Chromatography - 33

Chromatography

An analytical technique that separates components in a mixture between a mobile phase & stationary phase

Mobile Phase

Liquid/Gas

Stationary Phase

Solid (TLC)

Liquid/Solid on a solid support (Gas chromatography)

Thin-Layer Chromatography

A plate is coated with a solid and a convent moves up the plate

Column Chromatography

A column is packed with a solid an a solvent moves down the column

Gas Chromatography

A column is packed with a solid/solid coated by a liquid & a gas is passed through the column under pressure at high temp

GC - Mobile phase

Inert gas - Nitrogen/Helium/Argon

GC - Stationary Phase

Liquid on an inert solid

Column Chromatography Separation

Dependent on the balance between solubility in the moving phase & retention in the stationary phase

Solid stationary phase separates by

Adsorption

Liquid Stationary phase separates by

Relative solubility

Identification

1. Retention times

2. Rf values

Polar Stationary Phase & Moving phase non polar

Non polar compounds would pass through the column faster that polar compounds (They’d have a greater solubility in the non polar moving phase)

TLC

a) Wearing gloves, draw a pencil line 1 cm above the bottom of a TLC plate and mark spots for each sample, equally spaced along line.

b) Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry.

c) Add solvent to a chamber or large beaker with a lid so that is no more than 1cm in depth

d) Place the TLC plate into the chamber, making sure that the level of the solvent is below the pencil line. Replace the lid to get a tight seal.

e) When the level of the solvent reaches about 1 cm from the top of the plate, remove the plate and mark the solvent level with a pencil. Allow the plate to dry in the fume cupboard.

f) Place the plate under a UV lamp in order to see the spots. Draw around them lightly in pencil.

g) Calculate the Rf values of the observed spots.

Justify:

1. Wear plastic gloves

2. Pencil line

3. Tiny drop

4. Depth of solvent

5. Lid

6. Solvent rising to the top

7. Dry in a fume cupboard

8. UV lamp

Wear plastic gloves - to prevent contamination from the hands to the plate

pencil line - will not dissolve in the solvent

tiny drop - too big a drop will cause different spots to merge

Depth of solvent - if the solvent is too deep it Will dissolve the sample spots from the plate

lid - to prevent evaporation of toxic solvent

Solvent reaching the top - Will get more accurate results if the solvent is allowed to rise to near the top of the plate but the Rf value can be calculated if the solvent front does not reach the top of the plate

Dry in a fume cupboard - the solvent is toxic

UV lamp used if the spots are colourless and not visible

Rf value

Distance moved by amino acid/distance moved by the solvent

Two Directional Chromatography (Two solvents are used)

Needed to separate a complex mixture where the components have similar solubilities in a solvent OR the mixture doesn’t separate with the first solvent

TDC Method

A spot of the mixture on a TLC plate is first separated with one solvent.

Then the TLC plate is rotated 90o and the plate is placed in a second solvent for a second

separation to take place

How many components are there

6

Simple Column Chromatography

• A glass tube is filled with the stationary phase usually silica or alumina in powder form to increase the surface area.

• A filter or plug is used to retain the solid in the tube. Solvent is added to cover all the powder.

• The mixture to be analyzed is dissolved in a minimum of a solvent and added to the column.

• A solvent or mixture of solvents is then run through the column.

• The time for each component in the mixture to reach the end of the column is recorded (retention time)

HPLC - High performance liquid chromatography

• Industry used column chromatography

• Solid silica = stationary phase

• Liquid = Mobile phase

GC

Used to separate mixtures of volatile liquids

Retention Time

Time taken for a particular compound to travel from the injection of the sample to where it leaves the column to the detector

Basic GC

Shows how many components they are in the number of peaks + the abundance of each substance (area under the peak is proportional to the abundance)

Inert Carrier Gas

Will not react with the components separated in the GC column

Factors affecting retention times in GC

GC column temperature, column length , flow rate.

If the temperature or the flow rate is higher then substance will move more quickly through the column to give shorter retention times.

Connection to IR/NMR/Mass spec

GC machine can be connected to any of the above = all components to be identified

Similar retention times In GC

Components wouldn’t be distinguished

GC+MS

Used in Analysis

1. Forensics

2. Environmental Analysis

3. Airport Security

4. Space probes

Explain why each amino acid has different Rf values

Each amino acid has different relative affinity in stationary and mobile phases