1. SEPARATIONOF PLANT CONSTITUENTSUSING CHROMATOGRAPHY

2. GROUP MEMBERS: ANEEQA KHAN FARHAT WAZIR SATTI MARIA ASHRAF SAHAR ASHFAQ UME-HABIBA 3. BACKGROUND In pharmacognosy we deal with drugs and drug products obtained from plant, animal and mineral sources. These drugs and drug products need refining: to identify to differentiate to extract the therapeutic constituents from non-therapeutic constituents 4. Chromatography is one of the extraction procedures to identify and to separate different constituents of crude plants, chemicals, and other substances etc. chromatography is the most frequently used analytical technique in pharmaceutical analysis. 5. CHROMATOGRAPHY The process of separation of individual components of a mixture based on their relative affinities towards the stationary or mobile phase OR It is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds 6. TERMS USED IN CHROMATOGRAPHY The ANALYTE is the substance to be separated during chromatography.It is also normally what is needed from mixture. The ELUATE is the mobile phase leaving the column. The ELUENT is the solvent that carries the analyte. A STATIONARY PHASE is the substance fixed in place for the chromatography.It may be a solid or liquid. The MOBILE PHASE is the phase that moves in a definite direction.It may be a liquid, a gas, or a superficial liquid. A CHROMATOGRAM is the visual output of the chromatography. 7. GENERAL PROCEDURE In chromatography a 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 mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases depending upon the Rf values. 8. Rf value Rf value is the retardation factor or the retention factor The ratio of distance travelled by the solute to the distance travelled by the solvent front 9. PARTITION COEFFICIENT Molar concentration of analyte in the stationary phase divided by molar concentration of the analyte in the mobile phase K = C (stationary phase) C (mobile phase) 10. TYPES OF CHROMATOGRAPHY chromatography adsorption chromatography thin layer chromatography papaer chromatography partition chromatography gas and column chromatography & HPLC 11. CHROMATOGRAPHIC TECHNIQUES There are two chromatographic techniques planar chromatographic technique paper chromatography thin layer chromatography columnar chromatographic technique column chromatography gas chromatography 12. PARTITION CHROMATOGRAPHY Partition chromatography is based on the differences in partition coefficients of the compounds of a mixture which have to be separated, between an aqueous & immiscible organic liquid. The stationary phase is normally aqueous, which is mixed with an inert carrier powder & packed into a glass column. 13. PROCEDURE: The mixture to be separated is dissolved in an organic solvent, & is introduced into the column & the chromatogram developed with more solvent, on different solvents of increasing eluting power. Diffusion of the mobile phase through the stationary phase occurs, & the different rates of travel of the constituents of the mixture are directly related to their partition coefficients between the mobile organic & stationary aqueous phase. 14. The finished chromatogram exhibits separated zones similar to those seen an adsorbent column. 15. TECHNIQUES IN PARTITION CHROMATOGRAPHY 16. USES preparative separation of plant pigments Purify individual chemical compounds 17. HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC) 18. Typical Applications of HPLC Field of Application Separation Pharmaceuticals Antibiotics, Sedatives, Steroids, Analgesics Biochemical Amino acids, Proteins, Carbohydrates, Lipids Food Products Artificial Sweeteners, Antioxidants, Preservatives Industrical Chemicals Condensed Aromatics, Surfactants, Propellants, Dyes Forensic Chemistry Drugs, Poisons, Blood Alcohol, narcotics Clinical Medicine Bile Acids, Drug Metabolites, Urine Extracts, Estrogens 19. GAS CHROMATOGRAPHY 20. USES analysis of oil mixtures detect drugs & steroids fruit esters 21. ADSORPTION CHROMATOGRAPGY 22. PRINCIPLE The surface phenomenon of adsorption is utilized. The finely-divided solids are capable of selective adsorption of other substances. The components of a mixture introduced onto a column of adsorbent are more or less strong adsorbed. Those which are least strongly adsorbed are carried down the column by the passage of solvent, & are the 1st to be eluted from the bottom of the column. The passage of the more strongly adsorbed substances is slower, & these are the last to elute. The fractions of eluant containing each component of the original mixture can then be separately analyzed. 23. ADSORPTION CHROMATOGRAPHY In its simplest form, this method of chromatography consists of passing a solution of the mixture of compounds needing to be separated, through a hollow glass column, packed with a finely divided absorbent powder, and collecting the solution (eluate). 24. TECHNIQUES OF ADSORPTION CHROMATOGRAPHY PAPER CHROMATOGRAPHY: It is a chromatographic analytical separation technique for complex mixtures involving the progressive adsorption of the dissolved component onto a special grade of paper. 25. PRINCIPLE The certain solvent are used to separate a mixture e.g., water, alcohol. With capillary action the solvent will move up to filter paper. Movement of a solvent will bring together component that are separated from the mixture. Every component that are separated will move to several velocity The moving components are depend on : a. Solubility solute in solvent b. Intermolecule forces c. Pore size of filter paper d. Size of solute 26. At the end of process, components that are separated will emerge to different distance on filter paper. Rf values are used to identification of each the component. 27. Spot Detection Color spot observed by naked eye Non color spot color reagent will give specific colors for different compound. Example: Ninhydrin amino acids 28. USES OF PAPER CHROMATOGRAPHY To isolate plant pigment from root and leaves. To isolate amino acid mixtures. To isolate food colors or dyes. 29. THIN LAYER CHROMATOGRAPHY(TLC) TLC is an example of adsorption chromatography, the stationary phase being a thin layer adsorbent held on a suitable backing. PRINCIPLE: Separation of the compounds present in the plant extract depends on the differences in their adsorptive/desorptive behaviour in respect of the stationary phase. 30. PROCEDURE TLC involves a thin layer of adsorbent, mixed with a binder such as, which is spread on a glass plate & allowed to dry. The plant mixture to be separated is applied as a spot near the base of the plate, which is then placed in a closed glass tank containing a layer of developing solvent. Now separated components of the mixture appear as spots on the finished plate (chromatogram), corresponding to the bands of the adsorbent column. 31. COMPONENTS OF THE TLC SYSTEM There are three components THE ADSORBENT Stationary Phase THE ELUENT (THE DEVELOPING SOLVENT) Mobile Phase THE SUBSTANCE REQUIRING SEPARATION Plant Sample 32. GENERAL RULE FOR TLC TLC is best used for moderately or weakly polar mixtures. 33. VISIBILITY OF SPOTS OR BANDS (COMPOUNDS) If the spots or bands are invisible on the developed chromatogram they are made visible either by; Heating for a specific period Examining under U.V light (if substances are florescent). Spraying the finished chromatogram with a suitable reagent e.g. iodine & Dragendorffs reagent are used as sprays for the general detection of alkaloids(although they are not specific for alkaloids). Ninhydrin solution amino acids Ferric chloride solution phenols Alkali anthraquinones Antimony trichloride in chloroform steroids & some volatile oil components Aniline hydrogen phthalate reagent - sugars 34. TLC Advantages over paper Its faster It gives a better separation. It is more versatile as the solid on the plate can be varied. USES: To detect amino acids. To check composition of dyes and food colors. 35. SEPARATION OF ALKALOIDS 36. Alkaloids need a moderately polar solvent for good separation (e.g. ether/ethanol: 95/5). A more polar solvent (e.g. pure methanol), would be preferentially adsorbed, & the alkaloids would be carried along by the passage of the solvent resulting in poor separation. On the other hand, a non-polar solvent (e.g. cyclohexane) would be unable to displace the alkaloids from the adsorbent layer & they would then remain at or near the base of origin. 37. ADDITIONAL FACTOR FOR SEPARATING ALKALOIDS If using an aluminium thin layer (neutral), a neutral solvent should be used. If using Silica-gel (slightly acidic due to the method of preparation), and alkaline solvent such as acetone/water/25%ammo nia: 90/7/3 makes for good separation. 38. SEPARATION OF VOLATILE OILS 39. The constituents of volatile oils are mainly non- polar (terpenes) & are therefore best separated with corresponding non-polar solvents such as chloroform/benzene mixtures. Certain oils may need more polar solvents (e.g. clove oil phenolic). 40. SEPARATION OF SUGARS & SUGAR ACID MIXTURES 41. These mixtures are produced by hydrolysing starch & gums. They are strongly polar & are therefore strongly adsorbed onto silica & aluminium layers. Although strongly polar solvents are used, separation on these thin layers is not generally satisfactory & better results are obtained using weakly polar adsorbents such as cellulose & a polar solvent such as butanol/ethanol/water: 5:4:1. Alternatively, paper chromatography could be used.