Mass Spectrometry

Prof. Dr. Artemis Karaali

Introduction
Mass spectrometry is unique among other spectroscopic techniques, because it works by placing a charge on a molecule using an electron beam, converting it to an ion(ionization). The ions are then resolved according to their mass to charge(m/z) ratios by subjecting them to an electrostatic field. The result of ion generation, seperation, fragmentation, detection is manifested as a mass spectrum that can be interpreted to yield molecular weight and molecule structural information. It is then interfaced with chromatographic techniques(i.e. GC or HPLC) to confirm the identity of compounds as they elute off the chromatographic column.

Historical background
J. J. Thomson was the first scientist to discover the principle of mass spectrometry while he was measuring the effects of electric and magnetic fields on ions generated by gases in cathode ray tubes. Thomson noticed that the generated ions moved through parabolic trajectories proportional to their "mass-to-charge" ratios.

This was the basic idea behind mass spectroscopy:

Producing ions by bombarding organic molecules with high-energy electrons (~70 eV) produced from a tungsten filament by heating with a direct current; Then accelerating these ions in a definite direction so that they can be separated according to their mass or velocity. The separated ions are then detected individually and their respective intensities are measured.

Every molecule has a characteristic pattern of splitting to

charged ions with different masses.The masses and charges of the fragments and their relative abundance reveals information about the structure of the molecule

Mass Spectrometry

Mass Spectrometer

Ion generation

separation

fragmentation

detection.

Mass SpectrometryTerminology
When the electron beam ionizes the molecule, the species that is formed is called a radical cation, and symbolized as M+. The radical cation M+ with highest mass number is called the molecular ion or parent ion. The mass of M+ represents the molecular weight of M. Because M is unstable, it decomposes to form fragments of radicals (cations that have lower molecular weights than M+). The mass spectrometer analyzes the masses of all cations. A mass spectrum is a plot of the amount of each cation (its relative abundance) versus its mass to charge ratio (m/z, where m is mass, and z is charge). Since z is almost always +1, m/z actually measures the mass (m) of the individual ions.

Mass Spectrometry

The tallest peak in the mass spectrum is called the base peak. The base peak is almost always the M peak(although this may not always be the case). Though most C atoms have an atomic mass of 12, 1.1% have a mass of 13. Thus, 13CH4 is responsible for the peak at m/z = 17. This is called the M + 1 peak.

Mass Spectrometry
Since the molecular ion is unstable, it fragments into other cations and radical cations (containing one, two, three, or four fewer hydrogen atoms than methane itself).These are called daughter ions. Thus, the peaks at m/z 15, 14, 13 and 12 are due to these lower molecular weight fragments.

A mass spectrum is a presentation of the masses of the positively charged fragments vs.

their abundance(relative concentration).

Masses are graphed or tabulated according to their relative abundance.

Mass spectrum of CO2. Molecular ion is seen at m/e=44. Separation of the positive charge ion fragment is on the basis of mass. (Mass/Charge)

Consider the CH3CH2CH2SH (n-propyl mercaptan) molecule . If a high-energy electron strikes this molecule and transfers its energy to it, the following ions may be produced from this molecule:
C3H5S+ (m/e=73) 0.9% (parent ion molecule)

C3H5+ (m/e= 41) 4.7%

CHS+ (m/e= 41) 4.7%

C3H8S+e

C3H5+ (m/e= 45) 11.2%

CH2S+ (m/e= 46) 42.8% CH3S+ (m/e= 47) 4.3%

All other negative and neutral ions:15.9%

The Nitrogen Rule

Hydrocarbons like methane (CH4) and hexane (C6H14), as well as compounds that contain only C, H, and O atoms, always have a molecular ion with an even mass. An odd molecular ion indicates that a compound has an odd number of nitrogen atoms. The effect of N atoms on the mass of the molecular ion in a mass spectrum is called the nitrogen rule: A compound that contains an odd number of N atoms gives an odd molecular ion. A compound that contains an even number of N atoms (including zero) gives an even molecular ion.

MS is used for:
Characterisation of unknown molecules( measuring the molecular weight and determining the molecular formula of organic compounds). Measurement of concentrations of a wide variety of known molecules(i.e. gases, proteins etc.). Discriminating isotopomers of molecules. Most elements have naturally abundant atoms of several different masses. Some are radioactive, but many are not. Non-radioactive isotopes are called stable isotopes. (Examples: 2H2 = deuterium = heavy hydrogen; 13C = Carbon 13). Organic molecules will therefore sometimes contain atoms of higher mass. The mass spectrometer can detect these and measure the proportion of each mass. This is also crucial to use of tracers in clinical research.

ISOTOPES

81Br

Isotopes are present in compounds in their usual natural abundance. All hydrocarbons contain 1.1% 13C, so there will be a small M+1 peak before M+ peak. . If Br is present, M+2 is equal to M+(50.5:49.5) If Cl is present, M+2 is one-third of M+(75.5:24.5) If Iodine is present, only M+ peak at 127. If N is present, M+ will be an odd number(15). If S is present, M+2 will be 4% of M(95:4.2) and there will also be a tiny M+1 peak(0.8%)

Mass Spectrometry with Isotopes

Alkyl Halides and the M + 2 Peak
Most elements have one major isotope. Chlorine has two common isotopes, 35Cl and occur naturally in a 3:1 ratio.
37Cl,

which

So, there are two peaks in a 3:1 ratio for the molecular ion of an alkyl chloride. The larger peak, the M peak, corresponds to the compound containing the 35Cl. The smaller peak, the M + 2 peak, corresponds to the compound containing 37Cl. So, when the molecular ion consists of two peaks (M and M + 2) in a 3:1 ratio, a Cl atom is present.

Br has two isotopes: 79Br and 81Br, in a ratio of ~1:1. Thus, when the molecular ion consists of two peaks (M+ and M + 2) in a 1:1 ratio, this means a Br atom is present.

Mass Spectrum with Sulfur

M+2 is ~4% of M+ There is also a M+1 peak

=>

Mass Spectrum with Chlorine

M+2 is 1/3 of M+

=>

Mass Spectrum with Bromine

M+2 is ~equal to M+

=>

Resolution of Mass Spectrometers

Low resolution mass spectrometers report m/z values to the nearest whole number. But, the mass of a given molecular ion can correspond to many different molecular formulas. High resolution mass spectrometers measure m/z ratios to four (or more) decimal places.

Consider a compound having a molecular ion at m/z = 60 using a low-resolution mass spectrometer. The molecule could have any one of the following molecular formulas.

High Resolution MS
Here the masses are measured to 1 part in 20,000. A molecule with mass of 44 could be C3H8, C2H4O, CO2, or CN2H4. If a more exact mass is 44.029, pick the correct structure from the table:
C3H8
44.06260

C2H4O
44.02620

CO2
43.98983

CN2H4
44.03740

All mass spectrometers have three major components:

ion source analyzer detector

Essential Features of Mass Spectrometer

(1) Sample Inlet System a) Direct Introduction Probe (DIP) - Solids and viscous liquids are introduced directly into the ion source of the mass spectrometer by a direct introduction probe. The sample is placed in a glass capillary and gently heated to produce the required vapor pressure without thermal decomposition. b) GC inlet system - The samples separated by gas chromatography are introduced into the ion source of the mass spectrometer.

Mass spectrometric analysis starts by the injection of the sample to the system. Sample is subjected to an ion source. If the sample is pure enough, there is no need for any additional equipment and procedure. So this method is called direct injection or DIP(direct introduction probe) method. In the direct injection probe method, the sample is put to the end of probe and this probe is inserted into the ion source. The source is heated until the solid vaporizes. But in the case of more complex samples, dynamic sample introduction methods are required where separation of sample can be done by GC or HPLC which is combined with mass spectrometer

Time-of-flight (TOF)mass analyzers

the lower the ion's mass, the greater the velocity and shorter its flight time.

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.

 Mass spectrum is a table of the intensity of different mass fragments that are formed after the ionization process. Mass spectrum contains the information about the structure of the molecules. Spectrum is shown as graph due to the abundances of all ions and they are displayed as percentages and most abundant ion has a value of %100.

Combined Modes
1. TandemMS: Mass Spectrometry/Mass

Spectrometry (MS/MS) 2. Interface Methods: -Gas Chromatography/Mass Spectrometry (GC/MS) - Liquid Chromatography/MassSpectrometry (LC/MS)

 MS/MS which is also called tandem mass spectrometry is a technique of ion fragmentation that is used in mixture analysis, for specific detection of target compounds or compound classes, and structure elucidation.

Gas Chromatography-Mass Spectrometry (GC-MS)

Gas Chromatography-Mass Spectrometry (GC-MS)

To analyze a urine sample for tetrahydrocannabinol, (THC) the main psychoactive component of marijuana, the organic compounds are extracted from urine, purified, concentrated and injected into the GC-MS. THC appears as a GC peak, and gives a molecular ion at 314, its molecular weight.

 Gives the maximum structural information for the smallest amount of sample. An MS device coupled with GC allows the identification and determination of unknown peaks. We can also determine the purity of each peak that is eluted from the column. Example: Aroma analysis

Sample is injected to the liquid chromotography column for the aim of separation to its components. Components are eluted through the liquid column and enter the mass spectrometry Following HPLC conditions should be optimised: mobile phase composition, gradient, physical characteristics of column, flow rate

 Substances that are naturally present in foods such as, proteins, lipids, olygosaccharides, vitamins, flavonoids, phenolic compounds and aroma compounds can be investigated with mass spectrometry. MS applications are also done on food materials through the analysis of pesticides, drug residues, toxines, amines and packaging migrants. One other important study area of mass spectrometry on food analysis is the detection of trace metals bound to organic molecules.

Bacteria Identification
Identify Proteins produced by bacteria Data base search of spectra identifies bacteria At this point, still no large consistent Data Base is available. research still continues.

 Mass spectrometry not only allows the precise determination of the molecular weight of peptides and of proteins but also the determination of their sequences.

 Classically,

protein identification requires total or partial determination of their sequences by the method of Edman degradation. But this technique requires a long analysis time and a large amount of sample. Without any doubt, mass spectrometry is now the most efficient way to identify proteins.

Advantages of MS are; involves shorter analysis time and less manpower requires a small amount of sample involves very little sample preparation gives reproducible data, which are easily accessible to computer processing

 Reliable, sensitive and rapid methods for fat-soluble vitamins determination in fortified food products are essential for nutritional reasons. MS is beginning to be recognized as an advantageous detection system

 Because of their poor volatility, most of the fat-soluble vitamins are not suitable for determination by GCMS. Particle beam LCMS has been used to identify Vitamins A and E in infant formula powder as well as for the simultaneous determination of Vitamins A, D3 and E in pharmaceutical preparations

Several multi-residue methods for determination of organo-phosphorus, organochlorine and organonitrogen pesticides in crops using gas chromatography for separation of individual compounds followed by

detection with MS on selective and sensitive detectors

 Mass spectrometry is a very sensitive and selective technique for both

multi-residue determination trace-level identification of a wide range of pesticides

 Gas chromatography combined with mass spectrometry is one of the appropriate seperation and characterization method for aroma compounds

Flavour studies on a large number of virgin olive oils of good quality Determinig of the off flavours with gas chromatographymass spectrometry methodology. Generation of the off flavours can be followed by the static headspace GC and the solid phase microextraction combined with GC-MS.

By using a semipermeable membrane, toxins can be extracted from the food compound into an organic phase. Acceptable recovery, reproducibility and linearity results are obtained. Both LCMS and GCMS can be used during the characterization of the fungal metabolites and for the determination and the confirmation purposes of mycotoxins. For selective determination of the shellfish poisoning toxins in seafoods, LC-MS is applicable and provides less reagent usegae in different steps of the analysis.

Mass spectrometers have become pivotal for a wide range of applications in the analysis of inorganic, organic, and bio-organic chemicals Mass spectrometry has gained noticable importance on food analysis, to determine, identify and quantify the naturally occuring substances in food samples:both nutritive and non-nutritive food components that are volatile, thermolabile or ionic compounds can be analysed with GCMS, LCMS and LCMSMS methods as appropriate. Mass spectrometry is being continually improved and has recently had significant advances in its application to molecular biology, where it is now possible to analyze proteins, DNA, and even viruses