Mass spectroscopy, Ionization techniques and types of mass analyzers

Spec trosc opySpec trosc opy

MASS SPECTROSCOPY

1

-: Presented By :-M Asif shaheenLecturer KEMU Lahore


Basic principleMass spectroscopy is the most accurate method for determining the molecular mass of the compound and its elemental composition.

In this technique, molecules are bombarded with a beam of energetic electrons.

The molecules are ionised and broken up into many fragments, some of which are positive ions.

Each kind of ion has a particular ratio of mass to charge, i.e. m/e ratio(value). For most ions, the charge is one and thus, m/e ratio is simply the molecular mass of the ion.

8


Principle and Instrumentation

9


IonisationThe atom is ionised by knocking one or more electrons off to give a positive ion. (Mass spectrometers always work with positive ions).

The particles in the sample (atoms or molecules) are bombarded with a stream of electrons to knock one or more electrons out of the sample particles to make positive ions. 10


Most of the positive ions formed will carry a charge of +1.

These positive ions are persuaded out into the rest of the machine by the ion repeller which is another metal plate carrying a slight positive charge.

11


Acceleration The ions are accelerated so that they all have the same kinetic energy.

12


The positive ions are repelled away from the positive ionisation chamber and pass through three slits with voltage in the decreasing order.

The middle slit carries some intermediate voltage and the final at ‘0’ volts.

All the ions are accelerated into a finely focused beam.

13


DeflectionThe ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected.

The amount of deflection also depends on the number of positive charges on the ion -The more the ion is charged, the more it gets deflected.

14


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

The mass of the ion: Lighter ions are deflected more than heavier ones.

The charge on the ion: Ions with 2 (or more) positive charges are deflected more than ones with only 1 positive charge.

15


DetectionThe beam of ions passing through the machine is detected electrically.

When an ion hits the metal box, its charge is neutralised by an electron jumping from the metal on to the ion. 16

Only ion stream B makes it right through the machine to the ion detector.

The other ions collide with the walls where they will pick up electrons and be neutralised.

They get removed from the mass spectrometer by the vacuum pump.


That leaves a space amongst the electrons in the metal, and the electrons in the wire shuffle along to fill it.

A flow of electrons in the wire is detected as an electric current which can be amplified and recorded. The more ions arriving, the greater the current.

17


InletIonsource

Mass Analyzer Detector

DataSystem

High Vacuum System

Mass Spectrometer Block Diagram


InletIonsource


DataSystem

High Vacuum System

Mass Spectrometer Block Diagram

Turbo molecular pumps


Inlet IonSource


DataSystem

High Vacuum System

HPLCFlow injectionSample plate

Sample Introduction


Inlet IonSource


DataSystem

High Vacuum System

MALDIESIFABLSIMSEICI

Ion Source


InletIonsource


DataSystem

High Vacuum System

Time of flight (TOF)QuadrupoleIon TrapMagnetic Sector

Mass Analyzer


The Sample Inlet System Batch Inlets The batch inlet system is considered the most

common and simplest inlet system. Normally, the inside of the system is lined with glass to elude losses of polar analyte by adsorption.

This system externally volatizes the sample which leaks into an empty ionization region. Boiling points up to 500 degrees C of gaseous and liquid samples can be used on typical systems.

The system's vacuum contains a sample pressure of 10-4 to 10-5 Torr. Liquids are introduced using a microliter syringe into a reservoir; gases are enclosed in a metering area that is confined between two valves before being expanded into a reservoir container.


Liquids that have boiling points lower than 500 degrees C can not be used in the system because the reservoir and tubing need to be kept at high temperatures by ovens and heating tapes. This is to ensure that the liquid samples are transformed to the gaseous phase and then leaked through a metal or glass diaphragm containing pinholes to the ionization area.


The Direct Probe Inlet: A direct probe inlet is for small quantities of

sample, solids, and nonvolatile liquids. Solids and nonvolatile liquids are injected through a probe, or sample holder.

The probe is inserted through a vacuum lock. Unlike the batch inlet, the sample will need to be cooled and/or heated on the probe.

The probe is placed extremely close (a few millimeters) to the ionization source, where the slit leads to the spectrometer,


Chromatographic and Capillary Electrophoretic Inlets

Chromatographic systems and Capillary Electrophoretic units are often coupled with mass spectrometers in order to allow separation and identification of the components in the sample. If these systems and units are linked with a mass spectrometer, then other specialized inlets,

Electrokinetic and Pressure injection, are required. Electrokinetic and pressure injection controls the

amount of volume injected by the duration of the injection, which typically range between 5 to 50 nL.


ION SOURCE

Since the mass analyzer utilizes only gaseous ions i.e., starting point of mass spectrometric analysis is formation of gaseous analyte ions.

• Non –Volatile solids are first converted in to gases and from the gaseous sample the ions are produced in a Box like enclosure called Ion Source.

Function Produces ion without mass discrimination of the

sample. Accelerates ions into the mass analyzer.


Desorption A phenomenon whereby a substance is released from or

through a surface. Sorption A process whereby one substance attached to another. It

can be of two types 1 Adsorption Adhesion of atoms ions or molecules from a gas liquid

or dissolved solid to a surface. This process create a adsorbate on the surface of adsorbent.

2 Absorption A process in which atoms ions or molecules are taken up

by a bulk phase i.e solid liquid or gas. Molecules are taken up by the volume not by the surface.


Catogories of Ion sources Gas Phase Sources � Electron Impact Ionization (EI) � Chemical Ionization (CI) � Field Ionizations (FI) Desorption Sources � Field Desorption (FD) � Electrospray Ionization (ESI) � Matrix assisted desorption/Ionisation (MALDI) � Plasma desorption (PD) � Fast Atom Bombardment (FAB) � Thermospray Ionization (TS) � Secondary Ion Mass Spectrometry (SIMS)


• Electron impact (EI) ionization

Electron impact (EI) is the classical ionization method in mass spectrometry.

• It is the most widely used and highly developed method.

• It is also known as Electron bombardment or Electron Ionization.


CONSTRUCTION & WORKING:

Electron impact ionization source consists of a ionizing chamber which is maintained at a pressure of 0.005 torr and temperature of 200 ± 0.25 degrees.

Electron gun is located perpendicular to chamber. Electrons are emitted from a glowing filament (tungsten or

rhenium) and accelerated by a potential of 70 V applied between the filament and anode.

These electrons are drawn in the ionization chamber through positively charged slits.

• The number of electrons is controlled by filament temperature and energy of energy is controlled by filament potential.

The sample is brought to a temperature high enough to produce molecular vapors.

• The gaseous Neutral molecules then pass through the molecular leaks and enter the ionization


The gaseous sample and the electrons collide at right angles in the chamber and ions are formed by exchange of energy during these collisions between electron beam and sample molecules

Since the ionization energy of most of the organic molecules is 15eV an electron is expelled to produce a radical cation.

At hard ionization event i.e at 70 e V molecule ions are fragmented.

The positive ions formed in the chamber are drawn out by a small potential difference (usually 5eV) between the large repeller plate (positively charged) and first accelerating plate (negatively charged).


ADVANTAGES Gives molecular mass and also the fragmentation

pattern of the sample. Extensive fragmentation and consequent large number

of peaks gives structural information. Gives reproducible mass spectra. DISADVANTAGES Sample must be thermally stable and volatile. A small amount of sample is ionized (1 in 1000

molecules). Unstable molecular ion fragments are formed so readily

that are absent from mass spectrum.


•EI works well only for thermally stable and volatile samples;


Schematic representation of an electron ionization ion source.

sample pressure in the ion source is about 10-5 torr

about every 1/1000 molecule is ionized

only cationsthe sample is heated up until a sufficient vapour

pressure is obtained


Chemical ionization

In chemical ionization, the ionization of the analyte is achieved by interaction of it’s molecules with ions of a reagent gas in the chamber or source.

Chemical ionization is carried out in an instrument similar to electron impact ion source with some modifications such as:-

Addition of a vacuum pump. Narrowing of exit slit to mass analyzer to maintain

reagent gas pressure of about 1 torr in the ionization chamber.

Providing a gas inlet.


It is a two part process. • In the first step A reagent gas is ionized by Electron Impact ionization

in the source. The primary ions of reagent gas react with additional

gas to produce stabilized reagent ions. In the second step, the reagent ions interact with

sample molecules to form molecular ions. • In this technique the sample is diluted with a large

excess of reagent. Gases commonly used as reagent are low molecular

weight compounds such as Methane, tertiary Isobutane, Ammonia, Nitrous oxide, oxygen and hydrogen etc.


TYPES OF CI: Depending upon the type of ions formed CI is

categorized as:- 1. Positive Chemical Ionization 2. Negative Chemical Ionization 1. Positive Chemical Ionization In this technique positive ions of the sample are

produced. In positive chemical ionization, gases such as Methane,

Ammonia, Isobutane etc are used.


For example, Ammonia is used as reagent gas. First ammonia radical cations are generated by electron

impact and this react with neutral ammonia to form ammonium cation (reactive species of ammonia CI).

NH4+ reacts with the sample molecules by proton transfer to produce sample ions


Negative Chemical Ionization Negative chemical ionization is counterpart of Positive

chemical ionization. In this technique, negative ions of the sample are

formed. Oxygen and Hydrogen are used as reagent gasses. This method is used for ionization of highly

electronegative samples.


ADVANTAGES Used for high molecular weight compounds. Used for samples which undergo rapid fragmentation in

EI. LIMITATIONS Not suitable for thermally unstable and non-volatile

samples. Relative less sensitive then EI ionization. Samples must be diluted with large excess of reagent gas

to prevent primary interaction between the electrons and sample molecules.


Field Ionization FI is used to produce ions from volatile compounds

that do not give molecular ions by EI. It produces molecular ions with little or no

fragmentation. Application of very strong electric field induces

emission of electrons. FI utilizes 10-micron diameter tungsten emitter wires

on which carbon whiskers, or dendrites, have been grown.

A high electric field gradient (1010 V/cm) at the tips of the whiskers produces ionization


ADVANTAGES As fragmentation is less, abundance of molecular ions

(M+) is enhanced, hence this method is useful for relative molecular mass and empirical formula determination.

DISADVANTAGES Not suitable for thermally unstable and non volatile

samples. Sensitivity is les than EI ion source. No structural information is produced as very little

fragmentation occurs.


Field Desorption Also known as offspring of field ionization. In field desorption method, a multitipped emitter

(made up of tungsten wire with carbon or silicon whiskers grown on its surface) similar to that used in FI is used.

The sample solution is deposited on the tip of the emitter whiskers either by

dipping the emitter into analyte solution or using a microsyringe. Then the sample is ionized by applying a high voltage

to the emitter.


ADVANTAGES � Works well for small organic molecules, low

molecular weight polymers and petrochemical fractions. � DISADVANTAGES � Sensitive to alkali metal contamination. � Sample must be soluble in a solvent. � Not suitable for thermally unstable and non volatile

samples. � Structural information is not obtained as very little

fragmentation occurs.


Electrospray ionization Electrospray ionization is a technique used in mass

spectrometry to produce ions from macromolecules such as proteins, polypeptides and oligonucleotides having molecular weights of 10,000 Da or more.

The method generates ions from solution of a sample by creating fine spray of charged droplets.

• A solution of sample is pumped through a fine, charged stainless steel capillary needle at a rate of few microlitres/minute.

The needle is maintained at a high electric field (several kilovolts) with respect to cylindrical electrode.

• The liquid pushes itself out of the capillary as a mist or aerosol of fine charged droplets.


These charged droplets are then passed through desolvating capillary where the solvent is evaporated in the vacuum and attachment of charge to the analyte molecules takes place.

Desolvating capillary uses warm nitrogen as nebulising gas.

The desolvating capillary is maintained under high pressure.

• As the droplets evaporate the analyte molecules comes closer together.


These molecules become unstable as the similarly charged molecules comes closer together and the droplets explode once again. This is referred as Coulombic fission.

• The process repeats itself until the analyte is free from solvent and is alone ion.

• The ion then moves to the mass analyzer.


ADVANTAGES Most important techniques for analysis of high

molecular weight biomolecules such as polypeptides, proteins, oligonucleotides and synthetic polymers.

Can be used along with LC and capillary electrophoresis.


High voltage applied to metal sheath (~4 kV)

Sample Inlet Nozzle(Lower Voltage)

Charged droplets

++

+++

+

++

++++

++

++++

+++

+++

+++

++

++

+

++

+

+

+

+++

+++

+++

MH+

MH3+

MH2+

Pressure = 1 atmInner tube diam. = 100 um

Sample in solution

N2

N2 gas

Partialvacuum


Plasma Desorption

Plasma desorption produces molecular ions from the samples coated on a thin foil when a highly energetic fission fragments from the Californium-252 “blast through” from the opposite side of the foil.

The fission of Californium-252 nucleus is highly exothermic and the energy is released.


When such a high energy fission fragments passes through the sample foil, extremely rapid localized heating occurs, producing a temperature in the range of 10000K.

•Consequently, the molecules in this plasma zone are desorbed.


Laser Desorption

Laser desorption methods involves interaction of pulsed laser beam with the sample to produce both vaporization and ionization.

Laser beam is usually of different wavelengths from far U.V to far IR depending upon the sample to be analyzed.

REQUIREMENTS Laser wavelength must be at absorption wavelength of

the molecule. In order to avoid decomposition absorbed energy must

be quickly dispersed in the molecules.


IONIZATION TECHNIQUE: • Ionization is carried out by two techniques :- Microprobe techniques Laser beam is focused to a very small spot on the back

side of a thin metal foil that holds a thin film of sample. Ions emerge out on the front side from a small cratered

hole in the foil. Bulk analysis techniques The technique uses a less focused beam and larger

samples. The laser beam produces microplasma that consists of

neutral fragments with elementary and fragment ions.


Matrix assisted laser desorption (MALDI)

Matrix assisted laser desorption is a technique in mass spectrometry for ionization of biomolecules (polymers such as proteins, polypeptides and sugars) and synthetic polymers that are more fragile and form fragments when ionized by conventional methods.

It consist of two components 1 Matrix : Matrix is used in MALDI to Absorb the laser energy. Prevent analyte agglomeration. Protect analyte from being destroyed by direct laser

beam


Matrix consists of a crystallized molecules of which the most commonly used are

Sinapinic acid) – cyano cinnamic acid ( –cyano or – matrix) α α α Dihydroxy benzoic acid (DHB) Nicotinic acid Matrix solution is then mixed with the analyte to be

investigated. The solution is then spotted in a air tight chamber on

the tip of the sample probe.


With a vacuum pump the air is removed and vacuum is created which leads to evaporation of the solvent leaving behind a layer of recrystalized matrix containing analyte molecules.

2 Laser The solid mixture is then exposed to pulsed laser

beam. The matrix absorbs the laser energy and transfers some

of this energy to the analyte molecules which results in the sublimation of sample molecules as ions or the matrix after

Absorbing the laser energy gets ionized and transfer part of this charge to the sample molecules and ionize it.


When the polymers form cations the cathode is placed right behind the sample and anode in front of the sample.

The cations get attracted towards the negatively charged anode. This acceleration is used to move the ion to the detector.

When the polymer forms anions the electrodes are interchanged.


Fast atom bombardment (FAB) is an ionization technique used in mass spectrometry in which a beam of high energy atoms strikes a surface to create ions.

When a beam of high energy ions is used instead of atoms (as in secondary ion mass spectrometry), the method is known as liquid secondary ion mass spectrometry (LSIMS)

https://en.wikipedia.org/wiki/Ionization

https://en.wikipedia.org/wiki/Mass_spectrometry

https://en.wikipedia.org/wiki/Atoms

https://en.wikipedia.org/wiki/Ion

https://en.wikipedia.org/wiki/Secondary_ion_mass_spectrometry


Quadrupole mass analyzer A quadrupole mass spectrometer contains four parallel

cylindrical rods which can scan or filter sample ions based on their mass-to-charge ratio.

Opposing rods are connected electrically and a radio frequency voltage is applied between the pairs of rods.

Ions travel between the rods and only ions with a specific mass-to-charge ratio will exit the quadrupole; other ions will collide with the rods.

The desired mass-to-charge ratio can be altered by changing the applied voltage.


Triple quadrupole mass spectrometry makes use of the same technology, but uses a linear series of three quadrupoles to improve sensitivity and selectivity.

This type of spectrometry is useful when studying particular ions of interest since it is able to stay tuned to a single ion for extended periods of time.


Ion trap mass analyzer

This analyzer employs similar principles as the quadrupole analyzer mentioned above, it uses an electric field for the separation of the ions by mass to charge ratios.

The analyzer is made with a ring electrode of a specific voltage and grounded end cap electrodes.

The ions enter the area between the electrodes through one of the end caps. After entry, the electric field in the cavity due to the electrodes causes the ions of certain m/z values to orbit in the space.

As the radio frequency voltage increases, heavier mass ion orbits become more stabilized and the light mass ions become less stabilized, causing them to collide with the wall, and eliminating the possibility of traveling to and being detected by the detector.


TOF Analyzers

TOF Analyzers separate ions by time without the use of an electric or magnetic field.

In a crude sense, TOF is similar to chromatography, except there is no stationary/ mobile phase, instead the separation is based on the kinetic energy and velocity of the ions.


Ions are accelerated by an electric field of known strength.This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio (heavier ions of the same charge reach lower speeds)

The time that it subsequently takes for the ion to reach a detector at a known distance is measured.

This time will depend on the velocity of the ion, and therefore is a measure of its mass-to-charge ratio. From this ratio and known experimental parameters, one can identify the ion.

https://en.wikipedia.org/wiki/Electric_field

https://en.wikipedia.org/wiki/Kinetic_energy

https://en.wikipedia.org/wiki/Mass-to-charge_ratio


Magnetic sector analyzers

Similar to time of flight (TOF) analyzer mentioned earlier,

In magnetic sector analyzers ions are accelerated through a flight tube.

Where the ions are separated by charge to mass ratios. The difference between magnetic sector and TOF is that a magnetic field is used to separate the ions.

As moving charges enter a magnetic field, the charge is deflected to a circular motion of a unique radius in a direction perpendicular to the applied magnetic field


THANK UUUUUUUUUUUU

Mass spectroscopy, Ionization techniques and types of mass analyzers

Science

Transcript of Mass spectroscopy, Ionization techniques and types of mass analyzers