1.CHROMATOGRAPHY

2. CHROMATOGRAPHY A laboratory technique in which thecomponents of a sample are separatedbased on how they distribute between twochemical or physical phases, one of whichis stationary and other of which is allowedto travel through the separation system. 3. CHROMATOGRAPHY Introduced first by the Russian botanistMikhail Semenovich Tswett. Mixtures of solutes dissolved in a commonsolvent are separated from one another bya differential distribution of the solutesbetween two phases. 4. PRINCIPLE Fractionalism of mixtures ofsubstances In the operation of thechromatogram, a mobile gaseous orliquid phase is use to wash thesubstances to be separated through acolumn of a porous material. 5. PRINCIPLE The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium. 6. DEFINITION OF TERMS: Capillary Action the movement of liquidwithin the spaces of a material due to theforces of adhesion, cohesion, and surfacetension. Retention time-the average time of thesubstance that is bound by the stationaryphase and will travel slowly through thecolumn and exit at some later time. Peak Viscosity 7. DEFINITION OF TERMS: VOCs volatile organic compounds Stationary phase- fixed phase that iscoated or bonded within the column Mobile phase-phase that is flowingthrough the column and causessample components to move towardsthe columns end. 8. COMPONENTS OF A CHROMATOGRAPH MOBILE PHASE A phase that is flowingthrough the column and causes samplecomponents to move toward the columns end. STATIONARY PHASE- A fixed phase that iscoated or bonded within the column; it alwaysremain in the system.It is responsible for delaying the movement of compounds as they travel through the column. SUPPORT- onto which the SP is coated orattached. 9. COMPONENTS OF A CHROMATOGRAPHORIGINAL SAMPLEAND MOBILE PHASE COLUMNSUPPORT ANDSTATIONARYPHASEReceiving vessel 10. TYPES OF CHROMATOGRAPHY CAN BE CLASSIFIEDACCORDING TO: MOBILE PHASE STATIONARY PHASE SUPPORT 11. GAS CHROMATOGRAPHY A type of Chromatography in which themobile phase is a GAS. First GC system was developed by ErikaCremer The presence of a gas mobile phase makesGC valuable for separating substances likeVOCs that occur naturally as gases that caneasily be placed into gaseous phase. 12. GAS CHROMATOGRAPHY It can separate nanograms orpictograms of volatile substances. It is principally a method for theseparation and quantitativedetermination of gases and volatileliquids and substances. 13. GAS CHROMATOGRAPHY HOW IS IT PERFORMED? A System called Gas Chromatograph is usedto perform GC. COMPONENTS:GAS SOURCEINJECTION SYSTEMCOLUMNDETECTOR 14. COLUMN DETECTOR MOBILE PHASE FLOW METER SOURCE ANDFLOW CONTROLINJECTORCOLUMN OVENDATA ACQUISITION 15. GAS CHROMATOGRAPHY GAS SOURCE- supplies the mobile phase. It istypically a gas cylinder equipped withpressure regulators to deliver the mobilephase at a controlled state. INJECTION SYSTEM- consists of a heatedloop or port into which the sample is placedand converted into a gaseous form. 16. GAS CHROMATOGRAPHY COLUMN- contains the stationary phase andsupport material for the separation ofcomponents in the sample. This column isheld in an enclosed area known as the columnoven. COLUMN OVEN- maintains the temperatureat a well-defined value. DETECTOR- monitors sample components asthey leave the column. 17. VIDEO 18. GAS CHROMATOGRAPHYFACTORS THAT AFFECT GC: Requirements for the Analyte Volatility and Thermal Stability Chemical Derivatization 19. GAS CHROMATOGRAPHYCommon Mobile Phases in GC: Hydrogen Helium Nitrogen Argon 20. GAS CHROMATOGRAPHYGC SUPPORT MATERIALS Packed Column filled with small support particles that acts as anadsorbent or that are coated with the desired stationaryphase. Open- Tubular Columnsstationary phase coated on or attached to its interiorsurface. 21. GAS CHROMATOGRAPHY GC STATIONARY PHASES Gas- Solid Chromatography ( solid adsorbents)Gas-Liquid Chromatography (liquids coated on solids)Bonded phases 22. GAS CHROMATOGRAPHY Gas-Solid Chromatographyo Solid adsorbent is used as a stationary phase.o Uses the same material as both the support andstationary phase, with retention occurring through theadsorption of analytes to the supports surface.o Example of support is a MOLECULAR SIEVE.o Other supports include:o ORGANIC POLYMERS - porous polystyreneo INORGANIC SUBSTANCES Silica or Alumina 23. GAS CHROMATOGRAPHYo Increasing the surface area of the GSC support willincrease the phase ratio and result in higher retention foranalyteso Pore size is important because only compounds smallerthan the pores are able to contact the surface are withinthe space.o Polarity of Support and its functional groups will alsoaffect how analytes will bind to them. 24. GAS CHROMATOGRAPHY Gas-Liquid Chromatographyo A chemical coating or layer is placed onto the supportand used as the stationary phase.o Most Common types of GC.o 100% dimethylpolysiloxane, 5%phenyl-95% methylpolysiloxane Examples of liquids that are used asStationary phase. 25. GAS CHROMATOGRAPHY Gas-Liquid Chromatographyo All of these liquids have High boiling points and lowvolatilities, which allows them to stay within thecolumn at relatively high temperatures that are oftenused in GC for sample injection and elution.o Liquids are also wettable- easy to place onto a supportin a thin, uniform layer. 26. GAS CHROMATOGRAPHY Gas-Liquid Chromatographyo All of these liquids have High boiling points and lowvolatilities, which allows them to stay within thecolumn at relatively high temperatures that are oftenused in GC for sample injection and elution.o Liquids are also wettable- easy to place onto a supportin a thin, uniform layer. 27. GAS CHROMATOGRAPHY Bonded Phaseso Column Bleed- most nonvolatile liquid will slowlyvaporize or break apart and leave the column overtime.o Column bleed changes the retention characteristics ofthe column.o It can also cause some GC detectors to have a highbackground and noisy signal as the stationary phaseleaves the column and enters the detector.o Use of bonded phase minimize column bleed. 28. GAS CHROMATOGRAPHY Bonded Phaseso Produced by reacting groups on a polysiloxane stationary phase with silanol groups that are located on the surface of a silica support. 29. GAS CHROMATOGRAPHYTypes of Gas Chromatography Detectors General Detectorsx Thermal Conductivity Detector-used for both organic and inorganic compounds-measures the ability of the eluting carrier gas andanalyte mixture to conduct heat away from hot-wirefilament-thermal conductivity.-example: Wheatstone bridge 30. GAS CHROMATOGRAPHY Flame Ionization Detector- detects organic compounds by measuringtheir ability to produce ions when they are burnedin flame. 31. GAS CHROMATOGRAPHY Selective Detectors x Nitrogen-Phosphorous Detector- selective for the determination of nitrogen- orphosphorous containing compounds.- Similar to FID, but does not use a flame for ionproduction. 32. GAS CHROMATOGRAPHY Electron capture detector- detects compounds that have electronegativeatoms or groups in their structure, such as halogen atoms( I,Br,Cl and F) and Nitro Groups.-can also be used to detect polynuclear aromaticcompounds, anhydrides and conjugated carbonylcompounds. 33. GAS CHROMATOGRAPHYApplications: Most effectively used for analyses of organiccompounds, space related, complex mixtures ofvolatile substances at column temperature ofless than -40 C to greater than 550 C. Geochemical research projects such asdetermination of various environmentalpollutants at extremely low concentrations. 34. GAS CHROMATOGRAPHYADVANTAGES:DISADVANTAGES:o Ability to provide qualitative o LIMITED to volatileinformation and quantitative samplesinformation o Not suitable foro FAST ANALYSIS thermally labile sampleso Efficient, providing high o Fairly difficult for largeresolutionpreparative sampleso Sensitive o Requires spectroscopyusually masso Nondestructivespectroscopy foro Requires small samplesconfirmation of peako Inexpensive identity 35. LIQUID CHROMATOGRAPHY A Chromatographic technique in which themobile phase is a liquid. Originally developed by Russian botanist MikhailTswett in 1903. Its popularity is due to the ability of this methodto work directly with liquid samples, whichmakes it valuable in such areas as food testing,environmental testing and biotechnology. 36. LIQUID CHROMATOGRAPHYHOW IS LIQUID CHROMATOGRAPHY PERFORMED? A System known as a Liquid Chromatograph isused to perform LC. 37. LIQUID CHROMATOGRAPHY Components of the LC System: Support enclosed in a Column Stationary phase- enclosed in a Column Liquid mobile phase-delivered to Column by means ofPump Injection device- on analytical applications it is beingused,to apply samples to the column. Collection Device (optional)- placed after the columnto capture analytes as they elute. 38. LIQUID CHROMATOGRAPHYFACTORS THAT AFFECT LIQUID CHROMATOGRAPHY:*requires both a difference in retention and good efficiencyfor it to separate two given chemicals*Sample*Analyte Requirements*Formats*Role played by the Mobile phase 39. LIQUID CHROMATOGRAPHYRequirements for the Analyte:Must be possible to place this chemicalinto a liquid that can be injected onto thecolumn.There must be a difference in retentionbetween the analytes to be prepared. 40. LIQUID CHROMATOGRAPHYTypes of Liquid Chromatography: Adsorption Chromatography Partition Chromatography Ion-Exchange Chromatography Size-Exclusion Chromatography Affinity Chromatography 41. LIQUID CHROMATOGRAPHY1. ADSORPTION CHROMATOGRAPHYA chromatographic technique that separates solutesbased on their adsorption to the surface of a support. Also known as Liquid-Solid Chromatography Equivalent method in GC is Gas-Solid Chromatography Uses the same material as both stationary phase andsupport. Retention process can be explained on the ff below: A+ n M-SurfaceA-Surface + n M 42. LIQUID CHROMATOGRAPHY Elutropic strength- strength of a mobilephase in adsorption chromatography It is a measure of how strongly a particularsolvent or liquid mixture will absorb to thesurface of a given support. Examples: silica and Alumina supports A liquid with large elutropic strength willstrongly adsorb to the given support, whichwill prevent the analyte from binding to thesupport. 43. LIQUID CHROMATOGRAPHY Stationary Phases and Mobile PhasesSilica (SiO2) - most popular support since it ispolar in nature, thus it will strongly retain polarcompounds.Alumina (Al2O3) general-purpose support, butcan retain some polar solutes so strongly thatthey are irreversibly absorbed onto its surface.Carbon-based Compounds- used as nonpolarsupports that retain nonpolar solutes and have astrong mobile phase that is nonpolar. 44. LIQUID CHROMATOGRAPHY Applications Help purify new compounds Separation of geometrical isomers and chemicals that belong to a given class of substances Remove undesired side-products during synthesizing AZT or other drugs 45. LIQUID CHROMATOGRAPHY 46. LIQUID CHROMATOGRAPHY2. PARTITION CHROMATOGRAPHYo It is a Liquid Chromatographic technique in which solutes are separated based on their partitioning between a liquid mobile phase and a stationary phase coated on a solid support.o Support used is usually Silicao Originally, it involves coating of support with a liquid stationary phase that was immiscible with the mobile phase 47. LIQUID CHROMATOGRAPHYTwo Main Categories of Partition Chromatography: Normal-phase- uses polar stationary phase Reversed phase-uses nonpolar stationary phase 48. LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Liquid Stationary Phases coated onto solid supports Bonded Phases widely used due to their better stabilityand efficiency compared to liquid stationary phases. Silica - often used as the support. To place bonded phaseson this support, silanol groups on the surface of silica arefirst treated with an organosilane that contains thedesired stationary phase as a side chain. Agents like triethylamine and trifluoroacetic acid can alsobe added to the mobile phase to prevent silanol groupsfrom binding to analytes. 49. LIQUID CHROMATOGRAPHYApplications: Used in analytical laboratories Separation of analytes in organic solvents and chemicals that contain polar functional groups. Employed in pharmaceutical industry as a method for separating and analyzing drugs during their testing and development. 50. LIQUID CHROMATOGRAPHY3. ION- EXCHANGE CHROMATOGRAPHYSolutes are separated by their adsorptiononto a support containing fixed charges on itssurface. Routinely used in Industry for the removal or replacement of Ions in products. Used for the separation of charged compounds( inorg. Ions, org. ions, AA, Proteins and NucleicAcids) 51. LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Cation-exchanger has negatively charged groupand is used to separate positive ions Anion-exchanger has positively charged groupand is used to separate negative ions 52. LIQUID CHROMATOGRAPHY Silica (SiO2) - most popular support since it is polar in nature, thus it will strongly retain polar compounds. Polystyrene used for small inorganic and organic ions Carbohydrate-based Gels useful in separation of biological compounds, which can have very strong, undesirable binding to organic polymeric resins like polysterene. Example: Agarose 53. LIQUID CHROMATOGRAPHY ApplicationsRemoval of certain types of ions from samples such asin water-purificationDirect separation and analysis of samples 54. LIQUID CHROMATOGRAPHY4. SIZE-EXCLUSION CHROMATOGRAPHYIt is a LC technique that separates substancesbased on different abilities of analytes to accessthe mobile phase within the pores of a support..o No true stationary phase is present in this systemo Uses a support that has a certain range of pore sizes.o A separation based on size or molar mass. 55. LIQUID CHROMATOGRAPHYStationary Phases and Mobile PhasesIdeal support consists of a porous materialthat does not interact directly with the injectedsolute.o Carbohydrate-based supports like dextran and andagarose in their underivatized form can be used in SECfor biological compounds and aqueous-basedsamples.o Polyacrylamide gel can also be usedo For organic solvents, Polystyrene can also be usedo Silica containing diol-bonded phase can be used onaqueous samples 56. LIQUID CHROMATOGRAPHYAPPLICATIONS: Used in biological samples Transfer of large analytes from one solution toanother Removal of salts from sample Separation of biomolecules and polymers Estimation of molar mass 57. LIQUID CHROMATOGRAPHY5. AFFINITY CHROMATOGRAPHYA Technique based on biologically relatedinteractions.o Makes use of the selective, reversible interactions thatcharacterize most biological systems.o Example: binding of enzyme with its substrate. 58. LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Stationary phase is represented by theaffinity ligand.o High specificity ligands- bind to only one or a few veryclosely related moleculeso Examples:antibodies for binding to foreign agents andsingle-stranded Nucleic Acids for separating and binding tocomplemetary strandso General ligands- bind to a family or class of relatedmolecules 59. LIQUID CHROMATOGRAPHYCarbohydrate gels like agarose or cellulose are commonly used with affinity ligands serve as supports.Silica can also be used with affinity ligands 60. LIQUID CHROMATOGRAPHYAPPLICATIONS:oLarge-Scale Purification method for enzymes and proteinsoSample preparationoDirect analysis of complex biological samples.oStudy of biological interactions. 61. CHROMATOGRAPHY 62. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY Uses a pressure for the pumping ofaqueous or organic solution through acolumn. The mobile phase is forced underpressure through a long, narrow column,yielding an excellent separation in arelatively short time. Highly sensitive and specific. 63. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY Become the primary means ofmonitoring the use of drugs and ofdetecting drug abuse. Also used to separate the compoundscontributing to the fragrance of theflowers. 64. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ADVANTAGES:An automated process that takes only a few minutes to produce results.Uses gravity instead of high speed pump to force compounds through the densely packed tubing.Results are of high resolution and are easy to read.Can be reproduce easily via automated process. 65. HIGH PERFORMANCE LIQUID CHROMATOGRAPHYDISADVANTAGES:Difficult to detect coelution, which may lead to inaccurate compound categorization.High cost for equipment needed to conduct HPLC.Operation is complex, requiring a trained technician to operate.Equipment has low sensitivity to some compounds because of the speed of the process. 66. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY Application: Use in biomedical research, routineclinical determination and drugresearching programs 67. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Instruments Fraction Collector Auto Sampler Pumping systems Columns & Packing Detectors Control Data & Processing 68. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 69. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHYApplication: Use in monitoring the use of therapeuticdrugs and detecting drug abuse. also use to separate compoundscontributing to the fragrance of flowers 70. OLD TECHNIQUES INCHROMATOGRAPHYA separation that takes place on a flatsurface or a plane: PAPER CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY 71. PAPER CHROMATOGRAPHY Based on nature of solvent,solubility of soluteand rate of diffusion. Uses paper as thestationary phase anda solvent as themobile phase. 72. PAPER CHROMATOGRAPHY Solvent movesthrough the paper bya capillary action Separation dependson the solubility ofsolute and solvents,the polarity of solvent,and polarity of solutesin the sample. 73. PAPER CHROMATOGRAPHYVisualization of theseparated sampleoccurs by chemicalreaction, whichproduces a colorchange. 74. PAPER CHROMATOGRAPHY Considered to be the simplest andthe most widely used of thechromatographic techniquesbecause its APPLICABILITY TO THEFOLLOWING: ISOLATION IDENTIFICATION AND QUANTITATIVEDETERMINATION OF ORGANICAND INORGANIC COMPOUNDS 75. THIN LAYER CHROMATOGRAPHY Used as a semi-quantitativescreening test screening test Uses as thin layer of silica gel,alumina gel, polyacrylamide gel, orstarch gel attached to glass plate asstationary phase and the mobilephase is liquid solvent. 76. THIN LAYER CHROMATOGRAPHY Fractions in the sample are generallyquite soluble in the solvent and movewith it up the stationary phase bycapillary action. Separated fractions are also developedin TLC by applying a chemical reactionwith the separated fractions to producecolor changes 77. THIN LAYER CHROMATOGRAPHY 78. THIN LAYER CHROMATOGRAPHYADVANTAGES: DISADVANTAGES: Simple and economical Spots are often faint Easy to perform since it TLC is difficult toonly involves spottingthe stationary phase reproducewith the sample & Not typicallyplacing one edge of theautomatedstationary phase plate inthe mobile phasereservoir. 79. THIN LAYER CHROMATOGRAPHY 80. THIN LAYER CHROMATOGRAPHY 81. Thanks a lot !