Peptide content versus purity — the distinction most buyers get wrong
Purity is a ratio of areas. Content is a fraction of mass. A 99 % pure vial can contain 78 % peptide, and the difference is the whole ball game.
Content and mass
Residual solvents by GC headspace
The mass in the cake nobody measures: DMF, DMSO, acetonitrile, DCM and TFA, to ICH Q3C(R8) classes and limits.
Peptide synthesis and purification use a specific and predictable set of solvents, and lyophilisation does not remove all of them. Residual solvent is mass in the vial that is not peptide, it is a mass-balance term, and in the index's dataset it is measured on almost nothing.
The solvents that turn up, and why
| Solvent | Q3C class | Limit | Origin in peptide work |
|---|---|---|---|
| Dichloromethane | Class 2 | 600 ppm | Resin washing, older cleavage protocols |
| N,N-dimethylformamide | Class 2 | 880 ppm | The principal SPPS coupling solvent |
| N-methylpyrrolidone | Class 2 | 530 ppm | Alternative coupling solvent for difficult sequences |
| Acetonitrile | Class 2 | 410 ppm | Preparative HPLC mobile phase |
| Methanol | Class 2 | 3000 ppm | Washing, recrystallisation |
| Pyridine | Class 2 | 200 ppm | Older activation chemistry |
| Dimethyl sulfoxide | Class 3 | 5000 ppm | Oxidative folding, sample dissolution |
| Acetic acid | Class 3 | 5000 ppm | Salt exchange, lyophilisation buffer |
| Diethyl ether | Class 3 | 5000 ppm | Crude peptide precipitation |
| tert-Butyl methyl ether | Class 3 | 5000 ppm | Crude peptide precipitation |
| Trifluoroacetic acid | not classified in Q3C | controlled as counter-ion | Cleavage and mobile phase; see the counter-ion page |
Method
Static headspace gas chromatography with flame-ionisation detection is the standard. A weighed sample is dissolved in a high-boiling diluent — dimethyl sulfoxide, dimethylacetamide or water depending on which solvents are being sought — sealed in a vial, equilibrated at 80–105 °C, and an aliquot of the vapour phase is injected onto a wide-bore capillary column. Quantification is by external standard or, better, by standard addition, because matrix effects on partition coefficients are substantial.
DMSO cannot be sought using DMSO as the diluent, which sounds obvious and is a recurring error. Water is the alternative, at the cost of sensitivity for the water-miscible solvents.
What the index holds
One laboratory offers GC headspace residual solvents. Across the whole index, fewer than 3 % of records carry a residual-solvent figure, and no supplier certificate in the dataset reports one. The index therefore estimates the residual term in its gravimetric mass balance rather than measuring it, and labels it as an estimate on every record — 0.14 to 1.02 % of cake mass, which is the range the measured subset supports.
Normative references
Cited because they are the documents the acceptance criteria above are taken from. The index applies them as written and states every deviation.
- ICH Q3C(R8) — Impurities: guideline for residual solvents (2021)
- USP <467> — Residual solvents
- Ph. Eur. 2.4.24 — Identification and control of residual solvents
Related methods
Counter-ion quantification
Acetate, trifluoroacetate, mesylate and the rest — by ion chromatography, with the stoichiometry check that tells you whether the number is plausible.
Content and mass
Water content by Karl Fischer titration
Coulometric KF on lyophilised peptides: sample handling, the two things that ruin the measurement, and what a high result actually tells you.
Content and mass