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How reverse-phase HPLC measures peptide purity, what the percentage represents, what the impurities usually are, and where the method's limits lie.
Reverse-phase HPLC pushes a sample through a column packed with a hydrophobic stationary phase, using a gradient of increasing organic solvent. Compounds that bind the column more strongly come off later.
For peptides the detector is almost always ultraviolet absorbance at 214 nm, which is where the peptide bond itself absorbs. That makes the response roughly proportional to the number of amide bonds, and therefore to peptide mass.
Purity is calculated as the area under the main peak divided by the total area under all peaks, expressed as a percentage. It is a relative measure of what the detector saw.
Because detection is at 214 nm, anything without a peptide bond is largely invisible to the method. Residual salts, counterion and water do not appear as peaks and are not part of the calculation.
The overwhelming majority of impurities in a synthetic peptide are synthesis-related rather than foreign contamination. Deletion sequences arise when a coupling step fails and the chain continues one residue short. Truncated sequences arise when a chain stops growing entirely.
Incomplete side-chain deprotection leaves protecting groups attached. Racemisation during coupling produces diastereomers, which elute very close to the target and are the hardest impurity class to resolve.
For research-grade material, 95% is a common floor and 98-99% is a good specification. Pushing beyond 99% requires progressively more aggressive preparative purification, and the yield cost rises steeply.
The difference between 98% and 99% is often not the limiting factor in an assay. Endotoxin level and correct identity confirmation frequently matter more.
HPLC purity is method-dependent. The same sample run on a different column, gradient or wavelength can give a different number. A certificate should state the method conditions so results are comparable.
It is also blind to anything that does not absorb at the detection wavelength and does not retain on the column. That is why identity confirmation by mass spectrometry is a separate line on the certificate rather than an inference from purity.
This guide is general laboratory reference material relating to the handling of research compounds. It is not medical, veterinary or clinical guidance, and it does not describe or imply any use in humans or animals. All PeptideSeed material is supplied for in-vitro laboratory research only — see the terms of supply.