Source: Column Chromatography Lab Manual (CC.1–CC.4) · Catalyst Education / University of Tennessee, Knoxville
Difficulty: Intermediate | Prerequisites: General Chemistry II, familiarity with intermolecular forces and polarity concepts.
Column chromatography is one of the core purification techniques in organic chemistry. This lab sits at the intersection of synthesis (making a new compound via nucleophilic aromatic substitution) and separation science (isolating that compound from leftover starting material and byproducts). You need a working understanding of polarity, intermolecular forces, and how "like dissolves like" before this will click. If you can explain why water and oil do not mix, you have the conceptual foundation; this lab applies the same logic to separating coloured compounds on a silica gel column.
You react tetrachloro-1,4-benzoquinone with aqueous ammonia to swap two chlorine atoms for amino groups, producing 2,5-diamino-3,6-dichloro-1,4-benzoquinone. The crude mixture is then purified by column chromatography on silica gel, using a 4:1 petroleum ether to ethyl acetate eluent, and fractions are checked by TLC to decide which ones contain pure product.
Chromatography
A separation technique that exploits differences in how components of a mixture interact with a stationary phase versus a mobile phase. Think of it as a race: compounds that stick more to the stationary phase move slowly, while those that prefer the mobile phase move quickly.
Stationary phase
The fixed solid medium through which the mixture passes. In this experiment, silica gel packed into a pipet column. In simple terms, it is the surface that grabs onto molecules as they flow past.
Mobile phase (eluent)
The solvent that carries the mixture through the stationary phase. Here, a 4:1 mixture of petroleum ether to ethyl acetate. Think of it as the current that sweeps compounds along the column.
Polarity
A measure of how unevenly electric charge is distributed in a molecule. Polar molecules have regions of partial positive and partial negative charge; nonpolar molecules do not. This property determines how strongly a compound interacts with silica gel.
Elution
The process of washing a compound through the column and off the stationary phase using the mobile phase. Less polar compounds elute first from silica gel; more polar compounds elute later.
Rf value (retention factor)
In TLC, the distance a compound travels divided by the distance the solvent front travels. Each compound has a characteristic Rf under a given set of conditions. In simple terms, it tells you how far up the plate a spot moved, as a fraction of the total solvent distance.
Thin-layer chromatography (TLC)
A quick analytical technique using a thin layer of silica on a plate to check purity and identify compounds by their Rf values. Think of it as a rapid preview of what column chromatography will do on a larger scale.
Nucleophilic aromatic substitution
A reaction in which a nucleophile (here, ammonia, NH₃) replaces a leaving group (here, chlorine) on an aromatic ring. In simple terms, ammonia kicks out chlorine atoms and takes their place on the ring.
Percent yield
The ratio of actual product obtained to the theoretical maximum, expressed as a percentage: (actual mass / theoretical mass) × 100. It tells you how efficient your reaction and purification were.
IR spectroscopy (infrared spectroscopy)
An analytical method that measures which infrared wavelengths a compound absorbs. Different functional groups absorb at characteristic frequencies, so the spectrum acts as a fingerprint confirming what bonds are present in your product.
In vacuo
Latin for "under vacuum." Concentrating in vacuo means removing solvent using reduced pressure (a rotary evaporator), which lowers the solvent's boiling point so it evaporates faster without overheating the product.
How column chromatography works
A vertical column is packed with a stationary phase (silica gel, SiO₂).
The mixture is loaded onto the top of the column, dissolved in a small volume of solvent.
Fresh eluent (mobile phase) is continuously added to the top, pushing compounds down through the silica.
Silica gel is polar. Polar compounds interact more strongly with silica and move slowly. Nonpolar compounds interact weakly and move quickly.
The result: compounds separate into bands as they travel down the column at different rates.
Polarity and elution order on silica gel
Silica gel has surface Si–OH (silanol) groups that are strongly polar.
Nonpolar compounds elute first because they have little affinity for the silica surface.
Polar compounds elute later because they are attracted to the silanols through hydrogen bonding, dipole-dipole interactions, or both.
Increasing the proportion of a polar solvent (e.g. ethyl acetate) in the eluent speeds up the elution of polar compounds.
The eluent system in this experiment
4:1 petroleum ether to ethyl acetate.
Petroleum ether is nonpolar and is the bulk of the mobile phase.
Ethyl acetate adds moderate polarity, giving the eluent enough strength to move the target product through the column in a reasonable time.
If you used pure petroleum ether, polar compounds would barely move. If you used pure ethyl acetate, everything would rush through with poor separation.
Fractions and TLC monitoring
As liquid drips from the bottom of the column, it is collected in small portions called fractions.
Each fraction is spotted on a TLC plate alongside the starting material for comparison.
Fractions showing only one spot (one colour) contain pure product.
Fractions with multiple spots contain a mixture and are set aside.
Pure fractions are combined, and the solvent is removed in vacuo to recover the solid product.
Starting material and product
Starting material: tetrachloro-1,4-benzoquinone (chloranil). A quinone ring bearing four chlorine substituents.
Product: 2,5-diamino-3,6-dichloro-1,4-benzoquinone. Two of the four chlorines have been replaced by amino (–NH₂) groups.
Reaction mechanism overview
This is a nucleophilic aromatic substitution (SNAr).
Ammonia (NH₃) acts as the nucleophile. It attacks the electron-poor carbon bearing a chlorine on the quinone ring.
The quinone's carbonyl groups are electron-withdrawing, which makes the ring carbons electrophilic and activates the ring toward nucleophilic attack.
Chloride departs as the leaving group.
Two of the four chlorines are replaced. The substitution is selective for the 2 and 5 positions, producing a symmetric product.
Why only two chlorines are replaced
After two amino groups are installed, they donate electron density back into the ring through resonance.
This partially deactivates the ring toward further nucleophilic attack, so the remaining two chlorines at the 3 and 6 positions are retained under these mild conditions.
Reaction conditions
0.4 mmol tetrachloro-1,4-benzoquinone in 1 mL ethyl acetate.
3 drops of concentrated aqueous ammonia (ammonium hydroxide).
The vial is capped and left to sit at room temperature.
The reaction progress is visible as a colour change: the starting material is yellow, and the product is a dark reddish-purple.
Calculating percent yield
Determine the theoretical yield: moles of limiting reagent × molar mass of product.
Weigh the actual product after purification and solvent removal.
Percent yield = (actual mass / theoretical mass) × 100.
Reaction equation
Tetrachloro-1,4-benzoquinone + 2 NH₃ (aq) → 2,5-diamino-3,6-dichloro-1,4-benzoquinone + 2 HCl
The starting material has the molecular formula C₆Cl₄O₂. The product has the molecular formula C₆H₄Cl₂N₂O₂.
Percent yield formula
\text{Percent yield} = \frac{\text{actual mass of product}}{\text{theoretical mass of product}} \times 100\%Rf value formula
R_f = \frac{\text{distance travelled by compound}}{\text{distance travelled by solvent front}}Column setup (bottom to top)
Cotton plug → ~5 cm silica gel → ~0.5 cm sand → liquid eluent layer (maintain 1–2 cm above the silica at all times).
Column chromatography is the workhorse purification method in pharmaceutical labs, natural product isolation, and synthetic chemistry research. When a chemist synthesises a new drug candidate, column chromatography is typically how they separate the desired compound from side products before testing it. The same principle, scaled up and automated as HPLC (high-performance liquid chromatography), is used in quality control for everything from food additives to blood serum analysis.
Students often think a more polar eluent always gives better separation. It does not. A very polar eluent can push everything through the column too quickly, so the bands overlap rather than separate. The eluent polarity must be tuned to the specific mixture.
Students sometimes assume the compound that appears first at the bottom of the column is the product. On silica gel, the first compound to elute is the least polar one, which may well be unreacted starting material or an impurity, not the target.
A common error is thinking TLC and column chromatography are different techniques. They rely on the same principle (differential adsorption on silica). TLC is the analytical version (small scale, quick check), and column chromatography is the preparative version (larger scale, actual isolation).
Students occasionally confuse Rf with polarity directly: a high Rf does not mean "more polar." On silica gel TLC, a high Rf means the compound is less polar (it traveled farther with the solvent because it interacted less with the polar silica).
⚠️ Be prepared to explain why compounds elute in a particular order on silica gel. The answer always comes back to polarity and intermolecular forces.
⚠️ Know how to calculate percent yield from moles, molar masses, and the actual mass recovered. This is a near-guaranteed calculation question.
⚠️ Understand what an Rf value tells you and how to use TLC to confirm purity (one spot = pure, multiple spots = mixture).
⚠️ Be able to explain why the reaction replaces only two of the four chlorines. The answer involves the electron-donating effect of the newly installed amino groups deactivating the ring.
⚠️ IR spectroscopy: know which peaks confirm N–H bonds (broad absorption around 3300–3500 cm⁻¹) and C=O stretches (strong absorption near 1650–1700 cm⁻¹) in the product.
True or False: On a silica gel column, polar compounds elute before nonpolar compounds. (False – nonpolar compounds elute first.)
Fill in the blank: The stationary phase in this experiment is ______, and the mobile phase is ______. (Silica gel; 4:1 petroleum ether to ethyl acetate.)
True or False: A TLC spot with an Rf of 0.9 corresponds to a more polar compound than a spot with an Rf of 0.2, assuming both are run on the same silica gel plate with the same solvent. (False – a higher Rf on silica means less polar.)
Fill in the blank: In the synthesis, ammonia acts as a ______ and replaces ______ on the aromatic ring. (Nucleophile; chlorine atoms.)
True or False: If your TLC plate shows two spots in a fraction, that fraction contains pure product. (False – two spots means it is a mixture.)
Q: Explain why, in column chromatography on silica gel, less polar compounds elute before more polar compounds.
A: Silica gel is a polar stationary phase. Nonpolar compounds have weak intermolecular interactions with the silanol (Si–OH) groups on the silica surface, so they spend most of their time dissolved in the mobile phase and travel through the column quickly. Polar compounds form stronger hydrogen bonds and dipole-dipole interactions with the silica, causing them to adsorb more tightly and elute later.
Q: A student obtains 0.040 g of 2,5-diamino-3,6-dichloro-1,4-benzoquinone from 0.4 mmol of tetrachloro-1,4-benzoquinone. Calculate the percent yield. (Molar mass of product = 206.03 g/mol.)
A: Theoretical yield = 0.4 mmol × 206.03 g/mol = 0.0004 mol × 206.03 g/mol = 0.0824 g. Percent yield = (0.040 g / 0.0824 g) × 100 = 48.5%.
Q: Why is it important not to let the silica gel column run dry during chromatography?
A: If the silica dries out, air pockets (channels) form in the packing. When eluent is added again, solvent preferentially flows through these channels rather than uniformly through the silica. This leads to uneven elution, band broadening, and poor separation of the compounds.
Q: You run a TLC plate and observe that your product has a lower Rf than your starting material. What does this tell you about the relative polarities of the two compounds?
A: The product is more polar than the starting material. A lower Rf on silica gel TLC means the compound interacted more strongly with the polar stationary phase and did not travel as far with the solvent front.
Q: What evidence from IR spectroscopy would confirm that the amination reaction was successful?
A: The appearance of a broad N–H stretching absorption in the region of approximately 3300–3500 cm⁻¹ in the product spectrum, which is absent in the starting material spectrum, would confirm that amino groups have been introduced. The C=O stretch of the quinone near 1650–1700 cm⁻¹ should still be present in both.
This material connects directly to intermolecular forces (Gen Chem II): the entire separation logic rests on polar vs. nonpolar interactions, hydrogen bonding, and dipole-dipole forces. If those concepts are shaky, review them before tackling chromatography.
The synthesis itself is an example of nucleophilic aromatic substitution, which connects to the broader topic of aromatic reactivity covered in Organic Chemistry I/II. Compare it with electrophilic aromatic substitution (Friedel-Crafts, halogenation) to understand why the mechanism here runs in the opposite direction: electron-withdrawing groups on the ring activate it toward nucleophiles rather than electrophiles.
IR spectroscopy will appear again in virtually every synthesis lab going forward. The skill of identifying functional groups from an IR spectrum is cumulative, so start building fluency now.
Column chromatography, silica gel chromatography, gravity column, flash chromatography, stationary phase, mobile phase, eluent, elution, polarity, intermolecular forces, adsorption chromatography, thin-layer chromatography, TLC, Rf value, retention factor, nucleophilic aromatic substitution, SNAr, amination, chloranil, tetrachloro-1,4-benzoquinone, 2,5-diamino-3,6-dichloro-1,4-benzoquinone, percent yield, theoretical yield, IR spectroscopy, infrared spectroscopy, N–H stretch, C=O stretch, purification, separation techniques, organic chemistry lab, microcolumn chromatography