What HPLC measures
High-performance liquid chromatography pushes a dissolved sample through a packed column. For peptides this is usually reversed-phase HPLC: a C18 column and a gradient of water and acetonitrile with a little acid, such as trifluoroacetic acid. Components leave the column at different times depending on how strongly they stick to it.
A UV detector watches what comes out. Peptide bonds absorb strongly at 214–220 nm, so every peptide-like component shows up as a peak. Purity is the area of the main peak as a share of the area of all the peaks.
What HPLC can't tell you
- Identity. The wrong peptide can give one clean, sharp peak. HPLC shows that something is pure, not what it is.
- Hidden impurities. Two components that leave the column at the same moment sit under one peak.
- Anything that doesn't absorb UV. Water, salts and counter-ions are invisible to the detector, so HPLC purity says nothing about how much peptide is in the vial. See Purity vs. peptide content.
What mass spec measures
A mass spectrometer turns molecules into ions and measures their mass-to-charge ratio. Electrospray ionization (ESI) usually puts several charges on one molecule, so a single peptide shows a series of peaks that software converts back to one molecular mass. MALDI usually adds a single charge, so the main peak sits at the molecular mass plus one proton.
If the observed mass matches the mass calculated from the sequence, within the instrument's tolerance, the identity is confirmed.
What mass spec can't tell you
- Purity. Different molecules ionize with different efficiency, so peak heights in a mass spectrum don't translate into percentages.
- Sequence order. Two peptides with the same residues in a different order have the same mass. Telling them apart takes fragmentation (MS/MS) or a reference standard.
- Mirror-image residues. A D-amino acid in place of an L-amino acid doesn't change the mass at all.
LC-MS: both at once
LC-MS runs the HPLC separation straight into a mass spectrometer, so every peak gets a mass. That turns an anonymous impurity peak into something with a name.
| Finding | Likely cause |
|---|---|
| Mass lower by one residue | Deletion sequence: one coupling step didn't complete |
| Mass of a shorter piece of the sequence | Truncated sequence: synthesis stopped early |
| +16 Da | Oxidation, most often of methionine |
| +0.98 Da | Deamidation of asparagine or glutamine |
| +56 Da | A tert-butyl protecting group left on after cleavage |
Why a report needs both
| Question | HPLC | Mass spec |
|---|---|---|
| Is it the right molecule? | No | Yes |
| How pure is it? | Yes | No |
| What are the impurities? | Shows they're there | Says what they are, when coupled to LC |
| How much peptide is in the vial? | No | No |
That last row is why some reports add a peptide content test. It has a guide of its own.