Analytical Techniques of chemistry BE.pdf

Chemistry

2. Lecture 1 Chromatography
•Chromatographyisananalyticaltechniquethatiswidelyusedfortheseparation,isolationand
identificationofcloselyrelatedchemicalcomponentsfortheseparationorganiccompoundslike
aminoacids,sugars,vitamins,hormones,plantpigmentsetc.
•Chromatographyisatechniqueofseparationandpurificationofcomponentsofamixtureby
theirdifferingaffinitiesfortwophases(states)ofmatterwithwhichtheycomeintocontact.
•ARussianbotanistMikhailTswettin1986inventedchromatographicmethodtoseparatevarious
plantpigmentssuchaschlorophyllsandXanthophyll'sandseveralothersubstancesbypassing
solutionofthesecompounds(vegetableextracts)throughaglasscolumnpackedwithfinely
dividedcalciumcarbonate.Hiscolumndevelopedbandsofcolor,andhenamedthisseparation
techniquechromatography,whichinGreekmeans"writtenincolor".
•Chromatographyistheprocessofseparatingthecomponentsofmixtures(solutes)thatare
distributedbetweenastationaryphaseandaflowingmobilephaseaccordingtotherateatwhich
theyaretransportedthroughthestationaryphase.
•AccordingtoA.I.M.Keulemans,"Chromatographyisaphysicalmethodofseparation,inwhichthe
componentstobeseparatedaredistributedbetweentwophases,oneofthesephase
constitutingastationarybedoflargesurfacearea,theotherbeingafluidthatpercolatesthrough
alongthestationarybed."

3. Principle of Chromatography
•Chromatographyisbasedontheabilityofacolumnoffinelypowderedsolidmaterial(suchas
Al
2O
3),thestationaryphase,toadsorbsubstancesfromasolution(themobilephase)thatis
allowedtotricklethroughit.
•Ingeneral,theattractiveforceofthesolidsurfacediffersfordifferentspeciesinsolution.The
substancethatsolidabsorbsmoststronglymovesthroughthestationaryphasemuchmore
slowlythandothosethatarenotsostronglyadsorbed.Thismeansthatalthoughthevarious
componentspresentinthemobilephasestartouttogether,whentheyfirstcomeincontactwith
thestationaryphase,theysoonbecomeseparated.
•Chromatographyisbasedontheprincipleofselectivedistributionofthedifferentcomponentsof
amixturebetweentwophases,namelystationaryphaseandmobile(ormoving)phase.The
stationaryphasecanbeasolidorliquid;whilethemobilephaseisaliquidorgas.Whenthe
stationaryphaseissolid,theselectivedistributionisbasedonadsorption,whilewhenitisa
liquid,thebasisofselectivedistributionispartition.
•Thestationaryphasemaybeeitherasolidorliquid;andthemovingphasemaybeeitheraliquid
oragas.
•Inallthechromatographictechniques,thesolutestobeseparatedmigratealongacolumn,andof
course,thebasisoftheseparationliesindifferentratesofmigrationforthedifferentsolutes.

4. Steps of Chromatographic methods
•1) Adsorption or retention of substances on the stationary
phase.
•2) Separation of the adsorbed substances by the mobile
phase.
•3) Recovery of the separated substances by a continuous flow
of mobile phase, the method is called elution.
•4) Qualitative and quantitative analysis of the eluted
substances.
No other separation process can match chromatography for
simplicity, efficiency, and range of applications. Therefore,
chromatography is a major analytical tool for chemists,
biologists, and workers in the related field.

5. Classification of Chromatographic Techniques
A) Mobile Phase arrangement
1) Liquid Chromatography (LC) -The mobile phase is liquid
2) Gas Chromatography (GC) -The mobile phase is gas

6. B) Stationary Phase arrangement
1) Column Chromatography –The stationary phase is placed in a column.
2) Planar technique
a) Paper Chromatography (PC) –Stationary Phase is a special paper
b) Thin Layer Chromatography (TLC) –Stationary Phase is spread on solid flat support (like glass
plate or aluminum foil)
Classification of Chromatographic Techniques

7. Classification of Chromatographic Techniques
C) Interaction of analytewith the stationary phase
1) Adsorption Chromatography: It is a technique in which small differences in the adsorption
behavior of substances between a moving solvent (liquid or gas) and a stationary solid phase are
utilized to achieve the separation.
When the mobile phase is a liquid, it is called liquid-solid chromatography
When the mobile phase is a gas, it is called gas-solid chromatography
2) Partition Chromatography: It is a technique in which mixtures of substances are separated using a
partition between a moving solvent and a stationary liquid held on a suitable solid support.
When a solvent (mobile phase) is a liquid, it is called liquid-liquid chromatography.
When a solvent (mobile phase) is gas, it is called vapor or gas-liquid chromatography.
3) Ion Exchange Chromatography
4) Size Exclusion Chromatography
5) Affinity Chromatography

8. Paper Chromatography
•Paper chromatography is a simple and widely used chromatographic technique. It was developed
in 1865 by Schonbein. Later on, it was further developed by Martin and Synge in 1944.
•In 1952, A. Martin and R. Synge won the Nobel Prize for Chemistry in 1952 for the development of
chromatographic techniques.
•This is a powerful method of liquid-liquid chromatography, used widely in separating free amino
acids (produced as a result of hydrolysis of protein material), sugars, peptides, and sugar
derivatives.

9. Principle of Paper Chromatography
•In paper chromatography, both the stationary phase and mobile phase are liquid. Specially designed
filter paper, composed primarily of cellulose materials, serves as the stationary phase, while an
appropriate immiscible organic solvent is the mobile phase.
•An ideal filter paper for chromatography is composed of 99% alpha-cellulose, 0.3% beta-cellulose,0.5%
pentasans, and 0.07% ash.
•The mixture of different components can pass through the paper using appropriate solvent systems.
The organic solvent moves in the filter paper by capillary action and adsorption on the filter paper.
•The components of the mixture exhibit differential adsorption due to their different partition
coefficients (the ratio of the concentration of a substance in one medium to the concentration of
another substance in which these concentrations are at equilibrium). The ratio of the distance traveled
by the solute to the distance traveled by the solvent is expressed in terms of the retardation factor (R
f).
The R
fvalue can be regarded as a function of the partition coefficient. Its value is always less than one.
•The retardation factor (R
f) is calculated as follows: R
f=
•The R
fvalue can be the basis of the separation of components of mixtures, as this value for a particular
substance is always the same under the given set of conditions.
Distancetraveledbysolute
Distancetraveledbythesolvent

10. A thin pencil line is drawn near one edge of a rectangular piece of filter paper and parallel to it.
The solid mixture, present in solution, is now applied as a small drop from a fine glass syringe along
the pencil line, and near one vertical edge of the paper.
If the problem is to identify the amino acids in a mixture, then small drops of separate solutions of
pure amino acids (thought to be present in the mixture) are also placed at intervals along the pencil
line.
Experiment:an example of separation of amino acids

11. The filter paper is now hung vertically in a glass tank, which contains the developing solvent and
the bottom edge of the filter paper is so positioned in the solvent that the pencil line is just clear
of it.
The tank is sealed with a lid to prevent the evaporation of solvent.
As the solvent travels up (due to capillary action) the paper, it moves the components in the
mixture, and the pure substances, at different rates.
Experiment continue

12. The paper is removed from the tank, and allowed to dry, once the solvent has almost reached
the top of the paper.
Since amino acids are colorless, their positions on the paper are located by spraying the whole
paper with a solution of ninhydrinin propanone.
After warming the paper in an oven set at a temperature of about 373 K the individual amino
acids show up as blue-lilac spots.
Experiment continue

13. •Under carefully controlled conditions, it is possible to characterize a particular compound separated from
a mixture by its so-called R
fvalue.
•This is defined to be
•R
fvalue = The distance moved by the pure substance /The distance moved by the solvent front.
•Its value for a particular compound depends upon the solvent used and the temperature. From its
definition above, it is clear that all R
fvalues are less than unity.
A
B
Experiment continue

14. In some cases, the use of one solvent may fail to separate two or more of the constituents present in
the mixture, ie, their R
f, values under these conditions are almost identical. The answer to this
problem is simple. The paper is turned through 90°and the whole process is repeated with an end
solvent. The logic being that it is most unlikely that two or more components will have identical R
f
values in a different solvent. This technique is called two-way chromatography.
Rotate +90°
Solvent BSolvent A
Experiment continue

15. Steps involved in the paper chromatography
a.Preparationofsolutionoforganiccompoundsoranalyte:
Propersolventisselectedforthepreparationofsolution.Commonly,concentratedsolutionpreparedforpaperchromatographytoavoiddiffusionprocess.
Itispreferabletoremovetheimpuritiestonullifytheeffectofinterferingentities.
b.Applicationofthesampleonthepaper
Usually,itispreferabletousearectangularfilterpaperwithdimension(15cmx30cmordifferentasperrequirement).Apencillineisdrawnabove2.5cm
formoneend.Howeversmalldimensionoffilterpaperpiececanalsobeprepared.Adropofabout1-2µLofsolutionofsampleisspottedfromacapillary
tipforthestudyofpaperchromatography.Onthesamplelines,otherstandardreferencescanbespottedforcomparativestudy.
c.Developmentofthechromatogram
Appropriatesolventischosenforthedevelopmentofchromatogram.Commonly,plesolventsareusedforthispurpose.Ifrequired,amixtureofsolvent
canbeusedforpreparationofachromatogram.Commonlyusedsolventsarepropanol,n-butanolandfuran.The
Solventisallowedtomoveupwardbyascendingthroughthefilterpaperinthesolventtank.
d.Dryingofthechromatogram
Afterpassingthesolventuptocertaindistanceandtheseparationofcomponentsisover,thechromatogramisremovedfromthesolventanddriedby
blowinghotair.
e.Locatingthecomponentsofthemixtureontheofchromatogram:
Theseparatedcomponentsarelocatedbyphysical(eg,fluorescence)orchemicalsprayingwithreagents)methods.
f.Elutionofthespots:
Theidentifiedareaofeachspotsarecutandextractedwithasuitablesolvent.

16. Applications of paper chromatography
•Identify and separate different components from an analyte.
•Check the pharmaceutical compounds.
•Check food adulteration.
•Analysis of cosmetics.
•Forensic studies (DNA and RNA sequences).

17. Thin Layer Chromatography
•propoundedbyLamaiterandSchwelt.
•developedbyStahlin1956.
•Inthischromatography,thefilterpaperisreplacedbyathinlayerofaninertadsorbent(like
Sephadex,cellulose,alumina,orsilicagel)spreadoverasquareplateofglassorplastic.
•Themethodisusedtoidentifythecomponentsinatinysampleofasolidmixture.
•Itsadvantageoverpaperchromatographyarisesfromthefactthatavarietyofthinfilms(eg,
alumina,Sephadex,silicagel)canbeuseddependinguponthenatureofthesubstancesused.
•Thethinfilmismorecompactthanfilterpaper,andthesamplespotsaresmallerandmore
concentrated.
•Theprocessisquiterapid.Thisrapidmethodisusefulinisolatingandidentifyingaminoacids,
nucleotides,triglycerides,otherlipids,sterols,sugars,andsugarderivatives.

18. Experimental details for carrying out thin-layer
chromatography
A slurry of silica gel, for
example, is made in
trichloromethanecontained in
an ordinary screw-cap bottle.
A previously cleaned
microscope slide is immersed in
the slurry and then carefully
withdrawn using a pair of
tweezers.
The coated glass plate
is now allowed to dry
in an upright position
and is then ready for
use.

19. At a distance of about 2.0-2.5 cm from the bottom of the
plate, a horizontal line is drawn.
Then, with the help of a syringe (or
micropipette), spots of the sample
solution and standards are applied.
Experimental details continue

20. The chromatogram is developed by the ascending technique in which the plate is immersed in the
developing solvent to a depth of 0.5 cm.
Development is allowed to proceed, until the solvent front has traveled the required distance (usually
10-15 cm).
The plate is removed from the chamber and the solvent front reached is marked with a pointer object.
The plate is then allowed to dry in a fume cupboard (or in air. oven).
Experimental details Continue

21. The positions of the separated solutes can be located by various methods.
Colored sub-stances can be seen directly when viewed against the stationary phase; whereas
colorless species usually detected by spraying the plate with an appropriate reagent that
produces colored areas in the regions which they occupy.
Some compounds may be located without spraying if they fluoresce under ultraviolet light.
R
f, values of different
components and pure substance
are measured
Experimental details Continue

22. •Thin layer chromatography is performed in glass slides or sheet of glass of aluminum
coated with thin layer of adsorbent guard intents could be silica gel of aluminum oxide.
•The layer of adsorbent acts as stationary phase and liquid serves as mobile phase.
•Different analytesascent onto the TLC plate with different rates and the separation of
either component is possible in this type of chromatography.
•In TLC, components of mixture partitioned between adsorbent (eg, silica gel on glass or
alumina support) and solvent (mobile phase).
•Moisture or water affects the development of adsorption chromatography. Therefore,
should be removed by putting the chromatoplatesin an oven at about 100-105 °C for
about half an hour. This step is termed as activation of chromatoplates.
Principle of TLC

23. Various steps involved in the separation of
compounds by TLC
•Preparation of sample: The analytesample is dissolved in appropriate solvent.
•Preparation of TLC plate: A rectangular TLC plate of appropriate dimension (e.g., 15 cm x 20 cm
or as per requirement) is prepared and a baseline is drawn above some distance (say 2.5 cm) from
one end. The sample under test is spotted on a plate followed by dried with hot air.
•Development of chromatogram: The chromatoplateis placed in the TLC chamber vertically such
that the spots of the plates remain just above the solvent surface. The atmosphere of the tank is
saturated with the vapor of the solvent. The lid of the tank is tightly closed. When the solvent
rises sufficiently 10-12 cm) abonentsof the mixture are taken out from the tank and dried.
•Locating the components of the moisture on the mixture of chromatogram: The separated
components are located by physical method or chemical method.
•Elution of the spots: The identified areas of each spot are cut and the components are extracted
with a suitable solvent.

24. Advantage of TLC
•It is simple and quick method relative to other methods of chromatography.
•Varieties of organic and inorganic compounds can be separated by this technique.
•Even a corrosive substance can be analyzed by this method.
•The chromatogram can be heated without damaging it.
•Thin layers of adsorbents are better adsorbed than paper.

25. Appropriateness of TLC
•When a substance to be separated or purified is nonvolatile and polar.
•If sample used for analysis is likely to damage the column of liquid chromatography or gas
chromatography.
•If substance in the material being analyzed cannot be detected by other methods of
chromatography like liquid chromatography (LC) or gas chromatography (GC).

26. Applications of TLC
•It is used to check the purity of substance.
•It is used to identify the compounds.
•It is used to purify the compounds.
•It is used to separate the components from the mixture.
•It is used to monitor the progress of reaction in research.

27. References
•Compiled by : NA [email protected]
•References:
•P.C. Jain, M. Jain, Engineering Chemistry, (16
th
Edition), 2013.
•BhishmaRaj Pandey, An Easy Approach to ANALYTICAL CHEMISTRY, (1
st
Edition), 2015.
•Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash Chandra Lohani,Engineering
Chemistry, (1
st
Edition), 2080.
•2080/11/10

28. Lecture 2 Mass Spectrometry
•Mass spectroscopy is an analytical tool that measures mass to charge
ratio of ions in the gas phase. It provides qualitative as well as
quantitative information. In this method, the material being analyzed
is vaporized and vapor is bombarded with a high-energy electron
beam whereby many molecular ions and fragments are formed.
M+ e M
+
+ 2 e
•Fragmentations are guided by their relative stabilities. The stability of
a carbocation is in the order
CH
3
+
<1°carbocation < 2 °carbocation < 3 °carbocation

29. Principle of Mass Spectroscopy
•In a mass spectrometer, organic molecules are vaporized and bombarded with a
beam of very high-energy electrons.
•The resulting collisions impart considerable energy to the molecule, emitting
electrons to produce positively charged ions.
•These ions possess so much energy that they often fragment through various
bond cleavages to produce new positively charged ions.
•ABC electron beam ABC
+
(Molecular ion) + AB
+
+ C
+
+ A
+
+ BC
+
•The positive ions are accelerated toward a negatively charged plate by passing
them through a magnetic field, which deflects the ions.
•The ions of lighter mass are deflected more than heavier ions.
•Each kind of ion has a particular mass to charge (m/e) ratio.
•For most ions produced in the fragmentation of the molecule, the charge is +1 so,
that m/e usually represents the mass of the ion.

30. •BasePeak:Thesetofionsproducedfromamoleculecanbeanalyzedsince
eachhasitsownm/eratio,andproducesasignalwhoseintensityisdueto
therelativeabundanceofthation.Thelargestpeakfoundinamass
spectrum(thatofthehighestintensity)iscalledthebasepeakandisgiven
thenumericalvalueof100.Theintensitiesofallotherpeaksareexpressed
relativetotheheightofthebasepeak.Amassspectrumishighly
characteristicofaparticularcompound.
•MolecularIon:Theionformedbyremovingoneelectronfromtheparent
moleculeiscalledamolecularionorparention.Themolecularionpeakis
usuallyrepresentedasM
+
.Itmayormaynotbethepeakofthehighest
intensity.Themolecularionisthemostimportantsinceitsmassisthe
MolecularWeightoftheparentmolecule.
Principle Continue

31. Schematic diagram of a Mass spectrometer.
Instrumentation of mass spectrometry

32. Instrumentation of mass spectrometry
a. Ion Source: It produces gaseous ions from the given sample.
b. Analyzer: It is used to analyze and separate the ions into their
characteristic mass according to their mass-to-charge ratio.
c. Detector: Detectors in mass spectrometry detect the ions and
maintain their relative abundance.
Apart from these, a sample introduction system is required to add the
sample to the ion source.

33. Source Analyzer DetectorInput
Output
Gas phase ion Ion sorting Ion detection
Block diagram of a Mass spectrometer.
Instrumentation of mass spectrometry

34. Mass Spectrum : Examples
A mass spectrum is a graphical representation of the distribution of ions as a function of their mass-to-charge
ratio (m/z). It is generated through a technique called mass spectrometry.

35. m/z 16 (Molecular ion, CH
4
+
​): Usually the most intense peak,
with a relative abundance set at 100% for normalization
purposes.
m/z 15 (Methyl cation, CH
3
+
​): Typically, less intense than the
molecular ion peak but still significant.
m/z 14 (Methylene cation, CH
2
+
​): Usually present but with
lower intensity compared to CH
3
+
​.
m/z 13 (Methyl cation radical, CH
+
): Typically, a minor peak
compared to CH
3
+
​ and CH
2
+​
m/z 12 (Carbon cation, C
+
): Usually present but with low
intensity.
m/z 17 (Methyl cation with hydrogen radical, CH
3
+
+ H
2​):
Typically, less intense than the main fragment ions.
Mass Spectrum of methane

36. Mass Spectrum : Examples

37. CH
3
+
(Methyl cation): m/z 15
C
2H
4
+
(Ethylene cation): m/z 28
CH
3CH
2
+
(Ethyl cation): m/z 29
CH
2OH
+
(Methoxycation): m/z 31 Base Peak
CH
3OH
+
: M
+
(Molecular ion peak, Methanol): m/z 32

38. Mass Spectrum : Examples

39. Applications of mass spectroscopy
a. Mass spectrum is useful to establish structure of a new compound.
b. It helps to find molecular formulas or at least a narrow list of possible compounds.
c. Mass spectrum is used for the analysis of gas, test of impurities in semiconductors.
d. Mass spectrum is used for the analysis of gas, test of impurities in understanding
reaction mechanisms.
e. Mass spectroscopy is the most reliable tool to determine accurate molecular mass.
f. Mass spectrum is highly characteristic of a particular compound. Therefore, the mass
spectrum is used to prove the identity of two compounds.
g. It can be used as a fingerprint in forensic science.

40. Drawback of mass spectrometry
a. One major drawback of mass spectrometry is that samples under
investigation are destroyed.
b. Installation cost of the mass spectrometer is high.(A high vacuum is
maintained (10
-5
-10
-8
torr) and a computer system is needed to store
the data well and to compare the obtained spectrum with the
reference one)

41. References
•Compiled by : NA [email protected]
•References:
•ArunBahl, B.S. Bahl, The Essentials of Physical Chemistry.
•Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash Chandra Lohani, Engineering
Chemistry, (1
st
Edition), 2080.

42. UV-Visible Spectroscopy
TheUV-visiblespectroscopyisanimportantanalyticaltoolforanalyzing
organicandinorganicchemicalspecies.Themethodisbasedonthe
absorptionoflightbytheanalyte.Therefore,UV-visiblespectroscopyisan
exampleofabsorptionspectroscopy.
Spectrophotometryisaspecialtechniqueofabsorptionphotometrywhere
theuseofaspectrophotometerismadeforabsorptionmeasurement.
Ultraviolet-visible(UV-vis)spectroscopyinvolvestheabsorptionof
ultraviolet/visiblelightbyachemicalsubstancecausingthepromotionofan
electronfromagroundelectronicstatetoanexcitedelectronicstate.
Inthisspectroscopy,thewavelengthregionof200-400nmintheUV
spectrumand400-750nminthevisiblespectrumareusedtogetsome
qualitativeandquantitativeinformationaboutgivenchemicalspecies.

43. Principle
ThefundamentalprincipleofUV-visiblespectroscopyistheinteractionofradiantenergywithmatter.
ThebasicprincipleofthisspectroscopydependsontheabsorptionofUV-visradiationthatcauses
electronswithinmoleculestobepromotedfromthegroundorlowerenergyleveltoahigher
electronicenergylevel.
UV-visibleradiationissufficientlyenergetictocausethepromotionoflooselyheldelectrons,suchas
nonbondingelectronsorelectronsinvolvedinaπ-bondoforganicmoleculestohigherenergylevels.
WhenanorganicmoleculeabsorbsUV-visradiation,itmeansthatthecompoundcontainsacarbonyl
grouporconjugateddoublebonds.Forexample,carbonylcompounds,conjugateddienes,and
aromaticcompoundscanabsorbUV-visradiation.
RadiationofUVandVisiblewavesissufficientlyenergetictocausethepromotionoflooselyheld
electrons,suchasnonbondingelectronsorelectronsinvolvedinaπ-bondtohigherenergylevels.
Forabsorptioninthisparticularregionoftheultravioletspectrum,themoleculemustcontain
conjugateddoublebonds.Iftheconjugationisextensive,themoleculewillabsorbinthevisibleregion.
Thewavelengthofmaximumabsorbanceisreferredtoasλmax.

44. A diagram showing the bonding, non-bonding, and antibonding orbital Molecules

45. The basic principle of the UV-visible spectrophotometer is based on the Lambert-Beer Law.
The effect of the thickness and concentration of the sample on the absorption is measured by
Lambert-Beer Law.
According to this law, the absorbance of light by a solution is directly proportional to its molar
concentration and path length.
According to this law, Absorbance (A)= Ɛbc.
Where,
A=absorbance,
Ɛ=molar absorption coefficient
b =path length (the path length is usually 1 cm)
c =molar concentration
Lambert-Beer Law: Basic Principle of UV-
visible spectrophotometer

46. Instrumentation
Lamp
The UV-Visible spectrophotometer can be equipped with a tungsten filament lamp (350-2500 nm) Deuterium
lamp (200-400 nm) or a xenon Arc lamp (200-1000 nm) which cover the whole range for UV-visible reasons.
Mirror
The radiation emanating from tungsten lamps and deuterium lamps are reflected to the mirror whereby these
get incident onto the monochroismator.
Monochromator
It is composed of prisms and slits. It selects the wavelength of radiation reflected from the mirror. The beam
selected by the slit is a monochromator.
Exit slit
It is the opening through which the monochromatic radiation passes into the beam splitter.
Beam splitter
It splits the beam emanating from the monochromatorand causes it to pass into the reference sample and
trust sample in a double-beam UV-Visible spectrophotometer. One of the two divided beams is passed through
the reference sample and another is passed through the test sample.

47. Sample
The test sample as well as the reference sample are put into the cuvette and the absorbance of the test sample
is recorded by computer after processing. Quartz cuvettes are referred to over a plastic of cuvette for sample
analysis as glass cuvettes can absorb radiation.
Detector
Two photocells act as detectors in UV-vis spectroscopy. One photocell receives the radiation from The
reference solution and another photocell receives the radiation from the test solution. The intensity of
radiation obtained from the reference solution is stronger than that from the test sample. The difference in
intensity of radiation generates the alternating current.
Amplifier
The current generated from the detector is too small and is amplified by the amplifier which can generate
signals.
Recorder
All the generated data is recorded and stored in the computer. In UV-visible spectroscopy, molecules undergo
electronic transition involving n and n electrons. The o electrons are present in saturated compounds, t
electrons are present in unsaturated compounds and n electrons are present in non-bonded electrons.

48. UV-Visible spectrophotometer

49. Instrumentation of UV-Visible spectrophotometer

50. Source Monochromator
Beam
Splitter
Sample
Reference
Detection Unit
Recorder
Block Diagram of UV-Visible spectrophotometer

51. UV-Vis Spectrum
AUV-Visspectrumgraphshowstheabsorptionoflightbyasampleatvariouswavelengthsintheultraviolet-
visibleregion,providinginsightsintotheelectronictransitionsoccurringwithinmoleculesoratoms.
Chromophore
Mostoftheabsorptionoforganiccompoundsresultsfromthepresenceofπ-bonds.Suchabondcontaining
amoleculeiscalledchromophorewhichmakesacompoundwithabsorptionbetween185and800nm.
Auxochrome
Conjugation of the double bond with additional double bonds increases both the intensity and the
wavelength of the absorption band. A group that extends the conjugation of a chromophore by sharing
nonbonding electrons is called auxochrome.
Anauxochromeisagroupofatomswithoneormorelonepairsofelectronsattachedtoachromophore
whichcanalterboththewavelengthandintensityofabsorption.Someexamplesofauxochromeare,-OH,-
OR,-NH2,-NHR,-SH
Auxochromescanaffectthemolecularstructureofacompoundbyinfluencingtheelectrondensityofthe
chromophore.
Forexample,inaniline,theaminogroupcanincreasetheelectrondensityofthebenzeneringwhichleadsto
theelectronshiftingofabsorptionspectratowardslongerwavelengthandincreasetheintensityofcolor.The
presenceofsubstituentsinamoleculecausestheUVabsorbanceband.

52. If the absorbance band shifts to a longer
wavelength (lower energy) region, it is termed a
bathochromic shift (redshift).
If the absorbance band shifts to a shorter
wavelength (higher energy) region, it is termed a
hypsochromicshift (blue shift).
If the intensity of the absorbance band gets
increased, it is termed a hyperchromicshift.
If the intensity of the absorbance band gets
decreased, it is termed a hypochromic effect.

54. Qualitative Application of uv-visible
spectroscopy
•The qualitative applicability of the absorption method is based on the
fact that the wavelength of absorbed light depends on the properties
of absorbing atoms, ions, or molecules.
•For a given species, an absorption spectrum (a single band) is
observed for a qualitative identification of compounds, λ max value is
calculated.
•λ max is the wavelength (in nm) in which maximum absorbance
occurs by the chemical species. For a chemical species, the λ max
value is always unique.

55. Qualitative analysis from UV Visible spectroscopy
What is this?

56. Quantitative Application of uv-visible
spectroscopy
•The quantitative applicability of the absorption of the number of
photons is directly proportional to the number of concentrations of
atoms, ions, and molecules.
•For Quantitative analysis, a calibration curve is obtained for the
known concentration vs Absorbance.
•From the Graph based on Beer-Lambert law, the concentration of the
unknown sample is calculated.

57. Quantitative analysis from UV Visible spectroscopy
How much is
this?

58. Strength of UV-Vis Spectroscopy
•Instruments are easy to handle, requiring little user training before
use.
•The technique allows the sample to be reused or proceed to further
processing.
•Measurements can be made easily and quickly.
•Data analysis generally requires minimal processing even completed
with little work braining.

59. Weakness of UV-Vis Spectroscopy
•Light may come from the environment and may cause errors.
•Light scattering can be termed out by suspended solids in liquid
samples, which may cause undesired measurement errors.
•Misaligned positioning of any one of the instrument's components,
especially the cuvette holding the sample, may yield inaccurate
results.

60. Applications of UV-vis spectroscopy
Qualitative analysis
Quantitative analysis
Structural analysis
UV-Visible spectroscopy is applied for structural analysis of compounds. It is possible
to measure the presence or absence of unsaturation in an organic compound.
Chromophore group analysis
It is applicable to ensure the presence of chromophore groups in molecules.
Photometric analysis
It is applicable in photometry

61. Detection of impurities
The presence of impurities can be detected by UV-Visible spectrometers. The
presence of impurities gives some additional peaks in UV-Visible spectra.
DNA and RNA analysis
UV-Visible spectrometer is used in the analysis of DNA and RNA. It is applicable in
protein analysis
Study of kinetics of photochemical reaction
The Kinetics of photochemical reactions can be studied using UV-Visible
spectroscopy.
Pharmaceutical analysis
This technique is widely used to ensure the purity of pharmaceutical raw materials.
Beverage Analysis
Visible spectroscopy can give a wide range of information regarding food and
beverage
.

62. Bacterial and cell culture
Bacteria and cell culture studies can be carried out by the UV-Vis method
Pollution control
absorbance can be useful in the study of the presence of contaminants like
dyes and heavy metal ions like Cr(VI) response to the UV-Vis light and their
absorbance can be ng UV-Vis spectroscopy. However, some metal ions like
iron need color complexing for its absorbance measurement.
Study the properties of nanoparticles of novel metals and quantum dots
Some nanoparticles have their characteristic absorbance band in the UV-Vis
region. For example, silver nanoparticles exhibit surface plasmonresonance
(SPR) phenomena at around 450 Particle size, and UV-Vis absorbance bands
are related in the case of many nanoparticles, and quantum dots.

63. •Compiled by: NA [email protected]
•References:
•Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash
Chandra Lohani, Engineering Chemistry, (1
st
Edition), 2080.
•P.C. Jain, M. Jain, Engineering Chemistry, (16
th
Edition), 2013.
•BhishmaRaj Pandey, An Easy Approach to ANALYTICAL CHEMISTRY,
(1
st
Edition), 2015.
•2080/11/27