Mass Spectrometry

Mass spectrometry is one of the analytical techniques for characterizing matter, based on the determination of atomic or molecular masses of individual species present in a sample. 

The sample under investigation is bombarded with a beam of high-energy electrons. Thus, the molecules are ionized and dissociated into various fragments, including positive ions. Each ion has a specific mass to charge ratio (m/z), by which they are separated and identified.

The electron impact on the molecule results in the formation of the molecular ion. However, excess energy from the electron may cause the molecular ion to break apart into several fragments. So the energy of the electron is chosen accordingly. 

As the molecular ion is often very unstable, many ions produce fragments with no molecular ion remaining. Moreover, fragment information is essential as it can help in structural determination. Additionally, 70 eV classical spectra are used for comparisons.

Theory of Mass Spectrometry

A mass spectrum has an array of peaks of different heights. In a mass spectrometer, a molecule is bombarded with electrons having energy greater than its ionization potential so the removal of an electron takes place by the reaction:

M(g) + e → M+(g) + 2e

This ionization requires an energy of about 70 eV (~1600 kcal mol-1). This is greater than the usual bond energies of organic molecules. In fact, the energy can be in the form of electrons, photons, heat, electric field, or electrical discharge.

Soon as the energy of the electron just equals the ionization potential, an electron is removed from the molecular orbital of the molecule to form the molecular or parent ion M+.

As the energy of the bombarding electron is increased, the probability of collision involving ionization increases. Furthermore, if the energy of the electron is high enough, the parent ion M+ may retain the excess energy to dissociate into a new ion N+ and a neutral fragment O.

M+ → N+ + O

The neutral fragment O cannot be detected in the mass spectrometer, because it cannot be deflected since it does not possess a charge.

Example

Neopentane (Mr: 72) can be used as an example to explain the theory.

C5H12 + e → C5H12+ + 2e

The molecular ion C5H12+ undergoes fragmentation as follows:

  • C4H9+ with an m/z of 57 (100% intensity).
  • C3H5+ with an m/z of 41 (41.5% intensity).
  • C2H5+ with an m/z of 29 (38.5% intensity).
  • C2H3+ with an m/z of 27 (15.7% intensity).

Like neopentane, each compound yields its characteristic series of fragments which is known as the fragmentation pattern or the cracking pattern.

Hence the mass spectrum is a record of the masses and the relative abundances (intensities) of the molecular ion and the positively charged fragments formed by the bombardment of electrons.

Nature of the Spectrum

The nature of the resulting spectrum depends on:

  1. Properties of molecules
  2. Ionization potential
  3. Sample Pressure
  4. Instrumental design

Important terminologies

Molecular/Parent ion – The ion formed by the loss of a single electron from the molecule.

Base peak – The most intense peak of the mass spectrum, assigned 100% intensity.

Fragment ions – Lighter cations obtained by the decay of the molecular ion. These are often stable carbo-cations having a specific mass-to-charge ratio.

Radical cation – Positively charged species containing an odd number of electrons.

Working of a Mass Spectrometer

In a mass spectrometer, particles are ionized into positive charges, accelerated, deflected by an electric or a magnetic field, and finally detected. The path followed by the deflected ions depends on their mass to charge ratio. Particles with a smaller m/z ratio value are deflected most whereas particles having a large m/z value are deflected least. 

A mass spectrometer consists of an ion source, an accelerator, an analyzer, and a detector. Likewise, the steps involved in mass spectrometry are ionization, acceleration, deflection, and detection.

The first process that occurs is the ionization of the gaseous particles by the bombardment of electrons from an electron gun. 1+ charged ions are formed from the neutral particles.

The second step involves the acceleration of the ions by an electric field.

Afterward, the accelerated charged particles are deflected with the help of an electric or magnetic field.

Finally, the ions are detected by electric or photographic methods.

Components of a Mass Spectrometer

1. Sample inlet system 

The sample under investigation may be in a solid, liquid, or gaseous state. An appropriate amount of sample must be taken to convert into the vapor state, to obtain the stream of molecules that flows into the ionization chamber.

The gaseous sample may be inserted directly into the ionization chamber, which is at a lower pressure than the partially evacuated inlet system. 

The vapors move through a small pinhole known as the molecular leak before entering the ionization chamber. This simple system can be used for volatile liquids or solids.

For less volatile substances, other inlet methods can be used:

a. Direct probe method: The sample is placed on a thin loop of wire or a pin at the tip of the probe, which is inserted through a vacuum lock into the ionization chamber. The probe is then heated causing the sample to produce vapors in the proximity of the electron beam. This method is suitable for substances with vapor pressure lower than 10-9 mmHg at room temperature.

b. Atmospheric pressure chemical ionization technique (APCI): The sample is placed in a stream of ionized gas or solvent aerosol between the ion source and the mass analyzer inlet.

c. Gas chromatography-mass spectrometry: The stream of gaseous molecules coming out of the gas chromatograph enters the tube through a molecular leak.

2. Ion Source (ionization techniques)

There are several ways to ionize the sample particles, choosing which depends on the nature of the material and the analytical requirements. 

Moreover, mass spectrometry can be directly linked with other analytical techniques such as gas or liquid chromatography, and emission spectroscopy. Some of the ionization techniques are provided below. 

a. Electron-impact Ionization (EI) utilizes a high-energy beam of electrons (~70 eV) emitted from a filament heated up to several thousand degrees Celsius. Collisions between electrons and vaporized analyte molecules result in the formation of molecular ions, which are essentially radical cations. The molecular ions then decompose into smaller fragments.

b. Chemical Ionization (CI) is a relatively softer technique than EI, with the molecular ion being produced by collisions between sample molecules and those of a pre-ionized gas. The gas may be methane or ammonia and is ionized by EI initially. The choice of reagent gas must be made carefully to match the proton affinity of the reagent gas with that of the sample so that efficient ionization of the sample takes place without excessive fragmentation.

In the CI method, a molecular species, MH+, that is one mass unit higher than the analyte, is formed.  When compared with EI, CI provides much less fragmentation and hence a simpler spectrum. However, the simpler spectra may not provide much structural information as a consequence.

c. Desorption ionization techniques: The desorption techniques are soft and give less fragmentation. In these techniques, the matrix should be volatile, relatively inert, and an electrolyte to allow the formation of ions. They are useful for compounds with a high molecular mass. In comparison, EI and CI both require a more volatile sample to be analyzed.

There are three desorption methods used for the ionization of large, non-volatile molecules in mass spectrometry.

  1. Secondary Ion Mass Spectrometry (SIMS)
  2. Fast Atom Bombardment (FAB)
  3. Matrix-Assisted Laser Desorption Ionization (MALDI)

The analyte is dissolved or dispersed in a matrix and bombarded with:

  1. High energy beam of ions (Ar+ or Cs+) in SIMS
  2. High energy beam of neutral atoms (Ar or Xe) in FAB
  3. High-intensity photons (Nitrogen laser at 337nm) in MALDI

The collision of these with the sample ionizes some of the sample particles and ejects them from the surface. The ejected ions are then accelerated towards the mass analyzer.

Some common matrices for SIMS and FAB include glycerol, thioglycerol, diethanolamine, and triethanolamine.

The matrices for MALDI are picolinic acid, 3-Hydroxypicolinic acid, and anthralin.

d. Electrospray/Thermospray Ionization: These two techniques are to ionize non-volatile compounds with a high molecular weight.

I. Electrospray Ionization (ESI): The sample solution is sprayed out of a fine capillary into a heated chamber at atmospheric pressure. The capillary has a high voltage potential across its surface which causes small charged droplets to form. These droplets are then expelled into the ionization chamber. The droplets are then subjected to the counter-flow of a drying gas (N2) that causes the solvent molecules to evaporate.

In this way, the charge density of the droplets increases until the electrostatic repulsive forces exceed the surface tension of the droplet. This causes the droplet to break into smaller droplets. The process continues until only sample ions are left in the gas phase, free of the solvent.

II. Thermospray Ionization (TSI): TSI occurs in a similar manner to ESI with the exception of using a heated capillary rather than one with electric potential. Thermospray is a soft ionization source by which sample solution passes through a thin, heated column to turn into a spray of fine droplets.

The droplets are ionized with the help of a low current discharge electrode to form a solvent-ion plasma. The charged particles are then directed by a repeller through the molecular leak, into the mass analyzer.

3. Accelerator

Accelerator mass spectrometry (AMS) is a type of mass spectrometry that involves accelerating ions to high kinetic energies before the analysis. The advantage lies in its power to separate a rare isotope from its abundant neighboring mass.

The positive ions produced are directed through a slit by applying the repeller potential and then are accelerated by applying an acceleration potential. The ions move through the analyzer portion with high velocities and are separated according to their mass to charge ratio.

4. Mass analyzer (deflection)

The accelerated ions then enter the mass analyzer, where they are separated according to their mass to charge ratios.

There are various types of mass analyzers:

a. Magnetic sector mass analyzer: At a given energy potential (1-10 kV), each ion will have kinetic energy

mv2 / 2 = zV

where m is the mass of the ion, v is velocity, V is the potential difference, and z is the charge on the ion.

As the ions enter a magnetic field, they move in a curved path, and the radius of this curvature is

r = mv / zB

where B is the strength of the magnetic field and r is the radius of the ion’s path.

Rearranging and combining the equations, we get

m / z = B2r2 / 2V

This equation indicates that an ion with a larger m/z value will move in a path with a larger radius (or will be deflected less). The analyzer has a fixed radius of curvature so only the particle with the correct m/z value will reach the detector.

This implies that particles having greater or lesser than the specified m/z value will strike the walls of the analyzer and not reach the detector. So a mass spectrum can be obtained by changing B at constant V (magnetic scanning) or by changing V at constant H (electric voltage scanning).

b. Double-focusing mass analyzer: A double-focusing mass analyzer utilizes an electrostatic separator along with the magnetic analyzer to improve the resolution. Ions having the same mass acquire a range of kinetic energies when accelerated, leading to overlapping signals. The application of an electrostatic field allows the selection of ions with the same kinetic energy only.

c. Quadrupole mass analyzer: It consists of four parallel metal rods close together but leaving a small space through the center. The ions are accelerated into this space. A DC potential and a high-frequency RF signal are applied across opposite pairs of rods. The ions of a specific m/z value pass straight through the space to the detector while others spiral towards the rods. Ions with different m/z ratios can be allowed to reach the detector by altering the DC and RF signals.

d. Ion-trap mass analyzer: It is a modified version of the quadrupole mass analyzer with a circular polarizable rod and end caps confining the ions in a central cavity in circular trajectories before allowing them to move to the detector in order of increasing m/z value. An important feature of the quadrupole and ion-trap analyzers is that they allow rapid scanning through a wide range of masses. This ability makes them ideal for monitoring chromatographic peaks.

e. Time-of-flight mass analyzer: The working of this analyzer is based on the idea that the velocities of two ions created at the same time with the same K.E, will depend on their masses. The heavier ion will possess a lower velocity. Therefore, the lighter ion will reach the detector first. 

One requirement is that the ions must be created in well-defined pulses so all ions start moving towards the detector at the same time. Another requirement is that the electronics must be fast enough to measure the ion flight times accurately in orders of microseconds. The advantages of TOF mass analyzers are that they have high sensitivity and rapid data acquisition, in addition to having no upper limit to their effective mass range.

5. Detector

a. Photographic plates: The earliest detectors had photographic plates at the end of the analyzers. All ions of a specific m/z value hit the same spot at the plate, making a spot. The darkness of the spot indicated the intensity of the particular m/z ion.

b. Electron multipliers: Contains more than 20 dynodes (Cu/Be surfaces) and an anode held at successively higher voltages. A burst of electrons is emitted when ions with energy or electrons hit the dynodes each time. The detector detects positive ions and works in a similar fashion to the photomultiplier tube in UV/Visible spectroscopy.

c. Faraday cup (cylinder electrode detector): It consists of a hollow collector open at one end and closed at the other to collect a beam of ions. The working principle is that the incident ion hits the dynode surface which causes an emission of electrons. A current is induced which is amplified and recorded.

It is surrounded by a cage that prevents the escape of reflected ions and ejected secondary electrons. The dynodes are made of secondary emitting material such as BeO, CsSb, or GaP. This detector is suited for isotope-ratio mass spectrometry (IRMS). The features of this detector are that it is very robust and inexpensive. It is also independent of the energy, mass, or nature of the ion.

Concepts Berg

What is the basic principle of mass spectrometry? 

Mass spectrometry works on the principle of ionization of particles by an appropriate method. This is followed by their deflection in an electric or magnetic field by their mass-to-charge ratio. Finally, the ions are detected using a suitable detector and spectrum of m/z against relative abundance or intensity plotted.

What are the four stages of mass spectrometry?

The four stages of mass spectrometry are ionization, acceleration, deflection, and detection.

What is the purpose of mass spectrometry?

Mass spectrometry by itself is used for qualitative analysis to analyze the composition of a sample using ionization, and then determine the structure with the help of its spectrum.

What are the uses of mass spectrometry?

Mass spectrometry is used for pharmaceutical analysis including pharmacokinetics, bioavailability studies, identifying drugs, screening of drugs, and characterization of drug candidates. It can also be used in environmental analysis for pesticides and soil or water contamination. Moreover, it can be utilized to characterize biomolecules like proteins and peptides. Furthermore, forensic or clinical analysis can also be supported by mass spectrometry. Mass spectrometry also finds its applications in carbon dating and isotope ratio determination.

What is detection in mass spectrometry?

After the particles are ionized, accelerated, and deflected by their mass-to-charge ratio, the final stage is the detection of the positive ions before a spectrum is plotted. A suitable detector can be used for this purpose, keeping in mind the nature of the sample. The first detectors were photographic plates on which dark spots were formed when ions hit. Some of the commonly used detectors are Faraday cup and electron multipliers.

What are the components of a mass spectrometer?

The components of a mass spectrometer include a sample inlet system, ion source, accelerator, mass analyzer (deflection), and detector.

How does a mass spectrometer work?

A mass spectrometer works by the ionization of the sample to form positive ions having a mass-to-charge ratio by which they are separated in the mass analyzer after they have been accelerated. The ions are then detected and a spectrum is plotted of the m/z value against its intensity.

How is a mass spectrometer used?

The sample to be analyzed could be present in the solid, liquid, or gaseous state. The gaseous sample can be directly injected into the ionization chamber. For less volatile substances, either of direct probe method or the APCI technique could be used. Alternatively, the mass spectrometer can be coupled with a gas chromatograph. Gaseous ions are formed with the help of a suitable technique. The ions are then accelerated. The mass analyzer deflects these ions by their mass-to-charge ratio and finally, the ions are detected. A spectrum is then shown.

Why mass spectrophotometry is never called mass spectroscopy?

Spectroscopy is used to define the measurements by interaction with electromagnetic radiation. Spectroscopies include ultraviolet/visible, infrared, and Raman; all of which utilize electromagnetic radiation of relevant frequencies. On the other hand, mass spectrometry works on the ionization and fragmentation of the sample, and not by its interaction with electromagnetic radiation.

References

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