APPLICATIONS OF

MASS,IR,NMR,UV-VISIBLE
SPECTROSCOPY

Presented by
Dr. A. Suneetha
Dept. of Pharm. Analysis
Hindu College of Pharmacy
 APPLICATIONS OF MASS
SPECTROSCOPY
• Molecular mass determination :
-mass spectroscopy is one of the best
method to determine the molecular mass
accurately
-when a substance bombarded with
moving electrons and the mass spectrum
is recorded, the mass of the peak at
highest m/e is the molecular mass
accurately
• Characterization of polymers :
- initially the polymer is pyrolised (decomposed by
heating) and then the pyrolised products are fed in to the
inlet of mass spectrometer and identified
• Impurity detection :
-mass spectroscopy is one of the best method to detect
the impurities,
-if the molecular weight of the impurities are much larger
than major components their detection is more easy
because their masses peaks are free from contribution
by those of the major components.
on the other hand if the molecular weight of
impurities are much lower then the major components ,
their detection is not easy job because of the formation
of common fragment ion
Distinction between cis and trans isomers :
-mass spectroscopy is successful in making distinction between cis
and trans isomers
-both cis – trans isomers yield similar mass spectra but the
molecular ion peak for the trans isomer is more intense than cis
isomer
Latent heat of vaporization of liquids :
- clausius-clapeyron eqution useful for measure the latent heat of
liquid

Log p = -ΔH/RT + constant

Particular temperature, pressure -peak height constant.

As increasing temperature change in the peak height. Based up on
peak height we can calculate the latent heat
ΔH directly proportional to saturation vapour pressure at
temperature T similarly the value of ‘p’ are determine from the height
of the peaks in the mass spectrum at different temperature
• Reaction kinetics :
Mass spectroscopy is very frequently used in
kinetic and mechanistic reaction studies.
The concentration of many of the unstable
species present in reaction vessel can be
determined . From the peak heights in mass
spectrum, the concentration of different species
can be determined.
The graph drawn between time ‘t’ and peak
height a straight line of slope -2.303/k is
evaluated
Eg: A(m/e120) + B(m/e240) → C(m/e360)
C(m/e360) + D(m/e140) → E(m/e500)
• Determination of ionisation potential
The minimum energy that the bombarding electron
must possess in order to produce molecular ion from the
neutral molecule is refer to as ionisation potential
we can determine ionisation potential by taking plots of
ion current against electron beam eV
AB → AB+ + e-
A → A++ e-
B → B- + e-
(AB is the molecule and A,B are atoms)
Ionisation potential of molecule are atom may be defined
as the energy required to remove an electron from the
molecule or the atom
• Quantitative analysis of mixtures
• Evaluation of heat of sublimation
• Identification of unknown compound
-conjugated olefins show more intense molecular ion peak as
compared to non conjugated olefins with the same number of
unsaturation
-unsaturated compound give more intense peak than compared
to saturated or cyclic molecule
- the molecular ion peak in aromatic compounds is relatively
much intense due presence of p electron system
-absence of molecular ion peak in mass spectrum means that the
compound under examination is highly branched or tertiary alcohols
primary and secondary alcohols gives small molecular ion peaks
• Also useful in geological information about
rocks(determination of 16O,18O ratio in CO2
produced from rock carbonated samples
• Mass spectroscopy Also useful in clinical,
toxological and forensic applications

Clinical : mass spectra coupled with LC this

instrument is useful for quantification of
pharmaceutical compounds in picogram level
-it also helpful in conduct the bioavailability and
bio equivalence studies
-it is also useful for estimation of substance in
biological fluids

Toxological :
Use ful for identification of toxic compounds like
organo phosphorus compounds
Also useful for estimation of narcotic substance
• It is useful for the study the environmental
gases present at planets (it is coupled with
satellites)
• Natural products :
It is useful to identify the compound
mass which is present in herbal extract
• Chemical criminology:
It is useful for identify the mass of at a
time 25 different ball pens
• Isotopic abandance
The isotopic abandace of easily
vapourisable elements can be determined
by using mass spectrometer when this
information coupled with precise isotopic
mass determination , values of atomic
weight of elements may be obtained
Eg: cl35 ,cl37 these two isotops
can add to the sample cl35 gives more
intense peak than cl37. it can give peaks in
1:3 ratio, based up on peak we can
identify the particular compound
 APPLICATIONS OF NMR
• Structural elucidation of organic compounds
Organic compounds invariably have
hydrogen atoms in their structure and the
environment of each proton is not same
CH3CH2CH2OH CH3CH(OH)CH3
(propanol-1) (propanol-2)

4 signals 3 signals
• Based upon the δ values we can elucidate
the structure of compound
• H1 –NMR is useful for identification of
nature of the carbon
• C13NMR is useful for identification of
nature of carbon
• 2D NMR is useful for identification of exact
position of carbon and hydrogen
• DEPT it is useful for identification of no. of
CH3 and CH2
• Structure determination of polyethylene
-It shows two types of lines one of
these is narrow while the other is broad,
narrow component is denote the
amorphous phase where as broad
component to the crystalline phase
• Determination of optical purity
• Study of molecular interactions like micelle
formation and drug macromolecule or drug
receptor interactions
• Detection of electro negative atom or group
It is known that the presence of an
electronegative atom or group in the
neighbourhood of the proton cause
deshielding and signal is shifted to down
field
Fluorine cause more downward shift
as compared to oxygen
Oxygen cause more downward shift
compared to nitrogen
• Distinction between cis-trans isomers
-The cis and trans isomers of compound can be easily
distinguished as the concerned proton have different
values of chemical shifts as well as coupling constants
• Elemental analysis
-NMR spectroscopy can be used for
determination of total concentration of given kind
of magnetic nucleus in a sample
• Rates of reactions
-An interesting special use of PMR spectroscopy is study of certain
reaction that are too fast for measurement by ordinary techniques
• Detection of hydrogen bonding
-By NMR we can differentiate whether the compound having
intermolecular and intramolecular hydrogen bonding
• Anti-gauche confirmation of a compound
• Quantitative analysis
Assay of components:
Single or multi component with out separation of
components, can be quantitatively estimated
Specific peak for each component is identified and
peak area /height ratio given by integral value is found
using standard and sample and the quantity can be
estimated
Iodine value :
which is measure of double or triple bond can be
known form the proportion of olefinic protons
Moisture analysis :
-since water can give characteristic peak, the % can
be known from the peak ratio of water peak and
component peak
APPLICATION OF INFRA-RED
SPECTROSCOPY
• Identification of drug substance
IR spectrum of sample and standard can be compared to identify
a substance, if the spectra same then the identify of the sample can
be confirmed
• Structure determination
This technique helps to establish the structure of a unknown
compound. all major functional groups absorb at their characteristic
wave number
• Identification of functional groups
The presence or absence of absorption bands help in predicting
the presence of certain functional groups in the compounds.

Fuctional group region 4000 -1600 cm-1 (streching vibrations occur)

Finger print region 1550-600 cm -1(bending vibrations occur)
• Identifying the impurities in a drug sample
Impurities having different chemical
nature when compared to the pure drug.
Hence these impurities give rise to
additional peaks than that of the pure
drug. By comparing these ,we can identify
the presence of impurities.
• Conformational analysis
This technique is quite useful in
determining the relative stability of various
conformations of cyclic compounds.
• Study of complex molecule
This technique is also useful to
establish the structure of complex
molecule.
eg: penicillin's
β-lactams do not absorb near 1770 cm-1
where as β-lactams fused to thioazolidine
ring exhibits a band 1770 cm-1. Since
penicillin gives a strong band at 1770cm-1
• Study of keto – enol tautomerism
diketones and keto esters exhibit keto-enol
tautomerism provided they have α-hydrogen atom in
them. The IR spectrum of a compound contain bands
due to C=O, O-H, C=C bonds.
• Quantitative analysis
It helps to make a quantitative estimation of an
organic mixture.
eg: xylene mixture have Ortho, Meta, Para xylenes.
The % composition of the mixture can be determined
by taking the IR spectrum of mixture.
Ortho – 740cm-1
Meta – 880cm-1
Para – 830cm-1
• Study of chemical reaction
This technique is quite useful for
studying chemical reactions, consider the
reduction of a saturated aliphatic ketone to
form secondary alcohol.
Ketone butane-2-ol
-1 -1
(band at 1710cm ) (band at 3300cm )
due to OH streching

During reaction C=O streching will be

missing and only a band due to O-H strech
is present.
• Rotational isomerism :
1,2-dichloro ethane it shows two
types of confirmations
At low temperature
-gauche form(1235cm-1)
At high temperature
-trans form(1291cm-1)
 APPLICATIONS OF UV-
VISIBLE SPECTROSCOPY
• Qualitative analysis
• A) detection of impurities
to detect the presence of impurities we can use UV
spectrophotometric measurements .additional peaks can
be due to impurities in the sample and can be compared
with that of standard raw material. also by absorbance
measurements at specific wavelength
• Eg: presence of phenones(310 nm) in adrenaline(280
nm) can be determined. The extinction of a 1cm layer of
0.2% w/v solution in 0.01N HCl at 310nm is not more
than 0.2 if it is more than 0.2 then the phenones then
phenones as impurities are present
• B) structure elucidation of organic
compounds
UV spectroscopy helps in structure
elucidation of organic molecules
Type of  n  n p pp
transition
Inference Saturated Hetero Hetero Unsaturate
compound atom with atom and d
C-C saturation double compound
C-O-H bond C=C,
C=O,S=O, _
C=S,C=N C=C
• By using following considerations we can
elucidate structure of organic compound
A) effect of conjugation
extended conjugation shifts the  max to longer (batho chromic
shift or red shift).
on the other hand reduction of the compound or saturation of
double bond leads to opposite effect (hypsochromic shift or blue shift)

Substance No. of double max

bonds
Ethylene 1 174nm
Butadiene 2 217nm
Butatriene 3 267nm
Vitamin A1 5 326nm
Vitamin A2 6 351nm
• B) effect of geometric isomerism
Cis and trans isomers,trans isomer
absorb at longer wavelength than cis
isomer.
Hence conversion from cis to trans
isomer results in bathochromic shift
Conversion from trans to cis results in
hypsochromic shift
• C) effect of cross conjugation
Cross conjugation has no effect on  max
• D) effect of alkyl substitution
Alkyl substitution shift the  max to longer 
bathochromic shift

Substance No.of alkyl Max

substituants
Crotanaldehyde 1 217nm
α- ionone 2 228nm
Progesteron 3 241nm
• Number of rings
The addition of rings causes bathochromic
shift

Substance No.of rings  max

Benzene 1 261nm
Napthalene 2 312nm
Anthracene 3 375nm
Napthacene 4 480nm
Pentacene 5 580nm
• Quantitative analysis
To determine the concentration and
amount of drug in sample solution and thus
%purity can be determine
Several quantitative methods are
A) using E1%1cm values
%purity = observed absorbance / E × 100/CONC
B) reference standard method
1)single standard or direct
comparision method
C2=C1 × A2/A1
2) multiple standard method or
caliberation curve
• Determination of molecular weight
• Chemical kinetic
kinetic of a reaction can also be
studied using UV spectroscopy. The UV
radiation is passed through reaction cell
And absorbance changes can be followed
• Keto-enol tautomerism
Keto-enol form of a substance have
different UV absorption pattern using this
principle the% of keto and enol form in
mixture can be calculated
• Examination of poly nuclear hydrocarbons
• Elucidation of the structure of vitamin A and
vitamin K
• Assay of medicinal substance

Drug Solvent Wavelength E1%1cm

values
Allopurinol 0.1N HCl 250 563
tablets

Riboflavin Acetate buffer 444 323

 REFERENCE
 INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS.
--B. K. Sharma

 INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS.

- - Chatwal

 ELEMENTARY ORGANIC SPECTROSCOPY

--Y. R. Sharma.

 www.wikipedia.com ,

 TEXT BOOK OF PHARMACEUTICAL ANALYSIS

-RAVI SHANKAR