Peptides, short chains of amino acids, are central to biochemistry, drug discovery and diagnostics. Most research peptides are made by solid-phase peptide synthesis (SPPS). This guide explains how SPPS works, which reagents and equipment it needs, how peptides are purified and checked, and what online peptide calculators do.
This is a laboratory chemistry guide. It is not about so-called “research peptides” sold online for personal use, which are unregulated, often mislabelled and not intended for human consumption.
Solid-phase peptide synthesis in brief
SPPS was invented by Robert Bruce Merrifield, who received the 1984 Nobel Prize in Chemistry for it. The peptide is built one amino acid at a time while attached to tiny insoluble resin beads. Excess reagents are simply washed away after each step, which makes the process fast and easy to automate.
Key ingredients
- Resin: the solid support, chosen to give a free acid or amide at the C-terminus.
- Protected amino acids: the amine is temporarily protected, usually with Fmoc (removed by a mild base) or, in older methods, Boc (removed by acid). Reactive side chains carry permanent protecting groups.
- Coupling reagents: carbodiimides with additives such as HOBt or Oxyma, or uronium/phosphonium reagents such as HATU and HBTU, plus a base such as DIPEA or NMM. See our guide to coupling reagents.
- Solvents: typically DMF or greener alternatives, in peptide synthesis grade.
- Cleavage cocktail: usually trifluoroacetic acid (TFA) with scavengers, handled in a fume cupboard with acid-resistant gloves.
Automation
Automated peptide synthesisers repeat the cycle unattended, and microwave-assisted systems shorten coupling times. Many labs also outsource longer or difficult sequences to custom peptide synthesis companies.
Purification and analysis
- Precipitation of the crude peptide, often in cold ether.
- Reversed-phase HPLC purification, usually with water/acetonitrile gradients containing a little TFA.
- Mass spectrometry to confirm the molecular weight.
- Analytical HPLC to determine purity.
- Freeze-drying to give a stable powder.
What do peptide calculators do?
Online peptide calculators take a sequence and estimate useful properties, including:
- Molecular weight, average and monoisotopic, for checking mass spec results;
- Net charge at a chosen pH and isoelectric point (pI), which help predict solubility and HPLC behaviour;
- Extinction coefficient at 280 nm, for estimating concentration by UV;
- Hydrophobicity, a hint of difficult sequences or aggregation.
They are estimates. Always confirm identity and purity experimentally.
Frequently asked questions
What is the difference between Fmoc and Boc SPPS?
Fmoc chemistry removes the temporary protecting group with a mild base and is the most common today; Boc chemistry uses repeated acid treatment and harsher final cleavage conditions.
How long a peptide can be made by SPPS?
Routinely up to roughly 50 residues; longer chains are possible but become harder and are often joined from fragments.
Why is TFA present in my peptide?
TFA from cleavage and HPLC often remains as a counter-ion. It can be exchanged for acetate or chloride if it interferes with experiments.
