Welcome to the first chapter of your Certificate Course on HPLC. Before we touch the instrument, we must understand the science behind it. In this chapter, we will cover the history of chromatography, the fundamental principles of separation, and the evolution from simple liquid chromatography to modern UHPLC.
Definition: Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (Stationary Phase) while the other moves in a definite direction (Mobile Phase).
The term “Chromatography” comes from the Greek words chroma (color) and graphein (to write). It was coined by the Russian botanist Mikhail Tswett in 1903.
Mikhail Tswett separated plant pigments (chlorophylls) using a glass column filled with calcium carbonate (chalk) and ethanol. He observed colored bands separating by gravity.
Fig 1: Tswett’s Gravity Column
Gravity was too slow. Scientists realized that pushing the solvent with High Pressure forced the separation to happen faster and allowed for smaller packing particles, which improved resolution.
How does the instrument actually separate compounds? It relies on the competition between the sample loving the Mobile Phase vs. the Stationary Phase.
In Reversed Phase Chromatography (Partition), if a compound really “likes” the stationary phase (C18 column), what happens to its retention time?
✗ Incorrect. If it likes the stationary phase, it will want to stay there, not move quickly.
✓ Correct! High affinity for the stationary phase means the compound “hangs out” in the column longer, resulting in a later peak.
The industry has moved towards smaller particles for better resolution. Smaller particles mean we can pack more “plates” (efficiency) into the column, but it creates much higher backpressure.
Standard HPLC
(5 µm)
UHPLC
(< 2 µm)
| Parameter | Traditional LC | HPLC | UHPLC (Ultra-High) |
|---|---|---|---|
| Particle Size | > 50 µm | 3 µm – 5 µm | < 2 µm |
| Pressure Limit | Gravity / Low | ~400 bar (6000 psi) | ~1000+ bar (15000 psi) |
| Run Time | Hours | 20 – 45 mins | 3 – 10 mins |
Important Note: UHPLC gives sharper peaks and faster results, but it requires much cleaner samples and solvents because the tiny tubing blocks easily!
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