Separation methods

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KWAME UNKRUMAH UNIVERSITY OF SCIENCE AND
TECHNOLOGY
FACULTY OF PHARMACEUTICAL SCIENCES
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
Name: Japhat Opoku Ofosu
Index Number: 8037112
Experiment: O.3.1.3
Instructor: Mr.Bright Yaw Vigbedor
Date: October 21, 2014.
Experiment Title: Basic Phytochemical Screening

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AIMSOBJECTIVES:
 To enlighten students on the separation method- chromatography.
 To separate a given sample into its various components.
 To calculate the value for a given sample.
LITERATURE REVIEW
The technique of paper chromatography, although of comparatively recent origin, has
assumed such importance in the last few years that it is now no longer a novelty and every
worker in the fields of chemistry and the biological sciences should have some knowledge
of the scope and limitations of the method. When Consden, Gordon and Martin, in 1944,
published their experiments with paper chromatograms, several branches of pure and
applied chemistry were almost revolutionised overnight. Problems which up to that time
had seemed virtually incapable of solution became amenable to attack and such was the
simplicity of the equipment required and the techniques involved that interest in the
method spread rapidly and its use is now commonplace in laboratories throughout the
world
INTRODUCTION:
Separation involves isolating and/ or purifying a chemical constituent or a group of
chemically related substances. Hence, during separation, either the interfering substance is
removed or the component required isolated. To achieve a successful and efficient
separation, the substances to be separated must differ in a chemical or physical property
due to which the separation can occur. There are lots of separation methods one can
choose from when about to do any separation, some of which include ion exchange,

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electrophoresis, absorption, adsorption, distillation, chromatography, decantation, filtration
and precipitation. In this experiment, the separation method employed is chromatography.
Chromatography from the Greek word (chroma: means -colour and graphein: means -to
write ) is an analytical technique, which involves passing a mixture dissolved in a mobile
phase through a stationary phase which separates the analyte to be measured from other
molecules in the mixture based on differential partitioning between the two immiscible
phases; mobile and stationary phase. This technique is based on differences in solubility.
The stationary phase consists of a fixed bed of small particles with a large surface area
while the mobile phase is a fluid that moves constantly through or over the face of the
stationary phase. Evaluation of the separation can be achieved by determining the
retardation of each separated compound. A factor namely the Retardation factor Rf , for
each eluted substance is introduced to enable the qualitative evaluation of the
chromatogram.
The retardation factor is defined as a measure of that fraction of the total elution time that
it spends in the mobile phase. Mathematically, it is defined as the ratio of the distance
travelled by the mobile phase in a particular time.
Thus, =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 (𝑖𝑛 𝑐𝑚)
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡(𝑖𝑛 𝑐𝑚)
Chromatographic processes are classified into two groups. First, classification according
to the mechanism involved in the separation. Under this class, we have Sorption
chromatography, partition chromatography, ion exchange chromatography and exclusion
chromatography. More than one of these four mechanisms may be occurring
simultaneously in any chromatographic separation. Secondly, classification according to
instrumentation or apparatus used. These include Column Chromatography, Paper
Chromatography, Thin-layer Chromatography, Gas Chromatography and High-pressure

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liquid Chromatography. Paper and Thin-layer chromatography are the chromatographic
techniques employed in this experiment.
Chromatographic Mechanisms
Chromatographic techniques are based on four different sorption mechanisms, surface
adsorption, partition, ion exchange and size exclusion.
Surface Adsorption Chromatography
The separation mechanism depends upon differences in polarity between the different feed
components. The more polar a molecule, the more strongly it will be adsorbed by a polar
stationary phase. Similarly, the more non-polar a molecule, the more strongly it will be
adsorbed by non-polar stationary phase.
During a surface adsorption chromatography process, there is competition for stationary
phase adsorption sites, between the materials to be separated and the mobile phase. Feed
molecules of low polarity spend proportionally more time in the mobile phase than those
molecules that are highly polar, which are retained longer. Therefore the components of a
mixture are eluted in order of increasing polarity.
Almost any polar solid can be employed as a polar stationary phase. The choice of
stationary phase is governed by the polarity of the feed components. If the feed
components are adsorbed too strongly, they may be difficult to remove. Weakly polar
mixtures should be separated on highly active absorbents, or little or no separation will
occur.
The choice of mobile phase is equally important. The polarity of the mobile phase should
be chosen to compliment the choice of stationary phase. In general, good separation is

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achieved by using fairly polar stationary phases and low polarity mobile phases such as
hexane. Water, it should be noted, is a very polar solvent.
The 2 most common adsorbents used in chromatography are porous alumina and porous
silica gel. Of lesser importance are carbon, magnesium oxide, and various carbonates.
Alumina is a polar adsorbent and is preferred for the separation of components that are
weakly or moderately polar, with the more polar components retained more selectively by
the adsorbent, and therefore eluted from the column last. In addition, alumina is a basic
adsorbent, thus preferentially retaining acidic compounds. Silica gel is less polar than
alumina and is an acidic adsorbent, thus preferentially retaining basic compounds. Carbon
is a non-polar (apolar) stationary phase with the highest attraction for larger non-polar
molecules.
Adsorbent-type sorbents are better suited for the separation of a mixture on the basis of
chemical type (e.g. olefins, esters, acids, aldehydes, alcohols) than for separation of
individual members of a homologous series. Partition chromatography is often preferred
for the latter, wherein an inert solid (often silica gel) is coated with a liquid phase
Hydrophobic interaction chromatography (HIC) is a special form of surface adsorption
chromatography. The materials to be separated should be at least partially hydrophobic in
nature. Separation is facilitated by differences in the relative strength of interaction
between these materials and a matrix substituted with suitably hydrophobic groups. This
type of process is extensively used for the preparative-scale separation of proteins.
[ Back on Top ]

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Partition Chromatography
Unique to chromatography is the liquid-supported or liquid-bonded solids, where the
mechanism is absorption into the liquid, also referred to as a partition mode of separation
or partition chromatography. With mobile liquid phases, there is a tendency for the
stationary liquid phase to be stripped or dissolved. Therefore, the stationary liquid phase
has to be chemically bonded to the solid bonding support.
In partition chromatography, the stationary liquid phase is coated onto a solid support such
as silica gel, cellulose powder, or kieselguhr (hydrated silica). Assuming that there is no
adsorption by the solid support, the feed components move through the system at rates
determined by their relative solubilities in the stationary and mobile phases.
In general, it is not necessary for the stationary and mobile phases to be totally immiscible,
but a low degree of mutual solubility is desirable. Hydrophilic stationary phase liquids are
generally used in conjunction with hydrophobic mobile phases (referred to as "normal-
phase chromatography"), or vice versa (referred to as a '"reverse- phase chromatography").
Suitable hydrophilic mobile phases include water, aqueous buffers and alcohols.
Hydrophobic mobile phases include hydrocarbons in combination with ethers, esters and
chlorinated solvents.
Ion Exchange Chromatography (IEC)
In this process, the stationary phase consists of an insoluble porous resinous material
containing fixed charge-carrying groups. Counter-ions of opposite charge are loosely
complexed with these groups.

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Passage of a liquid mobile phase, containing ionised or partially ionised molecules of the
same charge as the counter-ions through the system, results in the reversible exchange of
these ions.
The degree of affinity between the stationary phase and feed ions dictates the rate of
migration and hence degree of separation between the different solute species.
The most widely used type of stationary phase is a synthetic copolymer of styrene and
divinyl benzene (DVB), produced as very small beads in the micrometer range. Careful
control over the amount of DVB added dictates the degree of cross-linking and hence the
porosity of the resinous structure.
Resins with a low degree of cross-linking have large pores that allow the diffusion of large
ions into the resin beads and facilitate rapid ion exchange. Highly cross- linked resins have
pores of sizes similar to those of small ions.
The choice of a particular resin will very much be dependent upon a given application.
Cation (+) or anion (-) exchange properties can be introduced by chemical modification of
the resin.
Ion exchange chromatography has found widespread uses in industrial processes. This
technique is used in the separation of transition metals, the removal of trace metals from
industrial effluents and in the purification of a wide range of organic compounds and
pharmaceuticals. The resin matrix is usually relatively inexpensive when compared with
other types of stationary phase. Ion exchange chromatography is probably the most widely
used large-scale chromatographic process, but is limited to ionisable, water soluble
molecules.

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[ Back on Top ]
Size Exclusion Chromatography (SEC)
In this process, also known as gel permeation chromatography, molecules of a feed
material are separated according to their size or molecular weight. The stationary phase
consists of a porous cross-linked polymeric gel.
The pores of the gel vary in size and shape such that large molecules tend to be excluded
by the smaller pores and move preferentially with the mobile phase. The smaller
molecules are able to diffuse into and out of the smaller pores and will thus be retarded in
the system.
The very smallest molecules will permeate the gel pores to the greatest extent and will
thus be most retarded by the system.
The components of a mixture therefore elute in order of decreasing size or molecular
weight.
The stationary phase gels can either be hydrophilic for separations in aqueous or polar
solvents, or hydrophobic for use with non-polar or weakly-polar solvents. Sephadex, a
cross-linked polysaccharide material available in bead form, is widely used with
polar/hydrophilic mobile phases. The degree of cross-linking can be varied to produce
beads with a range of pore sizes to fractionate samples over different molecular weight
ranges. Hydrophobic gels are made by cross-linking polystyrene with DVB and are
therefore similar to ion exchange resins but without the ionic groups.

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SEC is used extensively in the biochemical industry to remove small molecules and
inorganic salts from valuable higher molecular weight products such as peptides, proteins
and enzymes.
Paper chromatography is essentially an analytical tool for separating chemical compounds,
and in this respect is comparable with distillation, crystallisation, precipitation, etc. The
latter methods all depend on differences in volatility or solubility of the substances
involved, whereas partition chromatography in general, and paper chromatography in
particular, rely on differences in another physical property — namely the partition
coefficient. If a pure substance is shaken up with two immiscible solvents the ratio of the
concentrations of the substance in the two liquid layers is known as the partition
coefficient. For a given solute and solvents at constant temperature the partition
coefficient is a constant, and, in particular, is independant of the actual concentration of
solute in the liquid phases. Partition coefficients vary from 0 to 1 and it is the small
differences in the partition coefficients of various substances which are utilised for their
separation.
Paper chromatographic methods possess several advantages compared with the more
conventional methods of separation. Perhaps the most important of these is the facility
with which substances which are extremely similar chemically can be separated. The
classical example of this kind is the separation of the amino-acids and of the
carbohydrates. So great are the difficulties associated with the separation of members of
either of these important groups of substances by methods other than chromatographic

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ones, that the analysis by conventional methods of even comparatively page 104 simple
mixtures was a time-consuming and intricate process requiring considerable amounts of
material. Accurate quantitative analysis was in general impossible. Paper chromatography
has made possible the analysis of mixtures of 18-20 amino-acids in a time of the order of
36 hours.
Another major asset is the small amount of material required. Qualitative analyses can be
carried out on amounts which may be as small as a few micrograms of each component,
and only slightly larger quantities are often sufficient for approximate quantitative results.
Few other analytical procedures can be adapted to this minute scale, yet in the case of
material of biological origin, amounts greater than a few milligrams may be almost
unobtainable.
The wide versatility of paper chromatographic methods is illustrated by the fact that they
may be successfully employed for separations in such diverse series as the amino-acids,
sugars, plant pigments, phenolic constituents of wood, etc., volatile fatty acids, sugar
phosphates, and related compounds, nucleic acid degradation products, compounds such
as adenosine triphosphate (A.T.P.) and adenosine diphosphate (A.D.P.), metallic ions, etc.
In view of this wide range of application and the fact that the equipment required is simple
and the technical skill required not great, it is not surprising that the methods of paper
chromatography have assumed such importance in a short space of time.
Partition of a solute or solutes between two immiscible liquids was utilised by Craig in his
elegant counter current distribution technique which is essentially a method of carring out
a series of extractions of one liquid phase with another in a definite order. However
counter current distribution studies often require considerable amounts of material, may

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require expensive precision equipment and are frequently time-consuming. Martin and
Synge conceived the idea of keeping one of the liquid phases stationary and allowing the
other liquid to pass continuously over it, thus essentially having an extremely large
number of extremely small-scale extractions. If one of the liquid phases is water it may be
held stationary by absorbing it on the surface of a material such as silica gel, diatomaceous
earth, starch or cellulose. This idea led to the development of partition chromatography
using columns of materials such as these saturated with water, with solvents such as
chloroform or butanol flowing slowly down the column. This technique is of great
importance and is used extensively for the separation of substances in amounts of the
order of 5-10 mg. upwards.
For smaller quantities Consden, Gordon and Martin showed that a sheet of filter paper
serves admirably to hold the stationary phase (water) and the moving phase may be made
to travel slowly up or down the paper by capillary action and/or gravity: thus paper
chromatography came into being.
In practice the mixture to be separated is applied in the form of a small spot a few
millimetres in diameter near one end of a strip of filter paper which is then hung in an
atmosphere saturated with both water and the page 105 solvent to be employed. The strip
may be suspended with the spot near the top from a trough containing the solvent
(descending technique) or alternatively hung upside down dipping in a trough or dish of
solvent (ascending technique). The mobile phase is allowed to move down or up the paper
for a time which may vary from minutes or hours to several days. During this time the
material originally placed on the paper will have moved a greater or lesser distance in the
direction of flow of the solvent and in the case of a mixture under ideal conditions the
original spot should be resolved into several spots distributed along the length of the strip.

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If the paper is then removed from the solvent and dried it should be possible to reveal the
position of the spots by spraying the paper with a suitable reagent or examining it in ultra-
violet light, etc.
The ratio Distance travelled by substance/Distance travelled by solvent, known as the RF
value, is a constant which, for a given solvent at a given temperature, is characteristic for
the substance, and thus ‗unknown‘ substances may be identified with some degree of
certainty by measuring their RF values preferably in several different solvents. RF values
should be reproducible, but sometimes careful control of conditions is necessary for this to
be the case, and it is normal practice to run standard spots simultaneously alongside the
‗unknowns‘.
A number of factors influence the ease with which paper chromatograms can be
successfully prepared, and of these brief mention may be made of the design of the
equipment, the type of paper, the nature of the solvent, and the method of detecting the
spots.
(a) Equipment
The original apparatus used by Consden, Gordon and Martin consisted of a drain pipe
closed at the lower end by immersion in a dish containing the aqueous phase and at the
upper end by a sheet of glass. A small glass trough resting on the shoulder near the top of
the pipe contained the solvent and the paper, held in the trough by means of a glass rod or
sheet, passed over a glass rod and hung down inside the pipe. If the paper was hung
directly over the rim of the trough difficulty was experienced with solvent syphoning out
between the paper and the trough. It is convenient to be able to follow the progress of the
solvent down the paper visually and a tall glass jar is a more satisfactory container. The

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trough must then be supported on some type of stand preferably of glass (to resist the
attack of solvents). Metal troughs have the great advantage of not being fragile but are
often unsatisfactory with strongly acidic or basic solvents. It is an advantage, for reasons
which will appear later, if the solvent can be run into the trough through a small hole in
the top cover so as to avoid opening the jar completely to the atmosphere.
The ascending technique requires less complicated apparatus and any container in which
the paper can be hung so that it dips into a pool of solvent at the bottom may be employed.
Again a glass container is page 106 desirable so that the movement of the solvent front
can be followed visually, although this is by no means essential. Pieces of wide bore glass
tubing closed at the top with a cork carrying some support may be used, or alternatively
bottles such as milk bottles, or large test-tubes. For accurate work it is desirable that the
solvent front may be able to move 30-40 cms., but for demonstration purposes, especially
when time is an important factor, shorter distances may be adequate.
In general the descending method is preferred for accurate work, although the ascending
technique is somewhat simpler and requires less complicated apparatus. The former
method has the advantage that the solvent may be allowed to run off the bottom of the
paper and, although RF values can not then be determined, substances with very low RF
values can be separated more completely. As mentioned above, standard spots are usually
run alongside the ‗unknown‘ so that the fact that RF values are not obtainable is not of
major importance. The ascending method, on the other hand, has the advantage that the
solvent cannot ‗run off‘ the paper and thus the chromatogram needs less attention.
A combination of the two methods in which the paper dips into a trough, runs up over a
glass rod and then hangs down is useful especially when handling large sheets of paper as

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it obviates the use of a large tall glass container and vessels such as glass aquarium tanks
may be employed Whatever the type of equipment used it is most desirable that it can be
sealed as completely as possible.
(b) Type of Paper
A large variety of types of paper have been tried, some of which possess advantages for
specialised work. Whatman No. 1 filter paper is most widely used and will give
satisfactory results in the majority of cases. Thicker papers such as blotting paper are not
usually suitable.
(c) Solvents
The choice of solvent depends on the type of substances to be separated, the most
common ones being alcohols such as butanol with or without the addition of acids such as
acetic acid or bases such as ammonia or amines, phenol, hydrocarbon solvents, collidine,
etc. The solvent phase is saturated with water except in certain cases where the solvent is
actually miscible with water. (Although it would appear at first sight that such a solvent
would not give any separation this is, for reasons which will not be discussed here, not
always the case.) It is perhaps unfortunate that the more common solvents are often
unpleasant to handle and toxic, and care should be taken to avoid unduly prolonged
exposure to their vapours.
(d) Detection of the Spots
The lower limit to the quantities of substances which can be separated usually depends on
the sensitivity of the method used for detecting the resulting spots. Detection is usually
carried out by spraying the dried page 107 paper with a suitable reagent which will

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develop colours with the substances present, often on gentle warming. A small atomiser or
throat spray or some such device is convenient for applying the detecting reagent. In
certain cases the paper may be dipped into a solution of the reagent instead of sprayed
with it. Although spraying is the most common method of detecting the spots, many
ingenious methods have been used as alternatives. Fluorescence in ultra-violet light and
absorption of ultra-violet light, have been utilised as have biological methods of detection
(for example for antibiotics) and radio-autographs (as for example in the analysis of
compounds present in the thyroid gland after the administration of radio-active iodine) to
mention but two.
General
As a general rule the spots applied to the paper should be small, preferably not more than
a few millimetres in diameter and the concentration of the individual substances should
not be too high or ‗tailing‘ and excessive spreading of the spots will take place. In most
cases a single small drop — say 5 microlitres — of a 1% solution is about the amount of
substance required. For quantitative work an accurately known amount must be applied to
the paper and for this a micro-pipette is required, but this elaboration is unnecessary for
qualitative studies.
It is often desirable though not essential that the paper after the application of the material
be allowed to stand in an atmosphere saturated with respect to both the aqueous and
mobile phases before the chromatography is commenced. For this reason it is desirable
that the solvent can be added to the trough or, in the case of ascending chromatograms, the
paper may be lowered into the solvent, with the least possible dismantling of the
apparatus.

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The paper must almost always be dried before spraying and this is best accomplished
either in a hot air oven or in a current of warm air such as that provided by a hair drier.
Because of the toxic nature of the solvents drying should be carried out in a fume
cupboard, or failing that in a well-ventilated place. Overheating of the papers should be
avoided.
The spray should be applied lightly so that the paper becomes merely damp and not
saturated in order to avoid undue migration and diffusion of the spots. For the same reason
most spraying reagents are made up in non-aqueous solution.
With complex mixtures it is often impossible to obtain complete resolution with any single
solvent and in such cases two-dimensional chromatograms may be resorted to. This may
lead to a very high degree of resolution of complex mixtures but it is not possible to run
standard substances simultaneously.
Quantitative paper chromatography may involve measurement of spot sizes, intensities of
coloured compounds developed by sprays or elution of page 108 the spots from the
chromatogram and subsequent determination by standard micro-chemical methods, to give
some examples. However, the techniques employed are often specialised and outside the
scope of this article.
The following practical directions should enable the beginner in the field to become
familiar with the general methods and the suggested materials for investigation should
provide a guide to those interested in further studies.

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Ascending Chromatograms in Test Tubes
Whatman No. 1 filter paper is cut into strips which will fit easily into an ordinary test-tube
and the strips are creased lengthwise to form a V-shaped trough. A line is drawn lightly
across the strip about 2 cm. from the end, and by means of a fine glass capillary tube or a
match-stick, a small spot of the sample (about 2 mm. in diameter) is placed on the line in
the centre of each half of the V. The spots are allowed to dry then the paper strip is placed,
spotted end down, in a test-tube containing 1-2 cc. of solvent in such a way that the strip
touches the side of the test-tube only at the outside top corners. It is essential that the
starting line marked on the strip be above the solvent surface, and the paper strips will stay
in position readily if the tube is placed in a slightly slanting position. The test-tube is
corked and set aside undisturbed until the solvent front rises almost to the top of the strip.
This may take up to about one hour, depending on the temperature and the nature of the
solvent. The strip is removed, the position of the solvent is marked, and the solvent is
evaporated either by allowing the strip to stand in the air or, preferably, in a hot air oven
or in a current of warm air. The chromatogram is sprayed lightly with the detecting
reagent and heated if necessary, the position of the spots is marked, and their RF values
determined. For this purpose the ‗distance moved by the solvent front‘ is the distance
between the starting line and the final position of the solvent front. The ‗distance moved
by the substance‘ is usually taken as the distance from the starting line to the ‗centre of
gravity‘ of the spot.
Ascending Chromatograms in Bottles
Wide-necked bottles such as milk-bottles are most satisfactory and the paper can be
suspended from a glass or wire rod passed through a cork or rubber stopper. If the support

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can be moved vertically it is possible to allow the paper strip to hang in the atmosphere
inside the bottle for up to 24 hours before it is lowered into the solvent, and if this is done
more regular spots may be obtained and there may be less tailing. The additional width of
the paper strip allows several samples to be run side by side. If this is done the individual
spots should be spaced at intervals of 1.5-2 cm. along the starting line.
Ascending Chromatograms with Large Sheets of Paper
When a large number of samples must be run side by side or for two-dimensional
chromatograms with sheets of paper up to 40-50 cms. page 109 square or larger it is often
convenient to clip the sheet in the form of a cylinder which can then be stood in a dish of
the solvent. In such cases a comparatively large container is required, or alternatively the
chromatography may be carried out under a large bell jar. With large-scale chromatograms
it is desirable to place in the container a dish containing water saturated with the solvent
so that the atmosphere remains saturated with respect to both liquid phases. The rate of
movement of the solvent front decreases with time and large-scale chromatograms may be
conveniently allowed to run overnight.
Descending Chromatograms
The spots are placed on a line 6-8 cm. from the end of the paper strip which should be
about 40-50 cm. long. The paper is arranged in the trough so that the starting line is well
clear of the edge of the trough, a dish of water saturated with solvent is placed in the
bottom of the container, the solvent is added to the trough and the container is closed.
Once again, if possible, it may be desirable to allow the paper to equilibrate in an
atmosphere saturated with both liquid phases before the solvent is added to the trough.
The chromatogram may be allowed to develop as long as desired and if necessary the

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solvent may be allowed to run off the lower edge of the paper. If this is to be done the
lower edge of the paper should be serrated to facilitate an even flow of the solvent. When
development is complete the paper is removed, dried and sprayed in the usual way.
Two-Dimensional Chromatograms
When complex mixtures have to be analysed two-dimensional chromatography is a
valuable modification because of the large increase in resolving power. A single spot of
the mixture is placed near one corner of a sheet of paper 30-50 cm. square and the
chromatogram is run in the usual way using either the descending or the ascending
technique. At this stage the mixture will have partially separated into a series of spots
distributed along a line near one edge of the sheet. The paper is removed and dried, then
run in the direction at right angles to the original with a different solvent, then dried and
sprayed in the usual way. The resulting chromatograms will show a series of spots
distributed over the paper, and although standard substances cannot be run simultaneously,
the identification of a spot is simplified by the fact that its position depends on a
combination of two RF values. ‗Maps‘ showing the positions which substances will appear
in, with various solvent combinations have been published or can easily be constructed
from tables of RF values. For two-dimensional chromatograms the amount of material
placed on the original spot should be rather greater than for single dimensional ones, and
the first solvent should be removed as completely as possible before running in the second
direction. It is frequently found that there is a preferred order in which the two solvent
combinations should be used.
page 110

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Materials
For demonstration purposes amino-acids are the most satisfactory although some sugars
can also be separated reasonably well in a comparatively short time.
The amino-acids can be applied as 0.2-1% solutions and mixtures are most readily
obtained by applying the individual amino-acids successively to the same spot, the paper
being allowed to dry between each application. For small-scale chromatograms in test-
tubes or bottles, 80% phenol water (W/W) is a suitable solvent (this is rather cheaper to
use than phenol saturated with water) and combinations of amino acids such as valine,
glycine and aspartic acid or leucine, threonine and glutamic acid separate readily.
0.2% Ninhydrin (triketohydrindene hydrate) in water-saturated butanol is the spraying
reagent, and the sprayed papers must be heated briefly at about 110° to develop the
(usually blue) spots.
Sugars and related substances may be separated using a solvent mixture containing 5
volumes of acetic acid, 25 volumes of water and 110 volumes of butanol, although RF
values are low and complete resolution of mixtures is difficult on a small scale. However,
mixtures of monosaccharides and disaccharides such as glucose and lactose separate
comparatively readily. The solvent mixture given above approximates in composition the
organic (upper) layer obtained by shaking together butanol, acetic acid and water in the
volume ratios 4: 1: 5 — a solvent which is very widely used in chromatographic work.
This solvent mixture slowly changes in composition due to esterification and should not
be kept indefinitely.

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As spraying reagent, ammoniacal silver nitrate or 2% aniline hydrogen phthalate (made
from 0.93 g. of aniline and 1.60 g. of phthalic acid in 100 ml. of water-saturated butanol)
may be used. With either spray the paper must be heated to 105-110° to develop the spots
Thin Layer Chromatography (TLC)
TLC is a simple, quick, and inexpensive procedure that gives the chemist 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 (preferably both run on the same TLC plate).
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a
solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed
is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a
solvent in a developing chamber so that only the very bottom of the plate is in the liquid.
This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by
capillary action.
As the solvent moves past the spot that was applied, an equilibrium is established for each
component of the mixture between the molecules of that component which are adsorbed
on the solid and the molecules which are in solution. In principle, the components will
differ in solubility and in the strength of their adsorption to the adsorbent and some
components will be carried farther up the plate than others. When the solvent has reached
the top of the plate, the plate is removed from the developing chamber, dried, and the
separated components of the mixture are visualized. If the compounds are colored,
visualization is straightforward. Usually the compounds are not colored, so a UV lamp is

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used to visualize the plates. (The plate itself contains a fluorescent dye which glows
everywhere except where an organic compound is on the plate.)
1. dried powdered plant(Unknown Sample)
2. Fehling‘s solution
3. conc. HCL
4. dil NH3
5. Ethanol
6. Antimony Trichloride
7. Chloroform
8. Test tubes
9. Beakers
10. Water bath
11. U.V. light
12. Filter paper
13. Spatula
14. dil H2SO4
15. NaOH
CHEMICALS AND APPARATUS
Chemicals ;
 Unknown sample
 Solvent

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Apparatus
 Capillary tube
 Measuring cylinder
 Whatman#1 filter paper
 A rule and a pencil
PROCEDURE
Solvent phase:
 10ml ammonium hydroxide and 20ml of isopropyl alcohol was measured into a
beaker.
 The beaker was covered tightly and the solvent vapour allowed to saturate the
atmosphere inside the beaker.
Stationary phase:
 A clean sheet of whatman#1 filter paper picked, and a light pencil line parallel to
the bottom of the paper was marked on the paper.
 Using the capillary tubes, a small amount of each appropriate solution was spotted
on the line on the filter paper and allowed to dry.
 A second portion of the unknown was added to the spot increasing the quantity
 The paper was dropped gently into the beaker containing the solvent with the spots
above the level of the solvent. It was then covered and left to stand for separation
to take place
 After some time, the paper was removed and the level at which the separations
were made was calculated and the of each was calculated.
PRECAUTIONS

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 It was ensured that spots made were above the solvent in the beaker when it was
lowered.
 It was ensured that the spot made was not larger than 2mm in diameter but of
sufficient quantity.
1.4 Procedure
1) TEST FOR SAPONINS
 2g of the sample was boiled in 10ml of distilled water for 3-5 minutes.
 The hot solution was filtered hot and the filtrate shaken vigorously.
 Separation of froth (foam) which persisted for some time was
indicative of saponins.
2) TEST FOR GENERAL GLYCOSIDES
 About 0.5g of sample was put into two separate beakers and dried at
60o
C.
 5.0ml of H2SO4 was added to one beaker and 5.0ml of distilled water
to the other.
 Both mixtures were heated on a boiling water bath for 3-5 minutes and
after, filtered into two separate test tubes.
 The cooled filtrates were made alkaline with NaOH solution and
heated with Fehling‘s solution for 3 minutes.

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 The formation of reddish-brown precipitate in the test tube containing
the filtrate from H2SO4 treatment and the absence of precipitate in the
other test tube indicated the presence of glycosides.
3) TEST FOR FLAVONOIDS
(A). To an alcoholic solution of the sample, a small amount of magnesium
ribbon was added followed by conc. HCL dropwise. A brick-red
colouration indicated flavanoids.
(B). A small amount of the sample was placed on a filter paper, moistened
with dilute NH3, and viewed under U.V. light. A blue colouration
indicated flavanoids.
4) TEST FOR TERPENOIDS AND STEROIDS
(A). The sample was extracted with ethanol. A small amount of the extract
was evaporated to dryness in a crucible and redissolved in chloroform.
A few drops of acetic anhydride followed by two drops of conc. H2SO4
were added. A reddish-pink colouration indicated the presence of
terpenoids and steroids.
(B). To the solution of the extract in chloroform, a small amount of conc.
H2SO4 was added. A red colouration indicated the presence of
terpenoids and steroids.
(A). TEST FOR CAROTENOIDS : About 5g of the material was
extracted with about 10ml of ethanol. To 2ml of the extract, 3ml of

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antimony trichloride was added. A dark blue colouration indicated
carotenoids.
About 5g of the material was extracted with about 10ml of ether. To about
2ml of the extract, conc. HCL (about 1ml) was carefully added to form a
layer under the ethereal solution. The presence of an intense dark-blue, blue-
violet, or greenish blue colour in the acid layer indicated the presence of
carotenoids
1.5 Table of Results
Sample : C Group 1
PROCEDURE OBSERVATION INFERENCE
1) Sample + 10ml of
distilled water +
heat
3-5minutes+filter
Light brown solution
and foam obtained after
filtering and shaking the
filtrate. The foam
persisted for some time.
Saponins present
2) Sample + 5ml
H2SO4 + heat
Heating filtrate with
Fehling‘s solution
Yellowish solution
obtained after filtering.
Yellowish solution
obtained.
Glycosides absent

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3) Sample + ethanol +
magnesium ribbon
+ HCL
Brick-red Colouration
observed
Flavanoids present
4) A) Evaporated
extract +
chloroform + acetic
anhydride +
B) H2SO4 Solution
of extract +
chloroform + small
conc. H2SO4
Solution turns Reddish –
pink when H2SO4 added
dropwise
Red colouration
observed
Terpenoids and Steroids
present
Terpenoids and Steroids
Present
5) A) 2ml of extract +
3ml Antimony
Trichloride
B) 2ml of extract
+ conc. H2SO4
Yellowish solution
obtained.
Colourless solution
obtained
Carotenoids absent
Carotenoids absent
1.7 Discussion
The screening of plant sample labeled C as seen from the results above
indicates the presence of Flavoniods, Streoids, Terpenoids, and Saponins
whilst Glycosides and Cartenoids abscent. With reference to literature, the

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plant C papaya show similar result when screened. C.papaya indicates
negative availability of glycosides and Carotenoids.The plant species , C.
Citratus and Acissampeloides also exhibits these phytochemicals but differ in
the sense that glycosides are available in them.
Flavonoids in general are thought to help reverse age-related declines in
cognitive function by increasing the number of connections among neurons
and improving blood flow to the brain,53,54 which protects vulnerable
neurons and enhances the functioning of existing neurons.53 The
consumption of flavonoid-rich foods such as berries and cocoa throughout
life may hold the potential to limit, prevent, or reverse normal or abnormal
deterioration in cognitive function in the aging brain.53.
It should be noted that steroidal compounds are of importance and interest in
pharmacy due to their relationship with such compounds as sex hormones.
The importance of saponins and terpenoids in various antibiotics used in
treating common pathogenic strains has recently been reported by
Kubmarawa.
1.8 Error Analysis
―Pre-mature‖ timing is expected to have altered the formation of the
phytochemicals.
An improper filtration method is a factor to be attributed to error analysis.

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1.9 Precaution
1. All chemicals were added to the reaction mixture in the fume-hood
2. Goggles and gloves were worn to prevent accidental contact of fluid to
the eye and the hands respectively.
2.0 Conclusion
It is true that there are metabolites (phytochemicals) in plants. The
metabolites available in the sample plant investigated are Saponins,
Flavonoids, Terpenoids and Steroids
2.1 Recommendation
It is highly recommended that since the presence of some of these
compounds has been confirmed by previous workers to have medicinal as
well as physiological activity.
The plant extracts could therefore be seen as a potential source for useful
drug. The continued traditional medicinal use of these plants is therefore
encouraged while it is suggested that further work should be carried out to
isolate, purify and possibly characterize the active constituents responsible
for the activity of this plant.

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2.1 Reference
1. Higdon, J. An Evidence – Based Approach to Dietary Phytochemicals.
200
2. Nostro A, Germanò MP, D'angelo V, Marino A, Cannatelli MA (2000)
Extraction
methods and bioautography for evaluation of medicinal plant antimicrobial
activity. Lett Appl Microbiol 30: 379-384.
3. Krishnaiah D, Sarbatly R, Bono A (2007) Phytochemical antioxidants for
health
and medicine: A move towards nature. Biotechnol Mol Biol Rev 1: 97-104.
4. Mahato SB, Sen S (1997) Advances in triterpenoid research, 1990-1994.
Phytochemistry Pp 44: 1185-1236.
5. Kappers IF, Aharoni A, van Herpen TW, Luckerhoff LL, Dicke M, et al.
(2005) Genetic engineering of terpenoid metabolism attracts bodyguards to
Arabidopsis. Science 309: 2070-2072.
6. Nmorsi OPG, Ukwandu NCD Egwunyenga AO.Antioxidant Status of
Nigerian Children with Plasmodium falciparum malaria. Afr. J.Microbial Res
2007; Pp 61-64.
7. World Health Organization. Severe Falciparum malaria. Tran. R. Soc.
Trop. Med. Hyg 2000;94(Suppl 1): 51-590.

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8. Government in action Report from presidential research and
communication unit, office of puplic communication, State House, Abuja
2005.

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