Mass Spectrometry

Dr. Kusum ahlawat Professor and Head Chemistry Department MMU

Mass Spectrometry
Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules. Detection of compounds can be accomplished with very minute quantities (as little as 10-12g, 10-15 moles for a compound of mass 1000 Daltons). This means that compounds can be identified at very low concentrations (one part in 1012) in chemically complex mixtures. Mass spectrometry provides valuable information to a wide range of professionals: physicians, astonomers, and biologists, to name a few.

Did you know that mass spectrometry is used to...

Detect and identify the use of steroids in athletes Monitor the breath of patients by anesthesiologists during surgery Determine the composition of molecular species found in space Determine whether honey is adulterated with corn syrup Locate oil deposits by measuring petroleum precursors in rock Monitor fermentation processes for the biotechnology industry Detect dioxins in contaminated fish Determine gene damage from environmental causes Establish the elemental composition of semiconductor materials

What can mass spectrometry do for you?

Identify structures of biomolecules, such as carbohydrates, nucleic acids and steriods Sequence biopolymers such as proteins and oligosaccharides Determine how drugs are used by the body Perform forensic analyses such as conformation and quantitation of drugs of abuse Analyze for environmental pollutants Determine the age and origins of specimens in geochemistry and archaeology Identify and quantitate compounds of complex organic mixtures Perform ultrasensitive multielement inorganic analyses

How did mass spectrometry originate?

The technique of mass spectrometry had its beginnings in J.J. Thomson's vacuum tube where in the early part of the century the existence of electrons and "positive rays" was demonstrated. Thomson, the physicist, observed in his book "Rays of Positive Electricity and Their Application to Chemical Analysis" that the new technique could be a used profitably by chemists to analyze chemicals. Despite this far-sighted observation, the primary application of mass spectrometry remain in the realm of physics for nearly thirty years. It was used to discover a number of isotopes, to determine the relative abundance of the isotopes, and to measure their "exact masses", i.e., atomic masses to within a precision of 1 part in 106 or better. These important fundamental measurements laid the foundation for later developments in diverse feilds ranging from geochronology to biochemical research. On these Web pages we discuss the basic principles of mass spectrometry and some important applications. We also provide a brief explanation of how a mass spectrometer works, a description of its various component parts, and a discussion of mass spectra and the kind of information they contain. In later sections we will discuss ionization techniques, data presentation as it relates to chromatography, and some highlights of recent breakthroughs. Because mass spectrometry is most often used to analyze organic molecules, the examples on these pages are drawn from this group of substances. The principles of mass spectrometry are, however , broadly applicable and our discussion will also include analyses of inorganic substances

What is a Mass Spectrometer?

A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted into ions, i.e., molecules that have been electrically charged. Since molecules are so small, it is not convenient to measure their masses is kilograms, or grams, or pounds. In fact, the mass of a single hydrogen atom is approximately 1.66 X 10-24 grams. We therefore need a more convenient unit for the mass of individual molecules. This unit of mass is often referred to by chemists and biochemists as the dalton (Da for short), and is defined as follows: 1 Da=(1/12) of the mass of a single atom of the isotope of carbon12(12C). This follows the accepted convention of defining the 12C isotope as having exactly 12 mass units. As will become clear in what follows, a mass spectrometer does not actually measure the molecular mass directly, but rather the mass-to-charge ratio of the ions formed from the molecules. For reasons similar to those discussed in the context of mass, it is inconvenient to measure the charge on an individual ion in units appropriate to the macroscopic everyday world. A useful unit for this purpose is the fundamental unit of charge, the magnitude of the charge on an electron. It follows that the charge on an ion is denoted by the integer number z of the fundamental unit of charge, and the mass-to-charge ratio m/z therefore represents daltons per fundamental unit of charge. In many cases, the ions encountered in mass spectrometry have just one charge (z=1) so the m/z value is numerically equal to the molecular (ionic) mass in Da. Mass spectrometrists often speak loosely of the "mass of an ion" when they really mean the m/z ratio, but this convenient way of speaking is useful only for the case of singly-charged ions. An actual mass spectrometer ranges in size from about the size of a home microwave oven to large research instruments that dominate entire rooms. The different functional units of a mass spectrometer are represented conceptually in the

 Formation of gas phase samples ions is an essential prerequisite to the mass sorting and detection processes that occur in a mass spectrometer. Early mass spectrometers required a sample to be a gas, but due to modern developments decribed below, the applicability of mass spectrometry has been extended to include samples in liquid solutions or embedded in a solid matrix. The sample, which may be a solid, liquid, or vapor, enters the vacuum chamber through an inlet. Depending on the type of inlet and ionization techniques used, the sample may already exist as ions in solution, or it may be ionized in conjunction with its volatilization or by other methods in the ion source.
The gas phase ions are sorted in the mass analyzer according to their mass-to-charge (m/z) ratios and then collected by a detector. In the detector the ion flux is coverted to a proportional electrical current. The data system records the magnitude of these electrical signals as a function of m/z and converts this information into a mass spectrum.

What are the characteristics of a mass spectrum?

A mass spectrum is a graph of ion intensity as a function of mass-tocharge ratio. Mass spectra are often depicted as simple histograms as shown in Figure 2. This record of ions and their intensities serve to establish the molecular weight and structure of the compound being mass analyzed. For example, Figure 2 shows a mass spectrum of the simple molecule carbon dioxide, CO2. In this example, all the ions are positively charged. (It is possible to generate and detect negative ions as well.) The ionized CO2 molecule (or molecular ion) appears at m/z 441. Since the ionization process breaks up or fragments some of the CO2 molecules, a fraction of the ions appear in the spectrum at m/z values less than the m/z value that corresponds to the molecular mass of CO2. Cleavage of a carbon-oxygen bond in the molecular ion to produce ionized carbon monoxide or ionized atomic oxygen result in the fragment ions at m/z 28 and 16; loss of two neutral oxygen atoms results in an additional fragment at m/z 12 for carbon. The molecular ion is designated as M+ or CO2+ and the fragment ions are designated as CO+, O+ and C+. 1 The ion is singly charged and the "nominal ion mass" is 44 Da: carbon=12 and oxygen=16 (in calculating nominal ion mass, atomic masses are rounded to the nearest integer).

The ion is singly charged and the "nominal ion mass" is 44 Da: carbon=12 and oxygen=16 (in calculating nominal ion mass, atomic masses are rounded to the nearest integer).
1

How is the sample introduced into the mass spectrometer?

For reasonably pure solids the sample can be placed on the tip of a rod that is inserted into the evacuated source region through a vacuum-tight seal. The sample is then evaporated or sublimed into the gas phase, usually by heating. Gases and liquids can be introduced through specially designed inlets with controlled flow.The gaseous molecules are then ionized (often with accompanying fragmentation) and the ions are mass analyzed. In some special techniques, volatilization and ionization occur at the same time. To obtain the mass spectrum of a single compound in a mixture, the individual components must be separated prior to analysis by mass spectrometry. Separation is necessary for unambiguous identification because two compounds present in the source region simultaneously create an overlapping or mixed spectrum and even simple compounds can generate many fragment ions. Since the 1960's gas chromatography (GC) has been coupled to mass spectrometry. This connection allows compounds already in the vapor phase to enter the mass spectrometer separated in time so that the components of mixtures can be detected and analyzed sequentially. More recently, liquid chromatographs, supercritical fluid chromatographs, and capillary electrophoresis devices connected to mass spectrometers have been used to separate components of complex mixtures prior to mass analysis.

How are the molecular and fragment ions produced in the ion source?
In the commonly used electron ionization (EI) source, (sometimes referred to as "electron impact" in older literature), ions are generated by bombarding the gaseous sample molecules with a beam of energetic electrons as illustrated in Figure 3. (Other methods of ionization will be discussed in a later section.) In the EI technique, a mixture of positive and negative ions, as well as neutral species, is generated. The energy of the bombarding electrons is generally much greater than that of the bonds which hold the molecule together. Thus when high energy electrons interact with the molecule not only does ionization occurs , but bonds are broken and fragments are formed , giving rise to the ions other than the intact molecular ion that appear in a mass spectrum. Although both positive and negative ions are generated in the ion source at the same time, only one polarity is recorded at a time; hence any given mass spectrum consists of either positve or negative ions. Molecules that are not ionized and neutral fragments are pumped away. Positive-ion EI mass spectra are most commonly recorded, because many fewer negative ions are formed by this particular ionization technique than positive ions. Positive ions are propelled into the analyzer by maintaining the ion source at an electrical potential positive relative to the analyzer and by focusing with voltages applied to a lens system located between the source and the analyzer. The repeller electrode assists in focusing the ions into the analyzer. Negative ions and electrons are attracted to the positively charged electron trap.

HOW DOES THE ANALYZER WORK?

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. 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-to-charge ratios can be focused on a detector.

Double focusing mass spectrometers 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 4, 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, such as C2H4, N2 and CO at m/z 28. The so called "exact masses", more properly "high precision masses", of C2H4, N2 and CO are 28.0313, 28.0061, and 27.9949 Daltons, respectively2.

Quadrupole mass filter

Another type of mass analyzer, called a quadrupole mass filter, consists of four parallel poles or rods. In this device (Figure 5), 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 than double focusing instruments but tend to be more easily interfaced to various inlet systems and to be less costly. The quadrupole ion trap mass spectrometer (Figure 6) operates on a principle similar to a quadrupole mass filter. However, it does not operate as a filter. Rather, the ion trap stores ions for subsequent experiments and analysis. It uses fields generated by rf (and sometimes dc) voltages applied to electrodes arranged in a sandwich geometry: a ring electrode in the middle with cap electrodes on each end. Within a selected range of mass-to-charge ratios determined by the applied voltages, the device traps ions in the space bounded by the electrodes. Typically, a mass spectrum is produced by scanning the applied rf voltages to eject ions sequentially of increasing mass-to-charge ratio through an end cap opening for detection.

detection.

Two other analyzers now being used frequently are the Fourier transform ion cyclotron resonance (FT-ICR) spectrometer and the time-of-flight (TOF) mass spectrometer. The unique capabilities of each of these mass analyzers make them especially useful as mass spectrometry moves into new areas of application. In an FT-ICR spectrometer (Figure 7) 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 radio-frequency 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 FTICR instrument can therefore be used to generate very high resolution spectra.

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 Time-of-flight mass analyzers (Figure 8) seperate 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