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The CHROMacademy Essential Guide

Mass Analyzers – Traps and TOF’s
  sponsored by Thermo scientific

The Essential Guide from LCGC’s CHROMacademy presents the second in our series of webcasts on Mass Analyzers.  In this session, Dr. John Langley (Head of Mass Spectrometry, University of Southampton, UK) and Tony Taylor (Technical Director, Crawford Scientific), present the fundamentals of Ion Trap and Time of Flight, mass analyzers supported by Interactive Multi-Media from The CHROMacademy.  The session will consider topics such as the anatomy of the analyzer types, working principles, the importance of mass accuracy and resolution, when to use the various analyzer types and the performance one might expect from each.  A must see for everyone working, or hoping to work, with Ion Traps, Linear Ion Traps, Orbitraps or Time of Flight instruments.

 
Tony Taylor
Technical Director
Crawford Scientific

Dr G John Langley
Head of Mass Spectrometry,
University of Southampton, UK

 

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Topics include

  • Why use an Ion Trap or Time of Flight mass analyzing Device
  • Anatomy of the analyzers
  • Importance of analyzer accuracy and resolution
  • Two and three dimensional Ion Traps
  • Orbitrap analyzers
  • Mass analyzing principles
  • Important operating parameters
  • Mass spectral experiments
  • Pro’s and con’s of the mass analyzers
  • Figures of merit

Who Should Attend:

  • Current or potential users of LC-MS or GC-MS instruments with Ion Traps, Linear Ion Traps, Orbitraps or Time of Flight analyzing devices.

Key Learning Objectives:

  • Understand when to use Ion Trap or Time of Flight mass analyzers
  • Understand the anatomy of these analyzer types
  • Understand the theoretical and working principles of the mass analyzers
  • Explore the importance of the relationship between mass accuracy and mass resolution and the power this brings to MS-based techniques
  • Understand the mass spectral experiments possible with the various analyzers
  • Explore figures of merit and pro’s / con’s of the various analyzer types
  Mass Analyzers

In its simplest form the process of mass analysis involves the separation or filtration of analyte ions (or fragments of them).  The abundance of analyte and fragment ions are plotted in terms of their mass-to-charge ratio (m/z) against the abundance of each mass to yield a mass spectrum of the analyte as shown.

 

» Figure 1.  A typical mass spectrum

There are several very popular types of mass analyser associated with routine laboratory analysis and all differ in the fundamental way in which they separate ions on a mass-to-charge basis.  The objective of this Essential Guide is to explain theory, dynamics and operational aspects of the following mass analyser types:

  • Two dimensional quadrupole traps
  • Three dimensional quadrupole traps
  • Time-of-Flight mass analysers

A brief introduction to electrostatic trapping of ions (with Orbitrap mass analysers) is also given.

 

 
sponsored by Thermo scientific
 
mass spectrum

 

 
 
 

Mass Accuracy and Resolution

The mass spectrum of analyte species is represented by a bar graph that plots signal abundance or relative intensity of each of the ions against their mass-to-charge ratio which is often abbreviated as m/z. This is the property of the ion that is measured by the mass analyser.[1]

 

» Figure 2 .  Some fundamental MS definitions

mass spectrum

 
sponsored by Thermo scientific
 
 
 

 

 

 

Mass Accuracy: is the measurement of the closeness of the given measurement to the true mass of the analyte.  In this instance Δmaccuracy denotes the difference between a measured value (mmeasured) and the true mass (mtrue) of a substance.

Δmaccuracy = mtrue - mmeasured

 

Where:

mtrue is calculated mass
mmeasured is measured (observed) mass

 

Exact mass measurement can be achieved if very high accuracy is obtained from a mass analyser.  In practice this requires high resolution in conjunction with high mass accuracy in order to rule out the possibility of isobaric interferences.

Mass accuracy is usually expressed in parts-per-million:

 

Mass accuracy (ppm) =

 

Example:  Calculate mass accuracy for the compound para-Fluorophenylpiperazine (C10H13FN2, 180.1063 Da) if the observed molecular weight was 180.1057.


Δmaccuracy = mtrue - mmeasured = 180.1063 -180.1057 = 0.6 mDa    

Or in parts-per-million (ppm):

 

Mass accuracy (ppm) =

 

Mass Range: The property measured by mass analysers is the mass-to-charge ratio (m/z) of ions.  The difference between the highest and lowest measurable m/z denotes the mass analyser range.

Mass Resolving Power (Rm): is the ability of a mass analyser to separate two adjacent masses.  This is typically calculated using the equation:

 

Note that the parameter Δm represents a difference but it is not the same parameter that appears in the definition for mass accuracy.

In practice the resolving power of a mass analyzer is determined by one of the following methods.

  • Valley method
  • Resolution of a single peak (FWHM)

These methods are further explained in Figure 3

 

 

mass spectrum

Figure 3.  Mass resolving power definitions and calculations

 
 

Resolution is usually defined in one of two ways depending on the mass spectrometer being used.

The 10% valley (intensity) definition states that two peaks are considered to be resolved when they are separated by a valley, which is 10% of the height of each. This definition is typically used with magnetic sector instruments.  The definition used with quadrupole, ion trap and time of flight mass spectrometers is based on a peak width measured at 50% peak height (full width half maximum or FWHM).

 

Tandem Mass Spectrometry

For more information, please check Part 1 of this Essential Guide.

Defined as any method involving at least two different stages of mass analysis, tandem mass spectrometry (MS/MS or MSn) can be achieved by using either one single mass analyser (like quadrupole ion traps, Orbitrap’s, etc.) or a combination of them (triple quad, Q-TOF, etc.).

In the most common MS/MS approach, two different analysers are used in series.  The first analyser is used to select the ion of interest (usually known as the “precursor ion”), which is subjected to fragmentation prior to its analysis in the second mass analyser.  This approach permits to select and characterize analytes coming from complex matrices.  There are basically two types of tandem MS:

  • Tandem in space: implemented by using combinations of mass analysers (like triple quadrupole instruments).  In order to obtain high order MSn, several analysers are required (increasing the cost and complexity of the instrument)
  • Tandem in time: implemented by using single mass analysers (like quadrupole ion traps).  High order MSn, can be achieved with one single mass analyser

The term n in MSn refers to the number of MS stages or generation of ions used during the analysis and in general rarely goes beyond n = 7.
 
 

 

In quadrupole mass analyzer devices electric fields are used to separate ions according to their mass-to-charge ratio (m/z) as they pass along the central axis of four parallel equidistant rods (or poles) that have fixed (DC) and alternating (RF) voltages applied to them.
 

Remember:

RC (Radio Frequency): The range of electromagnetic frequencies above the audio range and below infrared (from 10 kHz to 300 GHz).

DC (Direct Current or Continuous Current): Is the constant flow of electric charge from high to low potential.

 
sponsored by Thermo scientific
 
 
 

The four rods with circular cross-section used to construct the quadrupole mass analyser are shown, along with the theoretically desired cross-section that is actually hyperbolic.[2] Circular cross-section rods are used for engineering convenience in most systems; however, the machining of the rods and their spatial positioning is critical for mass accuracy and resolution.

 

» Figure 4.  Quadrupole mass analyser device

 

  mass spectrum
 
 

Unlike simple deflection or acceleration of ions in magnetic and electric fields, the trajectory of ions in the quadrupolar field is complex.

Each rod is paired with its diametric opposite and has a potential
of +(U+Vcos( (ω t)) or -(U+Vcos( (ω t)) where U is the DC voltage (either positive or negative), and Vcos( (ω t) is a voltage which oscillates with a radio frequency ω in the time domain t.[3]

Note that each rod pair is successively positive and negative, allowing ions to be both attracted and repelled away from the rods or the central axis of the mass analyser.  Along the central axis of the quadrupole assembly and the planes shown, the resultant electric field is zero. Also note that for each pair of rods the peak maximum current in the positive or negative direction will be different. This can be explained by considering the permanent bias of each pair of rods (i.e. + and - U).

 

» Animation 1.  Quadrupole voltages

 

 

 
 
 

The region denoted by the letter A (called the first stability region) in Figure 5 represents the traditional operating region for quadrupole mass analyzers. Notice the symmetry of region A around the q axis. In practice only the top half of this diagram is considered, with the bottom half accessible by simply swapping the electrical connections to the quadrupole.

 

» Figure 5.  The Mathieu stability diagram in two dimensions (au (U) and qu (V)) for a quadrupole device (mass filter or linear trap).

 

 

  mass spectrum
 
 
 

 

Linear Ion Traps

Depending upon the magnitude of fixed direct current (DC) (U) and alternating radio frequency (RF) (V) voltages, it can be arranged that only ions of certain mass are allowed to pass the whole length of the quadrupole. Passing out of the quadrupole and into a detector, the other ions being deflected onto trajectories which cause them to collide with the rods and pass out of the analysing device.

» Figure 6. Ion trajectories within quadrupole mass analysers 

 

  mass spectrum
 
 
Linear quadrupole ion traps (LIT), also known as two dimensional quadrupole ion traps, have been developed from the conventional quadrupole mass analyser and can be used to collect and inject pulses of ions coming from continuous sources.
 
 
     
Figure 7.  Size comparison of a typical
quadrupole device and a watch (to scale)
  Figure 8.  Commercial quadrupole. 
Courtesy of Reliance Inc.
 
A linear ion trap includes two pair of rods which collect ions by using radio frequencies. Simple plate lenses at the ends of the quadruple provide the DC trapping field to keep the ions confined within the mass analyser.[4]
 
 
     
Figure 9.  Digital Light Processing (DLP) linear ion trap for high
mass range applications. Courtesy of University of Liverpool
  Animation 2.  Commercial quadrupole.  Courtesy of Reliance Inc.
 

The linear ion traps utilize an electrostatic potential for confinement of the ion beam. This device has two electrodes placed on either side of a linear space to form the electrostatic potential for the ion confinement. The ion beam is reflected repeatedly between the two electrodes.  A slot, made in one of the rods, allows ions to be radially ejected.

 

Linear quadrupole ion traps have some reported advantages over traditional (3D) ion traps:

  • Higher ion storage volume (reduced space charge effects)
  • Enhanced sensitivity for externally injected ions

 

However, the performance of a linear quadrupole ion trap is highly dependent upon mechanical errors of construction. If the rods are not completely parallel, then ions at different positions will experience different field strength, which in turn will cause different ejection times and loss of sensitivity and resolution.

The most common linear trapping devices can be quadrupoles, hexapoles or octapoles.  The following table shows the mathematical form of the potential in selected trapping devices.

 
Instrument Field (ΦN) Multipole Order (N)
Quadrupole 2
Hexapole 3
Octapole 4

Table 1.  Potentials used in selected linear trapping devices.
Note that the equations of motion for quadrupole mass filtering devices and quadrupole linear trapping devices are the same.

 
 

In a similar manner, it is possible to derive expressions that describe the movement of ions within hexapoles and octapoles; however, the movement of ions in these cases is highly coupled and the solution of the equations of motion is highly dependent upon initial conditions.  Therefore, there are no stability diagrams for multipoles of order 3 or above.  For low values of the trapping voltages in RF high order multipoles the ion trajectories are approximately determined by the following expression.

 

Where:

U(r): is the effective potential
Ve: electric effective potential
e electron charge
z: is the number of electrostatic charges the ion carries
N: multipole order (see table 1)

 
 

mass spectrumCurved Traps

Using curved quadrupole rods, curved traps work in a similar way to their linear counterparts.

The purpose of this modification is to force the ion beam to traverse a curved path prior to the detector or a subsequent mass analyser; by doing this, the incidence of photons (coming from the ion source) and of any other potentially interfering particles (neutrals) that could reach the detector is minimized. As expected, this reduction results in a substantial improvement in the signal to noise ratio.[5]

In the representation below, only ions with the desired mass-to-charge ratio will reach the detector (right hand side). Note how, if the collision cell was linear, then photons and neutrals would be able to reach the detector (right hand side).


» Figure 10.  Curved quadrupole rods

 

 
 

As with traditional multipole devices, different rod geometries are currently available with circular, hyperbolic or even square cross sectional areas.[6]

Curved multipoles have been used not only as a means of transporting and separating ions but they are also important in acting as collision cells in tandem mass spectrometry.[7, 8]

Curved linear traps function in the same way as their linear counterparts with the added advantage that ions can be focused in narrow areas to be ejected as compact bursts of ions.  This is usually achieved by altering the trapping fields of the electrodes at the front and rear of the instrument.

The presence of a collision gas (or bath gas) will reduce the kinetic energy of the ions within the trap.

In the animation below, ions are focused into a narrow area of the trap and then ejected through a slot in one of the electrodes; however, ions can also be axially ejected.


» Animation 3.   Curvilinear trapping device

 

Curved linear traps provide an efficient way to feed bursts of ions into other mass analysers, they are currently found in combination with many modern mass analysers.

 

 
 

Image Current Detection

Trapped ions can be detected in situ by measuring the image current they produce. The main advantage of this technique is the ability to re-measuring ion populations.

The image current detection technique has been successfully implemented not only in conventional ion traps but in high mass resolution analysers such as FT-ICRs and Orbitraps. [9, 10]

Ions populating the analyser describe different trajectories (according to the analyser itself). A coherent excitation (DC pulse) voltage is imposed on the ions in such a way that ions of the same mass-to-charge ratio move coherently. Following the DC excitation, the image current is measured using the detector electrodes.

The trajectories described for the ions through the quadrupole are complex and cannot be described in a simple way; however, after a coherent excitation is imposed (usually a pulse variation in the electrostatic field between rods) populations of ions will perform coherent oscillations between two opposite rods. The movement of ions between the rods will induce an electric current that can be measured. The intensity of the current is proportional to the number of ions. The principle of image current detection is further explained below.

Ion detection is followed by a Fast Fourier Transform (FFT) algorithm to convert the recorded time domain signal into a conventional mass-to-charge spectrum.

» Animation 4.  Principle of image current detection

 

 

 
 

MS/MS With Linear Traps

Linear traps are currently used as a means of achieving MS/MS, the most commonly used configuration is the triple quadrupole, described in our previous essential guide.  In spite of being called “triple quadrupole mass analysers”, most devices implement an additional RF quadrupole (Q0) to provide collisional cooling and focusing of the ions entering the instrument.  In addition, the collision cell (Q2 in the diagram below) can be either a traditional quadrupole or any high order multipole (hexapole, octapole,…).

mass spectrum

» Figure 11: Triple quadrupole

 

 

Triple quadrupole instruments also implement curved linear traps.

 

mass spectrum

 

» Figure 12: Triple quadrupole system capable of performing MS3 (AB SCIEX QTRAP® 5500).  Courtesy of Applied Biosystems

 
 
 

 

Also known as three dimensional ion traps, quadrupole ion traps (QIT) use oscillating electric fields (RF) to trap ions in a controlled manner.  The construction of a typical quadrupole ion trap mass spectrometer is shown and consists of a ring electrode with a hyperbolic inner surface and two electrically common hyperbolic end-cap electrodes.

The ion trap is operated by applying a sinusoidal potential (fixed RF frequency) to the ring electrode whilst the end-cap electrodes may be grounded, biased to a constant DC current (usually = 0), or maintained at an oscillating AC potential depending upon the mode of operation.[11, 12] Combinations of RF and AC potentials applied to the ring and end-cap electrodes may be used to:

  • Trap all ions within a specific m/z range
  • Trap all ions above a specified m/z value
  • Trap ions of a specified m/z
  • Eject ions of specified m/z values

All of these operations may be useful in various analytical applications and the usefulness of ion trap mass analysers in qualitative analysis is widely documented.

» Animation 5.  Principle of image current detection

 

 

 
 
 
 

Figure 13.  Cut away side view of a commercial three dimensional
ion trap.  Reproduced with permission from reference [13]

 

Figure 14.  Size comparison of an ion trap with a coin (to scale).  Reproduced with permission from reference [13]

 
 

Ion motion in the ion trap can be described by Mathieu equations.[14]  Because DC potentials can be applied to the trap, stability diagrams based on a (U) and q (V) can be used to determine which m/z values will remain stable within the trap and which will be ejected under particular sets of experimental parameters.

When solving the equations of motion for ions within the analyser, three parameters of fundamental importance emerge a, q and β according to the following relationships:

Where the subscripts Z and r represent axial and radial motion performed by the ions, U is the DC bias on the end-cap electrode, V is the RF amplitude applied to the ring electrode, ro is the radius of the ring electrode, ξo is the distance from the centre to the trap to one end-cap electrode, ω is the RF angular frequency and m/z the mass-to-charge ratio of the ion.  See figure below.

 

» Figure 15.  Ion trap representation

 

Ions within the mass analyser perform oscillations, the amplitude and frequency of these oscillations are closely related to the β parameter. The mathematical form of β is complex, but it depends upon both the RF and DC components of the storage field.

The expanded stability diagram shows values of a, q and β (in both axial and radial directions). The β values are important as they relate the extent to which any particular ion may follow the imposed RF field.[14]

Experimentally, it has been found that the ion storage process has a maximum efficiency when βr values are close to one and βz lies between zero and 0.3. In the same way, it has been reported that in order to obtain selective and controlled ion ejection, the magnetic field parameters of the mass analyser should be chosen in such a way that the βz parameter present values between 0.6 and 1.

When the operating line is chosen in such a way that the scan line passes close to an apex of the stability region, then only ions with a very narrow range of specific masses will have a stable trajectories.

 

» Figure 16.  Ion trap stability diagram

 

 
 
 

Ion Trap Space Charge Effects

The ion trap has potential to achieve very high sensitivity, but care is required to achieve this. The trap is limited in its capacity to trap charges (regardless of the m/z value), and as such the operating conditions should be optimized to ensure that only ions of interest are introduced into the trap.[15] In fact, ions due to natural background and matrix may be excluded using ion traps with external ion sources that are capable of excluding specified unwanted ions.

The resolution of the trap will diminish rapidly as the ion density increases. The trap has the capacity for only a limited number of ions at any one time before repulsive charges (known as the space charge effect) cause the excess ions to be ejected. Concentrations of ions above the space charge limit lead to poor performance in terms of grossly deteriorated resolution and shifts in the mass axis calibration.

To avoid the effects of space charge, the number of ions within the trap are regulated to an optimum (sometimes referred to as the ‘1/10th level’) to achieve optimum mass-to-charge analysis. This control occurs by calculating the optimum ion-injection time.

 

» Animation 6.  Principle of image current detection

 

 
 
 

Ion Trap Ion Manipulation

An early Thermo Finnegan (San Jose, Ca, USA) ESI - QIT (quadrupole ion trap) instrument is shown.

 

Figure 17.  Commercial ion trap instrument

 

Vacuum levels of 1.33 x 10-3 mbar and 3.33 x 10-5 torr are maintained by roughing and dual port turbo-pump systems.  This instrument is unique in that the ions are directly transferred from the end of the second octopole into the end-cap of the ion trap analyser.

At the outlet of the sampling capillary (200oC, 400 μm, 11.5cm), positive ions are transmitted through the skimmer by pulsing the tube lens between 0 and 200 V.  The sampled ions are collected by the first RF octople ion bridge and transmitted to the second octopole via an interoctopole lens.  Each octopole is 5 cm long (ro=3.3mm) and both are operated at 2.5 MHz and 400 V.

Octopole ion bridges are used as they improve transmission relative to static lens systems (mainly due to a decrease in gas scattering) and the octopole regions may be differentially pumped, each being capable of different DC potential offsets for optimum ion transmission.[16]

The interoctopole lens may be used to reduce output spikes due to cluster ions hitting the analyser via the application of several hundred volts between the ion injection and scan periods of the mass analysis.

 

Ion Trap Scanning Experiments

Ion trap spectral experiments are composed of several steps occurring sequentially, this chain of events being called a micro-scan. The different events in a micro-scan include ion injection, isolation, excitation, and analysis. In addition, very often an initial pre-scan is performed to determine the ideal injection time and to avoid space charge effects.[17]

After the ions are injected into the trap from the source, a suitable RF voltage on the ring electrode confines them to stable trajectories. An isolation scan can be performed subsequently in order to selectively accumulate a specific ion (or range of ions). The next optional step is ion excitation (in the case of tandem MS), where a voltage is applied to cause the trapped ions to oscillate with higher energy, ultimately causing collisions with the background gas and fragmentation. Finally, ions are analyzed by ejection from the ion trap to the detector through openings in the exit end cap electrode.

Ions are trapped within the analyzer by applying a RF voltage on the ring electrode. In order to increase efficiency, ions must be focused near the centre where the trapping fields are closest to the ideal, maximizing resolution and sensitivity. This is achieved by introducing a damping gas (which is usually helium) that cools injected ions by collisions, damping down their oscillations until they stabilize.

 

» Animation 7.  MS/MS experiments on a 3D ion trap

 

 
 

Other Ion Experiments

It is also possible to select specific m/z values to be ejected from amongst all of the ions within the trap. This is usually achieved by superimposing an AC voltage generated by the ring electrode to cause instability of only the selected ion by causing increasing amplitude resonance in the z axis. This techniques is often termed ‘resonant ejection’.[17]

Perhaps the most significant use of ion trap analysers involves sequential trapping and fragmentation of specific ions to produce MSn spectral data that is highly specific. This is achieved using collision induced dissociation of selected precursor ions held within the trap at increased background gas pressures. The product ions are then resonance ejected and monitored.

One or many of the product ions may be retained within the trap volume for further fragmentation. In this operating mode a series of precursor fragmentations may be carried out allowing MSn theoretical fragmentations.

 

» Animation 8.  MS/MS experiments on a 3D trap

 

 

 
 
 

MS/MS With Hybrid Quadrupole Traps

As previously stated, the number of ions that can be efficiently stored in a quadrupole ion trap is a decisive limiting factor for these instruments (charge space effects).  One way to overcome this problem is combining a linear ion trap (2D) with a quadrupole ion trap (3D).  In this way, the 2D trap provides the means to feed only ions of interest into the 3D trap.  Remember that linear traps have higher ion storage volume (reduced space charge effects) than their 3D counterparts.

 

Figure 18.  Typical quadrupole ion trap configuration

It has been reported that sensitivity in quadrupole 3D traps is usually higher than in 2D traps.

 

Figure 19.  MS/MS of lauric acid methyl ester (m/z 214). with a quadrupole ion trap.[18]

 
In spite of their low resolving power, quadrupole traps are widely used in the modern analytical laboratory.  Even in the very demanding world of proteomics, quadrupole mass analysers are in a very strong position.  The main reason being that multiple strategies to simplify the analysis of highly complex molecules have been developed (including protein digestion, isotopic labeling, etc.); these techniques had created new opportunities for relatively low resolution mass analysers.

Figure 20.  MS/MS spectra of a β-endorphin with a quadrupole ion trap.[19]

 
Finally, there are many research fronts where quadrupole instrumentation is still unrivaled (they are good value for money), especially in low mass range applications such as gas analysis, contamination monitoring, etc.
 
 
 

 

The basic principles of time-of-flight mass analysers are relatively straightforward in comparison to many of the other typical mass analyzing devices. Ions are extracted (or produced) in short bursts or packets within the ion source and subjected to an accelerating voltage. The ions then ‘drift’ or ‘fly’ down an evacuated tube of a set length.[20]

There are no complicated ion focusing devices necessary to constrain the ions to a particular flight path and as such the ions pass in a straight line, at constant speed towards the detector where a plot of abundance against time-of-flight may be recorded. The flight times are correlated against at least two known masses from an infused tune compound allowing a simple conversion to obtain a typical abundance versus mass- to -charge (m/z) spectrum.

 

» Animation 9.  The TOF mass analyser

 
 
 

Equations of motion

It is important to extract the ions in pulses or ‘packets’ into the mass analyser because as the ions are separated on basis of flight time, all ions must begin the flight at the same time in order to be able to calculate differences in arrival times.  After extraction the ions are subjected to an accelerating electric field (V1 volts –typically 3000 eV).  The kinetic energy of each of the ions may be expressed as:

Where:
m = mass of the ion
z = number of fundamental electrostatic charges on the ion
e = charge of an electron
v = final velocity

 

If the distance from the ion source to the detector is d, then the time (t) taken for the ion to travel through the drift tube to the detector is:

 

In practice the flight tube length (d) is fixed as is the value of V1.

The flight time of an ion (t) is directly related to the square root of m/z:

 

Where  is a constant based on d, e and V1.

An ion of m/z 100 will take twice as long to travel the distance d as an ion of m/z 25:

 

 

Resolution

Resolution in time-of-flight instruments is limited by two important factors, which are more evident at high m/z values.[21, 22]

The first problem is inherent in the nature of the analytical technique.  The difference in flight times between two ions of unit mass difference can be expressed as:

 

Where  is a constant based on d (tube length), e (electron charge) and accelerating electric field V1

Therefore the arrival time at the detector becomes smaller as the mass of the ions arriving at the detector increases and the ions are increasingly more difficult to differentiate via the detection system.

 

» Animation 10. 
The molecular weight of a given particle will affect its flight time

 
 

TOF - Issues with High Mass Resolution

The second problem regarding resolution lies with the method of ion production and separation in the instrument. Not all ions of the same m/z value arrive at the detector simultaneously due to a distribution of the kinetic energies they acquire in the accelerating voltage and are not all accelerated from the same point within the ion source. Therefore, even for ions of the same m/z value, there is a distribution of arrival times at the detector. When attempting to resolve masses that are contiguous, the arrival time of these masses at the detector may overlap and reduce spectral resolution. This problem may be particularly troublesome for ions of high mass.

 

» Animation11. 
High mass resolution issues with TOF mass analyzers

 

 

 

 

TOF - The Reflectron

Resolution in TOF instruments can often be improved through the use of a reflectron device.

A reflectron is a series of electrostatic lenses which create a homogeneous electrostatic field at the end of the (usual) flight path of the ions and has the same polarity as the incumbent ions. In this way, for example, positive ions will be quickly slowed within the electrostatic field of the reflectron, come to a brief standstill and are then accelerated in the opposite direction. The reflectron is often referred to as an “ion-mirror” due to the reversal of the ion flight path.

Ions having a greater amount of kinetic energy (faster ions) will penetrate the reflectron to a greater depth and so spend slightly longer in the device than ions with lower kinetic energy. In this way isobaric ions can be caused to bunch together to reduce the distribution of flight times and greatly enhance the resolving power of the instrument.

 

» Animation12.  The Reflectron

 
 
 

TOF - Increased Resolution Using Reflectrons

The reflectron may expel ions in the same path (off axis) as the arriving ions, but it is more usual to expel ions on a slightly deviated path, for convenience of instrument design and positioning of ion-production and detection devices.[23, 24]

Clearly, the longer the flight tube, the longer it will take for ions to traverse it. If the flight path length (d) is doubled, then ions previously arriving at the detector with times t1 and t2 will now have arrival times of 2t1 and 2t2 and the time difference between sequential ions arriving at the detector has doubled. The longer the time difference (usually in the order of nanoseconds), the greater the resolving power of the instrument. This is a further advantage when using the reflectron in TOF analysers as the flight path length of all ions is automatically doubled.

Traditional linear TOF instruments may achieve resolutions of a few 1000s or so but an increase to 10,000 or greater resolving power may be achieved through the use of a reflectron. The disadvantage of using reflectron technology is a slight decrease in the sensitivity of the analyser due to ion loss and dispersion of the ion beam, the problem being accentuated for ions of high mass. Due to these problems, the reflectron may be turned off when carrying out analysis of high mass ions at very low concentrations, detection being carried out in the linear mode.

Use the slider to watch the ion travel through the TOF flight tube.

 

» Figure 21.  Flight path comparison

 
 
 

Performance Limitations

There is no theoretical upper mass limitation in TOF analysis as all ions may be made to proceed from the ion source to the detector. In practice, however, there is a mass limitation in that it becomes increasingly difficult to discriminate between arrival times at the detector when m/z becomes large as has been discussed. This effect coupled with the inherent distribution of ions at nominal m/z values means that discrimination between unit masses becomes difficult over approximately 3000 m/z. At 50,000 m/z the mass overlap may be as much as 50 m/z units, however, this may be good enough for many routine applications.

The recording and storage of TOF data can be problematical as the data is produced at such high frequency. An instrument with a 2 m flight tube and ion energy of 2 keV may produce a complete mass spectrum from m/z 1 to m/z 800 in 90 μs.

Time array detection records full mass range spectra for each transient (or ion ‘packet’), a number of which are then integrated (summed), to produce a spectrum whose intensity reflects the sum of all transients. To store all data prior to integration two processors are normally required to ensure no transients are ‘missed’ due to the fast recording time (theoretically 10,000 transient spectra per second may be recorded).

 

» Animation 13.  Performance limitations of TOF instruments

 

 
 

Orthogonally Accelerated TOF Instruments

The direct coupling of API sources with TOF analysers can be problematical, since API techniques yield a continuous ion beam, whereas the TOF analyser operates on a pulsed process.

The classical method of overcoming this problem is to direct the ion beam past a slit placed at the ion source entrance, thereby obtaining a pulsed inlet. However, sensitivity of this type of system is poor as most ions are lost.

Ions may be collected in an ion storage device using a decelerating field, where they are extracted into the flight tube, which is perpendicular to the incident ion beam. The extraction procedure may be run at up to 1 kHz and the perpendicular orientation of the ion storage device helps to reduce kinetic energy dispersion, allowing resolution of up to 25,000 for high mass ions. The most usual approach to solving the interfacing problem between a continuous ion beam source and a TOF analyser is to use orthogonally accelerated TOF (OATOF).

 

Pusher Electrode

The ion beam from a continuous source emerges into the mass analyser and is accelerated through a voltage V1. The ion beam is a range of ions whose moments are proportional to the charge (z), mass (m) and accelerating voltage V1.

As the ion beam is produced continuously there is no separation of the ions in time. If a second electrode is placed at 90o to the emergent continuous ion beam with an applied potential of V2 volts, then by switching this electrode rapidly on and off a pulse of ions that is passing the electrode surface will be given a momentum of:



in a direction at right angles to the continuous beam.

The resulting vector of the pulse of ions produced is shown in the animation below.

The slowest pulsing speed can be anything the operator chooses and may be advantageous in terms of data storage space. However, sensitivity may be reduced if the pulsing speed is too low.


Effectively a section of the main beam has been selected and pulsed away.[25, 26]  All ions in this selected ‘packet’ will start into the flight tube at the same instant – a requirement for TOF analysis.

The magnitude of the pushing potential (V2) can be adjusted so that the injected ion packet will enter the axis of the flight tube. The pulsing electrode may be turned on and off at any frequency but there are some practical constraints.

At the fastest pulsing speeds there is little point in pulsing at such a rate that the previous pulse of ions have not arrived at the detector before the next pulse is generated. A practical maximum upper pulse frequency of around 30 kHz is usual.

 

Animation 14.  Pusher electrode

 

Deflecting Voltage

The ion beam may have to be directed a few degrees away from the axis of the instrument after leaving the acceleration region so that the reflected ions may strike the detector in an off axis position.[27]  Effectively the ion packet has to be ‘steered’ in the correct direction to either enter the reflectron at the correct angle or to impinge on the array detector in a linear instrument. If the initial component of ion velocity (i.e. the velocity of the ions in the axis of the ion source prior to orthogonal acceleration) is not enough to deflect the orthogonal ion packet from the axis of the analyser, then ions must be deflected using a small potential difference (100 V), otherwise resolution of the instrument will be compromised.  The ion storage region of the interface will accept ions with energies in the region 2.5 to 10 eV per charge for an accelerating voltage of 4 kV.

Because a significant proportion of the ion’s kinetic energy comes from the free jet expansion that takes place at the ion source, the optimum deflection voltage will have a mass component —that is, the heavier the ion the greater the deflection voltage required. This imposes some restrictions on the range of m/z values that may be simultaneously detected, without mass discrimination.

These effects may be reduced by using an RF only ion bridge at relatively high pressure (0.01 - 1 Torr) where collisional damping will take place and the energy of ions within the ion beam will be ‘normalized’ to reduce mass discrimination.[28]

 

» Figure 22.  Deflecting voltage effect on ion trajectory

 

 

 
 

Interfacing Details

Two approaches to interfacing are possible between the API ion source and the TOF analyser: one involving the use of skimmers with decreasing potential and increasing vacuum, the other involving the use of RF only ion bridges to transport the continuous ion beam from the ion source into the modulator of the mass analyser.

Dodonov et al,[2] at the University of Manitoba report a very direct approach to interfacing using a three stage pumped interface after Chait et al.[14]

 

» Figure 23.  TOF interfacing details

 

 

 

 

 
 
 

The Q-TOF Hybrid Analyser

The quadrupole-time of flight (Q-TOF) configuration is a hybrid mass analyser type consisting of a quadrupole or a system of them prior to the entrance of a time of flight mass analyser.  This hybrid device can be rationalized as a triple quadrupole where the last quadrupole (Q3) has been replaced with a TOF mass analyser (usually a reflectron).  These devices usually implement an additional RF-only quadrupole (Q0) to provide collisional cooling and focusing of the ions entering the instrument.  See diagram below:

 

» Figure 24.  Hybrid triple quadrupole-time of flight Q-TOF configuration

 

 
 

Other Factors Affecting Q-TOF Performance

It is well known that gases are heavily influenced by temperature and pressure; therefore, it is not surprising that temperature control is of overriding importance in QTOF-MS, as temperature variations will render drifts in mass calibration, usually affecting both the power supply output and the flight path of the ions within the flight tube (kinetic theory of gases).  Users shouldn’t be too worry about this issue, because, in general, it can be corrected with a single point calibration.

Gases are influenced by the pressure conditions at which they are confined, as a matter of fact, many Q-TOF systems have been developed for optimum indoor use only until certain altitude (note that atmospheric pressure is related to the altitude).

In addition to the above considerations, humidity and airborne dust can adversely affect your Q-TOF instrument response.  For more information contact your system manufacturer.

It is highly advisable to implement air conditioning control (including temperature, humidity and dust) whenever possible as well as making sure your lab location meet the altitude requirements for your system to operate.

Last but not least, make sure that the appropriate electrical power and power outlets for your Q-TOF system are in place.  In case of doubt, check your manuals or look for expert advice.

     
 

Figure 25.  Hybrid triple quadrupole-time of flight Q-TOF configuration

 
 

MS and MS/MS Experiments with Q-TOF Mass Analysers

During a single MS experiment, both Q0 and Q1 are operated in the RF only mode (note that Q0 is always operated in the RF only mode) while no fragmentation is promoted across Q2.  Alternatively, it is possible to scan Q1 for ion signals of interest.  Finally, ions exiting Q2 are orthogonally infused into the TOF mass analyser. As expected, in both situations, the final spectra will benefit from the high resolution and accuracy of the TOF instrument.

Note that MS experiments can be performed with or without collision gas in Q2.  If no collision gas is used, then the whole instrument is operated as in MS/MS mode but the fragmentation energy is kept low (below 10 eV) to avoid analyte fragmentation.  In this way the collision cell is used mainly for ion focusing.

During MS/ MS experiments, Q1 is set to transmit only the precursor ions of interest. Ions are accelerated under an energy of between 20 and 200 eV before their infusion into the collision cell Q2 where collision induced dissociation (CID) against neutral gas molecules (typically argon or nitrogen) renders analyte fragmentation.  The fragments of ions thus produced are finally infused into the TOF mass analyser.

Some instruments implement quadrupole ion traps before the TOF mass analyser device.

Figure 26.  Hybrid quadrupole ion trap-time of flight Q-TOF configuration

 
 
 

 

Quadrupoles and ion traps are instruments with constant bandwidth, which means that resolution decreases when increasing the mass-to-charge ratio of the ion.  As an illustration, let us consider that a bandwidth of 0.1 was found for certain ion.  If the m/z is equal to 100, then:

If the m/z is equal to 1000, then:

Note that in the second case we are facing a situation where most quadrupole mass analysers fail, as resolution for these instruments is typically below 2,000 (up to 10,000).

In the case of time of flight mass analysers, resolution remains constant when the mass-to-charge ratio is changed.  Due to their high resolution (up to 30,000 but 15,000 being typical) TOF’s instruments are suitable for challenging situations where quadrupoles tend to fail; however, they are almost as twice as expensive.

In the case of Orbitrap mass analysers the resolving power (Rs) is inversely proportional to the square root of m/z.  As an illustration, if a resolution of 100,000 was found for certain ion of m/z = 500, then the following resolution should be expected if the selected to mass ratio was 1,500:

Please bear in mind that the resolving power depends upon the mass-to-charge ratio and scan speed at which the instrument is operating.  It is also important to note that sensitivity also depends upon the ionisation efficiency.

 

Figure 27: Comparison of selected mass analysers

 
 

The selection of mass analyser for a particular application will depend upon the analytical needs and budget.  As expected, there is a considerable number of variables to consider when selecting a mass analyser (resolution, mass accuracy, capability of MS/MS, etc.).  The table below, will help with your mass analyser selection.

The table below lists uses and compares selected mass analysers

 
Analyser Selected Applications Resolution (ppm) Mass Accuracy (ppm) Sensitivity (mol)
Triple Quadrupole with
Linear Ion Trap
  • Bottom-up protein identification
  • MS/MS capabilities
2,000 100 1 × 10-15
Quadrupole Ion Trap
  • Bottom-up protein identification
  • MS/MS capabilities
  • Protein quantification
2,000 100 1 × 10-18
Q-TOF
  • Bottom-up and top-down protein identification
  • LC-MS/MS capabilities
  • Identification of posttranslational modifications
  • MS/MS capabilities
10,000 1 - 4 1 × 10-18
 
Table 2.  Typical proteomics uses and performance of selected mass analysers
 
 

The table below lists and reports typical figures of merit for selected mass analysers working.

 
Characteristic Magnetic Sector Quadrupole Quadrupole Ion Trap (QIT) Linear Ion Trap (LIT) Time of Flight (TOF)
Mass Resolving Power (FWMH) 1,000 - 100,000
  • Unit resolution
  • 100 – 10,000
Up to 10,000 1,000 – 10,000 Over 30,000 but 15,000 being typical
Mass Accuracy (ppm) 1 – 5 50 - 100 40 - 100

50 – 100

2 – 50
Mass Range (m/z) 10,000 Up to 4,000 but usually below 2,000 Up to 4,000 but usually below 2,000 Up to 4,000 but usually below 2,000 Over 100,000
Linear Dynamic Range 109 107 100 – 100,000 100-100,000 100 – 1’000,000
Scan Speed (seconds) ~ 1 ~ 1 ~ 1 ~ 1 0.001
Scan Frequency (Hz) 0.1 – 20 1 – 20 1 – 30 1–300 10 - 1’000,000
Sensitivity (g) 10-12 10-15 10-15 10-15 10-12
Efficiency Less than 1% 1% to 95% 1% to 95% 1% to 99% 1% to 95%
Operating Pressure (torr) 10-6 10-3 10-3 10-3 10-6
Advantages
  • High resolution
  • Very reliable
  • Tolerant of high pressures
  • Small size
  • Price
  • Tolerant of high pressures
  • Small size
  • Relatively low cost
  • Well designed for tandem MS
  • Tolerant of high pressures
  • Small size
  • Well designed for tandem MS
  • Price
  • Highest mass range
  • Very fast scan speed
  • Good adaptability to MALDI
  • Price
Disadvantages
  • Not well suited for LC(CE)-MS
  • Not tolerant of high pressures
  • Instrumentation is massive
  • Expensive
  • Low resolution
  • Low scanning speed
  • Poor adaptability to MALDI
  • Limited mass range, however, progress is being made
  • Low scanning speed
  • Limited mass range, however, progress is being made
  • Low scanning speed
  • Low resolution
Difficult LC(CE)-MS coupling
Laboratory requirements MS laboratory Bench top Bench top Bench top Bench top or floor standing
Cost Moderate to high Low Low Low to moderate Low to high
           
 

Table 3.  Figures of merit of selected mass analysers

 

 

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  29. LCMS-IT-TOFTM.  A revolutionary quadrupole ion trap – time of flight instrument.  Shimadzu.
 
 
 

 

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