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 liquid withinthe spaces of a material due to the forces ofadhesion, cohesion, and surface tension. Retention time-the average time of thesubstance that is bound by the stationary phaseand will travel slowly through the column andexit at some later time. 7. DEFINITION OF TERMS: Peak-the width of the region that contains eachcompound Viscosity- measure of the resistance of a fluidwhich is being deformed by either shear stress ortensile stress. VOCs volatile organic compounds made up oflarge group of small organic compounds withboiling point below 200 oC. 8. DEFINITION OF TERMS: 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. 9. DEFINITION OF TERMS: Partition Coefficient- ratioof concentrations of a compound inthe two phases of a mixture oftwo immisciblesolvents at equilibrium. Volatility- measure of the tendency ofa substance to vaporize 10. DEFINITION OF TERMS: Eluate- the separated components Elution- Eluent- the carrier portion of the mobile phase. 11. THE CHROMATOGRAPH 12. 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. 13. COMPONENTS OF A CHROMATOGRAPHORIGINAL SAMPLEAND MOBILE PHASE COLUMNSUPPORT ANDSTATIONARYPHASEReceiving vessel 14. TYPES OF CHROMATOGRAPHY CAN BE CLASSIFIEDACCORDING TO: MOBILE PHASE STATIONARY PHASE SUPPORT 15. GASCHROMATOGRAPHY 16. 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. 17. GAS CHROMATOGRAPHY It can separate nanograms orpicograms of volatile substances. It is principally a method for theseparation and quantitativedetermination of gases and volatileliquids and substances. 18. GAS CHROMATOGRAPHY HOW IS IT PERFORMED? A System called Gas Chromatograph is usedto perform GC. COMPONENTS:GAS SOURCEINJECTION SYSTEMCOLUMNDETECTOR 19. GAS CHROMATOGRAPHY 20. GAS CHROMATOGRAPHY GAS SOURCE- supplies the mobile phase. It istypically a gas cylinder equipped withpressure regulators to deliver the mobilephase at a controlled state. TWO STAGE REGULATOR- for the pressure 21. GAS CHROMATOGRAPHY INJECTION SYSTEM- consists of a heatedloop or port into which the sample is placedand converted into a gaseous form. COLUMN- contains the stationary phase andsupport material for the separation ofcomponents in the sample. This column is held in an enclosed area known as the column oven 22. GAS CHROMATOGRAPHY COLUMN OVEN- maintains the temperature at a well-defined value. DETECTOR- monitors sample components as they leave the column. DATA SYSTEM 23. Video 24. TypicalCHROMATOGRAM 25. Peak LabelNameMass Boiling Point (oC)1 Ethane 30.0694 -88.62Ethylene28.0536 -103.73Propane 44.0962-42.064Propylene 42.0804 -47.45Isobutane 58.123-11.76N-Butane58.123-0.457Acetylene 26.0378 -28.1 (sublimes) 26. GAS CHROMATOGRAPHYFACTORS THAT AFFECT GC: Requirements for the Analyte Volatility and Thermal Stability Chemical Derivatization 27. GAS CHROMATOGRAPHYCommon Mobile Phases in GC: Hydrogen Helium Nitrogen Argon 28. GAS CHROMATOGRAPHYELUTION METHODS IN GC: THE GENERAL ELUTION PROBLEM x Works well if the sample is relatively simple or has only a few known compounds. x Generally used for samples containing only relatively volatile analytes x Difficulty to find a single set of conditions that can separate all the components of a complex sample with adequate resolution and in a reasonable amount of time. 29. GAS CHROMATOGRAPHYELUTION METHODS IN GC: Temperature Programming*vary the temperature of the column overtime.*temperature increases; retentiondecreases 30. GAS CHROMATOGRAPHYGC SUPPORT MATERIALS Packed Columnfilled with small support particles that acts as an adsorbent or that are coated with the desired stationary phase. 31. GAS CHROMATOGRAPHYGC SUPPORT MATERIALS Packed Column Diatomaceous earth is a common support placed on packed columns. This material is formed from fossilized diatoms and mainly consists of Silicon dioxide or silica. 32. GAS CHROMATOGRAPHY Open- Tubular Columnsstationary phase coated on or attached to its interiorsurface. 33. GAS CHROMATOGRAPHY Open- Tubular ColumnsA coating of polyimide is present on the outside ofthis column to give it better strength and flexibility forhandling and storage. 34. GAS CHROMATOGRAPHYThree types of open-tubular columns in GCBased on how the stationary phase is placedin the column. 35. GAS CHROMATOGRAPHY1.Wall-Coated Open-Tubular (WCOT) ColumnThin film of a liquid stationary phase is placeddirectly on the wall of the column. 36. GAS CHROMATOGRAPHY2. Support- Coated Open-Tubular (SCOT) column Has an interior wall that is coated with a thinlayer of a particulate support, plus a thin film of aliquid stationary phase that is coated onto thissupport layer. 37. GAS CHROMATOGRAPHY3. Porous-layer Open-Tubular (PLOT) columnIt also contains a porous material that isdeposited on the columns interior wall, but thesurface of this material is now used directly as thestationary phase without any additional coating. 38. GAS CHROMATOGRAPHY GC STATIONARY PHASES Gas- Solid Chromatography ( solid adsorbents)Gas-Liquid Chromatography (liquids coated on solids)Bonded phases 39. 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 40. 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. 41. 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. 42. 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. 43. 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. 44. 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. 45. 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 46. GAS CHROMATOGRAPHY Flame Ionization Detector- detects organic compounds by measuringtheir ability to produce ions when they are burnedin flame. 47. 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. 48. 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. 49. GAS CHROMATOGRAPHY/MASSSPECTROMETRY A POWERFUL TOOL for both measuring andidentifying analytes as they elute from a GCcolumn. Converts a portion of the eluting analytes intogas-phase ions that can be separated anddetected. electron-impact ionization or chemicalionization can be used to create ions. 50. GAS CHROMATOGRAPHY/MASSSPECTROMETRY 51. SAMPLE INJECTION AND PRETREATMENTTGAS SAMPLES Natural candidates for this technique Should be possible to directly sample and injectthis gas onto a system. It is often necessary to collect and concentratethese analytes before they are examined by GC. Another way of collecting is using a cold trap. 52. SAMPLE INJECTION AND PRETREATMENTTLIQUID SAMPLES Direct injection can be used for a liquid samplewhich uses a calibrated microsyringe to applythe desired volume of sample to the system. 53. SAMPLE INJECTION AND PRETREATMENTTSolid samples Extraction of compounds of interest from thesolid material. Can be accomplished by using liquid-liquidextraction or super critical fluid extraction. 54. GAS CHROMATOGRAPHYADVANTAGES:o Ability to provide qualitative informationand quantitative informationo FAST ANALYSIS, HIGH SPEEDo Efficient, providing high resolutiono Sensitiveo Requires small sampleso Inexpensiveo EASY, WELL KNOWN 55. GAS CHROMATOGRAPHYDISADVANTAGES: o LIMITED to volatile samples o Dirty samples require clean up o Some training/experience is necessary o Most use another instrument for confirmation of Identity (e.g Mass Spectrometry) 56. 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. 57. LIQUIDCHROMATOGRAPHY 58. LIQUID CHROMATOGRAPHY A Chromatographic technique in which themobile phase is a liquid. Originally developed by Russian botanist MikhailTswett in 1903. Works directly with liquid samples, which makesit valuable in such areas as foodtesting, environmental testing andbiotechnology. 59. 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. 60. LIQUID CHROMATOGRAPHYFACTORS THAT AFFECT LIQUID CHROMATOGRAPHY:*requires both a difference in retention and good efficiencyfor it to separate two given chemicals*Sample*Analyte Requirements*Column Efficiency*Role of the Mobile phase 61. LIQUID CHROMATOGRAPHYTypes of Liquid Chromatography: Adsorption Chromatography Partition Chromatography Ion-Exchange Chromatography Size-Exclusion Chromatography Affinity Chromatography 62. 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 63. 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. 64. 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. 65. 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 66. LIQUID CHROMATOGRAPHY 67. 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 68. LIQUID CHROMATOGRAPHYTwo Main Categories of Partition Chromatography: Normal-phase- uses polar stationary phase Reversed phase-uses nonpolar stationary phase 69. 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. 70. 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. 71. 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) 72. 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 73. 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 74. LIQUID CHROMATOGRAPHY ApplicationsRemoval of certain types of ions from samples such asin water-purificationDirect separation and analysis of samples 75. Ion Exchange Column.mp4 76. Factors to be considered in Ion-Exchange ChromatographyBufferspHSalt Gradient 77. 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. 78. 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 79. 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 80. 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. 81. 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 82. LIQUID CHROMATOGRAPHYCarbohydrate gels like agarose or cellulose are commonly used with affinity ligands serve as supports.Silica can also be used with affinity ligands 83. LIQUID CHROMATOGRAPHYAPPLICATIONS:oLarge-Scale Purification method for enzymes and proteinsoSample preparationoDirect analysis of complex biological samples.oStudy of biological interactions. 84. CHROMATOGRAPHY 85. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY Uses a pressure for the pumping ofaqueous or organic solution through acolumn. The mobile phase is forced underpressure through a long, narrowcolumn, yielding an excellent separationin a relatively short time. Highly sensitive and specific. 86. 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. 87. 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. 88. 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. 89. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY Applications: Use in biomedical research, routineclinical determination and drugresearching programs 90. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Instruments Fraction Collector Auto Sampler Pumping systems Columns & Packing Detectors Control Data & Processing 91. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 92. HIGH PERFORMANCE LIQUIDCHROMATOGRAPHYApplications: Use in monitoring the use of therapeuticdrugs and detecting drug abuse. also use to separate compoundscontributing to the fragrance of flowers 93. OLD TECHNIQUES INCHROMATOGRAPHYA separation that takes place on a flatsurface or a plane: PAPER CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY 94. PAPER CHROMATOGRAPHY Based on nature of solvent,solubility of soluteand rate of diffusion. Uses paper as thestationary phase anda solvent as themobile phase. 95. PAPER CHROMATOGRAPHY Solvent movesthrough the paper bya capillary action Separation dependson the solubility ofsolute andsolvents, the polarityof solvent, andpolarity of solutes inthe sample. 96. PAPER CHROMATOGRAPHYVisualization of theseparated sampleoccurs by chemicalreaction, whichproduces a colorchange. 97. 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 98. THIN LAYER CHROMATOGRAPHY Used as a semi-quantitativescreening test screening test Uses as thin layer of silicagel, alumina gel, polyacrylamidegel, or starch gel attached to glassplate as stationary phase and themobile phase is liquid solvent. 99. 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 100. THIN LAYER CHROMATOGRAPHY 101. 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. 102. THIN LAYER CHROMATOGRAPHY 103. THIN LAYER CHROMATOGRAPHY 104. General Categories of Chromatographic Methods Gas ChromatographyType of Stationary Phase Gas-Solid ChromatographySolid, Underivatized Support Gas-Liquid Chromatography Liquid-coated supportLiquid ChromatographyType of Stationary Phase Adsorption Chromatography Solid, Underivatized SupportPartition ChromatographyLiquid-coated support or derivatized supportIon-Exchange ChromatographySupport Containing Fixed Charges Exclusion ChromatographyPorous, Inert SupportAffinity Chromatography Support with Immobilized Biological Ligand 105. Prepared by:Algarne, Den MarionArtates, Michael JosanHosillos, Ianniel EddynLipardo, Fredrik ThomasTapiculin, JamaicaBMLS3A