This are the Plant Chemical Constituent contain in Primary Metabolite and Secondary Metabolite and there Techniques of Isolation

1. Phytochemicals
Mr. Sagar Kishor Savale
(Department of Pharmaceutics, North Maharashtra University, college of
R.C.Patel Institute of Pharmaceutical Education and Research, Shirpur, 425405,
Dist.Dhule, Maharashtra.)
avengersagar16@gmail.com
1. Phytochemical
The plant chemical constituents
2. Preliminary phytochemical scribing
The test which are used in identification purpose known as preliminary phytochemical scribing
3. Phytochemical screening
The technique which are used in separation of number of (one or more) plant chemical
constituents is known as phytochemical screening.
4. Secondary metabolites
4.1 Steroids- the compound which can contain 4 & 5 ring structure known as steroids.
4.2 Terpinoides- the compound which can composed carbon, hydrogen, & oxygenated
hydrocarbon known as Terpinoides. (Group of isoprene unit (C5H8)) (5 ring or more than 5
ring are present)
4.3 Alkaloids- the compound contain in nitrogen atom is present in side chain & heterocyclic
ring is obtain for amino acid or other acid.
4.4 Glycosides –the compound which are is made by glycan (sugar) & glycan (non-sugar)
moiety is jointed to the strong glycosides bond or co valet bond known as glycosides.

2. 4.5 Resin-it is complex mix. Of sugar, acid, gums, volatile oil, & carbon, hydrogen, oxygen,
known as resin.
5. Solubility of the compounds-
5.1 Polar solvent- water, dichloromethane.
5.2 Semi-polar- alcohol, acetone.
5.3 Slightly non polar- chloroform.
5.4 Moderate non polar – benzene, toluene.
5.5 Strongly non polar - cyclohexane, petroleum ether.
Table 1: solubility of compound in different solvents
Chemical cons. Nature of solvent Choice of solvent
Fatty acid, lipid & fixed oil,
polysaccharide.
Strongly non polar Cyclohexane, diethyl ether
Ptro.ether
Steroids Moderate non polar Benzene, toluene.
Terpinoides Moderate non polar Ethyl acetate
Alkaloids Slightly non polar Chloroform
Glycosides Semi polar alcohol ,acetone
Flavonoids Semi polar alcohol ,acetone
Tannin Polar Water, dichloromethane.
Carbohydrate Polar water ,dichloromethane
Protein Polar water ,dichloromethane
Enzyme Polar water ,dichloromethane
Gums Polar water ,dichloromethane
Mucilage Polar water ,dichloromethane
Resin Semi polar alcohol ,acetone
6. Successive solvent extraction
The same plant material extracted with solvent by increasing order of polarity.
Drug material extracted with petro. Ether & cyclohexane

3. Extract contain in residue treated with
Fatty acid, polysaccharide benzene & toluene
Extract - Steroids, Terpinoides residue can treated
Treated with chloroform
Extract – alkaloids residue treated
With alcohol & acetone
Extract – glycosides, resin residue treated with water
Extract contain in
Carbohydrate, protein, gum

4. 7. General approach
Drug material extracted with petro. Ether & cyclohexane
Extract contain in residue treated with
Fatty acid, polysaccharide chloroform
Extract residue- treated alcohol and
Acetone
Chloroform layer acid layer (salt from of layer)
Basic from of alkaloid
Extract –glycosides & resin residue – treated with Water
Extract contain in carbohydrate, protein
Gum, enzyme

5. 8. Purification test
8.1 Distillation- technique which are used in separation of volatile oil.
8.2 Sublimation- solid directly converted into vapor without converted into liquid.
8.3 Crystaliasation- spontaneous arrangement of particle in regulate geometric Patten
8.4 Precipitation – insolubility of compound in given environment
8.5 Liberation- PH dependent solubility e.g. alkaloids
8.6 Chromatography- separation of active constituents of complex mix.
9. Chemical test for identification of phytochemical
9.1 Carbohydrate – molish test (alpha naphtha + sulfuric acid)
Saccharide 1) monosaccharide and disaccharide can separated by barfode test
2) Monosaccharide 1) reducing sugar – benedict test, toluene test, felling test
2) Non reducing sugar – selleminose test
3) Polysaccharide – iodine test
4) Glucose & fructose – furfural test, selleminose test.
9.2 Protein - Biuret test, melons test, picric acid.
9.3 Alkaloids -
1) Drangndrf reagent - (potassium bismuth iodide) --- brown color
2) Mayors reagent – (potassium mercuric iodide) ---- creamy color
3) Wagner reagent – (iodine potassium iodide) ----- brown color
4) picric acid – (hugger’s reagent) -------- yellow color.

6. 9.4 steroids-
Liebermann brunched test- drug sample + acetic a hydride + concentrated sulfuric acid from
side wall of test tube + shows red color (tri-Terpinoides)
But, shows green color (steroids)
9.5 Glycosides – RBC Breakdown test, Fish Poisoning, foam test (spooning).
9.6 Flavonoids-
Shinoda test – drug sample + concentrated sulfuric acid + addition of magnesium turnings ----
shows pink color.
10. Chromatography
-the technique which are used to separation, identification, purification, of active constituents
in complex mix. Is known as chromatography.
"Chroma" is Greek for "color".
"Graphing" is Greek for "to write".
Originally, chromatography was used to separate plant pigments. "Colors" developed and
separated as plant homogenates were passed through calcium carbonate-packed columns.
• The separation of a mixture by distribution of its components between a mobile and
stationary phase over time
– mobile phase = solvent
– stationary phase = column packing material
10.1 Purpose of Chromatography

7. Analytical - determine chemical composition of a sample
Preparative - purify and collect one or more components of a sample
10.2 Types of chromatography
10.2.1 Adoption chromatography principle — solubility of components
10.2.2 Partition chromatography principle — solubility of components
10.2.3 Ion exchange chromatography principle — molecular charge
10.2.4 Gel fermentation chromatography principle — molecular size
10.2.5 Gas chromatography principle — melting point.

8. Gel fermentation (molecular exclusion chromatography)
Basic Principle of chromatographic methods
1. Adsorption chromatography Different analyses have different binding affinities for
the stationary phase.
 Their movement through the stationary phase will be retarded by varying degrees, affecting
separation.
2. Partition between Two Phases
A. Separator Funnel
Two immiscible liquids -- solutes soluble in both
1. Partition Coefficient, K = CU / CL after
equilibration
2. K depends upon:

9. a. Solute -- polarity, ionization, etc.
B. liquid phases -- polarity etc.
1. If K >> 1 or K << 1 for a particular solute ==> can purify the solute pretty well
with a single extraction (or relatively few extractions). But what if K is
approximately equal to 1?
- Column
- Glass or Plastic
- Packed with a solid phase and
Equilibrated by passing the mobile phase
Through the column.
10.3 HPTLC
High Performance Thin Layer Chromatography (HPTLC)
-Synonym- Planar chromatography
- Modern, powerful, analytical technique with separation power, performance and
reproducibility superior to classic TLC
- TLC plates- particle size 3-5 microns.
- Precise instruments for each step of process.
- Computer controlled.
- Qualitative and quantitative analysis.
Advantages

10. • Unsurpassed flexibility by design being an offline technique HPTLC is extremely
flexible.
• Choice of detection
• Cost and time efficiency
• User friendliness and result presentation
• One time use of the TLC plate
10.4 High Performance Thin Layer Chromatography
Important steps
1. Sample preparation: Slice gel/alumina percolated plates, non-polar and volatile
solvents are used.
2. Selection of chromatographic layer: Commonly silica gel 60F, alumina, cellulose are
used materials coated on hand made plates with or without binders
3. Plates: Generally plates of 20 X 20 cm size is used. Particle size of coated material in
TLC is 5 - 40µm, and 4 – 8 µm in HPTLC.
4. Prewashing: Plates need to be prewashed to remove waters vapors or other volatile
impurities. These gives dirty zones and spots on the plates. Plates are cleaned by using
methanol
5. Conditioning: Pre washed plates are activated by placing in oven at 1200
C for 15 -20
mines.
6. Sample application: 1 – 1.5 µl is most satisfactory in HPTLC application of sample and
standard as band separation. Application is carried out by Lineman type applicator.
7. Pre conditioning (chamber saturation): This has profound influence on effective
separation of sample.

11. 10.5 HPLC
 High pressure liquid chromatography
 High performance liquid chromatography
 High-performance liquid chromatography (HPLC) is a form of liquid chromatography
to separate compounds that are dissolved in solution
• The different components in the mixture pass through the column at different rates due
to differences in their adsorption /partitioning behavior between the mobile liquid phase
and the solid / liquid stationary phase.
• This requires sophisticated instrument.
• Provides specific, sensitive and precise method for analysis of different complicated
samples.
Advantages of HPLC
 Sensitive method for analysis of different complicated samples
 There is ease of sample preparation and introduction
 There is speed of analysis
 Accuracy and precision
 Operating at low temperature so can be used for separation of thermo labile, high
polarity substances
Instrumentation in HPLC
 Mobile phase reservoir
 Pump and Gradient Controller

12.  Mixing Chamber
 Sample Conditioning Column
 Sample injector unit
 Pre column
 Analytical column
 Detectors
10.6 Application of chromatography
Identification
Purification
Separation
Quantitative and qualitative analysis
11. Extraction:
• It is technique which are used for separation active chemical constituents from
crud drug from complex mixture.
• Extract: Extracts can be defined as preparations of crude drugs which contain all the
constituents which are soluble in the solvent used in making the extract.
11.1 Type of extracts
• Dry extract: mfg. of tab, cap. E.g. belladonna extract
• Soft: mfg. of ointment, suppository e.g. glycyrrhizin ext.
• Liquid: as tinct.

13. Marc:
Men strum:
Solvent:
11.2 Mechanism
Solute is transfer into solvent against concentration gradients is known as Diffusion
11.3 Extraction process
• There are many procedures for obtaining extracts like:
1. Infusion
2. Maceration
3. Percolation
4. Digestion
5. Decoction
6. Continuous hot extraction
7. Solvent-solvent precipitation
8. Liquid-liquid extraction
9. Distillation
10. Specific procedures
A. extraction with solicitor
B. supercritical fluid extraction

14. Infusion
• In this method, the plant material (herbal tea) is placed in a pot and wetted with cold
water.
• Immediately afterwards, boiling water is poured over it, then left to stand, covered with
a lid, for about fifteen minutes after which the tea is poured off.
Decoction
If the plant material is boiled for ten minutes or if boiling water is poured over it and allowed
to stand for thirty minutes, the result is called decoction.
Digestion
• This method is suitable for hard barks or woods which are difficult for water to
penetrate.
• Digestion is also considered as maceration but, at a relatively elevated temperature.
• As a general rule the temperature of the extracting medium should be in the range from
35-40 but not exceeding 50.
Maceration
• This method is used frequently for water soluble active constituents. It consists of
macerating the plant material in cold water (15-20) for several hours.
• Types of maceration
• Simple maceration: for organized and unorganized C. drug
• Double maceration
• Triple maceration

15. • the maceration process may be carried out with help of heat or stirring
Percolation
• Percolate: down ward displacement
• In this method, the ground plant material is subjected to a slow flow of fresh solvent.
Apparatus used:
Percolator:
• Conical
• Cylindrical
Steps of percolation process
Size reduction of drug
Imbibition
Packing
Maceration
Percolation

16. 11.4 Continuous hot Extraction: Sox let Extractor -
: Stirrer bar
2: Still pot (the still pot should not be overfilled and the volume of solvent in the still pot should
be 3 to 4 times the volume of the sox let chamber)
3: Distillation path
4: Thimble
5: Solid
6: Siphon top
7: Siphon exit 8: Expansion adapter
9: Condenser
Maceration Percolation
• Time consuming and also extraction is
not complete
• Not required skilled operator
• For certain substances which are very
less soluble in solvent and requires
only prolonged contact with solvent.
• Suitable method for less potent and
cheap drugs
• short time and more complete
extraction
• Skilled operator is required
• Special attention should be paid on
particle size of material and
throughout process.
• Suitable method for potent and costly
drugs

17. 10: Cooling water in
11: Cooling water out
Advantages
• Large amount of drug can be extracted with much smaller quantity of solvent
• Tremendous economy in terms of time, energy & ultimately financial inputs
• Small scale used a batch-process
• Becomes more economical when converted into continuous extraction procedure on
large scale
Limitation:
Physical nature of drug.
Solvent.
Chemical constituent of drug.
11.5 Liquid-liquid extraction:
• partitioning,
• In this method separation compounds based on their relative solubility in two different
immiscible liquids, usually water and an organic solvent.
• Raw material in wet condition is pulverized
• Slurry is moved in one direction within a cylindrical extractor where it comes in contact
with the extracting solvent falling against it.
• Complete extraction possible with optimization of quantities of solvent and the material and
their flow rates.

18. • Process is efficient requiring least time with no risk of high temperature
• It is an extraction of a substance from one liquid phase into another liquid phase.
• Liquid-liquid extraction is a basic technique in phytochemical laboratories, where it is
performed using a separator funnel in small scale
In large scale Inc. It consist of small tube of narrow diameter and more length, 200 or more
tubes are linked to each other for complete extraction
Complete extraction possible with optimization of quantities of solvent and the material and
their flow rates.
• Process is efficient requiring least time with no risk of high temperature
• It is an extraction of a substance from one liquid phase into another liquid phase.
• Liquid-liquid extraction is a basic technique in phytochemical laboratories, where it is
performed using a separator funnel in small scale
• In large scale Inc. It consist of small tube of narrow diameter and more length, 200 or
more tubes are linked to each other for complete extraction.
DISTILLATION

19. DISTILLATION: traditional method of separation of constituents of volatile mixtures
(isolation of components of volatile oils).
ii. STEAM DISTILLATION: used to isolate volatile oils and hydrocyanic acid from plant
material.
• Solicitor
• Use of ultrasound with frequencies ranging from 20 KHz to 2000 KHz increases
permeability of cell wall to produce cavitation
• Powdered crude drug is solicited with an appropriate solvent to extract out solvent
soluble components (e.g. Rawolfia serpentine extract)

20. • ? Deleterious effect of ultrasound energy (more than 20,000 Hz) on the active
constituents of the medicinal plant through formation of free radicals and consequently
in the drug molecules
12. Supercritical Fluid Extraction
For every substance, there is a critical temperature (Tc) and pressure (Pc) above which no
applied pressure can force the substance into its liquid phase. If the temperature and pressure
of a substance are both higher than the Tc and Pc for that substance, the substance is defined
as a supercritical fluid.
Choice of SCFs solvent
• Good solubilizing and penetrating property
• Inert to the product
• Easy separation from the product
• Cheap

21. • Low CP because of economic reasons Carbon dioxide is the most commonly used SCF,
due primarily to its low critical parameters (31.1°C, 73.8 bar),
• Low cost and non-toxicity.
• However, several other SCFs have been used in both commercial and development
processes. The critical properties of some commonly used SCFs are ;
12.1 Supercritical Fluid Extraction Process
1. The oldest typical and most common supercritical fluid extraction from solids is
performed as a batch process, with a continuous flow of SCF.
2. An extraction medium (going to be SCF) stored in the feed tank and liquid SCF is
pumped from a reservoir; it is heated and pressurized to reach the supercritical
conditions.
Supercritical SCF enters the extraction chamber where contact with crude drug bed occurs and
the more volatile substances are dissolved into the supercritical fluid.
Solute and SCF leave extractor and extract is precipitated in separators, where SCF becomes
gaseous. Gas is recycled by condensation before returning to liquid reservoir

22. Advantages of Supercritical Fluid Extraction
Dissolving power of the SCF is controlled by pressure and/or temperature
SCF is easily recoverable from the extract due to its volatility
Non-toxic solvents/gases leave no harmful residue
High boiling components are extracted at relatively low temperatures
Separations not possible by more traditional processes can sometimes be effected
Thermally labile compounds can be extracted with minimal damage as low temperatures
can be employed by the extraction
ntages of Supercritical Fluid Extraction
Disadvantages of Supercritical Fluid Extraction
• Elevated pressure required
• High capital investment for equipment
12.2 Applications of Supercritical Fluid Extraction
1. Food and flavouring
SFE is applied in food and flavouring industry as the residual solvent. The biggest application
is the decaffeinication of tea and coffee. Other important areas are the extraction of essential
oils and aroma materials from spices. The method is used in extracting some edible oils and
producing cholesterine-free egg powder.
2. Pharmaceutical industy
Producing of active ingradients from herbal plants for avoiding thermo or chemical
degradation. Elimination of residual solvents from the products.

23. 3. Other plant extractions
Production of denicotined tobacco.
4. Enviromental protection
12.3 Applications of SCF include
Recovery of organics from oil shale
Separations of biological fluids
Bio separation
Petroleum recovery
Crude dew axing
Coal processing (reactive extraction and liquefaction)
Selective extraction of fragrances, oils and impurities from agricultural and food products
Pollution control
Combustion and many other applications
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