A purity number is only as good as the separation behind it. If the HPLC method cannot pull an impurity apart from the main peak, that impurity is counted as "pure" peptide. This post explains how a reversed-phase column and a solvent gradient actually separate peptides, which method details matter, and what they mean when you read a COA. It builds on post #2 (how purity is measured), post #23 (reading a chromatogram) and post #26 (UV detection wavelength). 1. What "reversed-phase" means • The column is a steel tube packed with tiny silica particles coated with oily hydrocarbon chains, usually C18 (18 carbons) or C8. • The liquid flowing through it (the mobile phase) is a mix of water and an organic solvent, usually acetonitrile. • Peptides stick to the oily coating through their hydrophobic (water-avoiding) parts. The more hydrophobic a molecule is, the more strongly it sticks. 2. How the gradient pushes peptides off the column • The run starts with mostly water, so peptides bind to the column. • Over the run, the share of acetonitrile slowly increases. This is the gradient. • As the solvent becomes more organic, molecules let go one by one, roughly in order of how hydrophobic they are. Each one leaves at its own retention time and shows up as a peak. 3. Why small differences can still separate • A deletion sequence missing one amino acid (post #16), an oxidized methionine (post #14), or a D-amino acid version (post #18) changes the molecule's hydrophobicity a little. • A small change is often enough to shift the retention time, so the impurity appears as its own peak next to the main one. • Some impurities are very close to the main peptide. A shallow gradient (a slow rise in acetonitrile) gives them more time to separate. 4. The role of TFA and other additives • Small amounts of TFA or formic acid are added to the mobile phase. They pair with charged groups on the peptide and give sharper, more symmetrical peaks. • Sharp peaks matter: a broad, smeared main peak can hide a small impurity sitting under its shoulder.

Every HPLC purity number depends on a detector, and for peptides that detector is almost always UV absorbance. The wavelength the lab chooses changes what the chromatogram can "see". This post explains why most peptide COAs list 214 nm or 220 nm, what changes at 254 or 280 nm, and why the detection wavelength belongs on every COA. It builds on post #2 (how purity is measured), post #23 (reading a chromatogram) and post #25 (COA red flags). 1. How a UV detector works • After the column separates the sample, the liquid flows through a small cell with a UV light beam passing through it. • Molecules that absorb light at the chosen wavelength reduce the light reaching the sensor. That drop is recorded as a peak. • The bigger the absorbance, the bigger the peak. Area percent purity is simply each peak's area divided by the total area of all peaks. 2. Why 214 to 220 nm for peptides • Every peptide has peptide bonds (the amide links between amino acids). These bonds absorb strongly in the low UV range, around 190 to 220 nm. • Because every amino acid in the chain contributes a peptide bond, almost any peptide or peptide fragment shows up at 214 to 220 nm. • That matters for purity: deletion sequences, truncations and other related impurities (post #16) also contain peptide bonds, so they become visible too. 3. What changes at 254 nm and 280 nm • At 280 nm, absorbance comes mainly from aromatic side chains, especially tryptophan and tyrosine. • A peptide with no Trp or Tyr can be nearly invisible at 280 nm. An impurity missing those residues can disappear as well. • Reading purity only at 254 or 280 nm can therefore make a sample look cleaner than it is, because fewer impurities absorb there. • Labs sometimes record a second wavelength as a cross-check, which is useful, but the main purity figure for peptides is normally taken in the 214 to 220 nm range. 4. Why the mobile phase matters at low wavelengths • Solvents and additives absorb low UV light too. Acetonitrile is preferred over methanol partly because it is more transparent near 210 to 220 nm.