How peptides are purified: preparative HPLC, ion exchange and size exclusion

When a peptide is made by solid-phase synthesis, the crude product is a mixture. It contains the intended sequence, plus shorter chains, chains with a residue missing, and other by-products. Purification removes as many of these as possible. This guide explains the main methods, how they're combined, and what the purification route means for the purity on a certificate of analysis (COA).

For how the crude is made, see solid-phase peptide synthesis. For the principles of chromatography, see how chromatography works. Terms are defined in the glossary.

What needs removing

After the peptide is cut from the resin, the crude contains:

• Deletion sequences: a residue is missing because one coupling step failed.
• Truncated sequences: the chain stopped growing early.
• Incompletely deprotected peptides: a protecting group is still attached.
• Modified forms: for example oxidised methionine or a rearranged aspartic acid.
• Small molecules: scavengers and cleavage reagents.

Many of these differ from the target by a single residue or a few daltons, so they behave almost the same way. That is what makes peptide purification demanding. See reading impurity peaks for how they show up on HPLC and mass spectrometry.

Step one: precipitation

Straight after cleavage, the peptide is usually precipitated by adding cold diethyl ether. The peptide comes out of solution as a solid, while many of the small-molecule scavengers and cleavage by-products stay dissolved. The solid is collected, often with a centrifuge, washed and dried. This is crude peptide, ready for chromatography.

Preparative reversed-phase HPLC

Most synthetic peptides are purified by preparative reversed-phase HPLC (RP-HPLC). It works on the same principle as the analytical HPLC used to measure purity. See how HPLC works.

• The column is packed with silica carrying non-polar chains, usually C18. C8 or C4 columns suit larger or more hydrophobic peptides.
• The mobile phase starts mostly water and becomes richer in acetonitrile over the run. Trifluoroacetic acid (TFA) is added at around 0.1% to sharpen the peaks. That is where the TFA counter-ion in most peptide powders comes from. See TFA counter-ions and salt exchange.
• Separation depends mainly on hydrophobicity. More hydrophobic components stay on the column longer.

How preparative differs from analytical

Fractions and pooling

As the peptide comes off the column, the eluent is collected in a series of fractions. Each fraction is checked by analytical HPLC, and often by mass spectrometry to confirm identity. Fractions that meet the purity target are pooled. Those at the edges of the main peak, where impurities overlap, are set aside or purified again (Bachem, Peptide User Guide).

This is where purity and yield trade off. A stricter purity target means discarding more of the edge fractions, so less peptide is recovered from the same crude. That is one reason higher-purity grades cost more.

The pooled fractions are then freeze-dried to remove the water, acetonitrile and free TFA, leaving the peptide as a dry powder. See what lyophilisation is.

Ion-exchange chromatography

Ion-exchange chromatography separates molecules by charge. The column is packed with a resin carrying charged groups.

• Cation exchange uses a negatively charged resin, which holds positively charged molecules. Most peptides are positively charged at acidic pH, so this is the more common choice.
• Anion exchange uses a positively charged resin, which holds negatively charged molecules.

Molecules are released by raising the salt concentration or changing the pH, so those with weaker charge come off first. How charged a peptide is at a given pH depends on its sequence. See isoelectric point and net charge.

Because ion exchange separates by charge rather than hydrophobicity, it can remove impurities that run alongside the target in reversed-phase HPLC, such as a deamidated form with one extra negative charge. Two methods that separate by different properties are called orthogonal. Ion exchange is sometimes used as a first step to reduce the load before reversed-phase HPLC. Its drawback is that the peptide comes off in a salt solution, and the salt then has to be removed (Phenomenex interview with Bachem).

Ion-exchange resins are also used to swap the counter-ion after purification, for example replacing TFA with acetate.

Size-exclusion chromatography

Size-exclusion chromatography (SEC), also called gel filtration, separates molecules by size. The column is packed with porous beads. Small molecules wander into the pores and take a longer path. Large molecules can't enter the pores and pass straight through, so they leave the column first.

For short peptides, SEC can't separate the target from impurities only one residue different in size. Its main uses are:

• Desalting: separating a peptide from salts and small molecules, for example after ion exchange.
• Buffer exchange: moving a peptide from one solution into another.
• Removing aggregates, which are much larger than single peptide molecules.

Other methods

• Solid-phase extraction uses a short cartridge, often with the same C18 material as an HPLC column, for a quick clean-up or to remove salt.
• Flash chromatography uses lower pressure and larger particles than HPLC. It's faster and cheaper per gram, with lower resolution, and can serve as a first pass on large batches.
• Mass-directed purification collects fractions when a mass spectrometer detects the target mass, rather than when a UV detector sees a peak.

What this means for a COA

• The purity figure comes from analytical HPLC of the final, freeze-dried material, not from the preparative run. See HPLC purity explained.
• The identity is confirmed separately, usually by mass spectrometry. See mass spectrometry and identity confirmation.
• The counter-ion depends on the last purification step. A peptide purified with TFA and not exchanged afterwards is a TFA salt.
• Impurities that run with the target in the purification method can also run with it in an analytical method of the same type. That is why an orthogonal method, such as a different column or mass spectrometry, adds confidence.
• Net peptide content is not the same as purity. A pure peptide powder still contains water and counter-ions. See net peptide content vs purity.

Related guides

For how to read the lot document as a whole, see what a certificate of analysis tells you. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.