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Ficus religiosa have been used to treat the parasitic infections in man and animals.The Whole parts of the plant exhibit wide spectrum ofactivities such as anticancer, antioxidant, anti diabetic,antimicrobial, anticonvulsant, anthelmintic, antiulcer,antiasthmatic, anti amnesic etc.

* The main aim of our study is to identify new phytochemicals by bark of  ficus religiosa.

* To study the T.L.C profile of chloroform, aqueous and organic layer extracts of  ficus religiosa.

* To study the pharmacognostic studies of.
A) T.S of bark  B) Analysis Of Bark Powder Of  Ficus Religiosa.

* To Study The Biological Activity (Analgesic activity) Of  Ficus Religiosa by TAIL IMMERSION METHOD.


5.1 introduction to chromatography

Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the "mobile phase", which carries it through a structure holding another material called the "stationary phase". The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.

Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.

Chromatography can be described as the process where analytes are separated due to their varying distribution between two phases, a stationary one (stationary phase) and one that moves (mobile phase).  Compounds traveling in the mobile phase interact with the stationary phase.  Those that are strongly retained by the stationary phase move slowly, while those that interact only weakly move rapidly. Compounds that move rapidly are thereby separated from the compounds that move slowly.  Most chromatography uses a stationary phase inside a column but the use of flat sheets of mobile phase (thin layer chromatography) are also used.

Chromatography is a powerful separation tool that is used in all branches of science, and is often the only means of separating components from complex mixtures.  Many types of chromatography have been developed.  High performance liquid chromatography (HPLC) is an extremely versatile technique where analytes are separated by passage through a column packed with micrometer-sized particles.  Gas chromatography separates compounds based on their volatility and their adsorption or solubility in the stationary phase.  Size exclusion chromatography separates molecules based on their size by passing the sample through a porous structure.  Ions can be separated based on their chemical charge using ion exchange chromatography.  Other types of chromatography include electrophoresis and supercritical fluid chromatography.

The stationary phase can also take two forms, solid and liquid, which provides two subgroups of GC and LC, namely; gas–solid chromatography (GSC) and gas–liquid chromatography (GLC), together with liquid solid chromatography (LSC) and liquid chromatography (LLC). The different forms of chromatography are summarized bellow. Most thin layer chromatography techniques are considered liquid-solid systems although the solute normally interacts with a liquid-like surface coating on the adsorbent or support or, in some cases an actual liquid coating.

Figure1.5 Classification of Chromatography

Thin layer chromatography is used to separate components of a plant extract, illustrating the experiment with plant pigments that gave chromatogram.

  • A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
  • Chromatographyis a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
  • The eluate is the mobile phase leaving the column.
  • The eluent is the solvent that will carry the analyte.
  • An eluotropic series is a list of solvents ranked according to their eluting power.
  • An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
  • The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and Capillary Electro chromatography (CEC)), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of  HPLC the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromotagraphy and the sample being separated
  • Preparative chromatographyis used to purify sufficient quantities of a substance for further use, rather than analysis.
  • The retention timeis the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also: Kovats' retention index
  • The solute refers to the sample components in partition chromatography.
  • The solvent refers to any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography.
  • The stationary phaseis the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography

Paper chromatography Is an analytical method technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layerchromatography; however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.


Rƒ value
The retention factor (Rƒ) may be defined as the ratio of the distance traveled by the substance to the distance traveled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead. If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front. To calculate the Rƒ value, take the distance traveled by the substance divided by the distance traveled by the solvent (as mentioned earlier in terms of ratios). For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, (2.1/2.8) the Rƒ value = 0.75

Thin layer chromatography
TLC is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieve.

Figure1.7 Thin Layer Chromatogram

Thin layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics , assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents

A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantization. This method is referred to as HPTLC, or "high performance TLC".

Plate preparation
TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC.

The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.

Figure1.8 Ellution Of Compounds On T.L.C

To run a thin layer chromatography, the following procedure is carried out:
A small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the bottom edge. The solvent is allowed to completely evaporate off, otherwise a very poor or no separation will be achieved

  • A small amount of an appropriate solvent (elutant) is poured in to a glass beaker or any other suitable transparent container (separation chamber) to a depth of less than 1 centimeter. A strip of filter paper (aka "wick") is put into the chamber, so that its bottom touches the solvent, and the paper lies on the chamber wall and reaches almost to the top of the container. The container is closed with a cover glass or any other lid and is left for a few minutes to let the solvent vapors ascend the filter paper and saturate the air in the chamber. (Failure to saturate the chamber will result in poor separation and non-reproducible results).
  • The TLC plate is then placed in the chamber so that the spot(s) of the sample do not touch the surface of the elutant in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate (elutes the sample). When the solvent front reaches no higher than the top of the filter paper in the chamber, the plate should be removed (continuation of the elution will give a misleading result) and dried.

Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling.

The classical preparative chromatography column, is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom. Two methods are generally used to prepare a column: the dry method, and the wet method.

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound




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