Mass spectrometry: An introduction, principle, instrumentation, working with its application.

What is Mass spectrometry?

schematic-diagram-of-mass-spectrometry

Mass spectrometry is one of the primary spectroscopic methods for molecular analysis available to organic chemists.  It requires only a few nano-moles of the sample to obtain characteristic information related to the structure and molecular weight of analytical material.

It is not concerned with non- destructive interaction between molecules and electromagnetic radiation. In most cases, the nascent molecular ion of the analytical material produced fragment ions by cleavage of the bond. The resulting fragmentation pattern constitutes the mass spectrum.

Introduction of Mass spectrometry-

Mass spectrometry is a subversive method which is used to measure molecular weight and provide data on molecular structure. It differs from the other spectroscopy methods because in that the sample is ionized and not subject to electromagnetic radiation like other spectrometers.

In this process, the ionized compounds are excited, which induces fragmentation. Analysis of such fragments provides information on the structure of molecules. Each fragment is characterized by the mass-to-charge ratio m/z, and devices are able to separate and detect such ions.

The mass or molecular weight of a compound can be found in several ways. This process is not only for the determination of Mass, but the technique can be used for structure elucidation, quantitative analysis, and even advanced studies could be done by using the Mass spectrum.

History of Mass spectrometry

The process of mass spectrometry has developed from the experiments and studies early in the 20th century. Tried to explain the behavior of charged particles in magnetic and electrostatic force fields in the analytical material.

The first mass spectral measurements were undertaken by J.J. Thomson in 1912, and, F.L. Arnot and J.C. Milligan in 1918 introduced the method of ionization and which pass through a magnetic sector. In 1946, W.E. Stephens assembled a time-of-flight (TOF) device.

In 1953–1958, W. Paul used a quadrupole analyzer. F.H. Field applied chemical ionization in 1966, which was followed by the ‘thermo spray’ method, introduced by the C.R. Blackley team in 1968.

Coupling with gas chromatography was performed by C. Gohlke and F. McLafferty, in 1973 the latter further introducing coupling with liquid chromatography. Since then, there is a continuous improvement is carrying in mass spectrometry techniques.

Principle of Mass spectrometry

It is based on Newton’s second law of motion and momentum, where a mass spectrometer uses this property of matter to plot ions of varying masses on a mass spectrum. From the law, we derive how much mass is relevant to the inertia and acceleration of a body.

This principle is applied to the aspect where ions with different mass to charge ratios are deflected by different angles in an electric or magnetic field.

Mass spectra are also called as Positive ion spectra or line spectra. Unlike other kinds of spectroscopy, we do not use any Electro Magnetic Radiation (EMR) for excitation. We use electron bombardment to convert a natural atom to a positively charged one.  Also, no ground or excited state is their like other types of spectroscopy in it.

Obtaining mass spectra consists of two steps-

-Conversion of the neutral molecule into a charged molecule, preferably to a positively charged molecule.

-Separation of the positively charged fragments formed which is based on their masses, by using an electrical or magnetic field or both.

The sample is bombarded with high energy electron beam (70eV). where an electron is knocked off from every molecule. Hence the molecule becomes positively charged. When a positive potential (accelerating potential) is applied, as the molecules are positively charged, they get repelled and travel with great speed, in a straight path.

Potential energy = Kinetic energy of the molecule

eV = 1/2 mv2

where e =charge of ion

V = acceleration voltage

m = mass

v = velocity after acceleration

When a magnetic field or an electric field is applied, the positively charged fragments which were traveling in a straight path now travel in a curved path.

When they travel in a curved path under the influence of the magnetic field, the fragments are separated into different masses because the radius of curvature depends upon their respective masses.

Under magnetic field,

Hev=mv2/r

Therefore- v = reH/m

where r = radius of ion path

H = strength of magnetic field

e, m, v = as described earlier

Substituting the value of v in the first equation, (i.e. eV = ½ mv2),

eV = ½ ×m×(reH/m)2

On simplifying- m/e = H2r2/2V

m/e r2 since H & V are maintained constant

Therefore- Mass ∝ (radius of ion path)2, since e = 1 (unit positive Charge)

You may read- Spectroscopy.

Theory of Mass spectrometry

Mass spectrometry is the most accurate method for determining the molecular mass of the compound and its composition. In this process, molecules are bombarded with a beam of energetic electrons. The molecules are ionized and broken up into many fragments, some of which are positive ions.

Each kind of ion has a particular ratio of mass to charge (m/z) ratio. For most ions, the charge is one, and thus, the m/z ratio is simply the molecular mass of the ion.  When one electron is removed from the parent molecule of the substance results in a parent ion.

Basic-principle-of-mass-spectrometer
Source-internet

The m/z ratio of the parent ion is equal to the molecular mass of the compound.  In a few cases, the parent ion peak can be the base peak and may be easily recognized, but in most cases, parent ion peak is not the base peak and is often of very small abundance.

Many elements occur naturally as isotopes, and out of these the lightest one greatly predominates.

The mass spectrometer is designed to perform three basic functions are-

-For vaporize compounds of varying volatility.

-For produce ions from the neutral compounds in the vapor phase.

-For separate ions according to their mass over charge ratio and for record them.

The plot of m/z values taken along the abscissa and their relative intensities along the ordinate is called the mass spectrum. Neutral particles (whether neutral molecules or radicals) are produced in the process of fragmentation that cannot be detected in the mass spectrometer.

Instrumentation of Mass spectrometry

Mass spectrometers consist of the basic components are- ion sources ionizer, accelerator, deflector, and a detector. Brief description of the various types of components are presented as-

Mass-spectrometer-with-label-diagram
Source-internet

Sample Inlet system-

In mass spectrometer the number of ion sources is extensive and each comes with its own advantages and disadvantages for analyzing different solids, liquids, and gasses. The method of ionization plays a key role in what type of samples can be analyzed. For example, for solid or liquid biological samples it is common to use techniques such as electrospray (ES) or matrix-assisted laser desorption/ionization (MALDI) while for analyzing compounds with high molecular weight a chemical ionization process may be more appropriate.

Solids– Directly into the chamber with low vapor pressure by means of probs. E.g.- Steroids, carbohydrates.

Liquid– Vaporized with the help of a heat inlet system. E.g.- Sugar, amino acids.

Gas– Directly inlet and vaporized.

Ion source-

Ionizer (Ionization)-

Ionizer, where the bombarding of the sample is done by the electrons. These electrons move between cathode and anode.  When the sample passes through the electron stream between the cathode and anode, electrons with high energy clatter electrons out of the sample and form ions.

The process of ionization in which the atoms are ionized by knocking one or more electrons off to give a positive ion. The process of mass spectrometers always works with positive ions.

ionization-in-mass-spectrometry

The particles in the sample like-atoms or molecules are bombarded with a stream of electrons to clatter one or more electrons out of the sample particles to make positive ions. Mostly the positive ions formed will carry a charge of plus 1.

These positive ions are persuaded out in the rest of the machine by the ion repeller which is another metal plate carrying a slight positive charge. Ionization can be achieved by-

Electron impact Ionization

Chemical Ionization

Field ionization

Laser desorption technique

Fast-acting bombardment

Accelerator (Acceleration)-

The accelerator is the ions placed between a set of charged parallel plates that get attracted to one plate and repel from the other plate. The acceleration speed can be controlled by adjusting the charge on the plates.

Acceleration-in-mass-spectrometry

The process of acceleration where the ions are accelerated so that they all have the same kinetic energy. The positive ions are repelled away from the positive ionization chamber and then pass through three slits with voltage in the decreasing order inside it.

The middle slot carries some intermediate voltage and the final at ‘0’ volts. All these ions are accelerated into a finely focused beam.

Mass analyzer-

Deflector (Deflection)-

Deflector, where the magnetic field deflects ions based on its charge and mass. If an ion is heavy or has two or more positive charges, then it is least deflected. If an ion is light or has one positive charge, then it is deflected the most.

The ions are then removed by a magnetic field according to their masses. Those are the lighter in weight, more they are deflected. The amount of deflection also depends on the number of positive charges on the ion – more the ion is charged, the more it gets deflected.

Deflection-in-mass-spectrometry

Different ions are deflected by the magnetic field in different amounts.  The amount of deflection depends on-

The mass of the ion– Lighter ions is deflected more than heavier ones.

The charge on the ion– Ions with two or more positive charges are deflected more than ones with only one positive charge.

Types of analyzers-

-Magnetic sector mass analyzers

Single focusing analyzer

Double focusing analyzers

-Quadrupole mass analyzers

-Time of Flight analyzers (TOF)

-Ion trap analyzer

-Ion cyclotron analyzer

Detector (Detection)-

The detector is those where the ions with correct charge and mass move and the ratio of mass to charge is analyzed through the ion that hits the detector. The beam of ions passing through the machine is detected electrically inside it.

Detection-in-mass-spectroscopy

When an ion hits to the metal box, then its charge is neutralized by an electron jumping from the metal on to the ion. It leaves a space amongst the electrons in the metal, and then the electrons in the wire dragging along to fill it. The flow of electrons in the wire is detected as an electric current, which can be amplified and recorded into the data system.

The more ions arriving, the greater the current. The detectors are used as-

Faraday cup detector

Photomultiplier detector

Photographic detector

Working-

It has a horseshoe-shaped glass tube which is evacuated. It has a sample inlet, an electron bombarding source, and accelerating plates on one end. At the other end, the collector slit is present. At the curvature of the tube, there is the provision to apply electrical or magnetic fields.

Sample in the form of is allowed through sample Inlet. This sample is bombarded with an electron beam at 70eV. This knocks off one electron from every molecule. As these molecules become positively charged, they are accelerated by accelerating plates which also have a positive charge.  These accelerated molecules travel in a straight path. But because of the application of either electrical field or magnetic field, they travel in a curved path. When they travel in a curved path, the positive charged molecular ions are separated according to their mass. These separated ions are collected using a collecting slit. The different fragments fall on the detector and a mass spectrum is recorded.

schematic-diagram-of-mass-spectrometry

In a regular mass spectrometer, firstly we have the material to be analyzed, but we need it to be ionized to pass through the spectrometer with enough required energy. Thus, the sample is bombarded by electrons to ionize it.

This ionized beam is now passed through a series of electric or magnetic fields depending on the type of the sample and its properties. The ions are deflected by the field through which they are passed through in such a way that the ions with the same mass to signal ratio will follow the same path to the detector.

These charged and deflected ions are now incident onto a detector which is capable of distinguishing the charged particles falling on it. Based on the mass spectrum produced by the charged ions, we can identify the atoms or molecules constituting the sample by comparing them with known masses or through a characteristic fragmentation pattern.

You may read- Infrared spectroscopy.

Types of peaks-

Parent peak, Mass peak, or molecular ion peak– (often called M+ or P+). The peak normally with the highest m/e value (x-axis) is called M+.

Base peak– The most intense peak (tallest on the y-axis) is called a base peak.

M+I peak– It occurs due to the presence of 13C 2H, 15N 33S.

M+2 peak- It occurs due to the presence of 180 34S 37Cl 81 Br.

Applications

Structure elucidation– Using Nitrogen rule, peak matching, the fragmentation pattern of compound, and % abundance of isotopes, structure elucidation of organic compounds can be done.

Detection of impurities- Impurities present can be detected by the additional peaks, the highest value of mass peaks than the compound itself, and from the fragmentation pattern.

Quantitative analysis- The intensity of peaks corresponds to the proportion of fragments. By comparing with the standard drug, the quantity of sample can be estimated.

Drug metabolism studies- By recording the mass spectrum of metabolite and that of pure drug, the metabolism of the drug can be known.

Clinical, Toxicological, and Forensic applications– Very minute quantities of the drug can be used to find out mass and hence the substance can be identified from the mass spectral pattern.


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