Note that
this is not so on tandem-in-time MS/MS instruments, where the selection of a
parent ion precedes (temporally, not spatially) the mass selection of the daughter
ion. On tandem-in-time MS/MS instruments such as ion traps, parent scans and
neutral loss scans are not available scan modes.

4. Conclusions
The quadrupole mass analyzer (and the RF-only quadrupole and multipole collision
cells) enjoy a central role in the success of mass spectrometric methods in pro-
teomics. Development of an understanding of the theoretical and practical aspects
of quadrupoles can improve ones ability to utilize single and triple quadrupole mass
spectrometers effectively to solve important problems in proteomics.

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