Giới thiệu về phổ khối Introduction to mass spectrometry

An Introduction to Mass Spectrometry

The following article is intended for biologists and biochemists who are interested in
knowing the basics of how mass spectrometers work. It provides a very general and
descriptive introduction to mass spectrometry, with an absolute minimum of math and
physics knowledge required.

1. Definition of Mass Spectrometry

Mass spectrometry is a chemical analysis technique which is based on the measurement
of the mass (atomic or molecular weight) of molecules or atoms.

2. Applications of Mass Spectrometry

Mass spectrometry is widely used today in many diverse areas. Some examples of current
applications are:
• environmental: analysis of air, water and soil samples for trace contaminants
• pharmaceutical: drug development and quality control
• biological research: determination of protein and peptide structure
• semiconductor electronics: determination of levels of additives and impurities in
silicon wafers
• metallurgy: determination of levels of trace elements in metals and metal ores
• astrochemistry: measurement of composition of planetary atmospheres and
surfaces (e.g. NASA Mars Rover)
• food: analysis of pesticide residues on fruits and vegetables
• security: explosives and contraband drug detection
• military: mobile detection of biological and chemical agents (e.g. bacteria, nerve
gas)
• medical: screening of newborn babies for genetic disorders
• sports: screening of athletes (and race horses) for performance-enhancing drugs

3. Basic Concepts and Definitions

Ion: a molecule (or atom) which has either a positive or negative electrical charge. The
amount of charge is "quantized", and is reported in integer units. For example, an ion
may have a charge of +1 or +2 or -3 units, but not + ½ or -1½ etc.

Proton: fundamental atomic particle which has, by definition, an electrical charge of +1
unit. A neutral molecule which (for example) gains one extra proton will have an overall
electrical charge of +1; if it gains 2 protons it will have a charge of +2; and so on. Protons
may be either gained or lost from molecules.

Electron: fundamental atomic particle which by definition has an electrical charge of -1
unit. Same behavior as "proton" above, but oppositely charged, i.e. if a neutral molecule
gains one electron (or alternatively loses one proton!) it attains a charge of -1.

Neutral: in mass spectrometry, this refers to any particle (usually a molecule) which has
no electrical charge.

Mass: in mass spectrometry, a synonym for "molecular weight" (or atomic weight);
usually symbolized by "m" in equations. Has units of "Da" (Daltons) or "amu" (atomic
mass units). One amu is defined as 1/12 of the mass of a carbon
12
atom (see "isotopes"
below). The prefix "k" denotes "1000" e.g. "40 kDa" indicates a molecular weight of
40,000 Da.

Charge: in mass spectrometry, the quantized amount of charge on an ion, e.g. +1, -2;
usually symbolized by the letter "z" in equations.

Mass to Charge Ratio: usually written as m/z, this is simply the molecular weight of an

ion divided by the number of charges it carries. (Note that even if the charge is negative,
i.e. -2, the value of m/z is still normally written as a positive number.)
All common mass spectrometric techniques are based on the use of electromagnetic fields
to separate ions. Ions are actually separated on the basis of their mass to charge ratio, not
on the basis of their mass. However if the charge on an ion is known, its mass can be
readily determined.

Mass Spectrum: the data output of a mass spectrometer is most frequently presented as a
graph of ion population versus mass (Figure 1 below). The largest peak in a mass
spectrum (e.g. at m/z 570.8 in the figure below) is referred to as the base peak.


Isotopes: Most elements consist of atoms with several stable masses or isotopes.
Different isotopes of any given element have the same number of protons, but different
numbers of neutrons. By far the most common example of this in biochemical mass
spectrometry is carbon. The most common stable isotope of carbon (~ 99% natural
abundance) has an atomic mass of 12; it is commonly referred to as "carbon 12" or C
12
.
There is also a stable isotope of carbon with a mass of 13 (~ 1% abundance), commonly
referred to as "carbon 13". Therefore in a mass spectrum of a carbon-containing
compound, peaks due to BOTH of these naturally occurring isotopes are observed; the
relative intensity of the peaks due to carbon-12 and -13 depends on the number of carbon
atoms in the molecule (ion) being analyzed (Figure 2, below).



Mass Resolution: the resolution, R, of a mass spectrometer is defined by R = m / Δm
where m is the ion mass and Δm is the width of the corresponding peak in the mass
spectrum. An instrument with a resolution of 1000 (at mass 1000) can clearly separate an

ion (peak) at mass 1000 from an adjacent peak at mass 1001 or 999.

AC: alternating current; an electrical potential which varies with time in a regular
periodic fashion. In the mass spec world, the term "RF" (radio frequency) is often used
interchangeably with the term AC.

DC: direct current; an electrical potential which does not vary in a periodic fashion; it
may, however, be "ramped"…increased or decreased in a smooth, controlled manner.




4. Essential Components of a Mass Spectrometer - (Figure 3, below)



Vacuum

Mass spectrometry is normally performed under high vacuum conditions. This is done
because the ion filtering techniques used in mass specs are only effective under
conditions where molecules do not undergo collisions with other molecules. Generally, a
pressure lower than about 5 x 10
-5
torr (torr = 1/760 of an atmosphere = 133.322 Pa) is
required for optimum performance of quadrupole and ion trap instruments; time of flight
instruments normally require even lower pressures for operation (10
-6
torr range) due to
their longer ion flight paths and higher mass ranges. If the pressure in the mass spec is too
high, both sensitivity and mass resolution will be compromised. In modern mass specs, a

combination of turbomolecular and roughing pumps are used to generate the required
high vacuum conditions.

It is worth noting that an ion separation technique known as Ion Mobility Spectrometry
(IMS), which is similar in some ways to mass spec, does not require a vacuum. Rather, it
is typically performed at atmospheric pressure. IMS offers sensitivity comparable to mass
spec, but it is rarely used in biochemical applications due to its limited resolution and
mass range. The beauty of IMS is that bulky and expensive vacuum pumps are not
required, making it ideal for mobile applications.



Ion Source and Vacuum Interface

Since mass specs filter ions only, the sample molecules of interest must be ionized before
they can be selected and detected. This is accomplished in an ion source, of which there
are many types…discussed in more detail later in Section 5.
Many ion sources operate at pressure higher than the pressure required by the mass spec
analyzer. In this case, a "vacuum interface" stage is required to transfer the ions from the
relatively high pressure of the source, to the very low pressure of the mass analyzer. A
vacuum interface is basically a device which separates ions (i.e., the sample) from
unwanted neutral gas molecules. There are many types of interfaces; most of the newer
ones incorporate "proprietary" technology. An exception is the traditional MALDI ion
source (discussed below), which forms ions under high vacuum conditions and therefore
does not require a vacuum interface. No interface is 100% efficient; some ions are always
lost.

Ion Separator (Mass Analyzer)

Ions are separated according to their mass-to-charge ratio. Common separation

techniques are discussed in detail below in Section 6.

Ion Detector

The most common types of ion detectors in use today are based on the collision of ions
with "active" surfaces. An active surface is most commonly a material which, when
struck by an ion with sufficient velocity, releases one or more electrons. These electrons
are then amplified and detected; the number of electrons produced and detected is
proportional to the number of ions striking the detector. Some detectors are based on
surfaces which emit light (photons) when they are struck by ions; the light is then
converted to electrons in a secondary process.

Data System

The data system (computer + software) is responsible for controlling the operating
parameters of the mass spec, and presenting the data to the operator. In the most basic
sense, the data system scans the ion separator (keeping track of the mass at any given
point in time), and correlates the quantity of ions detected with the selected mass.

Additional Mass Spec Stages, Components and Peripherals

Many mass spec components may be employed beyond the basics described above.

For example, fragmentation / reaction stages are often employed to "break up" large ions
into smaller fragments; this yields additional structural information beyond the simple
molecular weight of the compound. In conjunction with this fragmentation, it is common
to employ multiple sequential stages of mass separation. Put simply, an ion of interest is
selected, fragmented, and the resulting ionized fragments are then analyzed in a second
mass analysis step.
This process is commonly referred to as MS/MS or (an older term) Tandem MS; (Figure

4 below).



Multiple Ion Sources: many mass specs can be fitted with interchangeable ion sources, to
optimize their performance for particular tasks or types of samples.

Sample Prep and Separation devices: mass specs are commonly used with sample pre-
treatment and pre-separation equipment such as: liquid chromatographs; gas
chromatographs; gel electrophoresis; and a host of automated peripherals such as gel
cutters, extractors, autosamplers, plate spotters, flow splitters, UV detectors…the list
goes on. Although the same mass spectrometer may be used to analyze widely varying
types of samples (e.g. air, blood samples, soil), the sample prep equipment and
introduction procedure normally must be optimized for each sample type. Also, a general
goal of sample prep is to present the mass spec with the cleanest sample possible (after
all, who wants mud in their ion source…?)

5. Types of Ion Sources

There are many types of mass spec ion sources. The two ion sources used most often in
biochemical applications are electrospray and MALDI.

Electrospray-type sources: these sources are designed for the direct analysis of liquids,
such as a continuous flow of effluent from an LC column or discrete liquid samples
produced by various separatory techniques (gel electrophoresis etc.).

In general, electrospray-type sources produce ions by spraying or atomizing a liquid
sample under the influence of a high DC voltage. For production of positive ions, the
sample is flowed through an electrically conductive tube of small inner diameter
(typically 100 um) under pressure from a liquid pump (LC pump, syringe pump etc.). A

high positive voltage (typically +5000 V) is applied to the tube, and the outlet of the tube
is positioned close to a metal “plate” which forms the first inlet stage of the mass spec;
the plate is kept at a much lower potential (typically +500 V). The liquid becomes
electrically charged by being in contact with the walls of the sprayer tube; once the liquid
reaches the exit of the tube, it is virtually “sucked out” of the tube by the strong
electrostatic attraction of the nearby plate. (In Figure 5 below, this electrostatic spraying
process is assisted by an additional inert "sprayer gas"…more details follow…)



The liquid droplets evaporate, and as they do, sample ions are ejected from the droplets;
this process is called ion evaporation…(Figure 6, below)



Electrospray details and Jargon:

• No single electrospray source design can operate with maximum efficiency over
the extremely wide range of liquid flow rates (and sample volumes) which need to
be analyzed in biochemical labs. Therefore, many variations of the basic
electrospray source have been developed over the years, each one optimized for
particular applications.

• The basic electrospray source was originally developed for use with liquid flow
rates in the low microliter-per-minute range (0.5 to 20 µL/min).

• In some source designs, pressurized gas is used to assist with the spraying of the
sample. This tends to give a more consistent and stable spray pattern, especially at
higher liquid flow rates (above 20 µL/minute), which in turn improves signal
stability, sensitivity and signal/noise ratio. Double-click the window below to see

gas-pressure-assisted electrospray in action…(Video 1)



• At higher liquid flow rates, the volume of liquid being sprayed is too great to
evaporate at normal lab temperatures. This results in low sensitivity and/or
unstable ion signals. The most common cure for this problem is the addition of
HEAT, to speed up the evaporation of the sample droplets. The higher the liquid
flow rate, and the greater the proportion of water in the sample, the more heat is
required. (Figure 7 below: example of a heated electrospray source.)


• Heat can be applied to the sample by various means; generally the goal is to heat
the gas surrounding the sample, to speed evaporation and desolvation. Most of the
commercially-available heated sources have proprietary designs, and come with
cool names such as TurboSpray, IonMax and so forth. By varying the amount of
heat applied, and applying a pressurized gas to assist with the spray process, the
flow rate range over which the electrospray source is efficient can be extended up
to 1 ml/minute and beyond; this allows the entire output of high-flow LC columns
to be analyzed without flow splitting.

• Going in the other direction: if the inner diameter of the electrospray tube is
reduced, along with the dead volume of the liquid handling system, the
operational flow rate can be reduced to the sub-microliter-per-minute range. With
a very fine spray tip, flow rates of a few nanoliters per minute can be achieved.
Electrospray sources of this type are often referred to as “nanospray” sources.
These sources are very efficient, since the low flow of liquid evaporates readily,
and the sprayer tip can be positioned very close to (or even inside) the sampling
orifice of the mass spec.

Laser Desorption sources: The often-used term "MALDI" is an acronym for Matrix
Assisted Laser Desorption Ionization. The basic principle of MALDI is that the sample
(analyte) is mixed with a compound called a matrix, the purpose of which is to strongly
absorb laser light. In almost all cases, the sample and the matrix are prepared in the form
of separate solutions; the two solutions are mixed together, and the mixture is then
deposited on a solid surface and allowed to dry (form crystals).

For analysis, the dried sample/matrix mixture is inserted into the source region of the
mass spec, which is (usually) maintained at a moderate to high vacuum. A pulsed laser
beam is focused onto a tiny area of the sample; the matrix compound is chosen so as to
strongly absorb the laser light. The laser pulse causes a small region of the matrix
compound to instantaneously vaporize, taking the sample with it. The matrix compound
also transfers energy into the sample molecules, sufficient to ionize them. The result of
each laser “shot” is a “plume” of ionized sample and matrix molecules; the ions are
directed into the mass spectrometer by electrostatic fields (lenses, grids etc. as required)
for mass filtering….see Figure 8 below.

Since MALDI is in general a pulsed ionization technique, it is well suited to time of flight
mass spectrometers, which by their nature require pulsed ion sources.

MALDI details and jargon:

• The surface upon which the sample/matrix mixture is deposited is usually called a
“plate”; the most common MALDI plate material is stainless steel, although many
other materials can also be used (glass, gold, silicon etc.)…(Figure 8 below )


• The samples are usually deposited onto the plate in microliter or sub-microliter
volumes; this process is called “spotting”. Spotting may be done by hand, or for
high throughput applications automated plate spotters are available.

• MALDI plates are generally re-usable many times over, although they need to be
cleaned thoroughly to avoid cross-contamination. Disposable plates are also
available.
• Most lasers used for MALDI produce light in the near UV region; either nitrogen
lasers (337 nm) or ND:YAG lasers (355nm). For some applications, infra-red
lasers are used.
• The most common matrix material used for biochemical applications is alpha-
cyano hydroxycinnamic acid, often called “CHC” or “alpha-cyano”. There are
dozens of other matrix compounds which can be used.
• Any sample area on the plate which is struck by the laser, is rapidly depleted.
Therefore the plate (or the laser beam) must be continually be moved to allow
fresh area of sample to be exposed to the laser. A camera/video monitor
combination is used to visualize the interaction of the sample plate with the laser;
normally “burn spots” in the dried sample make it easy to see which areas have
been desorbed. The screen shot below shows an example of typical MALDI
source control software which incorporates a video image of an individual sample
spot, along with a "roadmap" of the entire sample plate (Figure 9 below)…



• MALDI plates containing dried (crystallized) samples can usually be kept for
long periods (days or weeks) without deterioration…for future re-analysis. Care
must be taken to protect stored plates from dust and contamination.
• The type of matrix used, as well as the laser energy, strongly influences the
amount of fragmentation which takes place during the ionization process.
• MALDI can also be done at atmospheric pressure (as opposed to in a vacuum).
This so-called “AP-MALDI” has various advantages (e.g. fast plate loading) and
disadvantages (e.g. more complex interface required, larger vacuum pumps etc.).
Most MALDI sources currently used in biochemical analysis are of the vacuum
type. Vacuum MALDI is very efficient and sensitive because it has no interface

losses (i.e. losses due to transfer of the sample from atmospheric pressure into
vacuum).

MALDI vs Electrospray (Nanospray)


• MALDI produces mostly singly charged ions; this yields simpler mass spectra,
especially for high mass compounds (large peptides and small proteins).
• ESI produces a lot of multiply charged ions, so the spectra of high mass
compounds can be very complex. BUT…a high mass range is not required to see
them. It is this multiple-charging aspect of ESI that allows large biomolecules to
be seen with quadrupole instruments of limited mass range; see Figure 10 below

• ESI does not give much source fragmentation, although the amount of
fragmentation can be varied to a certain degree by adjusting the interface
parameters (voltages).
• With MALDI, the laser energy density and type of matrix used can be used to
control the degree of fragmentation.
• If the amount of sample is extremely limited, MALDI is a good choice, not only
because of its high sensitivity, but because sample consumption is easily
controlled and unused sample deposited on the plate is easily stored for re-
analysis.

Other Types of Ion Sources Used in Mass Spectrometry:

Photoionization: photoionization involves the use of ultraviolet light to ionize the sample.
The distinction from MALDI is that in photoionization the sample absorbs the light
directly whereas in MALDI the matrix absorbs the light. Photoionization sources usually
employ a continuous UV light source (e.g. mercury lamp) rather than a pulsed laser.
Photoionization is useful for some classes of compounds which do not ionize efficiently

by electrospray, e.g. steroids, and polycyclic aromatic hydrocarbons.

ICP: this is an acronym for Inductively Coupled Plasma, a type of source used for
inorganic analysis (e.g. metallomics). The sample is typically dissolved in water and
introduced as a fine spray (mixed with argon gas), which is then dissociated and ionized
by application of a very intense RF electric field at atmospheric pressure. The resulting
argon plasma has a brilliant flame-like appearance. Compounds in the plasma are fully
dissociated to form atomic ions. ICP sources are typically used for trace metals analysis,
and for measuring levels of inorganic impurities and additives in silicon semiconductors.
Figure 11 below shows the basic components of a typical ICP source.



Thermal Desorption: a general term for sources which use heat to convert solid samples
to gas phase samples (and ions). There are many variations on this theme; sources may
operate at atmospheric pressure, or in vacuum. Usually the source uses a secondary
process (such as corona discharge APCI…see below) to generate ions. Thermal
desorption sources are most often used for environmental and security screening
applications, e.g. analysis of soils, dusts, fingerprints, and for polymer analysis.

API: this is an acronym for Atmospheric Pressure Ionization, which encompasses several
sub-types of ion sources. The electrospray source is a type of API (since it operates at
atmospheric pressure), but electrospray is so popular that it is usually considered to be in
a separate class by itself. The sources in use today, which are commonly referred to as
“API sources” e.g. APCI tend to use an electrical discharge as the primary means by
which ions are formed, while APPI (atmospheric photo ionization) uses photons. (In
electrospray, there is NO electrical discharge, ions are formed by evaporation of charged
droplets.) API sources require the sample to be in the gas phase before it can be ionized.

Some common types of CI sources are:

• APCI: Atmospheric Pressure Chemical Ionization. A continuous electrical
“corona” discharge is generated in the ion source, which causes the air molecules
(nitrogen and oxygen) in the source to ionize. These ionized air molecules in turn
transfer their energy to the sample molecules. This is a very “soft” ionization
process, i.e., it causes minimal fragmentation of most sample molecules. APCI
sources can analyze liquid samples (provided the liquid can be evaporated), and
are the preferred source for direct ambient air analysis (for environmental and
security applications).
• CI: Chemical ionization. This is very similar to APCI, except that the ionization
involves energy transfer to the sample from molecules other than air. That is, the
electrical discharge ionizes an additive compound, or CI reagent, which in turn
ionizes the sample. Depending on the additive used, the characteristics of the
ionization may be varied, e.g. to selectively ionize only a certain class of
compounds while leaving others as neutrals. CI reagents such as benzene, toluene,
dichloromethane etc. have been used for specific applications. The level of CI
reagent added is generally very low, in the parts per thousand to parts per million
range.
• Some types of CI sources run at reduced pressures; this allows a stronger
electrical discharge to be produced, which in turn allows more inert compounds
(such as pcb’s) to be ionized. CI sources are popular for environmental analysis
(e.g. measurement of dioxins in soil and water).

EI: Electron Ionization or Electron Impact. The earliest type of mass spec ion source; this
is the source you will see in old mass spec textbooks. It is a rugged "workhorse" device
with few adjustments and little to go wrong.
The EI source is designed to ionize gas phase samples in a moderate to high vacuum. It
works by bombarding the sample molecules with a beam of electrons. The electron beam
tends to "knock off" electrons from sample molecules, forming positive ions. The energy
of the electron beam is adjustable, but the "standard" setting is 70 eV…enough energy to
ionize and fragment any organic molecule. In reality, the sample is usually extensively

fragmented and the parent ion is often unseen. The EI source has a very low efficiency
for producing negative ions.
Due to the extensive fragmentation this type of source produces, it is rarely if ever used
today for the analysis of biomolecules, although it is useful for the analysis of things like
pcb's and dioxins.

And the list goes on: even more mass spec ion sources, in brief…
• FAB: Fast Atom Bombardment…a beam of high-energy atoms (usually Argon or
Xenon) is directed onto a liquid-phase sample. The sample is usually mixed with
a liquid "matrix" such as glycerol. The impact of the fast atoms causes desorption
(ejection) and ionization of sample molecules from the matrix. FAB is similar to
MALDI in that it generally produces a prominent parent ion peak with little
fragmentation; it is useful for determining the molecular weights of large
biomolecules.
• MAB: Metastable Atom Bombardment…similar to FAB.
• FD: Field Desorption… a solid sample is ionized and desorbed from a specially-
prepared surface by application of a very high electric field.

• Laser Ionization or Laser Ablation… this is useful for analysis of metal surfaces.
A pulsed laser beam is focused tightly onto a solid surface; this causes both
vaporization and ionization of a thin layer of the sample surface.

6. Types of Mass Analyzers

Now that the introductory material is out of the way, you are ready to learn some details
about the different types of mass specs in use today. In no time at all, you will be familiar
with all sorts of cool acronyms and what they mean. Prepare to impress your colleagues
with your new-found knowledge!!

Time of Flight (TOF)

The basic principle of Time of Flight (TOF) mass spectrometry is: a mixture of ions of
varying mass and charge, contained within a small area within a high vacuum, is
subjected to a strong electric field for a very short period of time (i.e., a "pulse"). This
pulsed field is applied such that all the ions begin to move in the same direction, due to
electrostatic force (attraction and/or repulsion). Uncharged molecules are not affected.

Consider Newton's third law: f = ma, or rearrange to get a = f/m. This just means that the
acceleration (a) of an object is equal to the applied force (f) divided by the mass (m). In
the case of ions, the applied force (f) due to the pulsed electric field is the same for all
ions which have the same charge. Ions which are, say, doubly charged, experience twice
the force as singly charged ions. And of course, we may have a wide range of masses (m)
for the ions in the mixture.

The end result is that, following application of a brief electric field pulse, a mixture of
ions of various masses and charge states is set into motion, in accordance with a = f/m;
therefore, ions with the lowest m are accelerated to the greatest speed during the duration
of the pulse. For equal m, ions with multiple charges are accelerated proportionally more
than singly charged ions (Figure 12 below).


If the ions are now allowed to "drift" through space under high vacuum conditions, they
will begin to separate (in space) according to the speed to which they were initially
accelerated by the pulse. The lighter (and/or more highly charged) ions are traveling
faster, and "pull ahead" of the heavier ions which are moving more slowly. (This is
analogous in some ways to the separation of compounds as they flow down the length of
a chromatographic column, although the mechanism of separation is of course different).

If we place an ion detector at a fixed distance from the ion source (pulsed field), and
monitor the arrival times of ions following the initial pulse, we find of course that the

lightest (and/or most highly charged) ions arrive first, followed in sequence by heavier or
less charged ions. This record of number of ions detected versus arrival time is the basis
of a time of flight mass spectrum.

Analogous to chromatography, a longer flight time generally results in greater separation
(resolution) between similar compounds (masses). In practice, the total ion flight distance
(path) is usually between 100 and 300 cm for commercial TOF mass spectrometers. This
length is a practical compromise based on the fact that lab instruments need to be of a
reasonable size, and also that there are many other factors influencing resolution besides
length of the flight path. Making the flight path longer offers minimal improvement in
resolution, beyond a certain point.

The common components of a time-of-flight mass spec are: (Figure 13 below)


• Ion Source: usually a MALDI-type source, but others may be used. See section on
ion sources for more details…
• Ion Accelerator: the unit which applies the pulsed electric field to the mixture of
ions from the source. Usually consists of an array of stacked metal plates and
metal meshes (grids) (Figure 14 below).


• Ion Reflector: sometimes called by other names, such as "ion mirror" or
"reflectron". Note that not all TOF mass specs use an ion reflector; when a
reflector is not used, the genre is known as "linear TOF". The main purpose of the
ion reflector is to lengthen the ion flight path (to improve mass resolution),
without making the instrument physically larger. Physically, the ion reflector
looks much like the accelerator, only much larger.
• Ion Detector: the ion detectors used in TOF are usually of the microchannel plate
(mcp) variety. An mcp is a very thin, flat glass plate with many microscopic

channels; the channels are coated internally with a material which emits electrons
when struck by ions (or electrons). Ions strike one side of the plate, causing
electrons to be released. The electrons "bounce" along through the channels in the
plate, eventually emerging from the other side, where they are collected and
counted. Two stacked mc plates are usually used, to give higher signal gain. See
photos and schematic diagram following.
• Note that the mcp detector responds best to ions which strike it at high velocity; if
an ion is traveling too slowly when it strikes the detector, it may not eject any
electrons, and therefore will not be detected.
• Timing and Data systems: in modern TOF instruments the timing of the
accelerating pulses, and detection of the ions, is all computer controlled. High
resolution TOF instruments require fast (and expensive) timing and data systems.



• High voltage power supplies: for large, heavy biomolecules in particular, very
strong electric fields are required to accelerate the ions to a reasonable velocity.
(Recall that "slow" ions are poorly detected.) In practice, this means that very
high voltages are usually applied in the accelerator and reflector
regions…generally in the range of 4 to 30 kV. The higher the accelerating
voltage, the better the performance for large molecules…but with a corresponding
increase in size and cost of the mass spec.

Types of TOF:

• linear: the first and simplest type of TOF mass spec; the ions travel in a straight
line from the ion source to the ion detector. Unless the flight path is very long,
resolution will be limited. However, this type of instrument is the simplest
(translation: lowest cost), is easy to tune and use, can have a very large mass
range (300k Daltons or more).

• reflectron: mentioned above. In addition to effectively lengthening the ion flight
path, the reflector also helps compensate for variations in ion motion in the
source, improving resolution even further. Multiple reflections of the ion beam
can be used (e.g. 3 reflectors can be used to create a "W" shaped flight path) to get
a very long flight path in a compact instrument. The drawback is cost, complexity,
sensitivity and difficulty in tuning the instrument (one or more reflectors). The
longer the flight path the better the resolution but sensitivity is decreased as some
ions are lost along the way.
• TOF-TOF: this is basically two separate TOF mass specs connected in series,
with an ion fragmentation stage in between. Following the initial TOF stage,
rather than striking an ion detector, ions enter a timed “selection stage” or “gate”,
which either allows ions to pass, or rejects them, based on their flight times (and
thus on their m/z ratio). Selected ions, which are allowed to pass this gate, enter a
fragmentation cell, where they collide with inert gas molecules. Following
fragmentation, the mixture of ions enters a second acceleration stage, after which
the flight path is similar to conventional TOF instruments (Figure 15 below):


• axial vs orthogonal ion injection: the traditional TOF instrument uses a pulsed
(MALDI) ion source which is located "in line" (on-axis or axial) with the initial
flight path of the ions; the ions travel in a straight line from the moment they are
created until they are accelerated into the flight tube; as in Figures 12 and 16. A
newer type of TOF instrument injects the ions into the accelerator stage at a 90
degree angle ("orthogonal") to the flight path, as in Figure 13 for example. This
allows the use of continuous ion sources (such as electrospray) in addition to
MALDI, and also offers some advantages in terms of improved resolution.

TOF in Biochemistry: Time of Flight mass specs are very popular in the biochemical
field due to their large mass range, very fast "scanning" and generally good resolution
and sensitivity. A typical high-end TOF instrument achieves a mass resolution of 10,000,

with very high mass accuracy, and femtomole detection limits for peptides.

Quadrupole

The basis of the quadrupole mass spec is a mass filter consisting of four parallel,
electrically-conductive electrodes or "rods". (In MS-speak, this mass filter assembly is
often called a "rod set" or a "quad".) The rods are most commonly cylindrical, although
sometimes they have a hyperbolic cross section (Figure 17 below).



A combination of alternating-current (AC) and direct-current (DC) voltages are applied
to these electrodes. The ions which are to be filtered (according to their mass-to-charge
ratio) are injected into one end of this electrode array, and (begin to) travel down the
central axis of the quad. Once inside the quad, the ions are influenced by the combined
electric field of the AC and DC voltages, and follow a complex pattern of motion as they
continue to travel down the length of the rod set. Figure 18 below is an "artist's concept"
of the flight path of the ions…


In the simplest mode of operation, the AC and DC voltages applied to the rods are kept at
a constant level; in this case, only one relatively narrow range of mass/charge ratios is
"stable" within the quad. These ions (if they exist) can pass freely through the quad and
exit the other end; all other ions are lost: either they are ejected out the sides of the quad
(i.e. between the gaps between the electrodes) or they strike the electrodes and are
neutralized.

Two other common modes of quadrupole operation are scanning (where the AC and DC
voltages are varied but the ratio between them is maintained at a constant value), and
peak hopping (where the voltages "jump" between a series of values which are stable for

particular ions of interest. The scanning mode of operation is the most common,
particularly for unknown samples, and produces what is immediately recognized as a
'mass spectrum". The peak hopping (or multiple ion monitoring) mode is used for
quantitation of samples in which the ions of interest are known, e.g. detection of
environmental pollutants.

The most popular configuration of the quadrupole mass spec is the so-called triple quad,
(see Figure 19 below). As the name implies, three quadrupoles are arranged in series, so
that sample ions pass through all of them sequentially on their path from the ion source to
the detector. The three quads are often referred to as Q1, Q2 and Q3. (Note that on some
instruments, the middle unit (Q2) is not actually a quadrupole (4-rod array): it can be a
"hexapole" (6 rods), "octopole" (8 rods), or some other structure, such as an array of ring-
shaped electrodes.). The vacuum interfaces used with triple quad instruments are usually
optimized for use with electrospray sources. (If quadrupoles are used in the interface
region, they are sometimes referred to as Q0 or Q00.)

Triple quad ion detectors come in various shapes and forms, but fundamentally are of
either the discrete-dynode variety, or the continuous-dynode (Channeltron) type (Figure
20).

_______________________________________________________________



The operation of a quadrupole MS/MS instrument is basically as follows. Ions of a
particular mass (m/z) of interest are selected in Q1; all other ions are rejected. The
selected ions (sometimes called parent ions or precursor ions) are injected into Q2,
which is called a collision cell (or something similar). An inert gas such as nitrogen or
argon is added to Q2, with the result that incoming ions collide with the inert gas
molecules and undergo fragmentation. The degree of fragmentation can be controlled by

varying (1) the amount of gas in Q2, (2) the type of gas in Q2, and (3) the speed with
which the ions enter Q2 (the "ion energy"). The ion fragments produced are sometimes
called daughter ions or product ions (but usually just fragment ions). Q2 is a non-
resolving quad, which means that it can only contain and transmit ions, but it cannot
mass-select them.

The fragment ions produced in Q2 are then passed into Q3, which is a resolving quad like
Q1. Here, masses of interest can again be selected…either by means of full mass scans,
or monitoring of selected ions. The power of triple quad MS/MS lies in its selectivity,
which results in a very low background noise level. Triple quad MS/MS is particularly
suited to quantitative pharmaceutical and environmental analysis at ultra-trace levels.

MS/MS is what made quadrupole mass specs famous; the first commercial MS/MS
instruments (developed in the early 1980s) were all triple quads. Today, TOF, Ion Trap,
and Hybrid mass specs also offer MS/MS capabilities, often combined with other
advantages such as fast scanning and wide mass ranges.

Quads in Biochemistry: Quadrupole mass specs are very popular for a number of reasons:
they are generally compact, and are available with a very wide range of price and
performance characteristics. The quadrupole MS is in general very rugged and reliable,
the "workhorse" of today's mass spec world. However in the world of biochemistry,
where samples with wide mass ranges are the order of the day, the quadrupole mass spec
is currently less popular than other MS techniques, such as MALDI-TOF and some types
of ion traps.

Ion Trap

Structurally, an ion trap mass spec is most closely related to quadrupole instruments. In
general, sample ions are injected into an electrode structure, where they are confined or
trapped by DC and AC electric fields. A fundamental difference between quadrupole and

ion trap systems is that the quadrupole mass filter is a "flow through" system, in which
the ions being filtered are (normally) in constant forward motion through the electrode
array. In the ion trap, by definition, ions are "trapped" for a period of time (which can be
up to several seconds), with no forward motion, before they are released for detection.
The selection or mass filtering of the ions occurs during the time in which they are
trapped. By varying (scanning) the intensity of the AC electric field(s), ions of specific
mass-to-charge ratios become resonant, and are selectively ejected (scanned out) from
the trap region. A unique advantage of most ion traps is that a stream of incoming ions
can be accumulated for a period of time, then scanned out all at once, in order to improve
sensitivity for ultra-trace analysis.

Types of ion traps: