Once a sample is ionized, it must be focused and drawn into the analyzer portion of the mass spectrometer, with charged drawout and a focusing lens electrically
62 LC/MS OVERVIEW Draw out
plate Entrance
lens Quadrupole rods
Focusing lens
Power supply RF/dc generator
FIGURE 7.2 Focus lens.
charged with the same charge type as the ions to concentrate them into a beam (Figure 7.2).
The direct-current (dc) charged surfaces of the analyzer are then swept with a changing radio-frequency (RF) signal that selects for different mass ions for each frequency, allowing them to follow a stable path to the detector. The stable ions at each frequency are then expelled to collide with the ion detector to be counted by striking the surface of the detector module, generating a signal that is amplified and sent to the computer.
The quadrupole analyzer has four cylindrical rods clamped in a tubular arrange- ment by ceramic collars. Opposing rods have the same dc charge applied to them, while adjacent rods have the opposite charge applied (Figure 7.3).
Ions are focused into the tunnel formed by the four rods and follow a corkscrew flight pattern down the rods as they are swept forward by the changing RF signal.
Ions masses that are not selected by the combined dc/RF signal follow unstable paths, collide with the inner walls of the analyzer rods, and are lost. The ion detector most commonly used with quadrupole and ion trap systems uses an impact/cascade detection mode (Figure 7.4).
When a charged particle strikes a detector’s membrane surface it causes an electron to be released from the other side. These electrons then strike the coated walls of the detector, releasing multiple electrons on each impact. This cascade of electrons amplifies the signal of a single contact for transfer to the data system.
In the data system, the data received are converted to a chromatogram of signal strength versus elapsed time as a total ion chromatogram (TIC).
The mass spectrometer also knows them/zvalue of each time point from the RF sweep signal sent to the analyzer. These data are combined for a number
ANALYZER AND ION DETECTOR DESIGNS 63
FIGURE 7.3 Quadrupole rods.
+ + + + + +
+ + Quadrupole
rods
Amu offset
Gamma rays
Surviving fragment
−2 kV
Conductive surface
Electron cascade
Signal FIGURE 7.4 Ion detector.
of scans and provide the molecular weight of the molecular ion. Them/zvalue determined for each peak may be displayed above the peak, but this depends on the software of the system being used. In other cases, a table is generated displayingm/zversus retention times versus total ion signal strength.
In ion trap systems all sample ions injected are held in a stable circular path in the analyzer by a constant-maintenance RF signal between a sandwich of dc charged plates of opposite polarity. The paths that the ions follow are said to
64 LC/MS OVERVIEW
resemble the stitching on a baseball. Changes in the radio-frequency signals as the dc/RF signal is swept to higher or lower frequencies cause each mass ion’s path in turn to become unstable, releasing it to the ion detector below the center of the bottom analyzed plate. Again the number of impacts at each frequency are measured, amplified, and sent to the computer. One advantage offered by an ion trap analyzer is the ability to trap and hold specific ions between the analyzer plates and then to induce collision fragmentation with gas introduced into the trapping chamber, followed by dc/RF frequency changes to release fragmentation ions to the ion detector. This allows a single analyzer to act as an inexpensive MS/MS system for fragmentation structure studies.
Time-of-flight analyzers are almost in a class by themselves. More conven- tional time-of-flight systems use a standard atmospheric pressure ionization inter- face and a trapping cell to provide bursts of ions to the flight tube for separation and detection by an ion detector. The molecular ions striking the detector surface induce a cascade of electrons within the detector body that amplify the single fragment signal, sending a stronger signal to the detector electronics. This signal can be amplified further with an electron multiplier tube, to provide a strong enough signal for data system processing.
In a MALDI/time-of-flight system, effluent sample is mixed with a chroma- phore, solvent is dried off, and the mixture is bombarded with a laser beam to form ions. Ions are released in a burst into a flight tube in which ions are separated by the flight times needed for each mass ion to reach the detector.
Again impacts on the detector are amplified and sent to the data system as signal strength versus flight time. Time-of-flight analyzers generally use a diode-array impact detector, with each element in the array being activated to detect at a specific time. This signal can be converted to signal versus mass by comparison to flight times of calibration standards.
The MS/MS analyzer usually combines two mass spectrometer analyzers with a collision cell. The target ion selected from the first analyzer is allowed to collide with inert gas molecules to induce fragmentation. The fragmentation ions are then passed into the second MS analyzer for separation and detection. The earliest of these, the triple-quadrupole or tandem mass spectrometer, used two scanning quadrupole modules, Q1 and Q3, units on either side of a quadrupole used as a collision cell, Q2 (Figure 7.5).
A triple-quadrupole LC/MS/MS system can be run in one of four modes to run a variety of experiments (Figure 7.6). Other MS/MS combinations can run in scan/scan mode, but may have trouble running all four of these experiments.
For instance, a three-dimensional ion trap has problems running a neutral loss scanning or MRM (multiple-reaction monitoring) experiment unless it is part of a hybrid system.
In the daughter mode (1), Q1 is scanned and all ions are sent to the frag- mentation chamber, Q3 is then parked at a specific m/z frequency to look for a specific fragmentation ion common to related Q1 ions. In the most common parent mode (2), Q1 is parked at a specific frequency to select only one ion to send to the collision chamber, and Q3 is scanned for fragmentation information
ANALYZER AND ION DETECTOR DESIGNS 65
Q1 Q2 Q3
dc/RF RF only,
no dc
dc/RF
+ ++ + ++ + ++ + + +
++ + + +
+
FIGURE 7.5 Triple-quadrupole MS/MS system. (Courtesy of Varian.)
66 LC/MS OVERVIEW
(1) Daughter mode All masses
Selected mass
Selected mass
Selected mass
SCAN XENON XENON SCAN
XENON
SIM SIM
SIM SIM
SCAN XENON SIM
Constant mass offset
Q1 Q2 Q3 Q1 Q2 Q3
Q1 Q2 Q3
Q1 Q2 Q3
(3) Neutral loss "link" scan
(2) Parent mode
All fragments
Select fragment
mass (4) Multiple reaction monitoring
FIGURE 7.6 MS/MS modes.
that can be used to identify the structure of the ion from Q1. In the neutral loss or linked scan mode, 3, both Q1 and Q3 are scanned with a specific frequency offset. Ions that are detected must have lost a common uncharged molecule that can provide information as to their fragmentation type. The last experiment (4), used to investigate trace compounds in complex mixtures, is less common. Q1 and Q3 are set at single-ion frequencies specific for the impurity to be analyzed and one of its daughter fragments. Since most of the signals being generated are ignored—like focusing only on specific trees in a forest—very specific signals can be generated for traces of material present.
MS/MS systems are not limited to combining analyzers of only a single type.
Hybrid systems can be made to take advantage of the strengths of different ana- lyzer types. One common analyzer combination uses a quadrupole, Q1, as a front end to feed a time-of-flight, Q3, analyzer. One of the newest combinations is the Qtrap system, combining a quadrupole module, Q1, with a linear ion trap, Q2.
The ion trap is based on a set of quadrupole rods and can be run either as a triple-quad system or used as a quad trap in trapping combinations that a triple quad cannot achieve.