Purity is not content
A purity figure says how clean the peptide is, not how much peptide is in the vial. For novel compounds we measure structure and content as well.
Two different numbers
HPLC gives two different numbers. Purity is relative: the main peak against all peaks that absorb UV light, with no standard needed. Content is found by comparing the sample against a reference standard of known content.
Purity ignores water and salts, because they do not appear in the UV trace. The amount of desired peptide in a vial is its content multiplied by its purity.
One vial, worked through
| Measurement | Result |
|---|---|
| HPLC purity | 99.2% |
| Water | 6% |
| Counter-ion | 14% |
| Residual solvent | 1% |
| Content, what is left | 79% |
| Desired peptide, content multiplied by purity | 78% |
A vial labelled 10 mg would hold about 7.8 mg of the peptide, with a certificate that truthfully says 99.2%.
Measuring what the UV trace does not show
Each part of the powder that HPLC cannot see is measured on its own: water by Karl Fischer titration, counter-ions such as TFA or acetate by ion chromatography, and residual solvents by gas chromatography. The total is cross-checked against qNMR, which measures content directly.
Necessary, not sufficient
HPLC and LC-MS come first on every batch. For a novel compound they do not settle structure or content on their own.
Sources
- Bachem: Quality control of amino acids and peptides: HPLC purity, counter-ions, water, residual solvents
- Bio-Synthesis: Peptide purity, peptide content and absolute amount: purity against content, content multiplied by purity
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