ASBASJSM COLLEGEBaba

Amar Shaheed OF PHARMACY (ANJujhar

Ajit Singh AUTONOMOUS COLLEGE) BELA
Singh Memorial
COLLEGE OF PHARMACY
(An Autonomous College)
BELA (Ropar) Punjab

Name of Unit Basics of Phytochemistry

Subject /Course Pharmacognosy and Photochemistry-II
Subject/Course ID BP 504T
Class: B.Pharm. Semester 5th
Course coordinator Dr.Satnam Singh
Mobile No. 9914544768
Email id [email protected]

Learning Outcome of Module 05

1. Modern methods of extraction
2. Application of latest techniques like spectroscopy, chromatography and Electrophoresis in
isolation, purification and identification of crude drugs.

Content Table
Topic
 Conventional methods of extraction viz. maceration, percolation,infusion,digestion,decoction.

 Soxhletextraction,Counter-current, sonication and modern methods like MAE, PLE,CLE.

 Application of Chromatographic techniques viz.TLC,HPTLC, PC, CC, GC, HPLC, Size

exclusion chromatographyfor isolation and purification of crude drugs.

 Application of latest techniques of spectroscopy like UV, Mass, IR,NMR for identification of

crude drugs.

 Application of Electrophoresis in separation and purification of crude drugs.

 Application of latest techniques like spectroscopy, chromatography and Electrophoresis in

isolation, purification and identification of crude drugs.

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BASICS OF PHYTOCHEMISTRY
A plant is a biosynthetic laboratory not only for fulfilling food needs of humans and animals but
also for production of muultitude of phytoconstituents i.e. primary and secondary metabolites
having commercial and medicinal importance. Phytochemistry is concerned with the study of the
chemicals produced by plants, particularly the secondary metabolites viz. alkaloids , glycosides,
terpenoids, tannins, resins etc. which are used in medicine for their pharmacological properties.
As has been discussed in chapter 1, they are not necessary for normal growth and development.
They are synthesized for self-defense against pests, herbivores and environmental hazards.
Phytochemistry involves:
1) Extraction of plant material by various methods viz. maceration, percolation, continuous hot
percolation or with modern methods of extraction like microwave assisted extraction, pressurized
liquid extraction etc.
2) Isolation and purification by various chromatographic methods, and
3) Identification (qualitatively and quantitatively) by performing qualitative tests and spectroscopic
methods.
Phytochemistry also takes into account the biosynthetic pathways, functions and it's medicinal,
industrial, and commercial applications.
EXTRACTION
It is the method of removing active constituents from a solid or liquid by means of liquid solvent.
It can be defined as the process of isolation of soluble material from an insoluble residue by
treatment with solvent.
It is done for separation of medicinally active portions of plant or animal tissues from the inactive
or inert components by using selective solvents.In this method the wanted components are
dissolved by the use of selective solvents known as menstrum and undissolved part is a marc.
After the extraction unwanted matter is removed by filtration.
Extraction is the process of efficiently dissolving and separating the desired constituents from the
crude drug with the use of solvent/s. Plant constituents are usually contained inside the cells.
Therefore, the solvent used for extraction must diffuse into the cell to dissolve the desired
compounds.
The choice of solvent depends upon the charactristics of secondary metabolites like polarity, pH
and thermal stability.
1. It should be highly selective for the compound to be extracted.
2. It should not react with the extracted compound or with other compounds in the plant material
3. Have a low price.

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4. Be harmless to man and to the environment.
Types
Solid extraction i.e. extraction of solid from solid plant material using a solvent and
Solvent extraction It‟s partitioning with immiscible solvent after solid extraction .
The conventional methods of extraction
1. Maceration (soaking)The whole or coarsely powdered crude drug is placed in a stoppered
container with the solvent.Allow it to stand at room temperature for a period of 7 days with
frequent agitation until the soluble matter gets dissolved. The mixture then is strained, the marc
(the damp solid material) is pressed. The combined liquids are clarified by filtration or
decantation after standing.This method is best suitable for use in case of the thermolabile drugs.
Modified maceration is used for unorganised drug like gums and resins The method can be
modified to increase the yield.
2. Percolation (pass through) It is continuous flow of the solvent through the bed of crude drug
material to get the extract. In this method a slow passage of menstrum under the influence of
gravity pass through column of drug powder and during this movement it goes on extracting the
drug molecules layerwise. In percolation the drug is exhaustively extracted by fresh menstrum.
Percolation consists of three stages viz. imbibition i.e. moistening, maceration and
percolation.Pack powered drug in percolator.Moisten the drug with sufficient quantity of
menstrum.Add suffiecient mensrtum to saturate the drug. Allow to stand for 4 hrs. Add sufficient
menstrum to form layer above drug,when liquid starts dropping, close the outlet.Allow to stand
for 24 hrs(maceration) in closed vessel. Collect extract (percolate).Clarify further if necessary.
Adjust final volume. Press marc and mix the liquid with extract Allow to percolate until 3/4th of
final extract volume.

It is used for extraction of vitamins, volatile ingredients and soft ingredients in which the
powdered drug is extracted with hot/cold water.
Some other conventional extraction methods are :

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3. Infusion Fresh infusions can be prepared by macerating the crude drug for a short period of time
with cold or boiling water. These are dilute solutions of the readily soluble constituents of crude
drugs.
4. Digestion This is a form of maceration in which gentle heat is used during the process of
extraction. The moderately elevated temperature is not objectionable. The solvent efficiency of
the menstruum is thereby increased.
5. Decoction In this process, the crude drug is boiled in a specified volume of water for a definite
time; it is then cooled and strained or filtered. This procedure is suitable for extracting water-
soluble, heat-stable constituents. The starting ratio of crude drug to water is fixed, e.g. 1:4 or 1:16;
the volume is then brought down to one-fourth its original volume by boiling during the
extraction procedure. Then, the concentrated extract is filtered and used as such or processed
further.
Sohxlet extraction (Continuous Hot Percolation) It‟s the most preferred method of extraction.
Same quantity of solvent is made to circulate through extractor of drug by evaporation and
subsequent condensation. Its suitable for extraction of thermostable constituents.
In this method, the finely ground crude drug is placed in a “thimble” made of fine filter paper,
which is placed in chamber extractor of the Soxhlet apparatus. The extracting solvent in flask RB
is heated, and its vapors condense in condenser. The condensed extractant drips into the thimble
containing the crude drug, and extracts it by contact. When the level of liquid in extracter rises to
the top of siphon tube, the liquid contents of extractor siphon into flask. This process is
continuous and is carried out until a drop of solvent from the siphon tube does not leave residue
when evaporated.

Advantages
The traditional extracting methods require a large amount of solvent, and they are time-
consuming and inefficient.

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The advantage of Soxhlet extraction is that it can be extracted multiple times. Compared with the
general extraction methods, it has the advantages of small solvent dosage, high efficiency and
complete extraction.
Counter-current extraction (CCE) In counter-current extraction pulverized raw material is used
to produce a fine slurry. In this process, the plant material generally in the form of a fine slurry, is
moved in one direction (within a cylindrical extractor) where it comes in contact with extraction
solvent. The further movements of material, produces more concentrated extract. Complete
extraction is thus possible when the quantities of solvent and material and their flow rates are
optimized. The process is highly efficient, requiring little time and posing no risk from high
temperature. Finally, sufficiently concentrated extract comes out at one end of the extractor while
the marc (practically free of visible solvent) falls out from the other end.

This extraction process has significant advantages:

i) A unit quantity of the plant material can be extracted with much smaller volume of solvent as
compared to other methods like maceration, decoction, percolation.
ii) CCE is commonly done at room temperature, which spares the thermolabile constituents from
exposure to heat which is employed in most other techniques.
iii) As the pulverization of the drug is done under wet conditions, the heat generated during
comminution is neutralized by water. This again spares the thermolabile constituents from
exposure to heat.
iv) The extraction procedure has been rated to be more efficient and effective than continuous hot
extraction.

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Sonication (Ultrasound Extraction) The procedure involves the use of ultrasound with
frequencies ranging from 20 kHz to 2000 kHz; this increases the permeability of cell walls and
produces cavitation. Although the process is useful in some cases, its large-scale application is
limited due to the higher costs.

One disadvantage of the procedure is the occasional deleterious effect of ultrasound energy on the
active constituents of medicinal plants through formation of free radicals and resulting
undesirable changes in the drug molecules.
Some common extraction methods of Volatile oils
1. By Hydrodistillation- It includes water distillation, water and steam distillation &
steamdistillation used for extraction of volatile oil from herbal drugs. The fresh crude drug is
subjected to hydrodistillation for volatile oil isolation. The apparatus used is Clevenger apparatus.
2. Enfluerage method- It is used for extraction of delicate perfumes. For this fresh flowerpetals are
mechanically spread on fatty material layer. It was allowed to imbibe and the exhausted petals are
replaced with fresh petals. The process is continued till the fatty material layer is saturated with
volatile oil which is further extracted with lipid solvents.
3. Ecuelle method- it is used for extraction of citrus oils. In this the oil cells are
rupturedmechanically using pointed projections by twisting raw material over them in clockwise
direction either mechanically or manually.
4. By using liquid Carbondioxide- CO2 is liquefied under pressure and it act as solvent
forextraction of essential oils. It reverses back to gaseous nature when pressure is reduced and
leaving no any residue of solvent

Modem Methods of Extraction

1. Microwave assisted extractionThe prefix „micro‟ indicates that these waves are
shorter than of radiowaves. Microwaves (frequency 300 MHz- 300 GHz) are nonioinizing
electromagnetic waves. The two types of oscillating perpendicular fields that generate

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microwaves are the electric field and magneticfield.The ionic conduction and dipole rotation
are responsible for heating of substances. When the microwaves interact with polar
solvents, heating of substance is caused due to any one of the above mentioned
phenomena, individually or simultaneously.
The electrophoretic migration of ions under the influence of the changing electric field is called
Ionic conduction.If the solution offers a resistance to this migration of ions, a friction is
generated and the solution is heated.
The realignment of the dipoles of the molecule with the rapidly changing electric field is
called Dipole rotation. At a frequency of 2450 MHz the process of heating occurs.
In this method high temperature is produced by microwaves evaporate the moisure in cell,
dehydrates cellulose causing rupturing of cellwallthereby facilitates quick extraction with solvent.
It‟s a rapid extraction method, completing extraction within 5-10 min.
Principle
The target for heating in dried plant material is the minute microscopic traces of moisture that
occurs in plant cells. The heating up of this moisture inside the plant cell due to microwave effect,
results in evaporation and generates tremendous pressure on the cell wall. The cell wall is pushed
from inside due to the pressure and the cell wall ruptures. Thus the exudation of active
constituents from the ruptured cells occurs, hence increasing the yield of phytoconstituents.The
yields from plant matrices can also be enhanced by using selective solvent. Higher yields can be
obtained also by increasing the temperature, which facilitates faster penetration of solvent into the
cell walls.The free water molecules present in the gland and vascular systems of plant matrices
are heated and this causes a localized heating and expansion of their walls, thus resulting into the
flow of constituents outside the cells.
In order to prevent the degradation of thermo-labile components the sample matrix is immersed in
a microwave transparent solvent like hexane.

Microwave Oven Design: The microwave extraction assembly comprises of four major
components
Magnetron is responsible for generation of microwaves
Wave guide is used to direct the propagation of microwave from the source to the microwave
cavity.
Applicator, where the sample holder along with the sample is placed.
In case of multi- mode systems the applicator is a closed cavity inside which a random dispersion
of microwaves is brought about.
Circulator which regulates the movement of microwaves only in the forward direction.

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Open systems Open vessels can be operated at atmospheric pressure. The major advantage of the
instrument is the ability to process large samples without the requirement of a cooling process.
The equipment can be obtained at a low cost and a complete automation with Open-vessel
operation can be done.

In a closed vessel system higher temperatures can be reached due to the increased pressure inside
the vessel that raises the boiling point of the solvents used thus increasing both speed and
efficiency of extraction. There is considerably no loss of volatile substances in a closed system
vessel and very less volume of solvent is required. There is no need of addition of solvent/s
repeatedly and hence risk of air-borne contamination is lowered.

Advantages
1. It‟s a rapid extraction method, completing extraction within 5-10 min.
2. The process can be automated .
3. Improved extraction yields .
4. Low solvent consumption and
5. The method is suitable for thermostable phytoconstituents.
Applications
1. Extraction of caffeine from the green tea leaves using 50% ethanol as solvent in just 4 minutes.
2. Extraction of Ginsenosides from the Ginseng root using ethanol as solvent in just 15 minutes.
3. Extraction of Anthraquinones from the Morindacitrifolia root using ethanol as solvent in just 15
minutes.

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2. Supercritical Fluid extraction (SCF extraction) At critical point, substance exists in vapour-
liquid equilibrium state. SCF offer liquid like density, gas like viscocity, gas like compressibilty
and high diffusivity than liquid. Carbon dioxide (CO2) is the most commonly used supercritical
fluid, sometimes modified by co- solvents. Extraction conditions for supercritical carbon dioxide
are above the critical temperature of 31 °C and critical pressure of 74 bar. Addition of modifiers
may slightly alter this.
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.

s
SFE used for obtaining residual solvent free extracts. It‟scan be used as a sample preparation step
for analytical purposes, or on a larger scale to either strip unwanted material from a product
(e.g. decaffeination), refining cooking oils or collect a desired product (e.g. essential oils) or
spices.
Advantages
Selective, fast and efficient method.
Disadvantages
High cost operation.
Due to non polar nature of CO2, the additional use of modifiers becomes necessity.

3. Pressurized Liquid extraction(PLE) Pressurized liquid extraction (PLE; also referred to

as pressurized solvent extraction, accelerated solvent extraction, high-pressure solvent extraction,
high-pressure, high-temperature solvent extraction, pressurized hot solvent extraction and
subcritical solvent extraction) is one of the most promising techniques in bioactive compound
extraction. The process uses high temperature and pressure. This technique is a solid–liquid

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extraction process performed at high temperatures (50–200 °C) with high pressures for short time
periods (5–10 min) in solid and semisolid matrices. The extraction taking less time and requiring
less solvent, and possibly also giving better analyte recovery. The elevated temperature is
employed to increase extraction efficiency of the analyte of interest. The elevated pressure (100-
200 bars) is used to keep the solvent in a liquid state as the temperature is increased above its
boiling point and also increases permeability of solvent. The temperatue and pressure is below
supercritical point. The CO2 is not used as solvent. It requires minutes to complete extraction
process. It‟s a solid liquid extraction technique which has been developed as an alternative to
current extraction methods such as Soxhlet, maceration, percolation or reflux, offering advantages
with respect to solvent consumption, extraction yields, extraction time and reproducibility.
The design of the extractor is such that it is capable enough to withstand high pressures, helps the
extraction temperature to be raised above the boiling point of the solvent used. The high pressure
helps in maintaining the solvent in a liquid state at a high temperature. Under such conditions, the
solvent has properties favoring the extraction process, such as high diffusion coefficients, low
viscosity and high solvent strength. This assists to attain a good dissolution processes and
favors desorption of analytes from the cellular matrix. Additionally, pressure allows the extraction
cell to be filled faster and helps to force liquid into the solid matrix. An increased temperature
augments diffusivity of the solvent resulting in an increased extraction rate.

The sample is placed in an extraction vesselas shown in figure made of stainless steel. Following
addition of the solvent, the cell is pressurized, heated to the desired temperature, and the sample is
extracted statically for a specific period of time. Next, the extract is removed from the cell and the
cell is flushed with fresh solvent. The cycle can be repeated. When the extraction is complete,
compressed nitrogen moves all of the solvent from the cell to the vial for analysis. The extract is
filtered prior to being collected in the receiver, thus eliminating the need for a separate filtration
step.
PLE has been used successfully for the extraction of analytes from medicinal plants, food,
environmental samples, etc.It can be used to flavonoids from flowers and polyphenols from fruits.
However this method is not suitable for thermolabilephytoconstituents.

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Phytonics Extraction
The Phytonics process involves the use of a novel non-toxic solvent based on hydrofluorocarbon -
134a, having a boiling point of - 25° C and a vapor pressure of 5.6 bar at ambient temperature and
technology to optimize its remarkable properties in the extraction of plant materials.
The products mostly extracted by this process are high quality natural fragrant essential oils,
flavors and biological extracts which can be directly used without further physical or chemical
treatment.
Advantages:
i) Unlike other processes that employ high temperatures, the phytonics process is cool and gentle
and its products are never damaged by exposure to temperatures.
ii) No vacuum stripping is needed which, in other processes, leads to the loss of precious volatiles.
iii) The technique is highly selective, offering a choice of operating conditions and hence a choice of
end products.
iv) It is less threatening to the environment.
v) It requires a minimum amount of electrical energy.
Disadvantages:
i) By most standards this is a poor solvent as the solvent does not mix with mineral oils or
triglycerides.
ii) The solvent does not dissolve plant wastes.
Applications:
1. The phytonics process can be used for extraction of the production of antibiotics, in the herbal
drug industry, in the food, essential oil and flavor industries, and in the production of other
pharmacologically active products.
2. In particular, it is used in the production of top quality pharmaceutical - grade extracts,
pharmacologically active intermediates, antibiotic extracts and phytopharmaceuticals.
3. The technique is also used in refining crude products obtained from other extraction processes.

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CHROMATOGRAPHY
It is a laboratory technique for the separation of a mixture of phytoconstituents.
„Chromatography‟is derived from two Greek words, chroma "color and graphein "to write.
Chromatography was first employed in Russia by the Italian born scientist Mikhail Tsvet in 1900.
In this technique the mixture is dissolved in a fluid called the mobile phase, which carries it
through a structure holding another material called the stationary phase. The separation is based
on differential partitioning between the mobile and stationary phases.
Principle: Chromatography usually consists of mobile phase and stationary phase. The mobile
phase is mixture of substances to be separated plus a liquid or a gas.The stationary phase is a
porous solid matrix through which the mobile phase along with sample percolates. The interaction
between the mobile phase and the stationary phase results in the separation of the compounds
from the mixture depending upon its partition coefficient between mobile phase and stationary
phase. Subtle differences in a compound's partition coefficient result in differential retention on
the stationary phase. Due to this various constituents of the mixture travel at different speeds
along with mobile phase, causing them to separate.
The chromatography may be preparative or analytical.The purpose of preparative chromatography
is to separate the components of a mixture for later use, and is thus a form of purification.
Analytical chromatography is done normally with smaller amounts of material and is for
establishing the presence or measuring the relative proportions of analytes in a mixture.
Classification of chromatography
I. On the basis of interaction of solute to the stationary phase
1. Adsorption Chromatography
2. Partition Chromatography
3. Ion Exchange Chromatography
4. Size Exclusion Chromatography
II. On the basis of physical state of mobile phase
1. Liquid Chromatography
2. Gas Chromatography
3. Super Critical Fluid Chromatography
Thin layer chromatography (TLC) It‟s is a widely employed laboratory technique and is similar
to paper chromatography. However, instead of using a stationary phase of paper, it involves a
stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose. Compared to
paper, it has the advantage of faster runs, better separations, and the choice between different

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adsorbents. The mobile phase runs upward along the plate due to capillary action and the
components get separated depending upon their polarity towards stationary phase.
Principle: It is based on the principle of adsorption chromatography or partition chromatography
or combination of both, depending on adsorbent, its treatment and nature of solvents employed.
The components with more affinity towards stationary phase travels slower andcomponents with
less affinity towards stationary phase travels faster.
Once separation occurs, the individual components are visualized as spots at a respective level of
travel on the plate. Their nature or character is identified by means of suitable detection
techniques.
TLC system components consists of:
1. TLC plates, preferably ready made with a stationary phase: These are stable and chemically
inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The
stationary phase on the plates is of uniform thickness and is in a fine particle size.
2. TLC chamber- This is used for the development of TLC plate. The chamber maintains a uniform
environment inside for proper development of spots. It also prevents the evaporation of solvents,
and keeps the process dust free.
3. Mobile phase- This comprises of a solvent or solvent mixture. The mobile phase used should be
particulate-free and of the highest purity for proper development of TLC spots. The solvents
recommended are chemically inert with the sample, a stationary phase.
The stationary phase is applied onto the plate uniformly and then allowed to dry and
stabilize. These days, however, ready-made plates are more commonly used.
1. With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.
2. Then, samples solutions are applied on the spots marked on the line in equal distances.
3. The mobile phase is poured into the TLC chamber to a leveled few centimeters above the
chamber bottom.
4. A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain
equal humidity (and also thereby avoids edge effect).
5. Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the
plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.
6. The plate is then immersed, such that the sample spots are well above the level of mobile phase
(but not immersed in the solvent) for development.
7. Sufficient time is given for the development of spots.
8. The plates are then removed and allowed to dry.

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9. The sample spots are then seen in a suitable UV light chamber, or any other methods as
recommended for the given sample.

Some common techniques for visualizing the results of a TLC plate include
1. UV light
2. Iodine chamber
Retention Factor (Rf ) Value: The behaviour of a compound on a TLC is usually described in
terms of its relative mobility or Rf value.
Rf = the distance travelled by solute/ the distance travelled by the solvent.
It‟s a simple, quick,cheap, qualitative and purity checkinglaboratory tool.
However it cannot be used for quantitative purposes.
Applications
TLC is used for separation and identification of alkaloids, glycosides, carbohydrates, protein and
peptides, terpenoids, fats and fatty acids etc.
High-performance thin-layer chromatography (HPTLC)
High-performance thin-layer chromatography is an enhanced form of thin-layer chromatography.
A number of enhancements can be made to the basic method of thin-layer chromatography to
automate the different steps, to increase the resolution achieved, and to allow more accurate
quantitative measurements.
The plates used in HPTLC contain silica gel particles with a very small size and the packing
density of the gel on the plate is high. The surface of the plate is smooth and gives efficient
separation. The analysis is faster than the TLC technique. The sensitivity of this method is also
high. In HPTLC, several different samples can be separated in the same plate without any sample
preparation step.

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HPTLC has a special instrument named HPTLC scanner which can scan developed TLC plates
and measure the optical density of separated spot and convert it into a graph called chromatogram.
These chromatograms are utilized for quantification of specific secondary metabolites.
HPTLC is suitable for both qualitative and quantitative analysis of herbal extracts and
formulation; the data can be stored digitally and being offline technique sample application,
scanning and data analysis can be done individually.
However mobile phase composition cannot be altered till operation is complete.
HPTLC is also useful stability testing of finished product and in finger printing profile of herbal
drugs.
Paper chromatography (PC)It‟s is a technique that involves placing a small dot or line of
sample solution onto a strip of chromatography paper.The paper is placed in a jar containing a
shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample
mixture, which starts to travel up the paper with the solvent. This paper is made of cellulose, a
polar substance, and the compounds within the mixture travel farther if they are non- polar. More
polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.
Retention factor :Rƒ = zero, - Solute remains in the stationary phase and thus it is immobile. Rƒ
= 1 - Solute has no affinity for the stationary phase and travels with the solvent front.
The technique and applications are similar to TLC, however drastic spraying reagents cannot be
used with PC

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.
Column chromatography (CC)
It‟s a technique which is used to separate a single chemical compound from a mixture dissolved
in a fluid. It separates substances based on differential adsorption of compounds to the adsorbent
as the compounds move through the column at different rates which allow them to get separated
in fractions. This technique can be used on a small scale as well as large scale to purify materials
that can be used in future experiments.
Principle
When the mobile phase along with the mixture that needs to be separated is introduced from the
top of the column, the movement of the individual components of the mixture is at different rates.
The components with lower adsorption and affinity to stationary phase travel faster when
compared to the greater adsorption and affinity with the stationary phase. The components that
move fast are removed first whereas the components that move slowly are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the
movement of the components is expressed as:
Rf = the distance travelled by solute/ the distance travelled by the solvent
Rf is the retardation factor
Column chromatography involves the following:
1. Adsorption/retention of substance on stationary phase
2. Separation of adsorbed substance using mobile phase.
3. Recovery of individual components by continuous flow of mobile phase. Stationary phase:
silica gel, alumina.

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Preparation of the Column
 The column mostly consists of a glass tube packed with a suitable stationary phase.
 A glass wool/cotton wool or an asbestos pad is placed at the botton of the column before packing
the stationary phase.
 After packing, a paper disc kept on the top, so that the stationary layer is not disturbed during the
introduction of sample or mobile phase.
There are two types of preparing the column, they are:
1. Dry packing / dry filling
In this the required quantity of adsorbent is poured as fine dry powder in the column and the
solvent is allowed to flow through the column till equilibrium is reached.
2. Wet packing / wet filling
In this, the slurry of adsorbent with the mobile phase is prepared and is poured into the column. It
is considered as the ideal technique for packing.
The various steps include:
1. Introduction of the Sample
The sample which is usually a mixture of components is dissolved in minimum quantity of the
mobile phase.The entire sample is introduced into the column at once and get adsorbed on the top
portion of the column.
2. Elution By elution technique, the individual components are separated out from the column.
It can be achieved by two techniques:
Isocratic elution technique: Same solvent composition or solvent of same polarity is used
throughout the process of separation.e.g. Use of chloroform alone.
Gradient elution technique: Solvents of gradually increasing polarity orincreasing elution
strength are used during the process of separation.e.g. initially benzene, then chloroform, then
ethyl acetate then chloroform
3. Detection of Components
If the compounds separated in a column chromatography procedure are colored, the progress of
the separation can simply be monitored visually and If the compounds to be isolated from column
chromatography are colorless then, small fractions of the eluent are collected sequentially in
labelled tubes and the composition of each fraction is analyzed by TLC.
This method needs large quantities of plant material and solvents. The isolated phytoconstituents
need further purification.

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Gas chromatography (GC) It‟s also sometimes known as Gas-Liquid chromatography, (GLC),
is a separation technique in which the mobile phase is a gas. It is the method of choice for the
separation of volatile substances or the volatile derivatives of certain non-volatile substances.
Stationary phase is an inert solid material (kieselgurh/ firebrick) impregnated with a non-volatile
liquid (silicon/PEG). This is packed in narrow column and maintained at high temperature around
2000C.
As sample is rapidly heated and vaporized at the injection port, the sample is transported through
the column by a mobile phase consisting of an inert gas (Ar/He/N). The sample components are
separated based on partioncoefficeint between mobile phase and staionary phase. The higher a
component's affinity for the stationary phase, the slower it comes off the column. The components
are then detected and represented as peaks on a chromatogram. It is commonly used for
quantitative estimation of lipids, drugs and vitamins.
Gas chromatography is mainly composed of the following parts:
1. Carrier gas in a high-pressure cylinder with attendant pressure regulators and flow meters e.g.
Helium, N2, H, Argon are used as carrier gases.
2. Sample injection system Liquid samples are injected by a microsyringe with a needle inserted
through a self-scaling, silicon-rubber septum into a heated metal block by a resistance
heater.Typical sample volumes range from 0.1 to 0.2 ml.
3. The separation column The heart of the gas chromatography is the column which is made of
metals (stainless steel )bent in U shape or coiled into an open spiral or a flat pancake shape.
4. Liquid phases No single phase will serve for all separation problems at all temperatures.
5. Detector These are either concentration-dependent or mass dependant.Detectors sense the arrival
of the separated components and provide a signal.

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6. Recorder The recorder should be generally 10 mv (full scale) fitted with a fast response pen (1
sec or less). The recorder should be connected with a series of good quality resistances connected
across the input to attenuate the large signals.

Step 1: Sample Injection and Vapourization

1. A small amount of liquid sample to be analyzed is drawn up into a syringe.
2. The syringe needle is positioned in the hot injection port of the gas chromatograph and the sample
is injected quickly.
3. The injection of the sample is considered to be a “point” in time, that is, it is assumed that the
entire sample enters the gas chromatograph at the same time, so the sample must be injected
quickly.
4. The temperature is set to be higher than the boiling points of the components of the mixture so
that the components will vaporize.
5. The vaporized components then mix with the inert gas mobile phase to be carried to the gas
chromatography column to be separated.

Step 2: Separation in the Column

 Components in the mixture are separated based on their abilities to adsorb on or bind to, the
stationary phase.
 A component that adsorbs most strongly to the stationary phase will spend the most time in the
column (will be retained in the column for the longest time) and will, therefore, have the longest
retention time (Rt). It will emerge from the gas chromatograph last.
 A component that adsorbs the least strongly to the stationary phase will spend the least time in the
column (will be retained in the column for the shortest time) and will, therefore, have the shortest
retention time (Rt). It will emerge from the gas chromatograph first.

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Step 3: Detecting and Recording Results
1. The components of the mixture reach the detector at different times due to differences in the time
they are retained in the column.
2. The component that is retained the shortest time in the column is detected first. The component
that is retained the longest time in the column is detected last.
3. The detector sends a signal to the chart recorder which results in a peak on the chart paper. The
component that is detected first is recorded first. The component that is detected last is recorded
last.
High Pressure Liquid Chromatography It‟s also called as high performance liquid
chromatography. It relies on pumps to pass a pressurized (5000-1000 psi) liquid solvent ie mobile
phase containing the sample mixture through a column filled with a solid adsorbent material. The
stationary phase-immobilized thin layer liquid on micro glass or plastic beads is tightly packed on
narrow column. The mobile phase- solvent system is passed under high pressure through column.
The eluents can be detected by detectors. It can be applied in the form of adsorption, ion
exchange, partition or molecular sieve chromatography. Due to rapidity in detection,its is used for
detection of amino acids, peptides, carbohydrates, proteins, lipids, nucleic acids, vitamins,
hormones, drugs, etc.

Principle
 The purification takes place in a separation column between a stationary and a mobile phase.
 The stationary phase is a granular material with very small porous particles in a separation
column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high
pressure through the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel,
the sample is injected into the mobile phase flow from the pump to the separation column using a
syringe.
 Subsequently, the individual components of the sample migrate through the column at different
rates because they are retained to a varying degree by interactions with the stationary phase.
 After leaving the column, the individual substances are detected by a suitable detector and passed
on as a signal to the HPLC software on the computer.
 At the end of this operation/run, a chromatogram in the HPLC software on the computer is
obtained.
 The chromatogram allows the identification and quantification of the different substances.

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Instrumentation
The Pump High-pressure generation is a “standard” requirement of pumps besides which, it
should also to be able to provide a consistent pressure at any condition and a controllable and
reproducible flow rate.
Injector An injector is placed next to the pump.The simplest method is to use a syringe, and the
sample is introduced to the flow of eluent.The use of the autosampler (auto-injector) system is
also widely used that allows repeated injections in a set scheduled-timing.
Columns The recent columns are often prepared in a stainless steel housing, instead of glass
columns.The packing material generally used is silica or polymer gels.
Detectors Separation of analytes is performed inside the column, whereas a detector is used to
observe the obtained separation.
Recorder There are various types of data processors; from a simple system consisting of the in-
built printer and word processor while those with software that are specifically designed for an
LC system which not only data acquisition but features like peak-fitting, baseline correction,
automatic concentration calculation, molecular weight determination, etc.
Types
1. Normal phase: Column packing is polar (e.g silica) and the mobile phase is non-polar. It is used
for water-sensitive compounds, geometric isomers, cis-trans isomers, and chiral compounds.
2. Reverse phase: The column packing is non-polar (e.g C18), the mobile phase is water+ miscible
solvent (e.g methanol). It can be used for polar, non-polar, ionizable and ionic samples.
3. Ion exchange:Column packing contains ionic groups and the mobile phase is buffer. It is used to
separate anions and cations.

Advantages
1. Speed
2. Efficiency
3. Accuracy

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4. Versatile and extremely precise when it comes to identifying and quantifying chemical
components.
Disadvantages
1. Cost: Despite its advantages, HPLC can be costly, requiring large quantities of expensive
organics.
2. Complexity of operation
3. Volatile substances are better separated by gas chromatography.

Size-exclusion chromatography (SEC) It‟s also known as gel permeation chromatography

(GPC) or gel filtration chromatography and separates molecules according to their size, shape &
molecular weight. It is also referred to as molecular sieving or molecular exclusion
chromatography. The chromatography column is packed with fine, porous beads which are
composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide. The pore
sizes of these beads are used to estimate the dimensions of macromolecules. Smaller molecules
are able to enter the pores of the media and, therefore, molecules are trapped and removed from
the flow of the mobile phase. The molecules that are larger than the average pore size of the
packing are excluded and thus suffer essentially no retention, the first to be eluted. This is how
the molecules are separated. It is also useful for determining the tertiary structure and quaternary
structure of purified proteins, especially since it can be carried out under native solution
conditions.
Some other methods of purification of isolated compounds include :
Sublimation, fractional crystallization, fractional distillation, fractional liberation, etc. Chemical
derivatization can also be employed based on groups or moieties present in the compound and
chemical reactions.
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.
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. It‟s largely used in the separation of
hydrocarbons from oxygenated volatile oil e.g. citral, eucalyptol.

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SPECTROSCOPY
Spectroscopy is concerned with the interaction between matter and electromagnetic radiation. It
can involve any interaction between light and matter, including absorption, emission, scattering,
etc. When a beam of electromagnetic radiation passes through a sample, the photons interact with
the sample. They may be absorbed, reflected, refracted, etc. The absorbed radiation affects the
electrons and chemical bonds in a sample. In some cases, the absorbed radiation leads to the
emission of lower- energy photons. Spectroscopy looks at how the incident radiation affects the
sample. Emitted and absorbed spectra can be used to gain information about the material.
Depending upon the wavelength or frequency or energy different types of electromagnetic
radiations are named as
1) Visible radiation
2) UV radiation
3) Infrared radiation
4) Radio rays
5) X-rays
6) Γ-rays
7) Microwaves
UV spectroscopy
Principle It is absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet (200-
400nm) and the full, adjacent visible spectral regions (400-800 nm). The compounds which are
colorless absorb radiation in UV range whereas the colord ones absorb light in visible range.
Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground
state to higher energy state. The absorption of energy is characteristic of a particular atom. It‟s
useful both for qualitative and quantitative determinations.
The absorption pattern (curve with wavelength on X axis vs. absorbance on Y-axis) is unique for
every colored compound. The wavelength at which maximum absorption of radiation takes place
is known as ʎmax, which is constant for a compound. This technique can be used for qualitative
determination.
The quantitative determination can be obtained by preparing calibration or standard curve
between concentration on X-axis and absorbance on Y-axis. So from absorbance, the unknown
concentration can be determined.
UV spectroscopy obeys the Beer-Lambert law, which states that when a beam of monochromatic
light is passed through a solution of an absorbing substance, the rate of decrease of intensity of

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radiation with thickness of the absorbing solution is proportional to the incident radiation as well
as the concentration of the solution. The expression of Beer-Lambert law is
A = log (I0/I) = Ecl
Where, A is absorbance, I0 is intensity of light incident upon sample cell, I is intensity of light
leaving sample cell, C is molar concentration of solute, L is length of sample cell (cm.) and E is
molar absorptivity
Chromophore is defined as any isolated covalently bonded group that shows a characteristic
absorption in the ultraviolet or visible region (200-800 nm). The chromophores can be divided
into two groups-
a) Chromophores which contain p electrons and which undergo pie to pie* transitions. e.g:
Ethylenes and acetylenes
b) Chromophores which contain both p and nonbonding electrons. They undergo two types of
transitions; pie to pie* and nonbonding to pie*. e.g: Carbonyl, nitriles,azo compounds, nitro
compounds.
Auxochromes- An auxochrome can be defined as any group which does not itself act as a
chromophore but whose presence brings about a shift of the absorption band towards the longer
wavelength of the spectrum. e.g: –OH,-OR,-NH2,-NHR, -SH etc.
IR spectroscopy (Infrared Spectroscopy) It deals with the infrared region of the
electromagnetic spectrum, i.e. light having a longer wavelength and a lower frequency than
visible light(wave number 400/cm to 4000/cm). The IR spectroscopy concept can generally be
analyzed in three ways by measuring reflection, emission, and absorption. The major use of
infrared spectroscopy is to determine the functional groups of molecules, relevant to both organic
and inorganic chemistry.
Principle The absorption of IR is restricted to compounds with small energy differences in the
possible vibrational and rotational states. For a molecule to absorb IR, the vibrations or rotations
within a molecule must cause a net change in the dipole moment of the molecule. The alternating
electrical field of the radiation (remember that electromagnetic radiation consists of an oscillating
electrical field and an oscillating magnetic field, perpendicular to each other) interacts with
fluctuations in the dipole moment of the molecule. If the frequency of the radiation matches the
vibrational frequency of the molecule (natural frequency of amolecule) then radiation will be
absorbed, with the generation of peak(IR spectra).
In IR, the region below 1500 cm-1 is rich in many absorption bands and the region is known as
fingerprint region. Here the number of bending vibrations are usually more than the number of
stretching vibrations. In this region, small difference in the structure and constitution of a

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molecule results significant changes in the absorption bands. Many compounds show unique
absorption bands in this region and which is very useful for the identification of the compound.
IR spectroscopy is useful for identification functional groups in a compound, identification of
compounds and detecting impurities by comparing with IR spectrum of a standard.
Mass spectrometry It‟ a powerful analytical technique used to determine molecular weight of a
compound, to identify unknown compounds within a sample, and to elucidate the structure and
chemical properties of different molecules.
Principle In MS the results are presented in the form of a graph known as mass spectrum.The
complete process involves the conversion of the sample into gaseous ions, with or without
fragmentation, which are then characterized by their mass to charge ratios (m/z) and relative
abundances. A mass spectrum is the plot of relative abundance (intensity) of ions against their
mass/charge ratio. Here no electromagnetic radiation is used for excitation to occur.
The instrument used carry out mass spectroscopy is called as Mass spectrometer. The basic aspect
of organic mass spectrometry consist of bombarding the vapour of an organic compound with a
beam of energetic electron accelarated from a filament to an energy of 70 eV to form positively
charged ions (molecular ions). The additional energy of the elecrons is dissipated in breaking the
bonds in the molecular ion, which undergoes fragmentation to yield several neutral or positively
charged species. The ions are then separated according to their mass to charge ration by virtue of
magnet and are thus detected by a detector.
Nuclear Magnetic Resonance (NMR) It‟sis the most powerful tool available for organic
structure determination.
Principle The spinning charged nucleus generates a magnetic field. When placed in an external
field, spinning protons act like bar magnets. The magnetic fields of the spinning nuclei will align
either with the external field, or against the field. A photon with the right amount of energy can be
absorbed and cause the spinning proton to flip. Energy difference is proportional to the magnetic
fieldstrength. This energy is provided through radio waves.It is used to study a wide variety of
nuclei like 1H, 13C, 15N, 19F, 31P etc.A nucleus with an odd atomic number or an odd mass
number has a nuclear spin.
Depending on their chemical environment, protons in a molecule are shielded by different
amounts. The number of signals shows how many different kinds of protons are present. The
location of the signals shows how shielded or deshielded the proton is. The intensity of the signal
shows the number of protons of that type. Signal splitting shows the number of protons on
adjacent atoms. Trimethylsilane (TMS) is added to the sample. Since silicon is less

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electronegative than carbon, TMS protons are highly shielded. Signal defined as zero.Organic
protons absorb downfield (to the left) of the TMS signal.
Electrophoresis
Electrophoresis is the movement of charged particles or molecules in a medium under the
influence of an applied electric field. It‟s a comprehensive term that refers to the migration of
charged particle of any size in liquid medium under the influence of an electric field. Depending
on kind of charge the molecule carry, they move towards either to cathode or to anode.It‟s a
cheaper separation technique as compared to others. The electrophoresis technique is extensively
used for the analysis of RNA, DNA and proteins.
An ampholyte become positively charged in acidic condition and migrate to cathode, in alkaline
condition they become negatively charge and migrate to anode. Its commonly used for isolation
of amino acids and proteins.
The rate of migration of an ion in electrical field depend on factors,
1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium and
5. Temperature of operation

Types of Electrophoresis
FreeElectrophoresis i.e. method performed without any support media.
Zone Electrophoresis The separation is carried out by using support media like paper or gel. The
components are separated as zones hence the named so. It‟s also known as electromatography.
Capillary Electrophoresis Here narrow bore tubes are used to effect separation of the samples.
It‟s a simple,quick, easyand more efficient method and requires less sample.
Gel Electrophoresis Here gels like agarose or sodium dodecyl polyacrylamide is used as a
support media. The technique is most suitable for separation of proteins.It can be used for
analytical purposes and as preparative technique for purification of molecules before before
subjecting to other analytical techniques.

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IMPORTANT QUESTIONS
2 MARKS
1. Write principle/applications ofSoxhlet extraction/ Counter-current/ Sonication/ MAE/ PLE/
CLE.
2. Write principle/applications of TLC/ HPTLC/ PC/ CC/ GC/ HPLC/ Size exclusion
chromatography
3. Write principle/applications of UV/ Mass/ IR/ NMR/ Electrophoresis.
5 MARKS
1. Write a note on Soxhlet extraction/ Counter-current/ Sonication/ MAE/ PLE/ CLE.
2. Write a note on Electrophoresis.
3. Discuss briefly TLC/ HPTLC/ PC/ CC/ GC/ HPLC/ Size exclusion chromatography
4. Discuss briefly UV/ Mass/ IR/ NMR/ Electrophoresis.

10 MARKS
1. Modern methods of extractionused in phytochemistry.
2. Discuss Chromatographic techniques used in phytochemistry.
3. Discuss spectroscopic techniques used in phytochemistry.
4. Discuss application of latest techniques of spectroscopy/ chromatography in isolation,
purification and identification of crude drugs.

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