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Mass Filters in Mass Spectrometry

Separations of ions based on properties of mass and charge.

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Mass Spec is “A Universal Technique” Analysis by MS does not require:

Chemical modification of the analyte Any unique or specific chemical properties In theory, MS is capable of measuring any gas-

phase molecule that carries a charge Analyzed molecules range in size from H+ to mega-

Dalton DNA and intact viruses As a result, the technique has found widespread use Organic, Elemental, Environmental, Forensic,

Biological, Reaction dynamics

All experiments have this basic backbone, but range of applications implies a diversity of experimental approaches.

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What does the mass filter do? A mass spectrometer determines the mass-to-

charge ratio (m/z) of gas-phase ions by subjecting them to known electric or magnetic fields and analyzing their resultant motion. Sectors – magnetic or electric Quadrupole Ion Trap Time of flight (TOF) Ion Cyclotron Resonance (FT-MS) Tandem system (MS-MS, MS-MS-MS, etc)

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Vacuum Requirement Mean Free Path: the average distance a molecule travels

between collisions. For typical MS conditions, can be estimated as:

L in cm, p in mTorr

Suggests pressures on the order of 10-5 torr to move a molecule across a meter without collision.

Requires moderately sophisticated, and moderately expensive systems of vacuum pumps

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Vacuum Systems All mass spectrometers operate at very low pressure (high

vacuum) to reduce the chance of ions colliding with other molecules in the mass analyzer collisions cause the ions to react, neutralize, scatter, or fragment. these processes will interfere with the mass spectrum

Experiments are conducted under high vacuum conditions (10-2 to 10-5 Pa or 10-4 to 10-7 torr)

Requires two pumping stages: mechanical pump - provides rough vacuum ~0.1 Pa (10-3 torr) second stage uses diffusion pumps or turbomolecular pumps to

provide high vacuum ICR instruments have even higher vacuum requirements

(often includes a cryogenic pump for a third pumping stage)

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The Mass Spectrum

A mass spectrum is a plot of signal intensity vs. m/z To compare different MS techniques we need to provide

numerical indications of how good the data is . . .

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Information from a Mass Spectrum

Identification of molecular mass Determination of structure Determination of elemental composition Determination of isotopic composition Quantification

Not inherent – requires consideration of ionization efficiencies, ion transmission, detector response …

Qualitative information is much easier to extract!

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m/z The mass-to-charge ratio is often referred to as m/z and

is typically considered to be unitless: m: mass number = atomic mass in u

with 1 u = 1/NA g

z: charge number = Q in e with 1 e = 1.6022×10-19 C

the Thompson has been proposed as a unit for m/z, but is only sometimes used

Historically, most ions in MS had z = 1 with new ionization techniques, this is no longer true

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Signal Intensity

Depending on the type of mass spectrometer, ions may be detected by direct impact with a detector or by monitoring of an induced current image.

Recorded signal can be measured in: Counts per unit time (Digital) Voltage per unit time (Analog) Power (Frequency domain)

To a first approximation, relative signal intensity reflects relative ion abundance

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Figures of Merit for the Mass Spec

Selection of appropriate MS instrumentation and conditions depends on analysis sought and key figures of merit.

SensitivityIon Transmission

Duty Cyclem/z Range

Mass Resolving PowerMass Accuracy

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Resolving Power

Mass peak width ( m 50%) Full width of mass spectral peak at half-maximum peak height

Mass resolution / Resolving Power (m / m 50%) Quantifies ability to isolated single mass spectral peak

Mass accuracy Mass accuracy is the ability to measure or calibrate the instrument response

against a known entity. Difference between measured and actual mass

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“The history of spectroscopy is the history of resolution …”- A. G. Marshall, et al, A. Chem., 74(9), 252A, 2002.

Different charge but the same mass

Differing in nominal closest-integer mass

Ions of the same chemical formula but different isotopic composition

Ions of the same nominal mass but different elemental composition

Note m/z dependence of necessary resolving power

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Mass Accuracy Mass accuracy is

linked to resolution. A low resolution

instrument cannot provide a high mass accuracy

High resolution and high mass accuracy enables determination of elemental composition based on exact mass

Possible because of elements’ mass defects

Requirements increase as m/z increases

Exact masses and corresponding formula for various ions of m/z 180 containing only C, H, N, and O atoms.

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Calculate resolution and accuracy

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Different types of mass filters

Analyzer System HighlightsTOF Theoretically, “no limitation” for

maximum m/z, high throughput

Quadrupole Unit mass resolution, fast, low cost

Ion Trap Unit mass resolution, fast, low cost

Sector (Magnetic and/or Electrostatic)

High resolution, exact mass

ICR (FT-MS) Very high resolution, exact mass, perform ion chemistry

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Time-of-Flight MS To determine m/z values

A packet of ions is accelerated by a known potential and the flight times of the ions are measured over a known distance.

Key Performance Notes Based on dispersion in time Measures all m/z simultaneously, implying potentially high duty cycle “Unlimited” mass range DC electric fields Small footprint Relatively inexpensive

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Time-of-Flight MS

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Time-of-Flight MS Ions accelerated by strong

field, E, within short source region, S.

Drift times recorded across long, field-free drift region, D

vD depends on starting position of ion – ideally all ions start from same plane.

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but there are complicating factors . . .

TOF = total recorded flight time of an ion to = Ion formation time after T0 of TOF measurement ta = Time in acceleration region, which depends on initial position and

initial energy tD = Time in drift region, which depends on initial position and initial

energy td = Response time of detector

For any m/z in a time-of-flight mass spectrum, the recorded peak will be the sum of signals corresponding to multiple, independent, ion arrival events

Each ion arrival will be recorded at a unique TOF, (see eqn above)

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Resolution in TOF

TOF’, which is the center of the peak in the mass spectrum, will be an average of all individual ion arrival TOFs

The width of TOF’, t, will depend on the distribution of the individual ion arrival TOFs (and other factors …)

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Improving Resolution in TOF MS

At ionization: U = U0 (initial ion energy)

At exit of extraction:

U = U0 + EextxqAt beginning of drift:

U = U0 + Eextxq + (V1-V2)qTune source voltages and/or delay to

compensate for U0 and create space focus at detector. Mass dependent.

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Improving Resolution in TOF MS - Reflectrons

Reflectron consists of a series of electrodes, forming a linear field in direction opposite of initial acceleration.

Ions are slowed by this field, eventually turning around and accelerating back in direction of detector.

Penetration depth depends on Us, which is function of U0 and acceleration field, E.

Reflectron voltages are tuned to create a space focus at the plane of the detector.

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Two means to the same end

In Delayed Extraction, we give ions different U to achieve same TOF.

In Reflectron, ions possess different U. We force them to travel different D to achieve same same TOF

In both cases resolution is enhanced!

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TOF is inherently a pulsed detector

TOFMS is an ideal detector for pulsed ionization methods If ionization event is synchronized with time zero, high duty cycle is achieve But not all sources are “pulsed” (ex. Electrospray, or stream ions from EI source, etc.) Because of pulsing, ions are wasted whenTOFMS is applied to a continuous source and . . . . Increased efficiency comes at the expense of mass range and mass

resolution Still, figures of merit and cost make thetechnique desirable

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Use “orthogonal” extraction Ions are extraction in a

direction orthogonal to original analyte stream trajectory

Extraction event is still rapid (t), but extraction volume is determined by length of gate region.

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orthogonalTOFMS (oTOFMS) Able to reduce average

initial energy in ToF direction to ~0 (resolution and accuracy)

Independent control of beam energy and drift energy, allows maximum duty cycle.

Want tightly collimated beam in extraction region

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A review question . . . . Suppose you are attempting to separate these two compounds by LC-MS. The first compoundto appear in your chromatogram has an intense peak at m/z = 344.1421. You know that your mass spectrometer has mass accuracy greater than 7 ppm. What conclusion can you make?

1. Compound A is the first to come off of the column2. Compound B is the first to come off of the column3. You are measuring an average of the two compounds that contains

mostly A4. Nothing -- You do not have sufficient mass accuracy to determine

which compound(s) you are measuring

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1. Compound A is the first to come off of the column

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Other detectors . . .

TOFMS Pulse packet of ions introduced into analyzer All m/z in packet reach detector (“simultaneous detection”) m/z determination based on dispersion Based on static, DC fields

Quadrupole MS Continuous introduction of ions into analyzer Transmit only specific m/z value to detector m/z determination based on band-pass filtering Based on time-vary, RF fields

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Quadrupole Geometry / operation

Quadrupole consists of four parallel rods

Typical length might be 10’s of cm

Precise dimensions and spacing

Rods connected diagonally in pairs

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Quadrupole Geometry / operation

Voltage of all rods have a DC component, U.

All rods have RF component of voltage with MHz frequency = ? /2p and amplitude Vo.

Potentials on the two sets are out of phase .

Quadrupole fields cause no acceleration along z axis.

V1 = V3= -Fo = -U – Vocos ? t

V2 = V4= Fo = U + Vocos ? t

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Quadrupole stability diagrams Stability diagram for fixed Rf

frequency, fixed m/z. An ion will have stable trajectory

through quadrupole if x and y are always less than radius of quadrupole.

(Sim A) With no RF and positive U, positive ion is stable along X (repelled to center), attracted to negative Y rod causes instability

(Sim C) RF field has stabilized Y trajectory.

Note that with increased U, need greater Vo to achieve this stability.

(Sim E) Instable along x-axis. Note that as U increases, lower Vo will

induce this instability.

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Forces – Generalizing for all m/z

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Stability Diagram for a Quadrupole

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Mass Selectivity Many conditions (U, V, m) fall within stability region – there is more than one

way for ion to pass through For selectivity, must also considerstability of other mass values Apex of generalized stability diagram is at a = 0.237, q = 0.706 To select transmit narrow mass window, adjust U and Vo such that a = 0.237, q = 0.706 (e.g., Ion B) For any value m we find

a/q = 2U/Vo

To scan values of m through narrow transmission window, hold other parameters constant and scan U andVo with constant ratio

U/Vo = ½(0.233 / 0.706)

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Mass Selectivity For ANY value m

a/q = 2U/Vo

For, example: Reduce U, Hold Still stable, slope of

“scan line” is reduced

What effect does this have on resolution?

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Mass Scanning

Scan line shows

U/Vo = ½(0.233 / 0.706)

Increase in mass requires proportional increases in U and Vo to maintain this ratio and these a and q values.

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Quadrupole PerformanceTypical Quadrupoles Maximum m/z ~ 4,000 Resolution ~ 3,000

Quadrupoles are low resolution instruments Usually operated at ‘Unit Mass Resolution’

Small, lightweight Easy to couple with chromatography

Rf-Only quadrupoles Operated with U = 0, quadrupole becomes a broad bandpass

filter Such “rf-only” quads are an important tool for transferring

ions between regions of mass spectrometers. Often denoted with small “q”

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Collisional Cooling A common application of rf-

only multipoles involves collisional cooling.

In an ESI source, the expansion into vacuum produces a ion beam with broad energy distribution

Ion optics and TOFMS experiments rely on precise control of ion energies

Desire strategies to dampen energy from external processes

Rf-induced trajectory in high pressure region yield collisions, and reduction in energy

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Collisional Cooling

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Triple Quadrupole Mass Spectrometer

Q1 selects parent; q2 CID fragmentation inside RF-only quad; Q3 fragment analysis; Detector

Fragment Ion Scan: Park Q1 on specific parent m/z; scan Q3 through all fragment m/z to determine make-up of Q1

Parent Ion Scan: Park Q3 on specific fragment m/z; scan Q1 through all parent m/z to determine source of fragment

Neutral Loss Scan: Scan Q1 and Q3 simultaneously, with constant difference, a, between transmitted m/z values (a = MQ1 – MQ3). Signal recorded if ion of m/z= MQ1 has undergone fragmentation producing a neutral of m = a.

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Quadrupole Ion Trap Quadrupole ion storage trap mass spectrometer (QUISTOR) - recently developed traps and analyzes all the ions produced in the source

the S/N is high. consists of a doughnut shaped ring electrode and two endcap electrodes A combination of RF and DC voltages is applied to the electrodes to

create a quadrupole electric field (similar to the electric field for quadrupole) electric field traps ions in a potential energy well scan the RF and DC fields the fields are scanned so that ions of increasing m/z value are ejected from the cell and detected The trap is then refilled with a new batch of ions to acquire the next mass spectrum

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Quadrupole Ion Trap

Several commercial instruments are available this analyzer is becoming more popular.

QUISTORs are very sensitive, relatively inexpensive, and scan fast enough for GC/MS experiments

The mass resolution of the ion trap is increased by adding a small amount 0.1 Pa (10-3 torr) of Helium as a bath gas. Collisions between the analyte ions and the inert bath

gas dampen the motion of the ions and increases the trapping efficiency of the analyzer

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Sector Instruments (Mag or Elec) Magnetic Sector: the first mass spectrometer, built by J.J.

Thompson in 1897, used a magnet to measure the m/z value of an electron

Magnetic sector instruments have evolved from this concept Sector instruments have higher resolution and greater mass

range than quadrupole instruments, but they require larger vacuum pumps and often scan more slowly

The typical mass range is to m/z 5000, but this may be

extended to m/z 30,000. Magnetic sector instruments are often used in series with an

electric sector, described below, for high resolution and tandem mass spectrometry experiments.

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Magnetic Sector explained Magnetic sector instruments separate ions in a magnetic field according to momentum and charge Ions are accelerated from the source intothe magnetic sector by a 1 to 10 kV electric field the radius of the arc (r) traveled depends upon the momentum of the ion, the charge of the ion (C) and the magnetic field strength (B). Ions with greater momentum follow a larger radius Ion velocity - determined by the acceleration voltage (V) and

mass to charge ratio (m/z) the m/z transmitted for a given radius, magnetic field, and acceleration voltage:

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Contribution by Electric Sectors An electric sector consists of two concentric curved plates.

A voltage is applied across these plates Ion beam bends as it travels through the analyzer voltage is set so the beam follows the curve of the analyzer The radius of the ion trajectory (r) depends upon the kinetic

energy of the ion (V) and the potential field (E) applied across the plates

an electric sector will not separate ions accelerated to a uniform kinetic energy

radius of the ion beam is independent of the ion's mass to charge ratio electric sector is not useful as a standalone mass analyzer

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Electric Sector/Double Focusing Mass Spectrometers

The mass resolution of a magnetic sector is limited by the kinetic energy distribution of the ion beam kinetic energy distribution results from variations in the

acceleration of ions produced at different locations in the source . . . . and

from the initial KE distribution of the molecules an electric sector significantly improves the

resolution of the magnetic sector by reducing the kinetic energy distribution of the ions

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Reverse Geometry Double Focusing Mass Spectrometer

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FT-Ion Cyclotron Resonance MS (FT-ICR MS) FT-ICR mass spectrometry exploits the cyclotron

frequency of the ions in a fixed magnetic field The ions are trapped in a Penning trap (a device for the

storage of charged particles using a constant magnetic field and a constant electric field) where they are excited to a larger cyclotron radius by an oscillating electric field perpendicular to the magnetic field.

The signal is detected as an image current as a function of time. After a Fourier transform, which converts a time-domain signal

(the image currents) to a frequency-domain spectrum (the mass spectrum), we can get a “traditional” mass spectrum

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The ICR trap explained

m/z = B/2f

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Advantages of FT-ICR MS

High Mass Resolution enhances sensitivity by making it possible to distinguish

between analyte and background species at or near the detection limit

narrow peak width allows the signals of two ions of similar mass to charge (m/z) to be detected as distinct ions. A peak at mass 800.000 Da can be distinguished from a peak at mass 800.001 Da

Has “almost unlimited” resolution M / M > 10,000,000 is possible,

M / M is in the range from 100,000 to 1,000,000 for most experiments

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Advantages of FT-ICR MS ultrahigh mass accuracy (1ppm)

offers an alternative to tandem mass spectrometry (MS/MS) for identification, an advantage if the amount of sample is limited.

The mass accuracy can be less than that of a single electron, so that chemical compounds with the same nominal molecular weight but different elemental compositions can be distinguished by ICRMS

wide mass range spectra are collected in a single scan over a wide mass

range without loss of sensitivity

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Advantages of FT-ICR MS detect different ions simultaneously, instead of one at a

time (scanning sectors) Thus, high speed

Multiple pulse / collection cycles can be used Signal averaging Time dependent studies of ion stability / ion reactions

The other particularity of the FTICR mass spectrometer is that new fragmentation techniques can be used, such as infrared laser activation, or electron capture dissociation. These techniques can be used in combination to fragment very large molecules such as whole proteins

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Limitations of FT-ICR MS The background pressure of an FTICR should be very

low to minimize ion-molecule reactions and ion-neutral collisions that damp the coherent ion motion. Strict low-pressure requirements mandate an external ion source for most analytical applications.

Need high magnetic field. A limit in the sensitivity of FTICR is caused by

broadband image current detection, requires approximately 100 charges to generate a measurable signal at a given m/z ratio. “Ion-counting” MS require fewer molecules to generate “signal”

Large and Expensive

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Applications of FT-ICR MS Macromolecules Multi-residue analysis Metabolomics Proteomics

by extracting proteins from cells or tissue, fragmenting them into shorter peptide segments, and then determining the masses of all fragments

Biomarkers Complex mixture, e.g. crude oil Fast and specific analyses of toxins Elemental composition Isotopes

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Ion detection - based on charge or momentum

large signals - faraday cup is used to collect ions and measure current

most modern detectors amplify the ion signal using a collector similar to a photomultiplier tube, for example: electron multipliers, channeltrons and multichannel plates.

gain controlled by changing HV applied to the detector detectors are selected for:

speed, dynamic range, gain, and geometry

some detectors are sensitive enough to detect single ions