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Certificates of Analysis reference specific testing methods, but those method names are not always explained. This page gives a plain-terms explanation of the two techniques most commonly used to test peptide purity and identity: high-performance liquid chromatography and mass spectrometry.
High-performance liquid chromatography, usually shortened to HPLC, is a technique used to separate the different components of a sample as it passes through a column under pressure. Different components travel through the column at different rates, which allows the main peptide to be distinguished from any minor impurities or side-products, and quantified as a percentage of the total sample.
Mass spectrometry measures the mass-to-charge ratio of molecules in a sample, which allows researchers to confirm the molecular identity of a peptide. Where HPLC is generally used to quantify purity, mass spectrometry is generally used to confirm that the molecule present actually matches the expected structure.
HPLC and mass spectrometry are often used together because they answer two different questions. HPLC establishes how much of the sample is the intended peptide, expressed as a purity percentage, while mass spectrometry confirms that the molecule detected is structurally correct. A Certificate of Analysis typically references results from both methods for this reason.
| Method | What it measures |
|---|---|
| HPLC | Purity, expressed as a percentage of the total sample |
| Mass spectrometry | Molecular identity, confirming the correct structure is present |
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