IDENTIFICATION OF

TERPENOIDS BY
VARIOUS METHODS

R.ANASUYA
TERPENOIDS
Organic compounds occur widely in plants
Most of them are essential oils
Used as flavouring agents, antibiotics,cosmetics
etc
Gen formula of (C5H8)n
TERPENES means derivatives of hydrocarbons
made up of ISOPRENE UNITS joined in a head
to tail way. Can be given by isoprene rule..
(C H )n
5 8

n=2 –mono terpenoids(ex: camphor)

n=3 –sesqui terpenoids(ex: santonin)
n=4 –di terpenoids( abietic acid)
n=5 –sester terpenoids
n=6 –tri terpenoids ( β-amyrin)
n=8 –tetra terpenoids (carotenoids)
n>8 –poly terpenoids
ISOLATION: are isolated from plants as essential
oils and terpenoids are seperated from essential
oils by different methods..
CAROTENOIDS
Polyene hydrocabons
Naturally occuring organic pigments
2 classes:
Xanthophylls - contain oxygen
Carotenes – purely hydrocarbons , contain
no oxygen
Ex: β- carotene
source: carrots,pumpkin
Spinach etc..
They will increase Why should we
immunity, cure take
heart diseases,act carotenes???
as anti oxidants,
treats cataract,and
prevents all forms
of cancer from
attacking our
body..
 It is a good
source of vitamin
A(retinol)and
nuetralizes free
radicals there by
protecting from
cancer
Antioxidant function of β carotene
Quenches singlet oxygen by dissipating
the energy as heat
Reacts with triplet oxygen to prevent the
formation of singlet oxygen.
 The quenching process does not destroy
beta-carotene.The mechanism is physical
rather than chemical.
Excess energy of the singlet oxygen, 1O2*,
is transferred to the carotenoid’s electron
 The carotenoid is excited by this added
energy into the “triplet” excited state, 3Car*,
and then relaxes into its ground state, 1Car,
loosing the extra energy as heat.
1O2* + 1Car → 3O2+ 3Car*
3Car* → 1Car + heat

A single molecule of beta-carotene can arrest

up to 1000 molecules of singlet oxygen.


 β-CAROTENE
• M.F: C H56
40

• STRUCTURE
• M.p: 183ºc
• Solubility: insoluble in H2O
soluble in fats, ether,chloroform
• Color : red to purple
• form: powder
• Stability: stable but sensitive to heat and light
 IDENTIFICATION TESTS
 Conc H2SO4: on treating with conc sulphuric
acid it gives blue colour, because of
conjugation a minimum of 6 double bonds.
 0.5 gms of crude was dissolved in 5 ml of
methanol +1 ml of 2, 4-dinitrophenyl
hydrazine dissolved in 100 ml of 2M HCl. A
yellow-orange coloration was observed as
an indication of terpenoids
• STUCTURE
ELUCIDATION
STRUCTURE ELUCIDATION
Presence of double bonds:
 On catalytic dehydrogenation:
C40H56 C40H78
H2 /pt

 Addition of malic anhydride forms an adduct

indicates presence of conjugation
Presence of β ionone str/-:
 By oxidation of a β carotene solution with alk. KMnO4 gives
b ionone
STRUCTURE ELUCIDATION
Presence of methyl side chains:
β carotene β ionone geranic acid αα- dimethyl succinic
acid acetic acid
Distillation of β carotene gives
toulene -
xylene

2,6 dimethyl naphthalene

BY USING CHROMATOGRAPHIC
TECHNIQUES
 THIN LAYER CHROMATOGRAPHY
 Glass plates: (20×20 cm)

were coated (0.5 mm) with silica gel

 Sample:5 mg/ml of each extract

dissolved in methanol.& chloroform

 Mobile phase: hexane and

acetone(60: 40)
 Conc H2SO4 sprayed

 Spots identified based on color

 The rf values of sampleare compared

with the reference

Extract β carotene from
samples nacl
carrot spinach
hexane
separate layers

Hexane layer aqueos layer

discard

Drops
for tlc
HPLC
It’s a reversed phase chromatographic
technique
Solvents used: acetonotrile
Column: 10µm novapak c18, 8 X 100 mm
mobile phase: acetonitrile : methanol: ethyl
acetate- 80: 18:2 v/v
 Uv detector : 450 nm
Rheodyne injector: 40- 50µl
Flow rate: 2 ml/ min
 samples:Retinol, a-tocoherol, lutein, all-
trans-lycopene, and a- and b-carotenes in
human plasma
 Retention time=17.7 min
UV SPECTROSCOPY

 The spectra shows a maxima at approximately 470nm, which

corresponds to the absorption of blue light and so the chemical
appears orange
WHY IS IT ORANGE?
 beta-carotene has delocalisation, with 11 carbon-carbon
double bonds conjugated together. The diagram shows
the structure of beta-carotene with the alternating double
and single bonds shown in red.
 The more delocalisation, the smaller the gap between the
highest energy bonding orbital and the lowest energy pi
anti-bonding orbital. To promote an electron therefore
takes less energy in beta-carotene- because the gap
between the levels is less.
 less energy means a lower frequency of light gets
absorbed - and that's equivalent to a longer wavelength.
 Beta-carotene absorbs throughout the ultra-violet region
into the violet - but particularly strongly in the visible
region between about 400 and 500 nm with a peak about
470 nm.
Colour voilet blue cyan green yellow orange red
region

Wave- 380 - 435- 500- 520- 565- 590- 625-

length 435 500 520 565 590 625 740
 Mass spectroscopy
 m/z = 536.4
 Peaks are observed at

m/z=92
m/z=106
for toulene and xylene
resp..
similarly at 69 and
56
IR SPECTROSCOPY

 1520 cmֺ1‫־‬- c=c stretching

 ~ 960 cm1‫־‬- C-H bonding for trans double
bonds
 H’ nmr :
7.95 δ – 8.15 δ
in chain molecules
8.31 -8-.44 δ (end of chain CH2)
 C13 NMR : 17 non equivalent carbons are
observed
Ir spectrum
β carotene….
STORAGE CONDITIONS
 Carotenoids are destroyed/altered to mixtures
of cis-trans isomers by the action of acids and
free halogens in the presence of light and high
temperature.
 They are also easily oxidised in the presence of
oxygen and other oxidising agents.
 For these reasons, care must be taken in
carotenoid analysis to remove light and limit
the exposure to oxygen
 Stability is effected due to ph, heat ,light
temperature etc..
Thank you!