The problem before 1963

Making a peptide is conceptually trivial: join amino acids in order. Doing it in solution is not. Every coupling step produces a mixture (your product, unreacted starting material, and side products), and each one has to be isolated before the next step can begin. Purification means precipitation, crystallisation or column chromatography, and every round loses material.

For a five residue peptide this is tedious. For a thirty residue peptide it is a research project measured in months, with a yield that rounds to nothing.

Tying it down

Merrifield's proposal was to attach the first amino acid (by its C-terminus) to an insoluble polystyrene bead. The growing chain stays bound to that bead throughout. Reagents are dissolved in solvent and poured over it; when the reaction is done, the solvent is simply drained away.

Purification stops being a chemical problem and becomes a plumbing one.

Because separation is now filtration, you can use a large excess of reagent to drive each coupling nearly to completion, something you would never do in solution, where the excess becomes tomorrow's purification headache. That single change is what made peptide synthesis automatable.

A resin bead anchoring a growing peptide chain, with the deprotect, couple and wash cycle marked above it
FIG. 1: The chain is anchored at its C-terminus and grows towards the N-terminus, the opposite direction to how sequences are conventionally written.

One cycle, repeated

Each residue is added by the same three step loop:

  1. Deprotect. The chain's N-terminus carries a temporary protecting group (commonly Fmoc), which is removed to expose a reactive amine.
  2. Couple. The next amino acid, itself protected and chemically activated, is added. Its carboxyl group forms a peptide bond with the exposed amine.
  3. Wash. Everything unreacted drains away.

Side chains carry their own, sturdier protecting groups so they cannot participate. Those come off at the end, in the same step that cleaves the finished chain from the resin.

A synthesiser runs this loop unattended. A 30 residue peptide is 30 cycles, roughly overnight.

Why length is expensive

The catch is compounding. Suppose each coupling proceeds with 99% efficiency, very good in practice. After 30 cycles the fraction of chains that received every residue correctly is 0.99³⁰, or about 74%. Drop to 98% per step and it falls to 55%.

The missing quarter to half is not waste in the ordinary sense. Those chains are still there, still attached to beads, still being carried through every subsequent cycle. They emerge at the end as deletion sequences: peptides identical to the target except for one absent residue.

This is why cost rises faster than length

Doubling the residue count does not double the price. It reduces the yield of correct chains and increases the burden on purification, both at once.

Where impurities come from

Deletion sequences are the largest category, but not the only one:

  • Truncated sequences: chains where coupling failed and never resumed, leaving a short fragment.
  • Racemisation: a residue flipping from the L to the D configuration during activation. The mass is unchanged, so mass spectrometry will not see it.
  • Incomplete deprotection: side chain protecting groups that survive the final cleavage and stay attached.
  • Oxidation: methionine and cysteine residues are vulnerable, and can oxidise during synthesis, cleavage or storage.
  • Aggregation: certain sequences fold on the resin mid synthesis and become physically inaccessible to reagents, causing a run of failed couplings.

What purification fixes, and what it does not

The crude product goes through preparative reverse phase HPLC, which separates by hydrophobicity. A deletion sequence missing a bulky hydrophobic residue behaves differently enough from the target to be separated cleanly.

A deletion missing a small residue such as glycine, in the middle of a long chain, may not. Neither will a racemised diastereomer, in many cases. This is the practical limit of the technique, and the reason a purity figure needs a method attached to be meaningful.

An HPLC chromatogram with one dominant product peak and several smaller impurity peaks, alongside a mass spectrometry inset
FIG. 2: A chromatogram reports what absorbs UV light at the detector wavelength. Anything that does not absorb at 214 nm is invisible to it, including most salts and solvents.

Why any of this matters

Two reasons, both practical.

First, it explains the price structure of the field. Peptide medicines are expensive not because of exotic ingredients but because stepwise chemistry has an unforgiving yield curve and the purification is genuinely difficult.

Second, it explains why "99% pure" is an incomplete statement. Pure by which method, at which wavelength, against which impurity classes? A certificate reporting HPLC purity alone has said nothing about stereochemistry, nothing about endotoxin, and nothing about how much of the vial's weight is peptide rather than water and counterions.

Those are separate tests, and they are the ones most often missing. We cover them in Part 06 of the foundations course, and the wider picture of what peptides are in What are peptides?.

A note on scope

This article describes how peptides are manufactured. It is not a guide to evaluating any particular product for personal use, and nothing here should be read as suggesting that a sufficiently good certificate makes an unapproved compound safe to take.