Activity 6. 1. Define and give the uses of chromatography.

Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the "mobile phase", which carries it through a structure holding another material called the "stationary phase". The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.

2. Types of Cromatography and give its principle a. Liquid Chromatography

b. Gas Chromatography
detect bombs in airports, identify and quantify such drugs as alcohol, used in forensics to compare fibres found on a victim Used to analyze volatile gases. Helium isused to move the gaseous mixture through a column of absorbent material.

c. Thin-Layer Chromatography
detecting pesticide or insecticide residues in food, also used in forensics to analyze the dye composition of fibers Uses an absorbent material on flat glass plates. This is a simple and rapid method tocheck the purity of the organic compound

d. Paper Chromatography
separating amino acids and anions, RNA fingerprinting, separating and testing histamines, antibiotics. The most common type of chromatography. The paper is the stationary phase. This uses capillary action to pull the solutes up through the paper and separate the solutes.

3. Give the meaning and use of Rf.

The distance travelled by a given component divided by the distance travelled by the solvent front. For a given system at a known temperature, it is a characteristic of the component and can be used to identify components. Its a relative value that shows how mobile your compound with respect to your solid phase and mobile phase (solvent). To calculate it you divide the distance your compound traveled from its initial starting position by the distance traveled by the solvent front. 4. Define the following terms

a. Chromatogram The word chromatogram comes from two Greek words meaning color and writing (writing, the noun) which, together, literally meanscolor writing. The term arose from the work of Tswett in his separation of plant pigments. Tswett separated some plant extracts on a calcium carbonate column using a dispersive solvent as the mobile phase. The different pigments aligned themselves along the column and, against the white chalk, gave a series of colored bands which Tswett called a chromatogram. In fact, the colored bands depicted the separation that he had obtained. From the same terminology the actual separation technique became known aschromatography vis color writing (writing the verb). To day all connotation between chromatogram and color has virtually been lost and a chromatogram is now understood to be a visual depiction of any chromatographic separation that has been developed. A chromatogram is usually a graph relating concentration (or mass per unit time) of solute leaving a chromatographic column, against time, and takes the form of a series of ever broadening peaks.. The first chromatograms of this form were traced on a recording milli-ammeter by Martin in the early 1950s. The recording milli-ammeter, however, was soon replaced by the potentiometric recorder. Today most chromatograms are presented on a computer screen and/or printed out on a computer printer. b. Solvent Front the wet moving edge of the solvent that progresses along the surface where the separation of the mixture is occurring. The solvent front in chromatography is the "signal" or the leading edge of the solvent as it moves through the matrix. In TLC, e.g., it will be the leading edge of the solvent as it moves up the plate. It HPLC, it is somewhat more problematic, in that it is the initial segment of solvent as it comes off the column, and may be large enough to interfere with the measurement of the compound of interest.
c. Staionary Phase

is the part of the chromatographic system though which the mobile phase flows where distribution of the solutes between the phases occurs. The stationary phase may be a solid or a liquid that is immobilized or adsorbed on a solid. In general immobilization by reaction of a liquid with a solid is used in liquid chromatography and absorbtion of a liquid on a solid is used in gas chromatography but there are many exceptions to both of these generalizations. The

stationary phase may consist of particles (porous or solid), the walls of a tube (eg. capillary) or a fibrous material (eg paper).

d. Mobile Phase
is the part of the chromatographic system which carries the solutes through the stationary phase. The mobile phases are either liquids or gases. The liquid mobile phases are used to adjust the chromatographic separation and retention in liquid chromatography and the temperature of the gas mobile phase is used to adjust the retention in gas chromatography.