Difficulty: Introductory
Prerequisites: Basic understanding of molecular polarity, intermolecular forces, and solution chemistry.
Column chromatography is one of the most widely used separation techniques in chemistry. It exploits differences in how strongly individual compounds interact with a stationary phase versus a mobile phase (the solvent flowing through the column). In this lab, you separated the red and blue food dyes in grape Kool-Aid using a Sep-Pak C18 cartridge and solvents of varying polarity. Understanding this technique is foundational for analytical chemistry, biochemistry, and any discipline where mixtures need to be broken into their components.
Grape Kool-Aid contains two dyes, Red 40 and Blue 1, which differ in polarity. A non-polar C18 column retains non-polar compounds more strongly, so by increasing the non-polarity of the solvent stepwise (5% then 25% isopropyl alcohol), you wash off the more polar dye first (Red 40) and the less polar dye second (Blue 1). Matching the separated fractions to known standards confirms their identity.
Column chromatography
A separation technique in which a mixture is dissolved in a mobile phase and passed through a column packed with a stationary phase. Different components travel at different rates depending on their affinity for each phase, causing them to separate.
In simple terms, this means you pour a mixture through a packed tube, and the ingredients come out at different times because some stick to the packing more than others.
Stationary phase
The fixed material inside the chromatography column that interacts with the components of the mixture. In this lab, the stationary phase is the C18 (octadecyl) packing of the Sep-Pak cartridge, which is non-polar.
Think of it as the surface the molecules can grab onto as solvent flows past.
Mobile phase (eluent)
The solvent or solvent mixture that carries the sample through the column. Changing the mobile phase composition changes which compounds are washed off the column.
Think of it as the moving stream that sweeps molecules along; a stronger eluent pries more stubborn molecules off the stationary phase.
Elution
The process of washing a compound off the stationary phase and out of the column using the mobile phase. A compound "elutes" when the mobile phase is strong enough to overcome its attraction to the stationary phase.
In simple terms, elution is the moment a dye lets go of the column packing and flows out with the solvent.
Sep-Pak C18 cartridge
A pre-packed, disposable chromatography column containing octadecylsilane (C18) bonded silica. The long hydrocarbon chains make the stationary phase very non-polar.
Think of it as a tiny column lined with waxy, non-polar chains that grab onto non-polar molecules.
Polarity
A measure of how unevenly electric charge is distributed in a molecule. Polar molecules have regions of partial positive and partial negative charge; non-polar molecules do not.
In simple terms, polar means the molecule has a lopsided charge distribution, which makes it attracted to water and other polar solvents.
"Like dissolves like"
The principle that polar solvents dissolve polar solutes, and non-polar solvents dissolve non-polar solutes. In chromatography, a more non-polar eluent will more effectively wash non-polar compounds off a non-polar stationary phase.
Think of it as: molecules prefer the company of molecules that share their polarity.
A mixture is loaded onto the top of a column packed with a stationary phase.
A mobile phase (solvent) is passed through the column.
Components that interact strongly with the stationary phase move slowly; components that prefer the mobile phase move quickly.
By collecting the liquid that exits the column in separate fractions, you isolate individual components.
The C18 stationary phase is very non-polar (long hydrocarbon chains bonded to silica).
Non-polar molecules are attracted to the C18 packing and stick to it.
Polar molecules prefer the polar mobile phase (water or dilute alcohol) and wash through earlier.
This is an example of reverse-phase chromatography: the stationary phase is non-polar, and separation is driven by increasing the non-polarity of the eluent.
Grape Kool-Aid contains two dyes: Red 40 (more polar) and Blue 1 (less polar).
Both dyes adsorb onto the non-polar C18 column when the sample is loaded.
Step 1, water wash: Pure water removes sugars, acids, and flavourings but not the dyes, which are held by the column.
Step 2, 5% isopropyl alcohol: This slightly non-polar eluent is strong enough to wash off Red 40 (the more polar dye) because Red 40 has less affinity for the non-polar stationary phase.
Step 3, 25% isopropyl alcohol: This more non-polar eluent is needed to dislodge Blue 1, which clings to the C18 packing more tightly due to its greater non-polarity.
Red 40 is more polar, so it is less attracted to the non-polar C18 stationary phase.
A mildly non-polar solvent (5% isopropyl alcohol) is enough to pull it away.
Blue 1 is less polar, so it binds the non-polar column more strongly and requires a more non-polar solvent (25% isopropyl alcohol) to elute.
This is the "like dissolves like" principle in action: non-polar Blue 1 needs a more non-polar solvent to be drawn off a non-polar surface.
If you ran 25% isopropyl alcohol first, Blue 1 would elute before Red 40.
The 25% solution is non-polar enough to pull Blue 1 off immediately.
Following with 5% isopropyl alcohol would then wash off Red 40.
The dyes would still separate; only the order of collection would reverse.
No quantitative formulas are required for this lab. The key conceptual diagram to keep in mind:
Picture a vertical tube packed with non-polar C18 material.
The mixture (grape Kool-Aid) sits at the top.
Solvent flows downward through the column.
The more polar dye (Red 40) travels faster because it has less attraction to the non-polar packing.
The less polar dye (Blue 1) lags behind, held by the stationary phase until a stronger (more non-polar) solvent is introduced.
Fractions are collected at the bottom in separate wells.
The separation factor depends on the relative polarity of the analyte, the stationary phase, and the mobile phase. No numerical calculation is tested, but you should be able to predict elution order from polarity alone.
Column chromatography is the workhorse of pharmaceutical purification: drug compounds are separated from reaction by-products using exactly this principle. Environmental chemists use similar C18 cartridges to extract trace pollutants from water samples before analysis.
Students often think "polar molecules stick to the C18 column." They do not. The C18 packing is non-polar, so non-polar molecules are the ones that stick. Polar molecules wash through with polar solvents.
Students sometimes assume that a stronger solvent means a more polar solvent. In reverse-phase chromatography, "stronger" means more non-polar (higher organic content), because that is what pulls non-polar analytes off the non-polar column.
Students may confuse normal-phase and reverse-phase chromatography. In normal-phase, the stationary phase is polar (e.g. silica) and you increase polarity to elute. In reverse-phase (this lab), the stationary phase is non-polar and you increase non-polarity to elute.
Students occasionally think the dyes are destroyed or chemically changed during separation. Chromatography is a physical separation; the dyes remain chemically intact.
You will almost certainly be asked to predict elution order given polarities of two or more analytes on a non-polar column. Know how to reason from "like dissolves like."
Expect questions that reverse the solvent order or change the column type (from C18 to plain silica) and ask you to predict what happens.
Be ready to explain why a clear fraction (like Well B in the lab) shows no significant absorbance peaks: it contains non-dye components (sugars, acids) that do not absorb in the visible range.
True or false: On a C18 column, the most polar compound elutes last. (False. The most polar compound elutes first because it has the least affinity for the non-polar stationary phase.)
Fill in the blank: Grape Kool-Aid contains two dyes, ______ and ______. (Red 40 and Blue 1.)
True or false: Increasing the percentage of isopropyl alcohol in the eluent makes it more polar. (False. Isopropyl alcohol is less polar than water, so increasing its proportion makes the eluent more non-polar.)
Fill in the blank: The principle that polar solvents dissolve polar solutes is called ______. ("Like dissolves like.")
True or false: If the Blue 2 Standard shows negative absorbance values, the data is likely due to instrument miscalibration. (True.)
Q: How many dyes are present in grape Kool-Aid, and what are they?
A: Two dyes are present: Red 40 and Blue 1.
Q: Which dye in grape Kool-Aid is more polar, and how can you determine this from the chromatography experiment?
A: Red 40 is more polar. It elutes first with a weaker (more polar) solvent (5% isopropyl alcohol), which indicates it has less affinity for the non-polar C18 stationary phase. Blue 1 requires a stronger (more non-polar) solvent (25% isopropyl alcohol) to elute, showing it interacts more with the non-polar column.
Q: Explain the principle of "like dissolves like" in the context of this experiment.
A: "Like dissolves like" means polar substances are more soluble in polar solvents and non-polar substances are more soluble in non-polar solvents. In this experiment, the non-polar Blue 1 dye is attracted to the non-polar C18 column and requires a more non-polar eluent to be washed off. The more polar Red 40 dye prefers the polar mobile phase and elutes with a less non-polar solvent.
Q: If you reversed the solvent order (25% isopropyl alcohol first, then 5%), predict what would happen.
A: Blue 1 would elute first because the 25% isopropyl alcohol is non-polar enough to dislodge it from the column immediately. Red 40 would elute second when the 5% isopropyl alcohol is passed through. The dyes would still separate cleanly; only the collection order would reverse.
Q: Why does the clear fraction (Well B) show no significant absorbance peaks in the visible spectrum?
A: The clear fraction contains non-dye components of Kool-Aid (sugars, citric acid, flavourings) that do not absorb light in the visible wavelength range (roughly 400 to 700 nm). Only the coloured dyes produce measurable absorbance peaks in that region.
This material connects directly to intermolecular forces (Chapter coverage of dipole-dipole, London dispersion, and hydrogen bonding), because the strength of those forces determines how strongly a dye interacts with the stationary phase. It also links to solution chemistry and solubility rules: "like dissolves like" is the same principle that governs why oil and water do not mix. If your course covers HPLC (high-performance liquid chromatography) later, that technique is simply a high-pressure, high-resolution version of exactly what this lab demonstrates.
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