Academy · Intermediate
Understanding HPLC Testing
How HPLC quantifies peptide purity: column chemistry, gradient methods, detection wavelengths, and how to read the resulting chromatogram.
Principle of separation
HPLC pushes a mobile phase through a packed column. Compounds in the sample interact differently with the stationary phase and so elute at different times. UV-Vis detection (usually 214 nm for peptide bonds) records each compound as a peak.
Method choices that matter
Reverse-phase C18 with a water/acetonitrile gradient is standard for peptide purity. Method validation parameters — column temperature, flow rate, injection volume — should be documented on the COA.
Reading a chromatogram
One dominant peak with minor shoulders is normal. Purity is the main peak area as a percentage of total area. Watch for unresolved shoulders that could indicate a deletion sequence or oxidation product.
Frequently asked questions
What wavelength do you trust?+
214 nm for general peptide bond detection; 280 nm if the sequence contains tryptophan or tyrosine.
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