IDENTIFICATION OF IDENTIFICATION OF TERPENOIDS BY VARIOUS TERPENOIDS BY VARIOUS

METHODS METHODS

R.ANASUYA

TERPENOIDSTERPENOIDSOrganic compounds occur widely in

plantsMost of them are essential oilsUsed as flavouring agents,

antibiotics,cosmetics etcGen 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..

(C5H8)n

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..

CAROTENOIDSCAROTENOIDSPolyene hydrocabonsNaturally occuring organic

pigments2 classes:Xanthophylls - contain oxygenCarotenes – purely hydrocarbons ,

contain no oxygenEx: β- carotenesource: carrots,pumpkinSpinach etc..

Why should we take

carotenes???

They will increase immunity, cure

heart diseases,act 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 Antioxidant function of ββ carotene caroteneQuenches singlet oxygen by dissipating

the energy as heatReacts 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: C40H56

• 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 ELUCIDATIONPresence of double bonds: On catalytic dehydrogenation:C40H56

Addition of malic anhydride forms an adduct indicates presence of conjugationPresence of β ionone str/-: By oxidation of a β carotene solution with alk. KMnO4

gives b ionone

C40H78H2 /pt

STRUCTURE ELUCIDATIONPresence of methyl side chains:β carotene β ionone geranic acid αα- dimethyl succinic

acid acetic acidDistillation of β carotene gives

toulene -

xylene

2,6 dimethyl naphthalene

BY USING CHROMATOGRAPHICBY USING CHROMATOGRAPHICTECHNIQUESTECHNIQUES

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

Drops for tlc

discard

HPLC HPLC It’s a reversed phase chromatographic

techniqueSolvents used: acetonotrileColumn: 10µm novapak c18, 8 X 100 mmmobile phase: acetonitrile : methanol: ethyl

acetate- 80: 18:2 v/v Uv detector : 450 nmRheodyne injector: 40- 50µlFlow 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.

Colourregion

voilet

blue cyan green yellow

orange

red

Wave-length

380 - 435

435-500

500-520

520-565

565-590

590-625

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 cmֺ 1־ - c=c stretching ~ 960 cm 1־ - 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!