IB Chemistry on High Performance Liquid Chromatography and Gas Liquid Chromatography

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 - 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


Separation analysis

Paper Chromatography

Thin Layer Chromatography

Adsorption Chromatography

Chromatography

Partition Chromatography




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

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



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


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-


Y

Y

Y

X

X


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



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-


Y

Y

Y

X

X



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

http://home.earthlink.net/~dayvdanls/photolab/photolab7.htm

Uses of Paper/TLC/Column chromatography •Separate dyes in food colourings/plant pigments • Separation/Preparative/Collection of pigments • Detection of amino acids in mixture


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


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


•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


•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


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


•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


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


•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

How GLC works

Polar column

Injection of non volatile sample

Sample move with mobile liquid phase

Elution of sample by using diff solvent polarity

Detection by UV (less sensitive)

Chromatogram - retention time for diff samples

Polar solute – greater retention time

Non polar solute – short retention time

http://lab-training.com/landing/free-hplc-training-programme-6/


Paper Chromatography Thin Layer Chromatography

Adsorption Chromatography Partition Chromatography




Y adsorb

strongly

X adsorb

strongly

GLC HPLC


Mobile phase – gas Stationary phase - liquid




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


Technique Stationary Phase Mobile Phase Application


Liquid on paper Liquid/Solvent - Detection of amino acids in a mixture - Different dyes in food colouring - Separation of plant pigments


Solid oxide coat Liquid/Solvent - Detection of amino acids in a mixture - Different dyes in food colouring - Separation of plant pigments


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

Click here to view TLC and Column Chromatography

Separation using TLC TLC techniques step by step

Column Chromatography Column chromatography separation

http://www.youtube.com/user/ChemToddler

http://www.youtube.com/watch?v=yoYECMP_1iI&feature=related

http://www.youtube.com/watch?feature=player_embedded&v=nCqwZuE4boY

http://www.youtube.com/watch?feature=player_embedded&v=clx8IfAgtXo

Click here to view HPLC and Gas Liquid Chromatography

HPLC works Gas Liquid Chromatography works

Click here to view Click here to view

Click here on column chromatography Click here to view amino acid separation

http://www.youtube.com/watch?v=kz_egMtdnL4

http://www.youtube.com/watch?v=Y2PB3KO06As

http://www.youtube.com/watch?v=kz_egMtdnL4

http://www.youtube.com/watch?v=Y2PB3KO06As

http://www.youtube.com/watch?v=9GiLjH9Oym8

http://www.youtube.com/watch?v=9GiLjH9Oym8

http://www.youtube.com/watch?v=tDaKxskUwA0

http://www.youtube.com/watch?v=tDaKxskUwA0

IB Chemistry on High Performance Liquid Chromatography and Gas Liquid Chromatography

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Transcript of IB Chemistry on High Performance Liquid Chromatography and Gas Liquid Chromatography