ThinThin--Layer ChromatographyLayer Chromatographyandand

Column ChromatographyColumn Chromatography

BackgroundBackground

Chromatography:Chromatography: refers to several related techniques for analyzing, refers to several related techniques for analyzing, identifying, or separating mixtures of compounds.identifying, or separating mixtures of compounds.

TwoTwo--part operation:part operation:The sample mixture is placed into a liquid or gas: The sample mixture is placed into a liquid or gas: mobile phasemobile phaseThe mobile phase carries the sample through a solid support: The mobile phase carries the sample through a solid support: stationary phasestationary phaseCompounds in the sample mixture move through the stationary Compounds in the sample mixture move through the stationary phase at different rates due to different attractions for the mophase at different rates due to different attractions for the mobile bile and stationary phasesand stationary phases

Choosing Adsorbents and EluentsChoosing Adsorbents and Eluents

Stationary phase:Stationary phase: AdsorbentsAdsorbents..

Alumina is generally used for chromatography of less Alumina is generally used for chromatography of less polar compounds.polar compounds.

Silica gel is better for compounds containing polar Silica gel is better for compounds containing polar functional groups.functional groups.

Mobile phase:Mobile phase: EluentsEluents (liquid)(liquid)

The more polar eluent = the greater eluting power: its The more polar eluent = the greater eluting power: its ability to move compounds over the adsorbent ability to move compounds over the adsorbent surface.surface.

Mixed eluents can be prepared by mixing low polarity Mixed eluents can be prepared by mixing low polarity and high polarity solvents and therefore creating any and high polarity solvents and therefore creating any eluting power needed.eluting power needed.

cyclohexanecyclohexane

petroleum etherpetroleum ether

hexanehexane

toluenetoluene

dichloromethanedichloromethane

ethyl acetateethyl acetate

ethanolethanol

acetoneacetone

methanolmethanol

least polarleast polar

most polarmost polar

TLCTLC

In TLC, In TLC, capillary actioncapillary action allows the mobile phase (eluent) to ascend the allows the mobile phase (eluent) to ascend the stationary phase (solid coated on a support stationary phase (solid coated on a support plateplate).).A sample is applied at the bottom of a dry TLC plate.A sample is applied at the bottom of a dry TLC plate.The plate is placed in a The plate is placed in a developing chamberdeveloping chamber..After the separation is complete, the TLC plate is called a After the separation is complete, the TLC plate is called a chromatogramchromatogram.

Before Development After Development

Solvent Front

Compound 1

Compound 2

Column ChromatographyColumn Chromatography

Column chromatography: technique that uses an adsorbent packed iColumn chromatography: technique that uses an adsorbent packed in a n a glass column, and a solvent that moves down slowly through the pglass column, and a solvent that moves down slowly through the packed acked column.column.

t = 0 t = 1 t = 2 t = 3 t = 4

TLC vs. Column ChromatographyTLC vs. Column Chromatography

What happens?What happens?AdsorbentAdsorbent adsorbs the mixture compounds.adsorbs the mixture compounds.EluentEluent travels up (or down for column) over the travels up (or down for column) over the adsorbent, the compounds within the mixture move at adsorbent, the compounds within the mixture move at different rates.different rates.

Why?Why?CompoundsCompounds with less attraction for the adsorbent with less attraction for the adsorbent move rapidly with the eluent.move rapidly with the eluent.CompoundsCompounds with more attraction for the adsorbent with more attraction for the adsorbent move slowly with the eluent.move slowly with the eluent.

Adsorbents vs. Eluents:Adsorbents vs. Eluents:Adsorbents are typically very polar. Adsorbents are typically very polar. The more polar is The more polar is a compound in the mixture, the more strongly it a compound in the mixture, the more strongly it adheres to the adsorbent and the more slowly it adheres to the adsorbent and the more slowly it moves.moves.The more polar the eluent, the more rapidly a The more polar the eluent, the more rapidly a compound movescompound moves.

Retention Factor (Rf)

Definition: ratio of the distance that a compound moves to the dDefinition: ratio of the distance that a compound moves to the distance that istance that the eluent front moves.the eluent front moves.

distance traveled by compound, mmdistance traveled by compound, mmRRff = = ————————————————————————————————————————

distance traveled by eluent front, mmdistance traveled by eluent front, mm

Eluent FrontEluent Front

SSAA11

OriginOrigin

AA11RfRf11 = = ————————

SS1

2

AA22

AA22RfRf22 = = ————————

SS

What You Need for What You Need for TLC ExperimentTLC Experiment

6 TLC Plates6 TLC Plates

5 5 microlitermicroliter pipets (one for each pipets (one for each solution, solution, do not mix themdo not mix them!)!)

4 clean test tubes4 clean test tubes

0.5 0.5 mLmL of of ““stock solutionstock solution””

0.5 0.5 mLmL of benzophenoneof benzophenone

0.5 0.5 mLmL of biphenylof biphenyl

0.5 0.5 mLmL of benzhydrolof benzhydrol

Vial with unknown (from TA)Vial with unknown (from TA)

O

OH

11-- OmitOmit22-- Preparing the Developing ChamberPreparing the Developing Chamber

Label 3 beakers (Label 3 beakers (ethyl acetate, ethyl acetate, hexanehexane, , toluenetoluene))Pour 5Pour 5--10 10 mLmL of the appropriate solvent into each beaker to moisten the of the appropriate solvent into each beaker to moisten the filter paper and to form a layer 3filter paper and to form a layer 3--5 mm deep5 mm deepCut a piece of filter paper to fit the beakers (used to saturateCut a piece of filter paper to fit the beakers (used to saturate the chamberthe chamber’’s s atmosphere with eluent vapor)atmosphere with eluent vapor)Cover each beaker with Cover each beaker with aluminum foilaluminum foil

33-- Spotting the TLC PlatesSpotting the TLC Plates

Obtain Obtain 3 TLC plates3 TLC plates and label them at the top with the name of one of the 3 and label them at the top with the name of one of the 3 eluents.eluents.Mark the Mark the origin lineorigin line across the plate across the plate 1 cm from the bottom1 cm from the bottom, and two cross , and two cross marks on the origin line to indicate where the solution will be marks on the origin line to indicate where the solution will be spotted (Use a spotted (Use a gentle pencil not to scratch the surface).gentle pencil not to scratch the surface).Place the end of the Place the end of the micropipetmicropipet into the into the stock solutionstock solution and allow the liquid and allow the liquid to rise by capillary action.to rise by capillary action.Spot the solution onto the plate by Spot the solution onto the plate by quickly and lightly touchingquickly and lightly touching the end of the the end of the micropipetmicropipet to the surface of the adsorbent. (diameter of the spot less thato the surface of the adsorbent. (diameter of the spot less than 2 n 2 mm)mm)Let it dry and apply a second time on one of the spotsLet it dry and apply a second time on one of the spots

Give 2 different Give 2 different concentrationsconcentrations

33-- Developing TLC PlatesDeveloping TLC Plates

Check that the level of eluent in each chamber is Check that the level of eluent in each chamber is below the origin linebelow the origin linePlace TLC plates into corresponding chambersPlace TLC plates into corresponding chambersCover the chambers with aluminum foil (do not Cover the chambers with aluminum foil (do not move the chamber after developing begins)move the chamber after developing begins)When the eluent front rises to within 1 cm of the When the eluent front rises to within 1 cm of the top of the plate, remove the plate and top of the plate, remove the plate and immediately mark the eluent front with a pencilimmediately mark the eluent front with a pencilAllow solvent to evaporate from the plate under a Allow solvent to evaporate from the plate under a fume hood fume hood (especially toluene: 15(especially toluene: 15--20 min)20 min)Examine the developed TLC under Examine the developed TLC under UV lightUV lightUse a pencil to circle spotsUse a pencil to circle spotsRecordRecord::

Whether a single application or double give Whether a single application or double give better resultsbetter resultsThe eluent that gives better separationThe eluent that gives better separation

Calculate Calculate RRff for each spot on the plate with the for each spot on the plate with the best separation (measure from center of spot)best separation (measure from center of spot)

44-- Identifying the Compounds in a MixtureIdentifying the Compounds in a Mixture

Using Using 2 TLC plates:2 TLC plates: 11st st -- stock solutionstock solution, , biphenylbiphenyl, , benzhydrolbenzhydrol22ndnd -- stock solutionstock solution, , benzophenonebenzophenone

Develop the plate with Develop the plate with the best solventthe best solvent / Visualize both plates under UV/ Visualize both plates under UVCalculate Calculate RRff for each compoundfor each compoundPlace one of the plates in an iodine chamber and record your obsPlace one of the plates in an iodine chamber and record your observations ervations (just for fun!)(just for fun!)

55-- Analyzing an Unknown MixtureAnalyzing an Unknown Mixture

Obtain an unknown solution from your instructorObtain an unknown solution from your instructorSpot your Spot your unknownunknown along with the along with the stock solutionstock solution ((1 TLC plate1 TLC plate))Develop the plate with your chosen solvent / Visualize under UVDevelop the plate with your chosen solvent / Visualize under UVRecord the Record the compound(scompound(s) present in the unknown) present in the unknown

TURN IN TURN IN ALLALL PLATES WITH YOUR REPORT (in an envelope or zip lock bag)PLATES WITH YOUR REPORT (in an envelope or zip lock bag)

Column Chromatography ExperimentColumn Chromatography Experiment

11-- Preparing a Dry Pack ColumnPreparing a Dry Pack Column

In a In a short stemshort stem 99--mm Pasteur pipet place: mm Pasteur pipet place: A small cotton plug at its tipA small cotton plug at its tipClamp the column Clamp the column verticallyverticallyA little bit of sand, then the alumina (height is A little bit of sand, then the alumina (height is important)important)90 mg90 mg of the of the already preparedalready prepared mixture: mixture: ferrocene/acetylferrocene/aluminaferrocene/acetylferrocene/aluminaA little bit of alumina, then sand on topA little bit of alumina, then sand on top

22-- Eluting Eluting FerroceneFerrocene -- HexaneHexane

Label a beaker hexane, fill with hexaneLabel a beaker hexane, fill with hexanePlace an empty Place an empty erlenmeyererlenmeyer flask (labeled hexane) under the columnflask (labeled hexane) under the columnWith a Pasteur pipet, add hexane to the top of the column (allowWith a Pasteur pipet, add hexane to the top of the column (allow the the liquid to flow down the side of the column, taking care not to dliquid to flow down the side of the column, taking care not to disturb isturb the alumina bed)the alumina bed)Collect the hexane as it elutes from the columnCollect the hexane as it elutes from the columnSwitch to a flask (labeled Switch to a flask (labeled ferroceneferrocene) to collect ferrocene) to collect ferrocene

33-- Eluting Eluting AcetylferroceneAcetylferrocene -- TBMETBME

Label a beaker Label a beaker TBME(TBME(tt--butyl methyl ether), fill with TBMEbutyl methyl ether), fill with TBMEPlace an empty Place an empty erlenmeyererlenmeyer flask (labeled TBME) under the columnflask (labeled TBME) under the columnWith another Pasteur pipet, add TBME to the top of the columnWith another Pasteur pipet, add TBME to the top of the columnCollect the TBME as it elutes from the columnCollect the TBME as it elutes from the columnSwitch to a flask (labeled Switch to a flask (labeled acetylferroceneacetylferrocene) to collect acetylferrocene) to collect acetylferroceneIf crystals form at the tip of the column, use TMBE to rinse intIf crystals form at the tip of the column, use TMBE to rinse into the flasko the flaskAfter all the acetylferrocene has eluted, stop adding solventAfter all the acetylferrocene has eluted, stop adding solvent

ObservationsObservations

Observe and record the Observe and record the colorcolor of the solutions containing the ferrocene and of the solutions containing the ferrocene and the acetylferrocenethe acetylferrocene

Check with TA and get his/her approvalCheck with TA and get his/her approval

Ferrocene in hexane

Acetylferrocenein TBME

Waste DisposalWaste Disposal

Waste solvents are collected in the hood.Waste solvents are collected in the hood.

Waste from Columns:Waste from Columns:

If you have time at the end of the experiment, please try to dryIf you have time at the end of the experiment, please try to dry out your out your columns using low level pressure from the house air.columns using low level pressure from the house air.

Dry column contents can be tapped out of the column into a paperDry column contents can be tapped out of the column into a paper towel towel and disposed in the collection flask in the hood.and disposed in the collection flask in the hood.

All glass All glass pipetspipets will be collected in the hood for proper disposal.will be collected in the hood for proper disposal.

Good to know if you want to have a good grade:Good to know if you want to have a good grade:

Your grade will suffer if you do not submit your Your grade will suffer if you do not submit your TLC platesTLC plates. 50% of your . 50% of your grade is based upon the results of your lab work and 50% upon yograde is based upon the results of your lab work and 50% upon your ur answers to lab questions and the writeanswers to lab questions and the write--up of your report.up of your report.

Have Fun!Have Fun!