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  2. 2. Contents… •What is chromatography? •History •Technical terms in chromatography. •Biochemistry of separations. •Selected chromatography techniques. •Advanced chromatography techniques. •Application of chromatography in Ayurveda.
  3. 3. What is chromatography??? Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction. Basic principle- components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent.
  4. 4. History •Chromatography was first employed by the Russian scientist Mikhail Tsvet in 1900 for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. •Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods.
  5. 5. Technical terminology Analyte It is the substance to be separated during chromatography. It is also normally what is needed from the mixture. Analytical chromatography Chromatography used to determine the existence and possibly also the concentration of analyte(s) in a sample. Chromatogram The visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
  6. 6. Eluate The mobile phase leaving the column. Eluent The solvent that carries the analyte. Mobile phase The phase that moves in a definite direction. It may be - Liquid - (LC and Capillary Electrochromatography (CEC)), - Gas - (GC) - supercritical fluid (supercritical-fluid chromatography, SFC). with the stationary phase and is separated.
  7. 7. Immobilized phase A stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing. Retention time The characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) Solute Refers to the sample components in partition chromatography.
  8. 8. Solvent Any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography. Stationary phase Substance fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography Detector Refers to the instrument used for qualitative and quantitative detection of analytes after separation.
  9. 9. Various modes of separation •Adsorption •Partition •Ion exchange •Molecular exclusion
  10. 10. Adsorption •It Has solid stationary phase and liquid or gaseous mobile phase •The best adsorbed solute travel slow. Eg: column chromatography
  11. 11. Partition •Stationary phase is a non volatile liquid held as a thin layer on the surface of an inert solid and a mobile phase which is gas or liquid. •The solute distribute themselves between the mobile and stationary phase. Eg paper chromatography
  12. 12. Ion exchange •Similar to partition chromatography. •It has a resin coated stationary phase and the mobile phase is always liquid. •When mobile phase is eluted through the resin the electrostaticaly bound ions are released and other ions are bonded preferentially. Eg, Domestic water softners.
  13. 13. Molecular exclusion •Different from all other chromatography. •Separation according to particle size. •Mixture passes as a gas or liquid through a porous gel. •Pore size allow large particles to pass through and small particles permeate the gel.
  14. 14. Planar chromatography Column chromatography According to the shape of chromatographic bed Classification According to the physical state of mobile phase Liquid chromatography Gas chromatography
  15. 15. According to the direction of flow of mobile phase . Ascending Descending According to the purpose Analytical Preparative
  16. 16. Selected chromatographic techniques •Planar chromatography- Thin Layer Chromatography(TLC) High Performance Thin Layer Chromatography(HPTLC) •Liquid chromatography- Column Chromatography(CC) Flash Column Chromatography High pressure liquid chromatography(HPLC) •Gas chromatography
  17. 17. THIN LAYER CHROMATOGRAPHY /TLC •Process of separation – Adsorption •Stationary phase- thin layer of solid as alumina or silica(5-50 micrometre) supported on an inert base such as glass, aluminum foil or insoluble plastic(20*20cm/20*10cm). •Mobile phase- mixture of organic solvents chosen by trial and error method. •Driving force for mobile phase- capillary action .
  18. 18. Procedure •Activation of plate – heat at 110 degree centigrade for 30 minutes. •Spot crude extract using micropipette. •Keep undisturbed in the pre saturated developing chamber containing appropriate solvent. •When the solvent front approaches the top of the plate remove it and record it before drying. •Calculate the Rf / retention factor.
  19. 19. Highly polar molecules interact strongly with the Si-O bonds of the adsorbent. Thus the weakly polar molecules move through the adsorbent rapidly . PRECAUTION- the technique is done in closed vessel to ensure that the atmosphere is saturated with solvent vapor.
  20. 20. Developing the plates. •Spraying visualizing agents e.g., iodine in petroleum ether for aromatic compounds, especially with electron donating groups . Ninhydrin for amino acids. •Specially prepared plates that fluoresce in UV light can be used.
  21. 21. Detection •Developed Plates / plates with out spraying are observed in UV chamber (254nm short UV,365nm long UV). •In Specially prepared plates the solutes mask the fluorescence so a dark spot will be observed. •Compounds with their own fluorescence can directly be identified. • Known solutes identified with Rf factor. •Radioactive solutes identified by passing plate under Geiger counter. •The plates can be scanned in UV/visible absorption along the lane which mixture has travelled the reflected light from the lane is then measured relative to the radiation outside the lane
  22. 22. High performance thin layer chromatography (HPTLC) Similar to TLC but require shorter time and better resolution. Stationary phase- Pre coated plates available . whatmann HPTLC plates are produced from 4-5 micrometre silica gel coated uniformly over aluminium sheet. Advantage •The use of smaller particle sized adsorbent helps increase resolution and sensitivity. •About 50-70mm solvent front enough for efficient separation which is achieved in about 4 minutes. •Very highly concentrated sample is taken for HPTLC thus it need to be applied in less amount.
  23. 23. Column Chromatography •Process of separation – Adsorption •Stationary phase- Inert solids such as silica gel (basic compounds), alumina (acidic compounds),cellulose, magnesia(unsaturated organic compounds), dextran(compounds like purines) supported in a glass column. •Mobile phase- A wide range of solvents from hydrocarbons to esters used. Mostly mixture of organic solvents chosen. Optimum solvent is chosen by running experiments on a small scale using TLC Plates •Driving force for mobile phase- Gravitational force .
  24. 24. Procedure •In the glass column initially place a glass wool plug to prevent the stationary phase from clogging the tap. •The silica/ appropriate stationary phase is mixed with solvent to form a slurry and the stationary phase is filled in the column with out any bubbles. •The column saturated with solvent is then topped with the solvent and the solvent is allowed to flow through. •The solvent is then brought to the exact level of the stationary phase.
  25. 25. •A porous flow adapter like sand is added above stationary phase so that the solute cannot mix with the solvent above them. • The sample is then loaded in the column. •Periodic saturation of column with solvent is needed. •All fractions of eluate is collected continuously in test tubes and analyzed using TLC. •Extract from liquid collected by evaporating the solvent.
  26. 26. Loading of the sample 1- wet loading 2- dry loading Precaution: The stationary phase should be saturated with solvent or else it may crack and lose its integrity. Bubbles hinder the smooth flow of solute in the column.
  27. 27. Flash Chromatography •Alternative technique similar to liquid / column chromatography. The difference is in the driving force for the solvent. •The solvent is forced through the column at a faster rate by applying pressure from nitrogen cylinder. •A valve is attached at the top of the column and nitrogen is introduced at a pressure up to 200000N/m sq. •The solvent is replaced by closing the tap and releasing the pressure. Before opening the tap after refill, the the valve is replaced and the gas supply is turned on.
  28. 28. High performance/presure liquid chromatography /HPLC •Process of separation – Adsorption if stationary phase is silica. Partition if the stationary phase contain bonded non volatile solutes to allow interaction of solutes with different polarities. •Stationary phase-uniform porous silica particles of 1 micrometre diametre and surface pores of nanometre range(high surface area). Coated particles also used. •Mobile phase- A wide range of solvents from hydrocarbons to esters used. Mostly mixture of organic solvents chosen. Optimum solvent is chosen by running experiments on a small scale using TLC Plates •Driving force for mobile phase- High pressure of 10 mega Newton/sq m or 100 atmosphere from twin cylinder reciprocal pumps.
  29. 29. Type of HPLC Stationary phase Mobile phase relevance Normal phase chromatography Polar Non-polar Non-polar compounds elude first Reverse phase chromatography Non-polar Polar Polar compound elude first. Used for separating plant constituents which are polar Types of HPLC
  30. 30. •High pressure means that the instrumentation has to be strong. The pump and the piping is constructed out of stainless steel. It will also be inert to the solvent and solutes. •Flow rate of HPLC column is slow 0.5-5 cc/min. •A small sample volume of 5- 20 cc is injected through the injection valve.
  31. 31. Detection of compound after HPLC Most compound separated by HPLC absorb UV radiation, the elute is passed through a small cell so that UV rays can be passed through the solute
  32. 32. Other methods used in conjunction with HPLC are:- 1. Mass spectrometry 2. Infra red spectroscopy 3. Visible spectroscopy 4. UV spectroscopy 5. Fluorescence spectroscopy 6. Conductivity measurement 7. Refractive index measurement
  33. 33. GAS CHROMATOGRAPHY •Process of separation – Partition b/w mobile gas and the stationary liquid phase. •Stationary phase- Solid or non volatile liquid coated in a narrow column in an oven . •According to the stationary phase it is classified to solid-gas or liquid-gas chromatography. •Mobile phase- gas .eg, Helium. • The sample must be capable of being volatilized without being decomposed.
  34. 34. Procedure •The sample is injected through a self sealing disc into a small heated chamber where it is vaporized. •The injector oven is 50-100 degree hotter than the start of the column. •Sample is taken through the column by an inert gas /carrier eg, helium, nitrogen. •The column is coiled to fit into the thermostatically controlled oven. •The nature of the stationary phase and the temperature is adjusted according to the polarity and volatility of the substance to be separated. •The solute pass to the end of the column to a detector.
  35. 35. •The carrier must be dry to avoid interference from water. •The carrier is dried by passing through anhydrous copper(II) Sulphate or self indicating silica and unwanted solvent vapors are dried by passing through activated charcoal.
  36. 36. Detectors •Thermal conductivity detectors •Respond to the change in the thermal conductivity of gas leaving the column. •A hot tungsten film is kept in an oven set at a given temperature . •Only hydrogen has thermal conductivity more than helium so any solute with helium will increase the temperature of the film.
  37. 37. Flame ionization detector. •Particularly used for detecting organic solvents. •The gas from the column is mixed with hydrogen and air and it is burned. •CH. Radicles formed on combustion are oxidised to CHO+ ions which allow a current to transmit via cathode. •This is then converted to signal.
  38. 38. Some advanced techniques of chromatography
  39. 39. Affinity chromatography based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. Supercritical fluid chromatography Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure Size-exclusion chromatography Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size.
  40. 40. Expanded Bed Adsorption (EBA) Chromatographic Separation Expanded Bed Adsorption (EBA) Chromatographic Separation captures a target protein from a crude feed stream when it passes through a chromatography column system containing adsorbent beads. With this technique the crude feedstock can be treated directly in the chromatographic column, avoiding the traditional clarification and pre- treatment steps. Simulated moving-bed chromatography The simulated moving bed (SMB) technique is a variant of high performance liquid chromatography; it is used to separate particles and/or chemical compounds that would be difficult or impossible to resolve otherwise. This increased separation is brought about by a valve-and-column arrangement that is used to lengthen the stationary phase indefinitely. In the moving bed technique of preparative chromatography .In the simulated moving bed technique instead of moving the bed, the sample inlet and the analyte exit positions are moved continuously, giving the impression of a moving bed
  41. 41. Pyrolysis gas chromatography Pyrolysis gas chromatography mass spectrometry is a method of chemical analysis in which the sample is heated to decomposition to produce smaller molecules that are separated by gas chromatography and detected using mass spectrometry. Fast protein liquid chromatography Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. Countercurrent chromatography Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. Chiral chromatography Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images.
  42. 42. CHROMATO GRAPHY IN AYURVEDA From recent researches Standardisation of Kutajarista by HPLC analysis based upon the presence of the biomarker conessine in the formulation. The Gas chromatography was set up for the quantitative estimation of alcohol. The method was developed by changing various parameters of gas chromatography. In ayurvedic preparation arishtha, the estimated alcohol (as self generated alcohol) was found to be 8.1% (khadirarishta), 10.2% (drakshakumari), 7.8% (saraswatarishta) and obeyed the label claim
  43. 43. It was observed that all commercial samples of Talishadi churna, and standard are similar in their organoleptic and qualitative chemical analysis but physical characteristic, fluorescence analysis and High Performance Thin Layer Chromatography (HPTLC) chromatogram of various formulations are not matching with each other, and it may be due to the raw material collection time, geographical variation, etc. HPTLC and HPLC fingerprinting analysis using a marker compound Bebeerine isolated from roots of Cissampelos pareira. The roots of Cissampelos pareira can be distinguished from other two plants by presence of high concentration of alkaloids especially the presence of high concentration of pharmacologically active alkaloid bebeerine, which was found to be present in very low concentration in Stephania japonica and absent in roots of Cyclea peltata.
  44. 44. Thirty Ayurvedic herbs and formulations have been screened for the presence of gallic acid by the use of silica gel thin layer chromatography. The samples showing presence of gallic acid in screening by the silica gel thin layer chromatography were quantitatively analyzed for gallic acid by ferric reducing antioxidant power (FRAP) assay.
  45. 45. CONCLUSION 1. Drug identification 2. Quantitative and qualitative standardization of crude drugs. 3. To Assess various stages of ongoing reaction like fermentation. 4. To develop Fingerprints for single and poly herbal formulation 5. Extraction of bio active substances for neo -formulations. 6. Discovery of new chemical compounds.
  46. 46. Ayurveda is a science that has its own rich fundamental principles and innate style of drug standardization thus it is always better to think twice before blindly implementing contemporary technical advancements.