Chromatography Techniques Paper Chromatography Thin Layer Chromatography Adsorption Chromatography Partition Chromatography Column Chromatography Partition Chromatography • Components distribute bet TWO immisible liquid phase • Depends on relative solubility bet TWO phase • Solutes bonds to stationary phase or mobile phase Adsorption Chromatography • Component adsorb on solid stationary phase • Depends on polarity of stationary phase, mobile phase and solutes • 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 Y adsorb strongly X adsorb strongly GLC HPLC Mobile phase – liquid Stationary phase - liquid Mobile phase – gas Stationary phase - liquid Mobile phase – liquid Stationary phase - liquid Mobile phase – liquid Stationary phase - solid Mobile phase – liquid Stationary phase - solid
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IB Chemistry on High Performance Liquid Chromatography and Gas Liquid Chromatography
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Partition Chromatography • Components distribute bet TWO immisible liquid phase • Depends on relative solubility bet TWO phase • Solutes bonds to stationary phase or mobile phase
Adsorption Chromatography • Component adsorb on solid stationary phase • Depends on polarity of stationary phase, mobile phase and solutes • 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
Y adsorb
strongly
X adsorb
strongly
GLC HPLC
Mobile phase – liquid Stationary phase - liquid
Mobile phase – gas Stationary phase - liquid
Mobile phase – liquid Stationary phase - liquid
Mobile phase – liquid Stationary phase - solid
Mobile phase – liquid Stationary phase - solid
Paper Chromatography, Thin Layer Chromatography
Colum Chromatography
Chromatography 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 Adsorption
Chromatography Techniques
Separation analysis
Paper Chromatography
Thin Layer Chromatography
Adsorption Chromatography
Chromatography
Partition Chromatography
Column Chromatography
Partition Chromatography • Components distribute bet TWO immisible liquid phase • Depends on relative solubility bet TWO phase • Solutes bonds to stationary phase or mobile phase
Adsorption Chromatography • Component adsorb on solid stationary phase • Depends on polarity of stationary phase, mobile phase and solutes • 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
Application • Detection of amino acids in a mixture • Different dyes in food colouring • Separation of plant pigments
Y adsorb
strongly
Application • Detection of amino acids in a mixture • Different dyes in food colouring • Separation of plant pigments
Application • Preparative/collection of sample of pigments
X adsorb
strongly
Chromatography Techniques
Chromatography
Partition Chromatography • components distribute between 2 immisible liquid phase • relative solubility in 2 phase • bonds strongly to mobile phase – move faster
Adsorption Chromatography • components adsorp on solid stationary phase
Stationary phase
has a layer of liquid
Mobile
liquid phase
containing
X and Y X
X
X
Y
Y
Y
Stationary
Liquid phase
Partition –distribution of solute X and Y between 2 liquid phase • X more soluble in mobile phase (move with mobile liquid phase) • Y less soluble in mobile phase (stay on stationary liquid phase)
Stationary phase
has a layer of liquid
Y
Y
Y
Y
X
X X
X X
X Mobile
Iiquid phase
containing X
Stationary phase
• solid
• AI2O3
• SiO2
O- O- O- O-
O-
Adsorption– solute X and Y adsorb temporary on the solid • Y adsorb strongly on solid phase, eluted slower • X in liquid mobile phase, eluted faster
Y
Y
Y
Y
Mobile
liquid phase
containing
X and Y
X
X
X
O- O- O-
Y Y
Y
Y
X X
X X
Mobile
liquid phase
containing X
Y adsorb strongly
X X
X
X
Y Y
Y
Y
Separation of X and Y
X X
X Y Y Y
Chromatography Techniques
Paper Chromatography
Partition chromatography • Distribution solute bet both liquid phase • Depends on relative solubility
Aqueous liquid phase on surface of stationary phase (paper)
Mobile liquid phase - solvent
Solvent move by capillary action
Thin Layer Chromatography
Adsorption chromatography • Solute adsorb on stationary solid phase
Stationary phase - solid silica SiO2 or alumina AI2O3
Separation depends on how strongly solute adsorb on solid
Mobile phase – liquid solvent move by capillary action
Adsorption of solute on stationary solid phase
Stationary phase - Cellulose paper
• absorb water on its surface
Mobile Liquid phase with solute X and Y
Y
Y
Y
X
X
Stationary phase solid
• AI2O3 or SiO2
O- O- O-
O- O-
Mobile Liquid phase with solute X and Y
Y
Y
Y
X
X
Chromatography Techniques
Solute in mobile phase adsorb on solid
Mobile phase – liquid solvent added from top (Eluant)
X - Strongly adsorb – travel slowly, eluted last Y - Weakly adsorb – travel fast with solvent, eluted first
Thin Layer Chromatography
Adsorption chromatography • Solute adsorb on stationary solid phase
Adsorption of solute on stationary solid phase
Mobile Liquid phase (solvent) with solute X and Y
Y Y Y
X
X Stationary phase solid
• AI2O3 or SiO2
O- O- O-
O- O-
Mobile Liquid phase with solute X and Y
Y
Y
Y
X
X
Column Chromatography
Adsorption chromatography • Solute adsorb on stationary solid phase
X
X
Y
Y
Stationary phase solid
AI2O3 or SiO2
Y Y Y
X - Adsorb strongly – travel slowly Y – Adsorb weakly – travel fast with solvent
X Y
Stationary phase - solid silica SiO2 or alumina AI2O3
Mobile phase – liquid solvent move by capillary action
Components 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 front
Uses of Paper/TLC/Column chromatography •Separate dyes in food colourings/plant pigments • Separation/Preparative/Collection of pigments • Detection of amino acids in mixture
Chromatography Techniques
Rf value for green spot = 3cm/12cm = 0.25
Rf value for blue spot = 6cm/12cm = 0.5
For colour formation of amino acids • Spray with ninhydrin for colour formation • Shine UV light for fluorescent compounds to form
High Performance Liquid Chromatography Gas Liquid Chromatography
High 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
Stationary phase
•solid. AI2O3 and SiO2
•layer of polar liquid
hydrocarbon
O- O- O-
O-
O- O-
Mobile phase
Non-polar liquid
with solute X and Y
X
X
X
Y
Y
Y
Y
O- O- O- O-
Y
Y
Y
Y Polar Y solute distribute
in polar stationary phase
(elute later)
X
X X
X
Non-polar solute X
in non polar mobile
phase (elute first)
X X X Y Y Y
Normal HPLC Reverse phase HPLC
Normal HPLC • non polar solvent (mobile phase) and polar stationary phase • polar solute – stay in polar stationary phase- elute later • non polar solute – stay in non polar mobile phase – elute first
Chromatography Techniques
Reverse phase HPLC • polar solvent (mobile phase) and non polar stationary phase • non polar solute – stay in non polar stationary phase- elute later • polar solute – stay in polar mobile phase – elute first
Stationary phase
•solid. AI2O3 and SiO2
•layer of non polar liquid
O-
O- O-
O- O- O- Mobile phase
Polar liquid
with solute X and Y
Y
Y
Y
X X
X
X
Y
Polar Y solute in polar
mobile phase (elute first) O- O-
O- O- X
X X
X
X
Y
Y Y Y
Y
Non-polar solute X in
non polar stationary phase
(elute last)
Y Y Y X X X
Gas 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
Stationary phase
•solid. AI2O3 and SiO2
•layer of non volatile liquid
hydrocarbon
O- O- O-
O-
O- O-
Mobile gas phase
with solute X and Y
X
X
X
Y
Y
Y
Y
O- O- O- O-
Y
Y
Y
Y Less volatile solute Y distribute
in liquid stationary phase
(elute later/longer retention time)
X
X X
X Solute X (volatile)
in mobile gas phase
(elute first)
X X X Y Y Y
GLC
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
Chromatography Techniques
GLC vs HPLC
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
Stationary phase
•solid. AI2O3 and SiO2
•layer of non volatile liquid
hydrocarbon
O- O- O-
O-
O- O-
Mobile gas phase
with solute X and Y
X
X
X
Y
Y
Y
Y
O- O- O- O-
Y
Y
Y
Y Less volatile solute Y distribute
in liquid stationary phase
(elute later/longer retention time)
X
X X
X Solute X (volatile)
in mobile gas phase
(elute first)
X X X Y Y Y
GLC HPLC
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
Chromatography Techniques
O-
O- O-
O- O- O- Y
Y
Y
X X
X
X
Y
O- O-
O- O- Y
Y Y
Y
Y X
X X
X X
X X X Y Y Y
Stationary phase
•solid. AI2O3 and SiO2
•layer of polar liquid
hydrocarbon
Mobile phase
Non-polar liquid
with solute X and Y
Polar Y solute distribute
in polar stationary phase
(elute later)
Non-polar solute X
in mobile phase
(elute first)
Normal HPLC • non polar solvent (mobile phase) and polar stationary phase • polar solute – stay in polar stationary phase- elute later • non polar solute – stay in non polar mobile phase – elute first
Chromatography Techniques How HPLC works
Injection and vaporization of volatile sample in Column chamber
Sample move with mobile gas phase
Detection by flame ionization detector FID (more sensitive)
Elution of sample by temp change
Chromatogram - retention time for diff samples
vs
Less volatile – greater retention time
More volatile – shorter retention time
Both can be use for identification, separation and quantification Quantification - Area under curve – represent amount solute
Partition Chromatography • Components distribute bet TWO immisible liquid phase • Depends on relative solubility bet TWO phase • Solutes bonds to stationary phase or mobile phase
Adsorption Chromatography • Component adsorb on solid stationary phase • Depends on polarity of stationary phase, mobile phase and solutes • 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
Y adsorb
strongly
X adsorb
strongly
GLC HPLC
Mobile phase – liquid Stationary phase - liquid
Mobile phase – gas Stationary phase - liquid
Mobile phase – liquid Stationary phase - liquid
Mobile phase – liquid Stationary phase - solid
Mobile phase – liquid Stationary phase - solid
Uses of GLC • Analysis of steroids, vegetable oils, alcohol in blood • Volatile/thermally stable compounds • Small amt volatile liquid, not sensitive to heat • More sensitive – Flame Ionization Detector • Use for identification/separation/quantification
Uses of HPLC • Analysis of sugar, additives, pesticides, herbicides • Non volatile/thermally unstable/sensitive to heat • Compounds decomposes upon heating ( Vit C, E , antioxidants) • Less sensitive – UV detector • Use for identification/separation/quantification
vs
Chromatography Techniques
Technique Stationary Phase Mobile Phase Application
Paper Chromatography
Liquid on paper Liquid/Solvent - Detection of amino acids in a mixture - Different dyes in food colouring - Separation of plant pigments
Thin Layer Chromatography
Solid oxide coat Liquid/Solvent - Detection of amino acids in a mixture - Different dyes in food colouring - Separation of plant pigments
Column Chromatography
Solid oxide coat Liquid/Solvent - Preparative/collection of pigments/dyes.
Gas Liquid Chromatography
Liquid on solid coat Gas - Analysis of steroids, vegetable oils, alcohol in blood - Volatile/thermally stable compounds - Small amt volatile liquid, not sensitive to heat - More sensitive – Flame Ionization Detector - Use for identification/separation/quantification
High Performance Liquid Chromatography
Liquid on solid oxide coat
Liquid/Solvent - Analysis of sugar, additives, pesticides, herbicides - Non volatile/thermally unstable/sensitive to heat - Compounds decomposes upon heating ( Vit C, antioxidants, vitamin E ) - Less sensitive – UV detector - Use for identification/separation/quantification