BioAnalytical Technologies (India) Plot No.

EL-72, Electronic Zone, TTC Industrial Area, Mahape, Navi Mumbai - 400 705

BioAnalytical Technologies (India) Pvt. Ltd.

BASICS OF LCMS

TABLE OF CONTENTS
1. PROLOGUE ............................................................................................................3 2. OVERVIEW OF LC ...............................................................................................4 3. OVERVIEW OF MASS SPECTROMETRY ....................................................15 4. SAMPLE INLET...................................................................................................21 5. IONIZATION TECHNIQUES ............................................................................24 6. MASS ANALYSERS ............................................................................................37 7. QUADRUPOLE OPERATIONS.........................................................................48 8. DETECTORS ........................................................................................................56 9. VACUUM...............................................................................................................67

LIST OF FIGURES
FIGURE 2.1 ..............................................................................................................................................................................4 FIGURE 2.2 ..............................................................................................................................................................................6 FIGURE 2.3 ..............................................................................................................................................................................6 FIGURE 2.4 ..............................................................................................................................................................................7 FIGURE 2.5 ..............................................................................................................................................................................9 FIGURE 2.6 ............................................................................................................................................................................10 FIGURE 2.7 ............................................................................................................................................................................10 FIGURE 2.8 ............................................................................................................................................................................11 FIGURE 2.9 ............................................................................................................................................................................11 FIGURE 2.10 ..........................................................................................................................................................................12 FIGURE 2.11 ..........................................................................................................................................................................13 FIGURE 3.1: BASIC DIAGRAM TO SHOW THE FLOW FOR MASS SPECTROMETER ....................................................................18 FIGURE 4.2: SCHEMATIC DIAGRAM OF A HPLC INSTRUMENT.............................................................................................22 FIGURE 5.1 ELECTRON IONIZATION ......................................................................................................................................26 FIGURE 5.2 ELECTRON IONIZATION PROCESS. ....................................................................................................................26 FIGURE 5.3 CH5 + ION...........................................................................................................................................................27 FIGURE 5.4 CHEMICAL IONIZATION ......................................................................................................................................28 FIGURE 5.5 ELECTROSPRAY IONIZATION SOURCE. ..............................................................................................................29 FIGURE 5.6 ATMOSPHERIC PRESSURE IONIZATION ..............................................................................................................30 FIGURE 5.7 TURBOIONSPRAY ...............................................................................................................................................31 FIGURE 5.8 TURBOIONSPRAY- FRONT VIEW .........................................................................................................................31 FIGURE 5.9 API 4000 TURBOIONSPRAY ...............................................................................................................................32 FIGURE 5.10 ATMOSPHERIC PRESSURE CHEMICAL IONIZATION..........................................................................................32 FIGURE 5.11 APCI PROCESS ..............................................................................................................................................32 FIGURE 5.12 ATMOSPHERIC PRESSURE PHOTO IONIZATION ................................................................................................33 FIGURE 5.13 APPI- CHARGE TRANSFER PROCESS ...............................................................................................................34 FIGURE 5.14 MALDI IONIZATION ........................................................................................................................................35 FIGURE 5.15 FAB IONIZATION .............................................................................................................................................35 FIGURE 5.16 TYPES OF IONIZATION ......................................................................................................................................36 FIGURE 6.5: SCHEMATIC DIAGRAM OF ION TRAP MASS ANALYZER ...................................................................................41 FIGURE 6.4. TIME-OF-FLIGHT MASS SPECTROMETER. ..........................................................................................................42 FIGURE 6.2: MAGNETIC SECTOR ..........................................................................................................................................43 FIGURE 6.6: T HE ION CYCLOTRON RESONANCE MASS SPECTROMETER .............................................................................46 FIG.7.1 ..................................................................................................................................................................................49 FIG.7.2 ..................................................................................................................................................................................50 FIG.7.3 ..................................................................................................................................................................................51 FIG. 7.4 .................................................................................................................................................................................51 FIG. 7.5 .................................................................................................................................................................................52 FIG.7.6 ..................................................................................................................................................................................52 FIG 7.7 QUADRUPOLE ANALYZER ........................................................................................................................................52 FIG 7.8 PRODUCT ION SCAN .................................................................................................................................................53 FIG. 7.9 PRECURSOR ION SCAN ............................................................................................................................................54 FIG. 7.10 NEUTRAL LOSS SCAN ............................................................................................................................................54 FIG. 7.11 SELECTED/MULTIPLE REACTION MONITORING SCAN ..........................................................................................55 FIG.7.12 GRAPH OF INTENSITY VS. TIME FOR MRM/SRM ..................................................................................................55 FIGURE 8.1: MECHANISM SHOWING CURRENT GENERATION BY A MOVING CHARGE...........................................................57 FIGURE 8.2: MECHANISM OF SECONDARY ELECTRON GENERATION ....................................................................................58 FIGURE 8.3: ILLUSTRATES A PHOTOPLATE WITH ION EXPOSURES........................................................................................60 FIGURE 4: FARADAY CUP .....................................................................................................................................................60 FIGURE 8.7: ELECTRON FLOW IN A CHANNEL ELECTRON MULTIPLIER .................................................................................63 FIGURE 8.10: PHOTOMULTIPLIER TUBE................................................................................................................................65

1. PROLOGUE
Welcome to BAT

The global development of Liquid Chromatography-Mass Spectrometry (LC-MS) has turned out to be a demanding and challenging task. In the recent years, many approaches to LC-MS have come up and still rapidly developing. The technique is expanding its power and attracting more and more users.

This course focuses on the working principles and strategies of LC and MS. This course provides comprehensive introduction and reviews all important aspects of this Hybrid Technology for beginners.

To start with, an overview of LC is explained followed by an overview of MS explaining each component at depth and a special mention about interfacing LC and MS to obtain LC-MS. How to interpret the data from MS is also explained elaborately with easy examples. A general touch about important applications is also mentioned. Finally, an overview of the software is covered with the further references.

2. OVERVIEW OF LC
The Ultimate Carving

Introduction to Liquid Chromatography

Liquid chromatography (LC) is an analytical technique of separation & identification (Figure 2.1) of the sample which are dissolved in a solvent. In Latin words, Chroma meaning color and graphein meaning to write an ion of interest. Since the instrument is not scanned the S/N improves, but any information about other ions is lost. Liquid chromatography (LC) is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column.

Separation
Solvent
A+B

Chromatography
Chromatogram - Detector signal vs. retention time or volume

Detector Signal

Packed column

detector
t0 t1 t2

A t3

B t4

Signal

A Time, t

time or volume

Figure 2.1 In any chromatographic mode the composition of the mobile phase provides the chemical environment for the interaction of the solutes with the stationary phase. Separation can be achieved by controlling and manipulating these interactions, which effects the relative retention time of the various sample components

Glossary of HPLC

1. 2. 3. 4. 5. 6. 7.

Column A tube that contains packed sorbents Mobile phase A liquid that carriers the sample through the column Retention The tendency of a solute to be retained by the stationary phase in the column. Resolution The degree of separation of 2 peaks Efficiency A measure of narrowness of peak widths produced by a column. Selectivity The ratio of retention of 2 adjacent peaks Sample capacity The maximum sample load for the column

History of Liquid Chromatography

In 1903, Column Chromatography was discovered by Russian botanist Mikhail Tswett at the turn of the century. He separated various plant pigments by passing solutions through a glass column containing calcium carbonate. British Chemists A.T.P.Martin and R.L.M. Synge developed partition chromatography in 1942; (Nobel prize in 1954) Since 1940s, chemists used gravity-fed silica columns to purify organic materials In late 1960s, LC turned high performance with small particle columns that require high pressure pumps.

Modes of Liquid chromatography

Adsorption Chromatography: Normal Phase, Reverse Phase. Partition Chromatography: Selective partition between 2 immiscible liquids.

Normal Phase or Liquid-solid Chromatography (NP, LSC)

LSC separation is based on adsorption/desorption of the analyte onto a polar surface (Figure 2.2). LSC separation uses Highly Polar Stationary Phase like Silica, Alumina, Amino-, Cyano- or Phenyl bonded phase. It uses relatively Non-Polar Solvent System like Hexane, Diethyl chloride modified with IPA. NP chromatography is excellent for non-polar analytes, isomers, groups separations and for sample clean up.

Figure 2.2

Reversed Phase Chromatography (RPC)

In Reversed Phase separations organic molecules are separated based on their degree of hydrophobicity. There is a correlation between the degree of lypophylicity and retention in the column. Separation is based on analytes partition coefficients between the mobile phase and the bonded stationary phase. It is also based on Partitioning of the analyte into Hydrophobic Stationary Phase bonded on Silica support. e.g. C18, C8, etc. (Figure 2.3) RPC uses polar mobile phase such as Methanol/ Acetonitrile and Water; Polar analyte elute first than nonpolar. RPC can be used for polar (water soluble), medium polarity and some non-polar analyte. Ionic analytes can be separated using ion-pairing reagents.

Figure 2.3

Types of Chromatography
There are three main types of chromatography, categorized by the mobile phase type: gas (GC), liquid (LC), supercritical fluid (SFC) (Figure 2.4).

Figure 2.4

Partition Chromatography
This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. Two types: 1.liquid-liquid chromatography 2. bonded-phase chromatography Liquid Liquid chromatography: Separation is mainly dependent upon the relative solubility of the compounds (Solutes) in the liquid attached to the support (Stationary phase) and in the liquid flowing through the stationary phase (Mobile phase).If the affinity of the solute for the stationary phase is high, the solute does not elute. Ex: Fat dissolves in oil but not in water. If oil is the stationary phase, a fatty molecule will only elute with an oily mobile phase. Therefore, one goes from a weak solvent (water) to a strong solvent (oil). Bonded Phase chromatography: A stationary phase chemically bonded to a support that is used for the separation. It is the most commonly used LC mode. The most popular support used is microparticulate silica gel. An organosilane, such as octadecyl (for reversed-phase chromatography), is the most accepted type of bonded phase. Approximately 70 percent of all HPLC is carried out on chemically bonded phases.

Adsorption chromatography
When the stationary phase is a solid, the type of equilibrium between this phase and the liquid mobile phase is termed adsorption. All of the pioneering work in chromatography was based upon adsorption methods, in which the stationary phase is a finely divided into the polar solid that contains surface sites for retention of analytes. Silica and alumina are the only stationary phases used, the former being preferred for most applications. The composition of the mobile phase is the main variable that affects the
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partitioning of analytes. Applications of adsorption chromatography include the separation of relatively non-polar water-insoluble organic compounds. Because of the polar nature of the stationary phase and the impact of subtle variations in mobile phase composition on the retention time, adsorption chromatography is very useful for the separation of isomers in a mixture.

Ion Exchange Chromatography

Ionic compounds are often better separated by Ion Exchange Chromatography (IEC). In this case, the stationary phase consists of acidic or basic functional groups bonded to the surface of a polymer matrix (resin or silica gel). Charged species in the mobile phase are attracted to appropriate functional groups on the ion exchanger and thereby separated. Ion pairing chromatography is an alternative to ion exchange chromatography. Mixtures of acids, bases and neutral substances are often difficult to separate by ion exchange techniques. In these cases ion pairing chromatography is applied. The stationary phases used are the same reversed phases as developed for reversed phase chromatography. Cation Exchange (CE) In which +vely charged ions bind to a vely charged resin. Anion Exchange (AE) - In which binding ions are ve and immobilized functional group is +ve

Size Exclusion Chromatography

In size exclusion chromatography molecules are separated based on their molecular size using a sieving effect. The bigger molecules (higher molecular weight) elute earlier. Size exclusion chromatography (SEC) or gel permeation chromatography (GPC) uses as the stationary phase a porous matrix which is permeated by mobile phase molecules. Sample molecules small enough to enter the pore structure are retarded, while larger molecules are excluded and therefore rapidly carried through the column. Thus size exclusion chromatography means separation of molecules by size.

High Performance Liquid Chromatography

HPLC is a physical separation technique in which a sample dissolved in liquid is injected into a column packed with small particles and is separated into its constituent components. It is the most important and widely used analytical technique for the quantitative analysis of organics and biomolecules. It can be applied to different types of samples. This technique is most useful for pharmaceuticals, biomolecules and labile organics.

Principle of HPLC
High Performance Liquid Chromatography is a separation technique utilizing differences in distribution coefficient of compounds between two phases, stationary phase and mobile phase. This technique involves stationary phase which are designates as a thin layer created on surface of fine silica particles. It also involves mobile phases which are the liquid flowing over the particles.

Basic Terminology

Figure 2.5 Chromatographic Plate A peak is characterised by the retention time (selectivity) and its shape (efficiency). Retention Time The total retention time (tR1or tR2) is the time, which is needed by a sample component to migrate from column inlet (sample injection) to the column end (detector). Plate Number (n) The number of theoretical plates in a given column is called the plate number of the column. It is a measure of the capacity of the column for restraining peak dispersion. The higher the plate number of the column, the more narrows the peaks. The plate number is calculated as 16 times the square of the ratio of the retention distance (the distance between the injection point and the peak maximum) to the peak width at the points of inflection (the points of inflection occur at 0.6065 of the peak height). The plate number has a maximum value at a particular mobile phase velocity called the optimum mobile phase velocity. The number of theoretical plates characterizes the quality of a column packing and mass transfer phenomena. The larger the n, the more complicated sample mixtures can be separated with the column. The height equivalent of a theoretical plate h, HETP, is the length, in which the chromatographic equilibrium between mobile and stationary phase is established. Since a large number of theoretical plates is desired, h should be as small as possible. There are, of course, no real plates in a chromatographic column, since the packing is homogeneous. The value of h is a criterion for the quality of a column. h values depend on the particle size, the flow velocity, the mobile phase (viscosity) and especially on the quality of a packing. Capacity Factor (k) A more useful measurement is the capacity factor, or k'. This is calculated from the retention time of a peak and the dead time of the column as shown at the right. This takes advantage of the fact that both dead time and retention time are affected the same way by changes in flow rate or column dimensions; these effects cancel so that the capacity factor is unaffected by flow rate or column dimensions; it is controlled entirely by the temperature and by the chemical characteristics of both the stationary phase and the mobile phase. The dead time of the system is unaffected by changes in mobile phase composition or column chemistry. (Figure 2.6).

Figure 2.6

K =

tR t 0 t0

K =

tR t0

k is a measure of peak retention or how many times the peak is retained vs. an unretained peak (t0). Selectivity Parameter If a sample has two or more peaks, we can also calculate a retention ratio ( ) for any pair of peaks. This is simply the ratio of capacity factors (k' of the second peak divided by k' of the first peak). It gives information about the selectivity of the chromatographic system. Like the capacity factors that comprise it, the retention ratio is unaffected by flow rate or column dimensions; it is controlled entirely by the temperature and by the chemical characteristics of the stationary phase and the mobile phase In chromatography, the selectivity of a phase system refers to its capacity to retain certain types of solutes to a significantly greater extent than others.

Figure 2.7 Resolution

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The difference between two peaks is known as the resolution and is the difference in retention time of the two solutes divided by their average peak width. (figure 2.8)

Figure 2.8

HPLC Instrumentation

Figure 2.9 An HPLC instrument has the elements which are shown Figure solvent reservoir, transfer line with frit, high-pressure pump, sample injection device, column, detector, and data recorder, usually together with data evaluation. Although the column is the most important part, it is usually the smallest one. For temperature-controlled separations it is enclosed in a thermostat. It is quite common to work with more

11

than one solvent, thus a mixer and controller are needed. If the data acquisition is done by a computer it can also be used for the control of the whole system. Solvent Reservoir The reservoir that holds the mobile phase is often no more than a glass bottle. Often, the reagent bottle that holds our HPLC solvent can be used as a reservoir. Solvent is delivered from the reservoir to the pump by means of Teflon tubing called the "inlet line" to the pump. The reservoirs in these systems may have additional features that allow the mobile phase to be degassed and isolated from contact with air. HPLC Pump There are a number of different types of pumps that can provide the necessary pressures and flow-rates required by the modern liquid chromatograph. In the early years of the LC renaissance, there were two types of pump in common use; they were the pneumatic pump, where the necessary high pressures were achieved by pneumatic amplification, and the syringe pump, which was simply a large, strongly constructed syringe with a plunger that was driven by a motor. Today the majority of modern chromatographs are fitted with reciprocating pumps fitted with either pistons or diaphragms. It provides precise and pulse free delivery of solvents. The flow rates ranges of 0.1mL-5mL/min for microbore and up to 30mL. It allows high pressure operation (up to 5000 psi). It can accommodate organic solvents, buffers, salts. Most HPLC pumps are reciprocating pumps. They are motor driven cam which drives with the help of piston to deliver solvent through the outlet check valve. It has unlimited capacity.

Figure 2.10 Sample Injection System Sample introduction can be accomplished in various ways. The simplest method is to use an injection valve. In more sophisticated LC systems, automatic sampling devices are incorporated where sample introduction is done with the help of auto samplers and microprocessors. In liquid chromatography, liquid samples may be injected directly and solid samples need only be dissolved in an appropriate solvent. The solvent need not be the mobile phase, but frequently it is judiciously chosen to avoid detector interference, column/component interference, and loss in efficiency or all of these. It is always best to remove particles from the sample by filtering, or centrifuging since continuous injections of particulate material will eventually cause blockage of injection devices or columns.

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Figure 2.11 Column Typical LC columns are 10, 15 and 25 cm in length and are fitted with extremely small diameter (3, 5 or 10 mm) particles. The internal diameter of the columns is usually 4 or 4.6 mm; this is considered the best compromise among sample capacity, mobile phase consumption, speed and resolution. However, if pure substances are to be collected (preparative scale), larger diameter columns may be needed Packing of the column tubing is done with the small diameter particles. In general, LC columns are fairly durable and one can expect a long service life unless they are used in some manner which is intrinsically destructive, as for example, with highly acidic or basic eluents, or with continual injections of 'dirty' biological or crude samples. It is wise to inject some test mixture (under fixed conditions) into a column when new, and to retain the chromatogram. Columns may be made of glass, stainless steel, plastic and many other materials. Some types of chromatography that utilize a column are gas chromatography and HPLC. Usually they are stainless steel, 1 - 5 mm diameter, ~5 mm packing. Most columns are packed with Silica that are used in HPLC. Detectors An HPLC detector is equipped with a small flow cell connected to the column outlet and monitors the concentration of the analyte component. An ideal detector should have a linear response, universal and sensitive. Common detectors are:

UV/V Fluorescence (Fl) Refractive index (RI) Mass spectrometry (MS) Other detector like Electrochemical (ECD), Conductivity and Infrared

Mobile Phase Characteristics & Composition Simple HPLC uses mobile phase of constant composition - isocratic elution.For more complex mixtures gradient elution is used.

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Typical Characteristics of Mobile Phase are as follows:

It should have desirable Physical properties It should have high purity, low cost, UV transparency, non-corrosive, low viscosity, non- flammable, sample solubility. It should have ionic strength and Selectivity It is related to polarity of solvent It depends on dipole moment, induced dipole, H-bonding, and dispersive characteristics of the solvents.

Advantages of HPLC Some common advantages of HPLC are:

It is Fast, Non destructive (detector dependent) technique Various types of columns can be used thus proving the versatility of the instrument. Wide range of Samples can be analyzed. It has High Resolution. It is good technique for quantitation. It operates at near ambient temperature.

Application of HPLC Important ranges of applications of HPLC are as follows: Field of Application Separation

Pharmaceuticals Biochemical Food Products Industrial Chemicals Forensic Chemistry Clinical Medicine Pollutants
Table 1: LC applications.

Antibiotics, Sedatives, Steroids, Analgesics Amino acids, Proteins, Carbohydrates, Lipids Artificial Preservatives Sweeteners, Antioxidants,

Condensed Aromatics, Surfactants, Propellants, Dyes Drugs, Poisons, Blood Alcohol, narcotics Bile Acids, Drug Metabolites, Urine Extracts, Estrogens Pesticides, Herbicides, Phenols, PCBs

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3. OVERVIEW OF MASS SPECTROMETRY

Feather in the Cap of Analytical Techniques

Introduction
Mass spectrometry is an analytical technique which determines the mass-to-charge (m/z) ratio of ions. It is most generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of the components of a sample. Applications of mass spectrometry includes identifying and quantitating pesticides in water samples, to identifying steroids in athletes, determining metals at p p q (Parts Per Quadrillion) levels in water samples. Mass spectrometry is essentially a technique for "weighing" molecules. Obviously, this is not done with a conventional balance or scale. Instead, mass spectrometry is based up on the motion of a charged p article, called an ion, in an electric or magnetic field. The mass to charge ratio (m/z) of the ion affects this motion. Since the charge of an electron is known, the mass to charge ratio a measurement of an ion's mass.

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Principle
The ions are generated in the ionization source by inducing either the loss or the gain of a charge (e.g. electron ejection, protonation, or deprotonation). Once the ions are formed in the gas phase they can be electrostatically directed into a mass analyzer, separated according to mass and finally detected. The result of ionization, ion separation, and detection is a mass spectrum that can provide molecular weight or even structural information. The first step in mass Spectroscopic analysis of compounds is the production of ions.

M + e M + + 2e
This molecular ion normally undergoes fragmentations. Because it is a radical cation with an odd no. of electrons, it can fragment to either a radical and an ion with an even no. of electrons, or a molecule and a new radical cation. The important difference between these two types of ions is

EE

+ R
+

even ion radical

OE

+ N
molecule

odd ion

These two types of ions have different chemical properties. Each primary product ion derived from molecular ion can, in turn, undergo fragmentation, and so on. All these ions are separated in the spectrometer according to their mass to charge ratio and are detected in proportion to their abundance. Most of the ions have a charge corresponding to the loss of only one electron. Multiple charged ions could also be obtained: they are detected according to the mass-to-charge ratio, the charge of the electron being taken as a charge unit.

History of Mass Spectrometery

A large no. of mass spectrometers have been developed according to the fundamental scheme since Thompsons experiments in 1897. Listed here are some of the highlights of the evolution. Pre-MS

1886: E. Goldstein discovers anode rays (positive gas ions) in gas discharge 1897: J.J. Thomson discovers the electron and determines its m/z ratio. Nobel Prize in 1906. 1898: W. Wien analyzes the anode rays by magnetic deflection, and establishes that they carry a positive charge. Nobel Prize in 1911. 1909: R.A. Millikan & H. Fletcher determines the elementary unit of charge.

MS Early Years

1912: First Mass Spectrometer (J.J. Thomson) 1919: Electron ionization and magnetic sector MS (A.J. Dempster) 1934: R. Corad applies mass spectroscopy to organic chemistry

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1942: The Consolidated Engineering Corp. builds the first commercial instrument dedicated to organic analysis. A.E. Cameron and D.F. Eggers publish a design and mass spectra for a linear Time-of-Flight (TOF) mass spectrometer.

MS Recent

1952: Theories of quassi-equlibrium explains the monomolecular fragmentation of ions. R.A. Marcus receives the Nobel Prize in 1992 1953: W. Paul and H.S. Steinwedel describes the Quadrupole Analyzer and the Ion Trap. W. Paul receives Nobel Prize in 1989. 1956: First GC-MS by F.W.McLafferty and R.S. Gokle 1968: Finnigan introduces the first commercial quadrupole. 1974: First high-performance liquid chromatography/mass spectroscopy coupling by P.J. Arpino, M.A. Baldwin, and F.W. McLafferty; E.C.Horning, D.I. Carroll and K.D.Haegele discovers APCI; M.D.Comisarov and A.G.Marshall develop FTICR mass spectroscopy. 1975: First commercial LC-MS with capillary columns 1990s: Explosive growth in biological MS, due to ESI &MALDI 2002: Nobel Prize to Fenn & Tanaka for ESI & MALDI

Mass/charge Ratio
The physical quantity mass/charge has the symbol m/q. The SI units would be kg/C, however, in mass spectrometry the Atomic Mass Unit u is used instead of the kilogram, and the atomic unit e is used instead of the Coulomb. e is the elementary charge. u is sometimes called Dalton (Da). [m/q] = u/e u/e is sometimes called Thomson (Th).

Components of Mass Spectrometry

The mass spectrometer always contains the following elements as shown in Figure 3.1.

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Figure 3.1: Basic diagram to show the flow for mass spectrometer The five different components along with their functions are as follows: 1. Sample inlet Its a device to introduce the sample that is to be analyzed. 2. Ionization source A source to produce ions from sample. 3. Analyzers Analyzers sort the ions according to their mass-to charge ratio. 4. Detector It counts the ions emerging from the analyzer and measures their abundance. 5. Data Recorder A computer is there to process the data, which produces the mass spectrum in a suitable form and controls the instrument through feedback. This data is then interpreted as per the desired application.

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Coupling LC to MS
Combining Liquid Chromatography with Mass Spectroscopy offers the possibility of taking advantage of both chromatography as a separation method and the mass spectrometry (MS) as an identification method. It is possible to fully exploit the intrinsic properties of these two methods; an extremely powerful combination would result. The history of LC-MS started by the end of the 1960s, when in various laboratories preliminary research started. Some breakthroughs in development reported in 1974 marked the actual beginning of history of LC-MS. Over the years, the technique has been extensively reviewed. MS is a universal detector for LC. The objectives behind the coupling are: For analysis of thermolabile and non-volatile analytes, which is not amenable to GC-MS, to avoid analyte derivatization and for identification of analytes. MS affords a low detection limits and helps in assessment of peak purity. Few specifications of LC-MS interface are:

There is no restriction to mobile phase composition Can use volatile and non-volatile additives There is free choice of LC column dimensions The flow rate can be between 1/min and 2/min The transfer efficiency is high There is no additional peak broadening and no loss of resolution There is a free choice for ionization methods and CI gas conditions.

The LC-MS interface and the MS operations put restrictions on the composition of mobile phase used in LC-MS.

LC Interface

MS

The general solution to LC-MS coupling is the development of interfaces that transfer the analyte from the LC column to the MS ion source and prepare the analyte for ionization, cope with the solvent from the LC, and bridge the pressure difference between column outlet and mass analyzer.

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The low cost, operational simplicity, low detection limits and quantitation are the positive sides of LC-MS coupling. Therefore, LC-MS has become an inevitable analytical technique. The following sections covers the basics of mass spectrometry, its components, and instrumentation and also introduces the interpretation of mass spectra

Summary
Mass Spectroscopys characteristics have raised it to an outstanding position among analytical methods because of its unequaled sensitivity and detection limits and diversity of its applications. Mass Spectroscopy has progressed extremely rapidly during the last decade: production, separation and detection of ions, data acquisition, data reduction, etc. This had led to the development of entirely new instrument and applications..

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4. SAMPLE INLET
Stepping in

Introduction
This topic provides a general idea on the different types of sample introduction systems used by MS. Sample inlet system acts as the interface, the samples are injected by different means to the mass spectrometer, from where it goes to the ionization source. The important sample inlet systems, their working and their characteristics are discussed below.

Sample inlets in LCMS

The selection of a sample inlet depends upon the sample and the sample matrix. Most ionization techniques are designed for gas phase molecules so the inlet must transfer the analyte into the source as a gas phase molecule. If the analyte is sufficiently volatile and thermally stable, a variety of inlets are available. Gases and samples with high vapor pressure are introduced directly into the source region. Liquids and solids are usually heated to increase the vapor pressure for analysis. If the analyte is thermally labile (it decomposes at high temperatures) or if it does not have a sufficient vapor pressure, the sample must be directly ionized from the condensed phase. These direct ionization techniques require special instrumentation and are more difficult to use. However, they greatly extend the range of compounds that may be analyzed by mass spectrometry. Commercial instruments are available that use direct ionization techniques to routinely analyze proteins and polymers with molecular weights greater than 100,000 Dalton.

Direct Vapor Inlet The simplest sample introduction method is a direct vapor inlet. The gas phase analyte is introduced directly into the source region of the mass spectrometer through a needle valve. Pump out lines are usually included to remove air from the sample. This inlet Works well for gases, liquids, or solids with a high vapor pressure. Samples with low vapor pressures are heated to increase the vap or pressure. Since this inlet is limited to stable compounds and modest temperatures, it only works for some samples.

Gas Chromatography Gas chromatography i s probably the most common technique for introducing samples into a mass spectrometer. Complex mixtures are routinely separated by gas chromatography and mass spectrometry is used to identify and quantitate the individual components. Several different interface designs are used to connect these two instruments. The most significant characteristics of the inlets
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are the amount of GC carrier gas that enters the mass spectrometer and the amount of analyte that enters the mass spectrometer. If a large flow of GC carrier gas enters the mass spectrometer it will increase the pressure in the source region. Maintaining the required source pressure will require larger and more expensive vacuum pumps. The amount of analyte that enters the mass spectrometer is important for imp roving the detection limits of the instrument. Ideally all the analyte and none of the GC carrier gas would enter the source region.

Figure 4.1: Block Diagram of a gas chromatograph. The most common GC/M S interface now uses a capillary GC column. Since the carrier gas flow rate is very small for these columns, the end of the capillary is inserted directly into the source region of the mass spectrometer. The entire flow from the GC enters the mass spectrometer. Since capillary columns are now very common, this inlet is widely used. However, wide bore capillaries and packed GC columns have higher flow rates. This significantly increases the pressure in the mass spectrometer. Several inlet designs are available to reduce the gas flow into the source. The simplest design splits the GC effluent so that only a small portion of the total flow enters the mass spectrometer. Although this inlet reduces the gas load on the vacuum system, it also reduces the amount of analyte. Effusive separators and membrane inlets are more selective and transport a higher fraction of the analyte into the source region. Each of these methods has efficiency and resolution drawbacks but they are necessary for some experiments.

Liquid Chromatography Liquid chromatography inlets are used to introduce thermally labile compounds not easily separated by gas chromatography. These inlets have undergone considerable development. Because these inlets are used for temperature sensitive compounds, the sample is ionized directly from the condensed phase. These inlets are discussed in greater detail in the section on ionization techniques.

Figure 4.2: Schematic Diagram of a HPLC instrument.

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Direct Insertion Probe The Direct Insertion Probe (DIP) is widely used to introduce low vapor pressure liquids and solids into the mass spectrometer. The sample is loaded into a short capillary tube at the end of a heated sleeve. This sleeve is then inserted through a vacuum lock so the sample is inside the source region. After the p robe is positioned, the temperature of the capillary tube is increased to vaporize the sample. This p robe is used at higher temperatures than are possible with a direct vapor inlet. In addition, the sample is under vacuum and located close to the source so that lower temperatures are required for analysis. This is important for analyzing temperature sensitive compounds. Although the direct insertion p robe is more cumbersome than the direct vapor inlet, it is useful for a wider range of samples.

A]

B]
Figure 4.3: A] and B] Direct Insertion Probe, for use on the mass spectrometers.

Direct Ionization of Sample. Unfortunately, some compounds either decompose when heated or have no significant vapor pressure. These samples may be introduced to the mass Spectrometer by direct ionization from the condensed phase. These direct ionization techniques are used for liquid chromatography/mass spectrometry, glow discharge mass spectrometry, fast atom bombardment and laser ablation. The development of new ionization techniques is an active research area and these techniques are rap idly evolving. Direct ionization is discussed in greater detail in the next section.

Summary
There are five basic sample inlet systems and the selection of these inlet systems depends on the sample and the sample matrix, and the ionization source. The important sample inlet systems are:

Direct Vapor Inlet. Gas Chromatography. Liquid Chromatography. Direct Insertion Probe.

23

5. IONIZATION TECHNIQUES
Activating the neutral molecules

Introduction
A variety of ionization techniques are used for mass spectrometry. Most ionization techniques excite the neutral analyte molecule which then ejects an electron to form a radical Cation (M+). Other ionization techniques involve ion molecule reactions that produce adduct ions (M H+). The most important considerations are the physical state of the analyte and the ionization energy. Electron ionization and chemical ionization are only suitable for gas phase ionization. Fast atom bombardment, secondary ion mass spectrometry, electrospray, and matrix assisted laser desorption are used to ionize condensed phase samples. The ionization energy is significant because it controls the amount of fragmentation observed in the mass spectrum. Although this fragmentation complicates the mass spectrum, it provides structural information for the identification of unknown compounds. Some ionization techniques are very soft and only produce molecular ions; other techniques are very energetic and cause ions to undergo extensive fragmentation. Although this fragmentation complicates the mass spectrum, it provides structural information for the identification of unknown compounds.

Glossary
Some common terms used frequently for Ionizations:
1.

Adduct ions: Adduct ions are produced by chemical reaction between an ion and a neutral molecule. Many of those reactions caused the addition of a proton (H+) to the molecule M and produced an adduct ion (MH+). M+ is molecular ion produced by removing single electron to form radical ion The SI unit for energy is t he Joule. The energetic of chemical reactions are typically expressed in kJ/mole. In many gas p has e experiment s (like mass spectrometry), t he mole is not a convenient unit. The electron volt is frequently used as an energy unit for single molecules or atoms. 1eV = 1.60217733 (49) x 1019 J. So that: 1eV (per molecule or atom) = 96.4152206 kJ/mole.

2. 3.

Types of Ionization
The two basic types of ionization are Soft Ionization & Hard Ionization.

24

Soft Ionization: It is a simple method of adding a charge on a molecule. The methods that are involved in soft ionization are CI, ESI, MALDI. E.g. Methanol CH3OH. electron CH3OH = CH3OH+.+ 2 electrons

Hard Ionization: It is a process where energy imparted by the ion source and, more importantly, instability in a molecular ion causes the ion to break into smaller pieces (fragments). It gives enough energy to rupture bonds and producing fragments. The methods that are involved in hard ionization are EI, FAB. E.g. Methanol CH3OH. CH3OH+.(molecular ion)=CH2OH+(fragment ion) + HCH3OH+.(molecular ion)= CH3+(fragment ion) + OH-

Ionization Process
Following is a brief description of the mechanism of ionization. It explains how ionization is done in MS.

An electron which strikes a molecule may impact enough energy to remove electron from that molecule. e.g. Methanol CH3OH. CH3OH + e- = CH3OH* + 2eThe sample is passed into the ionization chamber .The heated coil gives off electrons. Particles that are in the sample and the electrons both bombard to give off ions. These ions then pass in the gas phase into the vacuum system of the mass spectrometer. The ions are moved into the analyzer and then to the detector.

Ionization Methods
Following are the Ionization methods used in Mass Spectrometry:
1. 2. 3.

Gas Phase Ionization Spray Ionization Desorption Ionization

25

1. Gas Phase Ionization

The solvent is removed from an aerosol of the liquid chromatographic effluent, and the resulting neutral analyte molecules are introduced into the ion source of the mass spectrometer in the gaseous form where they are ionized by electron ionization (EI) or chemical ionization (CI). 1.1 Electron Impact (EI) 1.2 Chemical Ionization (CI)

1.1 Electron Ionization (EI)

It is the most common ionization technique used for mass spectrometry. EI works well for many gas phase molecules, but it does have some limitations. Although the mass spectra are very reproducible and are widely used for spectral libraries, EI causes extensive fragmentation so that the molecular ion is not observed for many compounds. Fragmentation is useful because it provides structural information for interpreting unknown spectra.

Fi lam ent

e-

e-

e-

Figure 5.1 Electron Ionization The electrons used for ionization are produced by passing a current through a wire filament (Figure5.1). The amount of current controls the number of electrons emitted by the filament. An electric field accelerates these electrons across the source region to produce a beam of high energy electrons. When an analyte molecule p asses through this electron beam, a valence shell electron can be removed from the molecule to produce an ion.

Figure 5.2 Electron Ionization Process. A) Ionizing electron approaches the electron cloud of a molecule; B) Electron cloud distorted by ionizing electron; C) Electron cloud further distorted by ionizing electron; D) Ionizing electron passes by the molecule; E) Electron cloud of molecule ejecting an electron; F) Molecular ion and ejected electron.

26

Ionization does not occur by electron capture, which is highly dependent up on molecular structure. Instead, EI produces positive ions by knocking a valence electron off the analyte molecule (Figure 5.2). As the electron p asses close to the molecule the negative charge of the electron repels and distorts the electron cloud surrounding the molecule. This distortion transfers kinetic energy from the fastmoving electron to the electron cloud of the molecule. If enough energy is transferred by the process, the molecule will eject a valence electron and form a radical cation (M + ). Since the ionization is produced by a single electron that is accelerated to 70 V, this is commonly referred to as 70eV EI. This is enough energy to cause extensive fragmentation, and at this level small changes in the electron energy do not significantly affect the fragmentation patterns. The amount of energy transferred during this process depends up on how fast the electron is traveling and how close it p asses to the molecule. In most 70eV EI experiments, approximately 1400 kJ/mole (15eV)* of energy is transferred during the ionization process. There is, however, a distribution of energy and as much as 2800 kJ/mole (30eV) is transferred to some molecules. Since approximately 960 kJ/mole (10eV) is required to ionize most organic Compounds and typical chemical bond energy is 290 kJ/mole (3eV), extensive fragmentation is often observed in 70eV EI mass spectra. The distribution of energy transferred during ionization and the large number of fragmentation pathway s results in a variety of products for a given analyte. Other electron voltages may be used to vary the amount of fragmentation produced during ionization. For most organic compounds the threshold energy for EI is about 10 eV. Because a mass spectrum is produced by ionizing many molecules, the spectrum is a distribution of the possible product ions. Intact molecular ions are observed from ions produced with little excess energy . Other molecular ions have more energy and undergo fragmentation in the source region. The abundance of the resulting fragments, often called product ions, is determined by the kinetics of the fragmentation pathway s and the ionization energy. Changing the ionization energy changes the observed distribution of fragment ions. This distribution provides the structural information for interpreting mass spectra and is discussed in detail in the section on interpretation.

1.2 Chemical Ionization

Chemical Ionization (CI) is a soft ionization technique that produces ions with little excess energy .As a result, less fragmentation is observed in the mass spectrum. Since this increases the abundance of the molecular ion, the technique is complimentary to 70eV EI. CI is often used to verify the molecular mass of an unknown. Only slight modifications of an EI source region are required for CI experiments. In Chemical Ionization the source is enclosed in a small cell with openings for the electron beam, the reagent gas and the sample. The reagent gas is added to this cell at approximately 10 Pa (0.1 torr) pressure. This is higher than the 10-3 Pa (10-5 torr) pressure typical for a mass spectrometer source. At 10-3 Pa the mean free path between collisions is approximately 2 meters and ion-molecule reactions are unlikely. In the CI source, however, the mean free path between collisions is only 10-4 eters and analyte molecules undergo many collisions with the reagent gas. The reagent gas in the CI source is ionized with an electron beam to produce a cloud of ions. The reagent gas ions in this cloud react and produce adduct ions like CH+ (Figure 5.3), which are excellent proton donors.

Figure 5.3 CH5 + ion. When analyte molecules (M) are introduced to a source region with this cloud of ions, the reagent gas ions donate a proton to the analyte molecule and produce M H+ ions. The energetics of the proton transfer is controlled by using different reagent gases. The most common reagent gases are methane, isobutane and ammonia. M ethane is the strongest proton donor commonly used with a proton affinity (PA) of 7.5eV. For softer ionization isobutane (PA8.5eV) and ammonia (9.0eV) are frequently used. Acid base chemistry is frequently used to describe the chemical ionization reactions.
27

The reagent gas must be a strong enough Bronsted acid to transfer a proton to the analyte. Fragmentation is minimized in CI by reducing the amount of excess energy produced by the reaction. Because the adduct ions have little excess energy and are relatively stable, CI is very useful for molecular mass determination. Some typical reactions in a CI source are shown in Figure 5.4. A) EI of reagent gas to form ions: CH4 + e- CH4+ + 2eB ) Reaction of reagent gas ions to form adducts: CH4+ + CH4 CH3+ + CH5+ OR CH4+ CH3+ + H CH3+ + CH4 C2 H5+ + H2 C) Reaction of reagent gas ions with Analyte molecules: CH5+ + M CH4 + MH+ C2H5+ + M C2H4 + MH+ CH3+ + M CH4 + (M-H)+ Figure 5.4 Chemical Ionization

2. Spray Ionization
The compound of interest in a volatile buffer mobile phase, is passed through heated, narrow bore tubing directly into the ion source of a mass spectrometer. The solution is vaporized in the tubing, and analyte ions desorb into the gas phase and pass into the mass analyzer. 2.1 Electrospray Ionization (EI) 2.2 Atmospheric Pressure Chemical Ionization (APCI). 2.3 Atmospheric Pressure photo Ionization (APPI)

2.1 ElectroSpray Ionization

Atmospheric Pressure Ionization (API) sources ionize the sample at atmospheric pressure and then transfer the ions into the mass spectrometer. These techniques are used to ionize thermally labile samples such as pep tides, proteins and polymers directly from the condensed phase. The sample is dissolved in an appropriate solvent and this solution is introduced into the mass spectrometer. With conventional inlets the solvent increases the pressure in the source region of the mass spectrometer. In addition, Joule-Thompson cooling of the liquid as it enters the vacuum causes the solvent droplets to freeze. The frozen clusters trap analyte molecules and reduce the sensitivity of the experiment. API sources introduce the sample through a series of differentially pumped stages. This maintains the large pressure difference between the ion source and the mass spectrometer (Figure 5.5) without using extremely large vacuum pumps. In addition a drying gas is used to break up the clusters that form as the solvent evaporates. Because the analyte molecules have more momentum than the solvent and air molecules, they travel through the pumping stages to the mass analyzer.

28

Figure 5.5 Electrospray Ionization Source. Electrospray Ionization (ESI) is the most common API application. It has undergone remarkable growth in recent y ears and is frequently used for LC/M S of thermally labile and high molecular weight compounds. The electrospray is created by applying a large potential between the metal inlet needle and the first skimmer in an API source (Figure 7). The mechanism for the ionization process is not well understood and there are several different theories that exp lain this ionization process. One theory is that as the liquid leaves the nozzle, the electric field induces a net charge on the small drop lets. As the solvent evaporates, the drop let shrinks and the charge density at the surface of the drop let increases. The drop let finally reaches a point where the columbic repulsion from this electric charge is greater than the surface tension holding it together. This causes the drop let to exp lode and produce multiply charged analyte ions. A typical ESI spectrum shows a distribution of molecular ions with different charge numbers. Because electrospray produces multiply charged ions, high molecular weight compounds are observed at lower m/z value. This increases the mass range of the analyzer so that higher molecular weight compounds may be analyzed with a less expensive mass spectrometer. An ion with a mass of 5000 u and a charge of +10 is observed at m/z 500 and is easily analyzed with an inexpensive quadruple analyzer. API Sources are also used for Inductively Coup led Plasma M ass Spectrometry (ICP/M S) and glow discharge experiments. In ICP/M S a nebulizer is used to introduce liquid samples into high temperature plasma. The temperature of the plasma is high enough to efficiently ionize most elements. These ions are introduced to the mass spectrometer using a series of differentially pumped regions similar to the electrospray source discussed above. Glow discharge experiments are similar, but used for solid samples. The high sensitivity and selectivity of the mass spectrometer provides rap id multielement detection at very low levels. Because the high temperature of the plasma destroy s any chemical bonds, these techniques are used for elemental analysis.

29

Figure 5.6 Atmospheric Pressure Ionization

Sample solution is sprayed across a high potential difference, from a needle into an orifice in the interface, by creating highly charged droplets in the presence of a strong electric field. The required electric field is 3.5 kV. ESI generates ions directly from solution by creating a fine spray of highly charged droplets. Ions are then electrostatically directed into the mass analyzer. The ions begin to leave the droplet through what is known as a "Taylor cone .Droplet decreases in size, the electric charge density on its surface increases. Mutual repulsion between like charges increases. The force becomes so strong that it exceeds the forces of surface tension. The ions leave the droplet in the form of Taylor Cone.

Advantages of ESI

ESI allows for large, non-volatile molecules to be analyzed directly from the liquid phase Good for charged, polar or basic compounds Permits the detection of high-mass compounds Best method for analyzing multiple charged compounds Very low chemical background leads to excellent detection limits Can control presence or absence of fragmentation by controlling the interface lens potentials The ionization efficiency is 103 104 times higher than that of Gas phase ionization The analysis of biomolecules, Oligonucleotides, pesticides, carbohydrates, Long chain fatty acids.
30

Disadvantages of ESI

Interpretation of Multiply charged species is sometimes be difficult (require mathematical transformation) Complementary to APCI. Not good for uncharged, non-basic, low-polarity compounds (e.g. steroids) Very sensitive to contaminants such as alkali metals or basic compounds. Relatively low ion currents Relatively complex hardware compared to other ion source

TURBOIONSPRAY (TIS): Modified Electrospray technique.

Figure 5.7 TurboionSpray The Turbo ion source maximizes ionization efficiency by combining embedded ceramic heater technology. The dual heater in the Turbo Ion source provides greater efficiency in ionization. Improved heat transfer and gas dynamics provide a tenfold increase in sensitivity.

Figure 5.8 TurboionSpray- front view

Error!

31

Figure 5.9 API 4000 TurboionSpray

2.2 Atmospheric Pressure Chemical Ionization

Figure 5.10 Atmospheric Pressure Chemical Ionization. Atmospheric pressure chemical ionization is a relative of ESI. A corona discharge is used to ionize the analyte in the atmospheric pressure region. Liquid effluent is introduced directly into APCI source. APCI source contains a heated vaporizer, facilitating rapid vaporization of the droplets. Vaporized sample carried through an ion-molecule reaction region at atmospheric pressure. Ionization occurs through a corona discharge, reacting reagent ions from the solvent vapor. The gasphase ionization in APCI is more effective than ESI for analyzing less-polar species.

How it works
1
Io n iz at io n p r o d u c e s p r e d o m in a n t l y s o lv e n io n s

T h e Cu r t a in Ga s h e lp s t r an sf e r io n s t o t h e m as s an aly z e r an d e f f e c t s io n d e c lu s t e r in g

C u rt a in Ga s

N e b u liz e r Ga s Sa m p le ( M) LC Ef f lu e n t X X M X Make-up Gas M

XH + XH + XH +

X X

X X
MH +

MH

XH

X ( - 1 2 0 C)

2
Co r o n a Dis c h ar g e Ne e d le X = s o lv e n t m o le c u le s , e .g . H 2 O , N H 3 , e t c

T h e s o lv e n t io n s r e ac t w i t h an a ly t e m o le c u le s , f o r m in g c l u s t e r s Curt a in Gas

Figure 5.11 APCI Process

32

Vaporization Atmosphere Ionized Charge Transfer Ion Molecule Reaction Proton Transfer

Advantages of APCI

APCI is good for less polar compounds It is excellent LCMS interface APCI is compatible with MSMS methods

Disadvantages of APCI

Sample should be volatile. Thermal. stability is required

2.3 Atmospheric Pressure Photo Ionization (APPI)

Figure 5.12 Atmospheric Pressure Photo Ionization APPI is a relatively new ionization technique that is complementary to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). The principal is to use photons to ionize gas phase molecules. Radiation from an ultraviolet light source ionizes a reagent compound (dopant) added to the sample stream. It creates ions by using a high fluoresce gas discharge lamp that produces 10eV energy photons. Dopant solvent is used to provide an a abundant source of ions to transfer charge or protons from the solvent to analytes. The ionized reagent molecules then undergo either charge exchange or proton transfer reactions to ionize the sample molecules. Use of a reagent compound greatly enhances the ionization efficiency for sample molecules since the reagent is present in large quantities and is chosen for its ease of photo-ionization

33

APPI technique

Figure 5.13 APPI- Charge Transfer Process

Advantages of APPI Ionization

It offers wide range of detection for polar compounds. It gives greater structural diversity, than the other two ionization techniques

Disadvantages of APPI

Sample should be volatile Thermal. stability is required

3. Desorption Ionization
The mixture is introduced via a capillary inlet onto a target within the ion source where it is bombarded with high energy (5-20keV) atoms or ions. There are two types of desorption ionization. 3.1 Matrix-Assisted Laser Desorption Ionization (MALDI) 3.2 Fast Atom Bombardment (FAB)

3.1 Matrix Assisted Laser Desorption/Ionization

Matrix Assisted Laser Desorption/Ionization (M ALDI) is used to analyze extremely large molecules. This technique directly ionizes and vaporizes the analyte from the condensed phase. MALDI is often used for the analysis of synthetic and natural polymers, proteins, and peptides. Analysis of compounds with molecular weights up to 200,000 Dalton is possible and this high mass limit is continually increasing. In M ALDI, both desorption and ionization is induced by a single laser pulse (Figure5.9).

34

The sample is prepared by mixing the analyte and a matrix compound chosen to absorb the laser wavelength. This is placed on a p robe tip and dried. A vacuum lock is used to insert the p robe into the source region of the mass spectrometer. A laser beam is then focused on this dried mixture and the energy from a laser pulse is absorbed by the matrix. This energy ejects analyte ions from the surface so that a mass spectrum is acquired for each laser pulse. The mechanism for this process is not well understood and is the subject of much controversy in the literature. This technique is more universal (works with more compounds) than other laser ionization techniques because the matrix absorbs the laser pulse. With other laser ionization techniques, the analyte must absorb at the laser wavelength. Typical MALDI spectra include the molecular ion; some multiply charged ions, and very few fragments.

Figure 5.14 MALDI Ionization 3.2 Fast Atom Bombardment and Secondary Ion Mass Spectrometry Fast Atom Bombardment (FAB) and Secondary Ion M ass Spectrometry (SIM S) both use high energy atoms to sp utter and ionize the sample in a single step. In these techniques, a beam of rare gas neutrals (FAB) or ions (SIM S) is focused on the liquid or solid sample. The imp act of this high energy beam causes the analyte molecules to sp utter into the gas phase and ionize in a single step (Figure5.10). The exact mechanism of this process is not well understood, but these techniques work well for compounds with molecular weights up to a few thousand Dalton. Since no heating is required, sputtering techniques (especially FAB) are useful for studying thermally labile compounds that decompose in conventional inlets.

Figure 5.15 FAB Ionization

35

Summary
In Mass Spectrometry, ionization techniques broadly categorized into three class viz. Gas Phase Ionization, Electrospray Ionization and Desorption Ionization. Selection of Ionization techniques depends on sample types to be ionized and types of fragmentation required.

Ionization Soft CI ESI APCI APPI MALDI

Figure 5.16 Types of Ionization

Hard EI FAB

36

6. MASSANALYSERS
Sorting of Ions

Introduction
Once the ions are produced, they need to be separated according to their masses, which must be determined. There are many different analyzers, just as there are a great variety of sources. This topic illustrates the technical principles and typical applications of the most common mass analyzers used in bioanalytical laboratories today. There are five basic types of mass analyzers found in modern MS: quadrupole analysers and a recent derivative quadrupole ion trap, time-offlight, magnetic sector and electric sector and ion cyclotron resonance. Rather than simply explaining the underlying physical principles, we concentrate on each mass analyzer's characteristic features, their limitations, and their applications. In addition, several unique techniques and unusual or exotic mass analyzer designs are highlighted throughout the text.

Glossary
1.

Angular frequency (): Angular frequency gives the frequency with which phase changes. The angular frequency is in radians per second. The unit Hertz (Hz) is in cycles per second where there are 2 radians per cycle. Resolution: Refers to the sharpness and clarity of an image Resolution in mass spectrometry refers to the separation of two ions where R=m/ m. These terms are defined several different ways. The most common are the 10% valley definition "Let two peaks of equal height in a mass spectrum at masses m and m- m be separated by a valley that at its lowest point is just 10% of the height of either peak." and the peak width definition "For a single peak made up of singly charged ions at mass m in a mass spectrum, the resolution may be expressed as m/ m, where m is the width of the peak at a height that is a specified fraction of the maximum peak height. Scan Rate: The scan rate of a mass spectrometer refers to how fast it scans a mass spectrum. This is important for chromatography applications where the entire mass spectrum must be scanned faster than the elution time of the chromatographic peak. Ideally, a minimum of ten complete mass spectra are acquired for a single chromatographic peak Signal to Noise Ratio: It is a measure of signal strength relative to background noise. The ratio is usually measured in decibels (dB). Unit resolution: Unit resolution of mass spectra distinguishes between ions separated by 1 m/z unit. The spectra, like those presented here, are commonly displayed as histograms. This is a common method for presenting spectra because it

2.

3.

4.

5.

37

results in much smaller data file size. Some mass analyzers can obtain spectra at much higher resolution.

Types of mass analyzers

Mass analyzers separate the ions according to their mass per charge (m/z). After ions are formed in the source region, they are accelerated into the mass analyzer by an electric field. The analyzer uses dispersion or filtering to sort ions according to their mass-to-charge ratios or a related property. The most widely used analyzers are magnetic sectors, quadrupole mass filters, quadrupole ion traps, Fourier transform ion cyclotron resonance spectrometers, and time-of-flight mass analyzers. The 1 selection of a mass analyzer depends up on the resolution, mass range, scan rate and detection limits required for an application. Each analyzer has very different operating characteristics and the selection of an instrument involves important trade offs. Analyzers are typically described as either continuous or pulsed.

Continuous analyzers: they include quadrupole filters and magnetic sectors. These analyzers are similar to a filter or mono-chromator used for optical spectroscopy. They transmit a single selected m/z to the detector and the mass spectrum is obtained by scanning the analyzer so that different mass to charge ratio ions is detected. While a certain m/z is selected, any ions at other m/z ratios are lost, reducing the Signal to Noise for continuous analyzers. Single Ion Monitoring (SIM) enhances the Signal to Noise by setting the mass spectrometer at the m/z for an ion of interest. Since the instrument is not scanned the S/N improves, but any information about other ions is lost. Pulsed mass analyzers: they are the other major class of mass analyzer. These are less common but they have some distinct advantages. These instruments collect an entire mass spectrum from a single pulse of ions. This results in a signal to noise advantage similar to Fourier transform or multi-channel spectroscopic techniques. Pulsed analyzers include time-of-flight, ion cyclotron resonance, and quadrupole ion trap mass spectrometers.

The different types of analysers are described below.

1. Quadrupole
The quadrupole is the most common and widely used analyzer due to its ease of use, mass range covered, good linearity for quantitative work, resolution and quality of mass spectra. The ion fragments are guided from the ion source into the quadrupole analyzer using lenses. Lenses are tiny tabs of metal or small, hollow cylinders that have a voltage running through them. By either attracting or repelling the ions, they guide the ions into the quadrupole analyzer. Quadrupole consists of four parallel poles or rods. Mass sorting depends on ion motion resulting from simultaneously applied constant (dc) and radio frequency electric (RF) electric fields. Scanning is accomplished by systematically changing the field strengths, thereby changing the m/z value that is transmitted through the analyzer. Quadrupole mass spectrometers provide lower resolution, but tend to be more easily interfaced to various inlet systems and to be less costly. Its compact size, fast scan rate, high transmission efficiency,2 and modest vacuum requirements are ideal for small inexpensive instruments. Most quadrupole instruments are limited to unit m/z resolution and have a mass range of m/z 1000. M any bench top instruments have a mass range of m/z 500 but research instruments are available with mass range up to m/z 4000.

Mass range refers to the highest mass to charge ratio transmitted by the mass spectrometer.

Transmission efficiency refers to how many of the ions produced in the source region actually reach the detector. This is an important measure of sensitivity for mass spectrometers.

38

A]

B]
Figure 6.1. Quadrupole Mass Analyser.

C]

A) Ion trajectory through the quadrupole, B) Ion focusing during first half of RF cycle, C) Ion focusing during second half of RF cycle.

The Radio Frequency (RF) and Direct Current (DC) voltages applied to the electrodes determine the m/z value, transmitted by the quadrupole. These voltages produce an oscillating electric field that functions as a band pass filter to transmit the selected m/z value. The RF voltage rejects or transmits ions according to their m/z value by alternately focusing them in different planes (Figure 6.1). The four electrodes are connected in p airs and the RF potential is applied between these two p airs of electrodes. During the first p art of the RF cycle the top and bottom rods are at a positive potential and the left and right rods are at a negative potential. This squeezes positive ions into the horizontal p lane. During the second half of the RF cycle the polarity of the rods is reversed. This changes the electric field and focuses the ions in the vertical p lane. The quadrupole field continues to alternate as the ions travel through the mass analyzer. This causes the ions to undergo a complex set of motions that produces a three-dimensional wave. The quadrupole field transmits selected ions because the amplitude of this three- dimensional wave depends up on the m/z value of the ion, the potentials applied, and the RF frequency. By selecting an appropriate RF frequency and potential, the quadrupole acts like a high pass filter, transmitting high
39

m/z ions and rejecting low m/z ions. The low m/z ions have a greater acceleration rate so the wave for these ions has greater amplitude. If this amplitude is great enough the ions will collide with the electrodes and cannot reach the detector. Adjusting the RF potential or the RF frequency changes the low m/z value cutoff of the quadrupole. Any ions with m/z greater than this cutoff are transmitted by the quadrupole. A DC voltage is also app lied across the rods of the analyzer. This potential combined with the RF potential acts like a low p ass filter to reject high m/z ions. Because they respond quickly to the changing RF field the motion of the low m/z ions is dominated by the RF potential. This motion stabilizes their trajectory by refocusing each time the RF potential changes polarity. Because low m/z ions are quickly refocused, the DC potential does not affect these ions. High m/z ions, however, do not refocus as quickly during the RF cycle. The DC potential has a greater influence on their trajectory and they slowly drift away from the center of the quadrupole. At the end of the analyzer, they are too far off-axis to strike the detector. The combination of high and low pass filters produced by the RF and DC potentials are adjusted to only transmit the selected m/z value. The quadrupole filter rejects all ions above or below the set m/z value. The RF and DC fields are scanned (either by potential or frequency) to collect a complete mass spectrum. Quadrupole mass analyzers are often called mass filters because of the similarity between m/z selection by a quadrupole and wavelength selection by an optical filter or frequency selection by an electronic filter. A series of three quadrupoles can be used; this is known as Triple Quadrupole mass spectrometry. The first and third quadrupoles are mass filters, and the middle one is a collision cell. This allows the study of fragments, useful in structural studies. For example, the first quadrupole may be set to "filter" for a drug ion of a known mass, which is fragmented in the second quadrupole. The third quadrupole can then be set to scan the entire m/z range, giving information on the sizes of the fragments made. Thus, the structure of the original ion can be deduced.

2. Quadrupole Ion Trap

The Quadrupole ion storage trap mass spectrometer is a recently developed mass analyzer with some special cap abilities. Several commercial instruments are available and this analyzer is becoming more popular. Quadrupole Ion Trap is very sensitive, relatively inexpensive, and scan fast enough for GC/M S experiments. The sensitivity of the Quadrupole Ion Trap results from trap ping and then analy zing all the ions produced in the source. Since all the ions are detected, the S/N is high. The quadrupole ion trap works on the same physical principles as the QMS, but the ions are trapped and sequentially ejected. Ions are created and trapped in a mainly quadrupole RF potential and separated by m/z, non-destructively or destructively. There are many mass/charge separation and isolation methods but most commonly used is the mass instability mode in which the RF potential is ramped so that the orbit of ions with a mass a > b are stable while ions with mass b become unstable and are ejected on the z-axis onto a detector. Ions may also be ejected by the resonance excitation method, whereby a supplemental oscillatory excitation voltage is applied to the endcap electrodes, and the trapping voltage amplitude and/or excitation voltage frequency is varied to bring ions into a resonance condition in order of their mass/charge ratio. The cylindrical ion trap mass spectrometer is a derivative of the quadrupole ion trap mass spectrometer. The Quadrupole Ion Trap consists of a doughnut shaped ring electrode and two end cap electrodes. A combination of RF and DC is applied to the electrodes to create a quadrupole electric field similar to the electric field for the quadrupole mass analyzer. This electric field trap s ions in a potential energy well at the center of the analyzer. The mass spectrum is acquired by scanning the RF and DC fields to destabilize low mass to charge ions. These destabilized ions are ejected through a hole in one end cap electrode and strike a detector. Scanning the fields so generates the mass spectrum that ions of increasing m/z value are ejected from the cell and detected. The trap is then refilled with a new batch of ions to acquire the next mass spectrum. Mass resolution of ion trap is increased by adding a small amount of Helium 0.1 Pa (10-3 torr), as a bath gas. Collisions between the analyte ions and the inert bath gas damp en the motion of the ions and increase the trap ping efficiency of the analyzer. The trap itself generally consists of two hyperbolic metal electrodes with their foci facing each other and a hyperbolic ring electrode halfway between the other two electrodes. The ions are trapped in the
40

space between these three electrodes by AC ~1MHz and DC (non-oscillating, static) electric fields. The AC radio frequency voltage oscillates between the two hyperbolic metal electrodes at the 'top' and 'bottom' of the trap ('top' and 'bottom' are in phase) and the hyperbolic ring electrode that forms the 'side' of the trap. The ions are first pulled up and down axially while being pushed in radially. The ions are then pulled out radially and pushed in axially (from the top and bottom). In this way the ions move in a complex motion that generally involves the cloud of ions being long and narrow and then short and wide, back and forth, oscillating between the two states Figure 6.5 shows the basic set up of a QIT mass analyzer. The ions, produced in the source of the instrument, enter into the trap through the inlet and are trapped through action of the three hyperbolic electrodes: the ring electrode and the entrance and exit end cap electrodes. Various voltages are applied to these electrodes, which results in the formation of a cavity in which ions are trapped. The ring electrode RF potential, an a.c. potential of constant frequency but variable amplitude, produces a 3D quadrupolar potential field within the trap. This traps the ions in a stable oscillating trajectory. The exact motion of the ions is dependent on the voltages applied and their individual mass-tocharge (m/z) ratios. For detection of the ions, the potentials are altered to destabilize the ion motions resulting in ejection of the ions through the exit end cap. The ions are usually ejected in order of increasing m/z by a gradual change in the potentials. This 'stream' of ions is focused onto the detector of the instrument to produce the mass spectrum

Figure 6.5: Schematic Diagram of Ion Trap Mass Analyzer The very nature of trapping and ejection makes a quadrupolar ion trap especially suited to performing MS experiments in structural elucidation studies. It is possible to selectively isolate a particular m/z in the trap by ejecting all the other ions from the trap. Fragmentation of this isolated precursor ion can then be induced by CID experiments. The isolation and fragmentation steps can be repeated a number or times and is only limited by the trapping efficiency of the instrument.

3. Time-of-flight
Time-of-flight mass analyzers separate ions by virtue of their different flight times over a known distance. A brief burst of ions is emitted from a source. These ions are accelerated so that ions of like charge have equal kinetic energy and then are directed into a flight tube. Since kinetic energy is equal to 1/2 mv2, where m is the mass of the ion and v is the ion velocity, the lower the ion's mass, the greater the velocity and shorter its flight time. The travel time from the ion source through the flight tube to the detector, measured in microseconds, can be transformed to the m/z value through the relationships described above. Because all ion masses are measured for each ion burst, TOF mass spectrometers offer high sensitivity as well as rapid scanning. They can provide mass data for very high-mass biomolecules. The time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight tube (Figure 6.4). This is a very simple mass spectrometer that uses fixed voltages and does not require a magnetic field. The greatest drawback is that TOF instruments have poor mass resolution, usually less than 500. These instruments have high transmission efficiency; no upper m/z
41

limit, very low detection limits, and fast scan rates. For some applications these advantages outweigh the low resolution. Recent developments in pulsed ionization techniques and new instrument designs with imp roved resolution have renewed interest in TOF-M S.

Figure 6.4. Time-of-Flight Mass Spectrometer. A time of flight mass spectrometer measures the mass-dependent time it takes ions of different masses to move from the ion source to the detector. This requires that the starting time (the time at which the ions leave the ion source) is well defined. Therefore, ions are either formed by a pulsed ionization method (usually matrix-assisted laser desorption ionization, or MALDI), or various kinds of rapid electric field switching are used as a 'gate' to release the ions from the ion source in a very short time. Recall that the kinetic energy of an ion leaving the ion source is . The ion velocity, v, is the length of the flight path, L, divided by the flight time, t: v =

L t

Substituting this expression for v into the kinetic energy relation, we can derive the working equation for the time-of-flight mass spectrometer:

m 2Vt 2 = 2 e L
m 1 e 2V

Or, rearranging the equation to solve for the time-of-flight: t = L

The ions leaving the ion source of a time-of-flight mass spectrometer have neither exactly the same starting times nor exactly the same kinetic energies (recall the "chromatic aberrations" discussed for magnetic sector mass spectrometers). Various time-of-flight mass spectrometer designs have been developed to compensate for these differences. A reflectron is an ion optic device in which ions in a time-of-flight mass spectrometer pass through a "mirror" or "reflectron" and their flight is reversed. A linear-field reflectron allows ions with greater kinetic energies to penetrate deeper into the reflectron than ions with smaller kinetic energies. The ions that penetrate deeper will take longer to return to the detector. If a packet of ions of a given mass-to-charge ratio contains ions with varying kinetic energies, then the reflectron will decrease the spread in the ion flight times, and therefore improve the resolution of the time-of-flight mass spectrometer.

42

A curved-field reflectron ensures that the ideal detector position for the time-of-flight mass spectrometer does not vary with mass-to-charge ratio. This also results in improved resolution for time-of-flight mass spectrometers

4. Magnetic sectors
Magnetic sectors bend the trajectories of ions into circular paths of radii that depend on the momentum-to-charge ratios of the ions. Ions of larger m/z follow larger radius paths than ions of smaller m/z values so ions of differing m/z values are dispersed in space. By changing the ion trajectories through variations of the magnetic field strength, ions of different nominal mass-tocharge ratios can be focused on a detector. The first mass spectrometer, built by J.J. Thompson in 1897, used a magnet to measure the m/z value of an electron. Magnetic sector instruments have evolved from this same concept. Sector instruments change the direction of ions that are flying through the mass analyzer, as shown in figure.6.2 The ions enter a magnetic or electric field which bends the ion paths depending on their mass-to-charge ratios (m/z), deflecting the more charged and faster-moving, lighter ions more. The analyzer can used to select a narrow range of m/z's or to scan through a range of m/z's to catalog the ions present. In a Magnetic Sector mass spectrometer, ions leaving the ion source are accelerated to a high velocity. The ions then pass through a magnetic sector in which the magnetic field is applied in a direction perpendicular to the direction of ion motion. From physics, we know that when acceleration is applied perpendicular to the direction of motion of an object, the object's velocity remains constant, but the object travels in a circular path. Therefore, the magnetic sector follows an arc; the radius and angle of the arc vary with different ion optical design

Figure 6.2: Magnetic Sector A magnetic sector will separate ions according to their mass-to-charge ratio, however, the resolution will be limited by the fact that ions leaving the ion source do not all have exactly the same energy and therefore do not have exactly the same velocity. To achieve better resolution, it is necessary to add an electric sector that focuses ions according to their kinetic energy. Like the magnetic sector, the electric sector applies a force perpendicular to the direction of ion motion, and therefore has the form of an arc. The simplest mode of operation of a magnetic sector mass spectrometer keeps the accelerating potential and the electric sector at a constant potential and varies the magnetic field. Ions that have a constant kinetic energy, but different mass-to-charge ratio are brought into focus at the detector slit at different magnetic field strengths. The dependence of mass-to-charge ratio on the electric and magnetic fields is easily derived. All ion formed in the ion source are accelerated to a kinetic energy, T of: t = eV =

mv 2 2
43

Solving for the velocity v we get: v =

2eV m

From the Lorentz force law, the magnetic field applies a force evB that must be equal to the centripetal force mv2/r as the ions move in an arc through the magnetic sector:

evB =

mv 2 r

Substituting for v, we arrive at the working equation for a magnetic sector mass spectrometer:

m B2r 2 = e 2V

As mentioned previously, the electric sector is usually held constant at a value, which passes only ions having the specific kinetic energy. V. Therefore the parameter that is most commonly varied is B, the magnetic field strength. The magnetic field is usually scanned exponentially or linearly to obtain the mass spectrum. A magnetic field scan can be used to cover a wide range of mass-to-charge ratios with a sensitivity that is essentially independent of the mass-to-charge ratio. The maximum ion transmission and sensitivity occur at the maximum working accelerating voltage for a given magnetic sector mass spectrometer. Decreasing the accelerating voltage, with a sensitivity that is roughly proportional to the accelerating voltage, can increase the effective mass range of the mass spectrometer. The resolving power of a magnetic sector mass spectrometer is determined by the slit widths. Decreasing the slit widths and thereby decreasing the number of ions that reach the detector obtain higher resolution. Sector instruments have higher resolution and greater mass range than quadrupole instruments, but they require larger vacuum p ump s and often scan more slowly .The typical mass range is to m/z 5000, but this may be extended to m/z 30,000. Older magnetic sector instruments use a photographic plate to simultaneously detect ions at different radii. Since each m/z has a different radius, they strike the photographic plate at a different location. Modern instruments have a set of slits at a fixed radius to transmit a single m/z to the detector. Changing the magnetic field or the acceleration voltage to transmit different m/z ions scans the mass spectrum. Some new instruments use multi-channel diode array detectors to simultaneously detect ions over a range of m/z values:

5. Electric Sector or Double focusing

Electric Sector or Double focusing mass spectrometers use a combination of magnetic and electrical fields to focus and sort ions. A common configuration for a sector instrument is the geometry shown in Figure 6.3, in which a magnetic "sector" follows an electric "sector". The slit acts as a filter to select for a specific m/z value. The electric sector focuses the ions with respect to differences in kinetic energy that they may have as they exit the source region. "Double focusing," this combination of "angular" or "directional" focusing and energy focusing, provide mass resolution high enough to separate ions of the same nominal mass but different chemical formulae. An electric sector consists of two concentric curved plates. A voltage is app lied across these plates to bend the ion beam as it travels through the analyzer. The voltage is set so that the beam follows the curve of the analyzer. The radius of the ion trajectory (r) depends up on the kinetic energy of the ion (V) and the potential field (E) applied across the plates, r =

2V E

The equation shows that an electric sector will not separate ions accelerated to a uniform kinetic energy. The radius of the ion beam is independent of the ion's mass to charge ratio so the electric sector

44

is not useful as a standalone mass analyzer, however electric sector3 is, useful in series with a magnetic sector. An electric sector significantly improves the resolution of the magnetic sector by reducing the kinetic energy distribution of the ions4. These high resolutions experiments are discussed in the section on mass spectral interpretation. The effect of the electric sector is shown in Figure 11 for a reverse geometry (BE) instrument with the magnetic sector (B) located before the electric sector (E).

Figure 6.3. Reverse Geometry Double Focusing Mass Spectrometer. A+ is a m/z 100.00 ion, B+ isa m/z

of 50.00 and C+ is a m/z of 50.01. A+ is rejected by the magnetic sector because of it's greater momentum. B+ and C+ are not resolved by the magnetic sector because they have the same momentum. To have the same momentum, however, B+ must have a more kinetic energy than C+ . As a result the electric sector separates these two ions.

6. Ion Cyclotron Resonance

In Ion Cyclotron Resonance ions are trapped electrostatically within a cubic cell in a constant magnetic field. A covalent orbital ("cyclotron") motion is induced by the application of a radiofrequency pulse between the excite plates. The orbiting ions generate a faint signal in the detect plates of the cell. The frequency of the signal from each ion is equal to its orbital frequency, which in turn is inversely related to its m/z value. The signal intensity of each frequency is proportional to the number of ions having that m/z value. The signal is amplified and all the frequency components are determined, yielding the mass spectrum. If the pressure in the cell is very low, the ion orbital motion can be maintained over many cycles and the frequency can be measured with very high precision. The instrument can therefore be used to generate very high-resolution spectra. Fourier transform mass spectrometry or more precisely Fourier transform ion cyclotron resonance mass spectrometry measures mass by detecting the image current produced by ions cyclotroning in the presence of a magnetic field. Instead of measuring the deflection of ions with a detector such as a electron multiplier, the ions are injected into a Penning trap (a static electric/magnetic ion trap) where they effectively form part of a circuit. Detectors at fixed positions in space measure the
3

The electric sector is a kinetic energy analyzer. In the source region of the mass spectrometer all ions are accelerated to the same kinetic energy. Because they have the same kinetic energy, they are not separated by an electric sector. A magnetic sector will resolve different mass ions accelerated to a uniform kinetic energy because it separates ions based upon their momentum (See eqs 1-3). Ion optics are complex and interested readers are referred to the literature for more detail. The model presented here provides a framework for understanding many high resolution and tandem mass spectrometry experiments. The article by Nier (25 ) provides an excellent introduction, a historical perspective, and many references for the development and theory of these instruments.

45

electrical signal of ions which pass near them over time producing cyclical signal. Since the frequency of the ions' cycling is determined by its mass to charge ratio, this can be deconvoluted by performing a Fourier transform on the signal. FTMS has the advantage of improved sensitivity (since each ion is 'counted' more than once) as well as much higher resolution and thus precision. Ion cyclotron resonance is an older mass analysis technique where ions are detected with a traditional detector. Ions trapped in a Penning trap are excited by an RF electric field until they impact the wall of the trap where the detector is located with ions of different mass being resolved in time.

Figure 6.6: The Ion Cyclotron Resonance mass spectrometer

The Ion Cyclotron Resonance mass spectrometer uses a superconducting magnet to trap ions in a small sample cell. This ty p e of mass analyzer has extremely high mass resolution (ca. 109 ) and is also useful for tandem mass spectrometry experiments. These instruments are very expensive and are typically used for specialized research applications. The ICR trap s ions in a magnetic field that causes ions travel in a circular path (Figure 6.6). This is similar to the path of an ion in a magnetic sector, but the ions are not traveling as fast and the magnetic field is stronger. As a result the ions are contained in the small volume of the trap .The ions cy clotron frequency () is the angular frequency of an ion's orbit. This frequency is determined by the magnetic field strength (B) and the m/z value of the ion.

Be m z

After ions are trap ped in this cell they are detected by measuring the signal at this cyclotron frequency. This signal is measured by p lacing electrodes on each side of the ions circular orbit. An RF voltage is applied to the transmitter electrodes at the cy clotron frequency of the ion of interest. This RF energy moves ions at the app lied frequency to a larger orbit. As these ions travels around the ICR cell they are close enough to the receiver electrodes to induce a capacitive current. This capacitive current oscillates at the cyclotron frequency and is detected as the signal. The ICR is also used as a Fourier Transform M ass Spectrometer (FT-M S). Instead of using a single excitation frequency, a fast RF pulse is applied to the transmitter electrodes. This simultaneously excites all the ions and produces a signal at the cyclotron frequency of each m/z ion p resent. A complete mass spectrum is obtained by using the Fourier transform to convert this signal from the time domain to the frequency domain. If several different masses are present, then one must apply an excitation pulse that contains components at all of the cyclotron frequencies. This is done by using a rapid frequency sweep ("chirp"), an "impulse" excitation, or a tailored waveform. The image currents induced in the receiver plates will contain frequency components from all of the mass-to-charge ratios. The various frequencies and their relative abundances can be extracted mathematically by using a Fourier

46

transform, which converts a time-domain signal (the image currents) to a frequency-domain spectrum (the mass spectrum). Most FTICR mass spectrometers use superconducting magnets, which provide a relatively stable calibration over a long period of time. Although some mass accuracy can be obtained without internal calibrant, mass accuracy and resolution are inversely proportional to m/z, and the best accurate mass measurements require an internal calibrant. Unlike the quadrupole ion trap, the FTICR mass spectrometer is not operated as a scanning device.

Summary
Mass analyzers separate the charged species in the ionized sample according to their m/z ratios, thus permitting the mass and abundance of each species to be determined. Six commonly used analyzers are the magnetic sector, electric sector, the quadrupole, the time of flight, Quadrupole ion trap and the Fourier transform analyzers. Scanning analyzers transmit ions of different masses successively along a time scale. These are the most frequently encountered in organic analysis. They are either magnetic sector instruments with a flight tube in the magnetic field, allowing only the ions of a given mass-to charge ratio to go through at a time, or a quadrupole instruments. Although mass analysers are generally scanning devices some allow the simultaneous transmission of all the ions, such as depressive magnetic analysers, the time-of-flight analysers, the ion trap or the ion cyclotron resonance instruments. The time of flight mass analysers require the ions to be produced in bundles and thus is easily well suited for pulsed laser sources

47

7. Quadrupole Operations
Monitoring the Ions
Introduction
The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. In a quadrupole mass spectrometer the quadrupole mass analyzer is the component of the instrument responsible for guiding sample ions to a detector, based on their mass/charge ratio (m/z). A quadrupole mass analyzer is essentially a mass filter that is capable of transmitting only the ion of choice. A mass spectrum is obtained by scanning through the mass range of interest over time. Quadrupole operations means application of Quadrupole where particular ions of interest are being studied because they can stay tuned on a single ion for extended periods of time. One place where this is useful is in liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry where they serve as exceptionally high specificity detectors. Quadrupole has a multiple scan modes options which are very useful to all mass spectrometer users.

Glossary
1.

Full Scan: A method of running a mass spectrometer so that the intensities of ions in a given range of masses are acquired. This method is used to obtain maximum information about a sample. MS/MS: Type of mass spectrometry in which there are two separate mass analyzer regions. Capable of operating in the normal, full scan and MI modes (using either analyzer) and in combined experiments where one mass (precursor) is selected in the first analyzer (Q1), fragmented in the collision cell, and the product ions analyzed by the second analyzer (Q3). Multiple Reaction Monitoring (MRM): Mode of operating an MS/MS system such as a triple quadrupole so that an ion of given mass (a precursor) must fragment (dissociate) to give a product ion of specific mass in order for a response to be detected. Product Ion: An MS/MS analysis where the quadrupole is fixed and the analyses a specified mass range. Q1 scan: A data acquisition using the quadrupole mass filter. The ion intensity is returned for every requested mass in the scan range. Scan Type (Quadrupole analyzer): A means of operating the instrument that implies the configuration of the quadrupoles, i.e. the resolution for each of the quadrupoles and also which quadrupole is analyzing and which is tracking. The available scan types are:Q1 scan , Q3 scan, Q1 MI, Q3 MI, MRM, Product ion scan, Precursor ion scan, Neutral loss/gain.

2.

3.

4.

5.

6.

48

7.

Single Ion Mode (SIM): A mode of running a mass spectrometer so that only certain expected masses are acquired Total Ion Current (TIC): A measure of the number of ions entering a mass spectrometer.

8.

Modes of acquiring and visualizing LC/MS data

Typically the mass spectrometer is set to scan a specific mass range. This mass scan can be wide as in the full scan analysis or can be very narrow as in selected ion monitoring. A single mass scan can take anywhere from 10 ms to 5 ms depending on the type of scan. Many scans are acquired during an LC/MS analysis. LC/MS data is represented by adding up the ion current in the individual mass scans and plotting that "totaled" ion current as an intensity point against time. The quadrupole can be used in two modes: 1 SIM (single ion monitoring) Scan: The SIM mode is also called SIR (Single Ion Recording). In SIM mode, the parameters (amplitude of the dc and rf voltages) are set to observe only a specific mass, or a selection of specific masses. This mode provides the highest sensitivity for users interested in specific ions or fragments, since more time can be spent on each mass. The term Selected Ion Monitoring is used to describe the operation of the mass spectrometer whereby the intensities of one or more specific ion beams are recorded rather than the entire mass spectrum. Maximum sensitivity & precision is obtained under SIM conditions and this mode of operation is used in quantitative analysis.

2 Full Scan mode: The amplitude of the dc and rf voltages are ramped (while keeping a constant rf/dc ratio), to obtain the mass spectrum over the required mass range. The sensitivity is a function of the scanned mass range, scan speed, and resolution. With most LC/MS instrument, it is possible to do positive/negative switching, in order to analyze in the same run molecules that will ionize in positive and negative modes.

Condition required for obtaining different Scan modes

Ions (of interest) must move from source to analyzer (different physical regions / Quadrupoles) where different functions take place to obtain the scan of interest.(Fig.7.1)

Fig.7.1 Below we will discuss the modes of scanning and acquiring MS data and their relevance to MS quantitation.
49

The most common modes of acquiring LC/MS data are:

1. 2. 3. 4. Full scan acquisition resulting in the typical total ion current plot (TIC) Selected Ion Monitoring(SIM) Product Ion mode Selected Reaction Monitoring (SRM) or multiple reactions monitoring (MRM). MRM and SRM are essentially the same experiment.

Fig.7.2

Different types of Quadrupole Operations

1.Single Quadrupole Operations: It is simple mass spectrometer made by combining the basic elements of ionization, ion transfer to vacuum, mass filtering and detection. We can operate it in both i.e. Full Scan and SIM Scan mode. Single quads provide low sensitivity and low selectivity.

1.1 Full Scan Analysis

Compounds of every mass are plotted in the TIC plot above. Finding the compound of interest can be difficult since many compounds have the same mass. The intact mass of a compound is not a unique identifier. Using the data set above a specific mass can be selectively plotted, however the sensitivity will be less than what is observed in the SIM experiment (Fig.7.3 & 7.4)

In this mode the full mass range of interest is set in Q1. All the masses in this range are passed from Q1 to detector. After detection the graph of Intensity vs. Time is plotted.

50

Q0

Fig.7.3

The total ion current full mass range plot from a PK analysis is a wild place. The MS total ion current plot is a plot much like an HPLC, UV traces except for the fact that the mass spectrometer can detect many more components, UV transparent components. The total ion current is a plot of the total ion current in each MS scan plotted as an intensity point.

Fig. 7.4

1.2 Selected Ion Monitoring:

The term Selected Ion Monitoring is used to describe the operation of the mass spectrometer whereby the intensities of one or more specific ion beams are recorded rather than the entire mass spectrum. (Fig.7.5 & 7.6)

In selected ion monitoring the mass spectrometer is set to scan over a very small mass range, typically one mass unit. The narrower the mass range the more specific the SIM assay. The SIM plot is a plot of the ion current resulting from this very small mass range. Only compounds with the selected mass are detected and plotted. The plots in Figures 1 and 2 can be from the very same sample however the plots look very different. The reason for this is because the peaks seen in the SIM plot may only be very minor components in the TIC plot above. The SIM plot is a more specific plot than the full scan TIC plot.

51

Q0

Fig. 7.5

Fig.7.6 2 Triple Quadrupole Operations: The analyzer of a triple quad instrument consists in two quadrupoles, separated by a collision cell. Such a configuration is often referred as a "tandem in space" instrument.(Shown in Fig 7.7) We can acquire the data in Full scan and SIM mode in the same manner as in Single Quadrupole for Triple Quadrupole also.

Fig 7.7 Quadrupole Analyzer But with Triple Quadrupole we can have some advanced scans.

Product Ion Scan: - Provides structural information. Precursor Ion Scan: - Provides structural information complementary to Product Ion Neutral loss scan: - Provides compound class specificity.
52

MRM: - Used for quantitation.

Note: In all MS/MS modes, 1) Q3 is a mass filter 2) Q3 may scan or be fixed at a given m/z 3) Q2 is a source of Product Ions entering in Q3

2.1 Product Ion Scan

Product or daughter ion scanning: the first analyser is used to select user-specified sample ions arising from a particular component; usually the molecular-related (i.e. (M+H) + or (M-H)-) ions(Fig7.8). These chosen ions pass into the collision cell, are bombarded by the gas molecules which cause fragment ions to be formed, and these fragment ions are analysed i.e. separated according to their mass to charge ratios, by the second analyser. All the fragment ions arise directly from the precursor ions specified in the experiment, and thus produce a fingerprint pattern specific to the compound under investigation. This type of experiment is particularly useful for providing structural information concerning small organic molecules and for generating peptide sequence information

Q1fixed

Q3 scanned Q3 Scan Fig 7.8 Product Ion Scan

Q1 set to transmit 1 m/z ratio

In Short,

It Allows Selection of single parent ion in Q1 It is followed by collision Induced dissociation in Q2 Q3 is set to full scan for daughter ions formed in Q2 It provides structural information about parent ion

2.2 Precursor Ion Scan

The first analyser allows the transmission of all sample ions, whilst the second analyser is set to monitor specific fragment ions, which are generated by bombardment of the sample ions with the collision gas in the collision cell.(Fig 7.9)
53

This type of experiment is particularly useful for monitoring groups of compounds contained within a mixture which fragment to produce common fragment ions, e.g. Glycosylated peptides in a tryptic digest mixture, aliphatic hydrocarbons in an oil sample, or Glucuronide conjugates in urine.

Q1 scanned Q1 Scan Q3 set to a specific m/z ratio Q2 is pressurized with N2

Q3 fixed

Fig. 7.9 Precursor Ion Scan

2.3 Neutral Loss Scan

When both analysers are scanned with a fixed mass difference (MS1 higher than MS2) only those ions that lose that specific mass in the collision cell will be detected. It provides compound class specificity.(Fig.7.10) e.g. this type of experiment could be used to monitor all of the carboxylic acids in a mixture. Carboxylic acids tend to fragment by losing a (neutral) molecule of carbon dioxide, CO2, which is equivalent to a loss of 44 Da or atomic mass units. All ions pass through the first analyser into the collision cell. The ions detected from the collision cell are those from which 44 Da have been lost.

Q1 scanned

Q3 scanned

Q1 Scan and Q3 are scanned simultaneously Q2 is pressurized with N2 Fig. 7.10 Neutral Loss Scan

2.4 Selected/Multiple Reactions Monitoring (MRM) Scan

Both of the analyzers are static in this case as user-selected specific ions are transmitted through the first analyzer and user-selected specific fragments arising from these ions are measured by the second analyzer. (Fig7.11) The compound under scrutiny must be known and have been well-characterized previously before this type of experiment is undertaken. This methodology is used to confirm unambiguously the presence of a compound in a matrix e.g. drug testing with blood or urine samples. It is not only a highly specific method but also has very high sensitivity The SRM experiment is accomplished by specifying the parent mass of the compound for MS/MS fragmentation and then specifically monitoring for a single fragment ion. One could think of this operation as the SIM of a fragment ion. The specific experiment in known as a "transition" and can be written (parent mass fragment mass). If we plot the graph of Intensity vs. Time.
54

(A-Y) (B-W) Q1 fixed Q1 Scan and Q3 are scanned simultaneously Q2 is pressurized with N2

(C-U) Q3 fixed

Fig. 7.11 Selected/Multiple Reaction Monitoring Scan

Fig.7.12 Graph of Intensity vs. Time for MRM/SRM In short,

It Allows Selection of single parent ion in Q1 It is followed by collision Induced dissociation in Q2 Q3 is used to select single daughter ion It provides unmatched selectivity and ultimate sensitivity It essentially eliminates background noise

Summary
Quadrupole Analyzer has been shown to be a versatile technique of high selectivity and high specificity. The theory of Quadrupole operation differs from those of other mass spectrometer analyzers and presents an exciting challenge & scope of analysis scan to the mass spectrometry community; it is hoped that this quadrupole operation scans will be very useful to its application in various fields.

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8. DETECTORS

Eyes of LCMS
Introduction
This chapter gives emphasis on-

Characteristics of detectors, Types of detectors explaining about the working and principle of each type of the detector.

Detection of ions is based up on their charge or momentum. For large signals a faraday cup is used to collect ions and measure the current. Older instruments used photographic plates to measure the ion abundance at each mass to charge ratio. Most detectors currently used amplify the ion signal using a collector similar to a photomultiplier tube. Mass analysis is the separation of bunches or streams of ions according to their individual mass-tocharge (m/z) ratio. Thus mass spectrometer consists of three processes: ion generation, ion separation and ion detection. The ion source generates ions, the mass analyzer sorts the ions according to their m/z ratio and the detector records the abundance of each m/z. So once the ion passes through the mass analyzer, it is then detected by the ion detector-the final element of the mass spectrometer. The detector allows a mass spectrometer to generate a signal current from incident ions by generating secondary electrons, which are further amplified. Alternatively, some detectors operate by inducing a current generated by a moving charge. Among the detectors described, the electron multiplier and scintillation counter are the most commonly used and convert the kinetic energy of incident ions into a cascade of secondary electrons.

Glossary
Some common terms used frequently for Data Interpretation:
1.

Analog Signal: Analog means 'copy'. An analog electronic signal is an electronic copy of an original signal as found in nature. For instance, an analog audio signal

56

follows the same pattern as the vibration in air pressure caused by the original sound, or a copy of the vibration of your eardrum as the air pressure hits it.
2.

Anode: (from the Greek word meaning 'going up') Is the electrode in a device that electrons flow out to return to the circuit. For electrons to flow through the anode a positive charge is applied to the anode (attracting electrons). Cathode: (Greek word meaning, 'going down') Is the electrode at which electrons go into a cell, tube or diode whether driven externally or internally. The flow of electrons is always from anodetocathode outside of the cell or device and from cathodetoanode inside the cell or device regardless of the cell or device type. Digital Signal: A digital signal is a signal that is both, discrete and quantized. Dynode: Is one of a series of electrodes within a photomultiplier tube. Each dynode is more positively charged than its predecessor. Secondary emission occurs at the surface of each dynode. Such an arrangement is able to amplify the tiny current emitted by the photocathode by typically one million

3.

4. 5.

Methods of ion detection

The detectors purpose is to translate the ion arrival into an electric signal measured by the electronic system of the mass spectrometer. The detector generates a signal from incident ions by two ways:

1. 2.

By inducing a current generated by a moving charge or By generating secondary electrons.

1. Current generated by a moving charge Only one ion stream makes it right through the machine to the ion detector. The other ions collide with the walls where they will pick up electrons and get neutralized. Eventually, they get removed from the mass spectrometer by the vacuum pump.

Figure 8.1: Mechanism showing current generation by a moving charge. When this specified ion stream hits the metal box, an electron jumping from the metal on to the ion neutralizes the charge of the ion. That leaves a space amongst the electrons in the metal, and the
57

electrons in the wire shuffle along to fill it, and during shuffling there is a flow of electrons. A flow of electrons in the wire is detected as an electric current, which can be amplified and recorded. The more ions arriving, the greater is the current. The mass of each ion being detected is related to the size of the magnetic field used to bring it on to the detector. An ion stream with a small value of m/z, to bring them on to the detector, they should be less deflected - by using a smaller magnetic field. To bring an ion stream with a larger m/z value on to the detector they should be deflected more by using a larger magnetic field. Thus if magnetic field is varied, each ion stream can be brought in to the detector to produce a current which is proportional to the number of ions arriving. The timing mechanisms, which integrate those signals with the scanning voltages, allow the instrument to report which m/z strikes the detector. Regular calibration of the m/z scale is necessary to maintain accuracy in the instrument. Introducing a well-known compound into the instrument and altering the circuits so that the compounds molecular ion and fragment ions are reported accurately perform calibration. 2. Current generated by secondary electrons Here the detector operates by producing a signal current from incident ions by generating secondary electrons, which are further amplified. The key part of such type of detectors is a dynode. Dynode is electron-multiplying electrode.

Figure 8.2: Mechanism of secondary electron generation Electrons are accelerated and caused to strike the surface of electrode or dynode. The energy deposited by the incident electrons can result in re- emission of more than one electron from the same surface. Electrons within the dynode material are excited by the passage of the energetic electron, originally incident on the surface, converting the kinetic energy of incident ions into a cascade of secondary electrons. It is possible for one electron incident on the first dynode to produce about 30 excited electrons per 100 V of accelerating voltage. Further amplification of these secondary electrons is done by using several dynones placed in series.

Characteristics of ion detectors

The characteristics of an ideal MS detectors are unity ion detection efficiency, low or no noise, simultaneous detection, wide mass range response, wide dynamic range, short recovery time and fast response, few operational attributes should be long life, low maintenance, low replacement cost etc. Certain of these characteristics are common to all detectors, such as high sensitivity and linear, quantitative response. However, some detectors are designed for very specific functions or applications. Many MS detectors must be able to perform at very high-count rates (>106 counts/s) with a minimal recovery time. TOF instruments require detectors with very rapid readout and response. Isotope-ratio mass spectrometers require highly stable, multiple, inter calibrated detectors that count or measure several ion masses simultaneously to provide extremely high precision ratio measurements. Low noise is of course required for good limits of detection, sensitivity, accuracy,
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and precision. Unfortunately, several sources of noise exist, and they may vary for the different types of detectors. These can arise from within the detector itself, its supporting electronics, the instrument, and the ever-present external sources. Biological MS demands an ability to uniformly detect ions in the range 102105 m/z. With conventional secondary-electron MS detectors, mass response falls off dramatically for larger ions, resulting in poorer detection and analytical performance for many macromolecules. This limitation is particularly severe for MALDI MS, in which intact, singly charged bio-molecules are often measured. Another distinction among detector types is the ability to count single or multiple masses. Many MS detectors are fixed-point detectors that only monitor single ion masses or sequentially scanned masses.

Types of Detectors
They are broadly categorized into two categories on the basis of their method of detection
1.

Photographic plate and Faraday cage detectors: These allow a direct measurement of the charges that reach the detector. These includes the following1.1 Photographic Plate Detector 1.2 Faraday Cup Detector

2.

Electron/ Photon Multiplier and Array detectors: These types increase the intensity of the signal and then detects. These includes the following2.1 Electron Multiplier 2.2 Channel Electron Multiplier 2.3 Microchannel Plate Ion Detector 2.4 Photomultiplier Detector

Details of Each Detectors

1.1 Photographic Plate Detector

The photoplate MS detector dates back to J. J. Thompsons work in which photographs of luminescent screens were first used to visualize and display mass spectra. Later, ion-sensitive photoplates were placed in the vacuum region of the MS instrument for direct ion detection. Various designs of the photographic plate concept were used for many years. New photographic plate instruments (e.g., SS MS) were still being produced into the 1980s, and some are still in use today. Not only was the photographic plate among the earliest ion detectors, it was and still is the largest ion detector. Ion-sensitive photo plates were placed in the vacuum region of the MS instrument for direct ion detection. The ion currents were recorded by allowing them to strike a photographic plate, which is subsequently developed. Ions of a given m/z would impact at the same place on the photographic plate making a spot. The darkness of the spot in the photoplate indicates the intensity of the ion current. MS instruments, the ions are dispersed along a rather lengthy focal plane (~25 mm); this provides adequate unit mass resolution across the entire spectral region. The photoplate thus offers simultaneous detection and signal integration features. Disadvantages for this type of detector are many, however, including poor-to-moderate sensitivity, a short linear dynamic range, off-line image development (in a photo lab or darkroom), off-line calibration (with an optical densitometer), poor precision and quantization, fragility, and (these days) a shortage of commercial photoplate suppliers.

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Figure 8.3: Illustrates a Photoplate with ion exposures

1.2 Faraday Cup Detector

The Faraday cup or cylinder electrode detector is very simple, a Faraday cup is a metal cup, it has a hollow collector, open at one end and closed at the other end, used to collect beams of ions. It is attached to an electrometer, which measures the ion-beam current.

Figure 4: Faraday Cup A Faraday cup operates on the basic principle that a change in charge on a metal plate results in a flow of electrons and therefore creates a current. One ion striking the dynode surface of the Faraday cup induces several secondary electrons to be ejected and temporarily displaced. This temporary emission of electrons induces a current in the cup and provides for a small amplification of signal when an ion strikes the cup. This detector is relatively insensitive, yet robust and simple in design. The dynode electrode is made of a secondary emitting material like CsSb, GaP or BeO. The Faraday cup is a relatively insensitive detector but is very robust. The outermost layer of the cup is a stainless steel shell, which serves to contain the inner components. A thin wire connects the inner components to a current meter. Since all the electrons in the beam are captured inside the insulating layer, and they have nowhere else to go but through the wire and into the current meter, a very precise measurement of the current in the accelerator beam can be made. When electrons hit the sample, some are back scattered, and some are absorbed. The relative proportion of back- scattered and absorbed electrons depends on the nature of the sample material. Light materials have low backscatter coefficients; heavy materials, like gold, backscatter a large proportion of the beam. Since a Faraday cup can only be used in an analog mode it is less sensitive than other detectors that are capable of operating in pulse-counting mode.
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2.1 Electron Multipliers

An electron multiplier is one of the most common means of detecting ions, especially when positive and negative ions need to be detected on the same instrument and achieving high sensitivity by extending the principle used with a Faraday cup. Whereas a Faraday cup uses one dynode, an electron multiplier is made up of a series of dynodes maintained at ever increasing potentials. Ions strike the dynode surface, resulting in the emission of electrons. These secondary electrons are then attracted to the next dynode where more secondary electrons are generated, ultimately resulting in a cascade of electrons. Typical amplification or current gain of an electron multiplier is one million.

Figure 8.4:Electron Multiplier - A series of dynodes at increasing potential produce a cascade of electrons. The task of the electron multiplier is to detect every ion of the selected mass passed by the mass filter. How efficiently the electron multiplier carries out this task represents a potentially limiting factor on the overall system sensitivity. Consequently the performance of the electron multiplier can have a major influence on the overall performance of the mass spectrometer. Electron multipliers exist in two major modifications: 2.1.1 Multiple-dynode types : It has series of dynodes maintained at increasing potentials resulting in a series of amplifications 2.1.2 Continuous-dynode types : It has a curved ('horn' shaped) continuous dynode where amplifications occur through repeated collisions with the dynode surface. In both cases, ions pass the conversion dynode (depending on their charge) and strike the initial amplification dynode surface producing an emission of secondary electrons, which are then attracted either to the second dynode, or into the continuous dynode where more secondary electrons are generated in a repeating process ultimately resulting in a cascade of electrons. The immediate advantage of electron multipliers is that, it has very high current gain, so it is sensitive and fast but the multiplier has a finite lifetime. It also requires a good vacuum to operate. Operation at pressures above 10-6 torr decreases the useful lifetime of the multiplier. Electron multipliers are widely used in Quadrupole and Ion trap Instruments.

2.1.1

Multiple-dynode type Electron Multiplier

It has series of dynodes maintained at increasing potentials resulting in a series of amplifications. Each incoming electron that reaches the first dynode ejects several other electrons by secondary emission. This process of multiplication is repeated at each succeeding dynode by applying a higher potential to it than to the preceding dynode. When the flow arrives at the anode, the electron flow is significantly amplified and can be measured.

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Figure 8.5: Electron Multiplier - Multiple-dynode - Type has series of dynodes maintained at increasing potentials

2.1.2 Continuous-dynode type Electron Multiplier

Figure 8.6: The Continuous dynode- Type has horn shaped tube where amplifications occur through repeated collisions with the dynode surface. The continuous-dynode type electron multiplier contains a glass pipe with a coating on its inner surface. This coating is made of a resistive material that provides a secondary emission for incoming electrons. The electron flow moves along the pipe, reflecting from the inner wall and progressively gaining electrons. The high voltage applied across the two ends of the pipe forms the electrical field accelerating the flow of electrons. The output signals are interpreted. There are several designs available, which are all based on the same accelerate-and-hit principle of electron multipliers. However, the dynodes may be replaced by a layer of metal oxides (lead and tin oxide) in a glass tube (channeltron), or by coated channels within a glass construction (channel plate). All these designs have in common that the voltage across the pathway of the electrons determines the overall gain. The user can adjust the voltage between the dynodes of the EMT in order to see the avalanche effect. An EMT has CuBeO dynodes for producing gain via secondary emission. The dynodes are connected by a built-in voltage divider (1M ohm per stage) and are supplied in an evacuated glass bulb. The first dynode can be replaced by a photocathode of CsI or KBr for use in VUV photometry. Electron multipliers are used in applications such as mass spectroscopy, field ion microscopy etc. Electron multipliers have high gain and low noise, making them suitable for the detection of very small or low energy particles by using the counting method. The multiplier tube is curved so as to prevent "ion feedback." Ion feedback occurs when residual gas molecules are inadvertently ionized and accelerated so they produce ions. A curved structure helps prevent this. By generating a large number of electrons, the electron multiplier amplifies the signal that was initially sent to the detector.

2.2 Channel Electron Multiplier

Channel Electron Multipliers have become the industry choice of the mass spectrometer industry. A channel electron multiplier is a modified continuous dynode type electron multiplier. It is comprised of "the channel," a hollow, cornucopia-shaped tube made of semi conductive glass.
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Figure 8.7: Electron flow in a channel electron multiplier The primary incoming ion passes through the inlet aperture and strikes the surface of the channel electron multiplier. If the energy of the collision is sufficient, at least one electron is ejected from the CEM wall. The ejected electrons are accelerated into the interior of the CEM by the local electric field developed by the bias voltage. If the magnitude of the bias voltage is sufficient, the accelerated electrons acquire enough energy to trigger more secondary electrons when they strike the CEM surface again. This process produces an avalanche of electrons, which are detected. The detection efficiency of a CEM with respect to a particle is defined as the probability of this particle or photon producing an output pulse. CEM can be used in a variety of analytical instruments and applications, including GC/MS, LC/MS, residual gas analyzers and other instrumentation. The high performance and long life electron multipliers are engineered for maximum reliability, from low gain analog amplification to high gain pulse counting requirements. If you want to do purely particle counting or current amplification then the channel electron multiplier is the device to use. The utility of channel electron multipliers as detectors in combination with integrating amplifiers and with ion counting techniques for organic mass spectrometry has been reported. There are two types of channel electron multipliers, analogue and pulse counting. The analogue multipliers have been designed for mass spectrometer. They have considerably lower resistance from the conventional pulse counting types and give maximum collection efficiency at the output of the multiplier. Most of this series of multipliers are provided with a collector assembly and have a mount that is specifically designed for mounting into the spectrometer system.

2.3 Microchannel plate ion detector

A Microchannel plate is a specially fabricated plate that amplifies electron signal similar to electron multiplier. Unlike electron multiplier, Microchannel plate has several million independent channels and each channel works as independent electron multiplier. It consists of an array of glass capillaries (10-25 um inner diameter) that are coated on the inside with an electron-emissive material. In other words, one can imagine Microchannel plate as an assembly of millions miniature electron multiplier. Microchannel plate consists of a two-dimensional periodic array of very-small diameter glass capillaries (channels) fused together and sliced in a thin plate. The capillaries are biased at a high voltage, an ion that strikes the inside wall one of the capillaries creates an avalanche of secondary electrons. This cascading effect creates and produces a current pulse at the output.

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Figure 8.8: A Microchannel Plate Ion Detector - An electron flow inside a single capillary A single incident particle (ion, electron, photon etc.) enters a channel and emits an electron from the channel wall. Secondary electrons are accelerated by an electric field developed by a voltage applied across the both ends of the Microchannel plate. They travel along their parabolic trajectories until they in turn strike the channel surface, thus producing more secondary electrons. This process is repeated many times along the channel; as a result, this cascade process yields a cloud of several thousand electrons, which emerge from the rear of the plate. If two or more Microchannel plate is operated in series, a single input event will generate a pulse of 108 or more electrons at the output. Microchannel Plates have a combination of unique properties like high gain, high spatial resolution and high temporal resolution. They can be used in a large variety of applications including, imaging spectroscopy, electron spectroscopy and microscopy, mass spectrometry etc. Most of these applications require only some of Microchannel Plates properties, for example Time-of-Flight Mass Spectrometry require high temporal resolution of Microchannel Plates, imaging of single atoms in field ion microscopes or X-ray imaging of the Sun require mainly spatial resolution. Detectors based on Microchannel Plates have variety of designs depending on the type of particles detected, throughput (counts/second), time and position resolution, imaging area, linearity and sensitivity, signal to noise ratio and other requirements. It's a challenge to detector developer to optimize detector design for particular application. In general, each detector that uses MCPs consists of three parts:

A Converter - it is responsible for conversion of initial particles into electrons that in turn efficiently interact with a Microchannel Plate. An Assembly of Microchannel Plates - a mechanism to amplify initial single electron or photon event into electron pulse and A Readout Device - a mechanism to detect the electron avalanche. For detection and particle counting applications where position resolution is not required, single metal anode can be used as a readout device. Microchannel Plates detectors with metal anode are widely used in mass-spectrometry.

Microchannel plate (MCP) detectors are widely used for detection and imaging of the electrons, ions, UV radiation, and soft X-ray fluxes. The MCP detectors provide high spatial and temporal resolution, direct conversion, high physical charge amplification, low noise, and pulse-counting capabilities. Advantages of this type of a detector are that is very fast and gives a high gain. But have a very poor resolution and have a short lifetime and very expensive.

2.4 Photomultiplier Detector

In the photomultiplier is similar to an electron multiplier. It is also called a Daly detector or scintillation counter. It has three parts; the dynode, a phosphor screen and a Photomultiplier tube.

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Figure 8.9: A Photomultiplier Detector - Showing the conversion of the ion signal into photon(s) which are then amplified and detected by the photomultiplier.

The ions initially strike a dynode, which results in electron emission. These electrons then strike a phosphorus screen, much like the screen on a television set, releases a burst of photons. The photons then pass into the photomultiplier where amplification occurs in a cascade fashion - much like with the electron multiplier. After the photomultiplier enters the photomultiplier tube. It converts the optical signal to an electrical one, and provides a large degree of amplification. The photomultiplier has a light sensitive electrode called the photocathode which emits electrons when photons strike it. These electrons are accelerated by a series of electrodes , called dynodes , towards a collector (or anode ). The electrons produce several secondary electrons each time they strike a dynode resulting in a multiplication of their number as they approach the anode. Fig. shows a photomultiplier tube. An external resistor chain connected to a stable power supply is used to produce the voltages which are applied to the dynodes.

Figure 8.10: Photomultiplier Tube Typically, each electron which strikes a dynode will produce about four secondary electrons . This means that if one electron is released from the photocathode, a phototube with 10 dynodes will deliver 104 electrons to the collector. This gain, of about one million, is critically dependent upon the dynode voltages which necessitate a very stable power supply. The primary advantage of the conversion dynode setup is that the photomultiplier tube is sealed in a vacuum (photons pass through sealed glass), unexposed to the internal environment of the mass spectrometer. Thus the possibility of contamination is removed. A five-year or greater lifetime is typical and, with sensitivity similar to electron multipliers, photomultiplier conversion dynode detectors are becoming more widely used in mass spectrometers.

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Recorder
After the detector detects the ions, they are in analogue mode; an interface is required to convert analogue data into digital format. The output is recorded and displayed in the monitor attached to the instrument.

Figure 8.11: ADC - Connected between detector and recorder. The interface receives conditions and digitizes the analog signals output by the detector and sends them to the recorder. An analog-to-digital converter (abbreviated ADC, A/D, or A to D) is a device that converts continuous signals to discrete digital numbers. Typically, an ADC is an electronic device that converts a voltage to a binary digital number.

Summary
Detectors are the eyes of the instrument, needed to correct, improve, and extend ion detection and, hence, our chemical vision. Despite the fundamental need to better see ions. The Ion detection technology, by contrast, has received less attention, advances in this technology have occurred, but they have been incremental rather than revolutionary. Very few new detection approaches have been devised. In addition, detector technology is often given brief or no mention in MS texts and reviews. Thus, it has remained an area of rather understated importance.

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9. V ACUUM

Linear Transmission of Ion

Introduction
All mass spectrometers require a vacuum. A vacuum is necessary to permit ions to reach the detector without colliding with other gaseous molecules. Such collisions would reduce the resolution and sensitivity of the instrument by increasing the kinetic energy distribution of the ion's inducing fragmentation, or by preventing the ions from reaching the detector. A well-maintained vacuum is essential to the function of a mass spectrometer. The major reason for maintaining high vacuum is to increase the mean-free path of ions. Mean-free path is the average distance that our ions and molecules will travel before colliding with another ion or molecules. A high mean free path ensures predictable and reproducible fragmentation, high sensitivity and reliable mass analysis. All mass spectrometers operate at very low pressure (high vacuum). This reduces the chance of ions colliding without molecules in the mass analyzer. Any collision can cause the ions to react, neutralize, scatter, or fragment. All these processes will interfere with the mass spectrum. To minimize collisions, experiment s are conducted under high vacuum conditions, typically 10 t o 10 Pa (10 to 10 torr) depending upon the geometry of the instrument. This 10-5 10-4 10-7 high vacuums requires two pumping stages. The first stage is a mechanical pump that provides rough vacuum down to 0.1 Pa (10 torr). The second stage uses diffusion pumps or 103 molecular pumps to provide high vacuum.

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Typical Vacuum System

Figure 9.1 A mass spectrometer is shown above (Fig.9.1) with three alternative pumping systems. All three systems are capable of producing a very high vacuum, and are all backed by a mechanical pump. Various mechanical configurations are now used to maintain the vacuum in a mass spectrometer while introducing the sample into the vacuum chamber. The mechanical pump serves as a general workhorse for most mass spectrometers and allows for an initial vacuum of about 10-3 torr to be obtained. A vacuum is produced using a combination of two pumps two stage vacuum system Roughing Pump & Turbomolecular Pump. A rotary pump serves as roughing pump. It can produce a vacuum of 102 to 10-4 torr. To achieve vacuums in the 10-5 torr range, turbomolecular or diffusion pump is used. Once a 10-3 torr vacuum is achieved, the other pumping systems can be activated to obtain pressures as low as 10-9 torr. The pumps used to maintain high vacuum actually acts like compressors.

Turbomolecular Pump

Figure 9.2

The pumping system uses a staged combination of turbomolecular (Fig.9.2) and rotatory vane pumps to maintain the high vacuum pressure in the vacuum chamber. Efficient operation of the two Turbo
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Pumps is assisted by the use of D8 rotary vane backing pump. A second D16 rotary vane pump, referred to as the interface pump, maintains the differentially pumped interface at a pressure below 2 torr. The turbo pumps are not connected directly to the system electronics, but are controlled by separate controllers, which are powered from the AC Distribution board. Various mechanical configurations are now used to maintain the vacuum in a mass spectrometer while introducing the sample into the vacuum chamber. One method is to introduce the sample in small quantities through a capillary column (e.g. gas chromatography) or through a small orifice directly into the instrument. A second method is to initially evacuate the sample chamber through a vacuum lock. Once a moderate vacuum is achieved (10-3 torr) using the vacuum lock in a prechamber, the sample can be introduced into the main vacuum chamber on the mass spectrometer. This method is usually employed with the direct insertion probes used with MALDI and FAB. Coupling any sample source to a mass spectrometer requires that the sample (at atmospheric pressure, 760 torr) be transferred into a region of high vacuum (~10-6 torr) without compromising the latter. The billion-fold difference in pressure between the atmosphere and the high vacuum was one of the first problems faced by the originators of mass spectrometry. Once the vacuum is compromised, sensitivity and resolution will be reduced.

Importance of Vacuum
In LCMS, vacuum plays very important role as,

A vacuum is necessary to permit ions to reach the detector. It reduces or eliminates the chances of ion collisions with mass analyzer. The major reason for maintaining high vacuum is to increase the mean-free path of ions. Increases sensitivity and resolution of Mass Spectrum.

Risk
At higher pressures the high voltages used in the instrument may discharge to ground, which can damage the instrument and/or the computer system running the instrument. Such a breakdown, which may arise from an extreme leak, is basically an implosion, and can seriously damage a mass spectrometer by destroying electrostatic lenses, coating the optics with pump oil, and damaging the detector. In general, maintaining a high vacuum is crucial to obtaining high quality spectra.

Summary
The typical mass detector ionization systems used in LC-MS work at atmospheric pressure, but ions does not travel well through gasses. Unnecessary collisions with gas molecules cause at best fragmentation, and at worst total loss of signal. Therefore a good vacuum is essential.

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