HPLC Purification Process — Technical Description

1. Principle

Reverse-phase high-performance liquid chromatography (RP-HPLC) separates peptides based on hydrophobic interactions between the peptide and the non-polar stationary phase. Peptides are eluted using a gradient of increasing organic solvent concentration.

2. Mobile Phase Systems

System	Aqueous Phase	Organic Phase	Modifier	Application
TFA system	0.1% TFA in H₂O	0.1% TFA in ACN	TFA	Standard peptides, good resolution
NH₄HCO₃ system	20 mM NH₄HCO₃	20 mM NH₄HCO₃ in 80% ACN	NH₄HCO₃	Mass-spec compatible

3. Gradient Design Parameters

Peptide Length	Gradient Slope	Typical Time	Comments
5 – 15 aa	1% ACN/min	20 – 30 min	Rapid separation
15 – 30 aa	0.5% ACN/min	30 – 50 min	Standard gradient
30 – 50 aa	0.3% ACN/min	40 – 60 min	Slow gradient for resolution
>50 aa	0.2% ACN/min	60 – 90 min	Long peptides, difficult

4. Column Selection

Parameter	Standard	Long Peptides	Hydrophobic Peptides
Stationary Phase	C18	C8 or C4	C18
Particle Size (µm)	10	10 – 15	10
Pore Size (Å)	100	300	100
Column Length (mm)	250	250	250

5. Fraction Collection Criteria

Purity Grade	Collection Threshold	Typical Yield
≥95%	Peak apex ±0.5 min	40 – 60%
≥98%	Peak apex ±0.3 min	25 – 45%
≥99%	Peak apex ±0.15 min	15 – 30%