Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu
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Separates mixtures (physical method)
Involves:
 Stationary Phase; Surface
 Mobile Phase; Solvent
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Quantifiable:
 Retention Factor / Rate of Flow (Rf)
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Can be:
 Preparative: separates mixture for further use
 Analytical: measures relative proportions of components
 Preparative more easily studied
Rf = Distance traveled by solute/Distance traveled by solvent
Mikhail Semenovich
Tsvett
 Separate Tints / Types of
Chromatography
 Trickled mixture through
glass tube with Calcium
Carbonate powder
 Pigments stuck to powder
 Different degrees of
strength  Coloured
bands
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Partition
 Based on the thin film formed on the
surface of a solid support by a liquid
stationary phase
 Solute equilibrates between the mobile
phase and the stationary liquid.
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Ion Exchange
 Resin is used to covalently attach
anions or cations by electrostatic
forces
 Solute Mobile Liquid Phase have
opposite charge
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Molecular Exclusion
 No attraction between stationary
phase and solute
 Liquid or Gaseous Mobile Phase
separates molecules according to
size
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Affinity
 Most selective
 Interaction between only 1
component of solute and
stationary phase molecule
 Only the specific component is
attracted to the stationary phase
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Adsorption: When a fluid
solute accumulates on
the surface of a solid or
liquid
Different amounts of the
components of a mixture
are adsorbed to the 2
phases
As mobile phase moves
through stationery phase,
the substances that are
easily adsorbed will ‘lag’
Separation created
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Archer JP Martin and
Richard LM Synge in 1941
Strip of porous filter paper
substituted for powder
Used to separate proteins
Later used to Separate and
Identify components of
various mixtures
In industrial settings, Paper
Chromatography has been
replaced by more
sophisticated methods
1)
2)
3)
4)
5)
Place a drop of a
mixture on the paper
Dip one edge of the
paper into the mobile
phase
Through capillary
action, adsorbent will
move up the paper
Once adsorption occurs
and the paper dries,
spray-on reagent
Reagent will reveal
change in color
•Purpose:
To predict the composition of various mixtures
using Paper Chromatography
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Pigment applied to Strip of
Chromatography Paper
Strips hung on rack at equal
lengths
Adsorbent added (70% Isopropyl
Alcohol)
Rack removed once solvent
travelled to the top of each strip
The final position of solvent
marked
The final position of pigments
marked
Distance travelled by the mobile
phase and each colour measured
View Demo using the ‘Play’ button
after each step. Do not proceed after
the Gas Chromatography Demo
appears. Use arrow keys for next slide.
The Mixture involving Red, Yellow, and Blue Food Coloring Pigments
Approximate elapsed time 1 minute
The Mixture involving Red, Yellow, and Blue Food Coloring Pigments
Approximate elapsed time 6 minutes
The Mixture involving Red, Yellow, and Blue Food Coloring Pigments
Approximate elapsed time 18 minutes
The Spinach, Pomegranate and Blue Pigments
Approximate elapsed time 0 minutes
The Spinach, Pomegranate and Blue Pigments
Approximate elapsed time 5 minutes
The Spinach, Pomegranate and Blue Pigments
Approximate elapsed time 13 minutes
Defined as Rate of
Flow or Retention
Factor
• Calculated by
dividing
“ Distance travelled by
a component” over
“ Distance travelled by
the mobile phase”
•
Component
Distance Travelled (cm)
Mobile Phase / Adsorbent (70% Isopropyl Rubbing Alcohol)
7.25
Red Food Coloring (In Mixture)
7
Yellow Food Coloring (In Mixture)
6.8
Blue Food Coloring (In Mixture)
7.1
Green Food Coloring (Yellow Component)
2.3
Green Food Coloring (Blue Component)
7.1
Spinach Juice (Yellow Component 1)
7
Spinach Juice (Yellow Component 2)
4
Spinach Juice (Green Component)
3.5
Pomegranate Juice (Orchid Purple Component)
6.1
Pomegranate Juice (Light Salmon Pink Component)
4.6
Component
Rf
Red Food Coloring (in Mixture)
0.97
Yellow Food Coloring (in Mixture)
0.94
Blue Food Coloring (in Mixture)
0.98
Green Food Coloring (Yellow Component)
0.32
Green Food Coloring (Blue Component)
0.98
Spinach Extract (Yellow Component 1)
0.97
Spinach Extract (Yellow Component 2)
0.55
Spinach Extract (Green Component)
0.45
Pomegranate Extract (Orchid Purple Component)
0.84
Pomegranate Extract (Light Salmon Pink
Component)
0.63
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Rf = Degree of Adsorption
Variances caused by Intermolecular Forces
– Between Solute and Mobile Phase
• Solute carried further by Adsorbent / More adsorption
when the forces between them are greater
– Forces between Solute and Stationary Phase
• More adsorbtion onto Stationary Phase when the forces
between them are greater
– In Both Cases:
• Polarity Major Factor
• H-bonding Larger Factor but capability limited
• London’s Dispersion Forces Weak
•
Mixture of Food Coloring
Component (in Food Rf
Coloring Mixture)
– Molecular Formulas:
• Red: C18H14N2Na2O8S2
Red Component
• Yellow: C16H10Na2O7S2N2
Yellow Component
• Blue: C16H10N2O2
Blue Component
– H-Bonding Pattern
– Relatively Similar Polarity
• Long Hydrocarbon Chain = Low Polarity
• Cellulose Chromatography Paper
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Green Coloring
Component (in Green
Food Coloring)
– Yellow and Blue Components
Yellow Component
• Yellow: C16H9N4Na3O9S2
• Blue: C16H10N2O2
Blue Component
– Same Blue Dye; Different Yellow Dye
• Higher Polarity Due to Presence of Sodium
• Higher Number of Nitrogen Atoms
0.97
0.94
0.98
Rf
0.32
0.98
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Spinach Extract
– Molecular Formulas:
• Yellow 1: Carotenes: C40H56
• Yellow 2: Xanthophylls:
C40H56O2
• Green: Chlorophyll B:
C55H70O6N4Mg
– H-Bonding common
– Carotenes Not-Polar
– Xanthophylls’ O2
Component (in
Spinach Extract)
Rf
Yellow Component 1
0.97
Yellow Component 2
0.55
Green Component
0.45
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Pomegranate Analysis
– Not Exact / Quantifiable
– Not Definite  Colours Subjective
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Components found in mixture can’t be re-used
– Neither Preparative nor Analytical
– Other Types such as Gas Chromatography provide the
components in usable form
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Prior Knowledge Required of Components
– Only Identifiable if known
– Incorrect Identifications
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All Rf Values are Relative
 Paper & Some Types of
Below: More sophisticated methods of
quantifying chromatography results
Thin Layer
Chromatography
 Other Methods
▪ Distribution Constant and
Concentration More Reliable
▪ Freundlich Equation for
Adsorption
▪ Kovats Retention Index
▪ Van Deemter Equation
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Safety Concerns
• Safety Goggles must be worn at all times
• Any contact with the alcohol must be avoided
• Alcohol is Very Flammable and requires a well-
ventilated area
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Modifications
• More Sample Pigments
• Various Adsorbents
• The more data, the more definite the results
•Thin-Layer Chromatography
•Gas Chromatography (In Column)
•Liquid Chromatography
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Thin-Layer Chromatography
 Mobile Phase: Solution; Stationary
Phase: Flat Sheet of Adsorbent
(e.g. Silica Gel)
 Analyzing organic reactions
▪ Components are separated using
TLC plates
▪ They can then be scraped off to be
analyzed
 Other Applications
▪ assaying radiochemical purity of
radiopharmaceuticals
▪ Plant pigments
▪ detection of pesticides or
insecticides
Click ‘Next Example’ to
View Gas
Chromatography Demo
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Gas Chromatography
(In Column)
 Mobile Phase: Gas;
Stationary Phase: Solid/Liquid
 Blood alcohol analysis in
forensics
▪ Blood analyzed using capillary
columns
▪ Column Pressure 20 psi
▪ Hydrogen is mobile phase
 Other Applications
▪ Environmental monitoring
▪ Drug, bomb detection
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Liquid
Chromatography
 Mobile Phase: Liquid;
Stationary Phase:
Solid/Liquid
 HPLC, NPLC
 Test for Water Pollution
▪ Analyze metal ions + organic
compounds in water
 Other Applications
▪ purification of a drug
product
• 2 Dimensional
• 2 separation stages
• Gas / Liquid
Chromatography
• Stationary Phase Rotated; 2
Mobile Phases Used
• RPLC
• Polar Mobile Phase Used
• Reverse Rf Analysis
This concludes the presentation.