Chem 237 Today The role of Prof Zimmerman is being played by Prof Hull Mystery Flask What would you do if you walked into the lab and were handed a ask What is in the ask Is it a single compound Can you isolate the compound How could you determine what is in the ask If it were a mixture of compounds how would you separate them USE CHROMATOGRAPHY Chromatography Chromatography is the collec ve term for a set of laboratory techniques for the separa on of mixtures The mixture is dissolved in a uid called the mobile phase which carries it through a structure holding another material called the sta onary phase The various cons tuents of the mixture travel at di erent speeds causing them to separate The separa on is based on di eren al par oning between the mobile and sta onary phases Subtle di erences in a compound s par on coe cient result in di eren al reten on on the sta onary phase and thus changing the separa on hIp en wikipedia org wiki Chromatography Thin layer chromatography TLC Separa on of a mixture of compounds based on polarity of the components Mobile phase solvent Concept Adsorbent stationary phase Glass or plastic A mobile phase solvent carries a mixture of compounds over a sta onary phase Di erent compounds have di erent a ni es for the sta onary phase Compounds with higher a nity for the adsorbent move more slowly are retained beIer http en wikipedia org wiki Thin layer chromatography Solute adsorbent interacMons Forces that bind solute to adsorbent Dipole dipole interac ons Hydrogen bonding O Ph H O H O OH Si O O H Ph Ph R O O H O Si O R Si H O glass Adsorbents Si O Si Silica acidic O Alumina Al2O3 basic glass http en wikipedia org wiki Silica gel Solute adsorbent interacMons Forces that bind solute to adsorbent Dipole dipole interac ons Hydrogen bonding polar solute nonpolar solute polar adsorbent solid support glass plastic mobile phase liquid flow polar adsorbent http en wikipedia org wiki Chromatography Experimental setup for TLC Sample prepara on and spoQng Dissolve sample in vola le solvent Draw horizontal line on TLC plate 0 5 cm from boIom pencil Mark your spot with a small ver cal line Spot a small amount of sample with capillary tube micropipet Prepare development tank Add small amount of development elu ng solvent or solvent mixture solvent system Height of solvent must be below line on plate where your sample is spoIed Experimental setup for TLC Develop TLC Place plate in beaker Allow solvent to move 90 of way up plate Immediately mark solvent front a er removing plate Visualiza on where did the components end up UV light don t look directly into the light Reversible stain I2 Irreversible stains with heat H2SO4 DNP vanillin anisaldehyde phosphomolybdic acid ceric ammonium molybdate Mark spots with pencil if needed Experimental setup for TLC Reten on factor Rf A B Rf Solvent selec on More polar solvents for more polar compounds Poten al problems Overloading Irregular spot shape distance spot moves A distance solvent moves B TLC E ect of varying solvent A A B cospot B TLC is extremely useful for Monitoring reactions Ph O A O H R O B R solvent conditions for determining column chromatography to purify a compound by column chromatography the Rf should be 0 25 0 35 TLC E ect of varying solvent solvent front elute with hexane A Co Mix Co B origin A Co Mix Co B solvent front elute with Hex EtOAc 3 2 A Co Mix Co B origin A Co Mix Co B Thin layer chromatography TLC Chromatography solvents Cl hexanes Cl Cl Cl Cl trichloroethylene O O O methylene chloride OH diethyl ether acetone methanol OH acetic acid Increasing ability to elute organic compounds Cl Cl Cl cyclohexane toluene chloroform O O OH H2O ethyl acetate ethanol water Column chromatography Packing the column amount of adsorbent 20 50 1 by weight of sample to be separated mount column perpendicular to laboratory bench column may have a frit on the bottom to hold in adsorbent fill with a few mL of eluent slurry silica gel gently pour into column allowing eluent to drain tap column to settle silica if needed add sand to top of silica gel lower eluent level to sand Column chromatography Applying mixture dissolve sample in minimum amount of eluent add gently to top of column rinse down sides using minimum amount of eluent lower solvent level to sand fill column with eluent Running the column flow through eluent collect fractions 1 10 or less of column volume analyze fractions by TLC change of solvent may be necessary gradient elution Column chromatography Importance of Rf http en wikipedia org wiki Column chromatography Column chromatography Achieving separaMon column fractions Isolation of components combine homogeneous fractions evaporate weigh do not discard adsorbent until mass balance is checked