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Column Chromatography Column Chromatography Column Chromatography Column chromatography 1- Apparatus: A) A vertical glass tube height /diameter ratio should be at least 20:1 or 50:1 B) A Reservoir of the solvent (eluent) C) Collecting vesseles 2- Chemicals : A- Adsorbent : 1) Insol. in solvents used. 2) Non-reactive with the sample or the solvent 3) White or colorless 4) Appropriate particle size 5) Uniform particle size Types of adsorbents 1- Alumina ( Al2O3 ) 2- Silica gel 3- Kieselguhr : (diatomaceous earth): Used as a carrier to the stationary phase in partition Chromatography. 4- Kaolin 5- Cellulose Powder (Partition Chromatography) 6- Sephadex gel 7- Polyamide (flavonoids) Types of columns 1- Gravity Columns: The mobile phase move through the stationary phase by gravity force. 2- Flash Columns (Air or nitrogen pressure): The mobile phase is pushed by stream of air or nitrogen using special device (Adaptors). 3- Low and Medium Pressure Columns (pumped): The movement of mobile phase is accelerated by using pumps that generate low or medium pressure. The increase in the flow rate shorten the time of separation. 4- High pressure Columns (HPLC): In this columns we use very fine silica gel so great increase in separation power. However, the flow rate of the mobile phase is severely decreased. High pressure pumps are used to push the solvent through the column which in this case must be made of stainless steel. I- Packing of the Column Dry Method: The stationary phase is packed dry and then wetted with the mobile phase. Wet Method: The stationary phase is made a slurry with the mobile phase, and is poured into the column & allowed to settle. 11 Mobile phase: It is a mixture of organic solvents (unusually one solvent only) the choice of the column mobile phase is achieved after TLC study in different solvent systems. Good solvent system must produce Rf value less than 0.6 for all materials to be separated by the column. If the system moves them more and produces higher Rf no separation will occur. Systems that do not move spots at all on TLC are not good for column separation. Isocratic system: Means using the same mobile phase from the beginning to the end of the separation. Gradient: The polarity of the system increased gradually during separation by increasing the proportion of the more polar solvent. A typical gradient may be start with CHCl3, followed by CHCl3/MeOH mixtures with gradual increase in % of MeOH till all spots are eluted from the system. II- Sample Application 1- Wet application: Dissolve the sample in the initial mobile phase and apply by pipette to the top of the column. This is very good method but in most of cases the samples are not soluble in the initial mobile phase. 2- Dry loading: Dissolve sample in any volatile solvent. The sample solution is then adsorbed on small weight of adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with the sample is then applied to the column. I- Packing of the Column II- Sample Application III- Mode of Development III- Modes of Chromatographic Separation 1- Frontal Analysis This type of chromatographic development is rarely used and probably is of academic interest only. The sample is fed continuously onto the column as a dilute solution in the mobile phase in contrast to displacement and elution development, where distinct samples are placed on the system and the separation subsequently processed. Frontal analysis can only separate part of the first compound in a relatively pure state, each subsequent component being mixed with those previously eluted. 2- Elution development Is best described as a series of absorption- extraction processes which are continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. Sample components travel through the column at rates determined by their retention on the solid packing Components are eluted from the column at different rates according to their distribution (or partition) coefficients. A complete separation is possible Elution Development E B + E E A + E E E= Eluate, B is more sorbed than A Elution analysis can be done in a number of ways: 1- Single-Solvent Elution : 2- Stepwise gradient elution Benzene Benzene – MeOH (9:1 ) Benzene – MeOH ( 8:2 ) Benzene – MeOH ( 7 : 3) 3- Continuous Gradient Elution : Advantages of gradient Elution 1- It minimize tailing and results in more discrete peaks and hence better resolution 2- Components of a mixture having widely different properties & polarities can be separated in a single operation . 3-Displacement Development Displacement chromatography employs three basically different mobile phases, such as the carrier, displacer, and regenerant (s). The carrier serves to facilitate the load; and to dissolve the sample. The function of the displacer is to displace the sample components from the stationary phase; 3-Displacement Development During this step the individual components also displace each other. By the end of the displacement step, the displacer occupies the stationary phase. Finally, the regenerant removes the displacer from the stationary phase and it prepares the column for the separation of the next sample. In Summary: •The developer is contained in the mobile phase. •The mobile phase must be more sorbed than any sample component •A single pure band of the first component is obtained •There is always an overlap zone for each succeeding component. Displacement analysis D B { A + B A Displacement analysis, D= Displacer which more sorbed than B, which more sorbed than A The following terms are used : Eluent: The chromatographic solvent used in elution analysis. Effluent: All liquid flowing out of the column Eluate: The volume of the effluent that contains eluted substance. Retention volume (Retardation volume): This is the volume of pure solvent flowing-out of the column, before the substance in question leaves it Monitoring the column: Usually fractions of certain volume are collected, evaporated to small volume and spotted on TLC. Similar fractions are collected together for more purification or crystallization. In bioassay guided fraction the fractions are monitored by the bioassay then by TLC. 31 Evaporation of similar collected fractions 32 Factors affecting separation Factors due to Stationary Phase: 1- Particle size of the stationary phase: Reducing the particle size increases the surface area and improve separation. However, reduction of the particle size will decrease the flow rate of the mobile phase. In HPLC we use very fine particles to get very good separation. The flow rate problem is solved by the use high pressure pumps to push the mobile phase through the stationary phase. Columns are made of stainless steel to withstand the high pressure. 2- Adsorbent type: The choice of the suitable adsorbent is very important. 3- Uniformity of packing of the column: If the stationary phase is not packed uniformly then the bands will be irregular and less uniform resulting in poor separation. 4- Concentration of the mixture: the proper ratio between sample to be separated and the amount of stationary phase is very important too much samples resulted in bad separation. Factors due to Mobile Phase: 1- Selection of the proper mobile phase: Very polar mobile phase will wash out all components without any separation. On the other hand very non polar mobile phase will result in broad band and poor separation. 2- Rate of flow: Slower flow rate usually resulted in a better separation and narrower bands. 3- Consistency of flow: The continuous flow of the mobile phase during the whole experiment gives better separation than interrupting the flow then continue it later. Factors due to Columns: Column dimensions: Increasing the length of the column improve separation. However, that usually leads to slower flow rate. Column temperature: Increasing the temperature usually reduces the adsorption power of the stationary phase and increase elution speed. This may leads to decrease in the efficiency of separation. Normal Phasa Chromatography Reversed Phase Chromatography Polar stationary Phase : Silica Non polar stationary Phase (ODS) Non polar mobile phase Octadecylsilane (C18) Polar mobile phase Non polar compounds elute faster polar compounds elute faster than than polar non polar When increase polarity of mobile When increase polarity of mobile phase elution time will increase phase elution time will decrease It can not be reused It can be reused Example of mobile phase: hexane – Example of mobile phase: methanol – chloroform – methylene chloride actonitrl- water .
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