Natural Product Chemistry T.Y.B.Pharm,SPPU(PUNE UNIVERSITY)

1. Characterization & Structure
elucidation of certain classes of
Secondary metabolites

2. Terpenoids
• General Chromatographic characteristics
• General means of structure elucidation by
chemical & Physical methods

3. Terpenoids
• The odor of a freshly crushed mint leaf, like many plant odors,
is due to the presence in the plant of volatile C10 and C15,
compounds, which are called terpenes.
• “Terpenoids are the hydrocarbons of plant origin of the
general formula (C5H8)n as well as their oxygenated,
hydrogenated and dehydrogenated derivatives.”

4. General Chromatographic characteristics
• Chromatography in its various forms has been widely used for
isolation and separation of terpenoids. Various chromatographic
techniques utilized for studying the isolated terpenoids
 TLC / HPTLC,
 Gas chromatography,
 HPLC,
 Column chromatography etc.

5. Thin Layer chromatography
• TLC is very sensitive technique for identifying volatile organic
compounds separated from natural products by gas
chromatography.
• Using specific colour producing spray we have been able to
detect quantities as small as 1 nannogram and to make some
conclusions regarding structure.
• TLC is also a powerful seperatoy tool and compounds have
been asigned to perticular chemical classes on the basis of
their Rfvalues using solvent system of varying polarity.
• The analysis of terpen hydrocarbons using TLC required the
use of non polar solvent. The choice of volatile hydrocarbon
solvent was obvious.

6. • This is rapid screening versatile tool, which finds its application extensively
for the analysis of various classes of terpenoids. Silica gel of mesh size 200-
300 is the most commonly employed stationary phase and some times for
better resolution 400 mesh size silica gel is used .
• The mobile phase consist of single solvent only in rare case monoterpens
and some sesquiterpens. In majority of cases,the mobile phase is a
combinations of polar and non polar solvent in different ratios.These
include
 Hexane:Acetone;
 Hexane:Ethyl acetate ;
 Hexane:Chloroform;
 Hexane :Diethyl ether;
 Chloroform:Methanol;
 Toluene :Ethyl acetate;
 Chloroform :Acetone;
 Acetic acid :n-Butanol;
 n-Butanol;Acetic acid:Water
• TLC can be employed for the isolation of miligram quantities of terpenoids
for further characterization .It is one of the quickest method for the
isolation of constituents and bioassay.
• Recently many models of High Performance thin layer chromatography
(HPTLC) are commercially available.

7. • Various spray reagents used for visualization of TLC spots are given
in table.
Reagents Types of compounds Colour
H2SO4 Limonene,camphene,α-
pinene,terpinyl acetate
Brown
α-terpinol,nopol,1,8-cineol Green
Geraniol Purple
Carvone Pink
4 % Vanillin in H2SO4 (W/V) Terpens Red and blue
5 % Phosphomolybdic acid in Ethanol Terpens Blue
10 % Ammonium molibdate H2SO Diterpens Blue
20 % Perchloric acid in water Terpens & Steroids Pink
O-Dianisidine Aldehyde Brown pink
Bromocresol green
(3 % bromocresol green in 50 % vol of MeOH to
which 8 drops of 30 % NaOH per 100 ml is added)
Acids Yellow spot on
green background

8. Column Chromatography
• Column chromatography is a routine but an important
tool,which finds extensive use in the seperation of various
terpenoids especially sesqui-,di-,tri-,and tetraterpenoids.
• Silica gel (60-120 mesh),a alumina cellulose,sephadex are
stationary phase used for column chromatography and of
these SiO2 is most common solid support used . Sometime for
better seperation SiO2 of mesh size 300-400 is also used
although elution becomes very slow.
• The ratio of product/ extract/fraction to be chromatographed
and solid phase SiO2 ranges from1:20 to 1:100 and higher ratio
the better is the separation.
• The separation of constituents depends on the judicious choice
of mobile phase,which consist of combination a non polar and
polar solvent with gradual increasing concentration of the

9. High Performance Liquid Chromatography
• HPLC is considered as one of the most versatile tool in recent years as it finds
increasing applications for the analysis of terpenods due to its excellent resolution
efficiency. It is the most reliable technique known today for the qualitative
analysis,quantitative estimation and isolation of various terpenoids.
• Five methods of HPLC known today are
 Partition chromatography,
 Liquid –solid absorption chromatography,
 Ion pair chromatography,
 Ion exchange chromatography,
 Size exclusion chromatography.
• Partition chromatography is very commonly used for analysis of terpenoids and is
carried out in normal phase or reverse phase mode.
• Normal phase chromatography
In this mode stationary phase is polar and mobile phase is non polar compounds
are retained for longer time.
• Reverse phase chromatography
In this stationary phase is non polar and mobile phase is polar and the polar
compound are eluted faster than the non polar.

10. General means of structure elucidation
by chemical & Physical methods
i) Molecular formula:
Molecular formula is determined by usual quantitative
analysis & mol.wt determination methods and by means of
mass spectrometry.
If terpenoid is optically active, its specific rotation can be
measured.
ii) Nature of oxygen atom present:
If oxygen is present in terpenoids its functional nature is
generally as alcohol aldehyde, ketone or carboxylic groups.

11. a) Presence of oxygen atom :
Presence of –OH group can be determined by the formation
of acetates with acetic anhydride and benzoyate with 3.5-
dinitirobenzoyl chloride.
Primary alcoholic group undergo esterification more readily
than secondary and tertiary alcohols.
Acetic anhydride
3.5-dinitirobenzoyl chloride Benzoate

12. b)Presence of >C=O group:
Terpenoids containing carbonyl function form crystalline
addition products like oxime, phenyl hydrazone and bisulphite
etc.
Carbonyl function gr Carboxylic acid
Carbonyl function gr Mixture of lesser
Phenyl Hydrazine
Hydroxylamine
Sodium Bisulfate
Oxidation
(Aldehyde) Without loss of any carbon atom of Aldehyde
Oxidation
(Ketone)

13. iii) Unsaturation:
The presence of olefinic double bond is confirmed by means of bromine,
and number of double bond determination by analysis of the bromide or
by quantitative hydrogenation or by titration with monoperpthalic acid.
Presence of double bond also confirmed by means of catalytic
hydrogenation or addition of halogen acids. Number of moles of HX
absorbed by one molecule is equal to number of double bonds present.
Addition of nitrosyl chloride (NOCl) (Tilden’s reagent) and epoxide
formation with peracid also gives idea about double bonds present in
terpenoid molecule.

14. iv) Dehydrogenation:
On dehydrogenation with sulphur, selenium, polonium or
palladium terpenoids converted to aromatic compounds.
Examination of these products the skelton structure and
position of side chain in the original terpenoids can be
determined.
For example α-terpenol on Se-dehydrogenation yields
pcymene.
Thus the carbon Skelton of terpenol is as follows.

15. v) Oxidative degradation:
Oxidative degradation has been the parallel tool for elucidating
the structure of terpenoids. Reagents for degradative oxidation
are ozone, acid, neutral or alkaline potassium permanganate,
chromic acid, sodium hypobromide, osmium tetroxide, nitric
acid, lead tetra acetate and peroxy acids.
Since oxidizing agents are selective, depending on a particular
group to be oxidized, the oxidizing agent is chosen with the
help of structure of degradation products.
vi) Number of the rings present:
With the help of general formula of corresponding parent
saturated hydrocarbon, number of rings present in that
molecule can be determined.

16. Vii) Relation between general formula of compound and type
of compounds: Table 2
For example: limonene (mol. formula. C10H16) absorbs
hydrogen to give tetrahydro limonene (mol. Formula C10H20)
corresponding to the general formula. CnH2n. It means
limonoene has monocyclic structure.
General formula of parent saturated Hydrocarbon Type of structure
CnH2n+2 Acyclic
CnH2n Monocyclic
CnH2n-2 Bicyclic
CnH2n-4 Tricyclic
CnH2n-6 Tetrayclic

17. viii) Spectroscopic studies:
All the spectroscopic methods are very helpful for the confirmation of
structure of natural terpenoids and also structure of degradation
products. The various methods for elucidating the structure of
terpenoids are;
a) UV Spectroscopy:
In terpenes containing conjugated dienes or α,β-unsaturated ketones,
UV spectroscopy is very useful tool. The values of λmax for various
types of terpenoids have been calculated by applying Woodward’s
empirical rules. There is generally good agreement between calculation
and observed values. Isolated double bonds, α,β-unsaturated esters ,
acids, lactones also have characteristic maxima.
b) IR Spectroscopy:
IR spectroscopy is useful in detecting group such as hydroxyl group
(~3400cm-1) or an oxo group (saturated 1750-1700cm-1). Isopropyl
group, cis and trans also have characteristic absorption peaks in IR
region.

18. c) NMR Spectroscopy:
This technique is useful to detect and identify double bonds, to
determine the nature of end group and also the number of rings
present, and also to reveal the orientation of methyl group in the
relative position of double bonds.
d) Mass Spectroscopy:
It is now being widely used as a means of elucidating structure of
terpenoids for determining mol. wt., mol. formula, and nature of
functional groups present and relative positions of double bonds.
ix) X-ray analysis:
This is very helpful technique for elucidating structure and
stereochemistry of terpenoids.
x) Synthesis:
Proposed structure is finally confirmed by synthesis. In terpenoid
chemistry, many of the synthesis are ambiguous and in such cases
analytical evidences are used in conjunction with the synthesis.

19. Alkaloids
General means of structure elucidation by
Chemical methods

20. General means of structure elucidation
by Chemical methods
The various chemical methods performed to determine the structure of
alkaloids is as follows:
• Molecular Formula Determination
The first step in structural elucidation is the determination of molecular
formula and optical rotatory power. Elemental composition and hence
the empirical formula is found by combustion analysis.
• Hydrolysis
Simple fragmentation by hydrolysis with water, acid or alkali yields
simple fragments which are then analysed separately.
e.g. Atropine Tropine + Tropic acid.
• Determination of Unsaturation
The unsaturation can be determined by adding bromine, halogen acids
or by hydroxylation with KMnO4 or by reduction (using either LiAlH4 or
NaBH4).
Hydrolysis

21. Functional Group Determination
By using the usual standard chemical tests or by infrared (IR)
spectroscopy, functional nature of the alkaloids is determined.
Functional nature of oxygen: The oxygen atom may be present in
the form of alcoholic hydroxyl (–OH), phenolic hydroxyl (–OH),
methoxyl (OCH3), acetoxy (–OCOH3), benzoxyl (–OCOC6H5),carboxyl
(–COOH), aldehyde (–CHO), ketone (C=O) and methylene dioxide
group (–O–CH2–O–).
These groups are characterized by the chemical tests as follows:
Phenolic hydroxyl group (=C–OH)
It is identifi ed by the following tests:
 Soluble in alkali and reprecipitation by CO2.
 Violet colouration with neutral ferric chloride.
 Yields ester on acetylation.
This reaction can be used to determine the number of phenolic–OH.
 Yields ether on reaction with alkyl halide.

22. • Alcoholic hydroxyl group (–C–OH)
It yields ester on acetylation and benzoylation (but negative
answer for phenolic –OH)—refer above tests.
This is confirmed by oxidation, dehydration, dehydrogenation
and by spectroscopy (IR and NMR).
Alcohols are of three different types: 1°, 2° and 3°, and they are
usually distinguished by their oxidation products.
Primary alcohol
Aldehyde with same
no. of ‘C’ as in alcohol
Carboxylic acid with same
no. of ‘C’ as in alco. & alde.

23. • Secondary alcohol
But in cyclic structure, 2° alcohol yields different oxidation products as
shown below:
• Tertiary alcohol
Ketone with same no.
of 'C' as in alcohol
Carboxylic acid with fewer no. of
'C' with respect to alco. and keto.
Ketone with same no.
of 'C' as in alcohol
Acid with same no. of
'C' as in alcohol
Ketone with lesser
no. of 'C' with respect
to alcohol
Carboxylic acid with fewer
no. of 'C' with respect to
alcohol and ketone

24. • The number of hydroxyl (OH) groups present in the compound is
determined by the following methods:
Acetylation method:
By determining the amount of acetic anhydride that reacted with
alcohol to form an ester, the number of hydroxyl groups is
determined.
Zerewitinoff active hydrogen determination method:
When alcohol is heated with CH3MgI, methane is obtained.
By measuring the methane so formed, the amount of alcohol can
be determined.
–OH = CH4 = 22.4 L of alcohol at normal temperature and pressure.

25. • Carbonyl group
The presence of aldehydes & ketones is detected by their reaction with
hydroxylamine, semicarbazide and phenylhydrazine to form the corresp. oxime,
semicarbazone and phenylhydrazone, respectively.
By determining the HCl formed, the ketones are estimated quantitatively.
The alde. and keto. are distinguished by their oxid. or red. pdts.
The carbonyl groups of aldehydes, ketones and carboxyl are further confirmed by
their spectral data such as IR, ultraviolet (UV) and NMR.
Hydroxylamine
Semicarbazide

26. • Carboxyl group (–COOH)
The presence of carboxyl group is determined by the following:
 Its solubility in weak bases such as NH3, NaHCO3 and Na2CO3.
 Esterifi cation with alcohols.
 Quantitatively by acid–alkali titration: Performed by titrating the carboxylic
acid with NaOH using phenolphthalein as an indicator. By knowing the
volume of NaOH consumed the number of –COOH groups are determined.
 Specific IR and NMR signals.
• Ester group (RCOOR)
Esters and related groups like amides and lactones are detected by their
reaction with water, dilute acids /alkali to the hydroxyl and acidic compounds.
By elucidating the acid and alcohol, the nature of alkaloids is determined.

27. • Alkoxy group (–OR)
Determined by Zeisel’s method—alkoxy group such as methoxy on
reacting with hydroiodic acid followed by silver nitrate yields equal
amount of silver iodide. From the amount of silver iodide formed, the
number of alkoxy groups is calculated.
Estimation of C-methyl group (Kuhn Roth method): By estimating the
acetic acid formed upon oxidation, the C-methyl groups are quantified.

28. Functional nature of nitrogen: Most alkaloids contain ‘N’ in their ring structure,
which may exist as 2° or 3°.
The 2°and 3° amines are distinguished as follows:
 2° Amines (acetylated or benzoylated) undergo Libermann’s nitroso reacn.
 2° Amines take up 2 moles of alkyl halide to form 4° ammonium salt.
 3° Amines take up 1 mole of alkyl halide to form 4° ammonium salt.
Alkaloid –Methylamine, Dimethylamine, Trimethylamine (indicates
the presence of 1 or 2 alkyl groups attached to amine ‘N’)
–Ammonia (indicates the presence of primary amine)
Further, the nature of ‘N’ is confirmed by degradation methods such as Hoffmann
Exhaustive Methylation (HEM).
The N-alkyl groups are estimated by Herzig–Meyer method:
*Differs form –OR (alkoxy group) estimation
From the amount of silver iodide formed, the number of N-alkyl groups is
calculated.
Distillation

29. Degradation of Alkaloids
Degradation of alkaloids gives rise to some identifiable
products of known structure and hence by knowing
structure of the degraded products and the changes
occurred during the degradation it is convenient to know
the structure of the original molecule. Different
degradation reactions carried out in elucidating the
structure of alkaloids are as follows:
1. HEM method
2. Emde method
3. Von Braun’s (VB) method for 3° cyclic amines
4. Reductive degradation
5. Oxidation
6. Zinc distillation
7. Alkali fusion
8. Dehydrogenation

30. 1. HEM method: Originally this method was applied by
Willstater in 1870 for naturally occurring alkaloids. It was
further developed by Hoffmann and hence it is known as
HEM. Principle of this method is that the quaternary
ammonium hydroxides yield olefi n with the cleavage of
carbon– nitrogen linkage upon heating with the loss of water
molecule (H from β-carbon atom with respect to N and OH
from the 4° ammonium hydroxide).
Quaternization is done by complete methylation of the amine
followed by hydrolysis with moist Ag2O or KOH.

31. This method can be applied to the reduced ring system but
fails with unsaturated analogues and hence, the unsaturated
rings are first saturated and then HEM is performed.

32. 2.Emde method: Emde modifi cation may be used in the above
two cases, where HEM failed. In this method, 4° ammonium
halide is reduced with sodium amalgam in aqueous ethanol or
Na–liquid NH3 or catalytically.

33. 3. VB method:
(a) For 3° cyclic amines: The 3° N atom in the ring upon reaction with CNBr
followed by hydrolysis yields brominated 2° amine.
This method is applied on compounds which do not respond to HEM. Ring
opening takes place differently in VB and HEM method which is shown in the
following degradation.
In general, CNBr cleaves the unsymmetrical amines to yield the bromides or
shorter bromides.
However, in the VB method only dealkylation may occur without ring cleavage
in some cases.

34. • (b) For 2° cyclic amines:
• 4. Reductive degradation: Ring system is opened by treating
with HI in many cases.

35. 5. Oxidation: Oxidation gives valuable information about the
fundamental structure of alkaloids and the position and nature of
functional groups, side chains, etc.
For example, picolinic acid obtained upon oxidation of coniine
indicates that the coniine is an α-substituted pyridine.
By varying the strength of oxidizing agents, a variety of products
may be obtained. Different types of oxidizing agents used are as
follows:
1. For mild oxidation: H2O2, O3, I2.
2. For moderate oxidation: acid or alkali KMnO4, CrO3 in CH3COOH.
3. For vigorous oxidation: K2Cr2O7–H2SO4, concentrated HNO3 or
MnO2–H2SO4.

36. 6. Zinc distillation: Distillation of alkaloid over zinc dust degrades it
into a stable aromatic derivative.
The reaction indicates that morphine is possessing phenanthrene
nucleus.
7. Alkali fusion: Fusion of alkaloids with solid KOH gives simple
fragments from which the nature of alkaloid can be derived.
The reaction indicates papaverine is containing isoquinoline
nucleus.
The reaction indicates adrenaline is a monosubstituted catechol
derivative.

37. 8. Dehydrogenation: Distillation of alkaloid with catalysts such as
S, Se and Pd yields simple and recognizable products from
which the gross skeleton of the alkaloid may be derived.
Thus with the help of degradation, nature of various
fragments obtained, nature of nucleus and type of linkages
are established. The fragments obtained are arranged in the
possible ways with the possible linkages and the one that will
explain all the properties is selected and confi rmed by
synthesis. Optical activity of an alkaloid helps greatly in
establishing the structure of alkaloid.

38. Flavonoids
General means of structure elucidation by
Spectral methods

39. Flavonoids
They have general structure of a 15 carbon skeleton, which
consist of 2 phenyl rings (A & B) and heterocyclic rings(C) .
This carbon structure can be abbreviated C6-C3 C6.
A
B
C

40. General means of structure
elucidation by Spectral methods
• Various spectrul methods used for structure
elucidation of flavonoids are as follows…
Ultraviolet Visible spectroscopy
Mass spectroscopy
NMR spectroscopy
IR specroscopy

41. Ultraviolet Visible spectroscopy
All phenolic compounds contain aromatic conjugated system that absorb
light in the UV Visible spectral region which enable their detection by
using UV visible detector.
Flavonoids consist of benzene ring A & B and their possible conjugation to
ring C it shows specific absorption resulting UV- Visible spectra .
All flavonoids have an absorption maximum at around 240-290 nm (Band
II),which is mostly affected by conjugation of ring A and substitution pattern
Some flavonoids have another absorbance maximum around 300-550 nm
(Band I) which is detected in flavoinoids where rings B and C are conjugated
via double bond between C-2 and C-3 I ring C.
The band I maximum is generally at a longer wavelength in flavanols than in
flavones which can help in diffreniate between these 2 types flavonoids.
For most flavonoids except for anthocynins and some aurones ,the above
mentioned Band I and II absorption maxima lie in the range of UV
radiation(i.e below 400nm) so the term UV spectrum of flavonoids is often
used instead of UV-vis spectrum.

43. Mass spectroscopy
The UV visible detectors only detect compound with UV-vis absorbing
chromophore.
Mass specrometers detect all kind of chemical compound that are ionised in the
analytical condition.
These 2 instruments are often coupled in line so that the eluent flow from LC
first ,passes through an UV-vis detector ,after which the eluent is directed to
MS(LC-DAD-MS).
In Mass specroscopy,analyte molecule are first ionised ,after which the ions are
analysed according to mass : chagre ratios.
Mass specrometer divided into 2 groups according to their ionisation tech, & mass
analyzers.
Ionisation tech used in flavonoid analysis - FAB (Fast Atom Bombardment)
MALDI (Matrix assisted laser
desorption/Ionization)

44. FAB
Flavonoid sample + Liquid matrix (to dissolve sample)
Dissolved Sample Fast atom (Ar/Xe)
Desorption of ions
Flavonoid sample dissolve in liquid matrix that is bombarded with
beam consisting of fast atoms(e.g.Ar/Xe) which causes desorption of
ions from sample matrix.
MALDI
Flavonoid sample + Liquid matrix (to dissolve sample)
Dissolved Sample
(Absorb energy from laser pulses)
Formation of plasma
(It causes desorption of ions from analyte molecule)
Bombadment

45. • Several mass analyzers are available in addition to different
ionization technique,It include –
Ion trap (IT)
Fourier transform ion cyclotron resonance (FT-ICR)
• Electro Spray Ionization technique-
Function as interface between LC and MS .
Eluent and analyte from LC sprayed through needle that is kept at high
potential or there is a potential difference between the needle & inlet of of
MS.
The applied potential causes formation of charged drying droplets that move
towards inlet of MS.
The charged droplets shrink as the assisting drying gas flow helps to evaporate
the solvent and repulsion of positive and negative charges break down the
droplets until ions are formed from the analyte molecule.
Inside the MS ,the ions are transferred to mass analyser ,where they are
analysed accordingly to their m/z ratios.
Both positive and negative ionization mode used to analysis of flavonoids with
ESI.The Negative ionization often gives better sensitivity for flavonoids
,probably becausetheir ionization is enhanced by deprotonation of acidic

46. • NMR spectroscopy
Sample in homogenous magnetic field
Radio frequency pulses are directed to the sample.
Different NMR active nuclei have characteristics frequencies
at which they absorb energy from these pulses, which is used
to measure NMR spectrum.
NMR spectra provide chemical shift & coupling constants of
NMR acive nuclei (most often 1H & 13 C)
Chemical shift –Information about chemical environment, and
neighboring groups and atoms of the NMR active nuclei.
Coupling constants-How nuclei coupled with each other.

47. In case of Flavonoid glycoside
1H NMR show the signal of an anomeric sugar proton whose coupling
constants to the neighboring sugar proton determines whether the
glycosyl is in α / β- anomeric configuration.The chemical shifts and
coupling constant of anomeric and other protons help to determine
the structure of glycosyl unit.
Heteronuclear multiple bond correlation (HMBC) spectra especially
useful in elucidating the attachment sites of glycosyl and acyl groups
which cannot be determined by the first order mass spectra of
flavonoids.
Total correlation spectroscopy (TOCSY) useful in the assignment of
glycosyl protons,especially in flavonoid oligoglycosides where the
proton signal of different glycosys are often overlapping.The position
of glycosyls units and other structural details of flavonoids can also
determined with 1H-1H experiments with nuclear overhauser
enhancement spectroscopy (NOESY) and rotating frame overhauser
effect spectroscopy(ROESY) that shows protons that are spatialy
close to each other in the molecule.

48. IR spectroscopy
IR specroscopy of all flavonoids and Isoflavonoids shows
absorption bands in th region 1500-1600 cm-1 due to
aromatic ring, along with carbonyl bands at 1620-1670 cm-1.
The presence of hydroxyl group in hydroxyflavonoids is
evidenced by absorption in the region 3300 -3450 cm-1.
The presence of dimethyl gr at 1400 cm-1.
The glycosidic nature of flavonoid is reflected by broad band
at 3250 & 1060 cm-1

49. High-resolution spectrometry
Provide information related to the molecular weight
,elemental composition, & molecular structure of natural
compounds.
Useful for providing accurate mass measurements.These
accurately masses of natural product used to determine
elemental composition for molecular & fragment ions.The
resolution of 10,000 is generally considered desirable for
accurate mass measurements.
This method often reffered as ‘High resolution ’.
The spectrum also provides more information such as isotopic
abundance,mass of small fragments or of lost neutrals.

50. • Relative Molecular Mass (Molecular Weight, Formula Mass,
Formula Weight)
Molecular compounds contain more than one type of atom,
example MgO. So the relatively molecular mass of a
compound is the addition of all the atomic masses of each
atom in the formula.
Molecule No of atom in
the Molecule
Relative
Atomic mass
Relative
Molecular
Mass
Water H2O H=2
O=1
H=1
O=16
2(1) +1(16)=18
Oxygen gas O2 O=2 O=16 2(16)=32
Carbon di
oxide (CO2)
C=1
O=2
C=12
O=16
1(12)+2(16)=44