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Quadrupoles, Use of in Mass Spectrometry
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1921
Q Quadrupoles, Use of in Mass Spectrometry PH Dawson, Iridian Spectreal Technologies Ltd., Ottawa, Ontario, Canada DJ Douglas, University of British Columbia, Vancouver, Canada
MASS SPECTROMETRY Methods & Instrumentation
Copyright © 1999 Academic Press
Quadrupole mass spectrometers (often referred to as quadrupole mass filters because of the way they operate) are the most successful example of the class of mass spectrometers called dynamic. Their performance depends upon a dynamic interaction of ions with time-varying electric fields. Most classes of spectrometers are static and use either the interaction of fixed magnetic and electric fields or time-offlight. The quadrupole mass spectrometer derives its name from the nature of the electric potential which is quadrupolar in the direction transverse to ion injection, i.e. dependent on the square of the distance from the centre of the field. The field is achieved by using four parallel rods as schematically illustrated in Figure 1. This article begins with a brief history. It then explains the principles of operation of this mass spectrometer. The idealized view of its operation has to be tempered by consideration of some real-world situations that influence performance, such as the finite length of the field, the inevitability of fringing fields and field imperfections. Observations of performance and its limitations are illustrated. The applications section deals with residual gas analysis, gas chromatography and liquid chromatography mass spectrometry (GC-MS and LC-MS), collision induced dissociation using triple quadrupoles (MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) used for elemental analysis.
A history of development The possibility of using quadrupole radiofrequency fields for mass analysis was first suggested in 1953
by Paul and Steinwedel and in a US government report by Post. The first practical implementation was by Paul and co-workers in 1958. This work became the foundation for the field. Wolfgang Paul was awarded a share of the Nobel Prize for physics in 1989 for the development of the ion trap technique that also used quadrupole fields. Early quadrupole mass filters were very limited in mass range and resolution but their physical simplicity and the absence of a magnet made them attractive for upper atmosphere and space applications. Major development occurred in the 1960s inspired by this application. This same period also coincided with a rising demand for residual gas analysers because ultrahigh vacuum technology began to have routine
Figure 1 A schematic illustration of a quadrupole mass spectrometer. Ions that are to be filtered to identify the presence of a particular mass are injected in the direction of the axis of the instrument. If the combination of radiofrequency and direct voltages applied to the rods is correctly chosen, only ions of one particular mass to charge ratio (m/z) will be successfully transmitted to the detector.
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application, first in research laboratories and later in production equipment, especially in semiconductor processing. Gradually the quadrupole mass filter became the dominant instrument for this application and remains so today. Quadrupole performance slowly improved but in the 1970s new applications to organic analysis and particularly the implementation of the combination of GC and MS placed increasing emphasis on better understanding of how the instruments worked in order to overcome their limitations. Computer simulations of performance became important. Combined with detailed experimental analysis, these led to a much improved knowledge of real-world quadrupoles with fringing fields and field imperfections. An important advance came with the application of phase space dynamics for calculating quadrupole performance. The new advances were incorporated in the classic textbook of the field written by Dawson and various collaborators and published in 1976. This book was re-issued by the American Institute of Physics in 1995 as a paper-back classic. In the 1980s and 1990s, the limits to quadrupole performance have been pushed back by precision manufacture and careful source design. A mass range of up to 2000 or more is commonly achieved with unit mass resolution. For LC-MS applications the mass range may reach 4000. There have been equally significant improvements in trace analysis capability based on a combination of sensitivity and more perfect peak shapes. High-performance quadrupole mass spectrometer manufacture has come to demand very high precision.
the applied voltages across the quadrupole are direct voltage U and an alternating radiofrequency voltage of V cos ω t and the minimum separation between opposite pairs of electrodes is 2ro. The charge to mass ratio of the ion is z/m. Time is expressed by ξ = ω t/2, where t is in seconds. For ion transmission the ions must have finite amplitude of oscillation in both x and y directions so that they do not strike the rods, i.e. the trajectories are both stable.
Principles of operation The perfect field
In a perfect quadrupole mass filter field, motion in the x and y (transverse) directions is independent. There is no field in the axial direction and motion is unchanged along the axis. Both x and y motion are governed by the Mathieu equation (see the Further reading section for the derivation of these equations);
i.e. where u represents x or y and where Figure 2 Zones for stable trajectories in both x and y transverse directions expressed in terms of the parameters a and q which are related to the m/z ratios of the ions. (A) General zones of stability, (B) a detail of the zone commonly used. The iso-beta lines are related to frequencies of ion oscillation.
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1923
Combinations of a and q values that give stable motion are shown in Figure 2. There are several areas of simultaneous stability. The one near the origin is commonly used but the higher zones have been examined both theoretically and experimentally especially in the search for peak shapes with more abrupt fall-off at the edges for applications in trace analysis. The quadrupole uses a sinusoidal alternating field. In principle any alternating field is possible but the higher harmonics may lead to complexity in the behaviour of the ions. The RF frequency is generally in the range of 12 MHz. Mass selection is obtained by choosing a ratio of q/a such that only a narrow region of the stability zone near the apex (a = 0.23699 and q = 0.706) is intersected by the operating line using q/a = constant. For a given RF and DC voltage, ions of different mass to charge ratio (m/z) appear at different points along the line. Mass scanning is carried out by altering the values of U and V while maintaining their ratio constant to bring ions of different m/z into the tip of the stability region. This would give a spectrum of constant resolution (M/∆M). In practice a resolution that increases with mass is preferred and the ratio q/a is adjusted electronically throughout the mass scan in order to achieve more or less constant peak width. An alternative to voltage scanning would be to scan the frequency but this is rarely done because of technical difficulties in covering a large mass range. This simple description implicitly assumes that the length of the quadrupole is infinite so that all ions with stable trajectories are differentiated from those with unstable trajectories. In practice, resolution may be limited by the length of the field. It is found that Rmax = n2/h, where Rmax is the maximum attainable resolution and n is the number of RF cycles the ions spend in the field. The parameter h depends upon the source and on the fringing fields. A value of 25 is not unusual; a value of 10 would be an excellent performance. High resolution is favoured for ions of low axial velocity (lower energy ions but, fortunately, also higher mass ions). One is limited, however, in reducing axial ion energies by the detrimental influence of fringing fields.
is to assume a linear increase of the x and y direction fields as the entrance is approached. The fringing fields extend over a distance comparable to the filter radius. The y-direction (a,q) values in the fringing field correspond to intrinsically unstable ion trajectories. If too much time is spent in the fringing fields, ion amplitudes will increase and the effective aperture of the spectrometer will be reduced. The finite diameter of the field (ro) means that ions are only accepted for transmission when they enter the field with a small initial transverse displacement from the axis and small transverse velocities. The combination of displacement and transverse velocities that are possible defines the acceptance of the instrument. The acceptance and so the overall sensitivity becomes smaller as the resolution is increased. The acceptance is calculated using phase space dynamics. The sensitivity of the mass spectrometer is best expressed in terms of a phase space diagram as shown in Figure 3. This shows the initial combinations of
The real world: transmission and fringing fields
In the real world, there are inevitably fringing fields at the entrance to and the exit from the mass filter. Ion motion in these fields can be very complex and the motion in each of the three coordinate directions becomes coupled. Very low energy ions may be reflected or even trapped in the fringing fields. However, a good theoretical approximation in most cases
Figure 3 Phase space acceptance diagrams showing the initial conditions of transverse displacement and velocities that lead to ion transmission at 100% and 50% of the initial phases of the RF field. (A) x-direction, (b) y-direction. This is for the centre of a peak and for ions spending two RF cycles passing through the fringing fields. The displacement from the axis is measured in units of r0 and the velocity in terms of r0 /ξ.
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transverse position and transverse velocity that will result in ion transmission. In practice, this depends upon the initial phase of the field when the ion first experiences the field. The Figure shows the x and y acceptances for transmission in 100% of the initial phases, 50%, and so on. At higher resolutions the acceptance area decreases. At a given resolution, combining the x and y acceptances together gives an indication of sensitivity for different numbers of RF cycles spent in the fringing fields giving a diagram such as that in Figure 4. This shows that fringing fields can be advantageous if properly chosen. There have been many attempts to tailor fringing fields for optimum performance, such as using retardation of ions after they enter the field or by adding RF only sections at the ion entry (the delayed DC ramp), or by using specially shaped electrodes. If the relative ion transmission is measured versus resolution for a particular mass number, there will be a curve such as that in Figure 5. If the source is
Figure 4 Combined phase space acceptance areas for x and y directions as function of the length of the fringing field (expressed as the number of RF cycles that the ions spend within the fringing field). This illustrates how sensitivity may vary depending on ion velocity.
evenly illuminated, at low resolution the source emittance will be less than the quadrupole acceptance and the transmission will vary little with resolution. Also the peaks will tend to be flat topped. At some point the acceptance will become limiting and transmission will tend to fall with the square of the resolution (taking fringing fields into account). Finally, at the length limitation of the quadrupole the transmission drops abruptly with resolution. Curves (a) and (b) illustrate different ion energies. In some cases, the source emittance may be rather diffuse or may not be evenly illuminated, then quite different transmission versus resolution behaviour will be observed. The real world: field imperfections
There are inevitably other departures from the perfect fields and these become more and more important at high resolution. Displacement of one or more rods from the ideal position is the simplest case. This leads to higher order terms in the expression for the electric potential. One rod displaced would give predominantly third-order or hexapole correction terms. Opposite rods displaced gives fourth-order or octopole terms. These imperfections limit the resolution that is attainable, i.e. even if n is increased, the resolution will not increase further. There have been various theoretical and more limited experimental studies of these effects. The field faults may also cause badly shaped or even split peaks because of nonlinear resonances in the ion oscillations at certain critical (a, q) values.
Figure 5 A typical example of transmission efficiency versus resolution for an instrument with a well-defined source emittance. There are three regions of transmittance as resolution is increased. (I) Sensitivity is source limited, (II) sensitivity is filter acceptance limited, (III) resolution is length limited (number of cycles in the field) and (IV) resolution is limited by field imperfection.
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1925
Other field faults may arise from nonparallel rods (bending or bowing) or from errors in the electrical waveform. It is common to substitute round rods in the quadrupole for the ideal hyperbolic ones. Round rods are easier to precision-manufacture. If the diameter and positioning of the rods is correctly matched, field faults are minimized and only sixth-order distortions are produced. These are not expected to significantly influence performance. However, there is controversy over this choice of round and hyperbolic rods with many assertions being made but little solid experimental evidence. One confusing factor is that other field faults (e.g. from rod positioning) may be more serious when using round rods. Observations of limits to performance
Figure 6 illustrates some of the data from an extensive examination of performance of a particular quadrupole. These data demonstrate most of the features discussed above. In addition, there is an ultimate limit to achievable resolution set by the perfection of the quadrupole field. Similar performance data have been generated for operation using other regions of the stability diagram such as near a = 0, q = 7.5 or a = 3, q = 3. These regions may have interest for specialized application.
Applications Residual gas analysis
One of the first uses of quadrupole mass filters was for residual gas analysis in high-vacuum chambers. An electron beam ionizes the background gas and a mass spectrum is recorded with the quadrupole. This allows determination of the composition of the background gas and, after calibration, the partial pressures of each of the components, such as N2, O2, H2O, etc. Because only light gases are generally involved, the mass range of the quadrupole need only be 100200 m/z. The compact and rugged construction of a quadrupole with purely electronic scanning make quadrupoles particularly attractive for this application. Often the quadrupole is mounted on a flange which is simply bolted onto the vacuum chamber. Sometimes a pressure reduction stage is used. Modern systems have computer controlled scanning and allow the possibility of searching libraries to match an unknown spectrum. GC-MS
The largest fraction of quadrupole mass spectrometers sold today are used as detectors for GC to identify
Figure 6 Some examples of performance measurement for a particular quadrupole mass filter showing how the limiting resolution varied with the number of RF cycles in the field. The ultimate resolution reached was dependent on frequency and mass number.
and quantify trace levels of organic compounds. Environmental analysis and drug testing of athletes, for example, rely extensively on GC-MS. Organic compounds separated by a gas chromatograph elute into the ion source of a quadrupole mass filter. Ions are formed either by electron impact (EI) or chemical ionization (CI). In EI, positive molecular and fragment ions are usually formed. The resulting mass spectrum gives a fingerprint of the compound. Unknown compounds can be identified by searching a library of spectra. In CI, analytes react with a reagent ion present in excess to produce either positive or
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negative molecular adduct ions, usually with minimal fragmentation. The combination of the retention time on the chromatograph and the appropriate molecular mass is often sufficient to identify trace analytes. Quadrupole mass filters have become the standard for GC-MS because they are easily interfaced to computers, scan rapidly on a time scale compatible with peaks eluting from a GC, require only medium vacuum (105 mbar), are compact, and are of comparatively low cost. Gas chromatography is restricted to relatively volatile compounds with moderate molecular masses and so the m/z range of a quadrupole used as a detector for GC is usually ∼5001000. A complete EI spectrum can be obtained on ∼10 pg of an analyte. If only ions of one m/z are monitored (single ion monitoring), with CI, the detection limits can be lowered to low femtogram levels. Alternatively a few selected m/z values (say four) corresponding to the major peaks in the spectrum of a targeted compound can be monitored by switching the quadrupole between these m/z values without scanning intervening regions (multiple ion monitoring). The ability to switch a quadrupole from full scans to single ion monitoring to improve detection limits is an advantage over other methods where a complete spectrum must be acquired (TOF, ion trap ICR). A quadrupole system dedicated to GC/MS can be quite compact, often smaller than the GC itself.
intermediate molecular masses produce ions with a few charges, depending on the number of basic residues and their pKa values. Conventional ESI operates best at flow rates of 1−10 µL min−1. LC flow rates are usually considerably higher (∼ 1 mL min−1) so the output of the LC is often split, with a fraction of the flow going to the ESI source. APCI and ESI produce ions at atmospheric pressure. These are transferred into the vacuum system of a quadrupole mass filter with two or more stages of differential pumping. The ability to operate a quadrupole at moderate pressures of ∼ 10−5 torr means only modest, lower cost vacuum pumps are required. ESI and APCI generally produce protonated molecular ions. As in GC-MS, for targeted compound analysis the quadrupole is operated in single ion mode to monitor the m/z of interest. Fragment ions can be formed by applying high electric fields to the ions in the ion sampling region. These fields accelerate the ions through the locally formed high density of gas causing collision-induced dissociation. If the system is operated in this mode a full scan over the spectrum of an analyte can be obtained. Alternatively multiple ion monitoring can be used for a few m/z values of interest and detection limits of a few picograms of organic compounds are possible. A major application of LC-MS is to identify proteins. The molecular mass of the protein is
LC-MS
To separate and detect less volatile, more polar, more labile or higher molecular mass compounds, the GC is replaced by a LC. A number of ion sources have been used for LC-MS but two dominate today: atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). In APCI, solvent and analytes eluting from the LC are sprayed into a heated tube at atmospheric pressure where they rapidly vaporize. Ions of the solvent are formed in a corona discharge. Typically in positive-ion mode these are protonated. These ions then transfer charge to analytes to produce molecular ions. In ESI the solution eluting from the LC is passed through a metal capillary that has a high voltage applied to it (30005000 V). Charged droplets emerge from the capillary tip at atmospheric pressure and lose solvent through evaporation, leading to the formation of gas phase ions characteristic of the ions in solution. Compounds that are present as simple ions in solution (M+, M− or MH+, MH−) give the same ion in the gas phase. Protein ions with molecular masses 5000100 000 acquire multiple charges to produce ions with m/z ratios of < 4000 (Figure 7) that can still be analysed by a quadrupole. Molecules with
Figure 7 The mass spectrum of the protein cytochrome c (Mr 12 200) obtained with an ESI ion source and quadrupole mass filter. The isotopic structure of the peaks is not resolved. Each peak is identified by the number of protons attached to the protein.
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1927
determined by ESI-MS. A proteolytic enzyme such as trypsin is then used to cleave the protein into peptides. These are separated by LC and their molecular masses determined. These molecular masses, the molecular mass of the protein and specifying the residues cleaved by the enzyme can be used to search libraries to identify an unknown protein. If this is insufficient, some sequence information on the peptides can be obtained by tandem MS (see below). Peptides often produce ions with two to four charges. Although quadrupoles are normally operated at unit resolution, sufficient to resolve peaks differing by one m/z, the resolution can be increased to separate the isotopic peaks of multiply charged ions. The spacing of these peaks allows the determination of the charge state directly (e.g. triply charged ions have isotopic peaks spaced by 0.33 m/z). Quadrupoles have demonstrated sufficient performance to resolve isotopic peaks of up to +4 ions in the range m/z < 2000. This is usually more than sufficient for peptide analysis. A higher mass range is required for LC-MS and this has pushed quadrupole performance to new limits. Current systems have an m/z range of 20004000. Triple quadrupole mass spectrometers (MS/MS)
In tandem mass spectrometry (MS/MS) a first mass analyser selects an ion from a mixture, the ion is fragmented by collision, and a second mass analyser produces a spectrum of the fragment ions. MS/MS is used to determine ion structure and to detect and quantify targeted compounds in complex mixtures. MS/MS can be carried out with a triple quadrupole system such as that shown in Figure 8. A first mass analysing quadrupole, Q1 mass selects a precursor ion from the ESI source. The ion enters the collision cell with energies typically 10500 eV. Here
collisions with a neutral gas such as N2 or Ar at a pressure 10−4 to 10−2 mbar transfer translational energy to the internal energy of the ion. It then undergoes unimolecular reaction to produce fragment or product ions (collision-induced dissociation). Ions are confined to the collision cell by a quadrupole, Q2, operated with only a radiofrequency voltage between the poles. A broad range of ions with q < 0.9 have stable trajectories and are transmitted to the exit of the collision cell. They are then mass analysed in quadrupole Q3. The system in Figure 8 shows an additional quadrupole Q0. This also operates in RF only mode and acts as an ion guide to transport ions from the skimmer to the first mass analyser. Early triple quadrupoles were unable to efficiently extract higher mass ions from Q2 and at the same time to attain unit resolution in Q3 because of high fragment ion energies. A solution to this problem was found when it was recognized that by increasing the pressure in the collision cell, product ions have additional collisions with neutrals. This causes them to lose radial and axial kinetic energy, i.e. to cool, and to move to the centre of the RF quadrupole. They are then well within the acceptance of Q3 and the transmission increases. In addition, product ions emerge from Q2 with energies and energy spreads of only about 1 eV. With a modern triple quadrupole system, at least 50% of the precursor ions that are transmitted by Q1 can be converted to fragment ions that are transmitted through Q3 with unit resolution or better. Resolution of the isotopic peaks of up to +4 ions has been demonstrated (Figure 9). The high collision pressure also minimizes any ion focusing effects which could lead to variable transmission. Hexapole or octopole fields have also been used to confine ions in the collision region. A triple quadrupole has many scan modes. In product ion scans, Q1 mass-selects an ion from a
Figure 8 A triple quadrupole mass spectrometer system with an electrospray ion source. S, electrospray source; N2, nitrogen curtain gas; O, ion sampling orifice; SK, skimmer; Q0, RF-only quadrupole ion guide; PF, delayed DC ramp ‘prefilters’; Q1 mass analysing quadrupole; Q2, RF quadrupole enclosed in a collision cell; Q3 mass analysing quadrupole; D, ion detector.
1928 QUADRUPOLES, USE OF IN MASS SPECTROMETRY
Figure 9 Mass spectra of multiply charged fragment ions of the peptide renin substrate tetradecapeptide. The precursor was the (M+4H+)4+ ion at m/z 440. The insets show the resolution of the isotopic peaks for the +1 to +4 ions. Reproduced with permission of The American Chemical Society from Thomson BA, Douglas DJ, Corr JJ, Hager JW, Jolliffe CL (1995) Analytical Chemistry 67: 1696– 1704.
mixture, it is fragmented in Q2 and a mass spectrum of product ions is obtained by scanning Q3. This scan mode is useful to obtain structural information of an ion such as sequence information of a peptide. In precursor ion scans, Q3 is fixed on a particular m/z value and Q1 scans through all the ions produced by the source. This scan is useful to identify those ions in the source that contain a particular functional group. In neutral loss scans, Q1 and Q3 scan together with a constant difference in m/z that corresponds to loss of a given neutral group. For example, if the loss corresponds to Cl2 (70 amu) this scan could identify ions that contain two or more chlorine atoms such as polychlorinated dioxins. For targeted compound analysis, the system can be run in single reaction monitoring mode. Here Q1 is fixed at the m/z of the precursor ion of a targeted compound and Q3 is set to a m/z value of a major fragment ion of that compound. There is a massive discrimination against other compounds. If still greater selectivity is required, multiple reaction monitoring can be done. Here Q1 is set to the m/z value of the precursor and Q3 peak hops to several (say four) m/z values of fragments that come from the targeted compound. If the intensity ratios of the fragments are correct the compound is identified. If there is an interference on one of the fragments, the remaining fragments can be used to identify and quantify the compound. The ability to independently
scan Q1 or Q3 under computer control makes a triple quadrupole MS/MS system a flexible tool for trace analysis. It has become the workhorse for LC-MS/MS and GC-MS/MS analysis. An example of the use of a triple quadrupole MS/ MS system to identify a protein is given here. The enzyme telomerase rebuilds the ends of chromosomes (telomeres) when cells divide. It consists of one RNA and two protein subunits of molecular mass 43 kDa and 123 kDa. The 123 kDa protein was separated on a 2D gel, extracted and digested to produce a mixture of peptides. Q1 of a triple quadrupole was scanned to produce a spectrum of all the peptides, shown in Figure 10A. To identify these peptides, Q1 was set to transmit a 2 m/z mass window and tandem mass spectra were collected with a 0.2 m/z step size. The fragment ion spectrum of a doubly charged peptide at m/z 830.4 is shown in Figure 10B. The doubly charged peptide ion fragments to singly charged ions to give fragments with m/z greater than the precursor. This mass spectrum along with esterification of the peptide allowed unambiguous assignment of the amino acid sequence. The sequences of eight of the peptides in the mass spectrum were determined. These amino acid sequences were then used to make DNA probes that led to identifying the gene containing the complete sequence of the protein. The total amount of
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1929
Figure 10 (A) Mass spectrum of the peptides obtained from digesting the 123 kDa subunit of telomerase. Peptides were not separated by chromatography. Peptides that were sequenced fully or partially are marked by T or t, respectively. (B) Tandem mass spectrum of the peptide at m/z 830.4. Reproduced with permission of The American Association for the Advancement of Science from Lingner J, Hughes TR, Shevchenko A, Mann M, Lundbald V, and Cech TR (1997) Science 276: 561–567.
protein available for this experiment was in the low picomole range. ICP-MS
Another major application area for quadrupoles is in ICP-MS systems for trace element analysis. A schematic of a system is shown in Figure 11. This ion source is an induction plasma in argon at atmospheric pressure with a temperature of 50007000 K contained in a torch. Samples are introduced to the plasma as aerosols, usually solutions that are sprayed. At the high plasma temperature dissolved solutes are vaporized, atomized and ionized. Most
Figure 11 A quadrupole ICP-MS system. T, torch; S, sampler; SK, skimmer; L, ion lenses; A, differential pumping aperture and lens; PF, delayed DC ramp ‘prefilter’; Q, quadrupole mass filter; D, ion detector.
elements of the Periodic Table are present in the plasma as singly charged atomic ions (the degree of ionization is typically 90% or more). The plasma expands through an orifice about 1 mm in diameter into a region at a pressure of a few mbar, and the centreline flow then passes through a skimmer into a region at a pressure of about 10− 4 torr. In this region ions are extracted from the rarefied plasma, pass through ion lenses and then are mass-analysed in a quadrupole. ICP-MS with a quadrupole gives simple mass spectra that are easy to interpret. Figure 12 for example shows the mass spectrum at unit resolution of some transition metals at a concentration of 100 ng mL1. By scanning a quadrupole over the range 7Li+ to 238U+, over 70 elements can be determined in a 1 min scan. Alternatively the quadrupole can peak hop to selected isotopes or elements, to improve the duty cycle. Detection limits are typically in the 10 pg mL1 region for elements in solution. Isotopic information is inherent in ICP-MS. With a quadrupole the precision on isotope ratios is typically 0.2% with a measurement time of a few minutes. This is insufficient for many geological dating applications but is more than adequate for many isotopic tracing experiments in nutrition and other studies. In addition, it greatly facilitates quantification of trace elements by isotope dilution. Ideally the ICP would produce only singly charged atomic ions of each element. However, it also
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Spectroscopy; Chemical Ionisation in Mass Spectrometry; Chromatography-MS, Methods; Hyphenated Techniques, Applications of in Mass Spectrometry; Inductively Coupled Plasma Mass Spectrometry, Methods; Mass Spectrometry, Historical Perspective; MS-MS and MSn; Photoacoustic Spectroscopy, Applications.
Further reading
Figure 12 Mass spectrum of transition metals each present at 100 ng ml–1 in solution. The peak at m/z 56 also includes a contribution from 40Ar16O+. Small peaks at m/z 63–68 indicate contamination by Cu and Zn at concentrations of few ng mL–1.
produces some molecular ions such as ArO+ which interfere with 56Fe+, or Ar2+ which interferes with 80Se+. Such interferences are most common at m/z < 80. To separate these interferences requires a resolution that is beyond the capabilities of quadrupoles operated conventionally, although the use of alternative stability regions is being investigated. The interferences are not prohibitive because in many cases an alternative isotope can be found that is free of interference. As with many applications it is the comparatively low cost and electronic control of quadrupoles that make them attractive for ICP-MS.
List of symbols a = 8zU/mZ2ro2; f = RF Frequency; m = mass of ion; n = number of RF cycles; q = 4zU/mZ2ro2; ro = separation between electrodes; Rlim = limiting resolution; Rmax = maximum resolution; t = time; u = x or y direction; U = direct voltage; V = voltage; z = charge of ion; [ = Zt/z; Z = zSf. See also: Atmospheric Pressure Ionization in Mass Spectrometry; Biomedical Applications of Atomic
Bruins AP (1994) Atmospheric pressure ionization mass spectrometry. Trends in Analytical Chemistry 13: 37 43; 8190. Busch KL, Glish GL and MacLuckey SA (1998) Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry. Weinheim: VCH. Cole RB (ed) (1997) Electrospray Ionization Mass Spectrometry. New York: Wiley. Dawson PH (ed) (1976) Quadrupole Mass Spectrometry and its Applications. Amsterdam: Elsevier: (Reissued as a paperback Dawson PH (ed) Quadrupole Mass Spectrometry and its Applications (1995) New York: American Institute of Physics Press. Dawson PH (1980) Mass filter design and performance. Advances in Electronics and Electron Physics 53: 153. Dawson PH and Bingqi Yu (1984) Performance comparison in conventional and higher stability regions. International Journal of Mass Spectrometry and Ion Processes 56: 41. Douglas DJ and Ying J-F (1996) High resolution ICP mass spectra with a quadrupole mass filter. Rapid Communications in Mass Spectrometry 10: 649652. Du Z, Olney TH and Douglas DJ (1997) Inductively coupled plasma mass spectrometry with a quadrupole operated in the third stability region. Journal of the American Society for Mass Spectrometry 8: 1230 1236. Heumann K (1982) Isotope dilution mass spectrometry for micro- and trace-element determination. Trends in Analytical Chemistry 1: 357361. Houk RS (1994) Elemental and isotopic analysis by inductively coupled plasma mass spectrometry. Accounts of Chemical Research 27: 333339. Paul W, Reinard HP and von Zahn U (1958) Zeitschrift für Physik 152: 143182. Yost RA and Enke CG (1979) Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation. Analytical Chemistry 51: 1251A1264A.
Q Quadrupoles, Use of in Mass Spectrometry PH Dawson, Iridian Spectreal Technologies Ltd., Ottawa, Ontario, Canada DJ Douglas, University of British Columbia, Vancouver, Canada
MASS SPECTROMETRY Methods & Instrumentation
Copyright © 1999 Academic Press
Quadrupole mass spectrometers (often referred to as quadrupole mass filters because of the way they operate) are the most successful example of the class of mass spectrometers called dynamic. Their performance depends upon a dynamic interaction of ions with time-varying electric fields. Most classes of spectrometers are static and use either the interaction of fixed magnetic and electric fields or time-offlight. The quadrupole mass spectrometer derives its name from the nature of the electric potential which is quadrupolar in the direction transverse to ion injection, i.e. dependent on the square of the distance from the centre of the field. The field is achieved by using four parallel rods as schematically illustrated in Figure 1. This article begins with a brief history. It then explains the principles of operation of this mass spectrometer. The idealized view of its operation has to be tempered by consideration of some real-world situations that influence performance, such as the finite length of the field, the inevitability of fringing fields and field imperfections. Observations of performance and its limitations are illustrated. The applications section deals with residual gas analysis, gas chromatography and liquid chromatography mass spectrometry (GC-MS and LC-MS), collision induced dissociation using triple quadrupoles (MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) used for elemental analysis.
A history of development The possibility of using quadrupole radiofrequency fields for mass analysis was first suggested in 1953
by Paul and Steinwedel and in a US government report by Post. The first practical implementation was by Paul and co-workers in 1958. This work became the foundation for the field. Wolfgang Paul was awarded a share of the Nobel Prize for physics in 1989 for the development of the ion trap technique that also used quadrupole fields. Early quadrupole mass filters were very limited in mass range and resolution but their physical simplicity and the absence of a magnet made them attractive for upper atmosphere and space applications. Major development occurred in the 1960s inspired by this application. This same period also coincided with a rising demand for residual gas analysers because ultrahigh vacuum technology began to have routine
Figure 1 A schematic illustration of a quadrupole mass spectrometer. Ions that are to be filtered to identify the presence of a particular mass are injected in the direction of the axis of the instrument. If the combination of radiofrequency and direct voltages applied to the rods is correctly chosen, only ions of one particular mass to charge ratio (m/z) will be successfully transmitted to the detector.
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application, first in research laboratories and later in production equipment, especially in semiconductor processing. Gradually the quadrupole mass filter became the dominant instrument for this application and remains so today. Quadrupole performance slowly improved but in the 1970s new applications to organic analysis and particularly the implementation of the combination of GC and MS placed increasing emphasis on better understanding of how the instruments worked in order to overcome their limitations. Computer simulations of performance became important. Combined with detailed experimental analysis, these led to a much improved knowledge of real-world quadrupoles with fringing fields and field imperfections. An important advance came with the application of phase space dynamics for calculating quadrupole performance. The new advances were incorporated in the classic textbook of the field written by Dawson and various collaborators and published in 1976. This book was re-issued by the American Institute of Physics in 1995 as a paper-back classic. In the 1980s and 1990s, the limits to quadrupole performance have been pushed back by precision manufacture and careful source design. A mass range of up to 2000 or more is commonly achieved with unit mass resolution. For LC-MS applications the mass range may reach 4000. There have been equally significant improvements in trace analysis capability based on a combination of sensitivity and more perfect peak shapes. High-performance quadrupole mass spectrometer manufacture has come to demand very high precision.
the applied voltages across the quadrupole are direct voltage U and an alternating radiofrequency voltage of V cos ω t and the minimum separation between opposite pairs of electrodes is 2ro. The charge to mass ratio of the ion is z/m. Time is expressed by ξ = ω t/2, where t is in seconds. For ion transmission the ions must have finite amplitude of oscillation in both x and y directions so that they do not strike the rods, i.e. the trajectories are both stable.
Principles of operation The perfect field
In a perfect quadrupole mass filter field, motion in the x and y (transverse) directions is independent. There is no field in the axial direction and motion is unchanged along the axis. Both x and y motion are governed by the Mathieu equation (see the Further reading section for the derivation of these equations);
i.e. where u represents x or y and where Figure 2 Zones for stable trajectories in both x and y transverse directions expressed in terms of the parameters a and q which are related to the m/z ratios of the ions. (A) General zones of stability, (B) a detail of the zone commonly used. The iso-beta lines are related to frequencies of ion oscillation.
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Combinations of a and q values that give stable motion are shown in Figure 2. There are several areas of simultaneous stability. The one near the origin is commonly used but the higher zones have been examined both theoretically and experimentally especially in the search for peak shapes with more abrupt fall-off at the edges for applications in trace analysis. The quadrupole uses a sinusoidal alternating field. In principle any alternating field is possible but the higher harmonics may lead to complexity in the behaviour of the ions. The RF frequency is generally in the range of 12 MHz. Mass selection is obtained by choosing a ratio of q/a such that only a narrow region of the stability zone near the apex (a = 0.23699 and q = 0.706) is intersected by the operating line using q/a = constant. For a given RF and DC voltage, ions of different mass to charge ratio (m/z) appear at different points along the line. Mass scanning is carried out by altering the values of U and V while maintaining their ratio constant to bring ions of different m/z into the tip of the stability region. This would give a spectrum of constant resolution (M/∆M). In practice a resolution that increases with mass is preferred and the ratio q/a is adjusted electronically throughout the mass scan in order to achieve more or less constant peak width. An alternative to voltage scanning would be to scan the frequency but this is rarely done because of technical difficulties in covering a large mass range. This simple description implicitly assumes that the length of the quadrupole is infinite so that all ions with stable trajectories are differentiated from those with unstable trajectories. In practice, resolution may be limited by the length of the field. It is found that Rmax = n2/h, where Rmax is the maximum attainable resolution and n is the number of RF cycles the ions spend in the field. The parameter h depends upon the source and on the fringing fields. A value of 25 is not unusual; a value of 10 would be an excellent performance. High resolution is favoured for ions of low axial velocity (lower energy ions but, fortunately, also higher mass ions). One is limited, however, in reducing axial ion energies by the detrimental influence of fringing fields.
is to assume a linear increase of the x and y direction fields as the entrance is approached. The fringing fields extend over a distance comparable to the filter radius. The y-direction (a,q) values in the fringing field correspond to intrinsically unstable ion trajectories. If too much time is spent in the fringing fields, ion amplitudes will increase and the effective aperture of the spectrometer will be reduced. The finite diameter of the field (ro) means that ions are only accepted for transmission when they enter the field with a small initial transverse displacement from the axis and small transverse velocities. The combination of displacement and transverse velocities that are possible defines the acceptance of the instrument. The acceptance and so the overall sensitivity becomes smaller as the resolution is increased. The acceptance is calculated using phase space dynamics. The sensitivity of the mass spectrometer is best expressed in terms of a phase space diagram as shown in Figure 3. This shows the initial combinations of
The real world: transmission and fringing fields
In the real world, there are inevitably fringing fields at the entrance to and the exit from the mass filter. Ion motion in these fields can be very complex and the motion in each of the three coordinate directions becomes coupled. Very low energy ions may be reflected or even trapped in the fringing fields. However, a good theoretical approximation in most cases
Figure 3 Phase space acceptance diagrams showing the initial conditions of transverse displacement and velocities that lead to ion transmission at 100% and 50% of the initial phases of the RF field. (A) x-direction, (b) y-direction. This is for the centre of a peak and for ions spending two RF cycles passing through the fringing fields. The displacement from the axis is measured in units of r0 and the velocity in terms of r0 /ξ.
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transverse position and transverse velocity that will result in ion transmission. In practice, this depends upon the initial phase of the field when the ion first experiences the field. The Figure shows the x and y acceptances for transmission in 100% of the initial phases, 50%, and so on. At higher resolutions the acceptance area decreases. At a given resolution, combining the x and y acceptances together gives an indication of sensitivity for different numbers of RF cycles spent in the fringing fields giving a diagram such as that in Figure 4. This shows that fringing fields can be advantageous if properly chosen. There have been many attempts to tailor fringing fields for optimum performance, such as using retardation of ions after they enter the field or by adding RF only sections at the ion entry (the delayed DC ramp), or by using specially shaped electrodes. If the relative ion transmission is measured versus resolution for a particular mass number, there will be a curve such as that in Figure 5. If the source is
Figure 4 Combined phase space acceptance areas for x and y directions as function of the length of the fringing field (expressed as the number of RF cycles that the ions spend within the fringing field). This illustrates how sensitivity may vary depending on ion velocity.
evenly illuminated, at low resolution the source emittance will be less than the quadrupole acceptance and the transmission will vary little with resolution. Also the peaks will tend to be flat topped. At some point the acceptance will become limiting and transmission will tend to fall with the square of the resolution (taking fringing fields into account). Finally, at the length limitation of the quadrupole the transmission drops abruptly with resolution. Curves (a) and (b) illustrate different ion energies. In some cases, the source emittance may be rather diffuse or may not be evenly illuminated, then quite different transmission versus resolution behaviour will be observed. The real world: field imperfections
There are inevitably other departures from the perfect fields and these become more and more important at high resolution. Displacement of one or more rods from the ideal position is the simplest case. This leads to higher order terms in the expression for the electric potential. One rod displaced would give predominantly third-order or hexapole correction terms. Opposite rods displaced gives fourth-order or octopole terms. These imperfections limit the resolution that is attainable, i.e. even if n is increased, the resolution will not increase further. There have been various theoretical and more limited experimental studies of these effects. The field faults may also cause badly shaped or even split peaks because of nonlinear resonances in the ion oscillations at certain critical (a, q) values.
Figure 5 A typical example of transmission efficiency versus resolution for an instrument with a well-defined source emittance. There are three regions of transmittance as resolution is increased. (I) Sensitivity is source limited, (II) sensitivity is filter acceptance limited, (III) resolution is length limited (number of cycles in the field) and (IV) resolution is limited by field imperfection.
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Other field faults may arise from nonparallel rods (bending or bowing) or from errors in the electrical waveform. It is common to substitute round rods in the quadrupole for the ideal hyperbolic ones. Round rods are easier to precision-manufacture. If the diameter and positioning of the rods is correctly matched, field faults are minimized and only sixth-order distortions are produced. These are not expected to significantly influence performance. However, there is controversy over this choice of round and hyperbolic rods with many assertions being made but little solid experimental evidence. One confusing factor is that other field faults (e.g. from rod positioning) may be more serious when using round rods. Observations of limits to performance
Figure 6 illustrates some of the data from an extensive examination of performance of a particular quadrupole. These data demonstrate most of the features discussed above. In addition, there is an ultimate limit to achievable resolution set by the perfection of the quadrupole field. Similar performance data have been generated for operation using other regions of the stability diagram such as near a = 0, q = 7.5 or a = 3, q = 3. These regions may have interest for specialized application.
Applications Residual gas analysis
One of the first uses of quadrupole mass filters was for residual gas analysis in high-vacuum chambers. An electron beam ionizes the background gas and a mass spectrum is recorded with the quadrupole. This allows determination of the composition of the background gas and, after calibration, the partial pressures of each of the components, such as N2, O2, H2O, etc. Because only light gases are generally involved, the mass range of the quadrupole need only be 100200 m/z. The compact and rugged construction of a quadrupole with purely electronic scanning make quadrupoles particularly attractive for this application. Often the quadrupole is mounted on a flange which is simply bolted onto the vacuum chamber. Sometimes a pressure reduction stage is used. Modern systems have computer controlled scanning and allow the possibility of searching libraries to match an unknown spectrum. GC-MS
The largest fraction of quadrupole mass spectrometers sold today are used as detectors for GC to identify
Figure 6 Some examples of performance measurement for a particular quadrupole mass filter showing how the limiting resolution varied with the number of RF cycles in the field. The ultimate resolution reached was dependent on frequency and mass number.
and quantify trace levels of organic compounds. Environmental analysis and drug testing of athletes, for example, rely extensively on GC-MS. Organic compounds separated by a gas chromatograph elute into the ion source of a quadrupole mass filter. Ions are formed either by electron impact (EI) or chemical ionization (CI). In EI, positive molecular and fragment ions are usually formed. The resulting mass spectrum gives a fingerprint of the compound. Unknown compounds can be identified by searching a library of spectra. In CI, analytes react with a reagent ion present in excess to produce either positive or
1926 QUADRUPOLES, USE OF IN MASS SPECTROMETRY
negative molecular adduct ions, usually with minimal fragmentation. The combination of the retention time on the chromatograph and the appropriate molecular mass is often sufficient to identify trace analytes. Quadrupole mass filters have become the standard for GC-MS because they are easily interfaced to computers, scan rapidly on a time scale compatible with peaks eluting from a GC, require only medium vacuum (105 mbar), are compact, and are of comparatively low cost. Gas chromatography is restricted to relatively volatile compounds with moderate molecular masses and so the m/z range of a quadrupole used as a detector for GC is usually ∼5001000. A complete EI spectrum can be obtained on ∼10 pg of an analyte. If only ions of one m/z are monitored (single ion monitoring), with CI, the detection limits can be lowered to low femtogram levels. Alternatively a few selected m/z values (say four) corresponding to the major peaks in the spectrum of a targeted compound can be monitored by switching the quadrupole between these m/z values without scanning intervening regions (multiple ion monitoring). The ability to switch a quadrupole from full scans to single ion monitoring to improve detection limits is an advantage over other methods where a complete spectrum must be acquired (TOF, ion trap ICR). A quadrupole system dedicated to GC/MS can be quite compact, often smaller than the GC itself.
intermediate molecular masses produce ions with a few charges, depending on the number of basic residues and their pKa values. Conventional ESI operates best at flow rates of 1−10 µL min−1. LC flow rates are usually considerably higher (∼ 1 mL min−1) so the output of the LC is often split, with a fraction of the flow going to the ESI source. APCI and ESI produce ions at atmospheric pressure. These are transferred into the vacuum system of a quadrupole mass filter with two or more stages of differential pumping. The ability to operate a quadrupole at moderate pressures of ∼ 10−5 torr means only modest, lower cost vacuum pumps are required. ESI and APCI generally produce protonated molecular ions. As in GC-MS, for targeted compound analysis the quadrupole is operated in single ion mode to monitor the m/z of interest. Fragment ions can be formed by applying high electric fields to the ions in the ion sampling region. These fields accelerate the ions through the locally formed high density of gas causing collision-induced dissociation. If the system is operated in this mode a full scan over the spectrum of an analyte can be obtained. Alternatively multiple ion monitoring can be used for a few m/z values of interest and detection limits of a few picograms of organic compounds are possible. A major application of LC-MS is to identify proteins. The molecular mass of the protein is
LC-MS
To separate and detect less volatile, more polar, more labile or higher molecular mass compounds, the GC is replaced by a LC. A number of ion sources have been used for LC-MS but two dominate today: atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). In APCI, solvent and analytes eluting from the LC are sprayed into a heated tube at atmospheric pressure where they rapidly vaporize. Ions of the solvent are formed in a corona discharge. Typically in positive-ion mode these are protonated. These ions then transfer charge to analytes to produce molecular ions. In ESI the solution eluting from the LC is passed through a metal capillary that has a high voltage applied to it (30005000 V). Charged droplets emerge from the capillary tip at atmospheric pressure and lose solvent through evaporation, leading to the formation of gas phase ions characteristic of the ions in solution. Compounds that are present as simple ions in solution (M+, M− or MH+, MH−) give the same ion in the gas phase. Protein ions with molecular masses 5000100 000 acquire multiple charges to produce ions with m/z ratios of < 4000 (Figure 7) that can still be analysed by a quadrupole. Molecules with
Figure 7 The mass spectrum of the protein cytochrome c (Mr 12 200) obtained with an ESI ion source and quadrupole mass filter. The isotopic structure of the peaks is not resolved. Each peak is identified by the number of protons attached to the protein.
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1927
determined by ESI-MS. A proteolytic enzyme such as trypsin is then used to cleave the protein into peptides. These are separated by LC and their molecular masses determined. These molecular masses, the molecular mass of the protein and specifying the residues cleaved by the enzyme can be used to search libraries to identify an unknown protein. If this is insufficient, some sequence information on the peptides can be obtained by tandem MS (see below). Peptides often produce ions with two to four charges. Although quadrupoles are normally operated at unit resolution, sufficient to resolve peaks differing by one m/z, the resolution can be increased to separate the isotopic peaks of multiply charged ions. The spacing of these peaks allows the determination of the charge state directly (e.g. triply charged ions have isotopic peaks spaced by 0.33 m/z). Quadrupoles have demonstrated sufficient performance to resolve isotopic peaks of up to +4 ions in the range m/z < 2000. This is usually more than sufficient for peptide analysis. A higher mass range is required for LC-MS and this has pushed quadrupole performance to new limits. Current systems have an m/z range of 20004000. Triple quadrupole mass spectrometers (MS/MS)
In tandem mass spectrometry (MS/MS) a first mass analyser selects an ion from a mixture, the ion is fragmented by collision, and a second mass analyser produces a spectrum of the fragment ions. MS/MS is used to determine ion structure and to detect and quantify targeted compounds in complex mixtures. MS/MS can be carried out with a triple quadrupole system such as that shown in Figure 8. A first mass analysing quadrupole, Q1 mass selects a precursor ion from the ESI source. The ion enters the collision cell with energies typically 10500 eV. Here
collisions with a neutral gas such as N2 or Ar at a pressure 10−4 to 10−2 mbar transfer translational energy to the internal energy of the ion. It then undergoes unimolecular reaction to produce fragment or product ions (collision-induced dissociation). Ions are confined to the collision cell by a quadrupole, Q2, operated with only a radiofrequency voltage between the poles. A broad range of ions with q < 0.9 have stable trajectories and are transmitted to the exit of the collision cell. They are then mass analysed in quadrupole Q3. The system in Figure 8 shows an additional quadrupole Q0. This also operates in RF only mode and acts as an ion guide to transport ions from the skimmer to the first mass analyser. Early triple quadrupoles were unable to efficiently extract higher mass ions from Q2 and at the same time to attain unit resolution in Q3 because of high fragment ion energies. A solution to this problem was found when it was recognized that by increasing the pressure in the collision cell, product ions have additional collisions with neutrals. This causes them to lose radial and axial kinetic energy, i.e. to cool, and to move to the centre of the RF quadrupole. They are then well within the acceptance of Q3 and the transmission increases. In addition, product ions emerge from Q2 with energies and energy spreads of only about 1 eV. With a modern triple quadrupole system, at least 50% of the precursor ions that are transmitted by Q1 can be converted to fragment ions that are transmitted through Q3 with unit resolution or better. Resolution of the isotopic peaks of up to +4 ions has been demonstrated (Figure 9). The high collision pressure also minimizes any ion focusing effects which could lead to variable transmission. Hexapole or octopole fields have also been used to confine ions in the collision region. A triple quadrupole has many scan modes. In product ion scans, Q1 mass-selects an ion from a
Figure 8 A triple quadrupole mass spectrometer system with an electrospray ion source. S, electrospray source; N2, nitrogen curtain gas; O, ion sampling orifice; SK, skimmer; Q0, RF-only quadrupole ion guide; PF, delayed DC ramp ‘prefilters’; Q1 mass analysing quadrupole; Q2, RF quadrupole enclosed in a collision cell; Q3 mass analysing quadrupole; D, ion detector.
1928 QUADRUPOLES, USE OF IN MASS SPECTROMETRY
Figure 9 Mass spectra of multiply charged fragment ions of the peptide renin substrate tetradecapeptide. The precursor was the (M+4H+)4+ ion at m/z 440. The insets show the resolution of the isotopic peaks for the +1 to +4 ions. Reproduced with permission of The American Chemical Society from Thomson BA, Douglas DJ, Corr JJ, Hager JW, Jolliffe CL (1995) Analytical Chemistry 67: 1696– 1704.
mixture, it is fragmented in Q2 and a mass spectrum of product ions is obtained by scanning Q3. This scan mode is useful to obtain structural information of an ion such as sequence information of a peptide. In precursor ion scans, Q3 is fixed on a particular m/z value and Q1 scans through all the ions produced by the source. This scan is useful to identify those ions in the source that contain a particular functional group. In neutral loss scans, Q1 and Q3 scan together with a constant difference in m/z that corresponds to loss of a given neutral group. For example, if the loss corresponds to Cl2 (70 amu) this scan could identify ions that contain two or more chlorine atoms such as polychlorinated dioxins. For targeted compound analysis, the system can be run in single reaction monitoring mode. Here Q1 is fixed at the m/z of the precursor ion of a targeted compound and Q3 is set to a m/z value of a major fragment ion of that compound. There is a massive discrimination against other compounds. If still greater selectivity is required, multiple reaction monitoring can be done. Here Q1 is set to the m/z value of the precursor and Q3 peak hops to several (say four) m/z values of fragments that come from the targeted compound. If the intensity ratios of the fragments are correct the compound is identified. If there is an interference on one of the fragments, the remaining fragments can be used to identify and quantify the compound. The ability to independently
scan Q1 or Q3 under computer control makes a triple quadrupole MS/MS system a flexible tool for trace analysis. It has become the workhorse for LC-MS/MS and GC-MS/MS analysis. An example of the use of a triple quadrupole MS/ MS system to identify a protein is given here. The enzyme telomerase rebuilds the ends of chromosomes (telomeres) when cells divide. It consists of one RNA and two protein subunits of molecular mass 43 kDa and 123 kDa. The 123 kDa protein was separated on a 2D gel, extracted and digested to produce a mixture of peptides. Q1 of a triple quadrupole was scanned to produce a spectrum of all the peptides, shown in Figure 10A. To identify these peptides, Q1 was set to transmit a 2 m/z mass window and tandem mass spectra were collected with a 0.2 m/z step size. The fragment ion spectrum of a doubly charged peptide at m/z 830.4 is shown in Figure 10B. The doubly charged peptide ion fragments to singly charged ions to give fragments with m/z greater than the precursor. This mass spectrum along with esterification of the peptide allowed unambiguous assignment of the amino acid sequence. The sequences of eight of the peptides in the mass spectrum were determined. These amino acid sequences were then used to make DNA probes that led to identifying the gene containing the complete sequence of the protein. The total amount of
QUADRUPOLES, USE OF IN MASS SPECTROMETRY 1929
Figure 10 (A) Mass spectrum of the peptides obtained from digesting the 123 kDa subunit of telomerase. Peptides were not separated by chromatography. Peptides that were sequenced fully or partially are marked by T or t, respectively. (B) Tandem mass spectrum of the peptide at m/z 830.4. Reproduced with permission of The American Association for the Advancement of Science from Lingner J, Hughes TR, Shevchenko A, Mann M, Lundbald V, and Cech TR (1997) Science 276: 561–567.
protein available for this experiment was in the low picomole range. ICP-MS
Another major application area for quadrupoles is in ICP-MS systems for trace element analysis. A schematic of a system is shown in Figure 11. This ion source is an induction plasma in argon at atmospheric pressure with a temperature of 50007000 K contained in a torch. Samples are introduced to the plasma as aerosols, usually solutions that are sprayed. At the high plasma temperature dissolved solutes are vaporized, atomized and ionized. Most
Figure 11 A quadrupole ICP-MS system. T, torch; S, sampler; SK, skimmer; L, ion lenses; A, differential pumping aperture and lens; PF, delayed DC ramp ‘prefilter’; Q, quadrupole mass filter; D, ion detector.
elements of the Periodic Table are present in the plasma as singly charged atomic ions (the degree of ionization is typically 90% or more). The plasma expands through an orifice about 1 mm in diameter into a region at a pressure of a few mbar, and the centreline flow then passes through a skimmer into a region at a pressure of about 10− 4 torr. In this region ions are extracted from the rarefied plasma, pass through ion lenses and then are mass-analysed in a quadrupole. ICP-MS with a quadrupole gives simple mass spectra that are easy to interpret. Figure 12 for example shows the mass spectrum at unit resolution of some transition metals at a concentration of 100 ng mL1. By scanning a quadrupole over the range 7Li+ to 238U+, over 70 elements can be determined in a 1 min scan. Alternatively the quadrupole can peak hop to selected isotopes or elements, to improve the duty cycle. Detection limits are typically in the 10 pg mL1 region for elements in solution. Isotopic information is inherent in ICP-MS. With a quadrupole the precision on isotope ratios is typically 0.2% with a measurement time of a few minutes. This is insufficient for many geological dating applications but is more than adequate for many isotopic tracing experiments in nutrition and other studies. In addition, it greatly facilitates quantification of trace elements by isotope dilution. Ideally the ICP would produce only singly charged atomic ions of each element. However, it also
1930 QUADRUPOLES, USE OF IN MASS SPECTROMETRY
Spectroscopy; Chemical Ionisation in Mass Spectrometry; Chromatography-MS, Methods; Hyphenated Techniques, Applications of in Mass Spectrometry; Inductively Coupled Plasma Mass Spectrometry, Methods; Mass Spectrometry, Historical Perspective; MS-MS and MSn; Photoacoustic Spectroscopy, Applications.
Further reading
Figure 12 Mass spectrum of transition metals each present at 100 ng ml–1 in solution. The peak at m/z 56 also includes a contribution from 40Ar16O+. Small peaks at m/z 63–68 indicate contamination by Cu and Zn at concentrations of few ng mL–1.
produces some molecular ions such as ArO+ which interfere with 56Fe+, or Ar2+ which interferes with 80Se+. Such interferences are most common at m/z < 80. To separate these interferences requires a resolution that is beyond the capabilities of quadrupoles operated conventionally, although the use of alternative stability regions is being investigated. The interferences are not prohibitive because in many cases an alternative isotope can be found that is free of interference. As with many applications it is the comparatively low cost and electronic control of quadrupoles that make them attractive for ICP-MS.
List of symbols a = 8zU/mZ2ro2; f = RF Frequency; m = mass of ion; n = number of RF cycles; q = 4zU/mZ2ro2; ro = separation between electrodes; Rlim = limiting resolution; Rmax = maximum resolution; t = time; u = x or y direction; U = direct voltage; V = voltage; z = charge of ion; [ = Zt/z; Z = zSf. See also: Atmospheric Pressure Ionization in Mass Spectrometry; Biomedical Applications of Atomic
Bruins AP (1994) Atmospheric pressure ionization mass spectrometry. Trends in Analytical Chemistry 13: 37 43; 8190. Busch KL, Glish GL and MacLuckey SA (1998) Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry. Weinheim: VCH. Cole RB (ed) (1997) Electrospray Ionization Mass Spectrometry. New York: Wiley. Dawson PH (ed) (1976) Quadrupole Mass Spectrometry and its Applications. Amsterdam: Elsevier: (Reissued as a paperback Dawson PH (ed) Quadrupole Mass Spectrometry and its Applications (1995) New York: American Institute of Physics Press. Dawson PH (1980) Mass filter design and performance. Advances in Electronics and Electron Physics 53: 153. Dawson PH and Bingqi Yu (1984) Performance comparison in conventional and higher stability regions. International Journal of Mass Spectrometry and Ion Processes 56: 41. Douglas DJ and Ying J-F (1996) High resolution ICP mass spectra with a quadrupole mass filter. Rapid Communications in Mass Spectrometry 10: 649652. Du Z, Olney TH and Douglas DJ (1997) Inductively coupled plasma mass spectrometry with a quadrupole operated in the third stability region. Journal of the American Society for Mass Spectrometry 8: 1230 1236. Heumann K (1982) Isotope dilution mass spectrometry for micro- and trace-element determination. Trends in Analytical Chemistry 1: 357361. Houk RS (1994) Elemental and isotopic analysis by inductively coupled plasma mass spectrometry. Accounts of Chemical Research 27: 333339. Paul W, Reinard HP and von Zahn U (1958) Zeitschrift für Physik 152: 143182. Yost RA and Enke CG (1979) Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation. Analytical Chemistry 51: 1251A1264A.