Lecture I. 1- Chromatographic methods A. Braithwait, E.J. Smith (1995) 2- Modern thin layer chromatography (chromatographic science services vol, 52)

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<p>474 PHG</p> <p>474 PHGLecture I1Recommended books 1- Chromatographic methodsA. Braithwait , E.J. Smith (1995) 2- Modern thin layer chromatography (chromatographic science services vol, 52) N. Grinberg (1990)3- Preparative chromatography techniqueHostettman K., Hostettman M. And Marston A. (1986)4- Organic structure analysis Crews P., Rodriguez J. And Jaspers M. (1998)5-Spectroscopic identification of organic compounds 6th ed Robert M. Silverstein, Francis X Webster (1996).</p> <p>2ChromatographyChromatography is a technique by which compounds of mixture are separated by differential migration of dissolved sample between two immiscible phases in a specified system.Two immiscible phases: Mobile phase is liquid or gasStationary phase is adsorbent or a second liquid 3Classification of chromatographyI- According to Mobile phaseA- Liquid chromatographyB- Supercritical fluid chromatographyC- Gas chromatographyII- According to stationary phaseA- Gas solid chromatographB- Liquid liquid chromatographC- Gas liquid chromatography4III- According mechanism of separationA- adsorption chromatography (Solid St. Phase)B- Partition chromatography (two immiscible liquids)C- Ion exchange chromatographyWhich is used for separation of charged molecules by using anaionic and cationic resin</p> <p>5D- Affinity chromatography in bio fluids (antigen-antibodies reaction)E- Gel filtration chromatography (steric exclusion, or molecular sieving )The column is packed with material having controlled pore sizes and the sample is screened or filtered according to its molecular size, there is no interaction between solute and stationary phase. The large molecules rapidly washed through the column, the smaller molecules penetrate inside the pores and elute later.</p> <p>Large moleculessmall molecules6IV- according to the equipment and the operational proceduresA- Column chromatography- The stationary phase is packed in tube and mobile phase pass through it by gravity or pressure -Column chromatography- HPLC- GC- SEC</p> <p>7B- Planar chromatographyThe stationary phase is solid coated onto glass, plastics foil (TLC) or supported by cellulose fibre of paper sheet (paper chromatography)</p> <p>8Terminology</p> <p>Adsorbent: finely divided homogenous solid having uniform particle size and large surface area which is capable of attracting molecules to its surface.Chromatogram : a record at the end of chromatographic separationDevelopment: description of the process of chr. (running of the mobile phase through the stationary phase)</p> <p>9Analyte; components of sample mixtureEluent: solvent used for separation in chromatographic techniquesEffluent: liquid out of the columnElution: seeping out of the components of the mixture in pure or partially mixed form.Resolution: the ability of any chromatographic process to separate pure compound.</p> <p>10Retention time: time taken to elute a particular soluteRate of flow: distance travelled by solute / distance travelled by solvent</p> <p>11Retention volume: volume of mobile phase required to elute a particular soluteTailing: disadvantage of chromatography and solute is eluted in several fractionVisualization: making the colourless bands visible</p> <p>12Modes of chromatographic separationI- Adsorption chr.I- Column chromatographyAll the major chr. Process are routinely carried out using column modeFor classical column chr. The following items are required</p> <p>13A- Adsorbent (stationary phase)The adsorption power of the adsorbent depends on</p> <p> e.g. Washing or heatingThe larger the particle size the lower is the back pressure the faster is the flow rate and more poor is the resolution (bad separation)The average particle diameter in open column chr. Is 10-2000um </p> <p>14The Ideal adsorbent must fulfill the following requirements:Insoluble in mobile phase. Inert to solutes (adsorptive).Colorless especially when work with colored mixtures. Suitable particle size enough to give good separation and reasonable flow rateDecrease particle size increases the surface area and consequently increases separation power.. 15</p> <p>16A- Silica gel is the most popular adsorbent with a general formula SiO2 (H2O)nThe active sites of silica gel are the hydroxyl groups attached to silicon atoms "Silanol groups" .</p> <p>17The selective adsorptivity of activated silica is attributed to the surface silanol group which form hydrogen bond solute with a particular</p> <p>Water is adsorbed by silica and inactivate it so we must activate it before using by heat at 190-200 C for two hours.Excessive heating leads to formation of non active siloxane</p> <p>18B- Alumina</p> <p>It is a porous polymer of Al2O3 available in various commercial varieties for both column and planar chr.</p> <p>Activation by heating at 200-400 C for 12 hrsTypes of commercial alumina : 1- Neutral alumna pH 7 7.5.</p> <p> 2- Acidic alumina pH 4. It is prepared by washing aluminum oxide with 2N HCl then with distilled water. </p> <p> 3- Basic alumina pH 10. This type is prepared by washing with NaOH then distilled water. </p> <p>19C- Magnesium silicate (Acidic adsorbent)It helps separate acetylated sugar steroids and essential oils.</p> <p>D- Kieselguhr it is prepared from the siliceous skeleton remains of microscopic marine animals.E- Charcoal colour, low sample recovery, non selective adsorptivity restrict of its use to limited application</p> <p>20B- Mobile phaseIt is moving solvent that percolate through the stationary phaseIdeal mobile phase- inert- low boiling point- Low toxicity- Low price- Low viscosity- Non volatile21C- Column construction 1- Column material and diameter2- Packing of column3- Sample loading4- Development5- Fractions collection</p> <p>221- column material, dimensionsColumns are made up of glass, stainless or synthetic polymer Tube-like glass column with length 10-100 times the internal diameter </p> <p>2- packing of the columnThere are two types of packing a- wet packing (slurry) </p> <p>- the specified amount of adsorbent is distributed in mobile phase in a beaker- the slurry is poured into the column after closing the bottom by cotton and close the tape23</p> <p>24- the solvent flow start by opening the tape (outlet) until the packing is settled. b- dry packing- dry adsorbent is poured directly in the columnsolvent pass through the adsorbent</p> <p>3- sample loading (application of sample)There are two methods of sample loadinga- dry methodThe sample is adsorbed onto small amount of stationary phase , dry, then delivered onto column top25</p> <p>262- wet methodDissolve the sample in a small volume of the mobile phase and delivered onto the top of the column.4- column development (elution) The process start by the continuous passage of suitable phase (mobile ph.) through the stationary phase</p> <p>27Elution techniques for column1- Gradient elution2- Isocratic elution28Gradient elutionIsocratic elutionThe mobile phase composition is changed during the separation process.</p> <p>The mobile phase composition remains constant throughout the separation procedure. </p> <p>29Gradient elution technique</p> <p>Advantages of gradient elution technique1- Shortening the time of analysis.2- Reduces tailing, gives sharp peak.3- Increases the sensitivity of analysis.4- Decreases the retention of the later-eluting components so that they elute faster.5- substances with different properties can be separated in one operation. </p> <p>30A- Tailing Is formation of diffusely bounded zone or band in the development Process.</p> <p>B- FrontingFronting is represented by extended diffused front portion of the peak and Sharpe tail.Disadvantages of isocratic elution </p> <p>Factors leading to tailingstrong interaction between solute and stationary phaseapplication of excessive amount of the sample to the columnpoor column packingimproper selection of mobile phase</p> <p>occurs when the interaction between solute molecules is strong relative to those between solute and stationary phase</p> <p>31</p>

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