Unit 13: Chromatography
Mastering the art of chemical separation based on differential distribution between two phases.
13.18 General Principles (Stationary & Mobile Phases)
Chromatography is a powerful laboratory technique used to separate the components of a mixture. The underlying principle of all chromatographic methods is the same: separation is achieved by distributing the components between two phases.
- The stationary phase is a substance that is fixed in place for the procedure. It can be a solid (like silica gel) or a liquid coated on a solid support.
- The mobile phase is a substance that moves through the stationary phase. It can be a liquid or a gas.
As the mobile phase carries the mixture through the stationary phase, the components of the mixture separate based on their differing affinities for the two phases.
- Components that have a stronger affinity for the stationary phase will move more slowly.
- Components that have a stronger affinity for the mobile phase (are more soluble in it) will move more quickly.
This difference in movement speed results in the separation of the components over time.
Solved Examples:
- What are the two phases in any chromatography
experiment?
Solution: The stationary phase and the mobile phase. - In paper chromatography, the paper is the stationary phase. What is the
mobile phase?
Solution: The liquid solvent that moves up the paper. - A mixture contains two components, A and B. Component A is more soluble in
the mobile phase than component B. Which component will travel
further?
Solution: Component A will travel further because it has a stronger affinity for the mobile phase and a weaker affinity for the stationary phase. - What does "affinity" mean in the context of
chromatography?
Solution: It refers to the tendency of a substance to be attracted to or interact with a particular phase, either through adsorption, solubility, or other intermolecular forces. - Why is chromatography considered a separation
technique?
Solution: Because its primary purpose is to separate a mixture into its individual components. - If a component in a mixture does not move from the starting line, what can
you conclude about its affinities?
Solution: You can conclude that it has a very strong affinity for the stationary phase and is essentially insoluble in the mobile phase. - What is the fundamental principle of separation in
chromatography?
Solution: The differential partitioning or distribution of components between a stationary phase and a mobile phase. - Can chromatography be used to identify
substances?
Solution: Yes. By comparing the distance a substance travels or the time it takes to pass through the system to that of a known standard, it can be identified. - Is the stationary phase always a
solid?
Solution: No. It can be a solid (adsorption chromatography) or a liquid coated onto a solid support (partition chromatography). - Is the mobile phase always a liquid?
Solution: No. It can be a liquid (as in TLC and HPLC) or a gas (as in Gas Chromatography).
13.19 Gas Chromatography (GC): Components, Retention Time, Applications
Gas Chromatography (GC) is a technique used to separate and analyze compounds that can be vaporized without decomposition.
- Mobile Phase: An inert gas, such as helium or nitrogen, called the carrier gas.
- Stationary Phase: A microscopic layer of a high-boiling-point liquid coated on a solid support, packed inside a long, thin, coiled tube called a column. The column is housed in an oven.
A small amount of the liquid mixture is injected into the instrument, where it is heated and vaporized. The carrier gas sweeps the vaporized components through the column. Separation occurs based on the components' boiling points and their solubility in the stationary phase. Volatile components with low boiling points that are less soluble in the stationary phase travel through the column faster.
The time it takes for a component to travel through the column and reach the detector is called its retention time. Each component in the mixture will have a characteristic retention time under a specific set of conditions. The output, a chromatogram, shows a peak for each separated component.
Solved Examples:
- What is the mobile phase in Gas
Chromatography?
Solution: An inert carrier gas, such as helium or nitrogen. - What is retention time?
Solution: The time taken for a specific compound to travel from the injection port, through the column, to the detector. - A mixture of hexane (b.p. 69 °C) and octane (b.p. 125 °C) is analyzed by GC.
Which compound will have a shorter retention
time?
Solution: Hexane will have a shorter retention time because it is more volatile (has a lower boiling point) and will therefore spend more time in the gas mobile phase. - What is a major limitation of GC?
Solution: It can only be used for samples that are volatile and thermally stable (i.e., they can be turned into a gas without breaking down). - How can GC be used for quantitative
analysis?
Solution: The area under each peak in the chromatogram is proportional to the amount of that component in the mixture. - What is the purpose of the oven in a GC
instrument?
Solution: To maintain the column at a precise temperature, ensuring that all components of the sample remain in the vapor phase as they travel through the column. - Why must the carrier gas be inert?
Solution: So that it does not react with the sample components or the stationary phase. Its only role is to carry the sample through the column. - If two compounds have very different retention times, what does this
imply?
Solution: It implies that they have significantly different volatilities and/or affinities for the stationary phase, and the GC column has separated them very effectively. - What is a common application of GC in
sports?
Solution: Drug testing. GC (often coupled with Mass Spectrometry, GC-MS) is used to separate and identify banned performance-enhancing drugs in athletes' urine samples. - What does the detector in a GC instrument
do?
Solution: It generates an electrical signal when a component elutes (exits) from the column. The size of the signal is proportional to the amount of the component.
13.20 High-Performance Liquid Chromatography (HPLC)
High-Performance Liquid Chromatography (HPLC) is a highly improved form of column chromatography. It is used to separate components of a mixture that are dissolved in a liquid.
- Mobile Phase: A liquid solvent or mixture of solvents.
- Stationary Phase: Very fine solid particles (like silica) packed tightly into a stainless steel column.
Because the stationary phase particles are so small and tightly packed, a high-pressure pump is required to force the mobile phase through the column at a constant rate. This tight packing provides a very large surface area for interactions, leading to much better and faster separations than traditional column chromatography.
Separation is based on the differential affinities of the components for the stationary and mobile phases. HPLC is extremely versatile and is a cornerstone of the pharmaceutical industry for separating and purifying complex mixtures.
Solved Examples:
- What does HPLC stand for?
Solution: High-Performance (or High-Pressure) Liquid Chromatography. - What is the main advantage of HPLC over traditional column
chromatography?
Solution: It is much faster and provides much higher resolution (better separation of components). - Why is a high-pressure pump necessary for
HPLC?
Solution: The stationary phase consists of very fine particles packed into a column, which creates a high resistance to flow. A high-pressure pump is needed to force the liquid mobile phase through the column at a steady rate. - What is the mobile phase in HPLC?
Solution: A liquid solvent or a mixture of solvents. - What is the stationary phase in HPLC?
Solution: Very fine solid particles, commonly silica, packed in a column. - Can HPLC be used to separate non-volatile
compounds?
Solution: Yes. Unlike GC, the sample does not need to be vaporized, so HPLC is ideal for separating non-volatile or thermally unstable compounds like proteins and large drug molecules. - What is a common application of HPLC?
Solution: Pharmaceutical analysis, such as determining the purity of a drug or separating the components of a complex biological sample. - In HPLC, if a compound has a long retention time, what does this say about
its affinity for the stationary phase?
Solution: It means the compound has a strong affinity for the stationary phase, causing it to move through the column more slowly. - What is "normal-phase" HPLC?
Solution: A type of HPLC where the stationary phase is polar (like silica) and the mobile phase is non-polar (like hexane). - What is "reverse-phase" HPLC?
Solution: The most common type of HPLC, where the stationary phase is non-polar (e.g., silica modified with C18 hydrocarbon chains) and the mobile phase is polar (like a water/methanol mixture).
13.21 Thin Layer Chromatography (TLC): Rf Values, Applications
Thin Layer Chromatography (TLC) is a simple, fast, and inexpensive chromatography technique used to separate the components of a mixture.
- Stationary Phase: A thin layer of an adsorbent solid (like silica gel or alumina) coated onto a flat, inert backing like a glass plate or plastic sheet.
- Mobile Phase: A shallow pool of a liquid solvent in a sealed container (the developing chamber).
A spot of the mixture is applied to the bottom of the TLC plate (the origin). The plate is then placed in the developing chamber. The solvent (mobile phase) moves up the plate by capillary action, carrying the mixture with it. Separation occurs as the components travel up the plate at different rates based on their affinity for the stationary phase and solubility in the mobile phase.
Retention Factor (Rf Value)
The result of a TLC experiment is quantified by the retention factor (Rf value) for each separated spot. $$ R_f = \frac{\text{Distance travelled by the spot}}{\text{Distance travelled by the solvent front}} $$ The Rf value is always between 0 and 1. Under a specific set of conditions (stationary phase, mobile phase, temperature), a particular compound will always have the same Rf value, which can be used for identification.
Solved Examples:
- What is the primary use of TLC in an organic chemistry
lab?
Solution: To quickly check the purity of a sample or to monitor the progress of a chemical reaction. - In a TLC experiment, the solvent front moved 8.0 cm from the origin. A
separated spot moved 4.8 cm. What is the Rf value for this
component?
Solution: $R_f = (4.8 \, cm) / (8.0 \, cm) = 0.60$. - If the spots are colorless, how can they be visualized on a TLC
plate?
Solution: The plate can be viewed under a UV lamp (if the compounds are UV-active, they will appear as dark spots), or it can be sprayed with a chemical stain (like potassium permanganate) that reacts to produce a colored spot. - In a TLC experiment with a silica gel plate (polar), a non-polar compound
will have a high or low Rf value?
Solution: It will have a high Rf value. The non-polar compound will have a weak affinity for the polar stationary phase and a strong affinity for the (typically less polar) mobile phase, so it will travel far up the plate. - Why must the developing chamber for TLC be
sealed?
Solution: To ensure the atmosphere inside the chamber is saturated with solvent vapor, which prevents the solvent from evaporating off the plate as it runs, ensuring consistent results. - What is the "origin" on a TLC plate?
Solution: It is the starting line near the bottom of the plate where the sample mixture is initially spotted. - What is the "solvent front"?
Solution: It is the highest point reached by the mobile phase (the solvent) on the stationary phase (the plate). - Two compounds have Rf values of 0.2 and 0.8 in the same solvent. Which
compound is more polar? (Assume a polar stationary phase like
silica).
Solution: The compound with the Rf value of 0.2 is more polar. It has a stronger attraction to the polar stationary phase and moves a shorter distance up the plate. - Can an Rf value be greater than 1?
Solution: No, because the spot can never travel further than the solvent front. - How can TLC be used to monitor a
reaction?
Solution: By taking small samples from the reaction mixture over time and spotting them on a TLC plate. As the reaction proceeds, the spot corresponding to the starting material will get smaller, and the spot for the product will get larger.