Thin Layer Chromatography & Paper Chromatography

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Thin Layer Chromatography & Paper Chromatography


<ul><li> 1. 1Thin Layer ChromatographyPaper ChromatographyJully TanSchool of EngineeringWhat is Chromatography?Chromatography is a technique for separating mixtures into their components inorder to analyze, identify, purify, and/or quantify the mixture or components.EP101 / EG101 Separate Analyze Identify PurifyMixture Components Quantify</li></ul> <p> 2. 2Uses for ChromatographyChromatography is used by scientists to: Analyze examine a mixture, its components, and their relations to one another Identify determine the identity of a mixture or components based on knowncomponents Purify separate components in order to isolate one of interest for further study Quantify determine the amount of the a mixture and/or the componentspresent in the sampleEP101 / EG101 Different Kinds of Chromatography Liquid chromatography separates liquid samples with a liquid solvent (mobile phase) and acolumn composed of solid beads (stationary phase).(includes column chromatography, thin-layer, and HPLC) Stationary phase: silica, alumina, etc. Mobile phase (moving phase): organic solventsEP101 / EG101Important properties: polarityPaper Chromatography separates dried liquid samples with a liquid solvent (mobile phase) and apaper strip (stationary phase)Thin-Layer Chromatography separates dried liquid samples with a liquid solvent (mobilephase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) Gas chromatography:separates vaporized samples with a carrier gas (mobile phase) and acolumn composed of a liquid or of solid beads (stationary phase) Stationary phase: a film of a polymer or a wax. The film must have a high boiling point Mobile phase: gas (Helium is the usual carrier gas) Important properties: boiling point 3. 3Illustration of ChromatographyStationary PhaseMixture ComponentsBlackRed EP101 / EG101Yellow Insoluble in MobilePhaseBlue ----------------Affinity to MobilePhaseAffinity to StationaryPhaseComponentsSeparationMobile PhaseThin Layer ChromatographyThe surface of the plate consists of a very thin layer of silica on a plastic oraluminum backing. The silica is very polar. This is the stationary phase. Spot thematerial at the origin (bottom) of the TLC plate.Place the plate into a glass jar with a small amount of a solvent in the glass jar.This solvent acts as the moving phase.Remove the plate from the bottle when the solvent is close to the top of the plate.Visualize the spots.Non-polar compounds will be less strongly attracted to the plate and will spendmore time in the moving phase. This compound will move faster and will appearcloser to the top of the plate.Polar compounds will be more strongly attracted to the plate and will spend lesstime in the moving phase and appear lower on the plate.EP101 / EG101 4. 4Forensic Analysis using Thin Layer Chromatography Ink analysis Determines the specific chemicals Uses organic solvents Results are compared to a database of pen inkSchool of Engineering EP101 / EG101TLC ProceduresPlate preparation Mix the absorbent, water and a binder such as calcium sulfate Silica gel, paper and alumina Spread a thin layer of absorbent on an unreactive hard surface Glass, plastic, thick aluminum Heat in oven at 110C for 30 mins to activate and dry the plateSchool of Engineering EP101 / EG101 5. 5TLC Procedure Place a small amount of solvent in a beaker In pencil, draw a straight line across the plateabout 1 cm from the end of the plate Place a drop of sample solution on the lineSchool of Engineering EP101 / EG101TLC procedure Add filter paper Place in solvent Sealed containerSchool of Engineering EP101 / EG101 6. 6How TLC works Sample solution is dissolved by solvent The solution sample will travel at different distances based on solubility, polarization,size Silica gel Polar substances do not move far Non polar substances move farther up the plateROSiOSiOSiOSchool of Engineering EP101 / EG101RHO OH OHR R RCalibration/Standards TLC No calibration Standards Compare to other known substances Rf valueSchool of Engineering EP101 / EG101 7. 7Solvents The solvent can be a mixture of compounds but the polar solvent propertieswill over take the non-polar one. 10-30% Methly tert-butyl ether, MTBE, in hexane, C6H14, works well 10-30% Methylene chloride, CH2Cl2, in hexane, C6H14, for a less polarmixture 10-30% Acetone, CH3COCH3, in Methylene chloride, CH2Cl2, for a morepolar mixture Trial and error is the best way to approach which solvent to use.School of Engineering EP101 / EG101Thin-Layer Chromatography: A Two-Component MixtureLess polar!More polar!EP101 / EG101 originmixturesolvent frontsolvent frontcomponent Bcomponent Aoriginsolvent frontcomponent Bcomponent AoriginIncreasing Development Time 8. 8Visualization Destructive visualization Spray plate with H2SO4, and then bake in the oven at 110C for 15-20minutes. Compound is destroyed but all spots will be visible Nondestructive visualization because of the use of a UV light the samplewill not be destroyed. Although, not all of the spots on the plate will bevisible. Long wave UV Short wave UV Semi-destructive visualizationSchool of Engineering EP101 / EG101VisualizationA plate under a UV light to display the compoundsafter they were developedSchool of Engineering EP101 / EG101 9. 9InterpretationCalculating the Rf valuef =R ValueDistance that the Spot TraveledSolvent Front DistanceSchool of Engineering EP101 / EG101Thin-Layer Chromatography: Determination of Rf Valuessolvent frontcomponent Bcomponent AorigindSdBdAEP101 / EG101 Rf of component A =dAdSRf of component B =dBdSThe Rf value is a decimalfraction, generally onlyreported to two decimalplaces 10. 10Calculate Rf ValueSchool of Engineering EP101 / EG101Rf Value The Rf value needs to be between 0.0 and 1.0 If the value is over 1.0 or less than 0.0, the calculation is wrong (yougoofed) If the Rf value is greater than 0.8 or lower than 0.2 the values are hard tointerpret, thus creating a larger error The best Rf values are 0.3 to 0.6School of Engineering EP101 / EG101 11. 11Rf Value The Rf value is not informative What affects the Rf value? Temperature Solvent Thickness and amount of spot Other compoundsSchool of Engineering EP101 / EG101Results Multiple spots from one sample can be achieved.School of Engineering EP101 / EG101 12. 12EP101 / EG101 Identification of spots The spots are usually identified by comparing of standards of known Rf values.Pros for TLC Sensitivity Speed InexpensiveSchool of Engineering EP101 / EG101 13. 13Cons for TLC Too little of sample Too much of sample SubjectiveSchool of Engineering EP101 / EG101Principles of Paper Chromatography Capillary Action the movement of liquid within the spaces of a porous material due to theforces of adhesion, cohesion, and surface tension. The liquid is able to move up the filterpaper because its attraction to itself is stronger than the force of gravity. Solubility the degree to which a material (solute) dissolves into a solvent. Solutesdissolve into solvents that have similar properties. (Like dissolves like) This allowsdifferent solutes to be separated by different combinations of solvents.Separation of components depends on both their solubility in the mobile phase and theirdifferential affinity to the mobile phase and the stationary phase.EP101 / EG101 14. 14Advantages of TLC over paper chromatography Greater resolving power because there is less diffusion of spots. Greater speed of separation Wide choice of materials as sorbentsEP101 / EG101</p>