Chromatographic Separation
Alina Guna
Ryan Young
Edward Sun
Jonathan Lam
Mr. Vincent SCH3U3
Contents

Introduction to Chromatography
 Adsorption
 Paper Chromatography

Experiment
 Materials
 Procedure
 Observations and Calculations
 Video


Applications
Conclusion
 Sources of Experimental Error
Introduction



Chromatography – Separating
Mixtures
Includes a mobile/stationary phase
Preparative or Analytical



Preparative: Separate to use
components
Analytical: Measure relative proportion
of a substance
5 different types of Chromatography:

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Adsorption
Partition
Ion Exchange
Molecular Exclusion
Affinity
Ex: Chromatography in Action
Adsorption Chromatography

When a mobile gas or liquid collects onto a stationary solid
 Forms a thin layer of molecules



Solvent travels up adsorbent to meet sample
Continues upwards as a solvent/solute solution (adsorbate)
Different compounds in sample
 Different solubilities
 Travels different distances
• Analyze
Separate
• Identify
• Purify
Mixture
Components
• Quantify
The other types …

Partition Chromatography
 Separation of similar substances by repeated
extraction by two immiscible liquids.

Ion Exchange
 Separation of ions and polar molecules

Molecular Exclusion
 Particles are separated based on size.
 Usually applied to large molecules such as
proteins and industrial polymers

Affinity
 Method of separating biochemical mixtures

We focus on Adsorption in our project.
Paper Chromatography

Stationary Phase
 Chromatography Paper

Mobile Phase
 Unreactive solvent


Primarily used to separate and
identify coloured mixtures
Two-way paper chromatography
 Complicated substances


Amino Acids
Solvent’s ability to move
upwards via capillary action
Process of Paper
Chromatography
Experiment
 Explain adsorption
 What it is
 How it is used to separate mixtures
 Demonstrate adsorption through paper chromatography
 Calculate retention factors of the substances
Safety Concerns


Safety Goggles
Rubbing Alcohol
 Spillage/Leakage
 Consumption

Ventilation of Area

Permanent Markers
 Condition
 Wastage
Materials








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Glass Jars
Chromatography Paper
Pencils
Rubbing alcohol (70% Isopropyl Alcohol)
Sharpie Pens of various colours
Measuring cup
Ruler
Scissors
Tape
Safety Goggles
Procedure

Use the scissors and ruler to cut equally long
strips of Chromatography Paper. With a pencil,
draw a horizontal line 1 cm above the bottom
edge of each strip.

Using each Sharpie pen, place a spot along the
line of the Chromatography Paper.

Tape each strip onto a different pencil, and
place the pencil onto the jar. Make sure that the
strip of Chromatography Paper is touching the
Isopropyl Alcohol inside.

Measure approximately 15 mL of Isopropyl
Alcohol and pour it in each jar.

Let the strips develop until the ascending
solution is approximately 2 cm from the top of
the paper. Then remove the strips and let them
air dry.

Record your observations and determine the
retention factor.
Data
Spot Colour
Adsorbate
Colour
Distances
Travelled
(Respective)
Distance
Travelled by
Solvent
Retention
Factor
(Respective)
Black
Blue, Orange,
Red
4.3cm, 1.6cm,
3cm
4.3cm
1, 0.3271,
0.6977
Gray
Purple
4.3cm
4.3cm
1
Blue
Blue
3.9cm
3.9cm
1
Green
Green, Yellow
4.1cm, 4.4cm
4.4cm
0.9318, 1
Light Green
Green, Yellow
4cm, 4.4cm
4.4cm
0.9090, 1
Orange
Orange, Red
3.1cm, 4cm
4cm
0.7750, 1
Burgundy
Burgundy
4cm
4cm
1
Green (2)
Green, Yellow
3.8cm, 4.2cm
4.2cm
0.9048, 1
Orange (2)
Orange, Red
3.2cm, 4.2cm
4.2cm
0.7619, 1
Burgundy (2)
Burgundy
4cm
4cm
1
Calculations
DistanceSubstance
Rf 
DistanceSolute
Black Ink
Blue Dye
Red Dye
Orange Dye
4.3
R 
1
f
4.3
3
R 
 0.697
f
4.3
1.6
R 
 0.3271
f
4.3
Applications


Anything involving separating a
solution
Medicine



Criminology & Forensic Science

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
Sports Medicine – Testing for drug
usage (Gas chromatography)
Pharmaceutical industries – Check if
the right reactants were used.
DNA sequencing (Capillary
electrophoresis)
Analyzing evidence (Thin-layer
chromatography)
Petrochemical Industries

Checking the purity of fuel (Gas
chromatography)
Sources of Experimental Error
 Chromatography Paper
Condition
 Measurements of Distance
 Marker Dot Size
Experiment Modifications
 More Solutes
 Different Colours
 Different Rf Values
 E.g. Food Colouring,
Homemade mixture
 Different Solvents
 Different Concentrations of
Isopropyl Alcohol
Experiment Modifications
 Testing known Solvents
 E.g. Known chemical formula
 Easier to find relationships

Record speed of Solution
 Relationships with respect to
time
Conclusion
 Intermolecular Forces
 Stronger forces = Slower speed
traveling upwards
 Longer time in stationary phase
 Retention Factor
 Can help identify an unknown
compound
 Surface tension of Markers
 All solutes tested were polar