8 Mass Spectrometry

Dr. Heena Rekhi Rekhi

  1. Description

Mass spectrometry deals with the most accurate method for determining the molecular mass of the compound and its elemental composition. The molecules are ionized when bombarded with the energetic beam of electrons and dissociate into several fragments. Each kind of an ion has a particular m/e ratio. Thus for the most ions m/e is the molecular mass of the ion.

 

The molecular ion is called parent ion and is designated as M+. The set of ions are analyzed in such a way that a signal is obtained for each value of m/e. The largest most intense peak in the structure is called base peak and its intensity is randomly assigned a value of 100%. A mass spectrum is a presentation of the masses of positively charged fragments versus their relative concentrations. It is produced as a result of a series of computing and consecutive unimolecular reactions. Mass spectrometry is not a true spectroscopic technique because the absorption of electromagnetic energy is not involved in any way. The advantages of mass spectrometry are its high sensitivity, accuracy, reproducibility and the amount of sample required for the mass spectral analysis.

 

A mass spectrometer should always perform the following processes:

  • Produce ions from the sample in the ionization source.
  • Separate these ions according to their mass-to-charge ratio in the mass analyser.
  • Eventually, fragment the selected ions and analyze the fragments in a second analyser.
  • Detect the ions emerging from the last analyser and measure their abundance with the detector that converts the ions into electrical signals.
  • Process the signals from the detector that are transmitted to the computer and control the instrument through feedback.

 

The fundamental requirement of a mass spectrometry analysis is that if an ion is formed with excess energy, bonds can be broken. In each unimolecular fragmentation reaction, one of the fragment will pick up the charge. If these secondary ions still have sufficient excess energy, they too can fragment. All of the ions are extracted from the ionization region by an applied electric potential and separated according to their mass to charge ratio. After the separation the ions are detected and a plot of abundance versus m/z is constructed. This leads to the representation of mass spectrum used to determine the molecular structure, to quantify the single compound or mixture of compounds or to study the ion molecule reactions. The basic functions of mass spectrometer are as follows:

Furthermore, the requirements and methodologies used to accomplish the analysis are modified day to day by the researchers working in this field to improve the routine analytical methods.

 

The small amount of sample is evaporated and vapors of it passed into the ionization chamber. The electron beam ionizes the vapor molecules and mainly yields positive electrons by the loss of valence electrons. The positive ions formed are forced to pass out from the ionization chamber by some voltage applied. Before they enter into the analyzer the ions left from the ionization chamber are accelerated by some electric field applied. The separation of ions takes place in mass analyser. The fast moving ions then follow the circulatory path due to Lorentz acceleration, whose radius is being determined by mass to charge ratio. Then they collide on the collector mode after passing through the exit slit. The resulting current is amplified and registered as a function of accelerating voltage. The mass distribution appeared is the characteristic of the given molecule. Figure 1 represents the essential components of an analytical mass spectrometer.

Instrumentation

 

Mass spectrometer instruments can be considered as a complex chemical reaction, as the resulting mass spectrum is directly related to the optimized experimental parameters. It has a wide range of applications in analysis and quantification of small to large molecular weight compounds due to all the highly informative data output from mass spectrometry. Common mass spectrometer consists of following components:

 

1. Sample introducing system

2. The ion source

3. Mass analyser

4. Ion collector

5. Recorder

 

1. Sample introducing system: Samples can be introduced inside the mass spectrometer in many ways. Solid sample can be placed on the tip of rod inserted into the vaccum chamber, evaporated or sublimed by heating. The ionic sample can be analyzed by fabricating spark gas electrodes from the sample itself. Other method of sample introduction is by coating on the filament which when heated  yields  positive  ions  directly.  The  external  sample  introducing  system  includes  direct insertion of only those samples which are volatile in nature into the ion beam source. In those cases in which catalytic decomposition of the sample on the metal surface is anticipated glass is used. Liquid samples can be introduced through special controlled flow inlets or they can be desorbed from the surface on which they are coated as a thin film. All the samples used for mass spectrometry must be pure because of the fragmentation that occurs causes the mixtures to be difficult to interpret.

 

2. The ion source: All the samples taken for analysis are ionized prior to the analysis in the mass spectrometer. The internal energy transferred during the ionization process and the physico-chemical properties of the analyte can be ionized. The most common way of producing the ions involves bombarding the analyte with energetic beam of electron from an ion gun. Some ionization techniques are very energetic causing extensive fragmentation whereas some are softer and produce ions of molecular species. The most common techniques used for the thermally volatile materials and non-volatile materials are as follows

 

Electron impact technique: It is also called as an electron ionization technique devised by dempster and improved by Bleakney and Nier.

 

This work well for the extensive fragmentation or for many gas phase molecules. Electrons are accelerated from the tungsten filament to an anode usually through a potential difference of 70V. In an electron impact source the ions are produced in the electron beam either by the loss of a radical or by loss of a molecule. A process of each kind is illustrating in the following example:

 

This example offers both pros and cons of electron impact spectrometry. The advantage includes extensive fragmentation giving rise to a pattern of fragment ions where as the disadvantage is absence of molecular ion even in the limited molecular weights range. EI spectra don’t produce the molecular ions of low abundance.

 

Chemical ionization mass spectrometry: Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection. Chemical ionization (CI) is a technique that produces ions with little excess energy. Thus this technique presents the advantage of yielding a spectrum with less fragmentation in which the molecular species is easily recognized.

 

Consequently, chemical ionization is complementary to electron ionization. In this technique a reagent gas is allowed to pass into the ion chamber at a high pressure of about 100 Nm-2. The gas is ionized using the electrons with energies upto 300 eV. For example

 

Chemical ionization comprises the production of ions by simply colliding the sample molecules with the primary ions present in the sample. Ion collision will be induced in the particular part of the source. Due to the collisions occurring in a CI source reduce the initial kinetic energies of the bombarding electrons to lower values at which they can be captured to give an anion, CI spectra gives satisfactory negative ion spectra. It can also be used for the molecules of lower molecular weight.

 

Field Desorption: In this technique a microgram of the sample is deposited on the wire. Since the needles on the wire are very sharp, the field at their tips may be as high as 108 Vcm-1. Electrons discharge from the sample into vacant orbital of metal leads to the creation of positive ions at the positive wire and M+ desorbed by the coulombic repulsion as shown in figure. As a result, bimolecular reactions may occur and field desorption spectra may show MH+ to give molecular weight information rather than M+. The different polar molecules can be determined by laser desorption, fast atom bombardment, secondary ion mass spectrometry and californium plasma desorption. These methods are based upon giving a large pulse of energy to the sample. In case of laser desorption energy is produced by a laser beam. In FAB microgram of a sample is dissolved in specific solvent like glycerol as a matrix and then bombarded with a fast beam of Xe atoms. The sample is desorbed after the impact of xenon atoms and then analysed by mass spectrometer. In some cases spark electrode and photons are used as a source of ionization. The ions produced in the ionization chamber are accelerated then fed to mass analyser for the analysis.

Mass analyser: The ions produced in ion chamber are accelerated by applying an acceleration potential. Further, these ions enter the mass analyser. In mass analyser the fragment ions produced are differentiated by mass to charge ratio. The ions formed have travelled in a circular path through an angle of 180° under the effect of magnetic field and fall on a collector. The amount of the energy required to remove an electron from an atom or a molecule is ionization potential. Suppose an ion having charge e accelerated through a voltage V. Thus the kinetic energy of the ion is given by: ½ mv2= eV; where m is mass of an ion, v is velocity of an ion, e is the charge and V is voltage. The charged particle moves in a circular path with radius r under the effect of magnetic field given by r= mv/eH. On combining we get;=   2 2  2

 

More the value of m/e more is the radius of a curvature in which ions are travelled. By varying either magnetic field or voltage the number of ions can be determined. Types of analysers used in mass spectrometry

 

  1. Ion detector: The ions separated by mass analyser are detected and measured electrically or photographically. The ions produced passes through the collecting slits one after the other and fall on detector for the determination. The recorder records all the peaks and the mass spectrum is scanned by going up the scale.

 

Interpretation of mass spectrum

 

 The mass spectrum of a compound consists of a series of lines corresponding to the ion fragments and relative abundance of these ions and also that of a parent ion. When a molecule A is bombarded with electrons of moderate energy, the initial processes which can occur are summarized as below:

When the bombarding energy equals to ionization potential, all of the electron’s energy transferred to molecule to remove an electron. But this probability to occur is less. When bombarding energy increases, the probability of collision increases which further increases the intensity of a peak. The acceleration potential of bombarding electron which is just enough to initiate the fragmentation is referred to as an appearance potential. Sometimes the rearrangement process occurs in mass spectrum complicates its interpretation.

 

5. Recorder: On passing through the slit ions enter the detector and a signal is recorded. The source and the path through which the ions pass must be kept under a vaccum of about 10-7 mm of mercury to provide a long mean free path for the ion.

 

Mass spectrometers:

 

1. Double Focussing mass spectrometer: A very high resolution is possible if all the ions have same m/e with same velocity. It is achieved by passing through an analyser before it enters into magnetic field. The precise mass measurement can be done by the use of a narrow exit and this kind is suitable for the separation and analysis of ions or the molecular weight determination. It uses both direction and velocity focusing, and therefore an ion beam initially diverging in direction and containing ions of different kinetic energies is separated into beams according to the mass/charge, these beams being focused on to a photographic plate or film. This technique is generally referred as MS/MS, since the molecular ion from an initial mass spectrum is selected and made to give a second mass spectrum.

 

2. Time of flight mass spectrometers: In case of time of flight mass spectrometers all the ions leave the accelerating field with same kinetic energies but with different velocities depending upon their masses. The ions are accelerated by a potential difference between A and B and pass into flight tube. They are separated according to their m/e values and collected at certain point. The time of flight is given by t = k√ . k is a constant depend upon length of flight time. The main benefits for time of flight mass analysers include fastest speed, high ion transmission, highest practical mass range of all mass analysers etc.

 

3. Quadrupole mass spectrometers: This kind of mass spectrometer also known as mass filter. Four electrode systems are used in which oppositely charged electrodes are connected together. A constant voltage and radiofrequency are applied between the oppositely pairs of four parallel rods. Mixture analysis can be achieved by the use of three quadrupole mass spectrometer connected in series. The first one present in series helps in separation of molecular ion, second as collision chamber and third separate the products of collision induced decomposition. The benefits of quadrupole includes reproducibility, low cost, low energy collisions induced dissociation (CID) MS/MS.

 

4. Radiofrequency spectrometer: In the presence of an electrostatic field the charged particles emerging from an ion source are accelerated to same energy. The parts are arranged in such a way that the ion having the maximum energy reach the detector. By varying the radiofrequency of the analyser, the mass spectrum can be obtained. Mathematically it can be deduced that:= 0.266

 

where, s is the spacing between adjacent grids

f is the frequency of the field

m is the mass range of radiofrequency spectrometer is from 2 to 100

 

5. Ion cyclotron resonance spectrometers: The ions are formed in an ionisation chamber which is further subjected to a constant magnetic field and an electrostatic field. Source of an ion formed in the ionisation chamber is accelerated in one way then in other way forming a circular path by magnetic field and the ions spiral outwards to the collector. For the good sensitivity the ions should kept resonating for times in the range of milliseconds to seconds. This method offers a good sensitivity and high resolving power. The working equation for ICR can be quickly derived by equating the centrifugal force and Lorentz force experience by an ion in a magnetic field.
2=

Solving the angular frequency which is equal to v/r

ω = =

A group of ions having same mass to charge ratio will have the same cyclotron frequency, but they will be moving independently and out of phase at roughly thermal energies. The main benefits of cyclotron analyser are the highest recorded mass resolution of all mass spectrometers. It has non destructive ion detection, ion measurement and a stable mass calibration in superconducting magnet systems.

 

Applications

 

Mass spectrometry has numerous applications in structure determination. Each compound has a characteristic fragmentation pattern that can be used to identify the compound. The fragmentation pattern indicated the stability of atomic group in a molecule. No two compounds can be ionized in exactly the same manner, the mass spectrum becomes a fingerprint for each compound and is unique. The molecular mass measured by mass spectrometry can correspond to the average mass calculated using the average atomic weight of each element of the molecule or to the monoisotopic mass calculated using the ‘exact mass’ of the most abundant isotope for each element. The type of mass measured by mass spectrometry depends largely on the resolution of the analyser. The second type of analytical information is the measurement of the mass with sufficient accuracy provides an unequivocal determination of the total elemental composition. Its various interdisciplinary applications are described in Table 1.

 

Table 1 simple examples illustrating contribution of mass spectrometry in interdisciplinary fields

 

Advantages of mass spectrometry

 

1. Mass spectrometry can be used in the study of fast reactions, free radicals and some exchange reactions.

2. Mass spectra have been recorded for the compounds with the molecular weight different form the normal compounds. These compounds have been synthesized from the heavier isotope elements.

3. It can be used in the petroleum industry for the characterization of paraffin, cycloparaffin, alkyl benzenes etc.

4. Ion molecule collision can produce peaks of higher mass number than the molecular ion peak.

5. In a molecule containing two equivalent bromine atoms, a triplet with ratios 1:2:1would result from the different combinations of isotopes in fragments containing two bromine atoms.

6. Impurities create a problem in case of fingerprint application because the major fragments of these impurities give rise to several low intensity peaks in the spectrum.

 

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