Separation By Chromatography (College Board AP® Chemistry)

Paper Chromatography

Principles of Paper Chromatography

• A solution is a mixture that cannot be separated by filtration, therefore an alternative separation technique must be used

• Paper chromatography is used to separate a mixture of liquids that have different intermolecular interactions in a given solvent (e.g. separate the coloured inks that were used to make black ink)

• The substances can be identified by the distance they travel across a sheet of chromatography paper

• The stronger the interaction with the solvent, the farther the distance the component will travel up the chromatography paper

• The solvent will travel up the paper because the liquid molecules will have adhesive and cohesive forces with the pores of the chromatography paper

  • Since the solvent moves up the paper, the solvent is called the mobile phase

  • Since the chromatography paper stays in a fixed position, it is called the stationary phase

• The different components of the mixture will be separated because of the different intermolecular interactions with the mobile and the stationary phases

Conducting a Paper Chromatography analysis

1. Draw a pencil line at the bottom of the chromatography paper. A sample of the mixture is placed as a spot on the top of the line. Additional spots of known reference compounds are placed to identify the components of the mixture

2. Place the paper into the solvent container, making sure that the line sits above the level of the solvent. This will prevent the samples from washing into the solvent container

3. Analyze the chromatography paper by comparing the sample with the reference compounds

Diagram to Show the Method of Chromatography

Analysis of the composition of ink that can be used using paper chromatography. Blue ink travels the most distance showing the strongest interaction with the solvent and the weakest interaction with the chromatography paper

• The component that travels the greatest  distance along the paper has the strongest intermolecular interaction with the mobile phase and the weakest interaction with the stationary phase

• The component that travels the shortest distance along the paper has the weakest intermolecular interaction with the mobile phase and the strongest interaction with the stationary phase

Thin Layer Chromatography

Principles of Thin Layer Chromatography

• Thin Layer Chromatography (TLC) is a separation technique used to analyze the components of a mixture by exploiting their different affinities towards a material

• In the same way as paper chromatography, there are two phases involved in TLC: an stationary phase and a mobile phase

Commonly used stationary phases

• The most common material is a thin metal sheet coated in silica (SiO₂) or alumina (Al₂O₃)

• The different components from the mixture will be separated because of the different intermolecular interactions with the coating

• Each component will travel a distance that depends on the interaction with the stationary phase

Commonly used mobile phases

• It could be polar or nonpolar liquids

  • Polar solvents (water or alcohol)

  • Nonpolar solvents (liquid alkanes)

• The mobile phase will flow over the stationary phase by adhesive and cohesive forces with the mobile phase

Conducting a TLC analysis

1. Draw a pencil line at the bottom of a TLC plate

2. A sample of the mixture is placed as a spot on the top of the line. Additional spots of known reference compounds are placed to identify the components of the mixture

3. Place the TLC plate into the solvent container and place a lid to cover the beaker, making sure that the line sits above the level of the solvent. This will prevent the samples from washing into the solvent container

4. The solvent will begin to travel up the plate

5. When the solvent almost reaches the top, draw another pencil line where the solvent has reached. This indicates the solvent font

6. Analyze the TLC plate and identify the components of the mixture by using the reference compounds

Method for TLC Analysis

A dot of the sample is placed on the baseline and allowed to separate as the mobile phase flows through the stationary phase; The reference compound/s will also move with the solvent

• The component that travels the most distance has the strongest intermolecular interaction with the mobile phase and the weakest interaction with the stationary phase

• The component that travels the less distance has the weakest intermolecular interaction with the mobile phase and the strongest interaction with the stationary phase

Rf values

• The TLC plate can be analyzed to calculate the Rf values for each compound

• These values gives important information about the identity of the components and their polarities

• Since the stationary phase is polar, the higher the R_f values the more polar the component

• The R_f value and the difference between the separated substances will also depend on their solubility in the solvent that acts as the mobile phase

Calculating the R_f value

R_f values can be calculated by taking 2 measurements from the TLC plate

Column Chromatography

Principles of Column Chromatography

• Column chromatography is the most sophisticated chromatography technique

• In the same way as paper chromatography and TLC, it separates a mixture in its components by exploiting their different affinities towards a stationary phase and a mobile phase

• This technique takes more time, but it allows to separate

Commonly used stationary phases

• In Column chromatography, silica (SiO₂) or alumina (Al₂O₃) are packed in the column

• The different components from the mixture will be separated because of the different intermolecular interactions with the polar packaging

• Each component will have different movement rates that depends on the interaction with the stationary phase

Commonly used mobile phases

• It could be polar or nonpolar liquids, and they need to be compatible with the packing of the

• The mobile phase will flow down the stationary phase

Conducting a Column Chromatography analysis

1. Pack the column with a stationary phase and ensure an even packing to facilitate uniform flow. Dissolve the sample in a solvent that is compatible with the stationary phase. Apply the sample onto the top of the column using a pipette, ensuring a concentrated and narrow band

2. Choose an appropriate solvent (mobile phase). Pour the solvent mixture into the column slowly to allow the sample to start moving down the column

3. As the sample moves down the column, different components will separate based on the interactions for the stationary phase

4. Collect the different mixture components in tubes or vials

5. Label and record all the components of the sample for later analysis

Column Chromatography

Steps for performing a Column chromatography analysis