1.Tutorial on Chromatographic Techniques,Paper Chromatography , ThinLayer, Column, HPLC and GLC. Prepared by Lawrence Kok http://lawrencekok.blogspot.com

2. Chromatography Techniques Partition Chromatography Adsorption ChromatographyPartition Chromatography Adsorption Chromatography Components distribute bet TWO immisible liquid phase Component adsorb on solid stationary phase Depends on relative solubility bet TWO phase Depends on polarity of stationary phase, mobile phase and solutes Solutes bonds to stationary phase or mobile phase Stationary phase is polar polar solutes adsorb strongly Stationary phase is non polar non polar solutes adsorb strongly Mobile phase is polar polar solutes stay in mobile phase Mobile phase is non polar non polar solutes stay in mobile phase 3. Chromatography Techniques Partition Chromatography Adsorption ChromatographyPartition Chromatography Adsorption Chromatography Components distribute bet TWO immisible liquid phase Component adsorb on solid stationary phase Depends on relative solubility bet TWO phase Depends on polarity of stationary phase, mobile phase and solutes Solutes bonds to stationary phase or mobile phase Stationary phase is polar polar solutes adsorb strongly Stationary phase is non polar non polar solutes adsorb strongly Mobile phase is polar polar solutes stay in mobile phase Mobile phase is non polar non polar solutes stay in mobile phasePaper Chromatography GLC HPLCThin Layer ChromatographyColumn ChromatographyY adsorbstrongly X adsorb strongly 4. Chromatography Techniques Partition Chromatography Adsorption ChromatographyPartition Chromatography Adsorption Chromatography Components distribute bet TWO immisible liquid phase Component adsorb on solid stationary phase Depends on relative solubility bet TWO phase Depends on polarity of stationary phase, mobile phase and solutes Solutes bonds to stationary phase or mobile phase Stationary phase is polar polar solutes adsorb strongly Stationary phase is non polar non polar solutes adsorb strongly Mobile phase is polar polar solutes stay in mobile phase Mobile phase is non polar non polar solutes stay in mobile phasePaper Chromatography GLCHPLCThin Layer Chromatography Column Chromatography Y adsorb strongly X adsorb stronglyMobile phase liquidMobile phase gas Mobile phase liquid Mobile phase liquid Mobile phase liquid Stationary phase - liquidStationary phase - liquidStationary phase - liquid Stationary phase - solid Stationary phase - solid 5. Paper Chromatography,Thin Layer Chromatographyand Colum Chromatography 6. Chromatography TechniquesChromatography Techniques Separation technique of mixture into their pure components Identify if sample is mixture or pure both quantitatively and qualitatively Interaction of substances between 2 phases - Stationary phase and Mobile phase Separation is based on Partition or AdsorptionSeparation analysis Chromatography Partition ChromatographyAdsorption ChromatographyPartition Chromatography Adsorption Chromatography Components distribute bet TWO immisible liquid phase Component adsorb on solid stationary phase Depends on relative solubility bet TWO phase Depends on polarity of stationary phase, mobile phase and solutes Solutes bonds to stationary phase or mobile phase Stationary phase is polar polar solutes adsorb strongly Stationary phase is non polar non polar solutes adsorb strongly Mobile phase is polar polar solutes stay in mobile phase Mobile phase is non polar non polar solutes stay in mobile phase Paper Chromatography Thin Layer Chromatography Column ChromatographyY adsorbstronglyX adsorbstronglyApplicationApplication Application Detection of amino acids in a mixture Detection of amino acids in a mixture Preparative/collection of sample Different dyes in food colouring Different dyes in food colouringof pigments Separation of plant pigments Separation of plant pigments 7. Chromatography Techniques ChromatographyPartition ChromatographyAdsorption Chromatography components distribute between 2 immisible liquid phase components adsorp on solid stationary phase relative solubility in 2 phase bonds strongly to mobile phase move faster Stationary Liquid phase MobileYMobile Y YStationary phaseliquid phaseXO-Stationary phase liquid phaseXhas a layer of liquid Y solidcontaining containingYO- AI2O3 X and YXX and YXYO- SiO2 Y X X O- O- 8. Chromatography Techniques ChromatographyPartition ChromatographyAdsorption Chromatography components distribute between 2 immisible liquid phase components adsorp on solid stationary phase relative solubility in 2 phase bonds strongly to mobile phase move faster Stationary Liquid phase MobileYMobile Y YStationary phaseliquid phaseXO-Stationary phase liquid phaseXhas a layer of liquid Y solidcontaining containingYO- AI2O3 X and YXX and YXYO- SiO2 Y X X O- O-Mobile Mobile O- Y Y Y adsorb stronglyXX YStationary phase Iiquid phase XXYhas a layer of liquidliquid phase O- Y containing XYcontaining XXXYX X O- Y X X 9. Chromatography Techniques ChromatographyPartition Chromatography Adsorption Chromatography components distribute between 2 immisible liquid phase components adsorp on solid stationary phase relative solubility in 2 phase bonds strongly to mobile phase move faster Stationary Liquid phase MobileYMobile Y YStationary phaseliquid phaseXO-Stationary phase liquid phaseXhas a layer of liquid Y solidcontaining containingYO- AI2O3 X and YXX and YXYO- SiO2 Y X X O- O-Mobile Mobile Y Y O-Y adsorb stronglyXX YStationary phase Iiquid phase XXYhas a layer of liquidliquid phase Y O- containing XYcontaining XXXYX X Y O- X XXX X X Separation of X and YX YYYY Y X X Y YPartition distribution of solute X and Y between 2 liquid phase Adsorption solute X and Y adsorb temporary on the solid X more soluble in mobile phase (move with mobile liquid phase) Y adsorb strongly on solid phase, eluted slower Y less soluble in mobile phase (stay on stationary liquid phase) X in liquid mobile phase, eluted faster 10. Chromatography Techniques Paper Chromatography Thin Layer Chromatography Partition chromatographyAdsorption chromatography Distribution solute bet both liquid phase Solute adsorb on stationary solid phase Depends on relative solubilityO-Stationary phase solid Stationary phase - Cellulose paper X O- AI2O3 or SiO2 absorb water on its surface Y YYO-X Y O-Y Y X O- XMobile Liquid phase with solute X and YMobile Liquid phase with solute X and Y 11. Chromatography Techniques Paper ChromatographyThin Layer Chromatography Partition chromatographyAdsorption chromatography Distribution solute bet both liquid phase Solute adsorb on stationary solid phase Depends on relative solubilityO-Stationary phase solid Stationary phase - Cellulose paper X O- AI2O3 or SiO2 absorb water on its surface Y YYO-X Y O-Y Y X O- XMobile Liquid phase with solute X and YMobile Liquid phase with solute X and Y Aqueous liquid phase on surface of stationary phase (paper) Stationary phase - solid silica SiO2 or alumina AI2O3Mobile phase liquid solvent move by capillary action Mobile liquid phase - solventSeparation depends on how strongly solute adsorb on solidSolvent move by capillary actionAdsorption of solute on stationary solid phase 12. Chromatography Techniques Paper ChromatographyThin Layer Chromatography Partition chromatographyAdsorption chromatography Distribution solute bet both liquid phase Solute adsorb on stationary solid phase Depends on relative solubilityO-Stationary phase solid Stationary phase - Cellulose paper X O- AI2O3 or SiO2 absorb water on its surface Y YYO-X Y O-Y Y X O- XMobile Liquid phase with solute X and YMobile Liquid phase with solute X and Y Aqueous liquid phase on surface of stationary phase (paper) Stationary phase - solid silica SiO2 or alumina AI2O3Mobile phase liquid solvent move by capillary action Mobile liquid phase - solventSeparation depends on how strongly solute adsorb on solidSolvent move by capillary actionAdsorption of solute on stationary solid phase 13. Chromatography TechniquesColumn Chromatography Thin Layer Chromatography Adsorption chromatography Adsorption chromatography Solute adsorb on stationary solid phase Solute adsorb on stationary solid phase Mobile Liquid phase (solvent) with solute X and YXStationary phase solid O-Stationary phase solid X X AI2O3 or SiO2Y YO- AI2O3 or SiO2 X Y YX O-X O-X O-YYY YYMobile Liquid phase with solute X and Y YYY 14. Chromatography TechniquesColumn ChromatographyThin Layer Chromatography Adsorption chromatography Adsorption chromatography Solute adsorb on stationary solid phase Solute adsorb on stationary solid phase Mobile Liquid phase (solvent) with solute X and YXStationary phase solidO-Stationary phase solid X X AI2O3 or SiO2 Y Y O- AI2O3 or SiO2 XY Y X O- X O- X O-YYY YY Mobile Liquid phase with solute X and YStationary phase - solid silica SiO2 or alumina AI2O3 YYYMobile phase liquid solvent move by capillary action Mobile phase liquid solvent added from top (Eluant)Adsorption of solute on stationary solid phaseSolute in mobile phase adsorb on solidX - Adsorb strongly travel slowly X - Strongly adsorb travel slowly, eluted last Y Adsorb weakly travel fast with solvent Y - Weakly adsorb travel fast with solvent, eluted first 15. Chromatography TechniquesColumn ChromatographyThin Layer Chromatography Adsorption chromatography Adsorption chromatography Solute adsorb on stationary solid phase Solute adsorb on stationary solid phase Mobile Liquid phase (solvent) with solute X and YXStationary phase solidO-Stationary phase solid X X AI2O3 or SiO2 Y Y O- AI2O3 or SiO2 XY Y X O- X O- X O-YYY YY Mobile Liquid phase with solute X and YStationary phase - solid silica SiO2 or alumina AI2O3 YYYMobile phase liquid solvent move by capillary action Mobile phase liquid solvent added from top (Eluant)Adsorption of solute on stationary solid phaseSolute in mobile phase adsorb on solidX - Adsorb strongly travel slowly X - Strongly adsorb travel slowly, eluted last Y Adsorb weakly travel fast with solvent Y - Weakly adsorb travel fast with solvent, eluted first 16. Chromatography TechniquesComponents separated by Paper/TLC can be identified using Rf value Rf = Retention factor for a given eluent. Measured the distance from original spot to the centre of a particular component to the solvent frontRf value for green spot Rf value for blue spot= 3cm/12cm= 6cm/12cm= 0.25= 0.5http://home.earthlink.net/~dayvdanls/photolab/photolab7.htm 17. Chromatography TechniquesComponents separated by Paper/TLC can be identified using Rf value Rf = Retention factor for a given eluent. Measured the distance from original spot to the centre of a particular component to the solvent frontRf value for green spot Rf value for blue spot= 3cm/12cm= 6cm/12cm= 0.25= 0.5 Uses of Paper/TLC/Column chromatography Separate dyes in food colourings/plant pigments Separation/Preparative/Collection of pigments Detection of amino acids in mixturehttp://home.earthlink.net/~dayvdanls/photolab/photolab7.htm 18. Chromatography TechniquesComponents separated by Paper/TLC can be identified using Rf value Rf = Retention factor for a given eluent. Measured the distance from original spot to the centre of a particular component to the solvent frontRf value for green spot Rf value for blue spot= 3cm/12cm= 6cm/12cm= 0.25= 0.5 Uses of Paper/TLC/Column chromatography Separate dyes in food colourings/plant pigments Separation/Preparative/Collection of pigments Detection of amino acids in mixturehttp://home.earthlink.net/~dayvdanls/photolab/photolab7.htm 19. Chromatography TechniquesComponents separated by Paper/TLC can be identified using Rf value Rf = Retention factor for a given eluent. Measured the distance from original spot to the centre of a particular component to the solvent frontRf value for green spot Rf value for blue spot= 3cm/12cm= 6cm/12cm= 0.25= 0.5 Uses of Paper/TLC/Column chromatography Separate dyes in food colourings/plant pigments Separation/Preparative/Collection of pigments Detection of amino acids in mixtureFor colour formation of amino acids Spray with ninhydrin for colour formation Shine UV light for fluorescent compounds to formhttp://home.earthlink.net/~dayvdanls/photolab/photolab7.htm 20. High Performance Liquid ChromatographyGas Liquid Chromatography 21. Chromatography TechniquesHigh Performance Liquid Chromatography Partition Chromatography Components distribute between 2 immisible liquid phase Mobile phase (liquid) with stationary phase (liquid) Uses high pressure liquid to force through a columnNormal HPLCReverse phase HPLCMobile phaseO-Stationary phase Mobile phase O-Stationary phaseNon-polar liquidYsolid. AI2O3 and SiO2Polar liquid Ywith solute X and Y O- layer of polar liquidwith solute X and Y X X O- solid. AI2O3 and SiO2 Ylayer of non polar liquidY X O- hydrocarbon Y Y O-X Y O- X O-X O- Y X O-O- O- 22. Chromatography TechniquesHigh Performance Liquid Chromatography Partition Chromatography Components distribute between 2 immisible liquid phase Mobile phase (liquid) with stationary phase (liquid) Uses high pressure liquid to force through a column Normal HPLCReverse phase HPLCMobile phaseO-Stationary phase Mobile phaseO-Stationary phaseNon-polar liquid Y solid. AI2O3 and SiO2Polar liquidYwith solute X and Y O- layer of polar liquidwith solute X and YX X O- solid. AI2O3 and SiO2Ylayer of non polar liquid Y XO- hydrocarbonY Y O- X YO-XO- XO-Y X O-O-O-Non-polar solute X inY O-Polar Y solute distributeX XO-non polar stationary phase YY in polar stationary phaseY O-(elute later)XXO-(elute last)XNon-polar solute X XX O- Polar Y solute in polarYO-in non polar mobileX phase (elute first) XO- mobile phase (elute first)YY YO- Y XXX YYYYYY XXX 23. Chromatography TechniquesHigh Performance Liquid Chromatography Partition Chromatography Components distribute between 2 immisible liquid phase Mobile phase (liquid) with stationary phase (liquid) Uses high pressure liquid to force through a column Normal HPLCReverse phase HPLCMobile phaseO- Stationary phaseMobile phaseO- Stationary phaseNon-polar liquid Ysolid. AI2O3 and SiO2 Polar liquidYwith solute X and Y O-layer of polar liquid with solute X and YX X O-solid. AI2O3 and SiO2 Ylayer of non polar liquidY X O-hydrocarbon Y Y O- X YO-XO- XO-Y X O-O-O- Non-polar solute X inY O-Polar Y solute distributeX XO- non polar stationary phase YY in polar stationary phaseY O-(elute later)XXO- (elute last)XNon-polar solute X XX O- Polar Y solute in polarYO-in non polar mobileX phase (elute first) XO- mobile phase (elute first)YY YO- Y XXXYYY YYY XXX Normal HPLCReverse phase HPLC non polar solvent (mobile phase) and polar stationary phase polar solvent (mobile phase) and non polar stationary phase polar solute stay in polar stationary phase- elute later non polar solute stay in non polar stationary phase- elute later non polar solute stay in non polar mobile phase elute first polar solute stay in polar mobile phase elute first 24. Chromatography TechniquesGas Liquid Chromatography Partition Chromatography Components distribute between TWO immisible phase Mobile phase (gas) with stationary phase (liquid) Mobile phase inert gas, helium, nitrogen Incresing Temp causes elution of solutes from stationary phase GLC Mobile gas phaseO-Stationary phase with solute X and Y Ysolid. AI2O3 and SiO2 O- layer of non volatile liquid Y YXO- hydrocarbon Y X O-XO- O- 25. Chromatography TechniquesGas Liquid Chromatography Partition Chromatography Components distribute between TWO immisible phase Mobile phase (gas) with stationary phase (liquid) Mobile phase inert gas, helium, nitrogen Incresing Temp causes elution of solutes from stationary phase GLC Mobile gas phase O- Stationary phase with solute X and Y Ysolid. AI2O3 and SiO2O-layer of non volatile liquid Y YX O-hydrocarbon Y XO-X O-O-Y O- Less volatile solute Y distribute YYin liquid stationary phaseY O- (elute later/longer retention time) Solute X (volatile) XX XO-in mobile gas phase (elute first) XO- XXX YYY 26. Chromatography TechniquesGas Liquid Chromatography Partition Chromatography Components distribute between TWO immisible phase Mobile phase (gas) with stationary phase (liquid) Mobile phase inert gas, helium, nitrogen Incresing Temp causes elution of solutes from stationary phase GLC Mobile gas phase O- Stationary phase with solute X and Y Ysolid. AI2O3 and SiO2O-layer of non volatile liquid Y YX O-hydrocarbon Y XO-X O-O-Y O- Less volatile solute Y distribute YYin liquid stationary phaseY O- (elute later/longer retention time)Solute X (volatile)XX XO- in mobile gas phase(elute first) XO- XXX YYY GLC Gas (mobile phase) and non volatile liquid on stationary phase Less volatile solute Y stay in stationary phase - elute later Volatile solute X stay in mobile phase elute first 27. Chromatography Techniques GLC vs HPLCPartition Chromatography Components distribute between TWO immisible phase Mobile phase (gas) with stationary phase (liquid) Mobile phase inert gas, helium, nitrogen Incresing Temp causes elution of solutes from stationary phaseGLC HPLCStationary phaseMobile gas phaseO-Stationary phase Mobile phase O-solid. AI2O3 and SiO2with solute X and Y Ysolid. AI2O3 and SiO2Non-polar liquidYO- layer of non volatile liquid with solute X and YX XO-layer of polar liquid Y hydrocarbonY X O- hydrocarbonY YO-X Y O-X O-X O-YX O-O- O- 28. Chromatography Techniques GLC vs HPLCPartition Chromatography Components distribute between TWO immisible phase Mobile phase (gas) with stationary phase (liquid) Mobile phase inert gas, helium, nitrogen Incresing Temp causes elution of solutes from stationary phaseGLC HPLC Stationary phaseMobile gas phaseO-Stationary phase Mobile phase O- solid. AI2O3 and SiO2with solute X and YY solid. AI2O3 and SiO2Non-polar liquidYO- layer of non volatile liquid with solute X and YX XO- layer of polar liquid YhydrocarbonY X O- hydrocarbonY YO-X Y O-X O-X O-YX O-O- O-Y O-Less volatile solute Y distributeY Y O- Polar Y solute distribute YY in liquid stationary phasein polar stationary phaseY O-(elute later/longer retention time)YY O- (elute later)Solute X (volatile)XX XO- Non-polar solute XXXX Y O- in mobile gas phase in mobile phase(elute first) XO- (elute first) XXO-XXX YYY XXX YYY 29. Chromatography Techniques GLC vs HPLCPartition Chromatography Components distribute between TWO immisible phase Mobile phase (gas) with stationary phase (liquid) Mobile phase inert gas, helium, nitrogen Incresing Temp causes elution of solutes from stationary phaseGLC HPLC Stationary phaseMobile gas phaseO-Stationary phase Mobile phase O- solid. AI2O3 and SiO2with solute X and YY solid. AI2O3 and SiO2Non-polar liquidYO- layer of non volatile liquid with solute X and YX XO- layer of polar liquid YhydrocarbonY X O- hydrocarbonY YO-X Y O-X O-X O-YX O-O- O-Y O-Less volatile solute Y distributeY Y O- Polar Y solute distribute YY in liquid stationary phasein polar stationary phaseY O-(elute later/longer retention time)YY O- (elute later)Solute X (volatile)XX XO- Non-polar solute XXXX Y O- in mobile gas phase in mobile phase(elute first) XO- (elute first) XXO-XXX YYY XXX YYY GLCNormal HPLC Gas (mobile phase) and non volatile liquid on stationary phase non polar solvent (mobile phase) and polar stationary phase Less volatile solute Y stay in stationary phase- elute later polar solute stay in polar stationary phase- elute later Volatile solute X stay in mobile phase elute first non polar solute stay in non polar mobile phase elute first 30. Chromatography TechniquesHow GLC works How HPLC worksvsPolar column 31. Chromatography TechniquesHow GLC worksHow HPLC worksvs Polar columnInjection and vaporization of volatile sample in Column chamber Injection of non volatile sample Sample move with mobile gas phase Sample move with mobile liquid phase Elution of sample by temp change Elution of sample by using diff solvent polarityDetection by flame ionization detector FID (more sensitive)Detection by UV (less sensitive)Chromatogram - retention time for diff samples Chromatogram - retention time for diff samples 32. Chromatography TechniquesHow GLC worksHow HPLC works vs Polar column Injection and vaporization of volatile sample in Column chamber Injection of non volatile sampleSample move with mobile gas phase Sample move with mobile liquid phaseElution of sample by temp change Elution of sample by using diff solvent polarity Detection by flame ionization detector FID (more sensitive) Detection by UV (less sensitive)Chromatogram - retention time for diff samples Chromatogram - retention time for diff samples More volatile shorter retention time Non polar solute Polar solute short retention timegreater retention time Less volatile greater retention timeBoth can be use for identification, separation and quantificationQuantification - Area under curve represent amount solutehttp://lab-training.com/landing/free-hplc-training-programme-6/ 33. Chromatography Techniques Partition Chromatography Adsorption ChromatographyPartition Chromatography Adsorption Chromatography Components distribute bet TWO immisible liquid phase Component adsorb on solid stationary phase Depends on relative solubility bet TWO phase Depends on polarity of stationary phase, mobile phase and solutes Solutes bonds to stationary phase or mobile phase Stationary phase is polar polar solutes adsorb strongly Stationary phase is non polar non polar solutes adsorb strongly Mobile phase is polar polar solutes stay in mobile phase Mobile phase is non polar non polar solutes stay in mobile phasePaper ChromatographyGLC HPLC Thin Layer ChromatographyColumn ChromatographyY adsorbstrongly X adsorb stronglyMobile phase liquidMobile phase gas Mobile phase liquidMobile phase liquidMobile phase liquid Stationary phase - liquidStationary phase - liquidStationary phase - liquidStationary phase - solidStationary phase - solidUses of GLC Uses of HPLC Analysis of steroids, vegetable oils, alcohol in blood Analysis of sugar, additives, pesticides, herbicides Volatile/thermally stable compounds Non volatile/thermally unstable/sensitive to heat Small amt volatile liquid, not sensitive to heat vs Compounds decomposes upon heating ( Vit C, E , antioxidants) More sensitive Flame Ionization Detector Less sensitive UV detector Use for identification/separation/quantification Use for identification/separation/quantification 34. Chromatography TechniquesTechnique Stationary Phase Mobile Phase ApplicationPaperLiquid on paper Liquid/Solvent - Detection of amino acids in a mixtureChromatography- Different dyes in food colouring- Separation of plant pigmentsThin Layer Solid oxide coatLiquid/Solvent - Detection of amino acids in a mixtureChromatography- Different dyes in food colouring- Separation of plant pigmentsColumn Solid oxide coatLiquid/Solvent - Preparative/collection of pigments/dyes.ChromatographyGas Liquid Liquid on solid coat Gas - Analysis of steroids, vegetable oils, alcohol in bloodChromatography- Volatile/thermally stable compounds- Small amt volatile liquid, not sensitive to heat- More sensitive Flame Ionization Detector- Use for identification/separation/quantificationHigh Performance Liquid on solid oxide Liquid/Solvent - Analysis of sugar, additives, pesticides, herbicides Liquidcoat - Non volatile/thermally unstable/sensitive to heatChromatography- Compounds decomposes upon heating ( Vit C, antioxidants, vitamin E )- Less sensitive UV detector- Use for identification/separation/quantification 35. Click here to view TLC and Column ChromatographySeparation using TLCTLC techniques step by stepColumn ChromatographyColumn chromatography separation 36. Click here to view HPLC and Gas Liquid ChromatographyHPLC worksGas Liquid Chromatography works Click here to view Click here to viewClick here on column chromatographyClick here to view amino acid separation 37. AcknowledgementsThanks to source of pictures and video used in this presentationThanks to Creative Commons for excellent contribution on licenseshttp://creativecommons.org/licenses/Prepared by Lawrence KokCheck out more video tutorials from my site and hope you enjoy this tutorialhttp://lawrencekok.blogspot.com