Mass spectrometry

MASS SPECTROMETRY

Arushe tickoo

B.Tech Biotech

Introduction

Mass spectrometry (MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the atoms or molecules comprising a sample of material.

It is a technique in which the molecules in the test sample are converted to gaseous ions that are subsequently separated in a mass spectrophotometer according to mass to charge(m/z) ratio

History

Mass spectroscopy was first performed at the cambridgeuniversity, in 1912 by J.J Thomson (1856-1940) when he obtained the mass spectra of O2, N2, CO

Mass spectroscopy took off in 1930s and advance technology resulted in the development of double focusing Mass spectrometers capable of accurate determination.

The modern techniques of mass spectrometry were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively. In 1989, half of the Nobel Prize in Physics was awarded to Hans Dehmelt and Wolfgang Paul for the development of the ion trap technique in the 1950s and 1960s. In 2002, the Nobel Prize in Chemistry was awarded to John Bennett Fenn for the development of electrospray ionization (ESI)

spectrophotometer

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Ionization Source Mass Analyser Detector

Sample under investigation has to be ionized inside the ionization chamber of the sample.

These ions are extracted into analyser where they are separated according to mass to charge (m/z) ratio.

Introduction

Components

A high vacuum system

An ion source : MALDI(matrix assisted low desorption ionization),

ESI(electrospray ionization), FD(field desorption), EI(electron impact)

A mass filter/analyser : TOF(time of flight), quadrupole

A detector : conversion dynode, electron multiplier

INLETION

SOURCEMASS FILTER DETECTOR

DATA SYSTEM

High vacuum system HIGH VACUUM SYSTEM

Vacuum system

All mass analysers operate under vacuum in order to minimize collision between ions and air molecules. Without a high vacuum , the ions produced in the source will not reach the detector.

Two vacuum pumps are used :

1. Rotary vane pump : It was invented by Charles C. Barnes of Sackville, New Brunswick,it is used to provide initial vacuum.

2. Turbomolecular pump : The turbomolecular pump was invented in 1958 by Becke, it provides high vacuum, it is a high speed gas turbine with moving blades and stators ( fixed blades ) whose rotation forces the molecules through the blade system.

Sample preparation

Sample can be introduced directly or through sometype of chromatographic techniques that is HPLC, GC or capillary electrophoresis.

Ionization sources :

1. ESI( electrospray ionization )

2. MALDI( matrix assisted laser desorption ionization )

3. FD( field desorption )

4. EI( electron impact )

Method of sample introduction often depends upon the ionization methods used

Hard and Soft ionization techniques

Field Ionization / Field Desorption

Possibly the first ‘soft’ ionization method was ‘Field Ionization’,involves desorption of ions from a surface in a very high electric field.

The salient feature of soft ionization is that analytes (even large ones) are transferred intact into the gas phase.

This includes:

1. Large Polymers

2. Nucleic Acids and non-covalent complexes

3. Proteins and non-covalent complexes

Examples: ESI, MALDI, Chemical ionization

Soft ionization

1. Leave excess energy in molecule – extensive fragmentation(the systematic rupturing of bonds acts to remove the excess energy, restoring stability to the resulting ion), resultant ions tend to have m/z lower than the molecular mass2. Common example of this ionization is electron impact ionization

Hard ionization

ESI (electrospray ionization )

This technique was developed by John Bennett Fenn.

Sample is dissolved in volatile solvent and pumped through a narrow and stainless steel capillary. A high voltage of 3-4 kv is applied,as a result of this strong electric field produced the sample emerging from tip is dispersed into aerosols of highly charged droplets.

Electrospray ionization (ESI) is a technique used in mass spectrometry to produce charged ions. The liquid containing the analyte(s) of interest is dispersed by electrospray, into a fine aerosol. Because the ion formation involves extensive solvent evaporation (also termed desolvation), the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol ,acetonitrile).Compounds that increase the conductivity (e.g. acetic acid) are added to the solution. These species also act to provide a source of protons to facilitate the ionization process. The aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary carrying a potential difference of approximately 3000V, which can be heated to aid further solvent evaporation from the charged droplets.

Electrospray ionization

MALDI( matrix assisted laser desorption ionization)

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, MALDI is a two-step process. First, desorption is triggered by a UV laser beam. Matrix material heavily absorbs UV laser light, leading to the adsorption of upper layer of the matrix material.

In desorption ionization the sample to be analysed is dissolved in a matrix and placed in a path of high energy beam of ions or high energy photons but in case of MALDI high intensity photons are used.

In this ionization matrix is used to protect the biomolecule from being destroyed by direct laser beam.

Solid matrix strongly absorb the laser radiations, with a strong pulse of laser light the analytes are analysed.

MALDI (matrix assisted laser desorption ionization)

The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (CHCA, alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added.

Matrix

Ionization mechanismMALDI produces gas phase protonated ions by the excitation of sample molecules from the energy of the laser transferred via a UV light absorbing matrix. The sample is mixed with the matrix and dried on the target plate where they co-crystallize on drying. Pulses of laser light of a few nano seconds causes rapid excitation and vapourisationof the crystalline matrix and ejection of analyte ions into the gas phase. This generates a plume of analyte ions that are analysed in a mass analyser.

Electron ionisation

Electron ionization (EI, formerly known as electron impact) is an ionization method in which energetic electrons interact with gas phase atoms or molecules to produce ions. This technique is widely used in mass spectrometry, particularly for gases and volatile organic molecules.

In an EI ion source, electrons are produced through thermionic emission by heating a wire filament that has electric current running through it. The electrons are accelerated to 70 eV in the region between the filament and the entrance to the ion source block. The accelerated electrons are then concentrated into a beam by being attracted to the trap electrode. The sample under investigation which contains the neutral molecules is introduced to the ion source in a perpendicular direction to the electron beam. Close passage of highly energetic electrons, referred to as a hard ionization source, causes large fluctuations in the electric field around the neutral molecules and induces ionization and fragmentation.[3] The radical cation products are then directed towards the mass analyzer by a repeller electrode.

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Chemical ionization

In chemical ionization a reagent gas, typically methane or ammonia is introduced into the mass spectrometer. Depending on the technique (positive CI or negative CI) chosen, this reagent gas will interact with the electrons and analyte and cause a 'soft' ionization of the molecule of interest.One of the main benefits of using chemical ionization is that a mass fragment closely corresponding to the molecular weight of the analyte of interest is produced.

In positive chemical ionization (PCI) the reagent gas interacts with the target molecule, In negative chemical ionization (NCI) the reagent gas decreases the impact of the free electrons on the target analyte.

Analysers Once the sample has been ionized, the beam of ions is accelerated by an

electric field and then passed into mass analyser, the region of mass spectrophotometer where the ions are separated according to the mass to charge ratio

Just like the ionization methods ,there are several types of mass analysers the most common type of mass analyser is TOF (time of flight)

once the ions are created and leave the ion source they pass into the mass analyser, the function of which is to separate the ions and measure their masses.

Types of analysers:

1. Quadrupole analyser

2. TOF(time of flight)

Quadrupole

The quadrupole mass analyzer is one type of mass analyzer used in mass

spectrometry. As the name implies, it consists of four cylindrical rods, set

parallel to each other. In a quadrupole mass spectrometer the quadrupole is

the component of the instrument responsible for filtering sample ions, based

on their mass-to-charge ratio (m/z). Ions are separated in a quadrupole based

on the stability of their trajectories in the oscillating electric fields that are

applied to the rods

The quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together electrically, and a radio frequency (RF) voltage is applied between one pair of rods and the other.

The ions are accelerated down the analyser towards the detector the varying electric field is precisely controlled so that during each stage , the ions of one particular mass to charge ratio pass down the length of analyser.

TOF (time of flight)

The TOF mass analyser measures the ion flight time.

It is based upon the simple idea that velocity of two ions with kinetic energy will vary depending upon the mass of ion that is lighter ions will have higher velocity

Mass spectrometry can be used to measure the mass of biomolecules well over 200,000 Da to provide sequence information of unknown peptide and proteins and to detect the non covalent complexes

Detector

1. Electron multipliers and conversion dynodes:

Electron multipliers are used as a detector in frequent combination with conversion dynode which is a device to increase sensitivity. Conversion dynode is surface that is held at high potential so that ions striking the surface produce secondary charged particles. The ion beam from the mass analyser are focused on the conversion diodes.

Positive or negative ions hit the conversion dynode, causing the emission of secondary particles. The secondary particles are accelerated into the dynodes of electron multiplier

Applications

1. Mass spectrometry is also used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare.

2. Mass spectrometry is an important method for the characterization and sequencing of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

3. Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been predominantly used in glycobiology for characterization and elucidation of glycan structures.

Disadvantages

A few of the disadvantages of the method is that often fails to distinguish between optical and geometrical isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring. Also, its scope is limited in identifying hydrocarbons that produce similar fragmented ions.