To study a protein, you usually need it pure. A cell contains thousands of different proteins; you need to pull out just one. Chromatography is the toolkit that makes this possible. Every column works the same way: a stationary phase in the column, a mobile phase (solvent) flowing through, and proteins that partition between the two based on some physical property.
Which technique separates by what
Separation
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Chromatography: separation in a column Electrophoresis: separation in a gel The property being exploited
Two-dimensional gelsRun isoelectric focusing across a strip, then SDS-PAGE down it. The first dimension sorts by pI, the second by mass, and the result is a scatter of spots where each protein has its own coordinates. It is how a whole proteome gets resolved on one gel.
Why size exclusion looks backwardsThe beads are porous. A small protein wanders into every pore and takes a long path; a large one cannot fit and takes the short route between the beads. Large elutes first. Almost every question on this topic is checking whether you know that.
Choosing oneIf the question mentions a tag, ligand, or antibody, it is affinity. If it mentions SDS or molecular weight, it is SDS-PAGE. If it mentions pH or pI, it is ion exchange or isoelectric focusing. If it mentions keeping the protein working, it is native PAGE or gel filtration.
Every one of these exploits exactly one physical property, and the technique is named after it. Work out which property the question changed and the technique names itself.
| Technique | Separates by | Who elutes first |
|-----------|-------------|-------------------|
| Ion exchange | Charge | Opposite-charge-to-column proteins stick; same-charge pass through |
| Size exclusion | Size | LARGE molecules elute first (they cannot enter the beads) |
| Affinity | Specific binding partner | Non-binders wash through; target comes off last |
| HPLC | Many properties, high resolution | Varies by column chemistry |
Ion Exchange Chromatography
The stationary phase is a resin with charged groups. Proteins with opposite charge stick to the resin; proteins with similar charge (or neutral charge) pass through.
Cation exchange column has negatively charged resin; it binds positively charged proteins.
Anion exchange column has positively charged resin; it binds negatively charged proteins.
To elute bound proteins, you raise the salt concentration (ions compete for binding) or change the pH (which changes the protein’s net charge).
Size Exclusion Chromatography (Gel Filtration)
The stationary phase is porous beads. Small proteins enter the pores and take a long, winding path. Large proteins cannot fit into the pores and flow straight through around the beads.
Counterintuitive result: large proteins elute first, small proteins elute last. Memorize this.
Affinity Chromatography
The most selective technique. The stationary phase has a specific binding partner for your protein of interest - an antibody, a substrate analog, or a tag-binding resin.
Your target protein binds tightly to the resin. Everything else washes through.
Elute the target by adding a competitor (the natural substrate, free tag, or changed pH/salt).
His-tag purification is the most common real-world example. You genetically engineer your protein to have a six-histidine tag. The His-tag binds tightly to a nickel (Ni2+)-charged resin. Wash away everything else. Add imidazole (which competes with histidine) to elute your pure, tagged protein.
High-Performance Liquid Chromatography (HPLC)
HPLC is not a different separation principle - it is a faster, higher-resolution apparatus that pushes solvent through tightly packed small-particle columns at high pressure. Any of the techniques above (ion exchange, size exclusion, affinity, reverse-phase) can be run as HPLC. Reverse-phase HPLC is common for peptides: a hydrophobic column separates by hydrophobicity, eluting with a water/acetonitrile gradient.
Combining Techniques
Real protein purification usually stacks two or three chromatography steps. A typical sequence: crude lysate → ion exchange → size exclusion → affinity. Each step enriches the target and removes different classes of contaminants.
In size exclusion chromatography, which proteins elute first and why?
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Large proteins elute first. They cannot fit into the pores of the gel beads, so they travel in the mobile phase around the beads and reach the column exit quickly. Small proteins enter the pores, taking a long, winding path inside the beads, and elute last. Students commonly guess the opposite.
How does a His-tag and Ni-NTA resin work in affinity chromatography?
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The protein of interest is genetically engineered with a polyhistidine tag. Histidine side chains coordinate Ni2+ tightly. A nickel-charged resin binds only His-tagged proteins; everything else washes through. Elution uses imidazole, which competes with histidine for Ni2+ binding and displaces the tagged protein.
If a protein has pI = 6 and you load it onto a cation exchange column (negatively charged resin) at pH 4, will it bind?
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Yes. At pH 4 (below its pI of 6), the protein has a net positive charge. The negatively charged cation exchange resin will bind it. To elute, you would either raise the pH above 6 (making the protein net negative and expelling it from the resin) or raise the salt concentration (ions compete for the resin).
