Mass Spectroscopy

MASS SPECTROSCOPY

- Branch of Spectroscopy

- Analytical technique that gives information concerning the molecular structure the of organic and inorganic compounds

- It can determine molecular weight of as high as 4000

- Qualitative analytical tool to characterize different organic substances

- The quantitative analysis of mixtures (gases or liquids and sometimes solids)

- Based on the simple principle

- Yet it is a very complex and very expensive instrument

- Commonly used because of its high speed and reliability

PRINCIPLE

The compounds under investigation are bombarded with a beam of electrons which produce an ionic molecules or ionic fragments of the original species then they are separated on the basis of the difference in their masses. Suppose ionization is as follows;

M + e^- ® M⁺ + 2e^-

Where:

M⁺ = an ionized molecule

e^- = an electron

The ions are then accelerated in an electric field at voltage V. Now the energy given to each particle is eV and this is equal to the kinetic energy of the ions.

½ mn² = e V

Or n = Ö 2e V/m

Where:

n = velocity of the particle of mass, m

e = charge on an electron

m = mass of the particle

V = accelerating voltage

All the particles possess the same energy, eV. Also, all particles have the same kinetic energy, ½ mn². As the value of m varies from particle to particle, the velocity of particle in motion also changes to meet the potential energy. E.g. For a particle with mass, m₁ and n₁

½ m₁n₁²= eV

Similarly, for particles of mass m₂, m₃, m₄,………………..m_n the velocities are n₁, n₂, n₃, …………….n_n. Now their kinetic energies are:

½ m₂n₂²= eV ½ m₃n₃²= eV ½ m₄n₄²= eV………………½ m_nn_n²= eV

From above, we have;

½ m₂n₂²= ½ m₃n₃²= ½ m₄n₄²= ………………½ m_nn_n²= eV

After the charged particles have been accelerated in an applied voltage, they enter into the magnetic field, H. This field attracts the particles and move in a circular around it. The attractive force due to magnetic field is Hen, where the balancing centrifugal force on the particle is mn²/r. When the particle starts moving uniformly around the circular path the two forces become equal. i.e.

mn²/r = H.e.n

r = mn / H.e

Where, r is the radius of circular path of the particle in the motion.

From above equations;

r = [m / H.e] Ö 2eV/m

On squaring both sides;

r² = [m² / H².e²] 2eV/m

\ m/e = [H² . r²]/ 2V

The radius of circular path of the particles depends on the accelerating voltage- V, the magnetic field-H and the ratio of mass upon charge-m/e. As e, V and H are constants; the radius of ionized molecule depends on the mass only which is actually the main basis of separation of particles.

INSTRUMENTATION

1. INLET SYSTEM:

- A Vapor form of sample is used

- Sample enters the ionization chamber at a constant rate

- Sample is changed into gaseous state in inlet system

- Pressure in inlet system is 30-50 torr

- Sample is injected through a pinhole of 0.013-0.05 mm size made up of gold foil

- Different inlet systems are used for different types of materials

- Sample size » 1 mmole (Micromole)

- Only few percent of sample enter ionization chamber and 0.1 % gets ionized

2. ION SOURCE:

- Electrically heated filament produces thermal electrons

- These electrons are accelerated by anode present just opposite to filament

- A beam of accelerated electrons intersects the flow of sample molecules

- Striking of electrons and sample molecule results in the formation of positively charged ions

- Ions are withdrawn by electric field, a charged repellor plate (anions are attracted to it), and accelerated toward other electrodes, having slits through which the ions pass as a beam

- Energy of electron beam is controlled by potential on Anode plate

- Low energy of 50-80 eV gives most reproducible results and doubly charged ions are rarely produced

3. ELECTROSTATIC ACCELERATING SYSTEM:

- Positively charged ions are formed in ionization chamber

- Withdrawn by electric field which exists in accelerator plates

- A strong electrostatic field of 400-4000 V is developed in those accelerators

- It accelerates m₁, m₂, m₃,……..m_n to their respective velocities which are finally escape through accelerating slits having kinetic energies i.e.

eV = ½ m₂n₂²= ½ m₃n₃²= ½ m₄n₄²= ………………½ m_nn_n²

- Initially the accelerating plates are charged at 4000 V, which is then gradually leak off to ground at a controlled rate over a period of 25 minutes

4. MAGNETIC FIELD:

- Accelerated particles enter the magnetic field

- Magnetic field is required to move the particles in their respective circular path

- When the ion beam experiences a strong magnetic field perpendicular to its direction of motion, the ions are deflected in an arc

- The radius of arc is inversely proportional to the mass of the ion. Lighter ions are deflected more than heavier ions.

- By varying the strength of the magnetic field, ions of different mass can be focused progressively on a detector fixed at the end of a curved tube (also under a high vacuum).

5. THE ION SEPERATOR OR ANALYZER:

- The main function of the mass analyzer is to separate, or resolve, the ions formed in the ionization source of the mass spectrometer according to their mass-to-charge (m/e) ratios.

- There are a number of mass analyzers currently available, the better known of which include quadrupoles, time-of-flight (TOF) analyzers, magnetic sectors, and both Fourier transform and quadrupole ion traps

The analyzer should have following features

High Resolution:- it should be able to differentiate C₁₆H₂₂O₂, MW = 246.1620 and C₁₇H₂₆O, MW = 246.1984

A high rate of transmission of ions:- The pore size in slit should be very minute

- When slits are made narrow, the resolution increases but the transmission of ions decreases. Therefore, both of these properties can’t be obtained in a single MS instrument

Single Focusing Magnetic Deflection:

Singly charged ions are given the same kinetic energy

Double Focusing:

Two beams from independent sources pass side by side through a common mass analyzer and deflected by separate collectors. It is used to compare a single sample under different ionizing conditions. It has high resolution

Time of Flight:

All ions leave the acceleration field with different velocities depending on their masses. Without the field, they will take different times to travel a given distance. It is based on the non-magnetic separator.

6. THE ION COLLECTOR OR DETECTOR:

- The detector monitors the ion current (10^-15-10^-19), amplifies it and the signal is then transmitted to the data system where it is recorded in the form of mass spectra.

- The m/e values of the ions are plotted against their intensities to show the number of components in the sample, the molecular mass of each component, and the relative abundance of the various components in the sample.

- The type of detector is supplied to suit the type of analyzer; the more common ones are the photomultiplier, the electron multiplier and the micro-channel plate detectors.

- Electron multiplier is necessary to amplify less than 10^-15 amp.

7. VACCUM SYSTEM:

- A very high vaccum system is maintained in the whole instrument.

- Presence of any gaseous or any other particle may interfere on the velocity of ionized molecule or sample.

- The inlet system is maintained at 0.015 torr while ion source at 10^-5 torr and analyzer tube at 10^-7 torr or as low as possible are maintained.

TYPES OF IONS PRODUCED IN MASS SPECTROSCOPY

Molecular ion or Parent peak:

· Electron beam of energy of 9-15 eV is used to generate it

· Molecular ion is produced by loss of a single electron from parent molecule

· A very simple mass spectra of one peak called parent peak is formed

Base Peak:

· Electron beam of energy 70 eV is used

· Molecule undergoes splitting to form many fragments

· Parent peak in mass spectra is called base peak. Heights of all other peaks of different fragments are measured with respect to it.

Dissociation peaks:

· Molecule undergoes dissociation into stable and ionic fragments

· Cleavage is favored at branching. E.g. Dissociation peaks of Methane (CH₄)

	Fragments	m/e ratio	Relative Abundance (%)
	H C CH CH2 CH3 CH4 13CH4	1 12 13 14 15 16 17	3.1 1.0 3.9 9.2 85 100 1.1

Rearrangement ions:

Multiple charge ions:

Negative ions:

Metastable ions:

GENERAL RULES FOR INTERPRETATION OF MASS SPECTRA

The Exact Molecular Weight:

· Exact molecular weight of a pure compound can be determined by identifying the parental peak.

· By using the molecular weight, molecular formula of a compound can be determined.

The Isotope Effects:

· It is used to determine the distribution of naturally occurring isotopes.

· Heavy isotopes of same atom exhibits peaks in a mass spectra m/e at one or more units higher than normal. Low abundance isotopes show small peaks at M+1, M+2 and so on.

· From the heights, the exact molecular weight and abundance can be calculated.

Nitrogen Rule:

· Once molecular weight is known, the number of Nitrogen per molecule can be determined from Nitrogen rule.

· RULE-All organic compounds having an even integral molecular weight must contain either none or even number of nitrogen atoms and all organic compounds with odd molecular weight must contain an odd number of Nitrogen atoms. Examples are-

	Compounds	Mol. Wt.	Number of Nitrogen atoms
	C3H5NO2, C2H7NS C6H17BrN2	87, 77 196	Contains only one nitrogen atom Contains two Nitrogen atoms

· This formula is applicable for all compounds which have only covalent bonds and contain combination of C, H, N, O, S, Si, As, P, halogens and alkaline earth metals

1. Ring Rule:

· If the molecular formula is known by MS, one can calculate the number of unsaturated sites in the compound from Ring rule

· RULE-The number of unsaturated sites R, is equal to the number of rings in the molecule plus the number of double bonds plus twice the number of triple bonds. For the compound with molecular formula C_wH_xN_yO_z

R= w+1+[(y-x)/2]

For compounds with halogens:

No. of sites = carbons + 1 –[(halogens –nitrogens )/2] –(halogens/ 2)

APPLICATIONS

Molecular Mass determination

Isotopic abundance and dilution method

Qualitative analysis of mixture / impurities

Identification of unknown compounds

Trace gas analysis

Protein Characterization

Determination of Ionization potential

Pharmacokinetics

Space Exploration

SOME EXAMPLES:

ISOTOPES: Since molecules of bromine have only two atoms, the spectrum on the left will come as a surprise if a single atomic mass of 80 amu is assumed for Br. The five peaks in this spectrum demonstrate clearly that natural bromine consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu respectively. Thus, the bromine molecule may be composed of two ⁷⁹Br atoms (mass 158 amu), two ⁸¹Br atoms (mass 162 amu) or the more probable combination of ⁷⁹Br-⁸¹Br (mass 160 amu). Fragmentation of Br₂ to a bromine cation then gives rise to equal sized ion peaks at 79 and 81 amu.

PROTEIN CHARATERIZATION: Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection of peptide products are then introduced to the mass analyzer. When the characteristic pattern of peptides is used for the identification of the protein the method is called peptide mass fingerprinting (PMF).

PHARMACOKINETICS: Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point data. The most common instrumentation used in this application is LC-MS spectrometer. Standard curves and internal standards are used for quantization of usually a single pharmaceutical in the samples. The samples represent different time points as a pharmaceutical is administered and then metabolized or cleared from the body. Blank or t=0 samples taken before administration are important in determining background and insuring data integrity with such complex sample matrices. Much attention is paid to the linearity of the standard curve; however it is not uncommon to use curve fitting with more complex functions such as quadratics since the response of most mass spectrometers is less than linear across large concentration ranges.

SPACE EXPLORATION: As a standard method for analysis, mass spectrometers have reached other planets and moons. This instrument analyzed atmospheric samples along its descent trajectory and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the probe had landed. These measurements compare the abundance of isotope(s) of each particle comparatively to earth's natural abundance. Mass spectrometers are also widely used in space missions to measure the composition of plasmas.