Chem 237 Today: The role of Prof. Zimmerman is being played by Prof. HullMystery'Flask' What%is%in%the%flask?% Is%it%a%single%compound?% Can%you%isolate%the%compound?%%What'would'you'do'if'you'walked'into'the'lab'and'were'handed'a'flask?' How%could%you%determine%what%is%in%the%flask?% 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%fluid%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%different%speeds,%causing%them%to%separate.%% The%separa?on%is%based%on%differen?al%par??oning%between%the%mobile%and%sta?onary%phases.% %Subtle%differences%in%a%compound's%par??on%coefficient%result%in%differen?al%reten?on%on%the%sta?onary%phase%and%thus%changing%the%separa?on.%hIp://en.wikipedia.org/wiki/Chromatography%ThinHlayer'chromatography'(TLC)' Separa?on%of%a%mixture%of%compounds%based%on%polarity%of%the%components%Mobile phase (solvent) Glass or plastic Adsorbent (stationary phase) Concept:% A%mobile%phase%(solvent)%carries%a%mixture%of%compounds%over%a%sta?onary%phase.% Different%compounds%have%different%affini?es%for%the%sta?onary%phase.% Compounds%with%higher%affinity%for%the%adsorbent%move%more%slowly%(are%retained%beIer).%%%http://en.wikipedia.org/wiki/Thin_layer_chromatographyglass' Forces%that%bind%solute%to%adsorbent% DipolePdipole%interac?ons% Hydrogen%bonding%%HOPhHOPhR OR'OAdsorbents: • Silica (acidic) • Alumina Al2O3 (basic) glassS iOSiOSiOS iOS iOO OH OHHHOPhSoluteHadsorbent'interac M ons'http://en.wikipedia.org/wiki/Silica_gelpolar adsorbent (solid)support (glass/plastic)mobile phase(liquid flow)polar adsorbentpolar solutenonpolar soluteSoluteHadsorbent'interac M ons' Forces%that%bind%solute%to%adsorbent% DipolePdipole%interac?ons% Hydrogen%bonding%%http://en.wikipedia.org/wiki/ChromatographySample%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'f or 'TLC 'Develop%TLC% Place%plate%in%beaker% Allow%solvent%to%move%%%%%~90%%of%way%up%plate% Immediately%mark%solvent%front%a\er%removing%plate%Experimental'setup'f or 'TLC ' 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%Reten?on%factor,%Rf% Solvent%selec?on% More%polar%solvents%for%%%%%%more%polar%compounds%% Poten?al%problems% Overloading% Irregular%spot%shape%%Rf = distance spot moves (A) distance solvent moves (B) B A Experimental'setup'f or 'TLC 'HOPhR OR'OAB TLC is extremely useful for: Monitoring reactions determining _________________ for column chromatography to purify a compound by column chromatography, the Rf should be _____________ solvent conditions 0.25 – 0.35 TLC:'Effect'of'varying'solvent'A B A + B cospotoriginoriginA Co Mix Co BA Co Mix Co Belute with hexanesolvent frontA Co Mix Co BA Co Mix Co Belute with Hex/EtOAc (3/2)solvent frontTLC:'Effect'of'varying'solvent'Chromatography solvents ThinHlayer'chromatography'(TLC)'ClClClClClClClClOOOOOHOHH2OOHOIncreasing ability to elute organic compoundshexanes trichloroethylene methylenechloridediethyl ether acetone methanol acetic acidcyclohexane toluenechloroformethylacetateethanolwaterPacking 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 Column'chromatography'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_chromatographyIsolation of components: combine homogeneous fractions evaporate; weigh do not discard adsorbent until mass balance is checked Column'chromatography:'Achieving'separaMon'column