Overview of means & methods used in structural elucidation of natural products. Presentation Topic Given by Faculty, It is compulsory because for this we are having our internal marks.

1. Overview of means &
methods used in
structural elucidation of
natural products

2. Index
a) Physical methods of characterization : M.P./B.P. ,
optical rotation, Refractive index.
b) Analytical methods of characterization : Elemental
composition, determination by combustion
analysis.
c) Chromatographic methods of characterization :
Constants derived from TLC/HPLC
d) Spectroscopic methods of characterization : UV, IR,
Proton NMR spectrum & Mass Spectrometry.

3. A.) Physical methods of determination :
 Melting Point :
The temperature at which solid changes state
from solid to liquid at atm pressure.
 At the melting point a solid & liquid phase exists in equilibrium.
 Pure substances usually melt sharply wherever in case of impure
substance usually softening occurs first.
 Dimorphous comp. &diff. crystalline forms of compound have diff.
melting point.
 X-ray analytical study reveled that the packing affects melting point.
 Melting points are largely influenced by various forces such as ion- ion
forces, dipole-dipole forces, Vander waals forces, molecular shape.
 There are some sub. Such as Theobroma oil, bisacodyl suppositories,
white beeswax, & wool alcohol for which the official process of M.P.
determination is modifies.

4.  Boiling Point :
• The B.P. of a liquid is that temp. at which the vapour
pressure is equal to that of the external pressure.
• Thus the increased pressure the boiling point also increases.
• The B.P. depends on the Vander waals forces if any
structural changes have been observed then this may
consequently lead to change the boiling point because of
change in Vander Waal force.
• The boiling point of a liquid varies depending upon
surrounding environment pressure.
• A liquid in a partial vacuum has a lower B.P. than when that
liquid is at atm pressure.
• Eg. Water boil at 100˚c (212˚F) at sea level but at 93.4˚c
(200.1˚c) at 1905 meters altitude.

5.  Optical Rotation :
 Structural requirements for the optical activity:
• Substances which refract or absorb right and left circularly
polarized light to different extents are “optically active” or show
optical rotatory power.
• Such substances have molecules that lack a plane or center of
symmetry and hence, they are said to be asymmetric.
• This absence of symmetry is the necessary criterion for optical
activity as per Pasteur.
• A carbon atom regular tetrahedron is said to be asymmetric.
• Consider the simplest molecule with its atoms C, A, B, D, E.
• This may be represented as (a) and (b) which are non-super
impossible mirror images of one another.

6. • The above figure shows two different molecules, each of which is
optically active and rotates the light to the same extent but in
opposite direction.
Such isomers are called optical isomers or optical antipodes or
enantiomers and the phenomenon is called as optical isomerism.
Enantiomers of the same substance (mirror images) have identical
physical and chemical properties except that they differ in the
sign of their optical rotations,
e.g. (α-amino propionic acid)

7. • Configuration :
The representation which shows the spatial arrangement of the groups of
atoms constituting a stereoisomer is known as its configuration.
Configuration of the optical isomers of lactic acid:
If the enantiomers contain an H atom attached to the central carbon atom
and the projection formula shows the horizontal group other than H on the
right hand of the C atom, it is said to have (+) D configuration. The other
isomer in which the group other than H points to the left hand is said to
have (-) L configuration.
Consider projection formula of Lactic Acid:

8. • Racemic Modification :
A mixture of
equimolar solutions of equal quantities of two optical isomers is
called racemic modification. Such a modification is optically
inactive since it is composed of equal number of molecules of
both dextrose (+) and molecules.
Diastereoisomers:
This term is applied to all molecules which
may be optically active and may exist in several asymmetric spatial
arrangements that are not enantiomeric.
In contrast to enantiomers, diastereomers have different physical
and chemical properties.

9.  Refractive Index :
When a beam of light passes from one medium to another medium of different density
,a change in the direction of beam of light is observed, due to difference in the velocity
of light beam in the two media. The change of direction of light in oblique passage
between media of different densities is refraction.
The angle between the incident ray and the normal ray to the dividing surface is called
the angle of incidence ( I ) whereas the angle between the refracted ray and the normal
to the dividing surface is called the angle of refraction.

10. • According to Snell’s law :
Sin I = constant = n
Sin r
The constant n is called the refractive angle of the medium.
The refractive index of a substance is also defined as the
ratio of velocities of light in vacuum to that in medium.
The refraction of light will occur when, the refractive indices
of the two media are different and the angle of incidence is
not zero.
The refractive index or organic liquid ranges from 1.2 to 1.8,
while those for organic solids from 1.3 to 2.5.

11. • Measurements of refractive index:
• Two types of instruments are used for measuring refractive index. They are:
1) Refractometry: This is based upon the measurement of positioning the
critical ray or by the displacement of an image. The instruments from this
category are very convenient to use for rapid measurement.
2) Interferometers: These instruments utilize the interference phenomenon to
get differential refractive indices. When the beam of light passes from a
rarer medium to a denser medium , the angle ‘r’ will be smaller than the
angle ‘I’.

12. B.) Analytical methods of characterization
Elemental Composition :
Elemental analysis is a
process where a sample of some material (eg. Soil waste,
Drinking water, bodily fluids, minerals, chemical
compounds) is analyzed for its elemental & sometime
isotopic composition.
Elemental analysis is the
classical method to obtain information about
the elemental composition of an unknown substance. A
known amount of unknown substance is converted to
simple, known compounds containing only the element to
be quantified.

13. • Determination of
elemental composition
1. Find the motor mass of all the elements in the compound in
grams per mole.
2. Find the molecular mass of the entire compounds.
3. Divide the components molar mass by the entire molecular
mass.
4. Multiply it by 100% to get percent composition.
Elemental analysis can be Qualitative
(determining what elements are present), & it can be
Quantitative (determining how much of each are present).
Elemental analysis falls within the ambit
of analytical chemistry, the set of instruments involved in
deciphering the chemical nature of our world.

14. • Quantitative Analysis :
 Determination of the mass of each element or compound present.
 Other quantitative methods include gravimetry optical atomic
spectroscopy, & neutron activation analysis.
 Gravimetry is where the Sample is dissolved & then the element of
interest is precipitated & its mass measured.
 Optical atomic spectroscopy include, flame atomic absorptiob, graphite
furnace, atomic absorption & inductively coupled plasma atomic
emission spectroscopy.
• Qualitative Analysis :
 Elements exist in a sample, The method are mass spectrometric
atomic spectroscopy such as inclusively coupled plasma mass
spectrometry.
 X-Ray Fluorescence, particle-induced x-ray emission, x-ray
photoelectron spectroscopy & Auger electron spectroscopy.

15. • Determination by combustion Analysis :
1. Combustion analysis is an elemental analysis technique used on solid &
liquid organic compound.
2. It is most common method to determine the elemental composition of
unknown hydrocarbon.
3. Combustion analysis involves burning of a small amount of unknown
comp. of an oxygen atmosphere.
4. It can determine the relative amount of carbon, hydrogen, oxygen in
comp. & occasionally can also identify the amount of nitrogen & sulfur
in comp.
Ex. Assume an unknown hydrocarbon is analyzed by combustion analysis.
Reaction Equation :
CxHy + O2  xCO2 + yH2O

16.  Organic Compounds are often
analyzed by combustion :
• All the carbon are converted to from the hydrocarbon to carbon dioxide.
• Measurement of CO2 provides a quantitative value for the amount of carbon in the substance.
• All the hydrogen are converted from the hydrocarbon to water.
• Measurement of the water provides a quantitative value for the amount of hydrogen in the
substance.
• The substance is heated to a high temp. with O2 to insure
complete combustion.
• The resulting CO2 & H2O are absorbed using appropriate
chemicals & The amount of CO2 & H2o are determined by
measuring the Increase in mass of the absorbance.

17. C.) Chromatographic methods of
characterization
 Constants derived from TLC :
The major application of chromatography
is separations compounds from the mixtures. Chromatography is a
separation tool which serves as a means of resolution of mixtures and for
the isolation the components. In chromatography, components to be
separated are distributed between stationary and mobile phase. Mass
transfer between mobile and stationary phase occur by adsorption
mechanism.
• Chromatographic Behavior
• Chromatographic behavior can be explained using following parameters :
1. Retention behavior :
It reflects the distribution of a solute between
the mobile and stationary phase. Retention volume (V) and Retention time
() are the two terms which describe the chromatographic behavior.

18. 1) Retention volume (VR) :
The volume of mobile phase necessary to convey a solute
band from the point of injection, through the column and to the detector is defined
as retention volume.
2) Retention time (tg) :
Distance travelled by the solute from the point of injection on
the chromatogram is retention time.
VR = tgFc
2) Partition Coefficient :
When a solute enters a chromatographic system, it
immediately distributes between stationary and mobile phases. If a mobile phase
flow is stopped at any time, the solute assumes an equilibrium distribution between
the two phases. The concentration in each phase is given by the thermodynamic
partition coefficient.
K = CS
CM

19. 3) Retention (Capacity) Factor (k) :
The retention (or capacity) factor (k) is a
means of measuring the retention of an analyte on the chromatographic column.
Higher value of k specifies that the sample is highly retained and has spent a
substantial amount of time interacting with the stationary phase. The retention
factor is equal to the ratio of retention time of the analyte on the column to the
retention time of a non retained compound.

20. 4) Relative retention (α) :
The relative retention (a) of two
solute, where solute 1 elutes before solute 2 is given by
following formula.
Relative retention depends on nature of stationary phase,
mobile phase and column operating temperature.
𝛼 =
𝑘2
′
𝑘1
′ =
𝑘2
𝑘1
=
𝑉𝑅2
′
𝑉𝑅1
′ =
𝑡 𝑅2
′
𝑡 𝑅1
′

21.  Other Important Parameters in
Chromatography :
1. Resolution :
The degree of separation or resolution of two adjacent bands
is defined as the distance between band peaks (or centers) divided by the
average band width. The resolution is the most important thing in HPLC. A
resolution value of 1.5 or greater between two peaks will ensure that the
sample components are well separated to a degree at which the area or
height of each peak may be accurately measured.
The resolution is affected by selectivity (separation
factor), Efficiency and retention (capacity factor).

22. 2. Plate height & Plate numbers :
 An important characteristics of a chromatographic system is its efficiency
expressed as a dimensionless quantity called the effective plate number, Net It
reflects the number of times the solute partition between the two phases during
its passage through the column.
 Plate number is an indication only of how well a column has been packed. Each
plate is the distance over which the sample components achieve one equilibration
between the stationary and mobile phase in the column. Therefore, the more
theoretical plates available within a column, the more equilibration are possible,
and the better quality the separation.

23. 3.) Plate height (H) :
 Plate height is the distance, a solute moves while undergoing one
partition.
H = L
N
 Plate height is a good way to express column efficiency in units of
length without specifying the length of column. H is small for an
efficient column. Plate height is often called Height Equivalent to
a Theoretical Plate (HETP).
 Each plate is the distance over which the sample components
achieve one equilibration between the stationary and mobile
phase in the column. Therefore, the more theoretical plates
available within a column, the more equilibrations are possible,
and the better quality the separation.

24. D) Spectroscopic methods of
characterization :
 Ultraviolet Visible Spectroscopy :
• It basically involves the spectroscopy of photons &
spectrophotometry.
• It uses light in visible & adjacent near ultraviolet (UV) & near
infrared (NIR) ranges.
• UV/Vis spectroscopy is routinely used in the quantitative
determination of solutions of transition metal ions & highly
conjugated organic compounds.
• For the quantitative measurements, Beer-Lambert law is
followed.
• The Beer-Lambert Law is useful for characterizing many
compounds but does not hold as a universal relationship for the
concentration & absorption of all substances. A 2nd order
polynomial relationship between absorption & concentration is
sometimes encountered for very large, complex molecules such as
organic dyes.

27.  IR Spectrophotometry :
Energy of molecules = Electronic energy + Vibrational
energy +
Rotational energy.
• IR Spectroscopyis concerned with the study of
absorbtion of infrared radiation, which causes
vibrartional transition in the molecule.
• Hence, IR spectroscopy also known as Vibrational
spectroscopy.
• IR spectra mainly used in structure elucidation to
determine the functional groups.

28. • Principale of IR Spectroscopy :
Infrared Spectroscopy is the analysis of infrared light interacting with a molecule.
This can be analyzed in three ways by measuring absorption, emission and
reflection. The main use of this technique is in organic and inorganic chemistry. It is
used by chemists to determine functional groups in molecules.

29.  Molecules are made up of atoms linked by chemical bonds.
The movement of atoms & the chemical bonds like spring &
balls (vibration).
 This characteristic vibration are called Natural frequency of
vibration.
 When energy in the form of infrared radiation is applied
then it causes the vibration between the atoms of the
molecules & when,
 Applied infrared frequency = Natural frequency of vibration
 Then, Absorption of IR radiation takes place & a peak is
observed.
 Different functional group absorb characteristic frequencies
of IR radiation. Hence gives the characteristic peak value.
 Therefore, IR spectrum of a chemical substance is a finger
print of a molecule for its identification.

30. IR region : 0.8 μm (800nm) to 1000 μm (1mm)
1. Near IR : 0.8-2 μm
2. Middle IR : 2-15 μm
3. Far IR : 15-1000 μm
Most of the analytical applications are confirmed to the middle
IR region because absorption of organic molecules are high in
this region.
Sub divided
into

31.  NMR Spectroscopy :
Introduction : -
Nuclear Magnetic Resonance (NMR) is a
spectroscopy technique which is based on the absorption of
electromagnetic radiation in the radio frequency region 4 to
900 MHz by nuclei of the atoms.
Proton Nuclear magnetic resonance
spectroscopy is one of the most powerful tools for elucidating
the number of hydrogen or protons in the compound.
It is used to study a wide variety of nuclei :
• H N
• F F
• C P

32.  If an external magnetic field is applied, an energy transfer (ΔE) is possible
between ground state to excited state.
 When the spin returns to its ground state level, the absorbed radiofrequency
energy is emitted at the same frequency level.
 The emitted radiofrequency signal that give the NMR spectrum of the
concerned nucleus.

34. • Principle of NMR :
 The theory behind NMR comes from the spin of a nucleus & it generates a
magnetic field. Without an external applied magnetic field, the nuclear spins are
random in directions.
 But when an external magnetic field (Ba), is present the nuclei align themselves
either with or against the field of the external magnet.

35.  Mass Spectroscopy :
 Mass spectroscopy is one of the primary spectroscopic methods for
molecular analysis available to organic chemist.
 It is a microanalytical technique requiring only a few nanomoles
of the sample to obtain characteristic information pertaining to
the structure & molecular weight of analyte.
 It is not concerned with non-destructive interaction between
molecules & electromagnetic radiation.
 Mass spectrometry (MS) is an analytical technique that measures
the mass-to-charge ratio of ions. The results are typically presented
as a mass spectrum, a plot of intensity as a function of the mass-to-
charge ratio.
 A mass spectrometer produces charged particles (ions) from the
chemical substances that are to be analyzed. The mass
spectrometer then uses electric and magnetic fields to measure
the mass ("weight") of the charged particles.

37. Basic Principle :
 Mass spectroscopy is the most accurate method for
determining the molecular mass of the compound & its
elemental composition.
 In this technique, molecules are bombarded with a beam of
energetic electrons.
 The molecules are ionized & broken up into many fragments,
some of which are positive ions.
 Each kind of ion has a particular ratio of mass to charge, i.e.
m/c ratio (value). For most ions, the charge is one & thus,
m/c ratio is simply the molecular mass of the ion.

38. OVERALL REVIEW :
A] Physical methods of determination :
1. Melting Point :
- Pure substances usually melt sharply wherever in
case of impure substance usually softening occurs first.
- Melting points are largely influenced by various
forces such as ion- ion forces, dipole-dipole forces, Vander waals forces,
molecular shape.
2. Boiling Point :
- The B.P. of a liquid is that temp. at which the
vapour pressure is equal to that of the external pressure.
- Thus the increased pressure the boiling point also
increases.
- The boiling point of a liquid varies depending upon
surrounding environment pressure.

39. 3. Optical Rotation :
- Substances which refract or absorb right and left circularly polarized light
different extents are “optically active” or show optical rotatory power.
This isomers of Carbon compound are called optical isomers or optical antipodes or enantiomers
and the phenomenon is called as optical isomerism.
4. Racemic Modification :
- A mixture of equimolar solutions of equal quantities of two optical isomers
called racemic modification.
5. Refractive Index :
- Refractive index, also called index of refraction, measure of the bending of
ray of light when passing from one medium into another. ... Refractive index is also equal to the
velocity of light c of a given wavelength in empty space divided by its velocity v in a substance.

40. B.) Analytical methods of characterization :
1. Elemental Composition :
- Elemental analysis is the classical method to obtain
information about the elemental composition of an unknown substance. A known amount of
unknown substance is converted to simple, known compounds containing only the element to
be quantified.
1. Quantitative Analysis :
- Determination of the mass of each element or compound
present.
- It include gravimetry optical atomic spectroscopy, &
neutron activation analysis.
2. Qualitative Analysis :
- X-Ray Fluorescence, particle-induced x-ray emission,
x-ray photoelectron spectroscopy & Auger electron
spectroscopy.

41. C.) Chromatographic methods of characterization
Chromatography is a separation tool which serves as a means of resolution of
mixtures and for the isolation the components. In chromatography, components to
be separated are distributed between stationary and mobile phase. Mass transfer
between mobile and stationary phase occur by adsorption mechanism.
Chromatographic behavior can be explained using following parameters :
1. Retention behavior
2. Partition Coefficient
3. Retention (Capacity) Factor (k)
4. Relative retention (α)
Other Important Parameters in Chromatography :
1. Resolution
2. Plate numbers
3. Plate height (H)

42. Spectroscopic methods of characterization
1. Ultraviolet Visible Spectroscopy :
- UV spectroscopy is type of absorption spectroscopy in which
light of ultra-violet region (200-400 nm.) is absorbed by the molecule.
- It basically involves the spectroscopy of photons &
spectrophotometry.
- It uses light in visible & adjacent near ultraviolet (UV) &
near infrared (NIR) ranges.
- UV/Vis spectroscopy is routinely used in the quantitative
determination of solutions.
- UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light
is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation
with thickness of the absorbing solution is proportional to the incident radiation as well as the
concentration of the solution.
2. IR Spectrophotometry :
- Infrared spectroscopy (IR spectroscopy) is the spectroscopy that deals
with the infrared region of the electromagnetic spectrum, that is light with a longer wavelength and
lower frequency than visible light. It covers a range of techniques, mostly based on
absorption spectroscopy.
- IR spectra mainly used in structure elucidation to determine the
functional groups.

43. 3. NMR Spectroscopy :
- Nuclear magnetic resonance spectroscopy, most commonly known
as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique to
observe local magnetic fields around atomic nuclei.
- The principle behind NMR is that many nuclei have spin and all nuclei
are electrically charged. If an external magnetic field is applied, an energy transfer is possible
between the base energy to a higher energy level (generally a single energy gap).
- It is based on the absorption of electromagnetic radiation in the radio
frequency region 4 to 900 MHz by nuclei of the atoms.
4. Mass Spectroscopy :
- Mass spectrometry (MS) is an analytical technique that measures
the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of
intensity as a function of the mass-to-charge ratio.
- A mass spectrometer generates multiple ions from the sample under
investigation, it then separates them according to their specific mass-to-charge ratio (m/z), and
then records the relative abundance of each ion type.
- Mass spectroscopy is one of the primary spectroscopic methods for
molecular analysis available to organic chemist.

44. Thank you