Documente Academic
Documente Profesional
Documente Cultură
CHROMATOGRAPHY
Chromatography is a family of analytical chemistry techniques for the separation of mixtures. It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who invented the first chromatography technique in 1901.
CHROMATOGRAPHY
The separation of molecules depends on differences of 1- size 2- shape 3- mass 4- charges 5- solubility 6- adsorption.
Types of Chromatography:
1. Adsorption chromatography. 2. Partition chromatography e.g. paper chromatography 3. Gel-filtration chromatography.
Uses of Chromatography:
* Government laboratories used to check for approved dyes in food that vegetables contained tiny amounts of pesticides and herbicides
The components will travel at different rates over paper depending on:
1. their solubility in the solvent 2. how well the dyes adsorb on the chromatography paper Generally, the more soluble the component is in the solvent and the less it adsorb onto the chromatography paper, the faster it would move with the solvent on the paper and hence the spot appears further up the paper
Stationary Phase
In paper chromatography, cellulose in the form of paper sheets makes an identical support medium. WHY? Because it has the ability to adsorb water molecules between cellulose fibers and forms a stationary hydrophilic phase. Paper: Watman No. 1 of high quality is the paper most frequently used for analytical purposes.
Mobile Phase
In paper chromatography, mobile phase is a mixture of solvents. The choice of solvent depends on the mixture investigated: 1- If the compounds move close to solvent (A) front >>> these compounds are highly soluble in solvent A 2- If the compounds are crowded around the origin >>> these compounds are not sufficiently soluble in solvent B. Therefore, a suitable solvent for separation would be an appropriate mixture of both solvent A & B. As a result R f values of the components of the mixture are spread across the length of the paper.
Retention Factor (R f ):
The retention is measured as the retention factor Rf, the run length of the compound divided by the run length of the solvent front: Unknown substances could be identified by the Rf values Rf = distance moved by the substance distance moved by the solvent
Retention Factor(R f ):
The Rf of a compound often differs considerably between experiments and laboratories due to variations of - the solvent, - the stationary phase, - temperature, and - the setup. It is therefore important to compare the retention of the test compound to that of one or more standard compounds under absolutely identical conditions.
Retention Factor (R f ):
Detection of Spots:
After development, the spots corresponding to different compounds may be located by their color However, most compounds are colorless and are visualized by: 1- Spraying the paper with specific reagents. 2- Dipping the paper in a solution of the reagent in a volatile solvent. 3- Fluorescent substances can be visualized by ultraviolet (UV) light.
The Rf value of an amino acid is the ratio of the distance traveled by the amino acid from the origin to the distance traveled by the solvent from the origin. Since Rf value for an amino acid is constant for a given chromatography system, an unknown amino acid can be identified by comparing its Rf value to those of known amino acids.
Applications
Materials: 1- Filter paper: Watman No.1. 2- Solvent system: Butanol: glacial acetic acid: water. 3- Ninhydrine reagent. 4- Standard amino acids and mixture of unknown.
Applications
Procedure: 1- Draw a light pencil line 1-2cm from the bottom of the paper. 2- Place a single drop of compound at intervals 2cm. 3- Dry with hair dryer. 4- Dip the paper in the jar with one of the edges of the paper to which the sample of the spot is adjacent into the solvent. 5- Allow to run. 6- Remove the paper. 7- Determine the solvent front. 8- Dry. 9- Spray the paper with ninhydrin. 10- Dry the paper.
Applications
Procedure: 1- Draw a light pencil line 1-2cm from the bottom of the paper. 2- Place a single drop of compound at intervals 2cm. 3- Dry with hair dryer. 4- Dip the paper in the jar with one of the edges of the paper to which the sample of the spot is adjacent into the solvent. 5- Allow to run. 6- Remove the paper. 7- Determine the solvent front. 8- Dry. 9- Spray the paper with ninhydrin. 10- Dry the paper.