Solid-phase peptide synthesis, explained

Every peptide in the Alphex catalogue is made by solid-phase peptide synthesis, and the method explains almost everything about the resulting material: why purity falls with length, which impurities appear, why counter-ions are present, and why the final product arrives lyophilised. This note is a plain explanation of SPPS for researchers who use peptides rather than make them. All Alphex products are for laboratory and in-vitro research only.

The core idea

Rather than building a peptide in solution and purifying at every step, SPPS anchors the growing chain to an insoluble resin bead. Reagents are washed in and out; the product stays put. Because excess reagent can be used and simply washed away, each coupling can be driven close to completion, and the whole cycle can be automated. Robert Bruce Merrifield's development of the approach won the 1984 Nobel Prize in Chemistry and it remains the basis of essentially all synthetic peptide manufacture.

The cycle

1. Loading. The C-terminal residue, with its side chain protected and its alpha-amino group blocked by a temporary group (usually Fmoc), is attached to the resin.
2. Deprotection. The Fmoc group is removed with piperidine, exposing the amine.
3. Coupling. The next protected amino acid, activated by a coupling reagent, forms the amide bond with that amine.
4. Washing. Excess reagents and by-products are washed away.
5. Capping (optional). Any chains that failed to couple are acetylated so they cannot grow further, converting deletion sequences into truncated ones that are easier to remove.
6. Repeat 2-5 for each residue, C-terminus to N-terminus.
7. Cleavage. Strong acid (usually TFA with scavengers) releases the peptide from the resin and removes side-chain protecting groups simultaneously.
8. Precipitation, purification and lyophilisation. The crude peptide is precipitated in cold ether, purified by preparative reverse-phase HPLC, and freeze-dried. See what lyophilised means.

Why purity falls with length

Each coupling is very good but not perfect. At 99.5% efficiency per step, a 15-residue peptide retains about 93% of chains as full-length; at 39 residues, about 82%. Real efficiencies vary by sequence (proline runs, beta-sheet-prone stretches and bulky residues are harder), so crude purity for a long peptide can be well below that. Everything not full-length is an impurity of the classes described in common peptide impurities, and purification has to remove it, which is why a 99+% specification costs more at length. See 98% versus 99+%.

What SPPS explains about your vial

• Counter-ions. Cleavage in TFA and purification in TFA-containing mobile phase leave the peptide as a TFA salt unless exchanged to acetate. See acetate versus TFA.
• Non-natural residues are straightforward. Any protected amino acid, D or L, natural or not, can be coupled, which is why Aib and naphthylalanine peptides are routine. See D-amino acids and unnatural residues.
• Modifications. N-terminal acetylation, C-terminal amidation (via the choice of resin) and side-chain lipidation are all done on-resin.
• Impurity profile is characteristic. Deletions cluster near the target on HPLC; protecting-group adducts elute later. Reading a chromatogram with this in mind is more informative than reading the percentage. See how to read a chromatogram.

From synthesis to the Alphex catalogue

Alphex specifies the sequence and the purity threshold; the synthesis partner performs SPPS, purification and vialling; an independent laboratory then tests the batch by HPLC before it is listed. A batch that does not meet specification is not listed. See how Alphex tests every batch and what happens when a batch fails.

Sources and further reading

• The Nobel Prize in Chemistry 1984 (R. Bruce Merrifield), nobelprize.org: the development of solid-phase peptide synthesis
• ICH Quality Guidelines (Q1A stability, Q3A/Q3B impurities, Q6 specifications): the reference framework for purity, impurity and stability testing

Alphex research peptides are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.

Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.

Published 21 April 2026, last reviewed 16 August 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.

Alphex Research Team

Alphex Limited, UK research peptide supplier

The Alphex research team writes and maintains these notes for UK laboratories buying research peptides. Every batch Alphex lists is verified by reverse-phase HPLC with a batch-matched Certificate of Analysis. Content covers laboratory and in-vitro research only.