3. Atomic Absorption spectroscopy
The technique was introduced in 1955 by Walsh in
Australia. The first commercial atomic absorption
spectrometer was introduced in 1959.
Definition: When we study the absorption of
energy by the atoms in the flame, we call this
technique asAtomicAbsorptionSpectroscopyAAS.
Atomic Absorption Spectroscopy (AAS) is an analytical
technique that measures the concentrations of metals
and metalloids in samples. It makes use of the
absorption of light by these elements, in order to
measure their concentration.
Principle:
Atomic-absorption spectroscopy quantifies or measures the
absorption of energy radiation by ground state atoms in the
gaseous state.
The absorptionof the ultravioletorvisible lightenergythathasthe
right wavelength causes the electrons of the sample to be
promotedfroma lowerenergy level to a higher energy level. The
analyte concentration is determined from the amount of
absorption.
The change inenergycan be calculatedas;
= −
=
=
So,the equationwillbe;
=
Where;
ΔE – Change in energy
E1 – Excitedenergy
E0 – groundstate
h – Planck’sconstant
c – velocityof light
– wavelength
4. Concentrationmeasurementsare usuallydeterminedfromaworkingcurve aftercalibratingthe
instrumentwithstandardsof knownconcentration.
The basic equationis; ∝
Instrumentation:
Atomicabsorptionspectrometerhave fourprincipalcomponents;
1. A lightsource (usuallyahollowcathode lamp)
2. Beamchopper
3. Atomizer
4. A Monochromator
5. A detector,andreadout device
Hollowcathode lamp:
The light source is usually a hollow cathode lamp of the element that is being measured. It contains a
tungsten anode and a hollow cylindrical cathode made of the element to be determined. These are
sealed in a glass tube filled with an inert gas (neon or argon). Each element has its own unique lamp
which must be used for that analysis.
5. Working:
Applyinga potential difference between the anode and the cathode leads to the ionization of some gas
atoms.
These gaseousionsbombardthe cathode and eject metal atoms from the cathode in a process
called sputtering. Some sputtered atoms are in excited states and emit radiation, as they fall
back to the ground state.
The shapeof the cathode which is hollowcylindrical concentrates the emitted radiation into a beam
which passes through a quartz window all the way to the vaporized sample.
Sinceatoms of different elements absorb characteristic wavelengths of light.Analyzinga sampleto see if
it contains a particular element means usinglightfrom that element.
Example:
A lampcontainingleademitslightfromexcitedleadatomsthatproduce the rightmix of wavelengths to
be absorbed by any lead atoms from the sample.
A beam of the electromagnetic radiation emitted from excited lead atoms is passed through the
vaporizedsample.Some of the radiation is absorbed by the lead atoms in the sample. The greater the
number of atoms there is in the vapor, the more radiation is absorbed.
Beam chopper:
It is present between the hollow cathode lamp and flame. It rotates and breaks the steady light into
intermittent light. This gives a pulsating current in photo cell.
Atomizer:
Elementstobe analyzedneedstobe inatomicsate.
Atomizationrefersto the separationof particlesintoindividual molecules and breaking molecules into
atoms .This is done by exposing the analyte to high temperatures in a flame or graphite furnace.
The role of the atom isto primarilydissolvate aliquidsample and then the solid particles are vaporized
into their free gaseous ground state form. In this form atoms will be available to absorb radiation
emitted from the light source and thus generate a measurable signal proportional to concentration.
There are twotypesof atomization;
Flame atomization
Graphitefurnace atomization
Atomization method or energy source is selected according to the sensitivity and selectivity of the sample.
6.
Flame atomization:
Flame Atomic absorption can only analyze liquids and solution samples, where it uses a burner to
increase the path length, and therefore to increase the total absorbance.
Sample solutions are usually introduced into a nebulizer by being sucked up by a capillary tube .In the
nebulizer the sample is dispersed into tiny droplets, which can be readily broken down in the flame.
The fine mist of droplets is mixed with fuel (acetylene), and oxidant (nitrous oxide) and burned. The
flame temperature isimportantbecause itinfluencesthe distributionof atoms.Itcan be manipulatedby
oxidant and fuel ratio. The technique is thus named as Flame atomic absorption spectroscopy.
Varioustypesof flamesusedinAtomicabsorptionSpectroscopy;
Fuel and Oxidant Temperature o
C
Gas : Air 1700o
C – 1900o
C
Gas : OxygenO2 2700 o
C – 2800 o
C
Hydrogen H2 : Air 2000 o
C – 2100 o
C
Hydrogen H2 : OxygenO2 2550 o
C – 2700 o
C
Acetylene :Air 2100 o
C – 2400 o
C
Acetylene :Oxygen O2 3050 o
C – 3150 o
C
Acetylene :N2O 2600 o
C –2 800 o
C
Process taking place inflame:
Followingisthe processthatoccursin the
flame;
Nebulization: conversionof
liquidsample intoafine spray.
Desolvation: Solidatomsare
mixedwiththe gaseousfuel.
Volatilization: Solidatomsare
convertedintotoa vapor ina
flame.
There are three typesof particlesthat
existinthe flame;
1. Atoms
2. Ions
3. Molecules
7.
Graphite furnace atomization:
Graphite furnace is used for the atomization of the
sample. The sample is dried then burned to ash and
finally atomized. The technique is thus named as
Graphite atomic absorption spectroscopy. This
technique should be used only when the sample size is
small and/ or when a greater sensitivity is needed.
Graphite atomic absorption can analyze liquid, solid,
semi-solid and solution samples. It should not be used
when ordinary flame AA would do as well, since there
are disadvantagesrelatingto sample size and precision.
Monochromator:
Thisis a veryimportantpart in an Atomic Absorption spectrometer. It is used to separate out all of the
thousands of lines. Without a good monochromator, detection limits are severely compromised.
A monochromatorisusedtoselectthe specificwavelengthof lightwhichisabsorbedbythe sample,and
to exclude otherwavelengths.The selectionof the specificlightallowsthe determinationof the selected
element in the presence of others.
Detector andreadout device:
The light selected by the monochromator is directed onto a
detectorthatis typicallyaPhotomultipliertube, whose function is
to convert the light signal into an electrical signal proportional to
the light intensity.
The processing of electrical signal is fulfilled by a signal amplifier.
The signal couldbe displayedforreadout,orfurtherfedinto a data
station for printout by the requested format.
Calibrationcurve:
A calibrationcurve isused to determine the unknown
concentration of an element in a solution. The
instrument is calibrated using several solutions of
knownconcentrations.The absorbance of eachknown
solution is measured and then a calibration curve of
concentration vs. absorbance is plotted.
The sample solutionisfedintothe instrument,andthe
absorbance of the element in this solution is
measured.The unknownconcentrationof the element
is then calculated from the calibration curve.
8. Relationof AAS withUV-Visible spectroscopy
UV-Visspectroscopy:isalsosimilartoAASinnumberof ways;
Have the similar basic principlewhich is promotingelectrons from lower energy level to a higher energy
level.
Both techniques usessimilarstepsto interpretresults.
Dissimilarities;
AAS– uses‘visible’partof the emissionspectrum
UV-Vis –uses ‘ultraviolet’ partoftheemission spectrum
Working of Atomic Absorptionspectrometer:
In actual practice;
A meterisadjustedtozeroabsorbance.Whena blank(unionizedwater)issprayedintothe flame
and unsaturatedlightof hollow cathode lampispassesontothe readoutdevice.
Next, when solution containing absorbing species is introduced, a part of light is absorbed, results in the
decrease light intensity falling on photomultiplier detector. And produce a deflection in meter needle.
Atomic absorption spectrum:
Spectrumof radiationsshowsaseriesof a dark linesina continuousband.
Absorption bands:
Regionsinspectrumfromwhere radiationshave beenabsorbedbythe substance insample.
Resonance spectral lines:
Theyare stable intense radiationsappearsaresonance spectral lines.Theyshouldbe narrowas
comparedto the widthof absorptionbands/lines.
9. Interference during AAS
The concentration of the analyte element is considered to be proportional to the ground state atom
populationinthe flame,anyfactorthataffectsthe groundstate atom populationcan be classified as an
interference.
Factors that mayaffect the ability of the instrument to read this parameter can also be classified as an
interference.The different interferences that are encountered in atomic absorption spectroscopy are;
Absorptionof Source Radiation:
Elementotherthanthe elementof interestmayabsorbthe wavelengthbeingused,andthus
interfere.
IonizationInterference:
The formationof ionsrather thanatoms causes’lowerabsorptionof radiation.Thisproblemis
overcome byaddingionizationsuppressorsornon-ionizingagents.
Example:
Certain,atomslike Na+
andK+
, theyionizesatlow temperature therefore non-ionizingagent
CsCl2 isused.
Self-Absorption:
The atoms of the same kind that areabsorbing radiation will absorb more at the center of the line
than at the wings and thus resulting in the change of shape of the line as well as its intensity.
Back ground Absorptionof Source Radiation:
Thisis causedbythe presence of aparticle fromincomplete atomization.Thisproblemis
overcome byincreasingthe flame temperature.
Physical Interference:
Physical propertiese.g.viscosity,surfacetension,vaporpressure,anddensity,of the sample
shouldbe similartothe standard.
Spectral interference:
It iscausedby overlappingof anyradiationswiththatof characteristicradiationsof testelement
to be estimated.Thistype of overlappingcanbe overcome byselectingotherspectral lines.
Chemical interference:
Certainmetalslike calciumCa+2
andmagnesiumMg+2
make strong bondswithphosphate PO-3
as Ca3(PO4)2 andMg3(PO4)2.Theninthiscase there will be noabsorbance byCa+2
andMg+2
. To
overcome suchproblem;
By increasingthetemperatureso thatbond dissociationoccur easily.
By addition of releasing agent (e.g. LaO2)
By addition ofchelating agent(e.g. EDTA)
10.
Back ground correction:
Whenlightspectraisreleased,monochromator (the wavelengthselector) selectsspecific
wavelengthof specificmetal.But,sometimessame wavesinterferewithspectra.
Therefore toovercome thisproblemablankisruninthe instrumentandcalculated.Andthenit
issubtractedfromthe readingsof the sample.
Advantages and disadvantages of Atomic AbsorptionSpectroscopy
The technique hasitsome meritsanddemeritsdiscussedbelow;
Advantages:
Precise andaccurate resultscanbe obtainedbythe usage of thistechnique.
It is a very sensitive. Itcan detect concentrations as small asa few parts to g / Litre (parts per million)
It is generally very specific as theset wavelength is strongly absorbed by the particularmetal ion being
analysed (and not by other components).
Only a littlequantity of thesampleisrequired about1ml –2ml.
It isa lifesavingtechnique.Injapanfrom1932 to 1968, AASwas usedtoidentifythe reason why
over3,000 residentswholivesnearthe MinimataBaystartedshowingneurogical problems and
pregnant women starts giving birth to impaired children. Scientist starts taking samples and
performingAASprocess;AASresultsshowsaveryhighconcentrationof mercury in their blood.
Thisresulton stoppingthe company,ChissoCorporationwhodumpedapproximately 27 tons of
mercury in the bay.
Disadvantages:
It isa cost effective technique.
Flame atomic absorption spectroscopy,can analyzeonly liquid sample.
Graphite atomic absorption spectroscopy should notbe used when ordinary flameAA would do as well,
sincethere are disadvantages relatingto samplesizeand precision.
11. Applications of Atomic Absorption Spectroscopy
There are manyapplicationsforatomicabsorption;
Clinical analysis:
By the use of this technique we can detect deficiencies / excessive amounts of certain metals in our
biological fluids such as; blood and urine.
Environmental analysis:
The technique iswidelyusedforthe monitoringof ourenvironment.Itisusedtoanalyze metal ionsthat
are polluting the soil, air and water. And thus to find out the levels of various elements in rivers,
seawater, drinking water, air, and petrol.
Pharmaceuticals:
In some pharmaceutical manufacturing processes, minute quantities of a catalyst used in the process
(usually a metal) are sometimes present in the final product. Therefore, by using AAS the amount of
catalyst present can be determined.
For example invitaminpreparations.
Industry:
Raw material analysis: Many rawmaterials areexamined and, AAS is widely used to check that the
major elements arepresent and that toxic impurities arelower than specified.
For example;inconcrete,where calciumisamajorconstituent,the leadlevel shouldbe low
because itistoxic.
Food industry:The technique is used for traceelements in food analysis. Where, it is used to track
harmful metals in our food/drinks.
Cosmeticsindustry: The technique is used for the trace element analysis of cosmetics in cosmetics
industry.
For example toanalyze the specificallergicmetal incosmetics.
Petroleumindustry:In petroleum industry the technique is used to analyze the metals present in
engine oils.
For example tocheckthe presence of anti-knockingagenttetraethyllead(TEL).
Mining:
By usingAASthe amountof metalssuchas goldinrocks can be determinedtosee whetheritisworth
miningthe rocksto extractthe gold.
12. Basic concepts
Emissionspectrum:
The collectionof spectral linesproducedbyanexcitedatomiscalledemissionspectrumandwillbe
characteristicof that atom.
Principle:
Whenthe elementisheatedinthe flame,the absorptionof energybythe groundstate electroninan
atom resultsinexcitationof some onthese electronstohigherenergyresultinginexcitation.
A solutionof sample tobe analyzedissprayedintoflame possessingthe thermal energyrequire to
excite the elementatwhichitwill radiate itscharacteristicbrightlineemission.
Types of emissionspectrum
There are three typesof emissionspectrum;
1. Line emissionspectrum
2. Band emissionspectrum
3. Continuousemissionspectrum
1. Line emissionspectrum:
It is consist of sharply defined and often widely and irregularly spaced individual lines of a single
wavelength. These spectra are characteristic of element. They are also called atomic spectrum.
2. Band emissionspectrum:
It consist of group of lines each of which has single wavelength that becomes closely spaced as they
approach the end of band. They are also called molecular spectrum.
3. Continuous spectrum:
Theyare obtainedwhensolids are heated to incandescence. They are characterized by absence of any
sharp lines as a function of wavelength. On the other hand when gases and vapors are heated to high
temp they yield a series of bands or lines.
13. Atomic emissionspectroscopy
Definition: When we study the emission of energy by
the atomsinthe flame, we call this technique as Atomic
Emission Spectroscopy AES.
Atomic Emission Spectroscopy (AES) is an analytical
technique thatmeasuresthe concentrationsof elements
insamples.Itmakesuse of the emissionof lightbythese
elements, in order to measure their concentration.
Principle:
In atomicemissionthe sampleisatomizedandthe analyte atoms are
excited to higher energy levels. The analyte concentration is
determined from the amount of emission.
The analyte atoms are promoted to a higher energy level by the
sufficient energy that is provided by the high temperature of the
atomizationsources.The excitedatomsdecaybackto lowerlevelsby
emitting light. Emissions are passed through monochromators or
filters prior to detection by photomultiplier tubes.
The change inenergycan be calculatedas;
= −
=
=
So,the equationwillbe;
=
Where;
ΔE – Change in energy
E2 – Excitedenergy
E1 – groundstate
h – Planck’sconstant
c – velocityof light
– wavelength
Concentrationmeasurementsare usuallydeterminedfromaworkingcurve aftercalibratingthe
instrumentwithstandardsof knownconcentration.
The basic equationis; ∝
14. Instrumentation:
The instrumentation of atomic emission spectroscopy is the same as that of atomic absorption, but
without the presence of a radiation source. Atomic absorption spectrometer have three principal
components;
1. Atomizer(Flame,Graphite furnace,ICP)
2. A Monochromator
3. A detector,andreadout device
Atomizer:
The major energysource inAtomicEmissionspectroscopyare;
Graphite furnace: Thistechniqueshouldbe usedonlywhenthe samplesize issmall and/or
whena greatersensitivityisneeded. The substance sample is dried at 200o
C for 60 sec. Then it
is burned at 1200o
C for 30 sec. to burn all organic sample. And then finally the atomization is
brought about by heating at 2700o
C for 60 sec.
A flame: The flame (1700 o
C – 3150o
C) is mostuseful forelementswithrelativelylow
excitation energieslike sodiumpotassiumandcalcium.
Inductively coupled plasma: The ICP(6000o
C – 8000o
C) has a veryhightemperature andis
useful forelementsof highexcitationenergies.
Electric arc: sample isheatedbyanelectricarc.
Electric spark: Sample isexcitedinhighvoltage spark.
15. Monochromator:
This is an important part in an Atomic Emission spectrometer. It is used to separate out all of the
thousands of lines. Without a good monochromator, detection limits are severely compromised.
A monochromator,inatomicemissionsepectroscopy,issimplyawavelengthselector.Itisusedtoselect
the specificwavelengthof lightwhichisemittedbythe sample, and to exclude other wavelengths. The
selection of the specific emitted radiation allows the determination of the selected element in the
presence of others.
Detector:
The selected wavelength by the monochromator is directed onto a detector that is typically a
Photomultipliertube, whose functionistoconvertthe light signal into an electrical signal proportional
to the light intensity.
The processing of electrical signal is fulfilled by a signal amplifier. The signal could be displayed for
readout, or further fed into a data station for printout by the requested format.
Calibrationcurve:
A calibrationcurve isused to determine the unknown
concentration of an element in a solution. The
instrument is calibrated using several solutions of
known concentrations. The emission of each known
solution is measured and then a calibration curve of
concentration vs. emission is plotted.
The sample solutionisfedintothe instrument,andthe
absorbance of the element in this solution is
measured.The unknownconcentrationof the element
is then calculated from the calibration curve
16. Comparison between Atomic Absorption and Emission Spectroscopy
Atomic Absorptionspectroscopy Atomic Emissionspectroscopy
Measure trace metal concentrationsincomplex Measure trace metal concentrationsincomplex
matrices. matrices.
Atomicabsorptiondependsuponthe numberof AtomicEmissiondependsuponthe numberof
groundstate atoms. excitedatoms.
Sensitivity:
Both techniquesare usedto
detectmetal ormetalloidsin
the sample.
The detectable elementsby
this technique are inpink
coloredinthe periodictable;
18. Introduction
Luminescence:
It isdefinedas,the emissionof photonsfromelectronicallyexcitedstate.
Luminescence is divided into two types, depending upon the nature of the ground and the excited states.
There are twostates;
1. Singletstate
2. Tripletstate
1. Singlet state
In a singletexcitedstate,the electron in the higher energy orbital has the opposite spin orientation as
the secondelectroninthe lowerorbital.These twoelectronsare saidtobe paired.Returntothe ground
state from an excited singlet state does not require an electron to change its spin orientation.
2. Triplet state:
In a tripletstate these electronsare unpaired,thatis,theirspinshave the same orientation. A change in
spinorientationisneededforatriplet state toreturnto the singletgroundstate.For example in case of
free radicles.
19. Types of Luminescence:
The luminescenceisdividedintotwomaintypes;
1. Photoluminescence
2. Chemiluminescence
1. Photoluminescence
The moleculesare excitedbythe interactionwithphotonsof radiation.Itisfurtherof twotypes;
Fluorescence:
It isfurtherof twotypes;
Prompt fluorescence: S1 S0 + h. The release of electromagnetic energy is
immediate or from the singlet state.
Delayed fluorescence: S0 S1 T1 S1 S0 + h. This results from two
intersystem crossings, first from the singlet to the triplet, then from the
triplet to the singlet.
Phospholuminescence: S0 S1 T1 S0 + h
A delayed release of electromagnetic energy from the triplet state. The intensity of
phosphorescence is low as electron loses some part of its energy during intersystem
crossing (S1 T1).
2. Chemiluminescence:
The excitationenergyisobtainedfromthe chemical energyof reaction.
Time frame of Processes Time (sec)
S0 S1 10-19
sec.
Interconversion(Intermediatestate stable S1 state) 10-15
sec
S1 S0 10-10
sec.
Phosphorescence 10-3
– some seconds.
20. Fluorescence spectroscopy
Fluorescence spectroscopy FS is an analytical
technique that measures the concentration of
fluorescent substance present in the sample. It
makes the use of the fluorescence in order to
measure their concentration.
While, fluorescence is the emission of the light by a
substance which absorbs light or Electromagnetic
radiation.
Principle:
Molecularfluorescence spectrometryisbasedonthe emissionof lightbymoleculesthathave become
electronicallyexcitedsubsequenttothe absorptionof electromagneticradiation.
It usesthe UV visible spectraalongwithsome partof IRregionof electromagneticradiation.Itisa
sensitivetechnique bywhichwe candetectthe sample ragingfromppm – ppt.
Jablonski Diagram:
Let usunderstandthisphenomenonfromJablonski
Diagram;
An electron was present in its ground state S0, it
absorbslightor electromagneticradiationandgets
excited to a high intermediate energy level.
Accordingto Kasha’s rule,the electronwill require
to attain a stable energy level near to that
intermediate level, so it moves to S1.
After attaining a stable energy state it jumps back to
its ground state So either in non-radiative fashion, or
in radiative fashion thus, producing fluorescence.
The non-radiative fashionisdue tothe lossof energyduringinterconversion.Ininterconversiononly
vibrational androtational changestakesplace withinthe molecules.
Properties of fluorescence:
Fluorescence isaspecificpropertyof eachmolecule anditdependsuponthe auxochromeof molecule.
The emittedlighthastwoimportantcharacteristics;
It is usually of longer wavelength (lower energy) than the excited light. This is becausepartof the energy
associated with S state is lostas heatenergy.
The emitted lightiscomposed of many wavelengthswhichresultsinfluorescencespectrum.
21. Instrumentation
The descriptionof variousinstrumental partsare givenbelow;
1. Light source:
Two typesof lightsourcesare used;
Mercury-arc lamp: inorganic mercury lamps areused to energize the electron from ground state to
excited state. They provide steady and homogenous supply of light.
Xenon-ArcLamp: Xenon gas is used as a source of light. It also gives steady supply. The wavelength
of light produce is about 300 – 1300 nm.
2. Excitationmonochromator:
Various types of monochromators can be used to select the specific type of wavelength to fall on the
sample so that the sample excites that is why it is also called as excitation monochromator. It is also
called wavelength selector.
Commonlyusedmonochromatorsare;
Prisms: varioussaltor glassprisms are usedforthe process.
Grating: In general, gratings are used in the design of the instrument and offer better resolution at
high frequency than the prisms. They offer much better resolution at low frequency.
3. Sample holder:
Cuvette or quarts cell is used as a sample holder. The cell is transparent from all sides so that the
fluorescence produced can be observed by the detector easily.
4. Monochromator:
A second monochromator is used to observe the produced fluorescence without difficulty. It acts as
wavelengthfilter.Itisplacedrightangle tothe firstmonochromator.Itdirectsthe fluorescence towards
the detector.
5. Detector:
The detectorobservesthe fluorescence produce bythe substance andgeneratesthe electrical signal.An
amplifier is attached that amplifies the intensity of the signal. The commonly used detectors are
phototube & photomultiplier tubes.
6. Readout devices:
The computerreadsthe detectedsignal andplotsagraph.
22. Schematic diagram of Fluorescence spectrophotometer:
The schematicdiagramof a simple fluorescence spectrophotometerisasfallows;
Applications of Fluorescence spectroscopy
Thoughthe Fluorescence isaspecificpropertyof substances,butthistype of spectroscopyhaslimite d
applicationsasnotall substancesshowfluorescence.
Some of its applicationare mentionedbelow;
1. Quinine wasthe firstmedicine thatshowedfluorescence.
2. The technique isusedforthe quantitative analysisaswell asforqualitative analysis.
3. The technique isalsouseful forproteinidentification.Thisisdone bytagginga fluorescent
molecule withinthe proteinandfluorescence isobserved.
4. Thistechnique isusedforthe assayof differentmedicationsthatshowsfluorescence.
For example;
Medication Absorption wavelength Emission wavelength
λabsorption λemission
AmphotericinB 340 427
Diphenhydramine 305 412
Flurazepam 375 475
Reserpine 390 510
23. Factors affecting Fluorescence
The variousfactors affectingfluorescence are discussedbelow;
1. Temperature:
Increase in temperature will lead to the decrease in intensity of fluorescence. It happens because by
increasing the temperature the collision between fluorescent and other molecules and it loses its energy.
2. Oxygen:
Presence of oxygen leads to the oxidation of substance and thus causes decreases the intensity of
fluorescence produced.
3. pH:
Fluorescence of various chemicals depends upon different pH values. Most of the compounds give
fluorescence on neutral pH and some on alkaline pH. For example; Aniline gives fluorescence at pH &
(neutral.)
4. Quenching:
There are some molecules which are called quenching molecule. These molecules decreases the
fluorescence either by collision or by any other way like by changing the viscosity etc.
5. RigidPlaner structure:
The fluorescence producedbyarigidmolecule isbetterascomparedtothe fluorescence produced by a
non-rigid structure.
For example; the fluorescence of Flourene is good as compared to the fluorescence produced by Biphenyl.
6. Electronwithdrawing group:
The fluorescence isdue tothe π- electrons,thusthe presence of anelectronwithdrawinggroupwilllead
to the decrease in fluorescence of the substance as it draw these π- electrons towards it.
For example;halogensX,Carboxylicgroup –COOH, –NO2 etc.
7. Electrondonating group:
Similarly, the presence of an electron donating group will result in increase in the intensity of fluorescence.
As itdonateselectrons.
For example;methyl group –CH3,aminogroup –NH2, hydroxyl group –OHetc.
25. Infrared spectroscopy
It deals with the region of electromagnetic spectrum that lies in
the infrared region (frequency range 500cm-1
– 670cm-1
)
IR radiations: Electromagnetic radiations that are lower in
energythanvisible radiations are called infrared radiations.
Absorption in IR region is due to the rotational and vibrational
levels. When radiations of frequency range less than 100 cm-1
are absorbed, molecular rotation takes place in the substance.
Therefore, on the bases of molecular rotation, the vibrational
spectra appears as vibrational-rotational bands.
WhenIR lightispassedthroughthe sample the vibrational androtational energiesof the moleculesare
increased.There are twotypesof vibrations;
1. Stretching vibration:
In thistype of vibrationthe distance betweenthe twoatomsincreasesordecreasesbutthe atoms
remainsat the same bondaxis.There are two typesof stretchingvibration;
Symmetric stretching: in this type the moment
of atoms with respect to the particular atoms
with respect to particular atom in the same
molecule is in the same direction.
Asymmetric stretching: in case of asymmetric
stretching one atom approaches the central atom
while the other departs from it.
2. Bending vibration:
In thistype of vibrationthe positionof atomschangeswithrespecttothe original bondaxis. We can say
that stretchingabsorptionof the bondappearsathigherfrequencies, (high energy) as compared to the
bending absorption of the same bond. There are four types of bending vibrations;
Scissoring: inthistype twoatoms approacheseachother.
Rocking: in this type the movement ofatoms takes place in the samedirection.
Wagging: in this type two atoms moves up and below the plane with respect to the central atom.
Twisting: in this type one of the atom moves up the planewhilethe other moves down the plane with
respect to central atom.
26. IntroductiontoIR Spectrometer:
The following figure gives the schematic representation or block diagram for the use of
spectrophotometer.
In this spectrometer the source of radiant energy is “Nernst glower.” It is consist of Zirconium and
Yttriumoxidesinthe shape of tube whichis electrically heated to 1500o
C to 2000o
C because IR rays are
not transmitted by glass.
A prism made of salt (NaCl) is used as a monochromator. The radiations from the Nernst glower are
polychromatic.Whenthese are passedthroughthe saltprism, the differentwavelength got separated a
slitisplacedinthe path of radiationemergingfromthe prismsothat only radiant energy of the desired
wavelength passed through and falls onto the solution under examination.
The radiant energy transmitted by the solution is then allowed to fall on a detector for measuring the
intensity of transmitted infrared radiations.
Thus, IR spectrometer can give the absorption spectrum of a substance. Therefore, by analysis the
spectrum and the information regarding the structure of the substance can be obtained.
Regions of IR:
There are three mainregionsof IR;
Near IR IR region Far IR
0.8 mµ to 2.5 mµ 2.5 mµ to 15 mµ 75 mµ to 200 mµ
AbsorptioninIRregionismainlydue tothe changesin the rotational andvibrational levelsof molecules.
27. Determination of IR spectrum of a substance
There are twomethodof determiningthe IRof the solidcompoundorsubstance;
1. MULL method
2. KBr discmethod
MULL method: (ParaffincommonlycalledNUJOL)
1. Take 15g to 20g of sample ina cleanpestle mortarandpowdereditthoroughly.
2. Nowadd to it2 dropsof purifiedparaffinandcontinue the triturationuntil verysmoothpasteof
uniformconsistencyisachieved.
3. Nowtransferthe slurryto NaCl window (tablet/window grooved)
4. Nowplace the otherwindowontothe cell partand finallyswitchonIRmachine toget various
spectrumof the sample.
KBr method:
1. Take the 100mg of the driedKBrin a cleanpestle andmortarand grindit thoroughlywith1mg
of sample.
2. Nowcarefullyplace the sample mixture intoprocessingchamberof the mold(dye).Insucha
mannerthat itis heldbetweenthe polishedsurface of the bottomandtop processingdyes.
3. Subsequently,attachthe chambertothe vacuum line andswitchonthe vacuumpump.
4. Initially applied a slight negative pressure to as to compact the powder and then gradually
increasingthe pressure lessthan15mm Hg for 30 seconds.Finallyenhance the pressing force to
10,000 pound/ inch2
pressure for 1 to 2 minutes.
5. Nowrelease the pressure andremove the dyesandtake outthe discfromthe moldand keptit
intoa positionontothe sample holdertostudythe variousspectrumof organiccompounds.
28. Interpretation of IR:
IR spectrumisdividedintotwoparts;
Functional groupregion
In case of functional groupregionthe range is4000 cm-1
– 1600 cm-1
.
Fingerprintregion
In case of fingerprintregionthe range is1600 cm-1
– 625 cm-1
.
As,the recentapproach isto examine the functional groupregionbecausemostthe compounds
have onlyfewstrongbondsdue to characteristicstretchingvibrationsof theirfunctional group.
Example:
The functional group such as C-H, O-H, and C-N absorbs in the region of 3700 cm-1
– 2500 cm-1
due to
their stretching vibrations.
The absorption due to triple bond C≡C, C≡N occurs in the region of 2300 cm-1
– 2100 cm-1
.
The absorptiondue to double bondthat is C=C, C=O occurs in the regionof 1900 cm-1
–1600 cm-1
.
The confirmationof functional groupshouldalsobe checkedinthe otherregionof spectrumsaswell.
Example: Analiphaticacetate absorbsnotonlyat 1740 cm-1
but alsoat 1240 cm-1
.
The characteristicbandscorrespondingtothe aromaticringsfallsinthe regionof 1600 cm-1
– 1450 cm-1
but aromaticcompoundsalsoshowsabsorptionbandsinthe regionof 900 cm-1
– 700 cm-1
.
The fingerprintregionthe range is1600 cm-1
– 625 cm-
1. It providesaset of absorptionbandsof each
compoundandserve as fingerprintregion.
The structural informationisderivedfromthe presence of orthe absence of characteristicabsorption
bandsof variousfunctional groups.
29. Instrumentation of IR Spectrophotometer:
The IR spectrophotometerare basedoneithersingle monochromatorordouble mnochromator.The
importantfeaturesof anIR spectrometerare asfollows;
1. IR source
2. Monochromator
3. Detector
4. Mode of operation
1. IR Source:
The most importantIRsourcesare electricallyheatedrodsof the followingtypes;
Sinteredmixturesof the oxidesof zirconium(Zr) &yttrium(Y),alsoknownasNernstglower.
Siliconcarbide (glowbar)
Variousceramicmaterial (clay)
2. Monochromator:
Three typesof substancesare normallyusedasmonochromator;
Metal Halide prisms: variousmetal halide prismsuchasPot.Bromide KBr andLithium
Fluoride LiFhave beenused.
NaCl prism: Sod.Chloride canbe usedfor the whole of the regionfrom4000cm-1
– 650cm-1
Grating: in general, gratings are used in the design of the instrument and offer better resolution at
high frequency than the prisms. They offer much better resolution at low frequency.
3. Detectors:
There are three typesof detectorsusedinIRregion.Theyare discussedbelow;
Thermocouple: (thermophiles) if dissimilar metals used are jointed head to tail then a
difference in temperature between head and tail causes a current flow in wires. This current
shall be directly proportional to the intensity of radiation falling on thermocouple and hence
they are used in the IR region.
Golay detector: in this detector the absorption of IR radiations affords expansion of an inert gas in a
cell chamber the current from the photocell is directly proportional to the incident radiations.
Bolometers: they are related with increase in resistance of a metal with increase in
temperature. For example; when two platinum foil are appropriately incorporated into a
weightstone bridge and radiations is allowed to fall on the foil and a change in resistance is
noted. Just like thermocouple the bolometers are used in IR region.
30. 4. Mode of operation:
The followingstepsare involvedforthe operationof the IRinstrument;
1. The lightfromIR source [A]is splittingequallyintotwobeamsone of which [B]is sample beam
ispassedthroughthe sample i.e.the sample beamwhile the otherservesasa reference beam.
2. These twobeamsare reflectedonarotatedsegmentedmirrorcalledChopper [C]whichhelpsto
pass the sample andreference beamtothe monochromatorgrating [D].
3. The grating rotatesslowlyandtransmitsindividual frequenciestothe detectorthermophile [E]
whichconvertsIRenergyto the electrical energy.
4. Whena sample hasabsorbeda certainquantumof lightof specificfrequency.The detectorshall
be receiving alternately from the chopper an intense beam (reference beam) and relatively
weak beam (sample beam). It will generate alternating current (AC Current) flowing from the
detector to the amplifier [F].
5. This out of balance signal received by the amplifier is coupled to a small servomotor [G]. That
gives an optical wedge [H] in the reference beam until the detector receives light of equal
intensity from sample and reference beam.
6. The slightestmovementof the wedge (attenuator) isfurthercoupledtoaninkpenrecorder [J]
and finelygivesthe variousabsorptionbands(signals)ona printedchart.
Schematic diagram of IR Spectrophotometer:
31. Applications of IR Spectrophotometer
The importantapplicationsof IRspectroscopyare discussedbelow;
1. Qualitative analysis:
We can identifythe functional groupof manyorganiccompoundsanddrugs.For example; benzoic acid,
salicylic acid, phenol, aniline, P-aminophenol.
We can identify the functional group as well as aromatic ring of many organic compounds by IR
spectroscopy. For example; acetanilide, Paracetamol, Aspirin etc.
2. Quantitative analysis:
By using IR spectrum of the sample of drug and by making the comparison of IR spectrum of standard
drug we can also determine the percentage purity of the drugs.
3. Hydrogenbonding:
By using IR spectroscopy we can determine the H-bonding of the organic molecules highly
electronegativeatomssuchas Nitrogen,OxygenandFluorine are involved in strong H-bond formation.
4. Purity of sample:
Generallythe pure sample showsfairlysharp spectrum whereas the material of high molecular weight
generally shows poor spectrum because of the presence of several kinds of functional groups.
5. Chromatographic separationstudies:
The process of chromatographic separation can readily be monitored by taking the spetra of selected
fractions.
6. Determinationof aromaticity:
By taking the IR spectrum of different organic aromatic drugs we can study the relative proportion of
saturated and unsaturated rings present in hydrocarbons.
7. Tatumerism:
Tatumeric equilibria can be studied with the help of IR spectroscopy. Most common system such as
keto-enol and Lactum to Lactum contain a group such as C=O, OH, NH2, C=S group which shows
characteristic frequency which make it possible for identification of particular drug.
8. Identificationof different groups andbonds:
By takingthe IR spectrumof differentfunctional groupswe canidentifyonthe basesof spectrumhaving
their specific frequency range.
32. Example:
Group Bond Frequency range cm-1
Alkyl C-H 2853 – 2962
Alcohol O-H 3590 – 3650
Amine N-H 3300 – 3500
Similarlyincase of bondand frequency;
Bond Frequency range cm-1
C=C 2100 – 2260
C=O 2220 – 2260
C≡C 1620 – 1680
C≡N 1630 – 1780
7. Organic reactions: We canstudythe IRspectrumof organicreactions.
8. Dipole moment: We can studythe dipole momentsof organiccompound.
9. Dissociation constant: We can study the degree of dissociation constant of organic compounds.
10. Complex formation: We can determinecomplex formationstudyof reactions.
11. Finger prints: We can studythe fingerprintregionof organiccompounds.
Some of the importantexamplesof rawmaterial anddosage formare as follows;
Raw material;
Paracetamol rawmaterial
Aspirin raw material
Ibuprofen raw material
Chlorocaine raw material
Chloroquinie raw material
Dosage forms;
Aspirintablet
Paracetamol Tabletand syrup
Ibuprofen tablet and syrup
Ponston tablet (Mefanamicacid)
Neubrol tablet(orphendrine+paracetamol)
34. Nuclear Magnetic Resonance NMR
Nuclearmagneticresonance spectroscopyisapowerful analytical techniqueusedto
characterize organicmoleculesbyidentifyingcarbon-hydrogenframeworkswithinmolecules.
It can be definedas,“Itis the studyof absorptionof electromagneticradiationsbyatomicnuclei inthe
radiofrequencyregioninthe presence of magneticfieldiscalledNMRspectroscopy.”
Principle:
The absorptionof electromagneticradiationinradiofrequencyregionbythe nucleusresultsin
the change in orientationof spinningnucleusinanappliedmagneticfield.
Types of NMR:
Two commontypesof NMR spectroscopyare usedto characterize organicstructure;
1
H NMR isusedto determine the type andnumberof Hatoms ina molecule.
13
C NMR is usedto determine the type ofcarbonatoms inthe molecule.
Basic theory:
A. Spinning nucleus:
Whena charged particle suchas a protonspinsaroundits axis,itcreatesa magneticfield.Thus,the
nucleusbehavesasatinybar magnet.
B. Behavior under applied magnetic field:
Normally,these tinybarmagnetsare randomlyorientedinspace.However,inthe presenceof an
external magneticfieldB0,theyare orientedwithoragainstthisappliedfield.More nuclei are oriented
withthe appliedfieldbecausethisarrangementislowerinenergy.
The energydifference betweenthesetwostatesisverysmall (<0.1cal).
35. C. Spin flipping:
In a magneticfield,thereare nowtwoenergystatesfor a proton;
1. α-spinstate: A lowerenergystate withthe nucleusalignedinthe same directionasB0
2. β-spinstate: A higherenergystate inwhichthe nucleusalignedagainstB0.
Transitionof a protonfrom α-spinstate toβ-spinstate bythe absorptionof radiofrequencyof
electromagneticradiationisreferredtoas “spinflipping.”
The energydifference betweenthesetwonuclearspinstatescorrespondstothe low frequencyRF
regionof the electromagneticspectrum.
D. NMR signals:
Thus, two variablescharacterize NMR;
An appliedmagneticfieldB0,the strengthof whichismeasuredintesla(T)
The frequency ofradiationusedfor resonance,measuredin hertz (Hz), or megahertz (MHz)
Both these variablesare proportionallyrelated;
Therefore,the NMRsignalscanoccur by keepingone of bothconstantandchangingthe second.
i. Fieldsweep(νisconstant&B0 isvaried)
ii. Frequencysweep(νisvaried&B0 isconstant)
Note: Onlynuclei thatcontain oddmass numbers (suchas 1
H, 13
C, 19
F and 31
P) or odd atomic
numbers (suchas 1H and7N) give rise toNMR signals.
36. NMR spectrum:
ModernNMR spectrometersuse aconstantmagneticfieldstrengthB0,andthena narrow range of
frequenciesisappliedtoachieve the resonanceof all protons.Protonsindifferentenvironmentsabsorb
at slightlydifferentfrequencies,sotheyare distinguishable byNMR.
An NMR spectrumcan be definedas,“itisa plotof the intensityof apeakagainstitschemical shift,
measuredinpartsper million(ppm).”
NMR absorptionsgenerallyappearassharppeaks.The increasingchemical shiftisplottedfromleft
to right.Most of the protonsabsorbbetween0-10 ppm.
The terms “upfield” and“downfield” describe the relativelocationof peaks.
Upfieldmeanstothe right.
Downfield means totheleft.
NMR absorptionsare measuredrelativetothe positionof areference peakat0 ppm onthe δ-scale due
to tetramethylsilane (TMS).
Followingfeaturesof a1
H NMR spectrumprovide informationaboutacompound’sstructure:
a. Numberof protons
b. Positionof protons(chemical shift)
c. Intensityof signals
d. Spin-spinsplitting
37. a. No. of protons:
The numberof NMR signalsequalsthe numberof differenttypesof protonsinacompound.
The equivalentprotonsgivethe same NMRsignal.
Protons indifferent environments give differentNMR signals.
To determine equivalent protons in cycloalkanes and alkenes, always draw all bonds to hydrogen.
In comparingtwoH atoms on a ringor double bond,twoprotonsare equivalentonlyif theyare
Cis(or Trans) to the same groups.
39. b. Positions of protons or Chemical shift:
The shiftinabsorptionpositionof NMRwhicharisesdue tothe shieldingordeshieldingof protonsby
electronsiscalledchemical shift.
Chemical shift tothe Right:
The proton isa molecule thatissurroundedbya cloudof electrons.Whenmagneticfieldisappliedit
inducesthe electrontocirculate aroundthe nucleusperpendiculartothe appliedmagneticfield.
These circulatingelectronsproducestheirownmagneticfield inadirectionopposite tothe applied
magneticfield.Since,the inducedmagneticfieldof electronsisinopposite directiontothe applied
magnetic field. The effective magnetic field experiences by the nucleus is reduced.
( ) = ( ) − ( )
Thus,the circulatingelectroncanpartiallyshieldthe nucleusfromthe appliedmagneticfield.
The nucleusissaidto be shieldedandsuch type of shieldingiscalled Diamagneticshielding.
Since,the nucleusisshielded therefore,itneedsalowerfrequencyto achieve resonance.Lower
frequencyistothe rightin an NMR spectrum, towarda lowerchemical shift,soshieldingshifts
the absorptionupfield.
Chemical shift tothe Left
The lessshieldedthe nucleusexperiencesthe more of the effective magneticfield(B0) it.
Thus,the deshieldednucleusexperiencesahighermagneticfield(B0) strength.Therefore,itneedsa
higherfrequencyto achieve resonance.Higherfrequencyistothe leftinanNMR spectrum, toward
higherchemical shift.So,deshieldingshiftsanabsorptiondownfield.Protonsnearelectronegative
atomsare deshielded,sotheyabsorbdownfield.
The effective fieldmaybe differentfordifferentnucleusbecause of shieldingeffect.Thus
theirresonance energywill differslightly.
41. Factors affecting Chemical shift
Followingare some factorswhichaffectthe chemical shift;
1. Electronegativity:
The extentof shieldingdependsuponthe electrondensityorelectroniccloudaroundthe nucleusand
electronsdependsuponthe electronegativity.Asthe electronegativityincreases,the electronswill be
drawnaway fromthe nucleusandshieldingwill be decreases.Thus,the nucleuswill be deshieldedinthe
case andexperiencesmore field.So,deshieldingshiftsanabsorptiondownfield.
2. H-bonding:
Increase inH-bondingwill leadtoincrease indeshielding.So,deshieldingshiftsanabsorptiondownfield.
3. Solvent effect:
Solventvariationresultsindramaticchange inchemical shift.Forexample;
Aromaticsolvents(benzene)cause deshielding.
Acidic solvents causes deshielding.
Non-aromaticsolvents causes shielding.
4. Magnetic anisotropic effect:
It isrelatedtothe geometry.A protonmayexperiencesadditional shieldingordeshielding
dependinguponitsorientationrelative tothe inducedmagneticfieldcausedbythe electronic
circulationsucheffectiscalledmagneticanisotropiceffect.
Example 1:
If the inducedmagneticfieldisinthe directionof appliedmagneticfieldthenthese bothfieldswill
reinforce eachother.The protonsthusfeel astrongermagneticfieldandahigherfrequencyisneeded
for resonance.Thustheyare deshieldedandabsorbdownfield.Incase of Benzene;
42. Example 2:
In some casesthe inducedmagneticfieldisinopposite directiontothe appliedmagneticfield.Thus,the
protonfeelsaweakermagneticfield,soalowerfrequencyisneededforresonance.The nucleusis
shieldedandthe absorptionisupfield.
5. Additionof Lanthanide shift reagent:
Theyare complexesof lanthanidesmetal whenwe addreagenttheyalterchemical shiftof
protonbyformingcomplexes.Theyare usedtosimplifythe NMRspectrum.
Formula:
= ( ) − ( ) =
ν(sample) =Frequencyof sample
ν(TMS) = Frequency ofTMS
ν0 = Frequency ofinstrument
The frequencyof sample andTMS is inHertz (Hz).While,the frequencyof the instrument ν0 isinMega
Hertz (MHz).That’s whywe take it inparts permillion(ppm).
= = =
43. c. Spin-spinsplitting or Spin-spincoupling:
“Splittingof resonance bandsinNMRintodoublets,triplets,quartetsetc.iscalledasspin-spinsplitting.”
Or
“The phenomenonthatdescribesmagneticinteractionbetweenneighboringnon-equivalentnucleiin
NMR.”
Peaksare oftensplitintomultiple peaksdue to magneticinteractions betweennonequivalentprotons
on adjacentcarbonsatoms.The splittingof absorptionsignal isasetof peaks.Itisa multiplet(2=
doublet,3= triplet,4= quartet,5=pentet,6=hextet,7=heptet…..)
Mechanismof Splitting or Coupling:
Let’sunderstandthiswiththe exampleof Tri bromoethane.
Whenit isplacedinan appliedmagneticfield(B0),the adjacentprotonsHa andHb can eachbe aligned
with() or against() B0.
Thus,the absorbingprotonfeelsthreeslightlydifferentmagneticfields;
1. One slightlylargerthanB0 (ab)
2. One slightlysmallerthanB0 (ab)
3. One the same strengthas B0 (ab)
As,the absorbingprotonfeelsthree differentmagneticfields,itabsorbsatthree differentfrequenciesin
the NMR spectrum,thussplittingasingle absorptionintoa tripletbecause there are twodifferentways
to alignone protonwithB0,and one protonagainstB0— i.e. ab and ab—the middle peakof the
tripletistwice asintense asthe twoouterpeaks,makingthe ratioof the areas underthe three peaks
1:2:1.
Thus,two adjacentprotonssplitanNMR signal intoa triplet.Whentwoprotonsspliteachother,they
are saidto be coupled.
Significance of coupling:
1. It helpsinidentifyingtypesof protons.
2. It ishelpful inidentifyingno.of protonsof each type.
3. It isa helpingtool inidentifyingno.of neighboringprotons.
44. Examples:
Coupling constant:
It isdefinedas,“the distance betweenthe centersof the adjacentpeaksina multipletisusuallya
constant,and it is known as coupling constant. It is represented by ‘J’. It varies from compound
to compound.
It is expressed in Hertz or Cycles/ sec. It is a constant that depends upon the structural relationship
between the coupled protons. It is independent of the applied field and the nature of solvent.
Significance:
It isuseful todistinguishbetweensingletanddoublet.
It also helps todifferentiatebetween 1 quartet and2 doublets.
45. Rules of Splitting
1. The splittingisintoone more peakthanthe numberof H’son the adjacentcarbon(s),Thisisthe
“n+1 rule”
2. The relative intensitiesare inproportionof abinomial distributiongivenby Pascal’sTriangle
1 singlet
1 1 doublet
1 2 1 triplet
1 3 3 1 quartet
1 4 6 4 1 pentet
1 5 10 10 5 1 hextet
16 15 20 15 6 1 heptet
3. Equivalent protons do not split each other.
4. Protonsthat are fartherthan two carbon atoms apart do not spliteachother
46. 5. If Ha and Hb are not equivalent,splittingisobservedwhen;
6. Splitting is not generally observed between protons separated by more than three bonds.
7. Complexsplitting:If there ismore than 2 interactinggroups,the bondmultiplicityof A asspitby
B & C isexpressedas;
(nB+1) (nC+1) = No. of lines
47. InstrumentationandData Analysis
The NMR instrumentconsistof followingparts;
1. Sample tube
2. Magnet
3. Fieldsweepcoil
4. RadiofrequencytransmitterorGenerator
5. Radiofrequencyreceiver
6. Amplifier&Readoutdevice
1. Sample tube:
The sample holderinNMR isnormallytube-shaped
and istherefore calledthe sampletube.Itisa tube of
15cm lengthand5mm diameter.
It ismade up of borosilicate glass.It is inert, durable
and transparent to radiofrequency radiations. Glass
or Pyrex tubes are commonly used. These are
sturdy, practical, and cheap.
48. 2. Magnet:
The magnetin NMR spectrometermustbe strong,stable,andproduce ahomogeneousfield.
Homogeneousinthiscontextmeansthatthe fielddoesnotvaryinstrengthor directionfrompointto
pointoverthe space occupiedbythe sample.
The magnetis so large that thisinstrumentcomeswithitsownstaircase sothatthe analystcan insert
and remove the sample tube fromthe topof the probe.
Three typesof magnatescan be employedinNMRspectroscopy;
a. Permanentmagnates
b. Electromagnets
c. Superconductingmagnets
a. Permanent:
It isCheapand it isconvenienttouse,butitnot frequentlyusedasitlacksflexibilityand
no variationscanbe made.
b. Electromagnets:
The type of magnetisinsensitive totemperature.The advantage of thismagnetisthat;
The flux densityorthe strengthof appliedmagneticfieldcanbe controlled.
The current passingthroughthe coil can be controlled.
c. Super conducting magnets:
Theyare mostpowerful magnetsascomparedtothe rest.They possesshighresolution.All
NMR spectrometershavingfrequencyabove than100 MHz are basedon Heliumcooled
superconductingmaterial.
3. Fieldsweepcoil:
It isa coil eitherwrappedaroundorplacedbetweenthe twopolesof the magnets.Itisusedtostable
the appliedmagneticfield,italsohomogenize the frequencyof magnets.
The strengthof appliedmagneticfieldcanbe variedbyvaryingthe passingcurrentinthe sweepcoil.
4. Radiofrequency generator:
It transmitsthe radiationsof controlledfrequencytothe sample inthe directiontothe magneticfield.
The radiationsare appliedinsucha way that theyare right angle toradiofrequencyreceiver.This
provide signalsrequire toinduce transitions.
5. Radiofrequency receiver:
49. It consistof a receivercoil.Itis coiledaroundthe sample tube atrightangle tothe appliedfieldas
well astransmittercoil.
6. Amplifier &readout device:
Signalsfromthe receivercoil isweak,therefore theyare amplifiedandrecordedmechanically,orpre-
calibratedchartswithrespecttoreference compounds.
7. Schematic diagramof NMR:
50. Reference standardfor NMR:
The position of absorption of various protons cannot be determined separately with accuracy. A
standard reference is used either an internal reference or external reference. Therefore, the NMR
absorptions are measured relative to the position of a reference peak at 0 ppm on the delta scale.
Reference isassumedtobe free of shieldingeffectanditrequireslessmagneticfield.The standards
usedforNMR mustbe chemicallyinert,misciblewithmostorganiccompoundsandmusthave low
meltingpoint.
The most commonlyusedreference standardsinNMRare;
1. TMS – Tetramethylsilane,isavolatileinertcompoundthatgivesasingle peakupfield.Itsboiling
pointof is 27o
C. It isgenerallyusedNMRstandardfororganic compounds.
2. DSS – Dimethyl silapentane sulfonicacid,ismore oftenusedinproteinexperimentsinwater.
Solvents usedinNMR
The solventswhichare mostlyusedforthe NMR spectroscopicanalysis are;
Carbontetra chloride CCl4
Carbon disulphide CS2
Deutrated Chloroform CDCl3
Deurated Benzene C6D6
Deutrated water or Heavy water D2O
Properties of agood solvent:
The solventusedforNMR musthave followingproperties;
It shouldbe cheapand easilyavailable.
It should benon-viscous.
It should bechemically inertto thesampleandsample holder.
It should dissolve maximum amount oforganic compound.
The solventmustdevoidofits ownprotons.
Magnetic anisotropy.
51. Working of NMR spectrometer:
The workingof NMR spectrophotometerconsistof followingsteps;
1. 0.5 ml of 15% sample solutionintesttube ismade.Thenaddfew dropsof reference standard
DSS or TMS.
2. The Sample tube isthenplacedbetweenpolesof amagnets.
3. Radiofrequencyradiationsismade tofall onsample byradiofrequencygenerator.
4. The field strength is increased by increasing the current in sweep coil. As a result precessional
frequency of each set of protons increase until resonance with radiofrequency source take place.
5. Nowthe detectorproducesa signal.
6. Signal fromthe detectorisamplifiedandthenrecorded.
Uses of NMR spectrometer:
1. It isusedin researchanddevelopment.
2. It isusedin biologyof biological studies.Forexample;tocheckthe structure of proteins.
3. It isusedin foodindustryformoisture analysis.
4. It isusedin agriculture.
5. It isusedin pharmaceutical analysisandindustry.
52. Applications of NMR
Spectrometer 1. Investigationof dynamic properties:
NMR can be usedto investigatethe dynamicpropertiesof moleculessuchas;
H-bonding
Molecular information
Conformationalisomer
Restricted rotation
2. Determinationof optical activity:
DiastereomersdiffersintheirNMRproperties.So,thistechniquecanbe usedforthe determination
the optical activity.
3. Keto-enol tautomerism:
It isa more powerful analytical techniqueforquantitative andqualitativeinvestigationof Keto-enol
equilibria.Forexample;keto-enol formsof Acetones.
4. Structure elucidation:
The structure of an unknown compoundcanbe determinedfrom;
No.of signals
Chemical shift (it indicates thetype ofelectronicenvironment ofthepeotons)
Spin-spin coupling
5. Quantitative analysis:
The technique hasbeenusedtodeterminethe molarratioof compoundina mixture.
Percentage of eachcomponent=area underpeakcomponent/total areaunderpeakX hundred
6. Elemental analysis:
To determine the giventype of magneticnucleusinthe sample.
7. Drug macromolecular interaction:
We can studymiscell formationof drugin the sample andmacromolecule interaction.
8. Study of isotops:
Several nuclei inadditiontoprotonsthathave magneticmovementscanbe studiedymagnetic
resonance technique.Forexample;fluorine,phosphorousetc.
9. Moisture analysis:
Water absorbedinbiological materialssuchasfoodproductsappear inNMR spectra as relativelysharp
bands.
10. Hydrogenanalysis:
Percentage of hydrogeninanunknownsample maybe determinedeasilyandrapidly.
53. 11. Cis-Tans isomers:
NMR can be usedto distinguishbetweenCis-transisomers.
12. Compound identification:
It can be used for compound identification that is, to confirm a compound in the sample.
13. NMR can alsobe usedto confirmthe Purity ofa chemical compound.
14. NMR is usedforthe determinationof surfactantchain length.
15. NMR is utilizedtofindthe Iodine value oftriglycerides
55. Basic concepts
Witt theory:
GermanscientistO.M.Witt in1876 suggestedthe chromophore auxochrome theoryfor colored organic
compounds. The various terms which are used as fallows;
Chromophore: The graphs which has unsaturation and electron withdrawing effect have an
appreciable effect on the absorption of light and well present in conjugation are responsible for the
color of compounds (due to absorption in visible region) such groups are known as chromophore.
Chromogen: The compoundscontainingchromophore groupsare known as chromogens. For example
N=O, -N=N, >C=S, >C=O, >C=C< etc.
Auxochrome: These groups are not responsible for colors but when present along chromophore groups are
responsible for deepening of the color. They are electron donating groups. For example -NH2, -OH.
Quinonoid theory:
According to this theory ortho and para quinonoid structures in a compound are responsible for its color.
Moderntheory:
This theory is basically the modification of Witt & Quinonoid theory. It explains the colors of organic
compounds based on resonance effect and its correlation with absorption of light.
The light waves in UV and visible region have high energy. When light falls on a compound it gets
absorbedandresultsinthree typesof excitationinthe moleculei.e.electronic,vibrational,rotational. If
E1 is the energy in ground stand and E2 is the energy in the excited state than ΔE energy required for
excitation will be;
ΔE = E2 – E1 =hν
The chromophore and auxochrome groups present in a compound causes deepening of color by
increasing the number of charged contributing structures during resonance effect.
The increasedconjugation(delocalization) inasystemshiftsthe absorption towards longer wavelength
regionlowerenergyandisknownas “Bathochromic shift.” Similarly shifting of the absorption towards
the shorter wavelength region (high energy) is known as “Hypsochromic shift.”
Example: Nitrobenzene is yellow in color due to presence
of nitro group. Whereas, para-nitroaniline is orange in
color. The nitro group is chromophore which produces
yellow color due to resonance effect. On the other hand
amino group is auxochrome which causes deepening of
color by increasing the no. of contributing structures
because of the presence of three nitro group.
56. UV-Visible Spectroscopy
Introduction:
There are tworegions;
UV region200nm to 400nm
Visibleregion 400nmto 850nm
Whena beam of sun lightispassedthrough a
prism it splits into seven colors i.e. red,
orange, yellow, green, blue, indigo, and
violet.Thissetof colors obtained by splitting
the white light is called spectrum. Different
colors are associated with different energy
and wavelength. For example red light has
smallest energy and longest wavelength.
Energygoeson increasingandwavelengthgoesondecreasingaswe move fromredto violetcolor.
Therefore,the violetcolorhasthe maximumenergyandminimumwavelength.
Electromagnetic spectrum:
In addition to white light with its seven different colors or radiations, there are many more different
typesof radiations.Some of themare more energeticandsome are lessenergeticthanthe visible white
light. The electromagnetic spectrum consist of the following radiations;
(i) Cosmic rays: these rayscome fromsun andare knownto be the radiationsof highest
energy. Theyhave wavelengthlessthan10-3
nm.
(ii) γ rays and X rays: theyare less energeticthancosmicraystheirwavelengthrange is
between10-3
nm to 10-1
nm.
(iii) UV light: these are the rayswhichare lessenergeticbutmore energeticthanvisible light.
The range of wavelengthof UV regionis200nm to 400nm.
(iv) Visible light: itis ordinarylightanditis composedof several differentradiations(fromred
to violet) andthe wavelengthrange is400nm to 850nm.
57. Principle:
Whena beam of electromagneticradiationispassedthroughacompoundcertainradiationsare
observed.There issome energyassociatedwithenergyradiationandisgivenbythe followingequation;
=
Where;
ν is the frequencyof radiations
h is the Plank’sconstant
The energy absorbed by the substance produces some changes within the molecule if the energy
absorbed is high it causes electronic excitation i.e. electrons are excited to higher level. The graph
betweenthe amountof radiationabsorbedbythe sample andthe wavelength of the radiation is called
absorption spectrum. It consist of bands with peaks of maximum intensity. The overall range of
electronic spectroscopy is from 180nm to 850nm.
Range of determination:
It records the spectra of compounds or drug molecules in the range of 180nm – 185nm. This range
consist of two parts;
180nm – 400nm, whichis range of UV radiation.
400nm – 850nm, whichis range of visiblelight
The spectra below180nm cannotbe takenbecause oxygenpresentinspectrophotometerabsorbsthese
radiationstorecord the spectraof substancesbelow 180nm.Special vacuumconditionsare needed.
58. Instrumentationandworking:
The UV-visible spectrometerconsistof followingparts;
1. Lightsource
2. Monochromator(Prismtype,Gratingtype)
3. Quarts cell
4. Detector(phototubes,photomultipliertubes)
5. Recorder
The followingfigure givesthe schematicrepresentationorblockdiagramforthe use of
spectrophotometer.
1. The solvent used to dissolve the sample are the ethanol, methanol, n-hexane and distilled
water.For recording the UV-visible spectrum the sample is dissolved in a solvent, which itself
don’t absorb light in that’s range.
2. A quartz cell made up of special glassof 1cm path lengthisusedasa containerforthe sample
solution(sampleholder).
3. The solutionisexposedtoUV-Visiblelightbythe prismselector.The prismselectorisrotating
continuouslytoemitlightsof differentwavelength.
Hydrogen lamp is used for emittingUVlight.
Tungsten lamp is used for emitting visiblelight.
4. The instrumentprovidesagraphbetweenwavelengthsof radiationsabsorbedandthe intensity
of absorption.
5. The wavelengthcorrespondingtotopof peakshowsthe maximumabsorption.The wavelength
isdenotedby λ and maximumabsorptionisdenotedby λmax
6. The intensityof absorptioncorrespondingtothe wavelengthiscalled‘Molarextinction
constant,’andit isdenotedbyepsilonor Ɛ.
59. Beer’s Lambart’s Law
Beer’s Law:
Whena beam of monochromaticlightispassedthroughasubstance dissolvedinanon-absorbing
mediumthe absorptionof lightisdirectlyproportionaltothe molarconcentrationof the substances.
Mathematically;
log10 ∝
Thisis referredasBeer’slaw.
Lambart’s Law:
Whena beam of lightis passedthroughsubstance the absorptionof lightisdirectlyproportionaltothe
path lengthof the substance.
log10 ∝
Thisis referredasLambart’slaw.
The two lawsare combinedtoobtainthe absorptionof lightbya substance therefore;
log10 ∝ .
log10 = Ɛ
Where;
Io standsfor intensityof incidentlight
I stands for transmittedlight
C stands for ‘concentrationofsubstance in centimeter
Ɛ stands for proportionality constantknown as molarextinctionconstant.
Expressionof absorbance is;
log10 = ( )
So,final equationcanbe writtenas;
= Ɛ
60. Spectrophotometricdeterminationof absorbance of
known solution;
Suppose we wantto determinethe amountof substancesinthe solutionbyspectrophotometric
methodthanthe procedure will be asfallows;
1. Place the solutioninaquartz cell.
2. Nowput the cell ina cell holderandmove itintoa positionsothat the solutioncomesinalight
path.
3. Go on changingthe wave lengthorthe incidentradiationandnote the absorbance foreach
value of the wave length.
4. Nowplota graph betweenabsorbance andwavelengthandfindoutthe value of λmax
5. Thiswavelengthisusedinthe measurementof absorbance throughoutthe experiment.
6. Prepare a seriesof differentconcentrationof solutionthatisforknownconcentrationmeasure
the absorbance of each knownconcentrationatthat wavelengthasmentionedinstep2.
7. Plota graph betweendifferentvaluesof absorbance andconcentration.Thisiscalleda
calibrationgraphor standardcurve.
61. Applications of UV-Visible spectroscopy
Applicationsof UV-Visible spectroscopyare asfallows;
1. Detection of conjugation: Withthe help of UV-visible spectroscopy figure out the presence
of double andtriple bondinacompound.The conjugationcanbe C=O,C=C, C≡C. the absorption
of that compound and by the observation of λmax values, we can also predict the location of
substituents.
2. Detection of functionalgroup: itispossible to detect certain functional group with the help
of UV-Visible spectroscopy.Absence of absorptionabove 200nm is indication of the absence of
the conjugation.
3. Detection of geometrical isomerism (Cis/Trans): when compounds shows geometrical
isomerism. The Trans isomer shows the absorption at higher wavelength with larger values of
extinction co-efficient. That is absorption as compared to Cis-isomers.
4. Qualitative analysis: (Identification of unknown compounds) the detection of unknown
compounds is carried out by making the comparison of unknown spectrum with the known
spectrum.
5. By using this technique the structures of some vitamins and organic compounds can be identified.
6. By the use of thistechnique the no.of chromophoressuchasaldehyde,isolateddouble bonds
can be identified.
7. By thistechnique we canalsoperformconformationalanalysisandwe can alsodeterminethe
rate of reaction.
8. By this technique we can also determine the quantitative analysis of drug having even low
concentrationinthe sample.The example of the drugsare paracetamol, vitamin B2 (riboflavin),
Vitamin C (ascorbic acid, Vitamin B12 cyanocobalamine.
9. We can alsodetermine the concentrationof the knowncompoundif itfallowsBeer’sLambart’s
law.
10. We can alsomeasure the kineticmeasurement;
i. The rate of chemical reactioncanbe determinedas,decreaseinabsorbance when
reactantsare absorbingspecies.
ii. Increase inabsorbance whenproductsare absorbingspecies.
11. We can alsodetermine the ionizationconstantof anacid andbase bymeasuringthe absorbance
at differentconcentrations.
62. Examples in aromatic systems:
In the polynuclearhydrocarbonsforexample suchasanthracine,naphthaleneandbenzene, anthracine
has highestλmax value because ithasa highlyextendedconjugatedsystem as compared to naphthalene
and benzene.
Benzene Naphthalene Anthracine
λmax (wavelength) 255nm 314nm 380nm
Ɛ (absorbance) 230nm 289nm 9000nm
Some of the pharmaceutical examplesare asfallows;
Ibuprofen(tabletandsuspension),Ponston (tabletandsuspension),Vitamin syrup and dexamethasone
Injection.
64. Chromatography
Mechial zwitt was the
chromatography is made
measurementsorwriting.
firstscientistwhodiscoveredchromatographytechnique.The wordupof
twoGreekwords ‘Khromatos’meansColorand‘Graphy’means
Definition:
It isa separationtechnique.Inwhichthe sample mixtureisseparatedintoitscomponentsbythe use of
propersolvent.
Chromatographyisa physical orchemical processor both;
It isa physical processasthere isno chemical reactionorformationof new compound.
It is a chemical process as thereis change in compositionbeforeandafter thechromatography.
Withchromatographywe can get accessto the compositionof the mixturetobe separated.
Compositionconsistsof;
Numberof components
Quantityof components
Chromatography is an analytical method used primarily for the separation of a sample of mixture. It
involvesthe distribution of the sample mixture between two phases i.e. stationary phase and mobile
phase.
Stationary phase:
It ismay be a solidorliquidsupportedasathinfilmonthe surface of an inertsolid.The phase
(solid/liquid) onwhichthe mobile phase flow iscalledstationaryphase.
Mobile phase:
The mobile phase flowingoverthe surface of the stationary phase that may be a gas or a liquid.
The solvent (gas / liquid) which is used to separate the components of the mixture is called
mobile phase.
Base of chromatography:
Polarityisthe base of chromatography.We are tryingto make unionizedandpolardrugs.
Order of polarity:
The order of polarityisthe sequence of solventswiththe increasingordecreasingorderpolarity.
Example: The givensequence of solventsformnon-polartopolar.
n-Hexane >Petroleumether>Chloroform> Ethyl acetate > Alcohol (Methanol >Ethanol > Propanol >
Butanol)
In case of alkanesincrease incarbonno.decrease inpolarityof alkanes.The mostpolarsolventisWater.
65. Gas Chromatography GC
Introduction:
It is an analytical technique. In this technique, liquid or gas
sample isusedforthe analysis.The particle size of the sample
mixture must be ranging 0.4µ – 0.5µ for the instrumentation.
In case of liquid sample it will be converted into vapors first
with the help of oven. The technique is used only when the
substance isheatstable because temperature is about 300o
C.
In thistechnique twophasesare used;
Mobile phase
Stationaryphase
Mobile phase:
The gas is the mobile phase. NormallyNitrogen N2 orHeliumHe gasis usedas mobile phase.Onlyone
gas can be usedat a time,bothcan’tbe usedsimultaneously.
NitrogenN2 isnon-reactiveingaseousphase.
Helium He is an inert gas. Helium is mostly usedbecauseit shows good results.
Stationary phase:
Columninthe formof coil isusedas stationaryphase.Itis commonlymade upor eithermetal orglass
tubing.There are twocommerciallyavailable columns;
There are twotypesof gas chromatographyon the basesof stationaryphase;
Gas solidchromatographyGSC
Gas liquid chromatography GLC
The columntubingcontainsolidsilica.ThiscolumnisusedinGassolidchromatography.
The column consist of two tubes. The outer tubing contain solid silica. The inner tubing contain either
polarpolyethyleneglycol ornon-polarpolysiloxaneinliquidformdependingupon the nature of sample
mixture. The column is used in Gas liquid chromatography.
66. Principle:
Polarityisthe principle of gaschromatography.We will findthe polarityof the stationaryphase,and
the sample tobe examinedandseparated.
Relationof Gas Chromatography with Fractional distillation:
Similarities;these bothtechniquesdependsuponthe boilingpointsof the componentmixtures.
Difference;Fractional distillationisusedonlarge scale ormacro scale.While,GCisusedfor Lab
scale purposes.
TLC:
TLC stands forthinlayerchromatography.
Advantage: Itisa cheapprocess.
Disadvantage: Ithas no temperature regulation.
HPLC:
It standsfor HighPerformance liquidchromatographyorHighPresure LiquidChromatography.
Advantages:
The resultsare precise.
Thermolaiblesubstances canbe analyzedby thetechnique.
Disadvantages:
Thermostable compoundsare notanalyzedupto40o
C temperature.
67. GC working:
A volume of mixture whichistobe separatedisinjectedintothe headof columnbythe syringe or micro
syringe.A mobile phase (gas) sweepsorcarriesthe mixture intothe column, this motion is inhibited by
the adsorption of each component of the mixture in the column. The rate at which the mixture or the
components of mixture progress or more along the column depends upon the strength of adsorption,
and the strength of adsorption depends upon the polarity of the mixture and the polarity of the
stationary phase.
So,in thiswayeach componentinthe mixture becomesseparatedinthe columnasthe mixture reaches
the end of the column at the different times (retention time).
Retention time: the time inbetweeninjectionof the sample anddetection of components.
Detectordetectthe componentsqualitativelyandquantitatively.It calculates the retention time of the
different components.
Flushingorwashingof the instrumentisdone onlybygas.The flow of the gas is maintained to get good
results so that the sample to be separated moves smoothly through the column.
Selectionof column:
It dependsupon;
Polarityof the mixture andpolarityof the column.
We alsoconsiderthe thickness,diameterandthe lengthof the column.
68. Column Temperature &Temperature program;
Columnisplacedinan oventhe temperature of whichisspeciallycontrolled.If there isnodifference in
initial andfinal temperature of the whole processsothisiscalled Isothermal process.
If there is difference ininitial andfinal temperature so,the initialtemperature,rate of increase in
temperature andfinal temperature iscalledas Temperature program.
Detectors:
Most commonlyuseddetectorsinGCis two;
Flame ionizationdetectorsFID
Thermal conductivity detector TCD
Amongthe othersare;
Catalyticcombustiondetector
Infrared detector
Atomic emission detector
Electron capturedetector
Helium ionizationdetector
Photo ionization detector
Data analysis:
Two typesof analysisare carriedoutby the GC;
Qualitative analysis: it refers to that which components arepresent in the sample mixture. The no.
of peaks gives the no. of components of mixture. For example; active ingredients.
If the retentiontime of the mixtureissimilar tothe reference thenqualitytestisperformed.
Quantitative analysis: it refers to that how much active ingredient is present in the sample mixture.
The greaterarea of the peakindicate the quantitativeanalysisof the component.
69. Applications:
We can isolate the compounds which are thermostable by using GC, but GC alone is not sufficient or
efficient to separate the mixture. Therefore, GC is connected with Mass spectrometer MS to get more
accurate results.
We can connectMS withGC,LC, HPLC, TLC, and we can connect MS withMS.
Mass Spec can be combinedwithgaschromatographytoanalyze mixturesof compounds.
GC separatesthe componentsof the mixture.
Each componentisanalyzedbythe Mass Spectrometer.
70. Introduction
Spectroscopy
It is the combination of two words spectrum & scope. Spectrum means, “a set of specific no. or set of
specific values or a set of digits,” while the word Scope means “Look or Watch.”
So,spectroscopymeans,studyof specificvaluesornumbers.Itdeals with the study of electromagnetic
radiations(radiationspossessingboththe propertiesof electrical andmagneticnature).Electromagnetic
magneticradiationsincludeultraviolet,infrared,microwave,radio waves, cosmic waves, visible waves
etc.
Everylightisa combinationof differentcolors.If the spectrumobtainedisfrom visible region then, it is
‘Photometry.’
Spectrometry:
It is the combination of two words spectrum & meter. Spectrum means, “a set of specific no. or set of
values or a set of digits,” while the word Meter means “measurement.”
So, Spectrometry means measurement of spectrum. When there is no absorption or emission of
electromagnetic radiations, then this study is known as spectrometry, but here the spectrum is not
made up by the electromagnetic radiations. The spectrum is Mass spectrum. If it has the concept of
mass then it is referred to as Mass spectrometer.
Mass Spectrometry
There is breakdown of a molecule into its different fragments that is why it is called as Mass spectrometry.
If a large molecule ispresentthen,there will be formation of too many fragments. So, that’s why MS is
used in combination with other techniques.
Attachment of MS withother techniques
Thisis the onlytechnique whichisusedincombination.Only MS is not so much efficient for separation
& identification of any molecule that’s why MS is attached to GC, HPLC, LC and TLC.
GC, LC, and HPLC firstly isolate compounds and then we connect them with MS and it is then attached
with digital library and compounds are identified.
It separatesthe componentsthenthe onlyone peakisobtained(make fragmentsof the compounds).
We can’t tell the mechanismof anydrugwithoutknowingits3Dstructure.We want onlyone compound
to come out that is why MS is combined with others.
71. Example:
For example; In case of morphine the possible fragments are,
benzene, cyclohexene etc.
They all got separated and different fragments are formed. The
fragment which has maximum quantity in the parent compound
forms the first peak and it is called base peak (100% peak).
The mass isinverselyproportional tothe distance.Therefore,the whole compound is broken down and
then the quantity of various fragments is checked.
Basic Theory
Mass Spectrometryisthe mostsensitive technique forstructural determination(elucidation). In MS the
molecule is broken down into fragments. These fragments are separated according to their mass to
charge ratio(m/e).Eachfragmentgive itspeak(response) onreachingtodetector.These fragments are
recorded as Mass spectrum.
Mass spectrum is the graphical representation and comparison in between mass to charge ratio vs.
relative intensity. The highest peak is recorded as 100% which is also called as base peak. The other
peaks are recorded with respect to the base peak (100% peak).
The total energy is 70 eV. Some amount of energy (10 eV) is utilized to ionize the compound and the
remainingenergy(70eV) isutilizedtobreakthe bonds.MS breakall bonds of different compounds and
fragments are formed (those compounds which have too much similarities).
If we give too much compounds to MS then it will mix all of them and we can’t identify the original
parent compound.
Highestpeak(base peak) representsthe highestquality.
Everyfragmentshows itsspecificresponse.
Detector provides a peak of every fragment. Detector detects the fragments on mass to charge ratio
(m/e) and their relative intensity use. Retention time is not checked over here as we use mass.
Recorder made comparison and it shows results. It is then attached with digital library. After
fragmentation the fragments are again combined to check the structure of the sample.
72. Instrumentation and Data analysis
There are five majorpartsof Mass
spectrometer,whichare asfallows;
1. Ionsource
2. Mass analyzer
3. VacuumPump
4. Detector
5. Recorder
Schematic diagram of MS:
73. 1. Ion source:
It isalso calledionizationchamberorionicchamberorionizationsource.Ionizationof the sample occurs
in ionization chamber.
Ionization potential: The minimumenergyrequiredtoionizeanatomor moleculesiscalled Ionization
potential. The energy may be supplied by different ways depending upon the physical or chemical
nature of sample.
Methods of ionization
Several methodsare employed forthe ionization of the sample depending upon the physical state and
chemical nature of the sample.
The ionizationmethodswhichare usedare discussedbelow;
i. Electronimpact EI
The firstand the common methodof ionizationof Electronimpactorelectronionization.Inthis method
electron beam is used for the purpose.
The sample is volatilized in a separate chamber and vapors at pressure 10-4
– 10-6
torr are allowed to
enter in the ionization chamber.
In the ionization chamber there is a bombardment of electron beam which can remove or eliminate
electron from sample creating Radical cations. The source of electronic beam is electrically heated
filament of tungsten.
On the opposite side of filament there is an anode plate which acts as “Electron trap.” Electron are drawn
towards trap and travel across the chamber with the help of magnates called as “Collimating magnates.”
The time span forthe productionof ions1 – 5 µs. Ionswhichare producedmove towardsmassanalyzer.
Detector signals are directly proportional to the no. of ions hitting to the detector. By adjusting the
magnates, ions of all the masses are collected and countered.
74. ii. Chemical ionizationCI
In chemical ionizationareagent(Methane,Ethane,Butane orisobutene) isintroducedintohigh
pressure source (0.1 – 1.0 torr) andionizedbyelectronsbombardment.
Primaryionsare produced.
CH4 + e-
CH4
+.
+ 2e
Due to the highpressure there isapossibilityorcollidingaprimaryionwiththe othermoleculesandthe
reactionwill be like;
CH4
+.
+ CH4 CH3
.
+ CH5
+
The introductionof small amountof sample inthe vaporphase inthe ionsource resultsinthe reaction
betweenreagentionsandsample molecules.
There mightbe hydrogenionH+
transferreaction.
CH5
+
+ MH MH2
+
+ CH4 (M + I)+
There mightbe hydride ionH-
transferreaction.
C2H5
+
+ MH M+
+ C2H6 (M- I)+
These reactionsare exothermicIn nature.The energyof exothermicityactsasinternal energywhich
may leadtofragmentationof the sample molecule.
iii. FieldionizationFI
It is also known as Field desorption FD. Many organic compounds are thermally unstable to meet the
requirement of electron ionization or chemical ionization, they can be ionized by Field ionization.
Sample inthe vaporphase can be ionizedwhenthe moleculesof the sample pass near the metal anode
carryingelectricfieldof 1010
V/m.The electronse-
are suckedoreliminatedfromthe sample molecules
intothe metal and the resultingmolecular ions are repelled towards the cathode slit. These molecular
ions are passed towards mass analyzer.
In FD or FI, sample in the vapor phase is directly deposited on metal anode and high electric field
produces ionization, fragmentation and desorption.
Application:
This method is used for the molecules having high molecular weight or naturally occurring complex
compounds. For example Carbohydrates.
75. iv. Fast AtomBombardment FAB
In this method the sample is dissolved in a viscous liquid typically Glycerol (matrix material) and
ionizationisachievedbythe bombardmentof sample matrix bythe beamof fast moving neutral atoms.
The bombarding atoms are mostly inert gases or Nobel gases, like Xenon (Xe) or Argon (Ar).
In order to achieve a very high kinetic energy K.E, the atoms are fast ionized and then these ions are
pass through electric field.
Aftergettingacceleratedthese ionsenterintoachambercontaining neutral atoms of the same gas and
there will be collision of ions and neutral atoms which result in the exchange of charge.
Xe+. (Fast) + Xe (Thermal) Xe (Fast) + Xe+. (Thermal)
2. Mass analyzer:
It isalso calledasionseparator.Followingare the commonlyusedmassanalyzersinMassspectrometer;
i. Magneticsector analyzer
ii. Time of flight
iii. Quadrupole massAnalyzer
i. Magnetic sector analyzer:
It isfurtherdividedintotwotypes;
Single focusinganalyzer
Double focusinganalyzer
Single focusing analyzer:
It is a curved metallic tube placed between two poles of electromagnet or magnet with a specific
magnetic field.
When an ion contains a charge Z is repelled by electrostatic field. Its potential energy is ZV due to
repulsion. Its potential energy is converted into Kinetic energy i.e. 1/2mv2
.
Whenionsentersinto magnetic field, magnetic field exerts centrifugal force on ions which is equal to
mv2
/r.Magnetic fieldalsoexertscentripetal force which is equal to ZHv. At equilibrium the two forces
becomes equal.
The instrumentinwhichionsare formedanalyze andseparatedonlybythe applicationof magneticfield
is called single focusing Mass spectrometer, and the analyzer is called as single focusing analyzer.
76. By comparingthe K.E and P.E;
. = .
1
2
2 =
2 = 2
By comparingthe centripetal andcentrifugalforce;
2 =
=
2 =
2 2 2
2
By comparingequation(i) andequation(ii);
2 2 2
= 2
2
2 2
= 2
2 = 2 2
By invertingthe equation;
2 2=2
∝ 2
It is clear from the above expression that mass to charge ratio (m/z) depends upon the radius of curvature.
Double focusing analyzers:
It isan instrumentinwhichelectrostaticanalyzeris used in addition to magnetic analyzer. Electrostatic
analyzer focuses or directs the ions towards the magnetic analyzer. For better results or resolution
monoenergatic (with same Kinetic energy) can be selected or can be obtained by magnetic analyzer.
77. ii. Time of flight:
The methodof analysisdealswiththe measurementof time requiredforaniontotravel fromthe ion
source to detector.
The sample is volatilized into the space between the first and second electrode and a burst of
electronsisallowedtoproduce ions.Anextractionpotential (E) isappliedforanothershorttime
period which will result in the ions being focused.
Afterfocusing,acceleratingpotential isapplied(V) foramuch shorter periodthanthat usedfor
ionproduction.Sothat all the ionsinthe ionsource are acceleratedsimultaneously.
The ion isthenpass throughthe thirdelectrode intothe driftzone andare eventuallycollected
by the sensingelectrode.
1
= (2 )2
Since all the ions have started from the ion source and gained the same K.E (1/2 mv2
= zeV). During
acceleration they began to separate according to their mass as they drift along the flight tube and
ions of different masses arrive at the detector at different times as per above equation.
iii. Quadrupole Mass analyzer:
Quadrupole massspectrometeriscompactruggedand easyto operate andconsequentlyisapopular
instrumentinuse.
Quadrupole massspectrometerconsistof fourrodswhichmustbe straight and parallel andso
arrangedthat the beamof ionsisdirectedaxiallybetweenthem.
A voltage comprisingaDCand radiofrequencycomponentisappliedbetweenadjacentrods,
opposite rodsbeingelectricallyconnected.
Ionsare acceleratedintothe centerbetweenthe rodswitharelativelysmall potentialranging
from10 to 20 volts.
Quadrupole massanalyzerisamass filter.CombinedDCandRF potentialsonthe quadrupole
rods can be setto pass onlya selectedm/e ratio.
All otherionsdonot have a stable trajectorythroughthe quadrupole massanalyzerandwill
collide withthe quadrupole rods,neverreachingtothe detector.
78. 3. Detector:
Three typesof commonlyuseddetectorsare discussedbelow;
i. Faraday cup detector
ii. Photographicplate detector
iii. Electronmultiplierdetector
i. Faraday cup detector:
It isa metalliccupwhichismaintainedatpotentialwhichallowsthe ionstobe capturedbythe cup.
ii. Photographic plate detector:
It iscupped at right angle to path of ions. So that, there is a linear formation of images and abundance
of ions is determined by the intensity of each image.
iii. Electronmultiplier detector:
It isa seriesof electrodes(dynodes) whichare connectedtoeachother.When ions hit the first dynode,
there isa release of large no.of electrons.These electronshitthe seconddynode andthenitwill release
large no. of electrons.Thisprocessgoesonthroughoutthe series of electrodes, which are normally 10.
As a resultof thissequence there isaproductionof electriccurrentwhichisamplified and presented in
the form of graph. The graph is called as Mass spectrum.
4. Vacuum system:
All massspectrometersoperate atlowpressure,justtoavoidthe ioncollision.Any collision of ions may
resulting ionic reactions; neutralization and scattering.
To minimize the collisionwhole procedure isoperatedatlow pressure orhighvacuum(10-4
– 10-8
torr).
The vacuum systemalsoattracts the ionstowardsthe detector.
5. Recording of Mass spectrum:
The most important method of recording the
mass spectrum is the use of online computer.
The whole system is known as data system.
A printer is also attached to the data system to
take out the print of recorded mass spectrum.
79. Applications of Mass spectrometer
MS isone on the most sensitive techniqueforthe qualitative analysis therefore it has various uses. The
various applications of the Mass spectroscopy are discussed below;
1. Preparationof isotopes:
MS isveryuseful forpreparationsof pure isotopes,highpolymersandnatural products can be analyzed
by this.
2. Cis-trans isomers:
It isalso usedtodistinguishbetweenCis&Trans isomers,since stabilityof ions produced may differ for
Cis & Trans ions significantly.
3. Study of free radicals:
It isuseful tostudyfree radicals,determinationof bondstrength,evaluationof heatof sublimationetc.
4. Study of closely relatedcompounds:
It is useful in the analysis of closely related compounds. For example; hydrocarbons, petroleum,
products, lubricating oils etc.
5. Identificationof Molecular formula:
It providesimportantinformationforidentificationbyhelpof molecularweight, molecular formula and
by fermentation pattern.
6. Determinationof molecular weight:
It is best tool for the determination of molecular weight of the substance (where substance is
bombarded with moving electrons and its mass spectra is recorded, the mass of peak at highest m/e
reveals molecular mass accurately also the molecular formula.
7. Trace analysis:
The inorganic trace analysis MS can used for trace analysis of elements in alloys and minerals and in
super conductors.
81. Potentiometery:
It is one of the volumetric technique of electro analytical chemistry which is used to measure electrochemical
potential of charged particles. An electrode system which is connected to potentiometer used to measure
this potential can detect ions while, other substances also present in the solution.
Measurements are always made when no current or very little current passes through the
potentiometriccircuit.So,the compositionof the solutionmeasuredisnotchangedmakingquantitative
analysis possible. So, potentiometric method involves two types of electrochemical analysis;
1st
method is used for the determination of pH based upon effect that difference of potential
between two suitable electrodes dipping into a solution containing hydronium ions which
depends upon concentration or activity of hydronium ions.
2nd
category ispotentiometrictitrationswhichinvolvesthe measurementsof changes in EMF of
the cell byaddinga titrant(thatis monitoringof the potentialservesonly to locate equivalence
point for a titration).
So the measurementswithelectrochemical cellsusedtocalculate ionsconcentrationinsolutionsby
electrode potential using Nernst equation which was discovered in the late 1800’s.
Types of Electrodes:
Electrodesusedformeasurementsinpotentiometery includes;
1. Reference electrode
2. Indicatorelectrode
Electrode usedforreference electrode are usuallyincludes Hydrogen ion electrode, Saturated calomel
electrode SCE,Mercury sulfate and Silver Chloride electrode.While,the indicatorelectrode is exposed
to the analyte solutionanditspotential variesdependinguponthe concentration of ions present in the
solution.
Each electrode is placed in a separate solution and connected to a single potentiometer, while, a salt
bridge is exposed to each sample completing an electrical circuit.
82. Electrode potential E (volts):
A cell ismade up of two partsor twohalf cells,eachcontaininganelectrode ineachhalf cell.There exist
a difference of potential betweenelectrodesandthe solutionin which it is dipping and this potential is
known as Electrode Potential E.
The electrode potential dependsupon;
The nature of electrodes
Concentrationof solution
Temperature
When temperature is 25o
C and the solution is one molar (which is unit activity for all species) or one
atmospheric partial pressure in case of gases than this potential is known as Standard Electrode
Potential Eo
and it is measured in Volts.
Application:
It isusedfor the determinationof thermodynamiccell potential.Forexample;EMF.
It is usedfor thecalculation ofequilibriumconstantfor redox reaction.
Limitations:
Electrode potential hascertainlimits,whichare asfallows;
Electrode potential willpredictwhetheragivenchemical reactionoccuror not,but they
indicate nothingaboutthe rate of reactionand alsodon’tassure the successof reaction.
Theyare useful topredictthata reactionwill notoccur if the potential differencesare not
sufficient.
Diagram:
84. Measurementof pH
It isbasedon the fact that difference of potential betweentwosuitableelectrodes dipping in a solution
congaing H3O+
or H+
ions. It depends on the concentration of or activity of H3O+
or H+
ions.
The developmentof potential is not a specific property of H3O+
. The solution of any ion will develop a
potential directlyproportional tothe concentrationof thation,if a suitable pairof electrode isplaced in
a solution.
The determinationof potential of cell orhalf-cellunder the conduction that no current flows through it
and potential across two electrodes is measured by potentiometric methods.
The electrode whose potential depends upon relative concentration of ion to be determined is called
indicator electrode.
pH:
Acidityoralkalinityof areactionisthe most importantfactoras it controlledthe;
Rate of reaction
Nature of speciespresent
Eventaste
pH can be definedas,“the negative logarithmof hydrogenorH+
ionconcentration.”
1
= − log[ +] = log[ +]
Similarnotationcanbe appliedtoOH- ionconcentrationas;
1
= − log[ −] = log [ −]
On thermodynamicground,these reactionsare modifiedas;
= − log +
= − log −
a H+
= is the activityor the effectiveconcentrationof H+
ions.
a OH- = is the activityor the effectiveconcentrationof OH-ions.
For dilute solution;
a H+
= [H+
]
a OH
-
= [OH-
]
So,the pH isthe measurementof the activityof the H+
or H3O+
ions.
Activityof the substance means,itseffective molarconcentration.
85. Molar concentration:
It referstomol/literof solution.
= − log[ (a +)]
= − log[ (a 3
+)]
pH scale:
Role of Buffer:
As manyreactionsdependsuponthe concentrationof H3O+
ion.Itis important to control the pH. This is
usuallyachievedbysolutionwhichhave definite pHto retain pH for long time and its pH is not affected
by addition of bases or acids or even with dilute is known as buffer solution.
It shouldbe notedthatweakbasesand theirsaltsacts as buffer.Some standardbuffersare;
Standard Buffers pH at 25o
C
0.1 M NH3 / 0.1M NH4Cl 9.25
0.025 M NaHCO3 10.012
0.025 M Na2CO3 10.012
0.1 M Borax 9.180
86. NERNST Equation:
The equationwasgivenbyWaltherH. Nernst,he wasawardedNobel Prize in1920.
The NERNST equationenablesustodetermine the;
Electromotive force EMFof many processes
Cell potential of asystemcalledGalvaniccell
Energyof a chemical reaction
The energyof a chemical systemdrivesthe charge tomove and drivingforce give rise tocell potential
therefore all these relationshipsare tiedtogetherinthe conceptof NERNSTequation.
(Reactants) A + B C+ D (Products)
= − ln
[ ] [ ]
[ ] [ ]
As we know;ln= 2.303 log10
= −
2.303
log10
[ ] [ ]
[ ] [ ]
By puttingthe valuesof R,T and F;
2.303
=
0.591
And,
[ ] [ ]
[ ] [ ] = = Reaction Quotient
So,
= −
0.591
log10
As, log10 = 1 so,final equationwillbe;
= −
0.591
Where,
E = Electrode potential
Eo
= Standard Electrode potential
R = General gasconstant 8.313 JK-1
mol-1
T = Temperature
n = No.of moleselectronsappearsinhalf cell
F = Faraday’sConstant9.648 X 104
JV-1
mol-1
In = Natural logarithm2.303 log10
87. NERNSTequationfor redox reaction:
A reactioninwhichboththe oxidationandreductionistakingplace iscalledoxidation-reduction
reactionor Redox reaction.
The NERNST equationforthe redox reactionisasfollowing;
General reaction
A ox + ne-
B red
As NERNSTequationis;
= −
0.591
log10
So the equationforredox reactionwill be,
= −
0.591
log10
Example:
In the followingreaction;
Ag+
+ e-
Ag
The NERNST equationwillbe;
= −
0.591 log10
[ ]
[ +]
88. Methods of calculation of pH
1. Calorimetermethod
2. Indicatormethod
3. pH metermethod
4. Glasselectrode pHmethod
pH Meter
It isa device whichisusedforthe determinationof pHof solution.
Principle:
The pH of the solutionmaybe determinedbymeasuringthe potential difference betweenapair
of electrodesimmersedinthe solution.
One of the electrode isanindicatorelectrode(e.g.glasselectrode) andotherone isareference
electrode (e.g.calomel electrode).
89. Reference Electrodes
A reference electrodeisahalf-cell havingaknownelectrode potential thatremainconstantandis
independentof the compositionof the analyte solution.Reference electrodeisalwaystreatedasanode.
Propertiesof an ideal Reference electrodeare asfallows;
It musthave a potential thatisaccuratelyknownandconstant.
It should becompletely intensiveto thecompositionofanalytesolution.
The electrodemustbe ruggedor easy toassemble.
It should maintain a constantpotentialwhilepassing a smallamount ofcurrent.
Commonlyusedreference electrodesare mentionedbelow;
1. Silver-silverelectrode
2. Saturatedcalomel electrode (SCE)
3. Mercury (I) sulfate electrode
4. StandardHydrogenelectrode (SHE)
1. Silver-SilverChloride Electrode:
A reference electrodesystemanaloguesof the calomel electrode consistof AgCl2immiscibleina
solutionof KCl thatis alsosaturatedwithsilvernitrate (AgNO3).
Construction:
The electrode is contained in a Pyrex tube fitterd with 10mm fritted
glass disc or porous drug. A plug of agar gel saturated with KCl is
formed on top of the disc to prevent loss of solution from half-cell.
The plug can be prepared by heating 4g – 6g of pure agar in 100ml of
wateruntil solutioniscompletelyform and then add about 35g of KCl.
A portionof thissuspensionispouredintotube uponcoolingitsolidify
to a gel with low electrical resistance.
A layerof solidKCl isplacedinthe gel and tube is filled with saturated
solution of salt. 1 to 2 drops of 1M silver nitrate is then added and a
heavy gauge 1mm – 2mm diameter. Silver wire is inserted in the
solution. The potential of electrode is governed by activation of
Chloride ion in KCl solution.
Reaction:
AgCl (s) + e-
Ag (s) + Cl-
(aq)
