MASS SPECTROMETRY / Mass Separation 397 Dong X, Gusev A, and Hercules DM (1998) Characterization of polysiloxanes with different functional

groups by time-of-flight secondary ion mass spectrometry. Journal of the American Society for Mass Spectrometry 9: 292–298. Farmer TB and Caprioli RM (1998) Determination of protein–protein interactions by matrix-assisted laser desorption/ionization mass spectrometry. Journal of Mass Spectrometry 33: 697–704. Festag R, Alexandratos SD, Joy DC, Wunderlich B, Annis B, and Cook KD (1998) Effects of molecular entanglements during electrospray of high molecular weight polymers. Journal of the American Society for Mass Spectrometry 9: 299–304. Harvey DJ (1999) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrometry Reviews 18: 349–451. Jackson AT, Yates HT, Scrivens JH, Green MR, and Bateman RH (1998) Matrix-assisted laser desorption/ionization-collision induced dissociation of poly(styrene). Journal of the American Society for Mass Spectrometry 9: 269–274. Merchant M and Weinberger SR (2000) Recent advancements in surface-enhanced laser desorption/ionizationtime of flight-mass spectrometry. Electrophoresis 21: 1164–1167. Montaudo G and Lattimer RP (eds.) (2002) Mass Spectrometry of Polymers. Boca Raton, FL: CRC Press. Parees DM, Hanton SD, Cornelio Clark PA, and Willcox DA (1998) Comparison of mass spectrometric techniques for generating molecular weight information on a class of ethoxylated oligomers. Journal of the American Society for Mass Spectrometry 9: 282–291. Tang N, Tornatore P, and Weinberger SR (2004) Current developments in SELDI affinity technology. Mass Spectrometry Reviews 23: 24–44. Zhu H, Yalcin T, and Liang L (1998) Analysis of the accuracy of determining average molecular weights of narrow polydispersity polymers by matrix-assisted laser desorption ionization time-of-flight mass sepctrometry. Journal of the American Society for Mass Spectrometry 9: 275–281.

Mass Separation
K J Welham, University of Hull, Hull, UK
& 2005, Elsevier Ltd. All Rights Reserved.

preferred method of mass analysis depends on the type of problem being investigated.

A mass spectrometer can be broken down into four basic components: sample introduction, ionization, mass separation, and detection/recording. This article will concentrate on mass separation, or ‘analysis’, although the components should not be treated in complete isolation as there are interactions between them. The modes of mass analysis that will be considered here can be classified as follows:

Types of Mass Analyzer
Magnetic Sectors



Continuous mode: * magnetic sectors, possibly with electric sectors * quadrupole mass filters Pulsed mode: * time-of-flight (TOF) analyzers Ion trapping devices: * quadrupole traps * Fourier-transform ion cyclotron resonance traps.

Magnetic sector instruments are relatively expensive but they generally give high performance when combined with electric sectors, especially in terms of resolution, mass measurement accuracy, sensitivity, and m/z range. They employ high accelerating potentials (several kilovolts) in comparison with quadrupole instruments. An ion of mass m and charge ze accelerated out of an ion source through potential V will acquire a velocity v, and under the influence of a magnetic field of intensity B will follow a circular path of radius r. The following equations apply:
Ion kinetic energy: mv2 =2 ¼ zeV Deflecting force: BzeV ¼ centrifugal force ¼ mv2 =r Combining these: m=z ¼ B2 r2 e=2V ½2Š ½3Š ½1Š

These various methods differ widely in cost, size, and complexity, and also in terms of performance (i.e., mass/charge range and resolving power). The

The number of charges, z, is usually equal to one, so eqn [3] shows that a spectrum of masses can be obtained by changing one of the three variables, B, r,

for example. Another potential source of loss of resolution is the angular broadening of the beam as it enters the magnetic field. i.  Change of radius.398 MASS SPECTROMETRY / Mass Separation Mass separation B S Angular focusing C Refocusing of single m/z value at single energy S Energy separation Figure 1 The variables for separation by a magnetic analyzer. path actually depends on the product of mass and velocity mv (momentum). reproducible single peak display or multiple peak monitoring. the ions rely on the kinetic energy imparted by the potential for effective transmission through the instrument. This is described by eqn [4] for an electric field of strength E: Deflecting force zeE¼ centrifugal force ¼ mv2 =r ½4Š Therefore.  Voltage scanning. different energies or V. and the reproducibility of the scan function as there is no hysteresis.. same energy r C Single m/z value. This method was used historically with photoplate detection. The field must be homogeneous and the ion beam must enter and leave the magnet at 901. in addition. as illustrated in Figure 1. Many commercial instruments combine different scan modes. although an electric sector has properties different from a magnetic sector. V: No theoretical limit to the mass range as the mass is inversely proportional to the accelerating potential. thereby reducing the resolution. The disadvantages of hysteresis and slow scanning associated with electromagnets have largely been overcome with modern laminated and/or air core magnet designs. B: Most commonly used. as in the case of voltage scanning (see below). and hence the performance deteriorates at high mass. High-performance magnetic scanning instruments are available with array detectors for increased sensitivity of detection over a restricted mass range (r). V Different m/z values. and as long as the source slit and collector slit are on a line passing through the center of the circle where the ion path through the magnet is an arc. although special applications such as accurate isotope ratio measurements use two or more fixed detectors. However. fast scanning. Advantages are the relatively low cost of permanent magnets (which do not. Ions with higher kinetic energy are deflected less than . and has been revived with microchannel plate photodiode array detectors. in practice the extraction efficiency of ions from the source depends on V. and is the reverse of the original design by Nier and Johnson. This example has ‘reversed’ geometry in that the magnet comes before the electric sector. provides wide mass range without loss of performance at high mass. A wedge-shaped magnet has a focusing action. independent of mass. using an electromagnet for normal scanning (B). Commercial instruments have been produced for each of these modes of scanning:  Magnetic scanning. and voltage scanning (V) for rapid. Ions of a common mass accelerated through the same potential V should all have the same momentum. Figure 3 shows the double focusing effect of a combination of a magnetic sector and an electric sector for a beam of ions of common mass but being both angularly and energetically divergent. This does not affect the focusing properties. r: Not possible with a fixed detector. require electronic circuits). of course. deflection being dependent upon the kinetic energy of the ions rather than the momentum. Angular and energy focusing are illustrated in Figure 2. Dividing eqn [2] by v shows that a magnetic sector is not a true mass analyzer as the radius of the ion S C Figure 2 The focusing properties of a magnetic sector. Double Focusing Magnetic Sector Instruments Ions can also be deflected by electric fields.e. this can improve signal-to-noise ratio as all the ions of interest can be monitored simultaneously and hence continuously. but in practice this will not be entirely true and a single-focusing magnetic sector instrument will give a broadened image at the collector slit. a divergent beam will be focused at the collector slit. the electric field can be used as a focusing element but does not need to be scanned to pass the full range of masses.

thereby bringing the energetically divergent ions back to a focus at the collector slit. There are other definitions that include 50% peak height. but at mass 10 000 it can only separate peaks differing by 10 Da. but the ability to separate peaks that differ by a constant amount deteriorates at high mass. although at reduced magnetic field. A high-resolution mass spectrometer allows the accurate determination of ionic masses to within B1 ppm. above which point the decreasing accuracy of mass measurement (as explained above) and the increasing number of possible compositions at each mass make the technique less useful. to give the familiar broad and rather weak metastable peaks in the normal mass spectra.031300). For a sector instrument the resolving power M/DM is constant throughout the mass range. hence the name double focusing. ions that fragment in the flight tube lose a fraction of their kinetic energy. In a reversed geometry instrument the electric sector will act as a barrier to these ions. or C2H4 (28. there can be other reasons for adopting a particular configuration. e. which is partitioned between the new ion and neutral fragment. This is illustrated in Figure 4.006148). giving pure metastable spectra. Thus. However. The choice of forward or reversed geometry for a double focusing mass spectrometer is not important for the attainment of high resolution.e. C those with lower kinetic energy. ions at þ m/z 28 can be CO þ (27. full-width at halfmaximum.MASS SPECTROMETRY / Mass Separation 399 100% E + B − 10% 5% M M+∆M Figure 4 Ten percent valley definition of resolving power. If these peaks differ in mass by one part in 60 000. the b-slit is usually quite wide for maximum transmission.g. which is the sort of performance that can be attained from a large modern double focusing instrument.994914). Ions that are accelerated out of the ion source by field potential V all have essentially the same kinetic energy and are transmitted by the electric sector at its ‘normal’ strength. The design affords a means of compensating for the spread of kinetic energies in the ion beam and allows high mass resolution to be attained by using narrow source and collector slits. especially for studies of metastable ions and tandem mass spectrometry. þ þ CH2N (28. and they can only pass through the electric sector at a reduced field strength. For both geometries it is possible to scan both sectors simultaneously in a ‘linked scan’. so that although the angularly divergent beams are brought to focus at the b-slit (which is equivalent to the collector slit in a single-focusing instrument). The resolving power required to separate a peak due to ions of mass M from a peak of mass M þ DM is defined as M/DM. e.g. which provides low-resolution tandem mass spectrometry. Consequently. However.. N2 (28. The mass defects in the atoms that make up organic molecules allow the atomic composition to be determined from the accurate mass.. Two peaks of equal height are defined as being resolved when the valley between them does not exceed 10% of the peak height (10% valley definition). However.. an instrument of resolving power 1000 can separate peaks differing in mass by less than 1 Da up to mass 1000. This works well up to Bm/z 500. the normal mass spectra show no metastable peaks. which for symmetrical peaks corresponds to defining the resolution relative to the peak width at 5% of the height. the resolving power needed will be 60 000. . i. The geometry of the instrument is accurately calculated so that the angular and energy foci coincide at the collector slit. S Figure 3 Double focusing mass spectrometer. which will not have sufficient energy for transmission. by reducing the electric field the ‘normal’ ions can be discriminated against and only the ionic products from the metastable fragmentations will be transmitted.018724). the spread of energies has broadened the beam and it cannot pass through a narrow slit without loss of sensitivity. Metastable ions breaking down in the second field-free region between the two sectors will be transmitted by the magnet in a forward geometry instrument. In the electric sector the higher energy ions take a longer path through the electric field and they are deflected more than the lower energy ions.

The maximum resolving power was B140. and shows the ions penetrating into an electric field and being repelled or reflected back out again. so that ions are introduced into the mass analyzer in bursts. and the masses being calculated from the flight times. Figure 7 shows the quadrupole analyzer. but circular rods are Reflector Drift region Detector Ion source 0V 0 − 270 V (Pulsed) −2. whereby all ions are formed in a very short space of time and can therefore start their flight through the drift tube simultaneously. This compensates for the shorter time they spend traversing the field-free region. In the original design shown in Figure 5. . the unrestricted mass range has been exploited in matrix-assisted laser desorption TOF mass spectrometers for protein analysis at masses up to 1 000 000 Da. More recently. the quadrupole analyzer is a true mass analyzer as it is unaffected by changes in ion velocity. Modern electronics provide much greater accuracy in gating and timing. ease of computer interfacing and mass calibration (as they have a linear scan law). and so eliminates the broadening associated with the spread of kinetic energies. the surfaces of which should actually be hyperbolic. simplicity.e. but they offer the advantages of flexibility. The development of reflectron instruments to compensate for differences in kinetic energies. ease of interfacing to liquid chromatography and gas chromatography (as they do not use high potentials in the ion source).400 MASS SPECTROMETRY / Mass Separation Time-of-Flight Analyzers TOF analyzers have recently enjoyed a considerable resurgence with the introduction of desorption ionization techniques such as laser desorption. the ions are formed in an electron impact source in which the electron beam is pulsed by applying a square wave voltage to a control grid. can respond extremely rapidly to changes in the sample composition as a spectrum can be recorded in milliseconds. in momentum or kinetic energy. 2. comparable to the performance of sector instruments. and therefore follow longer path lengths requiring longer times. The output. With these improvements it is possible to obtain resolving powers of 20 000 with TOF instruments. The acceleration of the ions into the flight tube is similarly pulsed. This is illustrated in Figure 6. lower-performance instruments. The major problem with early designs was associated with poor resolution. so that flight times can be recorded with nanosecond precision. Ud + Urefl Figure 5 TOF mass spectrometer. Quadrupole Analyzers Control grid Drift region 1m Quadrupole mass spectrometers are generally lessexpensive. The higher energy ions penetrate further into the field before being reflected. However. This has been further enhanced by the use of a gating voltage to delay the ion extraction and has given rise to the current high resolution (up to 20 000) for modern TOF instruments. which consists of four parallel rods. The pulsed nature of ion formation using techniques such as laser desorption. arising from the length of the gating pulses and the spread of kinetic energies due to thermal energies and local differences in the accelerating field.. 3. Improvements in resolving power have come from several developments: 1. the magnetic sector is actually a momentum analyzer and the electric sector is a kinetic energy analyzer. although both can give mass analysis under certain conditions. the lighter ions reaching the collector before the heavier ones. which in turn restricted the useful mass range although in theory the mass range of a TOF instrument is infinite. which is stored in a fast digitizer and sent onto the control computer for display. not capable of high resolution. As explained above. leading to time-focusing.8 kV 0V Multiplier Slow Fast Source U (V) Ud Figure 6 The reflectron TOF mass spectrometer. i. and speed of scanning and stepping between peaks for multiple ion monitoring. The accelerating potential of B3 kV gives all the ions the same kinetic energy: zeV ¼ mv2 =2 ½5Š ½6Š thus m=zN1=V 2 Nt2 The ions travel down a 1 m linear drift tube and are separated according to mass.

a=q ¼ 2U=V ½10Š 0 y −U −V Figure 8 Ac and dc voltages.MASS SPECTROMETRY / Mass Separation 401 −(U +V cos t ) No transmission Optimum x r x z y (U +V cos t ) Figure 7 The quadrupole analyzer. The gradient a/q is therefore selected to cut the shaded triangle near its apex and give the optimum combination of transmission and mass resolution.e. The behavior of the ions is defined by the stability diagram shown in Figure 9. The performance of the quadrupole cannot be defined by the M/DM relationship that applies to sectors. easier to manufacture and give satisfactory performance. Within the shaded triangle the ions follow stable orbits and they are transmitted through the analyzer.. As the gradient of the scan line is increased. thus. for which the actual peak width increases linearly with mass. Modern research grade instruments are now available with resolutions up to 5000. The gradient of the scan line determines the proportion of ions transmitted. With a quadrupole the peak width is constant throughout the mass range. The rods are electrically connected in diagonally opposite pairs and mass separation is achieved by applying a dc potential þ U to one pair (x) and À U to the other (y). In scanning the instrument the ratio a/q is kept constant by maintaining a fixed ratio between U and V (eqn [10]). and superimposing a radio-frequency ac potential V cos ot.e. and if the instrument is set up to resolve adjacent mass numbers it is described as having unit resolution. as shown in Figure 8. so the ‘positive’ pairs are sometimes negative. which are scanned simultaneously to give a spectrum that is linear in mass. i. + ac − + dc − dc/ac ratio Sweep y a Poor resolution y unstable x unstable q Figure 9 Stability diagram for quadrupole analyzer. which differs in phase by 1801 between the pairs of rods. m is linearly proportional to U and V. no dc voltage. as U is increased relative to V. If the scan line is too steep it will not intersect with the triangle at all and no ions will be transmitted. the proportion of ions following stable orbits decreases and the mass resolution increases. i. Low kinetic energy ions (B20 eV) are introduced and under the influence of the oscillating field follow a spiral path through the analyzer between the rods (along the z-axis). If U ¼ 0. which are of the form shown below for x (eqn [7]).. whereas outside this triangle the orbits become infinitely large and the ions are lost. +V +U x 0 The terms a and q are defined as follows: a ¼ 8eU=mr2 o2 ½8Š ½9Š and q ¼ 4eV=mr2 o2 where r is the radius from rod surface to center line. with an identical equation for y: d2 x dðot=2Þ2 þ ða þ 2q cos otÞx ¼ 0 ½7Š If the ac frequency is kept constant. . Their motion on the x and y axes is defined by the Mathieu equations. and vice versa. all ions will pass through and there will be no mass separation. The peak value of the ac voltage is greater than the dc voltage.

However. Increasing the ac voltage causes the ion trajectories to increase and to approach the end-caps. Helium is used as a buffer gas in the trap at about 10 À 3 Torr to dampen out the more violent motions and to give improved spectral stability. Fourier-Transform Ion Cyclotron Resonance Mass Spectrometers These are essentially a three-dimensional quadrupole in that ions move in the x and y directions but there is no z direction. and those that emerge through apertures in the lower end-cap are detected by the electron multiplier. This allows ion/molecule reactions to occur and may be used to effect chemical ionization (CI). Quadrupoles can be combined with several other types of analyzers in ‘hybrid’ instruments. Any thermal energy they have will cause them to spiral close to the center of the cube with cyclotron frequencies oc and velocities v inversely proportional to m/z: oc ¼ v=r ¼ zeb=m ½11Š Figure 10 The quadrupole ion storage trap (QUISTOR). or ions may be formed in a second adjacent cell or more remotely in a separate ionization region and then injected into the analyzer. It is constructed from a doughnutshaped ring electrode with two end-caps. the ions oscillate around a fixed point (Figure 10). and in the ac-only mode there is no mass discrimination. there are stability criteria for the ion trajectories in a manner analogous to the quadrupole. contained in a homogenous magnetic field generated by a superconducting magnet. whereas the second quadrupole is ac-only and is contained within a collision chamber to encourage fragmentation. Ions can be formed by a variety of techniques either externally in an ion source or directly within the trap.. A typical high-performance quadrupole will have rods of 1 cm diameter and 25 cm in length. possibly preceded by an ac-only quadrupole prefilter. It is ideal as a lowcost mass-selective detector for chromatography. an instrument known as the triple quadrupole with three quadrupoles in series is routinely used in tandem mass spectrometry. A radio frequency ac voltage of B1 kV at 1. which induces the oscillatory motion of the ions. Consequently. 2. and the importance of pulsed desorption ionization methods and are now in the forefront of mass spectrometer development. The storage time can be increased by delaying the start of the voltage ramp. The ac voltage will be in the megahertz frequency range and will have a maximum peak–peak voltage of 10 kV or more. Filament Control grid ac +100 V Multiplier −100 V Control grid Sample inlet ac voltage Ion cyclotron resonance (ICR) mass spectrometers have become increasingly popular with the use of Fourier transform (FT) techniques. A small potential applied to two opposite plates (trapping plates) parallel to the axis of the magnetic field traps the ions within the field.5 cm2. Sample gas molecules may be ionized within the cell by pulsed electron bombardment.1 MHz is applied to the ring electrode. all having cylindrically symmetric hyperbolic surfaces. an ac-only quadrupole gives no mass selection and therefore will transmit such ions.402 MASS SPECTROMETRY / Mass Separation Quadrupoles offer very fast scanning limited only by the impedance involved in changing electric potentials. This is a device for storing ions and has been called the QUISTOR (quadrupole ion storage trap). the development of superconducting magnets. i. In its simplest form it consists of a cubic cell made up of six isolated plates. A quadrupole mass analyzer will not transmit the ionic products of metastable ions breaking down in the analyzer as the change in mass causes the ion orbits to become unstable. The mass analyzer in an Fourier-transform ion cyclotron resonance (FT-ICR) (or Fourier-transform mass spectrometer (FTMS) instrument is illustrated in Figure 11. The circular electrode has an inner diameter of B2 cm. and the application of the ac signal stores ions of all masses down to a low cut-off value of m/z. The first and third quadrupoles are mass selective. . In the trap.e. as explained above. Quadrupole Ion Traps Ions flow into the trap controlled by the gating electrode. and the whole device is compact and inexpensive compared with most analyzers. More sophisticated versions are available for which other scan regimes allow different modes of operation for tandem and other studies including MSn. As this voltage is scanned upwards the ions are ejected from the trap in order of increasing m/z. The equations presented for the quadrupole analyzer also apply here. and are therefore ideally suited to interfacing with high-resolution chromatographic techniques. and furthermore will do so with high efficiency as the quadrupole is insensitive to ion kinetic energy and is relatively unaffected by angular distribution.

The dynamic range achievable with a sector instrument can be greater than 106:1. This limits the ionization methods available unless the ions are prepared remotely and injected into the cell.) (1995) Practical Aspects of Ion Trap Mass Spectrometry: Ion Trap Instrumentation. and is carried out by Fourier transformation. Vertes A. March RE and Todd JFJ (eds. some ions can be excited to such large orbits that they strike the sides of the cube and are destroyed. New York: Wiley. De Hoffman E.. The mass range for FTMS instruments depends on the strength of the magnetic field. From this the mass spectrum is derived.) (2001) Advances in Mass Spectrometry. Stroobant V. and so is lower for high mass ions. Alternatively. This signal is amplified. The instrument can be operated in a number of different modes. The dynamic range is poor compared with sector instruments as there is a space-charge limit to the number of ions that can be stored simultaneously. The application of a burst of low-amplitude radio frequency power to the side plates (excitation plates) will excite the ions into larger orbits.) (1991) FT-ICR/MS: Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. It is possible to vary the time between ion formation and excitation for CI or to study the kinetics of unimolecular fragmentation or ion/molecule reactions. the measurement of the cyclotron frequency can be very precise. or they can be re-excited to produce a new signal that allows spectrum averaging in order to obtain an enhanced signal-to-noise ratio. New York: Wiley. New York: VCH Publishers. New York: Wiley. Further Reading Asamoto B (ed.e. and stored in a computer for subsequent processing. American Chemical Society. and if the residual pressure is low and the density of ions within the cell is small. it is nondestructive. and so will induce an electrical signal in the circuit connecting these plates. Time-of-Flight. a key feature of this means of detection is that.. McNeal CJ (ed. The excited ions will gradually lose kinetic energy and will fall back toward the center of the cell. digitized.) (1995) Quadrupole Mass Spectrometry and its Applications. They can be ejected (quenched) by applying a voltage pulse to the trapping plates before new ions are produced. The pressure in the FTMS cell must remain low as ions will collide with residual gas molecules. Chichester: Wiley. In a 4. The cyclotron frequencies range from megahertz down to kilohertz. the cell can contain both positive and negative ions simultaneously.g. ACS Symposium Series.) (1993) Time-of-Flight Mass Spectrometry.) (1986) Mass Spectrometry in the Analysis of Large Molecules. by application of only certain frequencies. This involves an analysis of the constituent frequencies of the complex waveform that arises from having a complete range of m/z values. i. They will now pass close to the top and bottom plates (receiver plates). and Cl þ can be resolved from Cl À . leading to a randomization of their motion. unlike all other mass spectrometers. Berlin: Springer. Multidimensional. Dawson PH (ed.) (1993) Laser Ionization Mass Analysis. Stable Isotope Ratio. Boca Raton: Interpharm/CRC. Cole RB (1997) Electrospray Ionization Mass Spectrometry: Fundamentals. the time domain signal measured for m/z 18 over 51 s has been recorded to give a resolving power of M/DM ¼ 100 000 000. ions repel each other and are lost.MASS SPECTROMETRY / Mass Separation 403 ap Tr Receive Electrons Excite B Figure 11 Fourier-transform mass analyzer. Resolving power for the FTMS is inversely proportional to mass. and Charette JJ (2001) Mass Spectrometry: Principles and Applications. in this respect performance is comparable to large sector instruments. Instrumentation and Applications. 2nd edn. and with large superconducting magnets 10 000 Da for singly charged ions is well within reach. Chapman JR (1993) Practical Organic Mass Spectrometry: A Guide for Chemical and Biochemical Analysis.7 T magnetic field at 10 À 8 Pa. Gelpi E (ed. . Cotter RJ (ed. e. and Adams F (eds. FTMS instruments can operate at very high resolving power. Gijbels R. a difference of two electrons (B0. See also: Mass Spectrometry: Ion Traps. Modern Mass Spectrometry Series. In FTMS.001 Da). in tandem mass spectrometry experiments. Chichester: Wiley. whereas FTMS gives only 103:1. This will leave only selected ions for further study. New York: Wiley. This allows very accurate measurement of m/z and very precise separation of adjacent peaks.