Principles of Separation
Every separation technique exploits a property difference between the components of a mixture. If the components have identical properties, they cannot be separated. The more differences you can exploit (or the larger the differences), the easier the separation.
Key Properties Exploited
| Property | Technique |
|---|---|
| Boiling point | Distillation |
| Polarity / solubility in water vs. organic | Extraction |
| Acid-base behavior | Acid-base extraction |
| Solubility at different temperatures | Recrystallization |
| Affinity for silica, alumina, reverse-phase | TLC, column chromatography |
| Volatility / gas-phase behavior | Gas chromatography |
| Polarity in a high-pressure mobile phase | HPLC |
| Size | Gel filtration, SDS-PAGE |
| Charge (for proteins) | Ion exchange, native gel |
Choosing the right technique means matching the biggest property difference in the mixture to the technique that exploits it.
Partition Coefficient
A key concept for liquid-liquid separations is the partition coefficient (K):
K = [A]_organic / [A]_aqueous
A high K means the molecule prefers the organic layer; a low K means it prefers the aqueous layer. By choosing appropriate solvents, you can drive a compound into one layer with high selectivity.
The partition coefficient depends on polarity, charge, and H-bonding ability. Neutral nonpolar compounds → high K (organic). Charged or highly polar compounds → low K (aqueous).
Orthogonal Separations
Complex mixtures often need TWO or more separation techniques applied in sequence, each exploiting a different property. This is called an orthogonal separation strategy:
- 2D-PAGE: separates proteins by isoelectric point (1st dimension) + molecular weight (2nd dimension).
- LC-MS: liquid chromatography separates by polarity, then mass spectrometry separates by mass.
- GC-MS: gas chromatography separates by volatility, then MS identifies by mass.
Orthogonal strategies resolve mixtures that a single technique cannot handle.
Efficiency Metrics
- Resolution (R): how well two adjacent peaks are separated. High R = baseline separation.
- Selectivity (α): the ratio of retention times of two components. Higher α = better inherent separation.
- Plate number (N): how “sharp” the peaks are. More plates = narrower peaks = better resolution.
For MCAT purposes, focus on qualitative concepts: what makes a separation good, what makes it fail, and how to improve it.
General Rules
- Like dissolves like: polar dissolves polar, nonpolar dissolves nonpolar. Basis of extraction and chromatography.
- Lower boiling point distills first: in simple distillation, the more volatile component is collected first.
- Smaller particles move faster in gel: size-exclusion and SDS-PAGE separate by size.
- Charged species move in electric fields: ion exchange and electrophoresis.