Apllications of UV-Visible spectroscopy Difference and derivative spectroscopy

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2. • CHOICE OF SOLVENTS AND SOLVENT EFFECTS
• APPLICATIONS
• DIFFERENCE SPECTROSCOPY
• DERIVATIVE SPECTROSCOPY
PRESENTED BY
DIVYA V
FIRST M PHARM
PHARMACEUTICAL CHEMISTRY
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4. REQUIREMENTS
• It does not interact with solute
• Does not show absorption at wavelength to be
determined
• Good solvent-for substance
• For inorganic compounds-Water
• For organic compounds-Organic solvent
DISADVANTAGE OF POLAR SOLVENT-
It destroys fine structure of spectra-so non polar
must be used
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5. • Solvent-Transparent in required wavelength
• E.g. 95% Ethanol-Transparent down 210nm
• Cyclohexane-210nm
• 1,4-dioxane-220nm
Solvent λmax(nm) Solvent Λmax(nm)
Methanol 210 Cyclohexane 212
Hexane 199 Chloroform 247
Heptane 200 Acetone 331
Ether 205 Acetic acid 270
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7. • Change in polarity-Position and intensity of peak
shifted for a particular chromophore
• ∏→∏*-Excited state(∏*)is more polar & is more
stabilized by polar solvent.Thus in going from
nonpolar →polar solvent-red shift occurs(increase
in λmax , decrease in ∆E).A small energy is needed
for such kind of transitions.
• n→∏*-n state is more stabilized by polar
solvent.Thus in going from nonpolar →polar
solvent-blue shift occurs(decrease in λmax ,
increase in ∆E)
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8. • n→σ*-This is very sensitive to H-bonding. Here
ground state is more polar than excited state.
Alcohols,amines form H-bonding with solvent
Molecules.Therefore such transition requires high
energy and blue shift occurs.
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9. ∏*
∏*
n
n
∏
∏
NON POLAR POLAR
Fig; Absorption shift with change in polarity of solvent
 When a group is more polar in ground state
increase in polarity causes blue shift
 When a group more polar in excited state
increase in polarity causes red shift
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11. 1.QUANTITATIVE ANALYSIS
 Concentration of a drug or absorbing species in a given
sample can be easily analyzed by measuring the
absorbance of the solution prepared in transparent
solution
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12. a) SINGLE COMPONENTS
 Only one component in the sample absorbs
significantly.
Methods used are :
1 Standard absorptivity value method
2 Single standard or direct comparison method
3 Multiple standard or Calibration curve method
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13. 1 Standard absorptivity value method
The concentration of unknown compound can be
determined by using the measured absorbance and
standard absorptivity value.
where ;
A- absorbance of the solution
a- standard absorptivity value
b-path length of sample cell
c-concentration of unknown
A = abc
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14. 2 Single standard method
 Absorbance of standard solution of known
concentration is compared with sample solution.
Where as,
c1 &c2 = Concentration of standard & sample
A1 & A2 = Absorbance of standard & sample
c2=c1*(A2/A1)
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15. 3 Calibration curve method
A calibration curve is plotted using
concentration vs. absorbance value of 5 or more
standard solutions.
A
C
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16. b) MULTIPLE COMPONENTS
 By this the actual amount of sample under
investigation can be easily calculated after
removing the irrelevant absorption.
METHODS
1. Using absorbance corrected for interference
2. After solvent extraction of the sample
3. Absorption ratio method
4. Simultaneous equation method
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17. 1) Absorbance corrected for interference
Corrected absorbance will be the difference
between the total absorbance and the absorbance
of the interfering substance
 E.g. : Determination of concentration of ephedrine
hydrochloride and chlorocresol in ephedrine
hydrochloride injection
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18. 2) After solvent extraction of the sample
 Separate the absorbing interferants from the
analyte by solvent extraction procedures.
 Appropriate for acidic and basic drugs.
 The concentration of the analyte can be obtained
by simple measurement of the absorbance of the
extract containing the analyte.
 E.g. : Assay of caffeine in aspirin and caffeine
tablets.
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19. 3) Absorption ratio method
 For a substance that obeys Beer`s law at all
wavelength,the ratio of absorbance at any two
wavelength is a constant which is independent of
concentration
 This ratio is Q value
E.g.;Assay of trimethoprim & sulphamethoxazole in
co-trimoxazole tablets
Q=A1/A2
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20. 4) Simultaneous equation method
 Based on the fact that total absorbance of solution
is Sum of absorbance of individual components
Cx & Cy=Concentration of drug X & Y in g/L
respectively
A1 & A2=Absorbance at λ1 & λ2
ax1 & ay1=Absorptivity of drug X & Y at λ1
ax2 & ay2=Absorptivity of drug X & Y at λ2
Cx =A2*ay1-A1*ay2/ax2*ay1-(ax2*ay2)
Cy =A1*ax2-A2*ax1/ax2*ay1-(ax2*ay2)
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21. 2. QUALITATIVE ANALYSIS
 Identification is done by comparing absorption
spectrum with spectra of standard compounds.
 Includes detection of impurities, structural
elucidation of organic compounds, detection of
conjugation, detection of geometrical isomers etc.
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22. a)Detection of functional groups
 Presence and absence of chromophore can be
easily detected.
 If the spectrum is transparent above 200 nm it
shows the absence of :
 Conjugation/ unsaturation
 Carbonyl groups like aldehydes and ketones
 Benzene or aromatic groups
 Bromo or iodo atoms
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23. 2.Structural elucidation
• Absorption spectrum of unknown compound is
compared with known compound, so that most
probable structure of compound may be obtained.
• Presence or absence of unsaturation,hetero atom
etc can be detected.
• E.g. Structural elucidation of vitamin A1 and A2
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24. λmax =325 nm
λmax =351 nm
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25. 3. Detection Of Extent of Conjugation
 Extension of conjugation in a unsaturated
compound shifts the λmax to longer wavelength-
Bathochromic shift /red shift
 Reduction of double bond shifts the λmax to
shorter wavelength – Hypsochromic shift / Blue
shift
E.g. Nitrobenzene – 252 nm
o- nitro toluene- 250 nm
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26. 4.Distinction of conjugated and non –
conjugated compounds
 It distinguishes between a conjugated and a non-
conjugated compound. The following isomers can
be readily distinguished since one is conjugated
and other is not.First having longer wavelength
than second
CH3
C
CH3
CH C CH3
O
C CH2 C CH3
O
CH3
CH2
(i)
(ii)
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27. The forbidden n→∏* band for the carbonyl group in
the compound
(a) will appear at longer wavelength compared to
that for the compound
(b)Alkyl substitution in an alkene causes
bathochromic shift
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28. 5.Detection of impurities
 If impurities are present then the absorption
spectra will differ from the characteristic spectra of
pure substances as the impurities will also absorb
light .
 Additional peaks will be obtained.
 E.g.: The drug adrenaline shows λmax at 280nm
and the presence of adrenalone in adrenaline can
be detected from its absorption maximum at
310nm.
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29. 6.Detection of polynuclear hydrocarbons
 Made comparison with unknown with known
spectra of polynuclear hydrocarbons
 Presence of substituents in the ring shifts the
λmax to longer wavelength
 Increase in number of benzene ring-shift λmax in
to longer wavelength
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30. Compound structure Compound name Absorption maxima
NAPHTHALENE 312 nm
ANTHRACENE 375 nm
NAPTHACENE 480 nm
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31. 7.Preference over two tautomeric forms
 If a molecule exists in two tautomeric forms
preference over the other can be detected by UV
spectroscopy
 2- hydroxy pyridine which exists in equilibrium
with its tautomeric form 2-pyridone
 The spectra of these two compounds were found
to favour 2-pyridone and clearly the equilibrium is
shifted towards the right ie,2- pyridone
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32. 2- hydroxy pyridine 2-pyridone
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33. 8.Identification of configuration
geometrical isomers
• cis-alkenes absorb at different wavelength as
compared to their corresponding trans-isomers
• When steric hindrance forced over isomers cis
suffers distortion to lower wavelength
H H
H H
H
H
Cis- Stilbene Trans- Stilbene
λmax =283 nm λmax =295 nm
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34. 9. Identification of a compound in
different solvents
 Sometimes, the structure of the compound
changes with the change in the solvent.
 Chloral hydrate shows an absorption maximum at
290nm in hexane while the absorption disappears
in the aqueous solution.
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35. 3. DETERMINATION OF MOLECULAR WEIGHT OF
AMINES
A known weight of amines is taken and converted
into amine picrate and the absorbance of the
solution is found measured.Using the
equation,molecular weight can be find out.
where A – absorbance of solution
c – concentration
t- path length
For most of the amine drugs, molecular extinction
co-efficient of amine picrate at 380 nm is 13,400.
Molecular weight =13,400*ct/A
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36. 4.DETERMINATIONOF DISSOCIATION COSTANT of
indicators
RH R- + H+ RH=Indicator
 Concentration vs. absorbance at different pH is
plotted and pKa can be calculated from the
equation.
pKa = pH – (log [R-]/[RH])
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37. 5. CHEMICAL KINETICS
 Change in concentration of either reactant or
product with time is measured.
 Since absorbance is proportional to concentration,
UV can be used to follow the course of a reaction
Fig;RAPID STOP-FLOW APPARATUS FOR
KINETIC STUDIES
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38. 6. SPECTROPHoTOMETRIC TITRATIONS
 It is the titration in which the absorption of the reactant
,product is followed as a function of titrant
 ADVANTAGE; Sharp end point,no interference from other
absorbing species
 CONDITIONS;Either product/substance/titrant
absorbs,Beer `s law must be obeyed
 E.g. Titrant only absorbs (substance-Ar(III))
A
endpoint
volume of bromate bromide 38

39. DIFFERENCE SPECTROSCOPY
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40. • The selectivity and accuracy of spectrophotometric
analysis of sample containing absorbing
interference may be markedly improved by the
technique of difference spectroscopy
• Sensitive method for detecting small changes in
environment of chromophore
• In this spectra of 2 samples of slightly different
composition or physical state can be compared
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41. FEATURE OF DIFFERENCE
SPECTROSCOPY
 Measured value is the difference in absorbance
(∆A) between two equimolar solution of analyte
in different chemical forms which exhibit spectral
characteristics
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42. CRITERIA FOR APPLYING DIFFERENCE
SPECTROPHOTOMETRY
• Reproducible changes may be induced in the
spectrum of the analyte by the addition of one or
more reagents.
• commonly employed technique for altering the
spectral properties is adjustment of pH by means of
aqueous solution of acid,alkali,or buffers.
• The uv-visible spectra of many substance containing
ionisable functional groups are depend on the state
of ionisation of the functional group and pH of the
solution.
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44. • Another simplest method for an increasing
selectivity is derivatisation of spectra.
• This operation allows to remove spectral
interferences and as a consequence leads to
increase selectivity of assay.
• Ability to detect and measure minor spectral
features enhanced.So similar spectra can be
distinguished
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45. • Involves conversion of a normal spectrum to it’s
first, second or higher derivative spectrum.
• Normal absorption spectrum - D0 spectrum
• The first derivative D1 spectrum - plot of the rate of
change of absorbance with wavelength against
wavelength (dA/dʎ VS λ)
• The second derivative spectrum - plot of the
curvature of the D0 spectrum against wavelength or
a plot of d2A/dʎ2 ʎ
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46. Types of derivative spectra
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47. INSTRUMENTATION
• Derivative spectra-generated by following
techniques,
# Modification of the optical system
• Spectrophotometers with dual
monochromators,photodetectors used
• Generates a signal with an amplitude proportional
to the slope of the spectrum over the wavelength
interval
• Disadvantage: Expensive ,Restricted to the
recording of first derivative spectra only.
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48. dual wavelength instrument with two monochromator
operated at the same speed but with a lag of few nm from
each other. A chopper will sequentially pass the beams from
both monochromators and thus their difference divided by
the constant nm lag value is recorded versus the average
wavelength.
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49. #Based upon microcomputers differentiation
• Micro computers incorporated in to or interfaced
with spectrophotometer
To provide
 Derivative spectra during or after scan
 To measure derivative amplitudes between
specified wavelengths
 To calculate concentrations and associated
statistics from the measured amplitudes
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50. APPLICATIONS
 Multicomponent analysis
used in pharmaceutical analysis for assaying of a
main ingredient in presence of others components
or its degradation product.
 Accurate determination of λmax
 Better qualitative analysis of & identification of
absorbing species in a sample
 Calculation of some physico-chemical constants,
e.g. reaction, complexation or binding constants
 Disadvantage: Poor reproducibility
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51. REFERENCES
 Hobart.H.Willard,Lynne.L.Merrit.Instrume-ntal
method of analysis.PBS Publications.2009;6:66-82
 H.Kaur.Instrumental methods of chemical
analysis.Pragati Prakashan.2013;9:279-363
 Gurdeep.R.Chatwal,Sham.k.Anand.Instrumental
methods of chemical analysis.Himalaya Publishing
home.2013;5:2.107-2.184
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