Thin-Layer Chromatography

andColumn

Chromatography

BackgroundBackground Chromatography:Chromatography: refers to several related techniques for refers to several related techniques for

analyzing, identifying, or separating mixtures of analyzing, identifying, or separating mixtures of compounds.compounds.

Two-part operation:Two-part operation: The sample mixture is placed into a moving liquid or The sample mixture is placed into a moving liquid or

gas: gas: mobile phasemobile phase The mobile phase carries the sample through a solid The mobile phase carries the sample through a solid

support: support: stationary phasestationary phase Compounds move through the stationary phase at Compounds move through the stationary phase at

different rates due to different attractions from the different rates due to different attractions from the mobile and stationary phasesmobile and stationary phases

Alumina and silica gel are the most Alumina and silica gel are the most

commonly used commonly used stationary phasestationary phase

(adsorbents): (adsorbents): They are extremely polar, so they have They are extremely polar, so they have

strong attractions to compoundsstrong attractions to compounds

Organic solvents with different polaritiesOrganic solvents with different polarities make themake the mobile phase (eluent): mobile phase (eluent): The more polar an eluent, the greater is The more polar an eluent, the greater is

its eluting power, that is, its ability to its eluting power, that is, its ability to move compounds through the adsorbent.move compounds through the adsorbent.

cyclohexanecyclohexane

petroleum etherpetroleum ether

hexanehexane

toluenetoluene

dichloromethanedichloromethane

ethyl acetateethyl acetate

ethanolethanol

acetoneacetone

methanolmethanol

least polarleast polar

most polarmost polar

TLC vs. Column TLC vs. Column ChromatographyChromatography

TLC vs. Column TLC vs. Column ChromatographyChromatography TLCTLC

TLC plate: a plastic plate coated with a thin layer of TLC plate: a plastic plate coated with a thin layer of silica gel.silica gel.

The silica gel (The silica gel (adsorbentadsorbent)) adsorbs the mixture adsorbs the mixture compounds strongly.compounds strongly.

As the solvent (As the solvent (eluenteluent)) travels up through the travels up through the adsorbent, the compounds in the mixture move with adsorbent, the compounds in the mixture move with the solvent.the solvent.

ColumnColumn Tube instead of a plate. Eluent travels down.Tube instead of a plate. Eluent travels down.

Adsorbent vs Eluent:Adsorbent vs Eluent: Adsorbents are typically much more polar than eluents Adsorbents are typically much more polar than eluents

The more polar a compound from the mixture is, The more polar a compound from the mixture is, the more strongly it adheres to the adsorbent and the more strongly it adheres to the adsorbent and the slower it moves.the slower it moves.

The more polar the eluent is, the less strongly a The more polar the eluent is, the less strongly a compound adheres to the adsorbent and the faster compound adheres to the adsorbent and the faster the compound movesthe compound moves.

Thin-Layer Chromatography

Spotting, Developing, and Spotting, Developing, and VisualizingVisualizing Spotting:Spotting:

Mark the origin by with a Mark the origin by with a pencilpencil (1 cm from the edge). (1 cm from the edge). Mark the spot(s) where sample(s) will be spotted.Mark the spot(s) where sample(s) will be spotted. Dissolve sample in a volatile solvent such as acetone or dichloromethane.Dissolve sample in a volatile solvent such as acetone or dichloromethane. Use a glass capillary tube to apply a Use a glass capillary tube to apply a small amountsmall amount of sample onto the of sample onto the

plate (too much sample leads to tailing).plate (too much sample leads to tailing).

Spotting, Developing, and Spotting, Developing, and VisualizingVisualizing Developing:Developing:

Place the plate in a developing chamber (beaker). Place the plate in a developing chamber (beaker). Solvent in the chamber should Solvent in the chamber should be lower than the origin on the platebe lower than the origin on the plate..

When the eluent front reaches about 1cm from the top of the plate, remove the When the eluent front reaches about 1cm from the top of the plate, remove the plate and mark the eluent front immediately. Then let it dry.plate and mark the eluent front immediately. Then let it dry.

Spotting, Developing, and Spotting, Developing, and VisualizingVisualizing Visualizing:Visualizing:

Not necessary for colored compoundsNot necessary for colored compounds UV for fluorescent compoundsUV for fluorescent compounds Iodine vapor to form a colored complex with many compounds.Iodine vapor to form a colored complex with many compounds.Note:Note: Mark the spots with a pencil. Mark the spots with a pencil.

UV Light

Iodine

Retention Factor (Rf) Definition: Definition:

distance traveled by compound, mmdistance traveled by compound, mm

RRff = = ————————————————————————————————————————

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

Eluent FrontEluent Front

SSAA11

OriginOrigin

AA11

RfRf11 = ———— = ————

SS

- As long as the stationary and mobile As long as the stationary and mobile phases and temperature are kept constant, phases and temperature are kept constant, RRff of individual compound is reproducible. of individual compound is reproducible.

- An increase in eluent polarity increases An increase in eluent polarity increases the Rthe Rff..

- Two different compounds can sometimes Two different compounds can sometimes have the same Rhave the same Rff in a given eluent, but in a given eluent, but

they are unlikely to have the same Rthey are unlikely to have the same Rff in in

another eluent of different polarityanother eluent of different polarity..

1

2

AA22

AA22

RfRf22 = ———— = ————

SS

What You Need for TLC What You Need for TLC ExperimentExperiment

6 TLC Plates6 TLC Plates 5 micropipets (one for each 5 micropipets (one for each

solution, solution, do not mix themdo not mix them!)!) 4 clean test tubes4 clean test tubes

0.5 mL of “stock solution”0.5 mL of “stock solution” 0.5 mL of benzophenone0.5 mL of benzophenone 0.5 mL of biphenyl0.5 mL of biphenyl 0.5 mL of benzhydrol0.5 mL of benzhydrol

Vial with unknownVial with unknown

O

OH

TLC ExperimentTLC Experiment

Omit Part 1.Omit Part 1. Part 2: Al foil is used instead of plastic wrap in Fig. 4.Part 2: Al foil is used instead of plastic wrap in Fig. 4. Part 4: Only ethyl acetate, hexane and toluene are used Part 4: Only ethyl acetate, hexane and toluene are used

(so only 3 plates are needed).(so only 3 plates are needed). Part 5: Use only 2 plates (ask TA how).Part 5: Use only 2 plates (ask TA how). Part 7: All solvents go into a waste bottle.Part 7: All solvents go into a waste bottle. Turn in ALL plates with your report (in an envelope or zip Turn in ALL plates with your report (in an envelope or zip

lock bag).lock bag).

Column Chromatography

1- Preparing a Dry Pack 1- Preparing a Dry Pack ColumnColumn In a short stem Pasteur pipet place: In a short stem Pasteur pipet place:

A small cotton plug at the tip (don’t make it too A small cotton plug at the tip (don’t make it too tight otherwise eluent won’t flow).tight otherwise eluent won’t flow).

Attach the column to a support stand using a Attach the column to a support stand using a piece of paper towel (piece of paper towel (make sure it is verticalmake sure it is vertical).).

Pour a little bit of sand, then the alumina powder Pour a little bit of sand, then the alumina powder (tap the side of the pipet to pack firmly).(tap the side of the pipet to pack firmly).

Add 90 mg of the already prepared mixture Add 90 mg of the already prepared mixture sample: ferrocene/acetylferrocene/alumina.sample: ferrocene/acetylferrocene/alumina.

Add a little bit more of alumina to protect the Add a little bit more of alumina to protect the surface of the sample from disruption when surface of the sample from disruption when adding the solvent.adding the solvent.

2- Eluting Ferrocene2- Eluting Ferrocene Label a beaker hexane, fill with hexaneLabel a beaker hexane, fill with hexane Place an empty beaker or erlenmeyer flask (labeled hexane) under the columnPlace an empty beaker or erlenmeyer flask (labeled hexane) under the column Add hexane to the top of the column (allow the liquid to flow down the side of Add hexane to the top of the column (allow the liquid to flow down the side of

the column, taking care not to disturb the alumina bed)the column, taking care not to disturb the alumina bed) Collect the hexane as it elutes from the columnCollect the hexane as it elutes from the column Switch to a flask (labeled ferrocene) to collect ferrocene (yellow)Switch to a flask (labeled ferrocene) to collect ferrocene (yellow)

3- Eluting Acetylferrocene3- Eluting Acetylferrocene Label a beaker TBME (Label a beaker TBME (tt-butyl methyl ether), fill with TBME-butyl methyl ether), fill with TBME Place an empty erlenmeyer flask (labeled TBME) under the columnPlace an empty erlenmeyer flask (labeled TBME) under the column Add TBME to the top of the columnAdd TBME to the top of the column Collect the TBME as it elutes from the columnCollect the TBME as it elutes from the column Switch to a flask (labled acetylferrocene) to collect acetylferrocene when it Switch to a flask (labled acetylferrocene) to collect acetylferrocene when it

comes out (orange).comes out (orange). After all the acetylferrocene has eluted, stop adding solventAfter all the acetylferrocene has eluted, stop adding solventNote: If crystals form at the tip of the column, use TMBE to rinse into the flaskNote: If crystals form at the tip of the column, use TMBE to rinse into the flask

ObservationsObservations Observe and record the color of the solutions containing the ferrocene

and the acetylferrocene Check with TA and get his approval Pour both solutions into the waste bottle

Ferrocene in hexane Acetylferrocene in TBME

ExperimentExperiment

Part 1: It’s unnecessary to weigh the flasks. Larger Part 1: It’s unnecessary to weigh the flasks. Larger Erlenmeyer flasks may be substituted for the 50 ml Erlenmeyer flasks may be substituted for the 50 ml flasks.flasks.

Part 3: “Preparing the Sample” has already been done. Part 3: “Preparing the Sample” has already been done. Weigh out 90 mg of the prepared Weigh out 90 mg of the prepared ferrocene/acetylferrocene/alumina sample.ferrocene/acetylferrocene/alumina sample.

Part 7: Omit. Only observe and record the color and Part 7: Omit. Only observe and record the color and show your TA.show your TA.

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

Column glass pipetsColumn glass pipets 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 . 50% of your grade is based upon the results of your lab work and 50% your grade is based upon the results of your lab work and 50% upon your answers to lab questions and the write-up of your report.upon your answers to lab questions and the write-up of your report.

Have fun!Have fun!