Chromatographic Separation

Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu

Chromatography 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

History 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

Types of Chromatography

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

Types of ChromatographyMolecular 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 Chromatography

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

Paper Chromatography• 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• In industrial settings, Paper Chromatography has been replaced by more sophisticated methods

Common Practice for Paper Chromatography

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

Experiment

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

Materials

Procedure• 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

Procedure

Observations - Pictures

The Mixture involving Red, Yellow, and Blue Food Coloring PigmentsApproximate elapsed time 1 minute

Observations - Pictures

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

Observations - Pictures

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

Observations - Pictures

The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 0 minutes

Observations - Pictures

The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 5 minutes

Observations - Pictures

The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 13 minutes

Rf• Defined as Rate of

Flow or Retention Factor

• Calculated by dividing

“ Distance travelled by a component” over

“ Distance travelled by the mobile phase”

Observations – Distance Travelled

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

Calculations – Rf ValuesComponent RfRed Food Coloring (in Mixture) 0.97Yellow Food Coloring (in Mixture) 0.94Blue Food Coloring (in Mixture) 0.98Green Food Coloring (Yellow Component) 0.32Green Food Coloring (Blue Component) 0.98Spinach Extract (Yellow Component 1) 0.97Spinach Extract (Yellow Component 2) 0.55Spinach Extract (Green Component) 0.45Pomegranate Extract (Orchid Purple Component)

0.84

Pomegranate Extract (Light Salmon Pink Component)

0.63

Analysis – Rf Values• 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

Analysis• 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 0.97Yellow Component

0.94

Blue Component 0.98

Component (in Green Food Coloring)

Rf

Yellow Component 0.32Blue Component 0.98

Analysis 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

Limitations of Paper Chromatography• 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

Limitations of Paper Chromatography

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

Experimental Notes

• 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

Applications of Adsorption Chromatography

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

Applications of Adsorption 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

Gas Chromatography

Applications of Adsorption Chromatography 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

Applications of Adsorption Chromatography

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

Modifications

• 2 Dimensional• 2 separation stages• Gas / Liquid

Chromatography• Stationary Phase Rotated; 2

Mobile Phases Used • RPLC

• Polar Mobile Phase Used• Reverse Rf Analysis

Any Questions?This concludes the presentation.