Chromotography

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Chromatography
Chromatography is a technique that is used to separate pure substances present in a mixture. It is mainly used for organic compounds. E.g. drugs present in blood, sugars in fruit juice, pesticides in water and soil.
- Chromotography can also be used to determine the concentration of a substance.
Stationary phase
The material that is on the structure placed in the mobile phase.
Mobile phase
The fluid that the structure containing the stationary phase is placed in.
How it works?
Imagine a piece of chalk dipped in ink in a glass of water. The stationary phase is the chalk and the mobile phase is the water. As the components in the water-soluble ink are swept forward over the stationary phase by the solvent (water) they undergo a continual process of adsorption onto the stationary phase and desorption back into the liquid mobile phase. Adsorption occurs when a substance forms a bond with a surface. Desorption is the breaking of this bond.
The rate of movement of each component depends mainly on:
•How strongly it adsorbs (intermolecular forces) onto the stationary phase;
•How readily it dissolves into the mobile phase.
•How larger the molecule is (molar mass)
Paper chromotography
High quality absorbent paper is used as the stationary phase. A solution of the sample is made up and a very small spot is placed onto one end of the paper with a capillary tube. The position of the spot is called the origin. The paper is then placed in a container so that the edge of the paper below the spot is submerged in a solvent. As the solvent rises up the paper, the components of each sample separate.
Thin layer chromatography (TLC)
Similar to paper chromatography except the stationary phase is a fine powder such as alumina spread on a glass or plastic plate.

Paper and TLC are useful for qualitative analysis.
Chromatograms
A chromatogram is the pattern of bands or spots formed on the plate or paper.
Determine individual chemicals
1. Running known standards on the same chromatogram as the unknown and comparing
o In this method it is necessary to have some idea of the chemical present in the sample
o The sample and standard must be run on the same chromatogram because the distances moved from the origin will depend on the distance moved by the solvent front

2. Calculating Rf values of the sample
Advantages of paper chromatography
•Cheap
•Little preparation
•More efficient for polar and water-soluble compounds
•Easy to handle and store
Advantages of thin layer chromatography
•Faster
•Detects smaller amounts
•Better separation of less polar compounds
•Corrosive material can be used
•A wide range of stationary phases is available
Rf value
•Rf values are always less than one
•The component most strongly adsorbed onto the stationary phase moves the shortest distance and has the lowest Rf value.
•Rf values are compared to a known list of Rf values to identify what is present in a sample.
Gas chromatography
It is the most sensitive chromatographic technique, but is limited to compounds that are readily vaporised without decomposing. (Usually Molar mass of less than 300).
Gas chromatography is used in the detection of illegal drugs in athletes.

There are two types of GC: Gas-Liquid (GLC) and Gas-Solid Chromatography (GSC). They both work the same way.
• The mobile phase is a gas, usually nitrogen, called the carrier gas.
• A small amount of sample is injected into the top of the column through an injection port.
• The injection port is heated and the sample vaporises. The sample is swept into the column.
• In the column, the temperature is lower and the sample condenses and adheres to the stationary phase. The temperature is gradually increased and as the boiling points of components are reached, they are swept along with the carrier gas, adsorbing and desorbing along the way.
• The least soluble component (those least attracted to the solid or liquid stationary phase) are eluted first.
High Performance Liquid Chromatography (HPLC)
Stationary phase: solids of very small particle size packed in a glass column.

Mobile phase: solvent which is dripped slowly onto the column from a reservoir above. A tap at
the bottom allows the solvent (called eluent) to leave the column at the same
rate it enters.

Sample: The sample is applied to the top of the
column and slowly makes its way through, separating as it goes according to how much it is attracted to the stationary and mobile phases.

The time taken for a sample to pass through the column is called the Retention time (Rt). This is similar to an Rf value however it is a record of how long the sample stayed (was retained) in the column.

HPLC has a digital recorder attached and the chromatogram produced has a peak for each component detected. The time taken for each peak to emerge determines what the component is and the area under the peak can be compared to a standard sample of known concentration to find the unknown concentration.
Rf
Rf = (Distance moved by solute)/Distance moved by solvent)