The workhorse purification mode for synthetic peptides — optimized for selectivity, yield, and documentation (GLP / ISO 13485 aligned, non-GMP)
Reversed-phase chromatography separates peptides primarily by hydrophobicity. We tune selectivity using stationary phase choice, gradient design, temperature, and mobile phase conditions to isolate target peptide from near-neighbor impurities.
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When to use How we optimize Typical workflow Deliverables Related pages
When RP-HPLC is the right choice
Best fit
- Most synthetic peptides (linear or moderately modified)
- Truncations and deletion products
- Hydrophobicity-shifted byproducts
- High-purity fraction isolation at mg–g scale (project dependent)
When to add an orthogonal step
- Co-elution or partial co-elution of near-neighbor impurities
- Highly polar peptides with weak RP retention
- Charge variants that look identical in RP
In these cases, we often pair RP-HPLC with IEX or HILIC.
How we optimize selectivity
Stationary phase
C18/C8 (and other chemistries when needed) to change retention and resolution.
Gradient design
Gradient slope and holds are tuned to separate target from critical impurities.
Temperature & additives
Temperature and mobile phase conditions can materially improve peak shape and separation.
Typical RP-HPLC purification workflow
- Screen: short analytical runs to identify conditions that resolve target vs impurities
- Prep run: scaled purification with fraction tracking and defined collection criteria
- Verify: analytical HPLC + MS identity verification (as scoped)
- Finalize: pool selection, solvent handling notes, and reporting
Deliverables
- Analytical HPLC chromatograms + purity assessment
- Mass spectrometry identity verification (project-appropriate)
- COA + supporting data package
Related purification pages