1.

Thin layer chromatography-

Thin layer chromatography (TLC) is used to separate the mixtures. This can be performed on various platforms such as
glass, plastic and aluminum foil coated with absorbents. The sample is first applied onto the surface and after that
solvent is absorbed on the surface of the plate. This technique may be utilized for monitoring the process and
identifying components and checking the purity of the compound. Polar compounds which are more polar has strong
interlinking with the motion phase whereas the less polar constituents’ spreads to the highest of the surface. If the
motion phase is swapped with more polar compound or solvent, it would be more useful for dispelling the analyses
from silica gel and it would move to the higher.

Merits: Microliter is the only sample required.

• This is simple method to separate the constituents.

• This technique is sensitive.

• The non-volatile compounds are separated by the TLC method.

• In comparison to other methods, very few equipment are used.

Demerits: Results obtained from thin-layer chromatography are hard to replicate.

• The only constituents which are possible are solubilized components.

• In this technique, no quantitative analysis is being done.

2. High performance thin layer chromatography:

This type of chromatography is automation kind of technique which has way better separating economy and
sophisticated detection parameters. This technique is also referred to as high pressure thin layer chromatography or
also known by either planar chromatography or else know by flatbed chromatography. This technique is measure of
analysis and equally desirable for quality and quantity. Partition coefficient or adsorption helps in the separation
process

Merits: Shorter developing time and analysis time.

• Parallel separation of many samples with minimal time requirement.

• Lower amount of mobile phase/solvent consumption.

Demerits: Too costly.

• Maintenance of instrument is difficult

3. High Performance liquid chromatography - This type of technique is a certain signifier of column chromatography
utilized in biochemical studies for distinct elements, to indicate and qualify the active compounds (Zweig et al.,1973). It
is also called a high-pressure liquid chromatography. It basically has a chromatographic column which deemed packed
corporeal which is the stationary phase, supplier which transfers the mobile phase through with the chromatographic
column. validated sample is being used, therefore hold backwards particularly by chemically or else physically
interlinked by the stable phase. Total no progressive is determined by the nature of the both stationary as well as
motion phase. time-period at what the particular analyze moves out from the last of the chromatographic column is
referred as retention time. Prevalent solvent utilized includes any sort of miscible consolidation of organic liquid and
water.

Example: The basic example is acetonitrile and methanol. Separating is done to check the composition of motion phase
during analysis. It moves apart the object of analysis solution i.e. analyze for the present mobile phase.

Merits:

Higher resolution and speed of analysis.

• Easy automation of instrument operation and data analysis.

• HPLC columns can be reused without repacking and regeneration.

Demerits

Cost Complexity.

Low sensitivity to some compounds

4. Gas chromatography-

This type of technique is basically used to give a visual display of the amount of the substance of variable segment in
the sample. The inspection done by a particular gas chromatograph is referred as gas chromatography (Wang et al.,
2003). This technique is basically a kind of technique which is utilized as a analytical science which separate and
examine aggravate which can be vaporized without splitting. This technique basically tries to immaculate specific
substances or splitting them. In a various situation this technique helps in identifying the compound. This results in pure
compound. This type of chromatography is used as a chemical analytical apparatus for isolating different types of
chemicals in specific sample solutions. A gas chromatography contains a tube called a column, into which different
chemical substances get into gas stream which rely upon distinctive physical or chemical properties interlinked with
column filling. As soon as the substances leave the end point of the column they are validated and examined. The
stationary phase which is situated within the column has the capacity to isolate different constituents.

Merits: Sets analysis, typical minutes.

• Efficient, provide high resolution.

• Inexpensive.

• Reliable and relatively simple.

Demerits: These are restricted to the substances which can

be easily converted into vapors.

• It is not at all suitable for thermal labile substances.

• They are difficult for huge samples

5. Paper chromatography –

This type of chromatography is basically used for the alkaloids identification, purity, and validation. For carrying out this
process a tissue paper is used and its color is very important for checking out and for comparing it with standards. For
the identification of alkaloids, the solution of alkaloids is being transferred onto the tissue paper. This technique is
referred to as both analytical methods i.e. qualitative and quantitative. In this chromatography technique the paper
which is being used in the process resembles to that of thin layer chromatography but that doesn’t make any difference
and there is no need of specific coating. In today’s time period various modern chromatography techniques are being
used and those are based on the basic principles of this method, in which we do consider various forma i.e. splitting
and actual behavior of the compounds and their constituents in the both phases i.e. stationary as well a mobile phase
(Casella, 2012).

Merits: This technique needs less quantity of material.

• This type of chromatography is less costly than others.

• Paper chromatography helps in the identification of various organic and inorganic substances.

Demerits: Paper chromatography cannot handle large quantities of samples.

This type of chromatography cannot separate complex substances

6. Column chromatography-

Chromatographic methods are used to purify the characteristics like size, shape, net charge, stationary phase used and
binding capacity of proteins Among these methods, is the most prominent method for purification of molecules. The
column present in this instrument is used for the placement of sample and then mobile phase respectively. There is a
fiberglass present inside the instrument where we ensure the flow of the material which was placed inside. The
materials at the bottom of the instrument are being collected in a definite time and volume.

Merits: This type of chromatography is used for analysis and its applications.

• This helps in the identification of the constituents present in the mixture.

• It is used for many types of mixtures for separation.

Demerits: Time-consuming process.

More amount of mobile phase required

7. Ion exchange chromatography-

This type of chromatographic method involves electronic interlinking between proteins which are charged and
supporting material which is solid in nature. The basic process of separation involving the charge of ions i.e. if the
material has an opposite charge as compared to protein then the probability of separating them is high. If we alters pH,
concentration of salts and ions of the solution then we can separate proteins from the column (Li et al., 2003).
Positively charged ion material is known as the anion exchange material which absorb negatively charged proteins.
While the negative charged ion-exchange material is known as the cation exchange material which absorb positively
charged proteins.

Merits: This type of chromatography helps in increasing the life of resins.

• This type of instrumentation is cheaper to maintain.

Demerits: Nature and properties of ion exchange resins.

Nature of exchanging ions.

8. Gel permeation chromatography-

In this method, dextran-containing material is used to separate macromolecules having different weights. This type of
chromatography technique is usually utilized for the purpose of identifying various parameters of proteins i.e. their
molecular weights and concentrations of salts. In this technique, stable phase constitutes substances with small holes.
It constitutes of a part called as column through which the substances pass simultaneously with perpetual flow (Liu et
al., 1993). The substances which are of size which are larger than the holes cannot move through the gel and they are
being restricted their itself. The substances which are larger can easily move from the pores whereas the substances
whose size is smaller than the holes they diffuse and moves out of the column. The solution which contains different
dimensions are passed continuously with a constant flow rate through the column.

Merits: Short analysis time.

• Well defines separation.

• There is no sample loss.

• Small amount of mobile phase required.

Demerits: In this type of chromatography filtration must be done before using it

Chromatography may be defined as a method of separating a mixture of components into individual components
through equilibrium distribution between two phases.

Essentially, the technique of chromatography is based on the differences in the rate at which the components of a
mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called
moving phase)

In 1930's chromatography in the form of thin layer chromatography and ion exchange chromatography was introduced
as a separation technique. In 1941, Martin and Synge introduced partition and paper chromatography. They introduced
gas chromatography in 1952.

The chromatographic method of separation, in general, involves the following steps:

1. Adsorption or retention of a substance or substances on the stationary phase.

2. Separation of the adsorbed substances by the mobile phase.

3. Recovery of the separated substances by a continuous flow of the mobile phase; the method being called elution.
4. Qualitative and quantitative analysis of the eluted substances.

A more recent development in liquid-liquid chromatography is Countercurrent Chromatography

which entirely eliminates the use of a solid matrix support. Another form of chromatography where the stationary
phase is a porous gel and the separation is according to the size of the molecule, is Gel (Exclusion) Chromatography.
Chromatography using gels modified to develop highly specific biochemical reactions for separations is termed as
Affinity Chromatography (also called bio affinity Chromatography). Other modifications of this technique are, Metal-
Chelate Chromatography, Ligand Exchange Chromatography, and Dye-Ligand Affinity Chromatography. A newer
technique which makes use of all the above principles permits very rapid separations is High Performance Liquid
Chromatography-(HPLC)

Chromatography is relatively a new technique which was first invented by

M. T sweet, a botanist in 1906 in Warsaw. In that year, he was successful in doing the separation of chlorophyll,
xanthophyll and several other colored substances by percolating vegetable extracts through a column of calcium
carbonate. The calcium carbonate column acted as an adsorbent and the different substances got adsorbed to different
extent and this gives rise to colored bands at different positions, on the column. T sweet termed this system of colored
bands as the chromatogram and the method as chromatography after the Greek words Chroma and graphs meaning
"color" and "writing" respectively. However, in the majority of chromatographic procedures no colored products are
formed and the term is a misnomer

Chromatography is probably the most important single analytical technique used today and will probably continue to
be so far the foreseeable future. It is a cornerstone of molecular analytical chemistry in particular. Recently its coupling
with atomic absorption spectroscopy has extended its application to elemental analysis

 Types of Chromatography

In chromatography, the stationary phase may be a solid or a liquid and the mobile phase may be liquid or a
combination gas. Depending on the stationary and the mobile phase used, separation occurs because of a of two or
more factors such as rates of migration, capillary action, extent of adsorption etc., Chromatographic methods can be
classified on the basis of the stationary and the mobile phases used. In column adsorption chromatography, a liquid
mobile phase is allowed to percolate down a solid
stationary phase generally packed in a vertical glass column. The stationary phase competes for the solute by
adsorption and the liquid mobile phase competes for the solute by dissolution. In this type of chromatography,
sometimes both the phases are liquids. The method, is then termed chromatography. The liquid used as the stationary
phase is essentially insoluble in the liquid used as the as partition mobile phase. Generally, partition chromatography is
performed by wetting a solid by a liquid to be used as the stationary phase and the wet solid is packed in a column. The
liquid thus gets immobilized in this case is due to the differences in the solubilities of the solutes in the two liquids. This
process is also

sometimes termed as column partition chromatography.

In thin layer chromatography (TLC), the stationary phase is a finely divided adsorbent solid spread

over a glass plate. The mobile phase is a liquid. In gas liquid chromatography (GLC), separation is achieved by partition
between a mobile gas phase and a stationary liquid phased packed in a column. Paper chromatography is essentially
the same as column chromatography, except that the packed column is replaced by a filter paper strip (liquid stationary
phase). The mobile phase is a liquid. If the separation is carried out by allowing the liquid mobile phase to descent, the
method is called descending paper chromatography and if the mobile phase is allowed to ascend then the method is
called ascending paper chromatography

Column Chromatography:-

INTRODUCTION

Column chromatography is 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 small scale as well as large scale to purify materials that can be used in future experiments. This method is a type of
adsorption chromatography technique.

In column chromatography, the stationary phase in the form of silica gel, alumina, elite or diatomaceous earth, is filled
in a glass column or a stainless steel column of uniform diameter. The normal length of the column (length of stationary
phase) is 50-500 cm and diameter of the column is 1 - 5 cm. The particle size of the stationary phase is in the range of
150-200 µm. The mobile phase used is either a single solvent or a mixture of solvents. A chromatographic technique
carried out by using a polar stationary phase and a less polar or a non- polar mobile phase is termed as normal phase
chromatography.

stationary phase is less polar or non-polar compared to the mobile phase, the technique is called as reverse phase
chromatography. For the reverse phase chromatography, instead of a direct use of silica gel, its modified surface is
made use of. The surface modification is carried out by forming a covalent bond between silica gel and a reagent like
chlorooctadecylsilane

 PRINCIPLE AND THEORY OF COLUMN CHROMATOGRAPHY

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 slow 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:
Distance travelled by solute

Rf ----------------------------------

Distance travelled by solvent

TYPES OF COLUMN CHROMATOGRAPHY

1. Adsorption column chromatography: Adsorption chromatography is a technique of separation, in which the

components of the mixture are adsorbed on the surface of the adsorbent.

2. Partition column chromatography: The stationary phase as well as mobile phase is liquid in partition
chromatography.

3. Gel column chromatography: In this method of chromatography, the separation takes place through a column
packed with gel. The stationary phase is a solvent held in the gap of a solvent.

4. Ion exchange column chromatography: In this chromatography technique, the stationary phase is always ion
exchange resin.

TLC

The principle of separation is adsorption. Compounds under the influence of the mobile phase, through capillary action,
travel over the surface of the stationary phase. During this movement, the compounds with higher affinity to stationary
phase travel slowly while the others travel faster. Thus, the separation of components in the mixture is achieved. Once
separation occurs, the individual components are visualized as spots at a different levels of travel on the plate. Their
nature or characters are identified using suitable detection techniques.

Ideal Properties of TLC: -

Ideally TLC has the following requirements

•It should have adequate purity and stability

•It should have low viscosity and linear partition isotherm.

•A vapor pressure that is neither very low nor very high.

•It should have very low toxicity

The choice of the mobile phase depends upon the following factors:

1. Nature of the substance to be separated.

2. Nature of the stationary phase used.

3. Mode of chromatography (Normal phase or reverse phase)

4. Separation to be achieved- Analytical or preparative.

5. The organic solvent mixture of low polarity is used.

Introduction: -

Thin-Layer Chromatography

Thin-layer chromatography is similar to paper chromatography, except that a thin (0.25 mm) layer of some inert
material, such as Al₂O, MgO, or SiO₂ is used as the substrate instead of paper. A layer of any one of these oxides is
made from a slurry of powder in a suitable inert solvent (The slurry is spread evenly over a flat surface (glass) and dried.
It may be spread manually or mechanically The advantage of using inert substrates instead of paper is that more
reactive developing reagents, such as strong acids, can be used to detect the compounds without destroying the
substrate.

Thin-layer chromatography may be used with reagents such as H₂SO₄, which would react with paper and is is especially
useful for the separation and analysis of high molecular weight biochemical compounds. A wide variety of mixtures
such as amino acids, dyes, food colorings, drugs, sugars, natural products, and insecticides may be separated and
identified TLC has also been applied to micro-analytical studies of inorganic ions and salts. For example, a mixture of Ru,
Rh, Pd, Ir, and Au was semi quantitatively separated by y TLC

The technique of thin-layer chromatography was first introduced by Izmailov and Shraiber in 1938 These workers used
this technique for separating plant extracts on 2mm thick and firm adhesive layer of alumina set on glass plates.
Consden, Gordon and Martin (1944) started using filter papers. Their work in the field of amino acid analysis met with
considerable success. Williams carried out chromate-graphy on adsorbent layer sandwiched between two glass plates,
one of the latter has a small hole through which solutions and developing solvents were applied to the layer. Attempts
were made using adsorption chromatography on impregnated filter paper and later glass-fiber paper coated with silicic
acid or alumina.

Kirchner in 1950 was one of the first to do this. He was able to separate and identify terpenes. TLC as a procedure for
analytical adsorption chromatography was first introduced by Stahl (1958) who was mainly responsible for bringing out
a standard equipment for preparing thin layers.

TLC is often named by other names such as drop, strip, spread layer, surface chromatography and open column
chromatography.

Superiority of TLC over other Chromatographic Technique

TLC not only combines the advantages of paper and column chromatography but in certain respects is superior to
either of the two. The main advantages of TLC are listed below:
(i) Simple equipment.: -

The TLC requires simple equipment.

(ii) Short development time.

:- In TLC, development time, is only about 1 hour or so for good separation on inorganic adsorbent layers. This is an
advantage of TLC over paper and column chromatography which require several hours or days. adsorption, partition
(including

(iii) Wide choice of stationary phase.:-

The method may be employed for reversed phase) or ion exchange chromatography. Early recovery of separated
components. It is possible to remove the powdery coating of the plates

(iv) Early recovery of separated components.: -

It is possible to remove the powdery coating of the plates by scraping with a knife. This means that a spot or zone may
be removed quantitatively,

(v) Separation effects:-

The separation effects achieved by thin layer chromatography are usually superior to those obtained by paper
chromatography.

(vi) Easy visualization of separated compounds.:-

Detection of fluorescence compounds under UV light is easier than on paper because the inorganic background does
not fluoresce.

(vii) Sensitivity:-

Extremely sharp delineated spots are obtained in TLC. Even the usual spray reagents will be able to detect much
smaller quantities than with the more diffused spots obtained in paper chromatography. This increase in sensitivity is of
the order of 10 to 100 folds as compared as that of paper chromatography.

(viii) Variable thickness of thin layers:-

The method used in TLC analysis may also be applied for the preparative separation with the aid of thicker layers as
well as to most separations by column chromatography.

(ix) Chemically inert stationary phase:-

The greatest merit of an inert stationary phase as used in TLC lies in affording vigorous means of detection including the
application of strong heat or of corrosive reagent such as conc. H2SO4.

TLC has its limitations and sources of error too. These will be mentioned at the appropriate places.
HPLC

High Performance Liquid Chromatography (HPLC) is now one of the most powerful tools in analytical chemistry. It is a
chromatographic separation based on the difference in the distribution of species between two non-miscible phases, in
which the mobile phase is a liquid, which percolates through a stationary phase contained in a column.

It has the ability to separate, identify and quantitate the compounds that are present in any sample that can be
dissolved in a liquid. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a
column filled with a solid adsorbent material

HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase. Separation is achieved by
partition, adsorption or ion exchange process, depending upon the type of stationary phase used. The compound to be
analyzed are dissolved in suitable solvent and separation takes place at room temperature. Thus, the non-volatile or
thermo labile drugs can be chromatographed without decomposition or the necessity of making their volatile derivative

Some advantages of HPLC chromatography:

1. HPLC has high resolution and speed of analysis.
2. High surface area.
3. It has high pressure gradient.
4. It has wide range of stationary phases.
5. Precise flow rate control.
6. Sensitive detection methods.
7. Low sample method requirement.
8. Accurate peak identification using HPLC.

Some disadvantages of HPLC chromatography

1. HPLC has high cost.
2. High quality components are needed.
3. The solvents and columns used in HPLC are expensive.
4. Regular maintenance and calibration is needed which add extra cost.
5. Sophisticated software is required for data analysis.
6. Research and development cost.
High Pressure Thin Layer Chromatography (HPTLC)

Thin layer chromatography (TLC) is very widely used for the analysis of complex mixtures of natural origin.

Comparison of TLC with HPTLC.

heit separation and becomes inconvenient and costly Thin layer chromatography is pacifically used under this situation.
In TLC this type of analysis can be carried out at a reasonably fare cost by providing a large number of solvent tanks
Instrumentation in HPTLC

The instrument used for the quantitating entimine of compounds by HPTLC is known as cussing deulonter. A canning
densitometer has the following compound

A light source A collimating

(m) A monochromates

(iv) A converging lens

(v) A detector

(1) A light source

A scanning densitometer makes use of a source of radiation required to be focused on the separated compounds. A
hydrogen lamp, a mercury vapor lamp or a xenon lamp is used for this purpose. The advantages and disadvantages of
these sources are as discussed under UV spectrometry (Chapter 2, Section 2.4.1)

(ii) A collimating lens

A beam of radiation coming out from an entrance shit is made parallel before it enters the monochromatic by passing it
through a collimating lens.

() A monochromatic

A monochrome in the form of there prism of & grating it commonly widen densitometers. Either a single or d
monochrome it so depending on the of densitometer A monowaves makes me of a single monochromathera dual
wavelength densitometer makes use of w monochromatic

(iv) A converging lens to concentrate or focus the beams

A beam of radiation coming out fum the monochromatic is converged by passing it drought a converging lens in order
to focus it on the compounds separated on the plate The Negm when gets reflected back, falls on the detection

(v) A detector

In order to find the concentration of the separated compounds accurately, an effective detector, nach as a
photomultiplier tube, is employed for the unmeant of the intensity of the reflected beam. A signal produced by the
detector indicates the concentration of a compound present in that spot

(vi) A stepping motor

In HPTLC, compounds in the sample are separated at different positions on the plate in order to bring each spot in the
focus of converged beam of radiation, an additional device called a stepping motor, is used in densitometers. A
Mapping move moves a plate under the focused

light beam in order to scan the plate.

Densitometers are of different types, which are

discussed below.
(A) A single beam densitometer (B) A double beam densitometer

(C) A dual wavelength densitometer

(A) A single beam densitometer

As shown in Fig. 9.46, in a single beam

densitometer, there is only a single beam

containing a narrow range of radiation, coming out

from the monochromatic. This beam when falls on

the plate, where sample spots are not present, produces an output corresponding to the blank reading. Then the beam
falls on the separated compounds and produces some output. The two signals produced are then processed by the
instrument so as to produce a net signal, which corresponds to the concentration of the compound in that spot

Entrance slit

Monochromatic

Detector

Converging

Plate

(B) A double beam densitometer

In a double beam densitometer, as shown in a single beam coming out from the monochromator falls on the beam
splitter, which splits it into two beams. One beam falls on the plate, where there are no spots separated and another
bear falls exactly at a point where a separated compound is present. These two beams reflect

Extraction and purification techniques of phytoconstituents

Medicinal plants provide unfailing opportunities for new phytopharmaceutical drug leads. They
contain several types of bioactive compounds responsible for their therapeutic activity; their
extraction, isolation, and purification need to be standardized and a defined methodology can be
followed to extract the targeted active compounds.

1. Primary Purification
 Conventional extraction
techniques

There are numerous methods which are used to prepare plant extract and are selected on the basis of
the target compound/s. The efficiencies of extraction methods mostly depend on the critical input
parameters; understanding the nature of plant matrix; chemistry of bioactive compounds and
scientific expertise. The most commonly used methods are:

i. Soxhlet extraction: Soxhlet extraction is also known as continuous hot extraction. The apparatus
used is known as Soxhlet extractor, which consists of a round bottom flask, extraction chamber,
extraction thimble, siphon tube, and condenser. The finely powdered plant material is placed
inside a porous bag (thimble) and this thimble is placed in extraction chamber. The extraction
solvent placed in the round bottom flask is continuously heated and its vapors condense in a
condenser and drip into the plant material in the thimble and extracts it by contact. When the
level of the solvent in thimble reaches the top of siphon tube, the contents in the extraction
chamber (extracted material and the solvent) siphon into the round bottom flask. It is a most
common method used for plant extraction, as the process consumes less time and solvent for
extraction, and no filtration is required. The entire process is continued till the solvent flowing
from extraction chamber does not leave any residue behind. The method is not suitable for the
extraction of thermo-labile bioactive components.

Figure. Soxhlet apparatus

 ii. Microwave-assisted extraction: It is one of the advanced techniques which uses mechanism of
dipole rotation and ionic transfer by ion displacement in the solvent and the plant material. This
method is mostly used to extract polar compounds. To the plant material dissolved in solvent,
electromagnetic radiation (300 MHz to 300 GHz) is applied, which got converted into heat when
absorbed by the plant material-solvent mixture resulting into the dipole rotation and ion
migration in the solvent which facilitates solvent penetration and extraction process. However,
this process is not useful when nonpolar solvent is used as a very small heat is produced due to
low dipole movement. This technique uses less solvent and time for extraction. However, it is
suitable only for phenolic compounds and flavonoids. Compounds such as tannins and
anthocyanin’s may get degraded due to their thermos-labile nature.
iii. Ultrasound-assisted extraction: This process involves application of ultrasound energy at a high
frequency (20 to 2000 KHz), which disrupts the integrity of plant cell wall, hence increases the
plant material surface area for solvent penetration. This leads to the release of secondary
metabolites into the solvent. The extraction process is applicable to small sample, reducing the
time of extraction and amount of solvent used, and maximizing the yield. The major
disadvantage of this method is its reproducibility, as well as high amount of energy applied that
may degrade the phytoconstituents.
iv. Super critical fluid extraction (SFE): It is one of the advanced extraction techniques which
utilizes the unique properties of supercritical fluids. Supercritical fluid is a substance above its
critical temperature and critical pressure and have properties between those of a gas and a
liquid, where distinct liquid and gaseous phase do not exist. Supercritical fluids are capable to
diffuse quickly into the porous solid substance, hence dissolving the materials quickly. For plant
extraction, supercritical carbon dioxide is mainly used, as it is an ideal solvent with low viscosity,
high diffusion rate, and high volatility. Due to its low critical temperature, it is also used to
extract thermo-labile compounds. The supercritical carbon dioxide is inefficient to extract polar
solutes due to its non-polar nature, for which volatile polar modifiers such as methyl acetate,
diethyl ether, methanol, or formic acid are added to extract polar compounds.

v. Pressurized liquid extraction (PLE)/ Static/ Sub critical extraction: Pressurized liquid extraction
is an advanced extraction technique, which employs solvent extraction at high temperatures
and pressures, below the critical points of the solvent; so that the solvent remains in the liquid
state during the whole extraction process. As high pressure and temperature is used during the
process, a change in physicochemical properties of the solvent occur, leading to the decrease in
solvent surface tension and viscosity. This increases the solubility of analysts in the respective
solvent and significantly higher extraction yields are obtained as compared with
 conventional extractions. Both SFE and PLE methods are highly expensive and rarely used for
plant extraction process.
vi. Table. Suggested solvents for extraction

Phytoconstituents Polarity Solvents used for extraction

Alkaloids Polar Methanol, Ethanol
Flavonoids Polar Methanol, Ethanol, Water
Polyphenols Highly Polar Methanol, Ethanol, Water
Tannins Moderate/Non-polar Diethyl ether, Hexane, Chloroform, Dichloromethane
Terpenoids Non-polar Hexane, Chloroform, Petroleum ether, Toluene
Glycosides Polar Water, Methanol, Ethanol
Anthocyanin’s Polar Water, Methanol, Ethanol
Coumarins Polar Water, Methanol, Ethanol
Anthraquinones Non-polar Hexane, Chloroform, Petroleum ether, Toluene
Steroids Highly Non-polar Hexane, Toluene, Petroleum ether

2. SECONDARY PURIFICATION

To obtain extracts enriched in the components of interest when a specific phytochemical

ingredient or compound class is the topic of an inquiry, the polarity of a solution can be altered to
cause specific chemical classes to precipitate, leaving undesirable molecules in solution.
Compounds containing primary, secondary, or tertiary amines, carboxylic acids, lactones, and
phenols can be selectively extracted using pH changes to modify their polarity or solubility.
Before subjecting to a large percentage of the plant sample or crude extract to one of these
potentially harmful procedures, it is important to assess the stability of the target chemicals on a
modest scale.

i. Acid-base extraction: This method is of liquid-liquid extraction type, typically used to separate
organic compounds based on their acid-base properties. The process is based on the hypothesis
that most organic compounds are more soluble in organic solvents than water. The compounds
containing acidic or basic functionalities can easily be converted into cations or anions, either by
adding acid or base making them more soluble in water. After filtration, the aqueous layer will
contain organic acid in salt form, which can be isolated upon neutralizing it with a base and
separating it in an organic solvent. The aqueous and the organic layers are immiscible liquids;
they will form two separate layers, making it easy to get separated.
ii. Crystallization: It is a unique separation technique used to obtain purified compounds out of the
super-saturated liquid. For natural crystallization, the plant extract is dissolved in sparingly
soluble solvent, in which heat is used to dissolve the extract. After complete dissolution,
the
 solution is allowed to cool, during which precipitation of the crystalline natural compounds
occur. As the natural compounds are mostly amorphous in solid phase, this method is rarely used
for compound separation.
iii. Sublimation: Sublimation is one of the phase transitions of a solid substance to gaseous state
without passing through liquid state. It is an endothermic process that occurs at temperatures
and pressures below a substance's triple point in its phase diagram. The reverse process of
sublimation is deposition or desublimation, in which a substance passes directly from a gas to a
solid phase, crystallized on a cold surface.
iv. Liquid-liquid partitioning extraction (LLC): In general, liquid-liquid partitioning is the most
efficient method for doing repeated extractions. This indicates that the separation features of
extraction systems may be translated to liquid-liquid chromatography. The practically infinite
number of phase systems available in LLC is a significant benefit such as the ability to retain and
to select a precise blend of components. The analyte is partitioned between two immiscible
solutions with differing polarities in LLC. The varying dissolution of the analyte components in
both liquid phases determines analyte retention.
The outcomes of this method can be calculated as partition coefficient denoted as log P.
The log P value for a compound is the logarithm (base 10) of the partition coefficient (P), which
is defined as the ratio of the compound’s organic (oil)-to-aqueous phase concentrations:
Partition Coefficient (P) = [Compound’s concentration in organic phase] / [Compound’s
concentration in aqueous]
However, the calculated log P value for a compound in water vs. a simple organic solvent like
octanol or hexane can provide a guideline for predicting its solubility characteristics in other
aqueous and organic solvents.
If a drug with a log P equal to 0 partitions at a 1:1 ratio in organic to aqueous phase; the drug is
equally soluble in both organic and aqueous solvents, the choice of which depends on the specific
application.

Polar combination Mid-Polar combination Non-Polar combination

n-BuOH: n-PrOH: H2O Heptane: EtOAc: EtOH: H2O Heptane’s: EtOAc: Ethanol:
(4:1:5) (1:1:1:1) H2O (10:5:5:1)
n-BuOH: n-PrOH: H2O EtOAc: EtOH: H2O (4:3:2) Heptane: CH3CN: Me
(2:1:3) OH (8:5:2)
EtOAc: n-BuOH: H2O
(3:2:5)
 Calculated log P (cLog P): log P values do not directly correlate with solubility limits; they
merely indicate whether compounds are more or less soluble in water compared to organic
solvents. Two compounds may have log P values equal to -1, indicating that they are more
soluble in aqueous than organic solvents; but they may nonetheless have very different solubility
limits in aqueous buffers.

For maximum solubility a solvent with log P value similar to the log P of the compound could be
selected. Many solvents that are commonly classified as ‘organic’ are miscible (mix with water),
rather than immiscible (do not mix with water). Generally, miscible solvents have negative log P
values. Therefore, the log P does not provide much information to predict whether a compound
will dissolve more easily in water.

v. Flash chromatography: Flash chromatography is a specialized and inexpensive purification

technique that is designed for rapid separation by using air pressure to drive the solvent through
the column as opposed to slow and simple gravity fed chromatography. It differs from the
conventional column technique by using slightly smaller silica gel particles (230 - 400 mesh) and
pressurized gas (nitrogen or air) at ≤ 20 psi. Although the technique has moderate resolution, it
is a rapid separation method which can be used to separate mg to g scale. The technique uses
binary solvent systems with one solvent having a higher polarity than the other as they allow for
easy adjustment of the average polarity of the eluent. The quantity of silica gel to be packed in
the column is directly proportional to the amount of the sample to be loaded. In general, for n
grams of sample, 30 to 100 n grams of silica gel is required. This technique is particularly
advantageous because it allows faster flow rates of the solvent, and is widely useful in the
separation of closely related organic compounds. It is to be noted that in this technique, silica
gel 60 (popular grade), and small particle size give poorest resolution. This technique has shown
its superiority in fractionating natural derived compounds viz. alkaloids, xanthones, flavonoids,
tocopherols and cannabinoids.

Figure: Setup of flash chromatography

vi. Vacuum liquid chromatography (VLC): It differs from flash chromatography as in
this method gradient elution is done, and the column is allowed to run dry after
each fraction is collected. The VLC method works extremely well and is very quick.
Usually, a TLC grade binder free silica gel (silica gel H) or aluminum oxide are used
for column packing, by loading the dry adsorbent into a sintered funnel. The
adsorbent was allowed to settle just like in column chromatography. Tight packing
can be done by subjecting the vacuum to the dry column. Column uniformity can
be checked by passing hexane through the dry column under vacuum. The solvent
must pass uniformly through the column otherwise it has to be repacked. Sample
is added either as slurry made in non-polar solvent, or in the liquid form,
uniformly loaded onto the tightly packed dried silica column in such a way that a
thin band of the sample was obtained. The column was then first subjected to the
non-polar solvent. The solvent polarity was then increased successively. and
increasing amounts of a more polar solvent were added to each successive solvent
fraction. The smaller the band, the better will be separation. The column is left
under vacuum for 5-10 mins to take out the polar solvent used to dissolve the
sample. This routine solvent elution scheme suffices to reduce complex mixtures
into fractions suitable for 1HNMR assessment. Interesting fractions are then re-
chromatographed on a smaller column using smaller fractions and more gradual
solvent polarity steps, especially near the solvent mixture which eluted the
fraction from the first column. In case, crude mixture cannot be dissolved (or
suspended) in light petroleum, it should be dissolved in a volatile solvent and
mixed with an equivalent weight of silica gel. The solvent is then carefully removed
under vacuum after which the dried powder is packed onto the top of the column.
Figure: Setup of vacuum liquid chromatography

Identification and characterization

Due to the fact that plant extracts usually occur as a combination of various type of bioactive compounds or
phytochemicals with different polarities, their separation still remains a big challenge for the process of
identification and

characterization of bioactive compounds. It is a common practice in isolation of these bioactive compounds that a
number of

different separation techniques such as TLC, column chromatography, flash chromatography, Sephadex
chromatography and

HPLC, should be used to obtain pure compounds. The pure compounds are then used for the determination of
structure and

biological activity. Beside that, non-chromatographic techniques such as immunoassay, which use monoclonal
antibodies

(MAbs), phytochemical screening assay, Fourier-transform infrared spectroscopy (FTIR), can also be used to obtain
and facilitate

the identification of the bioactive compounds.

Chromatographic technique Thin-layer chromatography (TLC) and Bio-autographic methods of Tlc is a simple,
quick, and inexpensive procedure that gives the researcher a quick answer as to how many

components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a
compound is

compared with the Rf of a known compound. Additional tests involve the spraying of phytochemical screening
reagents, which

cause color changes according to the phytochemicals existing in a plants extract; or by viewing the plate under the
UV light.

This has also been used for confirmation of purity and identity of isolated compounds.

Bio-autography is a useful technique to determine bioactive compound with antimicrobial activity from plant
extract.

TLC bioautographic methods combine chromatographic separation and in situ activity determination facilitating
the localization

and target-directed isolation of active constituents in a mixture. Traditionally, bioautographic technique has used
the growth

inhibition of microorganisms to detect anti-microbial components of extracts chromatographed on a TLC layer.

This

methodology has been considered as the most efficacious assay for the detection of anti-microbial compounds
(Shah Verdi, 2007).

Bio-autography localizes antimicrobial activity on a chromatogram using three approaches: (i) direct bio-
autography, where the

micro-organism grows directly on the thin-layer chromatographic (TLC) plate, (ii) contact bio-autography, where
the

antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate through direct contact and
(iii) agar

overlay bio-autography, where a seeded agar medium is applied directly onto the TLC plate (Hamburger and
Cordell, 1987;

Rah Alison et al., 1991). The inhibition zones produced on TLC plates by one of the above bioautographic technique
will be use to

visualize the position of the bioactive compound with antimicrobial activity in the TLC fingerprint with reference to
Rf values

(Homans and Fuchs, 1970). Preparative TLC plates with a thickness of 1mm were prepared using the same
stationary and mobile
phases as above, with the objective of isolating the bioactive components that exhibited the antimicrobial activity
against the test

strain. These areas were scraped from the plates, and the substance eluted from the silica with ethanol or
methanol. Eluted

samples were further purified using the above preparative chromatography method. Finally, the components were
identified by

HPLC, LCMS and GCMS. Although it has high sensitivity, its applicability is limited to micro-organisms that easily
grow on

TLC plates. Other problems are the need for complete removal of residual low volatile solvents, such as n-BuOH,
trifluoroacetic

acid and ammonia and the transfer of the active compounds from the stationary phase into the agar layer by
diffusion (Cos et al.,

2006). Because bio-autography allows localizing antimicrobial activities of an extract on the chromatogram, it
supports a quick

search for new antimicrobial agents through bioassay-guided isolation (Cos et al., 2006). The bioautography agar
overlay method

is advantageous in that, firstly it uses very little amount of sample when compared to the normal disc diffusion
method and hence,

it can be used for bioassay-guided isolation of compounds. Secondly, since the crude extract is resolved into its
different

components, this technique simplifies the process of identification and isolation of the bioactive compounds (Rah
Alison et al.,

1991).

High performance liquid chromatography

High performance liquid chromatography (HPLC) is a versatile, robust, and widely used technique for the
isolation of

natural products (Cannel, 1998). Currently, this technique is gaining popularity among various analytical
techniques as the main

choice for fingerprinting study for the quality control of herbal plants (Fan et al., 2006). Natural products
are frequently isolated

following the evaluation of a relatively crude extract in a biological assay in order to fully characterize
the active entity. The
biologically active entity is often present only as minor component in the extract and the resolving
power of HPLC is ideally

suited to the rapid processing of such multicomponent samples on both an analytical and preparative
scale. Many bench top

HPLC instruments now are modular in design and comprise a solvent delivery pump, a sample
introduction device such as an

auto-sampler or manual injection valve, an analytical column, a guard column, detector and a recorder
or a printer.

Chemical separations can be accomplished using HPLC by utilizing the fact that certain compounds have
different

migration rates given a particular column and mobile phase. The extent or degree of separation is
mostly determined by the

choice of stationary phase and mobile phase. Generally the identification and separation of
phytochemicals can be accomplished

using isocratic system (using single unchanging mobile phase system). Gradient elution in which the
proportion of organic

solvent to water is altered with time may be desirable if more than one sample component is being
studied and differ from each

other significantly in retention under the conditions employed.

Purification of the compound of interest using HPLC is the process of separating or extracting the target
compound

from other (possibly structurally related) compounds or contaminants. Each compound should have a
characteristic peak under

certain chromatographic conditions. Depending on what needs to be separated and how closely related
the samples are, the

chromatographer may choose the conditions, such as the proper mobile phase, flow rate, suitable
detectors and columns to get an

optimum separation.

Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any
compound by
HPLC, a detector must first be selected. Once the detector is selected and is set to optimal detection
settings, a separation assay

must be developed. The parameters of this assay should be such that a clean peak of the known sample
is observed from the

chromatograph. The identifying peak should have a reasonable retention time and should be well
separated from extraneous

peaks at the detection levels which the assay will be performed. UV detectors are popular among all the
detectors because they

offer high sensitivity (Lia et al., 2004) and also because majority of naturally occurring compounds
encountered have some UV

absorbance at low wavelengths (190-210 nm) (Cannel, 1998). The high sensitivity of UV detection is
bonus if a compound of

interest is only present in small amounts within the sample. Besides UV, other detection methods are
also being employed to

detect phytochemicals among which is the diode array detector (DAD) coupled with mass spectrometer
(MS) (Tsao and Deng,

2004). Liquid chromatography coupled with mass spectrometry (LC/MS) is also a powerful technique for
the analysis of complex

botanical extracts (Cai et al., 2002; He, 2000). It provides abundant information for structural elucidation
of the compounds when

tandem mass spectrometry (MSn

) is applied. Therefore, the combination of HPLC and MS facilitates rapid and accurate

identification of chemical compounds in medicinal herbs, especially when a pure standard is unavailable
(Ye et al., 2007).

The processing of a crude source material to provide a sample suitable for HPLC analysis as well as the
choice of

solvent for sample reconstitution can have a significant bearing on the overall success of natural product
isolation. The source

material, e.g., dried powdered plant, will initially need to be treated in such a way as to ensure that the
compound of interest is
efficiently liberated into solution. In the case of dried plant material, an organic solvent (e.g., methanol,
chloroform) may be used

as the initial extractant and following a period of maceration, solid material is then removed by
decanting off the extract by

filtration. The filtrate is then concentrated and injected into HPLC for separation. The usage of guard
columns is necessary in

the analysis of crude extract. Many natural product materials contain significant level of strongly binding
components, such as

chlorophyll and other endogenous materials that may in the long term compromise the performance of
analytical columns.

Therefore, the guard columns will significantly protect the lifespan of the analytical columns.

Non-chromatographic techniques

Immunoassay

Immunoassays, which use monoclonal antibodies against drugs and low molecular weight natural bioactive
compounds,

are becoming important tools in bioactive compound analyses. They show high specificity and sensitivity for
receptor binding

analyses, enzyme assays and qualitative as well as quantitative analytical techniques. Enzyme-linked
immunosorbent essay

(ELISA) based on MAbs are in many cases more sensitive than conventional HPLC methods. Monoclonal antibodies
can be

produced in specialized cells through a technique known as hybridoma technology (Shoyama et al., 2006). The
following steps

are involved in the production of monoclonal antibodies via hybridoma technology against plant drugs:

(i) A rabbit is immunized through repeated injection of specific plant drugs for the production of specific antibody,
facilitated due

to proliferation of the desired B cells.

(ii) Tumors are produced in a mouse or a rabbit.

(iii) From the above two types of animals, spleen cell (these cells are rich in B cells and T cells) are cultured
separately. The

separately cultured spleen cells produce specific antibodies against the plants drug, and against myeloma cells that
produce
tumors.

(iv) The production of hybridoma by fusion of spleen cells to myeloma cells is induced using polyethylene glycol
(PEG). The

hybrid cells are grown in selective hypoxanthine aminopterin thymidine (HAT) medium.

(v) The desired hybridoma is selected for cloning and antibody production against a plant drug. This process is
facilitated by

preparing single cell colonies that will grow and can be used for screening of antibody producing hybridomas.

(vi) The selected hybridoma cells are cultured for the production of monoclonal antibodies in large quantity against
the specific

plants drugs.

(vii) The monoclonal antibodies are used to determine similar drugs in the plants extract mixture through enzyme-
linked

immunosorbent essay (ELISA).

Phytochemical screening assay

Phytochemicals are chemicals derived from plants and the term is often used to describe the large number of
secondary

metabolic compounds found in plants. Phytochemical screening assay is a simple, quick, and inexpensive
procedure that gives

the researcher a quick answer to the various types of phytochemicals in a mixture and an important tool in
bioactive compound

analyses. A brief summary of the experimental procedures for the various phytochemical screening methods for
the secondary

metabolites is shown in Table 2. After obtaining the crude extract or active fraction from plant material,
phytochemical screening

can be performed with the appropriate tests as shown in the Table 2 to get an idea regarding the type of
phytochemicals existing

in the extract mixture or fraction.

Fourier-transform infrared spectroscopy (FTIR)

FTIR has proven to be a valuable tool for the characterization and identification of compounds or functional groups

(chemical bonds) present in an unknown mixture of plants extract (Eberhard et al., 2007; Hazra et al., 2007). In
addition, FTIR
spectra of pure compounds are usually so unique that they are like a molecular "fingerprint". For most common
plant compounds,

the spectrum of an unknown compound can be identified by comparison to a library of known compounds.
Samples for FTIR can

be prepared in a number of ways. For liquid samples, the easiest is to place one drop of sample between two
plates of sodium

chloride. The drop forms a thin film between the plates. Solid samples can be milled with potassium bromide (KBr)
to and then

compressed into a thin pellet which can be analyzed. Otherwise, solid samples can be dissolved in a solvent such as
methylene

chloride, and the solution then placed onto a single salt plate. The solvent is then evaporated off, leaving a thin
film of the

original material on the plate

REFRENCES:-

1. Rakesh K. Sindhu*, Prabhjot Kaur, Sanjana, Manshu, Parneet Kaur, Anju Goyal, Rajni Bala and
Amanpreet Sandhu; PHYTOCHEMICALS: EXTRACTION, ISOLATION METHODS, IDENTIFICATION AND
THERAPEUTIC USES: A REVIEW; Plant Archives Vol. 21, Supplement 1, 2021 pp. 174-184
https://www.researchgate.net/publication/349182048_PHYTOCHEMICALS_EXTRACTION_ISOLATION_M
ETHODSIDENTIFICATION_AND_THERAPEUTIC_USES_A_REVIEW