Source: Column Chromatography Lab Manual (CC.1–CC.4) · Catalyst Education / University of Tennessee, Knoxville
Difficulty: Intermediate | Prerequisites: Part 1 study notes (chromatography theory, polarity, Rf values). Hands-on familiarity with basic lab glassware is helpful.
This document covers the practical, hands-on side of the column chromatography experiment. Where Part 1 explained why compounds separate and what the reaction produces, this part walks through how you actually set up the reaction, build and run the column, analyse your fractions, and confirm product identity. These are transferable lab skills: packing a column, running TLC, and interpreting IR spectra will come up repeatedly throughout organic chemistry coursework.
You dissolve chloranil in ethyl acetate, add ammonia, and let the reaction sit while you pack a silica gel microcolumn in a disposable pipet. Once the reaction finishes, you dilute the crude mixture with petroleum ether, load it onto the column, collect small fractions, check them by TLC, combine the pure ones, evaporate the solvent, and weigh the product for yield and IR analysis.
Microcolumn
A small-scale chromatography column built inside a disposable glass pipet. It uses less solvent and silica than a full-sized column, which makes it practical for small quantities of material.
Fraction
A small portion of eluent collected from the bottom of the column. Each fraction may contain different compounds or mixtures, depending on when it was collected during the separation.
Scintillation vial
A small glass vial (20 mL in this experiment) used as a reaction vessel. Named for its original use in scintillation counting; in teaching labs it serves as a convenient, cappable container.
Pasteur pipet (pasteur pipette)
A disposable glass pipette used for transferring small volumes of liquid. In this experiment, it doubles as the column body itself.
Cotton plug
A small wad of cotton pushed into the narrow end of the pipet to prevent the silica gel from falling through. It acts as a frit, holding the packing in place while allowing liquid to pass.
Sand layer
A thin (~0.5 cm) layer of sand placed on top of the silica gel. It protects the flat, even surface of the silica from being disturbed when you add the sample or more eluent.
Crude reaction mixture
The raw product of a reaction before purification. It contains the desired product along with unreacted starting material, byproducts, and solvent.
Concentrate in vacuo
To remove solvent under reduced pressure, typically using a rotary evaporator. The low pressure lowers the boiling point so the solvent evaporates quickly at mild temperatures, leaving the solid product behind.
Steps
Weigh out 0.4 mmol of tetrachloro-1,4-benzoquinone (chloranil) into a 20 mL scintillation vial.
Add 1 mL of ethyl acetate to dissolve the solid. Swirl gently.
Carefully add 3 drops of concentrated aqueous ammonia solution (ammonium hydroxide).
Cap the vial immediately and allow it to sit while you prepare the column (Part B).
Check the colour every few minutes. The mixture should gradually shift from yellow (starting material) toward a dark reddish-purple (product forming).
Tips and things to watch
The ammonia is concentrated and corrosive. Add it in a fume hood and avoid inhaling the vapour.
Capping the vial matters: ammonia is volatile, and an open vial loses reagent to evaporation, reducing your yield.
The colour change is your qualitative reaction monitor. If no colour change occurs after several minutes, the ammonia may not have been added properly.
Steps
Take a disposable glass Pasteur pipet. Push a small wad of cotton gently into the narrow tip using a piece of wire. The cotton should be snug but not compressed so tightly that liquid cannot pass through.
Add approximately 5 cm of silica gel on top of the cotton plug.
Clamp the pipet vertically to a ring stand inside the fume hood. Position it high enough that you can place a test tube beneath the tip to catch the eluent.
Add approximately 0.5 cm of sand on top of the silica gel. The sand protects the silica surface from being disturbed when you load the sample.
Add 4:1 petroleum ether to ethyl acetate (the chromatography solvent) until the silica is fully wetted. There should be 1 to 2 cm of liquid sitting above the top of the silica at all times.
Keep the silica wet from this point forward. One partner should continuously re-wet the column while the other finishes Part A.
Tips and things to watch
If the cotton plug is too loose, silica will leak out the bottom. If it is too tight, flow rate drops to nothing. Aim for a gentle resistance when you push it in.
Never let the silica go dry once it has been wetted. Drying creates cracks and air channels that ruin the separation.
The column should be perfectly vertical. A tilted column gives uneven band migration and poor separation.
Pack the silica evenly. Tapping the pipet gently can help settle the silica, but avoid vigorous shaking, which can create layers of different density.
Loading the sample
Using a Pasteur pipet, add approximately 1 mL of petroleum ether to your crude reaction mixture in the scintillation vial. Swirl to mix.
Wait until the liquid level in the column drops to the top of the sand layer.
Carefully pipette approximately 0.5 mL of the diluted reaction mixture onto the sand. Do not disturb the sand surface.
Let this drain until the liquid level is again even with the sand.
Gently add chromatography solvent (4:1 petroleum ether to ethyl acetate) to fill the column. Avoid splashing, which disturbs the bands.
Collecting fractions
Place a test tube beneath the column to collect the eluent.
Once you have collected approximately 0.5 cm of liquid in the tube, swap it for the next empty test tube.
Continue collecting 0.5 cm fractions until there is no colour visible in the liquid coming through, or until you have collected 10 fractions.
Label each tube in order (1, 2, 3 ...) so you know the sequence.
Add more eluent to the top of the column as needed. Never let the liquid level drop below the sand.
Critical rule: do not let the column run dry. If the silica dries out, air enters the packing, and the separation is compromised beyond recovery. Keep eluent flowing.
Deciding which fractions to spot
5 tubes or fewer: spot every fraction.
6 to 10 tubes: spot every other fraction.
Use the same 4:1 petroleum ether to ethyl acetate solvent as the mobile phase for TLC.
Running the TLC plate
Draw a faint pencil line near the bottom of the plate (the origin).
Spot a small amount of each selected fraction on the origin, spaced evenly.
Also spot the pure starting material as a reference, if available.
Develop the plate in a TLC chamber containing the chromatography solvent.
Mark the solvent front immediately when you remove the plate.
Reading the results
Fractions showing a single spot of one colour contain pure product.
Fractions with two or more spots contain a mixture of product and impurity (or starting material).
Compare Rf values to distinguish product from starting material. The product (more polar, with –NH₂ groups) will have a lower Rf on silica than the starting material.
Combine the fractions that contain only the pure product spot.
Recovering the product
Combine the pure fractions (those with a single TLC spot matching the product) into one flask.
Remove the solvent by concentrating in vacuo (rotary evaporator under reduced pressure).
Weigh the dried solid to get your actual product mass.
Calculate percent yield using the formula from Part 1.
IR analysis
Obtain an IR spectrum of your purified product.
Key absorptions to look for:
N–H stretching: a broad band around 3300–3500 cm⁻¹. This confirms the amino groups are present and the substitution worked.
C=O stretching: a strong absorption near 1650–1700 cm⁻¹. This confirms the quinone carbonyls are intact.
C–Cl stretching: absorptions in the 500–800 cm⁻¹ region. Two chlorines remain on the product.
Compare your product spectrum with the starting material spectrum. The key difference is the appearance of the N–H bands, which are absent in chloranil.
Tetrachloro-1,4-benzoquinone (chloranil): Causes skin irritation. Very toxic to aquatic life. Wear gloves; avoid skin contact.
2,5-Diamino-3,6-dichloro-1,4-benzoquinone (product): Toxic if swallowed. Very toxic to aquatic life.
Concentrated aqueous ammonia (ammonium hydroxide): Causes severe skin burns and eye damage. Harmful if inhaled or swallowed. May corrode metals. Always handle in a fume hood with gloves and eye protection.
Ethyl acetate: Extremely flammable liquid and vapour. Harmful if swallowed. Keep away from ignition sources.
Petroleum ether: Extremely flammable liquid and vapour. Harmful if swallowed. Use only in the fume hood.
Silica gel: Harmful if swallowed. Avoid inhaling the fine dust when pouring.
All work with these chemicals should be carried out in the fume hood wearing appropriate personal protective equipment (lab coat, safety goggles, nitrile gloves).
Students often think you can simply pour the crude mixture directly onto the column without diluting it first. You cannot. Adding petroleum ether to the crude mixture before loading ensures the sample is in a low-polarity solvent, which helps it adsorb onto the top of the column as a tight, narrow band rather than a wide streak.
A frequent mistake is loading too much sample at once. Overloading the column causes the bands to overlap no matter how well you run it. In this experiment, you load only about 0.5 mL of the diluted mixture.
Some students think that if the column dries for just a moment, you can simply re-wet it and carry on. Once air channels form in the silica, the packing is compromised. There is no reliable fix; prevention is the only strategy.
Students sometimes discard fractions that look colourless, assuming they contain nothing useful. Colourless fractions may still contain impurities or small amounts of product. TLC is the tool that tells you what is actually in each fraction, not your eyes alone.
⚠️ Know the order of column assembly from bottom to top: cotton plug, silica gel, sand, liquid. This is a common short-answer question.
⚠️ Be able to explain why the column must not run dry and what happens to separation quality if it does.
⚠️ Understand why you dilute the crude mixture with petroleum ether before loading it onto the column.
⚠️ Know how to decide which fractions to combine based on TLC results: only fractions showing a single spot at the product's Rf.
⚠️ The post-lab report will ask you to draw your TLC plate results, calculate theoretical and percent yield, and discuss your IR spectrum. Practise these before the report is due.
True or False: The sand layer in the column goes below the silica gel. (False – it goes on top of the silica gel.)
Fill in the blank: Before loading the sample onto the column, you dilute the crude mixture with ______ to ensure it adsorbs as a tight band. (Petroleum ether.)
True or False: You should collect all your fractions in one large flask for efficiency. (False – fractions must be collected in separate, labelled tubes to allow individual analysis.)
Fill in the blank: On the TLC plate, fractions containing pure product show ______ spot(s). (One.)
True or False: The N–H stretch in the IR spectrum of the product appears near 1700 cm⁻¹. (False – the N–H stretch is a broad band around 3300–3500 cm⁻¹. The 1700 cm⁻¹ region corresponds to C=O.)
Q: Describe the correct order of materials in a packed microcolumn, from the bottom of the pipet upward, and explain the purpose of each layer.
A: From bottom to top: (1) a cotton plug, which acts as a frit to hold the silica in place while allowing liquid through; (2) approximately 5 cm of silica gel, which is the stationary phase that performs the separation; (3) approximately 0.5 cm of sand, which protects the flat surface of the silica from being disturbed when sample or eluent is added; (4) a layer of eluent (1–2 cm of liquid above the silica), which keeps the column wet and maintains uniform packing.
Q: A student loaded the crude reaction mixture directly onto the column without diluting it with petroleum ether. What problem is likely to result, and why?
A: The crude mixture in ethyl acetate is relatively polar. When a polar sample solution is applied to the column, the compounds do not adsorb tightly onto the top of the silica; instead they begin to spread immediately. This produces broad, overlapping bands and poor separation. Diluting with petroleum ether (a nonpolar solvent) ensures the compounds adsorb as a tight, narrow band at the top of the column before elution begins.
Q: After running TLC on your collected fractions, you find that fractions 3 and 4 each show a single spot at the same Rf, and fraction 5 shows two spots. What should you do?
A: Combine fractions 3 and 4, which contain pure product. Do not include fraction 5, as its two spots indicate it contains a mixture of product and another compound. Concentrate the combined pure fractions in vacuo, weigh the solid, and proceed to IR analysis.
Q: Explain how you would use IR spectroscopy to confirm that your purified product is 2,5-diamino-3,6-dichloro-1,4-benzoquinone rather than unreacted starting material.
A: The product contains N–H bonds from its amino groups, which produce a broad absorption band around 3300–3500 cm⁻¹ that is entirely absent in the starting material (tetrachloro-1,4-benzoquinone has no nitrogen). Both compounds show C=O stretching near 1650–1700 cm⁻¹ from the quinone carbonyls. The presence of the N–H band and the absence of additional C–Cl absorptions (the product has two fewer C–Cl bonds) together confirm the identity.
The column packing and fraction collection skills here transfer directly to flash chromatography, which is the same technique run under air pressure for faster flow. Most research labs use flash columns rather than gravity columns, so this experiment is your introduction to the workflow.
TLC monitoring connects to every synthesis experiment you will do in organic chemistry. You will use TLC to track reaction progress (spotting reaction aliquots over time) and to check purity of isolated products.
IR spectroscopy analysis builds on the electromagnetic radiation and molecular vibration topics from physical chemistry or spectroscopy units. The ability to assign peaks to functional groups is tested in nearly every organic chemistry exam.
Column chromatography procedure, packing a column, microcolumn, gravity column, silica gel column, cotton plug, sand layer, loading the sample, collecting fractions, fraction analysis, TLC spotting, TLC plate, developing a TLC plate, Rf comparison, combining fractions, concentrating in vacuo, rotary evaporator, rotovap, IR spectrum interpretation, N–H stretch, C=O stretch, post-lab report, percent yield calculation, organic chemistry lab technique, purification procedure, column drying, band broadening, overloading a column, waste disposal organic lab