Source: Practice Quiz 2B (Key)
Difficulty: Intermediate | Prerequisites: General chemistry fundamentals, basic understanding of intermolecular forces (polarity, hydrogen bonding), familiarity with organic functional groups.
Chromatography is the family of techniques used to separate mixtures into their individual components. In organic chemistry lab, you will use it constantly to purify products and to confirm that a reaction worked. The three methods covered here, thin layer chromatography (TLC), column chromatography, and gas chromatography (GC), differ in speed, scale, and what you learn from them, but they all rely on the same principle: components partition between a stationary phase and a mobile phase at different rates. If you are comfortable with polarity and intermolecular forces, the logic of chromatography follows directly.
TLC, column chromatography, and gas chromatography all separate mixtures by exploiting how compounds distribute between a stationary phase and a mobile phase. More polar compounds stick to polar stationary phases and elute last. TLC is quick and qualitative, column chromatography lets you collect purified fractions, and GC gives precise qualitative or quantitative data for volatile mixtures.
Stationary phase
The fixed material that stays in place during the separation. In TLC and column chromatography this is typically silica gel (SiO₂) or alumina (Al₂O₃). In GC it is a high-boiling liquid coated on the inside of a capillary column. Think of it as the surface that compounds cling to.
Mobile phase (eluent)
The solvent or gas that moves through the stationary phase, carrying the mixture with it. In TLC and column chromatography, this is an organic solvent or solvent mixture (e.g. ethyl acetate/hexane). In GC, it is an inert carrier gas such as helium or nitrogen. In simple terms, this is the river that carries your compounds along.
Rf value (retention factor)
The ratio of the distance a compound travels to the distance the solvent front travels on a TLC plate. Rf = distance travelled by compound / distance travelled by solvent front. It ranges from 0 to 1. A higher Rf means the compound moved further up the plate, which means it interacts more with the mobile phase (less polar on a silica plate with a nonpolar eluent).
Elution
The process of washing a compound through a chromatographic system. Compounds that interact weakly with the stationary phase elute first.
Retention time
In GC, the time it takes for a compound to pass through the column and reach the detector. Longer retention time means stronger interaction with the stationary phase (higher boiling point, greater polarity match). Think of it as how long the column "holds on to" each compound.
TLC (thin layer chromatography)
A quick, small-scale technique where a thin coating of silica gel on a glass or plastic plate acts as the stationary phase. Primarily qualitative: you see how many components are present, estimate their polarity, and monitor reactions.
Column chromatography
A preparative technique that separates gram-scale quantities. A glass column is packed with silica gel, and the mixture is pushed through with solvent. Collected fractions can be qualitative or quantitative.
Gas chromatography (GC)
A technique for separating volatile compounds. The sample is vaporised and carried through a heated capillary column by an inert gas. A detector at the end produces a chromatogram. Primarily qualitative, but can be quantitative with proper calibration.
KMnO₄ stain (permanganate stain)
A visualisation reagent for TLC plates. Potassium permanganate is a strong oxidiser that reacts with most organic compounds, producing yellow or brown spots on a purple background. It is useful for detecting compounds that are not UV-active.
UV activity
Compounds with conjugated pi systems (such as aromatic rings or extended double-bond networks) absorb UV light and appear as dark spots under a UV lamp on a fluorescent TLC plate. Compounds without conjugation are UV-invisible and need a chemical stain.
Technique | Stationary Phase | Mobile Phase | Qualitative or Quantitative |
|---|---|---|---|
Thin Layer Chromatography (TLC) | Solid: silica gel (SiO₂) or alumina (Al₂O₃) | Liquid: organic solvent (e.g. ethyl acetate/hexane) | Qualitative |
Column Chromatography | Solid: silica gel | Liquid: organic solvent | Qualitative or quantitative |
Gas Chromatography (capillary) | Liquid: high-boiling oil (e.g. methylsilicone) coated inside column | Gas: helium or nitrogen (carrier gas) | Qualitative or quantitative |
A small spot of the mixture is applied near the bottom of the plate. The plate is placed upright in a shallow pool of solvent (the eluent).
Solvent wicks up by capillary action. Compounds travel at different rates depending on their polarity relative to the stationary and mobile phases.
On a silica plate with a relatively nonpolar eluent, nonpolar compounds travel further (higher Rf) because they interact less with the polar silica.
After the run, spots are visualised under a UV lamp (for conjugated/aromatic compounds) or with a chemical stain such as KMnO₄.
Spearmint oil contains three major components used in this quiz:
Carvone (A): contains a carbonyl (C=O) and a conjugated system. Boiling point 230 °C at 760 torr. Moderately polar. UV-active because of conjugation.
Limonene (B): a hydrocarbon with no oxygen-containing functional groups. Boiling point 178 °C at 760 torr. Least polar of the three.
Dihydrocarveol (C): contains a hydroxyl group (OH). Boiling point 240 °C at 760 torr. Most polar of the three.
On a silica TLC plate with 10% ethyl acetate/hexane as eluent, the elution order from highest Rf to lowest Rf is: B (limonene, least polar, travels furthest) > A (carvone, moderate polarity) > C (dihydrocarveol, most polar, barely moves).
The spot with Rf = 0.8 corresponds to B (limonene), the one that travelled furthest.
Of A, B, and C, only carvone (A) is UV-active. Carvone has a conjugated enone system (C=C conjugated with C=O), which absorbs UV light.
Limonene has an isolated double bond (not enough conjugation for strong UV absorption). Dihydrocarveol has no double bond conjugation either.
All three compounds can be visualised with a KMnO₄ stain because permanganate is a strong oxidiser. It reacts with double bonds (in A and B) and can oxidise the alcohol in C as well.
For 1.0 g of mixture, use roughly 10 to 20 g of silica gel (a standard ratio of 10:1 to 20:1 by mass).
Packing the column from bottom to top: place a layer of sand and glass wool at the bottom as a support. Pour in a slurry of silica gel in solvent, making sure it is uniform with no air bubbles. Top the silica with another thin layer of sand to protect the surface. Never let the silica gel go dry, as air bubbles will form and ruin the separation.
Elution order from the column (same eluent): B (limonene) first, then A (carvone), then C (dihydrocarveol) last. The most polar compound is held most strongly by the silica and elutes last.
Three instrument parameters that must be set for a GC separation:
Gas flow rate (carrier gas velocity)
Oven temperature (or temperature programme)
Type of packing material / length of column
The compound with the longest GC retention time is dihydrocarveol (C), because it has the highest boiling point (240 °C) and the strongest intermolecular forces (hydrogen bonding from the OH group).
R_f = \frac{\text{distance travelled by compound}}{\text{distance travelled by solvent front}}Rf ranges from 0 (compound stayed at the origin) to 1 (compound moved with the solvent front).
Higher Rf on a silica plate with a nonpolar eluent = less polar compound.
Silica gel to sample ratio for column chromatography: approximately 10:1 to 20:1 by mass.
Gas chromatography is the standard method for analysing the composition of essential oils, perfumes, and food flavourings. Forensic labs use GC to identify accelerants in arson investigations and drugs in biological samples. Column chromatography is used routinely in pharmaceutical labs to purify synthetic drug candidates. TLC is the quick "spot check" bench chemists use mid-reaction to see whether starting material has been consumed.
Students often think that a higher Rf means a compound is more polar. On a silica plate with a nonpolar eluent, the opposite is true: the less polar compound travels further and has a higher Rf.
Students sometimes assume every organic compound is visible under a UV lamp. Only compounds with extended conjugation (aromatic rings, conjugated dienes/enones) absorb UV. You need a chemical stain such as KMnO₄ for the rest.
Letting the silica gel dry out during column chromatography is a common lab error, not just a "minor issue." Air channels destroy the uniformity of the packing, and the separation fails. This is a point instructors test.
Students confuse elution order with boiling point. In column chromatography, elution order is governed by polarity and interaction with the stationary phase. In GC, boiling point matters more. The two techniques can give different orders for the same set of compounds.
⚠️ Know the stationary phase, mobile phase, and qualitative/quantitative classification for all three techniques cold. This is a standard table question.
⚠️ Be able to predict elution order from polarity for both column chromatography and TLC, and from boiling point for GC.
⚠️ Rf calculation and spot assignment on a TLC plate are near-certain exam questions.
⚠️ Explain why a compound is or is not UV-active (conjugation is the key). Expect a question that asks you to pick the UV-active compound from a set of structures.
⚠️ Column packing procedure (layers, slurry technique, never let it dry) is tested as a short-answer or fill-in.
⚠️ Name at least three GC parameters that affect separation (gas flow, oven temperature, column packing/length).
True or False: In TLC on silica gel, a compound with a higher Rf value is more polar than one with a lower Rf.
Fill in the blank: The mobile phase in gas chromatography is an inert ______ such as helium.
True or False: KMnO₄ stain can only visualise compounds that contain double bonds.
Fill in the blank: For column chromatography, you should use roughly ______ to ______ grams of silica gel per gram of sample.
True or False: Letting the silica gel dry out during column packing is acceptable as long as you add more solvent later.
Answers: 1. False (less polar = higher Rf on silica). 2. Gas. 3. False (it can also oxidise alcohols and other functional groups). 4. 10, 20. 5. False (air bubbles form and ruin the separation).
Q: A TLC plate run in 10% ethyl acetate/hexane shows three spots. Compound X has an Rf of 0.8, compound Y has an Rf of 0.5, and compound Z has an Rf of 0.1. Rank them from least polar to most polar.
A: X (least polar), Y, Z (most polar). On silica gel, less polar compounds travel further and have higher Rf values.
Q: You are running a silica gel column to separate carvone, limonene, and dihydrocarveol. Which compound elutes first, and why?
A: Limonene elutes first. It is the least polar compound (no oxygen-containing functional groups), so it has the weakest interaction with the polar silica stationary phase.
Q: Only one of carvone, limonene, and dihydrocarveol is visible under a UV lamp. Which one, and why?
A: Carvone. It has a conjugated enone system (C=C conjugated with C=O), which absorbs UV light. Limonene has only an isolated double bond, and dihydrocarveol has a saturated ring with an OH group.
Q: Name three GC instrument parameters you must set before analysing a mixture.
A: Gas flow rate, oven temperature, and column packing material or length.
Q: Why should you never let the silica gel dry out when packing a chromatography column?
A: Drying causes air bubbles to form in the packing. These create channels where the solvent flows unevenly, destroying the separation.
Chromatography links directly to intermolecular forces (Chapter on IMFs): the strength of dipole-dipole, hydrogen bonding, and London dispersion interactions determines how strongly each compound sticks to the stationary phase. It also connects to IR spectroscopy, because TLC fractions are often confirmed by running an IR spectrum. Stereochemistry comes in when chiral columns are used for enantiomeric separations, though that is beyond this quiz.
TLC, thin layer chromatography, column chromatography, gas chromatography, GC, capillary GC, silica gel, alumina, stationary phase, mobile phase, eluent, Rf value, retention factor, retention time, elution order, KMnO₄ stain, permanganate stain, UV visualisation, UV-active, conjugation, spearmint oil, carvone, limonene, dihydrocarveol, column packing, carrier gas, ethyl acetate hexane, separation techniques, CHEM 2311, organic chemistry lab