Chromatography
High-Yield Summary
- Chromatography separates compounds via a stationary phase (fixed, typically polar solid) and a mobile phase (liquid or gas flowing through/over it) — the more a compound resembles/interacts with a phase, the more it sticks to it (partitioning) and the slower it moves.
- TLC/paper chromatography: polar stationary phase (silica/alumina/cellulose), nonpolar mobile solvent, movement driven by capillary action. More nonpolar compounds travel further (prefer mobile phase).
- Rf value = distance traveled by compound ÷ distance traveled by solvent front; unitless, 0 to 1. Lower Rf = more stationary-phase interaction.
- Reverse-phase chromatography flips the setup: nonpolar stationary phase, polar mobile phase — polar compounds move faster, nonpolar compounds stick.
- Column chromatography variants target different properties: ion-exchange (charge), size-exclusion (size — larger molecules elute FIRST), affinity (specific binding). Gas chromatography separates volatile compounds via an inert gas mobile phase; HPLC uses high pressure for faster, higher-resolution liquid separation.
Rf Value
Rf = distance traveled by compound ÷ distance traveled by solvent front
- Rf = Retardation factor — unitless, always between 0 and 1
- Lower Rf → compound interacted more with the (polar) stationary phase.
- Higher Rf → compound preferred the (nonpolar) mobile phase, traveled further.
Column Chromatography Variants
| Type | Separates by / elution order |
|---|---|
| Ion-exchange | Charge — beads coated with charged groups retain opposite-charge compounds longer |
| Size-exclusion | Size — larger molecules elute FIRST (too big for pores); smaller molecules trapped, elute later |
| Affinity | Specific binding (antibodies/receptors/ligands) — target binds tightly, released later by elution solution |
Key Terms
- Partitioning
- A compound distributing itself between stationary and mobile phases based on polarity, size, charge, or other interactions.
- Capillary action
- Natural tendency of liquid to climb through the pores of a material — drives solvent movement in TLC/paper chromatography.
- Origin
- The spot near the bottom of a TLC plate/paper where the sample is applied.
- Developing
- Removing the plate/paper once the solvent front has traveled and marking that front.
Common MCAT Trap
- Size-exclusion chromatography elutes LARGER molecules first, not last — they're too big to enter the pores and pass around them, while smaller molecules get trapped and slowed down. This is the opposite of most students' first instinct.
- Don't assume the stationary phase is always polar — reverse-phase chromatography deliberately flips this (nonpolar stationary, polar mobile), reversing which compounds move fast vs. slow.
- Rf value depends on the specific solvent system used — the same compound can have a different Rf in a different mobile phase, so Rf values are only comparable within the same experimental conditions.
Quick Recall
A compound travels 3 cm while the solvent front travels 6 cm. What is its Rf value?
In size-exclusion chromatography, do larger or smaller molecules elute first?
What's the key difference between standard column chromatography and HPLC?