Unit 3 T. Y. B. Pharm

1. ISOLATION, IDENTIFICATION AND
ANALYSIS OF PHYTOCONSTITUENTS
PRESENTED BY-
MRS. POONAM NILESH CHOUGULE
ASSISTANT PROFESSOR
PHARMACOGNOSY DEPT. AMCP

2.  Selection
 Collection
 Separation of genuine raw material
 Drying
 Extraction
 Incorporation of desired active constituent into desired
dosage form and large scale production.
 Quality control
 Packaging and storage
 Delivery to the consumer market.

3. EXTRACTION
Factors affecting extraction
 Size reduction
 Agitation
 Temperature
 Suitable Solvent.

4. DRYING OF CRUDE DRUGS
 Shade drying
 Sun drying
 Far-infrared drying
 Vacuum drying
 Oven/hot air drying

5. CHOICE OF SOLVENT
 Depends on characteristics of the Sec. metabolites
like
--- Polarity, pH and thermal stability
 The solvent should be
--- Non-inflammable
--- Inert
--- Non-toxic
--- Easy to remove
--- Should dissolve the maximum amount of
desired phytoconstituents.

7. METHODS OF EXTRACTIONS
 Maceration
Modified maceration
Multiple maceration
Vacuum maceration
Kinetic maceration
Circulation maceration
Re-maceration
Solvent is continuously circulated by pumps

8. PERCOLATION
 It is continuous flow of the solvent
through the bed of crude drug material
to get the extract
 It implies a slow passage of menstrum
under the influence of gravity through
column of drug powder and during this
movement it goes on extracting the drug
molecules layer wise.
 In percolation the drug is exhaustively
extracted by fresh menstrum
11/24/2021
Mrs
Pradnya
Wadekar
Bapat,
Asst.
Prof.,ABCP,Sangli

9. MODIFICATIONS
 Re-percolation: drug is divided into 4/5 lots
percolation followed percolate is used a as
menstrum for next lot. Same menstrum for every
new lot
 Hot percolation: percolation at elevated
temperature increases the efficiency
 Reserved percolation: first part of percolate is
reserved and subsequent percolate is collected
separately. Most of active constituents are in first
percolate. Second percolate contains less active
constituents

10. HOT CONTINUOUS EXTRACTION

11. SUPERCRITICAL FLUID EXTRACTION
 Supercritical fluids.
 They have a high penetration powers and
extraction efficiency.
 Carbon dioxide (CO2) is the most used supercritical
fluid,.
 The gases like carbon dioxide are held as a
supercritical fluid at the critical point of 73.83 bar
pressure and 31.6 °C temperature

12. 12

14.  The system must contain a pump for the CO2, a pressure cell to contain the sample, a
means of maintaining pressure in the system and a collecting vessel.
 The liquid is pumped to a heating zone, where it is heated to supercritical conditions.
 It then passes into the extraction vessel, where it rapidly diffuses into the solid matrix
and dissolves the material to be extracted.
 The dissolved material is swept from the extraction cell into a separator at lower
pressure, and the extracted material settles out.
 The CO2 can then be cooled, re-compressed and recycled, or discharged to
atmosphere.

15.  Also called as sonication
extraction
 Sound waves of high frequency
pulse of 20 kHz are generated
in ultrasonic bath
 Sample with solvent is kept in
suitable vessel.
 The ultrasound increases the
cell wall permeabilty
maximum extraction
 Not suitable for large scale
ULTRASOUND EXTRACTION:

16. MICROWAVE ASSISTED EXTRACTION (MAE):
 Microwaves are (frequency 300 MHz- 300 GHz) non-ioinizing
electromagnetic waves.
 High temperature produced by microwaves evaporate the
moisture in celldehydrates cellulose ruptures
cellwallextraction with solvent
 Rapid extraction within 5-10 min

17. EXTRACTION OF VOLATILE OILS
 Water distillation for oils
 Steam distillation for oils
 Enflurage for delicate oils
 Clavengers apparatus

18. ISOLATION AND PURIFICATION
FRACTIONAL CRYSTALLISATION
 It is an important method for the purification of
compounds from mixture.
 In fractional crystallization the compound is mixed
with a solvent, heated, and then gradually cooled
so that, as each of its constituent components
crystallizes, it can be removed in its pure form from
the solution.
 Many natural products are crystalline in nature even
in mixture, process such as concentration, slow
evaporation, refrigeration are used for
crystallization

19. FRACTIONAL DISTILLATION
 Fractional distillation is
a process by which components
in a chemical mixture are
separated into different parts
(called fractions) according to
their different boiling points.
 This method is used for the
separation of the components
from volatile mixtures
 Largely using in the separation
of hydrocarbons from
oxygenated volatile oil eg citral,
eucalyptol

20. FRACTIONAL LIBERATION
 In this process the groups of compounds having the tendency
of precipitation from the solution.
 This process is often used in separation of cinchona alkaloids,
morphine etc.
 Some groups of compounds lend themselves to fractional
liberation from a mixture , ex : a mixture of alkaloid salts in
aqueous solution when treated with aliquots of alkali , will give
first the weakest base in the free state followed by base
liberation in ascending order of basicity .
 If the mixture is shaken with an organic solvent after each
addition , then a fractionated series of bases will be obtained.
 A similar scheme can be used for organic acids soluble in
water – immiscible solvents ; in this case, starting with a
mixture of the acid salts , it is possible to fractionally liberate
the acids by addition of mineral acids .

21. CHEMICAL METHODS USED FOR SEPARATION
 Acid hydrolysis- Glycosides like diosgenin, hecogenin,
solasodine and other sterdial aglycon.
 Enzymatic hydrolysis- β glactosidase, pectanase,
anthocyanase.
 Alkaline hydrolysis - sodium hydroxide or potassium
hydroxide
 Salt formation
Morphine Morphine hydrochloride
Eugenol Potassium eugenolate
 Acetylation- pyridine and acetic anhydrate is general
reagent
R - OH R - O - COCH3

22. CAFFEINE
10gms. Tea leaves
Transfer to 250 ml distilled water
Boil the water for 30 min. with occasional stirring.
Allow to cool and filter the solution.
Take filtrate in a separating funnel
Add 100 ml chloroform. Shake vigorously
Separate and evaporate the chloroform layer
White caffeine crystals at the bottom

23. CAFFEINE
Tea powder extracted with boiling water
Warm extract is treated with lead acetate and filter the ppt.
Addition of Sulphuric acid.
Filter the solution add charcoal to remove coloring matter
Filter again and extract the filtrate with three portions of chloroform
Separate and combine the chloroform layers
Evaporate to precipitate white crystals of caffeine
Recrystallise the crystals with alcohol and dried

24.  Identification test.
1) Caffeine + HCL and Potassium chlorate, heated to
dryness.
A purple colour is obtained by exposing the residue to
vapours of dil. Ammonia.
Colour is lost on addition of fixed alkali.
2) Caffeine + tannic acid solution= White ppt.
 M.P.- 235-2370 C

25. TLC METHOD
 Stationary phase: Silica gel
 Mobile phase: Benzene: Methanol: Ammonia
75:25:025
 Detection: Iodine vapours or UV
 Rf value: 0.7

26. ATROPINE
Powder of belladonna leaves
Extract with 95% ethanol
Conc. Under vacuum to syrupy mass.
Add 1% HCL to syrupy mass (resinous matter)
Add pet. Ether to remove the chlorophyll pigments and impurities
Shake well and make aqueous solution alkaline with ammonia
Extract the mixture with chloroformx3
Combine the Chloroform extracts and conc.
Add oxalic acid which will separate crystals of atropine and hyoscyamine oxalate.
Dissolve the crystals in acetone or ether filter, residue will be of atropine oxalate.

27. Vitali-morin test- Violet colouration.
TLC Method
Stationary phase- Silica gel
Mobile phase-
Butyl acetate: Formic acid: Chloroform
(60:40:20)
Detection- Iodine vapours.
Rf- 0.2

28. SENNOSIDES
Coarse powder of senna leaves
Extract with benzene on an electric shaker for 2 hours.
Filter, extract the dried marc with 70%methanol for 5 hours.
Filter and again extract the mark with fresh methanol for 2 hours.
Filter and combine both extracts and concentrates to 1/4th of its original volume.
Add sufficient quantity of HCl to obtain pH to 3.2 and keep aside for 2 hours at 50 C.
Filter and add alcoholic anhydrous calcium chloride with continuous stirring.
Add ammonia to bring the pH to 8 and keep aside for 2 hours.
Filter and collect the ppt. of calcium sennosides.
Dry the ppt. in desiccator and calculate the % yield.

29.  Borntrager test- Pink to red colour.
 TLC Study-
Stationary phase- Silica gel
Mobile phase- n-propanol:ethyl acetate:water
(50:30:20)
Detecting agents- Potassium hydroxide reagent
Rf Valus- A= 0.4, B= 0.2, C=0.7 and D= 0.5

30. Course powder of Dioscorea tubers.
Extract the powder with methanol for 6 hours. Filter and conc. the filtrate to obtain a semisolid
mass.
Add dilute HCL and stir for 6 hours.
Precipitate of diosgenin will be observed.
Filter and purify the ppt with alcohol.
DIOSGENIN

31. DIOSGENIN
Coarse powder of Dioscorea tubers
Refluxed with 2N mineral acid for 2 hrs.
Filter and dry the marc and again extracted this dry mass with methanol for 6 hrs.
Filter and evaporate the solvent to 1/4th of its volume.
Keep the concentrated liquid in a refrigerator for 2hrs to form the crystals of diosgenin.

32.  Libermann- Burchard test- Blue green to red orange
 Salkowaski test- Chloroform layer red colour
Acid layer green flurescence.
TLC Method-
Stationary phase: Silica gel
Mobile phase: Benzene:Ethyl acetate(85:15)
Detecting agent:Anisaldehyde sulphuric acid
Rf value: 0.42

33. MENTHOL
Course powder of Mentha piperita parts just before flowering
Extract the peppermint oil by hydro distillation or steam distillation method
Peppermint oil is subjected for cooling to -220C
Crystal s of menthol crystallize out
Crystals are separated by centrifugation

34. TLC Method
Stationary phase- Silica gel
Mobile phase- Chloroform
Detection- Vanillin-sulphuric acid reagent and heat
the plate at 1100C
Rf - 0.62

35. CURCUMIN
Turmeric powder
Extract with n-hexane for 2 hrs..
Discard the n- hexane extract and extract marc with acetone for 2 hrs.
Distill off the acetone and dry the crystals of curcumin
Recrystallise the curcumin from hot ethanol

36. CURCUMIN
Turmeric powder
Extract with 95% alcohol in a soxhlet assembly .
Distill off alcoholic extract to semi-solid brown coloured mass.
Dissolve the crude extract in benzene
Extract twice with equal volume of 0.1%sodium hydroxide solution
Combine the alkaline extract and acidify with dil.HCL
A yellow ppt is formed allow to settle for about 15 mins.
Conc. the extract by boiling on water bath
Filter the solution in hot condition and concentrate the filtrate to very small volume
Finally cool to get curcumin

37.  M.P.- 183oC
 TLC Study-
Stationary phase- Silica gel
Mobile phase- Chloroform: Methanol
(95:5)
Detecting agents- 366nm
Rf Valus- A= 0.75, B= 0.55, C=0.27

38. CITRAL
Take Lemon grass fresh parts just before flowering
Extract the oil by hydro distillation or steam distillation method also separated by using
ecuelle method.
Crystals are separated by fractional distillation.

39. TLC Method-
Sample preparation- 1gm of citral in 1ml of
methanol.
Stationary phase- Silica gel
Mobile phase- Chloroform
Detecting agents- 2,4-dinitrophenyl hydrazine
reagent.
Rf Valus- 0.51

40. GLYCYRRHETINIC ACID
Liquorice powder extracted with chloroform
Chloroform extract is discarded
Marc is again extracted with 0.5M Sulphuric acid.
The acid extract is cooled and shaken with chloroform
Chloroform extract concentrated to get dry Ghycyrrhetinic acid.

41.  TLC Method-
Sample preparation- 1gm of Glydyrrhetinic acid in 1ml
of methanol and chloroform (1:1).
Stationary phase- Silica gel -G
Mobile phase- Toluene: Ethyl acetate : Glacial acid
(12.5: 7.5 : 0.5)
Detecting agents- Anisaldehyde sulphuric acid and
heat plates for 10 mins at 110ᵒ C
Rf Valus- 0.41