Methods used in structural elucidation 1. Physical Methods 2. Analytical Methods 3. Chromatographic Methods 4. Spectroscopic Methods

1. Mr. S. P. Shinde
Assistant Professor
Pune- Maharashtra.

2. Mr. S. P. Shinde
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A. Physical Methods of Characterization
o Physical methods are applied more frequently to the active constituents
of the natural products such as volatile oil, fixed oil, alkaloids,
glycosides.
o The physical methods are determined after separation of these
constituents.
o A variation in the proportion of constituents, or the presence of
adulterants, changes the value of physical constants considerably.

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Melting Point
o It is one of the parameter to judge the purity of crude drug.
o In case of pure chemical or phytochemicals , melting point are very
sharp and constant.
o Since the crude drug from animals and plants origin contain the mixed
chemicals .
o Purity of crude drug can be determined by their melting points.
Ingredients M.P.
COLOPHONY 75-85°C
WOOL FAT 34-44°C
COCOA BUTTER 30-33°C
BEES WAX 62-65°C

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o Determination of refractive index is a significant parameter for the
evaluation of the essential oils and fixed oils.
o The refractive index changes, if the genuine oil is adulterated with
other oils.
o It is a ratio between the velocity of light in air and its velocity in the
oil, or the substance under study.
o The ratio can also be expressed as sine of the angle of incidence
divided by the sine of the angle of refraction.
o It is generally studied at 20°C using Abbe's Refractometer.
Refractive Index

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o Many volatile oils and other substances of natural origin show an
ability to rotate the plane of the polarised light to right or to left side
and likewise they are called dextrorotatory or laevorotatory,
respectively.
o This detection of optical rotation and its magnitude is an important
criteria for evaluation of certain drugs.
o It is studied by using polarimeter, at 20°C.
o The specific rotation is given by:
where α is observed rotation, L is length of observed layer in drug, d is
density and p is gm of substance in 100g of solution.
Optical Rotation
p
Ld
100
solution
of
ml
per
substance
of
grams
solution
of
dm
per
rotation
angular 
=

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Drug Optical Rotation Refractive Index
Cinnamon oil 00 to - 20 1.573 to 1.595
Terpeneless lemon oil - 40 to +10 1.428 to 1.485
Lemon oil +570 to +650 1.474 to 1.476
Caraway oil +700 to +800 1.4838 to 1.4858
Clove oil 00 to - 1.50 1.5300 to 1.5310
Castor oil +3.50 1.4758 to 1.4798
Refractive index and optical rotation of essential oils and fixed oils

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B. Analytical Methods of Characterization
o Characterization and analytical techniques are methods used to identify,
isolate or quantify chemicals or materials, or to characterize their
physical properties.
o They include microscopy, light or radiation scattering, spectroscopy,
calorimetry, chromatography, gravimetric and other measurements used
in chemistry and materials science.

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o Combustion analysis is a method used in both organic chemistry and
analytical chemistry to determine the elemental composition (more
precisely empirical formula) of a pure organic compound.
o Combusting the sample under conditions where the resulting
combustion products can be quantitatively analyzed.
o Once the number of moles of each combustion product has been
determined the empirical formula or a partial empirical formula of the
original compound can be calculated.
Elemental composition determination by
combustion analysis

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o Applications for combustion analysis involve only the elements of
carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) as combustion
of materials containing them convert these elements to their oxidized
form (CO2, H2O, NO or NO2, and SO2) under high temperature high
oxygen conditions.
o Notable interests for these elements involve measuring total nitrogen
in food or feed to determine protein percentage, measuring sulfur in
petroleum products, or measuring total organic carbon (TOC) in water.
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o A combustion train is an analytical tool for the determination of
elemental composition of a chemical compound.
o With knowledge of elemental composition a chemical formula can be
derived.
o The combustion train allows the determination of carbon and
hydrogen in a succession of steps-
• combustion of the sample at high temperatures with Copper(II)
oxide as the oxidizing agent,
• collection of the resulting gas in a hygroscopic agent (magnesium
perchlorate or calcium chloride) to trap generated water,
• collection of the remainder gas in a strong base (for instant
potassium hydroxide) to trap generated carbon dioxide.
Combustion Process

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o Analytical determination of the amounts of water and carbon dioxide
produced from a known amount of sample gives the empirical formula.
o For every hydrogen atom in the compound 1/2 equivalent of water is
produced, and for every carbon atom in the compound 1 equivalent
of carbon dioxide is produced.
o Nowadays, modern instruments are sufficiently automated to be able to
do these analyses routinely. Samples required are also extremely small
0.5 mg of sample can be sufficient to give satisfactory CHN analysis.
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C. Chromatographic Methods of
Characterization
Chromatography is a physical method of separation in which the components
to be separated are distributed between two phases one of which is stationary
phase while the other mobile phase moves through it in a definite direction.

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o Thin layer chromatography (TLC) Is a method for identifying substances
and testing the purity of compounds.
o TLC is a useful technique because it is relatively quick and requires small
quantities of material.
o Separations in TLC involve distributing a mixture of two or more
substances between a stationary phase and a mobile phase.
o The stationary phase: is a thin layer of adsorbent (usually silica gel or
alumina) coated on a plate.
o The mobile phase: is a developing liquid which travels up the stationary
phase, carrying the samples with it.
o Components of the samples will separate on the stationary phase
according to how much they adsorb on the stationary phase versus how
much they dissolve in the mobile phase.
Thin Layer Chromatography (TLC)

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o Visualizing Agents for TLC
• Alkaloids: Dragendorff’s reagent
• Cardiac glycosides: Antimony trichloride
• Sugar: Aniline phthalate
• Amino acids: Ninhydrin
o Interpreting the Data
• The Rf (retention factor) value for each spot should be calculated.
• It is characteristic for any given compound on the same stationary phase
using the same mobile phase for development of the plates.
• Hence, known Rf values can be compared to those of unknown
substances to aid in their identifications.

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o HPTLC technique is widely employed in pharmaceutical industry in
process development, identification and detection of adulterants in
herbal product and helps in identification of pesticide content,
mycotoxins and in quality control of herbs and health Food.
o Several samples can be run simultaneously by use of a smaller quantity
of mobile phase than in HPLC.
o It has also been reported that mobile phases of pH 8 and above can be
used for HPTLC.
o Another advantage of HPTLC is the repeated detection (scanning) of
the chromatogram with the same or different conditions.
o Consequently, HPTLC has been investigated for simultaneous assay of
several components in a multi-component formulation.
High Performance Thin Layer Chromatography (HPTLC)

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o With this technique, authentication of various species of plant possible,
as well as the evaluation of stability and consistency of their
preparations from different manufactures.
o Various workers have developed HPTLC method for phytoconstituents
in crude drugs or herbal formulations such as bergenin, catechine and
gallic acid in Bergenia cilliata and Bergenia lingulata
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D. Spectroscopic Methods of Characterization
o The instrumental methods which provide spectral data are
complementary to each other.
o There is not a single spectrometric method which can provide
complete information regarding the structural features of drug
molecules.
o The spectral data of one model compound, camphor, has been
provided here with the comments for its interpretation.
o Interpretation of molecular spectra is generally based on empirical
correlation of spectral data with reasonable assurance to a particular
group or arrangement of atoms in the molecule.

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o The absorption of light energy by organic compound in the UV (200-
400 u) range and visible (400-800 u) range involves promotion of
electrons from the ground state to higher energy states.
o The compounds with chromophore, auxochrome and conjugated system
are dissolved in suitable solvent.
o The UV spectrum obtained shows absorption bands which gives
valuable information regarding the nature of compound.
o UV spectrum of camphor in chloroform exhibits a of 290 nm, which
indicates its ability to absorb light energy in UV region.
Ultraviolet-visible Spectrum (UV)

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Infrared Spectrum (IR)
o The constantly vibrating molecules stretch and bend their bonds with
respect to one another, by absorbing infrared light.
o IR spectrum is highly characteristic to establish the identity of
compounds.
o The identity of two samples that have identical spectra in the finger print
region give conclusive identification of compounds. In IR spectrum -OH
group absorbs at 3200 to 3600 cm-1 aliphatic and aromatic C-H stretching
at 3100-2800 cm-1, carbonyl group at 1700-1800 cm-1.
o Many more stretching and bending vibrations are found at 1600-700-1 cm.
o Camphor shows C-H stretching at 2959-1 cm and C=0 strong stretching at
1744-1cm with other characteristic bands at 1418, 1021 and 751 cm-1,
which indicates presence of a saturated ketone.

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Nuclear Magnetic Resonance (NMR) Spectrum
o Electron, the nuclei of certain atoms like 1H and 13C act like a tiny bar
of magnet, spin and generate a magnetic moment along the axis of spin.
o If such protons are placed in an external magnetic field, its magnetic
movement can be aligned either with or against the external field.
o Alignments with the field are more stable and energy must be absorbed
to ‘flip’ the proton over to the less stable alignments against the field.
o At some value of the field strength, the energy required to flip the
proton matches the energy of the radiation.
o Absorption occurs, and the signals are observed in the form of NMR
spectrum.
o Since the 1H and 13C nuclei are involved informing the spectrum, it is
sometimes called as 1H NMR and 13C NMR spectrum, respectively.

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Mass Spectrum (NMR)
o In mass spectrometry, molecules are bombarded with electrons.
o The molecules are ionised and broken up into fragments.
o Each kind of ion has a mass to charge (m/e) value.
o For most ions, charge is 1, so m/e becomes the mass of the ion.
o The mass spectral data of camphor indicates the molecular ion peak and
other major and minor fragments.
o The data helps to establish the structure of camphor, by giving exact
molecular weight, molecular formula and an idea about its structural
type.

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