The progress in the techniques for isolation and analysis has led to the identification of many unknown compounds. Various processes are involved in the isolation of the particular compound from its plant material. The isolation process possibly will depend on the nature of the active constituent present in the crude drug. For example, trapping of the components is done for the volatile chemicals while extraction of nonvolatile compounds using organic solvents is also done. The isolation of components is done for both known constituents and also for the components which are unknown and the process of the separation, purification and identification of compounds coupled with biological screening is a demanding task. After the extraction of the required crude extract from the plant, the need of the marker component to be isolated and identified is also equally important for its study with respect to chemical nature or even for the development of newer formulations. The advances in the field of chromatographic techniques have enabled the separation and purification of compounds.

1. Contents……………….
1. Introduction
2. Fractional Crystallization
3. Fractional Distillation
4. Fractional Liberation
5. Sublimation
6. Introduction: Chromatography
7. Classification of Chromatography
8. Paper Chromatography
9. Thin Layer Chromatography (TLC)
10. High performance Liquid Chromatography (HPLC)
11. High performance Thin Layer Chromatography (HPTLC)
12. Gas Liquid Chromatography (GLC)
13. Column Chromatography
14. Flash Column Chromatography
15. Ultra Performance Liquid Chromatography (UPLC)

2. Chromatographic Techniques
Presented by
Sonali S Gadge
Assistant Professor
P R Patil Institute of Pharmacy, Talegaon (SP), Dist-
Wardha

3. • The progress in the techniques for isolation and analysis has led to the
identification of many unknown compounds.
• Various processes are involved in the isolation of the particular compound
from its plant material.
• The isolation process possibly will depend on the nature of the active
constituent present in the crude drug.
• For example, trapping of the components is done for the volatile chemicals
while extraction of nonvolatile compounds using organic solvents is also
done.
• The isolation of components is done for both known constituents and also for
the components which are unknown and the process of the separation,
purification and identification of compounds coupled with biological
screening is a demanding task.
• After the extraction of the required crude extract from the plant, the need of
the marker component to be isolated and identified is also equally important
for its study with respect to chemical nature or even for the development of
newer formulations.
• The advances in the field of chromatographic techniques have enabled the
separation and purification of compounds.

4. Fractional Crystallization
• Crystallization is an old but a very important method for the
purification of the compounds from the mixture.
• Crystallization mostly depends upon the inherent character of the
compound which forms crystals at the point of supersaturation in
the solvent in which it is soluble.
• Many phytopharmaceuticals and natural products are crystalline
compounds which tend to crystallize even in the mixtures.
• Compounds, such as sugars, glycosides, alkaloids, steroids,
triterpenoids, flavonoids, etc., also show the crystalline nature.
• The processes, such as concentration, slow evaporation,
refrigeration are used for crystallizing the products.
• In case of sugars, osazone formation leads to the crystallization of
the derivatives in the form of various types of crystals enabling the
analysis of the sugars.

5. Fractional Distillation
• For the distillation the component should have volatile nature.
Therefore, fractional distillation is mostly used for the separation
of essential oil components.
• Most of the volatile components are steam volatile and if the
process of fractional distillation is skillfully used, various low-
boiling and high-boiling components can be separated from the
total oil.
• This process is largely used for the separation of hydrocarbons
from the oxygenated volatile oil components— the product
referred to as terpenless essential oils.
The components like citral, citronellal and eucalyptol are even now
separated by fractional distillation.
• It is used in the separation of hydrocyanic acid from plant
material.

6. Fractional Liberation
• In the process of fractional liberation, groups of the compounds
having the tendency of precipitation come out of the solution.
• In certain cases the nature of the compound such as alkaloids is
modified by converting to their salt form or free bases.
• If such alkaloidal compounds are more in number with variation in
basic nature, with such conversions base liberation, these may be
brought about from a weaker base to relatively stronger base.
• The process is often used even at the industrial level for the
separation of cinchona alkaloid quinine, isolation of morphine and
many other alkaloids.
• By using the similar processes phenols, organic acids, like
compounds are liberated from the solution.

7. Sublimation
• As a matter of fact there are very few natural products which have
sublimating nature.
• In this process the compound if subjected to heating, changes from
solid state to gaseous state directly without passing through a
phase of liquid.
• Such compounds from the gaseous state get deposited on the
cooler surface in the form of crystals or cake.
• The process is traditionally used for the separation of camphor
from the chips of wood of Cinnamomum camphora to obtain solid
sublimate of camphor.
• Sublimation can also be used for the isolation of caffeine from tea
or for the purification of material present in a crude extract.
• In the inorganic compounds, sublimation is the well-known
process for the isolation and purification of sulphur.

8. • These methods are effective in separation, purification and identification
of many compounds.
• However, difficulty arises in case of compounds where individual
components have similar physical and chemical properties i.e. mixture
of liquids having very close boiling points, etc.
• However, these methods are not satisfactory in biological materials.
• Chromatographic methods represent the most useful and powerful
technique for these problems.
• They are used for the separation of components of a complex mixture.
• Because of the rapidity and effectiveness, chromatography has been
used in all the fields e.g. chemistry, biology, medicine, dyes, forensics
and clinical studies with the advantage over other methods.
• Similarly in plant extract there are various phytoconstituents are present.
Chromatography will be helpful to separate this phytoconstituents into
its individuals.

9. Advantages
• They are relatively gentle and disallow the decomposition of
substances.
• This is important for the labile substances and substances of
biological origin.
• Separations can be carried out on micro or semi micro scale ,i.e.
a small quantity of sample is required for analysis.
• Chromatographic techniques are simple, rapid and require simple
apparatus.
• The complex mixtures can be handled with comparative ease.
• In the chromatographic techniques, there are two phases, one
phase is Stationary phase, and the other is mobile phase.
• The mobile phase passes over the stationary phase and transports
components of the mixture at different speeds in the direction of
the flow of mobile phase.

10. Definition
The term chromatography was derived from the two Greek words
(Chroma- colour, Graphos- writing or Recording) meaning colour
writing.
Tswett (1906) defined chromatography as “the method in which
components of a mixture are separated on an adsorbent
column in a flowing system.”
IUPAC has defined Chromatography as “the method used
primarily for the separation of the components of a sample, in
which the components are distributed between two phases, one
of which is stationary while the other is mobile.”
The stationary phase may be a solid or liquid supported on a solid or
a gel and may be packed in a column, spread as a layer or
distributed as a film. The mobile phase may be gaseous or liquid.

11. History
Chromatography is a family of analytical chemistry techniques for
the separation of mixtures.
The study of chromatography was started in the eighteenth
century.
• It was the Russian botanist, Mikhail Tswett, who invented the
first chromatography technique in 1901, which was based on
adsorption principle.
• He used a glass column of calcium carbonate for separation of
chlorophyll pigments from plant by using Petroleum ether.
The pigments, according to their adsorption patterns, were
resolved into various coloured zones; he then separated and
estimated them.
• In 1952, Archer John Porter Martin and Richard Laurence
Millington Synge were the two scientists who identified partition
chromatography.

12. The theorotical aspects of chromatography were first studied by
Wilson in 1949, he discussed the quantitative aspects in terms of
diffusion, rate of adsorption and isotherm, non-linearity, etc.
Glueckauf in 1949, described the column performance in terms of
stationary phase, particle size and diffusion.
However, it was Van Deemter and co-workers who in 1956
developed the rate theory to describe the separation process.
There has been continuous development in chromatography
particularly in techniques, materials and requirements of
instrumentation which has resulted in the efficient, reliable and
sensitive chromatographic methods in use today.

13. Classification
• If the right adsorbent material, mobile fluid and operating conditions are
employed, any soluble or volatile component can be separated using
chromatography.
• Even structurally very similar components can be separated with
chromatography. The principle of chromatography differs according to
the stationary and mobile phase used.
• Thus depending upon the two phases, the chromatographic methods are
classified as:
Partition Chromatography
• This form of chromatography is based on a thin film formed on the
surface of a solid support by a liquid stationary phase. Solute
equilibrates between the mobile phase and the stationary liquid.
• This involves liquid or gas as mobile phase and another liquid or solid as
a stationary phase.
The operations include:
1. Partition column chromatography 2. Paper chromatography
3. Thin layer chromatography 4. Gas-liquid chromatography
5. High Performance Liquid Chromatography

14. Adsorption Chromatography
• Adsorption chromatography is probably one of the oldest types
of chromatography around.
• It utilizes a mobile liquid or gaseous phase that is adsorbed onto the
surface of a stationary solid phase. The equilibration between the
mobile and stationary phase accounts for the separation of different
solutes.
• This involves liquid or gas as mobile phase and adsorbent solid as
stationary phase.
The types under this category includes:
1. Adsorption column chromatography
2. Thin layer chromatography
3. Gas-Solid chromatography

15. Ion Exchange Chromatography
In this type of chromatography, the use of a resin (the stationary solid
phase) is used to covalently attach anions or cations on to it. Solute
ions of the opposite charge in the mobile liquid phase are attracted
to the resin by electrostatic forces.
Molecular Exclusion Chromatography
Also known as gel permeation or gel filtration, this type of
chromatography lacks an attractive interaction between the
stationary phase and solute.
• The liquid or gaseous phase passes through a porous gel which
separates the molecules according to its size.
• The pores are normally small and exclude the larger solute
molecules, but allow smaller molecules to enter the gel, causing
them to flow through a larger volume.
• This causes the larger molecules to pass through the column at a
faster rate than the smaller ones.
• The gels are of different types like Rigid gels, Semi-rigid gels and
Soft-gel

16. Affinity chromatography
• This technique is mainly used for the separation of proteins,
peptides, enzymes, antigens and antibodies, nucleic acids,
ribosomes, etc.
• The adsorbent used is one of the biological substance (which may
be termed as receptor) having the specific affinity for other
substances.
• These two substances are the biologically interacting pairs.
• Such adsorbent is attached to a porous stationary phase and placed
in a column, when a mixture containing other component of the
adsorbent (interacting pair) is passed through the stationary phase,
selective separation process.

17. • Affinity chromatography is efficiently applied for the separation of
enzymes, utilizing the technique for enzyme-substrate interaction.
• In the place of substrate, the ligand which is a competitive inhibitor
for enzyme is used.
• This technique is applied for the separation of m-RNA, purification
of enzymes, isolation of antibodies, etc.

19. Paper Chromatography
• Paper chromatography is carried out by specially designed filter
paper. The principle of separation may be here partition or
adsorption.
• If the filter paper is impregnated with alumina or silica, the
adsorption principle will be applied for separation whereas if
moisture/water present in the pores of cellulose fibre it works as
stationery phase and solvent as mobile phase then the principle of
separation will be partition.
• In general paper chromatography refers to the partition and
adsorption principle.
• Various types of filter paper are used for paper chromatography. It
may be Whatmann, paper of different grade, acid or base wash filter
paper, paper modified with glycol, formamide, methanol, glass fibre
type paper, hydrophobic paper (OH group can be acetylated) or
paper can be impregnated with alumina, silica or ion exchange
resin.

20. • The size of the paper should be suitable for the size of the chamber
and apply the sample by using capillary or micro pipette.
• The sample should be dissolved in the mobile phase and applied
with low concentration with small zone.
• In the mobile phase pure solvent or mixture of solvent or buffer
solution can be used.
• There may be ascending, descending, circular, two dimensional and
ascending descending type different development techniques.
• Ascending development technique is conventional technique in
which the mobile phase moves against the gravity and spot the
sample at bottom portion.
• While in descending development the mobile phase kept at the top
and the solvent flow down the paper.
• Here the samples applied at the top and the development is fast due
to gravity assisted solvent flow.

23. After development of the chromatogram the isolated compound can be
visualized by detecting agents. Detecting agents can be two types:
(a) Destructive type
(b) Non destructive type
• In destructive type the sample cannot be recovered or it will be
destroyed due to chemical reaction of spraying reagent with sample.
• While in non destructive method sample can be recovered. In non
destructive method sample can be detected by UV chamber method,
densitometric method or iodine chamber method.
• Paper chromatography can be use for both qualitative and
quantitative purpose. For qualitative purpose Rf value can be
determined.
Rf (Retardation factor) = Distance travelled by solute/ Distance travelled by solvent front
• For quantitative purpose the density of the spot can be measured or the spot can
be eluted with the solvent and analysed by conventional techniques like
spectrophotometric method or electrochemical methods

24. Operational technique
The following points should be taken into account in paper
chromatography
1. Choice of filter paper
• Chromatography paper is a specially manufactured paper.
Whatman filter paper is used extensively.
• In general, this filter paper contains 98-99% of the α-cellulose.
• The mineral content may vary from 0.07-0.01%.
• Besides this, β-cellulose, ether soluble matter, ammonia and
Lipophilic substances (waxes, fats, etc) are also present.
• Chromatographic papers are available in packs of 100 and 500
sheets of 46×57 cm or 58×68 cm and are cut to required size.
• The rectangular or square papers are cut from the sheet for
separation of substances.
• There are various grades and types of paper available for
separation of sample.

25. • The proper choice of paper depends upon the sample and solvent
system used.
• Another important factor that governs the choice of the paper is
whether the paper is to be used for the Quantitative, Qualitative or
preparative chromatographic analysis.
• The choice of the paper is also based on the thickness, flow rate,
purity and net strength.
2. Modified filter papers
• For efficient separation of certain substances, specially treated or
modified filter papers are used, i.e. buffered or treated papers like
Whitman- phosphate, whitman-citrate or paper treated with
alumina, silicic acid, etc.
• In case of reversed phase chromatography, paper is impregnated
with mobile phase, dried and then used.

26. 3. Preparation of paper
• Once the type of paper is decided, it is cut in desired size and shape
depending upon the work to be carried out.
• Generally, rectangular or square shapes are used.
• After noting the direction of run on paper, start line is marked.
• While storing, paper should be kept away from any fume
(especially ammonia) and should not be subjected to large changes
in humidity.

27. 4. Preparation of sample
• The mixture is to be separated is applied to the paper as a solution.
• It is important to choose proper solvent for making solution.
• Generally, a weighed amount of mixture is dissolved in volatile solvent and a
minimum volume of concentrated solution is applied on the paper avoiding
diffusion of spot.
5. Application of the sample
• The starting line is marked on the paper with an ordinary pencil some 5 cm from
the bottom edge.
• On the starting line marks are made about 2 cm apart from each other.
• Micropipette or glass capillary or platinum loop is used for application of sample.
• The micro-syringe is also used for application of sample.
• The sample may be applied as spots or bands. Generally, size of the spot should
be as small as possible. Diameter of the spot should not exceed 5 mm.
• When solution is very dilute, it can be concentrated on paper by applying a series
of drops to the same spot, allowing the each drop to evaporate before applying
the next.
• Drying of the spotted chromatogram should be carried out carefully in air.
• Hot air is not advisable particularly for acid solution as it may cause blackening
of the paper.

28. 6. Solvents
• A number of solvents can be used in the paper chromatography.
• The selection of proper solvent depends upon the nature of
substance to be separated.
• Factors which affects the selection are viscosity, polarity, surface
tension, etc.
• In general, one-phase system is used for development in paper
chromatography, avoiding the two-phase system.
• The solvent should be inexpensive and very pure.

29. 7. Chromatographic tank or chambers
• The chromatographic tanks are made from many materials like glass,
plastic or stainless steel.
• Glass tanks are preferred and are most commonly used.
• They are available in various sizes depending upon the length and
breadth of paper and type of development.
• The chambers of tanks have a lid with a hole (closed during
development) for introducing solvent through it.
8. Development of chromatogram
For proper development following points are to be considered:
1. Sufficient amount of solvent should be present in the chamber.
2. During development, paper should be freely suspended and should be
vertical.
3. Large temperature changes and exposure to light should be avoided.
4. The atmosphere of the chamber should be saturated with the solvent’s
vapor.

31. • The paper is so dipped in the solvent that the spots will not dip
completely into the solvent.
• The solvent will run over the paper by capillary action.
• It is allowed to maximum distance not exceeding two-third of total
height of paper for better and efficient resolution.
• After development is complete paper is taken out of the chamber
carefully.
9. Drying of the chromatogram
• The wet chromatogram after development is dried in special
cabinet heated electrically with temperature controls.
• The drying can also be carried out by transferring chromatograms
to racks and putting in drying cabinet.
• They are dried by cold or hot air depending on the volatility of the
solvent.
• A simple hair dryer is a convenient device to dry chromatograms.

32. 10. Location of spots
Once the developed chromatogram is dried, next step is to locate the
spot.
If the substances are coloured, they are visually detected easily, but
for colourless substances, various methods are used:
1. Physical method:
• Physical methods have the advantage that substances on paper are
not converted into other compounds can be covered for further
studies.
• Physical methods used are observation under UV light, detection
of fluorescence and radioisotope measurements.
2. Chemical methods:
• In this, chemical treatment is used to develop a colour
chromatograms are exposed to vapors or gases of chemicals or
sprayed with reagent.
• The chemicals and reagents so used are called “locating agents”.

33. Thin Layer Chromatography
The thin layer chromatography is a widely used, fast technique
for the qualitative analysis of a mixture of compounds.
• Stahl in 1958 developed the standard equipment and technique for
analysis by thin layer chromatography, used for separation of plant
extract.
• Before that in 1938 Izmailou and Shraiber separate the
phytoconstituent using 2 mm thick alumina on glass plate.
• Principle of separation totally depend upon the coating substance
apply on the plate.
• If silica or alumina is apply as coating substance adsorption
principle will take place.
• If coating substance is sephadex molecular exclusion will be
principle of separation.
• Ion exchange will be the mechanism of separation if resin will be
the coating substance and cellulose like coating substance adopt
partition mechanism.

34. • The stationary phase consists of a thin layer of adsorbent like silica gel,
alumina, or cellulose on a flat carrier like a glass plate, a thick aluminium
foil, or a plastic sheet.
• Silica gel G is one of the most common adsorbent uses for the coating of
plate.
• Here G represents the gypsum (CaSO4) which is around 15 percent of
the silica gel and act as binder.
• The other adsorbent which are use in the stationary phase is alumina,
cellulose, kieselguhr, polyamide powder and others.
Inorganic adsorbents like Alumina, Kieselghur, Magnesia, Magnesium
silicate, Calcium silicate, calcium carbonate, calcium hydroxide, etc. are
used.
Organic adsorbents like Cellulose and its salicylates, charcoal and activated
carbon, dextran gels, polyamides, polyethylene powders, etc are used.
• Most often the adsorbent are applied on the glass plate.
• The dimension of the glass plate may be vary from 20×20 cm to 20×5 cm.
Sometime even microscopic plates are also used to examine the progress
the chemical reaction by TLC method.

35. The glasses are coated with adsorbent with different techniques like:
1. Pouring, 2. Dipping, 3. Spraying, 4. Spreading techniques.
In pouring technique the prepared slurry is poured onto the glass
plate and try to maintain an equal level surface.
In dipping technique two plates are stick together and dip into the
slurry and then remove from the slurry. Separate both plates, one
surface will be coated and other one will be dried.
In the spray technique adsorbent is sprayed on glass plate using a
machine sprayer.
The best applying technique is spreading technique in which the
slurry is kept in the TLC spreader box.
• The glass plates of particular size are kept on base plate. The
prepared slurry kept in the reservoir of spreader box.
• Adjust the thickness of slurry by rotating the knob present in the
spreader box then spreader box is moved over the glass plate.
• Allow them for air drying and activate them at 100-120°C for one
hour.

36. • Sample can be applied by micro pipette or capillary tube. It should
be spotted at least 2 cm above the base of or on such height that
spotted area should not immersed in mobile phase.
• The area of the spotted sample should be minimum with sufficient
concentration.
• Mobile phase and TLC plate should be kept in the development
chamber.
• Chamber should be saturated with mobile phase otherwise edge
effect may found in which the solvent front in the middle of plate
move faster than the edge. The mobile phase selected on the basis
of phytoconstituent which have to be separated and the nature of
stationary phase.
• It may be single or the mixture of the solvent. The polarity of the
solvent adjusted such that it easily separated the phytoconstituents.

37. • The different development techniques used in the TLC are one
dimensional, two dimensional, horizontal and multiple
developmental techniques.
• In one dimensional or vertical technique, the mobile phase flows
against the gravity due to the capillary action.
• After development of the chromatogram the isolated compound can
be visualized by detecting agents.
• Several methods exist to make colourless spots visible.
• Often a small amount of a fluorescent dye is added to the adsorbent
that allows the visualization of UV absorbing spots under a black
light (UV254).
• Even UV light without fluorescent dye could scan the compounds,
both in long (365 nm) and short (254 nm) wavelength ultraviolet
light.
• Iodine vapors are a general unspecific colour reagent.

38. • Specific colour reagents exist into which the TLC plate is dipped or
which are sprayed onto the plate.
• Dragendorff ’s reagents are used in the form of sprays for the
general detection of alkaloids.
• Antimony trichloride in chloroform is used as a spray reagent for
steroidal compounds and other terpenoids.
• Ammonia vapour can be used for free anthraquinone compounds
and Fast Blue Salt B ‘Merck’ for cannabinoids and phloroglucides.
• This chromatography can be use for both qualitative and
quantitative purpose. For qualitative purpose Rf value can be
determine.
Rf (Retardation factor) =Distance travelled by solute/Distance travelled by solvent front
• For quantitative purpose the density of the spot can be measured or the spot can be eluted
with the solvent and analysed by conventional techniques like spectrophotometric method
or electrochemical methods.

39. • It is effective, comparatively cheap as relatively small amounts of
analyte, adsorbent and solvents are required.
• The use of appropriate developing agents can help understand the
compound properties and can be quantified by careful
standardization of procedures.

41. 1. Paper chromatography technique can be used for-
(a) Quantitative analysis only
(b) Qualitative analysis only
(c) Both
(d) None of the above
2. The stationary phase used in TLC is:
(a) Liquid held between glass plate and silica gel
(b) Silica gel
(c) Glass plate
(d) None of the above
3. The stationary phase in TLC is:
(a) Adsorbent
(b) Liquid held between glass plate and adsorbent
(c) Glass plate
(d) None of the above
4. In Quantitative TLC radioactive material can be studied by:
(a) Visual comparison
(b) Densitometer
(c) Gravimetry
(d) Geiger counter
5. semi-rigid gel used for exclusion chromatography
(a) Sephadex
(b) Gelatin
(c) Cellulose
(d) Alumina

42. High performance Liquid Chromatography
• This is a versatile natural product isolation technique which is
similar to flash chromatography; however, high pressure (up to
4,000–5,000 psi) is applied to the system to move the mobile phase
through the smaller particle sized (2–10 μm) stationary phase bed.
• The column is stainless steel to withstand the high pressure.
• It employs relatively narrow columns about 5 mm diameter for
analytical work, operating at ambient temperature or up to about
200°C
• HPLC run under normal phase mode or reverse phase mode which
depends upon the polarity of stationary phase and mobile phase. In
normal phase mode the mobile phase is non polar and stationary
phase is polar while in the reverse phase mode mobile phase is
polar and stationary phase is non polar..

43. • The separation technique in HPLC may isocratic or gradient.
• In the isocratic separation technique the polarity of mobile phase
is maintained same throughout the process while in gradient
separation the polarity of mobile phase can be gradually change
by changing the ratio of the different solvent present in the mobile
phase.
• The HPLC unit consists of different parts like pump, mixing unit,
injector, guard column, analytical column, detectors and recorder.
The solvent pushed into the column under very high pressure
(1000 to 5000 psi) because the particle size of the stationary phase
is very small (2-10 μm) so the resistance force by mobile phase is
very high.

44. To generate such pressure mechanical or pneumatic pumps are used.
Mechanical pump works under constant flow rate and for generally
analytical purpose.
Pneumatic pumps work under constant pressure with highly compressed
air.
• Flow rate and back pressure is controlled by the check valve.
• The pulse generate by the pump is dampen by pulse dampeners.
• In the mobile phase there may be single solvent or may be more than
one solvent.
• To mix properly more than one solvent there is a mixing unit in HPLC
apparatus.
It may be two types
a) low pressure mixing chamber
b) high pressure mixing chamber.
• In low pressure mixing chamber helium is used for degassing the
solvent. The quality of solvent should be very good or may be HPLC
grade.

45. • The sample can be introduced into the HPLC by manual method or
auto injection method.
• It can be septum injection, stop flow (on line) injection or loop
valve (Rheodyne injectors).
• In septum injection one rubber septum is use to inject the sample
but the septum should withstand to high pressure.
• Sometime stop the flow of mobile phase and introduce the sample
through a valve device but the most popular is loop valve
(Rheodyne) injector type.
• In this first sample is loaded on the injector (20- 50 μl) which is the
load position and then inject the sample without disturbing the flow
of mobile phase through inject mode.

46. Columns
• A very important part of the HPLC is the analytical column but
before the analytical column one guard column is also provided
which is comparatively cheaper to the analytical column and
helpful to increase the life of analytical column by removing the
entry of unwanted material in the analytical column.
• The performance of the analytical column decides the efficiency of
separation. Column can be made of polyether ketone, stainless
steel, polyethylene or sometime glass.
• Stainless steel can be most widely used column because it can
withstand the high pressure.
• The column length can vary from 5-30 cm with diameter 2-50 mm
using adsorbent particle size 1 μm to 20 μm with uniform spherical
porous material. 1gm stationary phase may have average 400 sqm
area.

48. Detectors
• The detector use in the HPLC can be divided into the two
categories, the solute property detector and bulk property detector.
• Bulk property detector also known as universal detector, measure
the characteristic to all analyte by analyzing mobile phase without
or with sample.
• The good examples of these are conductivity detector or refractive
index detector.
• Solute property detector corresponds to the particular unique
property of the analyte like UV detector, Flourimetric detector,
Photodiode array detector.

49. Recorder
• The response of the separated constituents can be recorded by the
recorder.
• Recorder amplifies the response which is detected by the detector.
• It record the time at which the constituent are separated or retention
time.
• Integrator measures the height and width of the peaks, peak area
and percentage of the area.
• HPLC can be use for the qualitative analysis by measuring the
retention time of sample under standard condition but generally it is
use for quantitative analysis by direct comparison method,
calibration curve method and by internal standard method.

50. HPLC Chromatogram of Curcumin from Curcuma longa extract
by Gradient system

51. Diagrammatic representation of HPLC

52. 1. In chromatographic separation, the different species in the sample, undergo the process of:
(a) Chemical interaction
(b) Partition
(c) Volatilisation
(d) ionisation
2. In HPLC, the analytical performance improves when:
(a) Particle diameter is increased
(b) Particle diameter is reduced
(c) Coarser particles are paired with shorter columns
(d) Successive injection of sample
3. Derivatisation techniques in HPLC are intended to enhance:
(a) Molecular weight
(b) Detectability
(c) Reversibility
(d) Reproducibility
4. Official method for the analysis of Ciprofloxacin is by:
(a) Potentiometry
(b) HPLC
(c) Gas Chromatography
(d) Non-aqueous titration

53. High Performance Thin Layer Chromatography
• The high-performance thin layer chromatography is a sophisticated and
automated form of TLC.
• It is useful in qualitative and quantitative analysis of natural products.
• The principle of separation is adsorption (same as that of TLC).
• In HPTLC, the precoated plates are used and the particle size of stationary
phase is less than 1μ in diameter.
• There is a wide choice of stationary phases like silica gel for normal
phase and C18, C8, etc., for reverse phase mode.
• HPTLC provides a higher efficiency than TLC because adsorbents used
are small and uniform in size.
• A very less amount of sample is spotted on the plate so the sample
prepared should be highly concentrated.
• The size of the sample spot should not be more than 1 mm in diameter.
• The samples are spotted by various techniques and commonly used
method is by semiautomatic linomet V apparatus.

54. Stationary phase, TLC plates and solvents
• Silica gel is a most popular stationary used in HPTLC afterwards
cellulose.
• Homemade sheets/plates show bigger particle and not shows
homogenous so it is not suitable for HPTLC analysis.
• Precoated HPTLC plates which are available in the market are more
appropriate for HPTLC analysis.
• Plastic sheet or aluminium foil supported HPTLC plate are now more
popular compare to the glass plate supported HPTLC plate because they
can easily cut into desired sizes as well as require less space to keep in the
lab but glass is more resistant to heat and chemical reaction compare to
the aluminium and plastic sheets.
• Mean particle size in HPTLC is 5-6μm compare to the 10-12μm in
classical TLC. The layer thickness is 100-200μm in HPTLC but 250 μm
in TLC. The relative humidity plays a crucial role to reproduce the result.
• The relative humidity is variable in the laboratory condition.
• To avoid such variation it is desirable to precondition (saturate the TLC
chamber with the vapours of mobile phase) the TLC chamber.
Preconditioning is more required with highly polar mobile phase.

55. • The environment of the lab contains various dirt particles, vapour of
various gases which can be deposited into the HPTLC plate. This
impurity can be removed by prewashing of the plate.
• Run the methanol or such other solvent without apply the sample
on HPTLC plate so that all dirt particle or impurities on the HPTLC
plate will be collected on the upper edge which can be removed.
• After prewashing the plate are kept in the oven for 15-20 min at
120°C which is known as conditioning.
Mobile Phase
• The mobile phase use for HPTLC should be utmost pure. Presence
of antioxidant and stabilizer altered the nature of chemical. They
should be kept in proper storage condition.
• Polarity, viscosity, volatility are some points which effect
chromatographic procedure.
• The mobile phase should be kept as simple as possible.

56. Chromatographic Development
• Chromatographic development is another important aspect in
HPTLC. Generally the glass development chambers are use for
such purpose.
• The various development chambers which can be used for
chromatographic development are:
1. Flat bottom chamber
2. Twin trough chamber
3. Sandwich chamber
4. Horizontal chamber
5. Automatic development chamber
6. Forced flow development chamber
7. Automatic multiple development chamber.
• Twin trough chamber uses less solvent. The linear development of
the chromatogram is the best method in which the HPTLC plate is
placed vertically in an appropriate chamber.
• The solvent is run by capillary action.

57. Detection
• After the development of chromatogram HPTLC plates are dried and
evaluated for following method:
Evaluation by non destructive method:
1. Direct visual method
2. Evaluation under UV light:
Reversible Reaction:
1. Iodine Vapour
2. Ammonia Vapour
Non Reversible Reaction:
1. Fluoroscent dye
2. pH indicator
3. Wetting/ Dipping
4. Spraying technique
All the above technique come under non reversible detection technique in
which the isolated constituents cannot be recovered and destroyed after
detection.

58. Scanning and Documentation
• After the development of spot the HPTLC plate are scanned at
selected UV regions wavelength and the selected can be measured
in the computer in the form of peak and can be compared with
standard compound or other constituents.
• The obtained band can be converted into the peak.
• The height and area peak of the peak correspond to the
concentration of isolated constituents.
• This document can be stored in the computer for the further
references.

59. Gas Liquid Chromatography (GLC)
• Gas chromatography generally divided into GSC (gas solid
chromatography) and GLC (gas liquid chromatography).
• In either type the gas is a mobile phase but the stationary phase vary in
GC (gas chromatography).
• In GLC stationary phase is liquid and in GSC it is solid.
• In GLC the principle of separation is partition while in GSC it is
adsorption. Most of the time when named GC it is GLC.
• Generally a liquid is coated on solid support used as stationary phase.
The mixture which has to separate into the individual constituent has to
be converted into the vapour and mix with mobile phase (gas).
• The constituent which have more affinity towards stationary phase
travels slowly compare to those which have high affinity towards mobile
phase.
• The constituents are separated out on the basis of their partition
coefficient.
• GLC is a good technique to detect those compounds which are volatile
and thermostable.

60. • The volatile compound should be mixed with the carrier gas.
• The carrier gas may be hydrogen, helium, nitrogen. Hydrogen is a good
option for carrier gas because of good thermal conductivity and low
density but it reacts with unsaturated compounds.
• Helium is also good choice but it is expensive. Nitrogen is inexpensive
but has reduced sensitivity.
• Gases are generally stored under high pressure. To blow the gases under
uniform pressure and flow rate there is a need of flow meter.
• Generally rotameter and soap bubble meter are used to control the flow
of gases.
• The sample can be introduced into any form like solid, liquid or gaseous
form. Valves are suitable device to introduce the gas sample.
• Solid samples are generally dissolved in appropriate solvent and then
injected through septum.
• Liquid samples can be dispensed through either loop or septum devices.
• Septum should be made of high quality silicone rubber and can tolerate
high temperature and suitable for repeated injection.

61. Column
• Another important part of GLC which effect the separation of the
constituent is column.
• Made of either glass or stainless steel
The stainless steel column - have long life ,easy to handle
but sometime it may react the constituent which are not in the case of
glass column
glass column- fragile and difficult to handle.
• The column may be analytical (length 1-1.5 mt, diameter 3-6 mm)
or preparative column (length 3-6 mt, outer diameter 6-9 mm).
• Depending upon its nature it may be packed column, open tubular
or golay or capillary column and support coated open tubular
column (SCOT).

62. The pre-heaters are required for converting the sample into vapour
form and mix with Carrier gas.
They are installed along with injecting device.
Detectors
Detector like kathrometer, FID (flame ionization detector), AID
(argon ionization detector), ECD (electron captured detector) are
used.
The most sensitive of them are ECD (10-12).
Recorders record the response and amplify it. Recorder record the
retention time, record base line and record all the peak. Heights,
width, area of the individual peak, percentage of area are
calculated by integrators.

63. Detection of the sample
• The separation or detection of the sample can be improved in GLC
by derivatisation techniques.
• It can be precolumn derivatisation or post column derivatisation.
In precolumn derivatisation the sample is converted into more
volatile and thermostable derivative (like carboxylic acid, phenols,
sugars are converted into less polar by reagent BSA bis trimethyl
silyl acetamide).
Post column derivatisation is done generally to improve the detector
response for isolated constituents.
Uses
GLC can be use for the qualitative analysis by measuring the retention
time of sample under standard condition but generally it is used for
quantitative analysis by direct comparison method, calibration
curve method and by internal standard method.

64. 1. Derivatisation is done in GC:
(A) To convert a less polar compound to more polar compound
(B) To make the compounds non-volatile
(C) To convert a polar compound to a less polar compound
(D) To liquify a solid
2. Gas chromatographic technique can be used for:
(A) Qualitative analysis only
(B) Quantitative analysis only
(C) Both
(D) None of the above
3. In gas chromatography, derivatisation is desirable to:
(P) Improve the thermal stability of the compounds
(Q) Enable interaction with carrier gas
(R) Introduce a detector oriented tag into the molecule
(S) Remove contaminants
(A) P,Q (B) Q,R (C) P,R (D) P,S
4. A/An ____ is the detector of choice for GC separations of halogenated compounds.
(A) Electron Capture Detector
(B) Flame Ionization Detector
(C) Thermal conductivity Detector
(D) Universal detector

65. Column Chromatography
• Column chromatography developed by the American Chemist D T
Day in 1900, M.S. Twsett in 1906 used adsorption columns in his
investigations of plant pigments.
• Column chromatography is one of the most useful methods for the
separation and purification of components.
• This is solid-liquid technique in which the stationary
phase is solid and mobile phase is liquid
• Column chromnatography works on the adsorption
principle.
• Adsorption column chromatography, the adsorbent
packed in a glass column, the solvent is used as mobile
phase is called as an eluent.

66. Adsorbents
The usual adsorbent used in column chromatography are silica,
alumina, calcium carbonate, calcium phosphate, Magnesia, starch,
etc.
Alumina is generally suitable for chromatography of less polar
compounds silica gel G gives good results with compounds
containing polar functional groups.
Adsorbents used in column chromatography should meet the
following criteria:
1. Particles should be spherical in shape and uniform in size.
2. They should not react chemically.
3. It should be useful for separation of wide variety of compounds.
4. It should be inexpensive.

67. Selection of mobile phase
Success of chromatography depends upon proper selection of
stationary phase, it depends on the following:
1. Removal of impurities
2. Number of components to be separated
3. Length of column used
4. Quality of adsorbent used
Mobile phase
They act as solvent, developer & eluent.
Different mobile phases used are Petroleum ether, carbon
tetrachloride, cyclohexane, ether, acetone, benzene, toluene, esters,
water, etc. It can be used in either pure form or as mixture of
solvents

68. Columns
The main function of all the columns is to support the stationary
phase.
The material of the column is mostly good quality neutral glass since it
shouldn’t be affected by solvents.
An ordinary burette can also be used as column for separation.
Column dimensions - length & diameter ratio (10:1,30:1 or 100:1)
Various accessories are attached to the top and bottom of the column
for maintenance of the elution process.
The length of the column depends upon:
• Number of compounds to be separated
• Type of adsorbent used
• Quantity of the sample
• Affinity of compounds towards the adsorbent used
Better separation will be obtained with a long narrow column than
short thick column.

69. Preparation of the column
It consists of a glass tube with bottom portion of the column – packed
with glass wool/cotton wool or may contain asbestos pad, above
which adsorbent is packed.
After packing a paper disc kept on the top, so that the adsorbent layer
is not disturbed during the introduction of sample or mobile phase.
There are two types of preparing the column, they are:
i. Dry packing / dry filling
ii. Wet packing / wet filling
• The column should be free from impurity, before using column, it
should be washed properly and dry it.
• Before filling column with stationary phase, cotton/glass wool is
kept.
• It should be uniformly filled.

70. Dry Packing Technique
Adsorbent is packed in the column in dry form. Fill the solvent, till
equilibrium is reached
DEMERIT: Air bubbles are entrapped between Mobile phase and
stationary phase, cracks appear in the adsorbent layer.
After filling tapping can be done to remove void spaces.
Wet Packing Technique
• Ideal & common technique.
• The material is slurried with solvent and generally added to the
column in portions.
• Stationary phase settles uniformly and no crack in the column of
adsorbent.
• solid settle down while the solvent remain upward.
• This solvent is removed then again cotton plug is placed.

71. Development technique
By elution technique, the individual components are separated out
from the column. The two techniques are:
(i) Isocratic elution technique :
In this elution technique , same solvent composition or solvent of
same polarity is used throughout the process of separation.
Example: chloroform only.
(i) Gradient elution techniques:
Initially benzene, then chloroform, then ethyl acetate then chloroform.

72. Detection of components
If the compounds separated in a column chromatography procedure
are colored, the progress of the separation can simply be monitored
visually.
If the compounds to be isolated from column chromatography are
colorless. In this case, small fractions of the eluent are collected
sequentially in labelled tubes and the composition of each fraction
is analyzed by TLC.
Applications
• Separation of mixture of compounds
• Purification process
• Isolation of active constituents
• Estimation of drugs in formulation
• Determination of primary and secondary glycosides in digitalis leaf.
• separation of diastereomers

73. Flash Column Chromatography
• It is also called medium pressure chromatography was popularized several years
ago by Clark Still of Columbia University.
• This is a fast, simple, widely used preparative separation technique, where the
stationary bed is packed in a long, narrow glass tube.
• Typically, silica is used as the stationary phase but other stationary beds such as
reverse phase silica or cellulose are also used depending on the nature of the
compounds to be separated.
• The flow rate of the mobile phase can be accelerated either by applying pressure
on the top of the column by applying suction from the lower end of the column
to decrease the time that the compounds spend in the column or to increase the
flow rate of the mobile phase.
• The particles size should be smaller than that of the column chromatography.
• Flash chromatography is usually carried out with a mixture of two solvents with
a polar and non-polar component.
One-component solvent system:
1. Hydrocarbons: Pentane, Petroleum ether, hexanes
2. Ethane and Dichloromethane
3. Ethyl acetate
Two component solvent systems:
Ether/petroleum ether, ether/hexane, ether/pentane
Ethyl acetate/hexane
Methanol/Dichloromethane

74. Various components of flash chromatography
These are some adsorbents which are used in flash chromatography:
1. silica: slightly acidic medium, best for ordinary compounds, good
separation is achieved.
2. Florisil: mild, neutral medium
3. Alumina: basic or neutral medium, can be effective for easy
separations and purification of amines.
Applications
• Purification of various peptides, antibiotics
• Separation of closely related organic compounds
• Purification of closely related drug intermediates
• High speed fractionation of natural products – tocopherols,
alkaloids, lignans, xanthones, stilbenes, flavonoids
• Agrochemistry

75. Ultra Performance Liquid Chromatography (UPLC)
• UPLC refers to the Ultra Performance Liquid Chromatography.
• In 2004, Waters launched and trademarked UPLC which is based
upon small, porous particles (2 μ).
• It is based on Van Deemter equation which correlates the
connection between linear velocity and plate height.
• The small particles require a high pressure to work with UPLC i.e.
6000 psi which is typically the upper limit of HPLC.
• It was observed that when the particle size is decreased below 2.5,
there is remarkable increase in the effectiveness.

76. Advantages
- More selective and sensitive with high resolution
- Reduces process cycle time
- Decreases consumption of the solvent
Disadvantages
- Higher back pressure compared to conventional HPLC which
decreases the life of the column.
- Particles less than 2 μ are mostly non-regenerable and have a
narrow use.
Sample injection
Conventional injection valves are used for injecting the sample.

77. UPLC Columns
Columns used for UPLC have been developed and manufactured
by the following different companies:
1. Waters: Acquity UPLC Columns and Vanguard Pre-columns
have been produced
2. Alilgent Technology provides highest performing columns that
provide fast and reproducible results.
These includes Poroshell 120 columns, ZOBRAX Eclipse Plus
columns and ZOBRAX Rapid Resolution High Definition
columns and ZOBRAX Rapid Reduction High Throughput
Columns
3. Altech Associate
4. Phenomenex provides Kinetex
Different types of columns being used in UPLC are packed with
particles which are produced through different technologies, are
as follows:

78. 1. Charged Surface Hybrid [CSH] particle technology
2. Ethylene Bridged Hybrid [BEH] particle technology
3. High Strength Silica [HSS] particle technology
4. Peptide Separation Technology
Applications
• UPLC can be used to identify and quantify procyanadines,
phenolic compounds, monomers, oligomers, isoflavones,
flavonoids, coumarins and alkaloids such as caffeine and
theobromine.
• UPLC also used for the separation of Amino acids. It separates
24 amino acids within 23 minutes.

79. References
1. Textbook of Pharmacognosy by C.K. Kokate, A. P. Purohit, S. B.
Gokhale, Thirty-Nineth Edition, Nirali Prakashan, Page No-114-
119.
2. Textbook of Pharmacognosy and Phytochemistry-II, By Dr P
Shukla, Dr S Alok, Dr P Shukla, Nirali Prakashan, First Edition,
Page No- 14.1-14.13.
3. Pharmaceutical Analysis, Instrumental Methods, Dr. A. V. Kasture,
Dr. K. R. Mahadik, Dr. S. G. Wadodkar, Dr. H. N. More, Volume II,
Nirali prakashan, Page No.: 2.1-2.4, 5.1-5.3, 5.8, 4.3, 4.12.
4. Pharmaceutical drug Analysis, Ashutosh Kar, New Age
International Publishers, Third Revised Edition, Page No. 488 495,
525. 549.
5. UPLC: Gita Chawla, Chanda ranjan, Principle, Instrumentation and
Applications of UPLC: A Novel Technique of Liquid
Chromatography, Open Chemistry Journal.