time of flight mass spectrometry: review

www.wjpps.com Vol 4, Issue 07, 2015.

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Sriveena et al. World Journal of Pharmacy and Pharmaceutical Sciences

TIME OF FLIGHT MASS SPECTROMETRY: REVIEW

T. Sriveena*, A.Srividya, A.Ajitha and V.Uma Maheswara Rao

Department of Pharmaceutical Analysis and Quality Assurance, CMR College of

Pharmacy, Kandlakoya (v), Medchal road, Hyderabad – 501401, T.S, India.

ABSTRACT

Time-of-flight instruments form a well-established group of mass

spectrometers, with its popularity still increasing. Methods of gating

ion populations and the special requirements in the detection and

digitization of the signals in the TOF-MS are of special importance.

Time of flight mass spectrometry (TOF-MS) has been an attractive

choice of instrument for many years due to its potentially unlimited

m/z range, high-speed acquisition, accurate mass measurement

capability and sensitivity. In which an ion's mass-to-charge ratio is

determined via a time measurement. Ions are accelerated by an electric

field of known strength.

KEY WORDS: TOF-MS, mass to charge ratio, high speed.

INTRODUCTION OF MASS SPECTROMETRY

Mass spectrometry (MS) is an analytical chemistry technique that

helps identify the amount and type of chemicals present in a sample by measuring the mass-

to-charge ratio and abundance of gas-phase ions. A mass spectrum (plural spectra) is a plot of

the ion signal as a function of the mass-to-charge ratio. The spectra are used to determine the

elemental or isotopic signature of a sample, the masses of particles and of molecules, and to

elucidate the chemical structures of molecules, such as peptides and other chemical

compounds. Mass spectrometry works by ionizing chemical compounds to generate charged

molecules or molecule fragments and measuring their mass-to-charge ratios.

In a typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized, for

example by bombarding it with electrons. This may cause some of the sample's molecules to

break into charged fragments. These ions are then separated according to their mass-to-

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Article Received on

10 May 2015,

Revised on 02 June 2015,

Accepted on 26 June 2015

*Correspondence for

Author

T. Sriveena

Department Of

Pharmaceutical Analysis

And Quality Assurance,

CMR College of

Pharmacy, Kandlakoya

(v), Medchal road,

Hyderabad – 501401, T.S,

India.


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charge ratio, typically by accelerating them and subjecting them to an electric or magnetic

field: ions of the same mass-to-charge ratio will undergo the same amount of deflection. The

ions are detected by a mechanism capable of detecting charged particles, such as an electron

multiplier. Results are displayed as spectra of the relative abundance of detected ions as a

function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified

by correlating known masses to the identified masses or through a characteristic

fragmentation pattern.

TIME OF FLIGHT MASS SPECTROMETRY

Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which

an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by

an electric field of known strength. This acceleration results in an ion having the

same kinetic energy as any other ion that has the same charge. The velocity of the ion

depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to

reach a detector at a known distance is measured. This time will depend on the mass-to-

charge ratio of the particle (heavier particles reach lower speeds). From this time and the

known experimental parameters one can find the mass-to-charge ratio of the ion.

Today's time-of-flight mass spectrometers offer excellent sensitivity and high mass range as

well as high-speed analysis. This has made TOFMS an essential instrument for biological

analysis applications - typically with MALDI and ESI ionization-particularly with the

development of high-resolution and hybrid instruments (for example Q-TOF and TOF-TOF

configurations). The TOFMS suitability for the analysis of fast transient signals has seen it

applied to studying fast gas-phase chemical reactions and to hyphenated flow or injection

analysis techniques such as ICP-TOF and GC-TOF. In addition time-of-flight mass

spectrometry is the dominant instrument for static SIMS.

Fig:1. Time of flight mass spectrometry


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A time-of-flight mass spectrometer uses the differences in transit time through a drift region

to separate ions of different masses. It operates in a pulsed mode so ions must be produced or

extracted in pulses. An electric field accelerates all ions into a field-free drift region with a

kinetic energy of qV, where q is the ion charge and V is the applied voltage. Since the ion

kinetic energy is 0.5mv2, lighter ions have a higher velocity than heavier ions and reach the

detector at the end of the drift region sooner.

Fig:2.Source of Time of flight mass spectrometry

In the source of a TOF analyzer, a packet of ions is formed by a very fast ionization pulse.

These ions are accelerated into the flight tube by an electric field (typically 2-25 kV) applied

between the sample plate and the charged grid. Since all the ions are accelerated across the

same distance by the same force, they have the same kinetic energy. Because velocity (v) is

dependent upon the kinetic energy (E ) and mass (m) lighter ions will travel faster.

K.E = mv2 ......... (1)

E is determined by the acceleration voltage of the instrument (V) and the charge of the ion (e

kinetic × z). Equation 2 rearranges to give the velocity of an ion (v) as a function of

acceleration voltage and m/z value.

v =√ ...........(2)

After the ions accelerate, they enter a 1 to 2 meter flight tube. The ions drift through this

field free region at the velocity reached during acceleration. At the end of the flight tube they

strike a detector. The time delay (t) from the formation of the ions to the time they reach the

detector dependents upon the length of the drift region (L), the mass to charge ratio of the

ion, and the acceleration voltage in the source

t = / ...........(3)


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From the above Equation 3 shows that low m/z ions will reach the detector first. The mass

spectrum is obtained by measuring the detector signal as a function of time for each pulse of

ions produced in the source region. Because all the ions are detected, TOF instruments have

very high transmission efficiency which increases the S/N level .

Time-of-flight mass spectrometry equation:

TF = L/v =L m/2zeV

Measurement of ion masses by Time-of-Flight Mass Spectrometry (TOF MS)

Time-of-flight mass spectrometers measure the time a sample molecule requires to fly a

known distance. Charged molecules of the sample are accelerated in an electric field with a

known energy.

The speed a molecule gains is then:

From this equation the mass can be calculated, by measuring the time such a molecule

requires to fly a known distance. Heavy ions will take longer than light ones.

Fig:3.Principle of time of flight mass spectrometry

This picture shows the working principle of a time of flight mass spectrometer. To allow the

ions to fly through the flight path without hitting anything else, all the air molecules have

been pumped out to create an ultra high vacuum. In the vacuum in the MS-200 chamber an

ion can fly on average 600 meters before it will hit an air molecule.

At the end of the ion flight path is a detector which produces electrical pulses as the ions

impact the detector. The time measurement is done by the timing electronics, which applies a

pulse of voltage to accelerate the ions and measures the time between this pulse, and the

electrical impulse from the detector.


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The MS-200 mass spectrometer has an flight path length of around 0.6m. A molecule with a

mass 26µm (atomic mass units) requires 6 µsec (6 x 10-6

seconds) to fly through the flight

path. For a sufficient distinction between different masses the timing electronics is capable of

measurement with a resolution of 2 nsec (2 x 10-9

seconds). Every 20 µsec the analyte in the

ionization area is accelerated and the masses of the molecules are recorded. In an experiment

of 1 second, 50,000 analysis cycles are performed. Therefore the gathered spectra is a good

representation of the sample.

PRINCIPLE

Fig:4.Basic principle representation of time-of-flight mass spectrometry

1. In a time-of-flight mass spectrometer the ions are formed in a similar manner by electron

bombardment, and the resulting ions accelerated between electrically charged plates.

2. Again, the sample must be a gas or vaporized and is bombarded with an electron beam or

laser beam to knock off electrons to produce positive ions.

3. However, the method of separation due to different m/e (mass/charge) values is then

dependent on how long it takes the ion to travel in the drift region' i.e. the region NOT

under the influence of an accelerating electric field.

4. The ions are accelerated in the same way between positive to negative plates in an electric

field of fixed strength i.e. constant potential difference.

5. The smaller the mass of the ionized particle (ionized atom, fragment or whole molecule)

the shorter the time of flight in the drift region where no electric field operates.

6. This is because for a given accelerating potential difference, a lighter particle is

accelerated more to a higher speed than a heavier ion, so the 'time of flight' down the tube

is shorter.

7. Therefore the ions are distinguished by different flight times NOT by different masses

being brought into focus with a magnetic field BUT the separation by time of flight is still

determined by the m/e (m/z) value of the ion.


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t = Kinst√(m/q)

t = time of flight, m = mass of ion, q = charge onion,

Kinst = a proportionality constant based on the instrument settings and characteristics e.g. the

electric field strength, length of analyzing tube etc.

Therefore t is proportional to the square root of the mass of the ion for particles carrying the

same charge - the bigger the mass the longer the 'flight time'.

KE = qV

the kinetic energy imparted to the ion is given by its charge x the potential difference of the

accelerating electric field.

The acceleration, for a fixed electric field, results in an ion having the same kinetic

energy (KE) as any other ion of the same charge q but the velocity v of the ion depends on

the m/e (m/z) value.

v = d/t

where v = velocity of accelerated particle in the drift region,

d = length of tube in the drift region. (or t = d/v)

KE = 1/2mv

2,

so the bigger m, the smaller is v in the drift region and hence the basis of detecting ions of

different mass by different 'flight times'.

INSTRUMENTATION AND OPERATION

Fig:5.Instrumentation of time-of-flight mass spectrometry

The TOFMS have subsystems

1. Vacuum system

2. Optical system

3. Source/analyzer

4. Reflectron

5. Electronics/data system


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Vacuum System

The vacuum system consists of a 52-in.3 coffin-type chamber machined from a block of

aluminum with a clear Lexan top and multiple polymer O-ring sealed vacuum ports (ISO-KF)

machined into its walls. A Pfeiffer combination turbo molecular/drag pump (model TPD011)

is used to evacuate the chamber into the low 106 torr range. With a pumping speed of 10 L/s

and a mass of 2.5 kg, the Pfeiffer pump is the smallest one of its kind that is commercially

available. This type of combination pump can exhaust into the relatively low vacuum of a

diaphragm pump. A KnF-Neuberger diaphragm pump (Model N84.4ANDC) capable of

operating at 1.5 torr and 4.8 L/min is used in this application. The chamber pressure is

measured with a Pfeiffer wide-range vacuum gauge (combination Pirani/cold-cathode gauge).

Optical System

The optical system of a typical MALDI TOFMS is designed to deliver a series of short

ultraviolet (UV) laser pulses to the source region of the TOF analyzer. The sample to be

analyzed is co-deposited with a UV-absorbing substance (matrix) onto a probe and inserted

into the source of the analyzer at the focal point of the optical system. The laser energy is

absorbed by the matrix and transferred to the analyte. Both analyte and matrix molecules are

desorbed, ionized, and accelerated into the TOF analyzer. Standard optical components

(lenses and windows) are used in nearly all commercial and custom TOF mass spectrometers.

In contrast, the APL Suitcase TOF uses fiber-optics to deliver the energy from a pulsed

nitrogen laser featuring a small, sealed plasma cartridge (LSI Model VSL-337, 140 J per

pulse, 5-ns pulse width, 337-nm wavelength, and 1- to 20-Hz pulse rate). This pulsed

nitrogen laser is also the smallest and lightest commercially available one that will work in

this application.

Fiber-optic components simplify the optical mounts and increase the overall ruggedness of

the instrument. The light is coupled into a 200um core UV, transmitted to a U-bracket (Oz

Optics Model UB-01), collimated and launched across the air gap of the U-bracket through a

linear-wedge neutral-density filter, and then coupled back into a second 200 m core UV fiber.

The linear-wedge 0–2 optical-density filter is used to attenuate the laser energy delivered to

the sample. A small lens pigtailed to the end of the second fiber focuses the light through a

vacuum window and onto the center of the sample probe. A simple sliding gimbal mount is

used to align the beam and set the spot size at the sample. Finally, a fast photodiode mounted

near the exit aperture of the laser detects scattered light to be used as the start trigger for the

TOF measurement.


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Source/Analyzer

Analyte and matrix ions formed in the electric field of the source region are all accelerated to

the same kinetic energy. A flat sample plate held at a high voltage (≈10 kV) separated by a

small distance from an extraction grid at ground potential typically defines a source. After

exiting the source, the ions drift in a field-free region until they strike a detector. Because all

the ions have the same kinetic energy, a more massive or heavier ion will have lower velocity

than a less massive or lighter ion. The velocity v and TOF t of the ions are related to their

mass m by the following equations:

v = [ 2zeEs/m]1/2,

t = [ m/2zeEs]1/2 D,

where z = the number of charges (e) on the ion,

E = electric field in the source region of width s,

D = the length of the drift region.

In a simple linear analyzer, the initial energy, temporal, and spatial distributions of ions in the

source lead to peak spreading at the detector. One method of correcting the initial energy

distribution and decreasing the peak width is to use a “reflectron,” a retarding electric field

that reflects the ions back along their original path to strike a detector placed at the ions’

spatial focus. Ions of the same mass but with slightly more energy enter the reflectron first,

penetrate slightly deeper, and exit later than less energetic ions. As they approach the

detector, the more energetic ions catch up with slower ones and come into a spatial focus at

the detector. The reflectron increases signal resolution, defined as (t/2Δt), by correcting for

the initial energy spread (decreasing Δt and increasing the effective drift length of the

analyzer (increasing t). An ion detector can be placed to the side of the original ion path or a

detector with a center hole can be placed coaxially with the original ion path so that the ions

pass through the center of the detector, are reflected by the ion mirror, and then are

accelerated back toward the active face of the detector. A reflectron with a linearly increasing

potential (constant electric field) corrects ion energy spreads to first order. Deviating from the

linear potentials typically used in commercial instruments to a slightly curved potential can

create higher-order corrections.7,8 APL’s Suitcase TOF analyzer is a miniature, nonlinear

reflectron TOF with a very efficient grid less focusing source and unique detectors that

produce very clean signals.


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Fine-mesh metallic grids are employed in most mass spectrometers to establish boundaries

between regions of differing electric fields. For example, the high electric field of the source

region is typically separated from the field-free drift region by a grid. In our Suitcase TOF,

we have eliminated all but one grid, which is located a few millimeters from the detector. The

grid less focusing source uses a very small three-element electrostatic lens to extract the ions

with a high field (≈5 kV/mm) and then slow them down and focus them for maximum

transmission through the center of the detector and into the reflectron. Typical reflectrons are

built by stacking many thin metal elements onto insulating rods and applying the reflector

potentials with a resistor network. We have simplified and ruggedized the reflectron by

defining the electrode structure on a flexible circuit board material, rolling it around a

mandrel, and encasing it in a fiber glass cylinder. A surface-mount resistor network is

soldered to pads on the end of the flexible circuit board. Finally, the analyzer uses a custom

micro channel plate (MCP) detector assembly with a novel anode structure that drastically

reduces the signal ringing that is often associated with coaxial detectors. A small polished pin

replaces the standard disk-shaped anode to collect the pulse of electrons generated by the

stack of MCPs. A reduction in the stray capacitance significantly improves the impedance

match between the anode and the 50-Ω transmission line, resulting in much less ambient

electrical noise coupling into the line and virtually no signal ringing.

Reflectron TOF

Fig:6.Representation of Reflectron TOFMS

The kinetic energy distribution in the direction of ion flight can be corrected by using a

reflectron. The reflectron uses a constant electrostatic field to reflect the ion beam toward the

detector. The more energetic ions penetrate deeper into the reflectron, and take a slightly

longer path to the detector. Less energetic ions of the same mass-to-charge ratio penetrate a

shorter distance into the reflectron and, correspondingly, take a shorter path to the detector.


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The flat surface of the ion detector (typically a micro channel plate, MCP) is placed at the

point where ions with different energies reflected by the reflectron hit a surface of the

detector at the same time counted with respect to the onset of the extraction pulse in the ion

source. A point of simultaneous arrival of ions of the same mass and charge but with different

energies is often referred as time-of-flight focus. An additional advantage to the re-TOF

arrangement is that twice the flight path is achieved in a given length of instrument.

Electronics/Data System

The current version of the Suitcase TOF uses a mix of commercial and custom electronics to

control the instrument and collect the data. Five high-voltage power supply modules are used

to manually control the source, reflectron, and detector voltages. A full-size LeCroy

oscilloscope (Model 9354, 500-MHz analog bandwidth, 500 mega samples/s) digitizes the

TOF signal and averages between 10 and 100 individual shots to produce one TOF mass

spectrum. A very small laptop computer (Panasonic Tough Book) running a National

Instruments Lab View program downloads the data from the oscilloscope and then processes,

formats, stores, and presents the mass spectra. Efforts at greatly reducing the size, weight,

power, and packaging of the electronics are being addressed in the next prototype.

BENEFITS

1. Fastest MS analyzer.

2. Well suited for pulsed ionization methods (method of choice for majority of MALDI

mass spectrometer systems).

3. High ion transmission.

4. MS/MS information from post-source decay.

5. Highest practical mass range of all MS analyzers

ADVANTAGES

1. Ii is Simplicity and Ruggedness.

2. Accessibility of the ion source and Unlimited mass range

3. Major advantages of the TOF technique over quadrapole and sector type analyzers are the

extremely high transmission, the parallel detection of all masses and the unlimited mass

range.

4. The 3-D ion trap time-of-flight mass spectrometer(LCMS-IT-TOF) is used to analyze

mass of components eluting from the HPLC column, and for structural elucidation.


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5. Time-of-flight (TOF) has quickly established itself as the preferred type of mass analyzer

for the characterization of synthetic macromolecules. TOF combines a high sensitivity

with a broad mass range and a high spectral resolution and accuracy.

DISADVANTAGES

1. Sensitivity

2. Resolution

LIMITATIONS

1. Requires pulsed ionization method or ion beam switching (duty cycle is a factor) .

2. Fast digitizers used in TOF can have limited dynamic range .

3. Limited precursor-ion selectivity for most MS/MS experiments.

APPLICATIONS

1. Very fast GC/MS systems

2. Matrix-assisted laser desorption ionization (MALDI) is a pulsed ionization technique that

is readily compatible with TOF MS.

3. Atom probe tomography also takes advantage of TOF mass spectrometry.

4. Photoelectron photo ion coincidence spectroscopy uses soft photo ionization for ion

internal energy selection and TOF mass spectrometry for mass analysis.

5. Common applications of TOF spectroscopy within pharmaceutical development include

metabolite profiling, surface analysis of organic and inorganic molecules, and biomarker

discovery

6. Matrix-assisted laser desorption/ionization (MALDI) TOF spectroscopy is efficient and

highly sensitive for analyzing polymeric compounds such as protein and polysaccharides.

7. Purchasing considerations for pharmaceutical TOF equipment include the molecular

stability of the compounds to be tested, requirements for instrument size, possible

instrument compatibility with quantification software, and the laboratory.

CONCLUSION

Advances in the technologies of ionization, ion detection as well as high speed signal and

data processing make TOF-MS and attractive alternative to conventional mass analyzers in

specific applications. Developments in ion optics and electronics have substantially removed

the limitations of early TOF instruments. In this many important instrumental and

performance of TOF instruments have been discussed. There is considerable scope for further


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development of the facilitating technologies to allow the speed of TOF to be exploited while

maintaining a dynamic range commensurate with scanning MS instruments.

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time of flight mass spectrometry: review

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