WK6:Purification

What is chromatography?

A physical method that separates mixtures by distributing components between a stationary phase and a mobile phase.

What is the stationary phase?

The material that stays still (e.g., silica, alumina, C18).

What is the mobile phase?

The solvent that moves through the stationary phase.

What does TLC stand for?

Thin Layer Chromatography.

What is Rf?

Distance compound moved divided by distance solvent moved.

If a compound has low Rf (0.1–0.3) on silica, is it polar or non-polar?

Polar (sticks strongly to silica).

If a compound has high Rf (0.7–0.9) on silica, is it polar or non-polar?

Non-polar (does not stick strongly).

What happens to Rf if you use a more polar solvent in normal phase TLC?

Rf increases (compounds move further).

Why is a low Rf (0.15–0.35) desirable for TLC method development?

It gives larger CV on column = better separation.

What are the three main types of chromatography?

TLC, column chromatography, HPLC.

What is column chromatography used for?

Purifying grams of material (50 mg to 5 g).

What is an advantage of column chromatography?

Cheap, easy to set up, scaleable.

What is a disadvantage of column chromatography?

Solvent hungry, labour intensive.

What is the stationary phase in normal phase chromatography?

Polar (silica or alumina).

What is the stationary phase in reverse phase chromatography?

Non-polar (C4, C8, C18).

In normal phase, which type of compound elutes first?

Non-polar compounds elute first.

In reverse phase, which type of compound elutes first?

Polar compounds elute first.

What is solvent strength (ε₀)?

A measure of how strongly a solvent competes with solutes for the stationary phase.

Name a weak solvent for normal phase chromatography.

Hexane (ε₀ = 0.01).

Name a strong solvent for normal phase chromatography.

Methanol (ε₀ = 0.95).

Calculate solvent strength for 50:50 hexane/ethyl acetate.

(0.5 × 0.01) + (0.5 × 0.58) = 0.30.

What is CV (column volume)?

The volume of solvent needed to elute a compound from a column.

What is the relationship between Rf and CV?

CV ≈ 1 ÷ Rf (low Rf = high CV).

What does HPLC stand for?

High Performance Liquid Chromatography.

What is a key difference between HPLC and column chromatography?

HPLC uses high pressure, small particles (3–10 μm), expensive.

What is retention time (tᵣ)?

Time from sample injection to when an analyte peak reaches the detector.

What is dead time (tᵢ)?

Time for the mobile phase to pass through the column (no compound).

What is band broadening?

Peaks getting wider as they travel through the column.

What are three causes of band broadening?

Eddy diffusion, longitudinal diffusion, resistance to mass transfer.

What is eddy diffusion?

Molecules take different length paths through the column.

How can eddy diffusion be minimised?

Smaller particle size, better column packing.

What is longitudinal diffusion?

Molecules diffuse from high to low concentration within the band.

How can longitudinal diffusion be minimised?

Higher flow rate (less time on column).

What is resistance to mass transfer?

Time taken for molecules to equilibrate between mobile and stationary phase.

How can resistance to mass transfer be minimised?

Lower flow rate (more time to equilibrate).

What is the elution problem?

Weak and strong compounds cannot be separated well with constant solvent strength.

What is isocratic elution?

Constant solvent strength throughout the run.

What is gradient elution?

Solvent strength increases gradually during the run.

Which is better for separating a mixture of weak and strong compounds?

Gradient elution.

What is an advantage of HPLC?

Very sensitive, quantitative, reproducible.

What is a disadvantage of HPLC?

Expensive, needs expertise, fragile columns.

What is an advantage of TLC?

Cheap, simple, fast.

What is a disadvantage of TLC?

Qualitative only, less reproducible.