Transcript Lecture 3
Partition chromatography Partition chromatography is carried out on sheets of filter paper, column or thin layer of powdered cellulose, moist silica gel or kieselguhr Silica acts as support and the water is stationary phase It is known that filter paper consists of numerous cellulose fibre which accomodate certain percentage of moisture we can consider that fibre + water constitute cells Partition of the substance between moisture in the cell and the solvent flowing over the cells which bring about separation Water of cell is the stationary phase and moving solvent is the mobile phase The extent to which the solute distribute itself between the two liquid is measured and is known as partition or distribution coefficient K= Conc. of solute in M. Ph Conc. of solute in St. Ph Partition chr. Includes A- liquid liquid extraction B- column chromatography C- paper chromatography D- TLC E- Gas liquid chromatography A- Liquid liquid extraction Solute distributes itself between equal volumes of two immiscible solvents. Methods of extraction 1- discontinuous extraction A- single contact In separating funnel using two immiscible liquid B- multiple contact Several times in separating funnel. 2- continuous liquid liquid extraction 1- single stage by using liquid liquid extraction app. It consists of round bottom Pyrex flask Extraction tube connected to the flask by another glass tube Condenser 2- multiple stage (counter current distribution) Several units of extraction app are specially constructed to transfer the upper phase to the subsequent tube keeping the lower stationary phase Substance distribute itself between lower stationary phase and upper according to distribution coefficient (K) Counter current extraction app. K= Conc. of solute in upper phase Conc. of solute in lower phase The procedure: 1- the app. Consists of 100 tubes affecting 100 equilibrations 2- fresh mob. Phase. Is transferred from reservoir to tube 1 3- after equilibrium mob. Phase move to fresh stationary phase, shacked and transferred to a tube containing stationary phase and so on.... It depends on 1- number of shacking 2- time allowed to stand for separation Number of equilibrium B- column (partition chromatography) As in column (partition chr.) the difference is the packing material which is porous material such as kieselguhr coated with layer of liquid, water, the stationary phase is the liquid layer and the solid serves as a support More soluble substance travel more slowly down the column Solid support as silica, diatomites, cellulose, cellulose acetate. Preparation of the column Mixture of support + stationary phase is stirred with some of mobile phase to be used initially The slurry is poured in a small portion into the tube which contain small portion of the mobile phase Application of the sample 1- by use of pipette with a bent tip which is placed against the column wall just above the adsorbent and the liquid is slowly drained from the pipette. 2- use of one or two small disks of filter paper which perforated with very small holes of a size which just to fit on the top of the column - Solute is dissolved in volatile solvent and adsorbed on one of the filter paper 3- Mixture is dissolved in some of the Mob. Ph. And mix with some of the stationary phase , the dry and put the powder on the top of the column. Elution As in adsorption column Identification of the separated substance as in adsorption column Applications of partition column 1- fractionation of many alkaloids and polyphenols 2- determination of amino end group of proteins and analysis of hormones 3- separation of methylated sugars on silica gel and free sugar on cellulose. Paper chromatography Stationary phase is water hold by adsorption on cellulose molecules which in turn are kept in a fixed position by the fibrous structure of the paper Apparatus and method Tightly covered jar Method 1- choice of the paper 2- purification of the sample 3- application of the sample on the paper 4- development 5- detection 6- identification 1- Choice of the paper Whatman No 1 or 3 for analytical Whatman No 3 or 3MM for preparative 2- choice of solvent system Depend on substances to be separated Amino acid..phenol/ water Butanol / water/ acetic acid Sugars...EtOAc/ pyridine/ water EtOAc/ i-prppanol/ water 3- sample purification By column or liquid liquid extraction 4- application of the sample to the paper Clear solution is prepared Take sample by capillary Apply the spot on the paper and dry it. 4- Development A- ascending the flow upward b- descending downward C- horizontal D- radial the flow in the radii of circular paper 1- ascending development Paper is stood vertically and immersed in the mobile phase Level of the spots should be above the liquid by 12 cm 2- descending development Jar is provided by a trough for the mobile phase in its upper part Paper suspended in trough wire fixed to side of the tank on which trough can fest Serrated edge to allow the solvent to flow uniformely off the paper Advantages of descending technique A- solvent flow faster B- long distance of development can be achieved 3- Horizontal development: Shallow tank of glass or metal The paper is placed horizontal on glass rods with one end of the paper is dipped in the mobile phase Direction of mobile phase paper support mobile phase -sample is spotted on a line on the horizontal part of the paper 1-2 cm from the edge - solvent is flows up by capillary forces 4- radial development The principle of this method is based on migration of the spot from the centre of the paper toward the periphery. . Detection of the resolved spots visualization 1- physical , UV, colour 2- chemical Aniline phthalate...........reducing sugars Dragendorff`s..................alkaloids FeCl3.....................phenolic compounds Ninhydrin...................amino acid, amino sugars Anisaldehyde H2SO4......terpene, sterols, sugars (Not used in paper as it cause charring of the paper) 3- biological enzymatic detection of amylase microbilogical detection of antibiotic Uses of paper chromatography 1- identification of components of mixture 2- detection of purity of components against authentic 3- major use is the separation of flavonoids and sugars