Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu

Separates mixtures (physical method) Involves:

Stationary Phase; Surface Mobile Phase; Solvent

Quantifiable: Retention Factor / Rate of Flow (Rf)

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

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.

Ion Exchange Resin is used to covalently

attach anions or cations by electrostatic forces

Solute Mobile Liquid Phase have opposite charge

Molecular Exclusion No attraction between

stationary phase and solute Liquid or Gaseous Mobile

Phase separates molecules according to size

Affinity Most selective Interaction between only 1

component of solute and stationary phase molecule

Only the specific component is attracted to the stationary phase

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

• 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• In industrial settings, Paper Chromatography has been replaced by more sophisticated methods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) Place a drop of a mixture on the paper

2) Dip one edge of the paper into the mobile phase

3) Through capillary action, adsorbent will move up the paper

4) Once adsorption occurs and the paper dries, spray-on reagent

5) Reagent will reveal change in color

•Purpose:To predict the composition of various mixtures using Paper Chromatography

• 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 PigmentsApproximate elapsed time 1 minute

The Mixture involving Red, Yellow, and Blue Food Coloring PigmentsApproximate elapsed time 6 minutes

The Mixture involving Red, Yellow, and Blue Food Coloring PigmentsApproximate elapsed time 18 minutes

The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 0 minutes

The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 5 minutes

The Spinach, Pomegranate and Blue PigmentsApproximate 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) 7Yellow Food Coloring (In Mixture) 6.8Blue Food Coloring (In Mixture) 7.1Green Food Coloring (Yellow Component) 2.3Green Food Coloring (Blue Component) 7.1Spinach Juice (Yellow Component 1) 7Spinach Juice (Yellow Component 2) 4Spinach Juice (Green Component) 3.5Pomegranate Juice (Orchid Purple Component) 6.1Pomegranate 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

• 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– Molecular Formulas:• Red: C18H14N2Na2O8S2

• Yellow: C16H10Na2O7S2N2

• Blue: C16H10N2O2 – H-Bonding Pattern– Relatively Similar Polarity• Long Hydrocarbon Chain = Low Polarity• Cellulose Chromatography Paper

• Green Coloring– Yellow and Blue Components• Yellow: C16H9N4Na3O9S2

• Blue: C16H10N2O2 – Same Blue Dye; Different Yellow Dye• Higher Polarity Due to Presence of Sodium• Higher Number of Nitrogen Atoms

Component (in Food Coloring Mixture)

Rf

Red Component 0.97

Yellow Component

0.94

Blue Component 0.98

Component (in Green Food Coloring)

Rf

Yellow Component 0.32

Blue Component 0.98

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

• Pomegranate Analysis– Not Exact / Quantifiable– Not Definite Colours Subjective

• 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• Prior Knowledge Required of Components– Only Identifiable if known– Incorrect Identifications

All Rf Values are Relative Paper & Some Types of

Thin Layer Chromatography

Other Methods▪ Distribution Constant and

Concentration More Reliable▪ Freundlich Equation for

Adsorption▪ Kovats Retention Index▪ Van Deemter Equation

Below: More sophisticated methods of quantifying chromatography

results

• 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• Modifications• More Sample Pigments• Various Adsorbents• The more data, the more definite the results

•Thin-Layer Chromatography•Gas Chromatography (In Column)•Liquid Chromatography

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

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

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.