In short
- HPLC measures how much of the detected material is the main compound; mass spec checks whether that compound has the right mass.
- HPLC purity is an area percentage that depends on the method, and impurities can hide under the main peak.
- Mass spec can't tell apart molecules with identical mass, such as scrambled sequences, and it isn't a purity test.
- A complete report shows an HPLC trace with a peak table and an observed mass next to the expected mass.
Almost every peptide certificate of analysis rests on two instruments: high-performance liquid chromatography, or HPLC, and mass spectrometry, often shortened to mass spec or MS. Suppliers quote the results constantly, but the attached reports can look like a page of squiggles and numbers.
This guide explains what each technique measures, how to read the trace or spectrum attached to a report, and where each one runs out of information. Once you see that the two tests answer different questions, it becomes clear why a good report includes both.
Two tests, two different questions
HPLC separates a sample into its components and measures how much of the detector signal belongs to each. It answers the question: how much of what is here is the main compound? That answer becomes the purity percentage.
Mass spectrometry weighs molecules. It answers a different question: does the main compound have the mass the intended sequence should have? That answer is the identity result.
Neither answer covers the other. A sample can be very pure and still be the wrong molecule, or be the right molecule mixed with a lot of something else. That is why a report with only one of the two is incomplete.
How reversed-phase HPLC works
Most peptide purity tests use reversed-phase HPLC. A small amount of dissolved sample is pumped through a narrow steel column packed with fine particles coated in oily carbon chains, most often a type called C18.
The liquid flowing through the column, called the mobile phase, starts mostly as water and gradually shifts toward an organic solvent, usually acetonitrile. A little acid, often trifluoroacetic acid (TFA) or formic acid, is added to keep peaks sharp. Compounds that cling more strongly to the coating wash off later. A peptide that is missing an amino acid, or that has an oxidized residue, behaves slightly differently from the intended sequence, so it tends to come off at a slightly different time.
As each compound leaves the column it passes a UV detector, typically set between 210 and 220 nm, where the peptide bond absorbs light. The detector's signal over time is the chromatogram.
Reading a chromatogram
The horizontal axis is retention time, in minutes from the start of the run. The vertical axis is detector response. Each compound shows up as a peak, and the area under a peak reflects how much light that compound absorbed as it passed.
Purity by area percent is the main peak's area divided by the total area of all the peaks the software counted. Peaks that come from the solvent or the instrument are excluded, and labs normally set a reporting threshold so that tiny peaks below it aren't counted. In the annotated example below, look for four things:
- The main peak: tall and reasonably symmetrical.
- A shoulder or close neighbor: a small peak right beside the main one, often a closely related impurity.
- Small peaks elsewhere: other impurities, each ideally small.
- The baseline: it should be flat. A drifting or noisy baseline makes small-peak areas, and therefore the purity figure, less reliable.
A full report also includes a peak table listing the retention time and area percent of each peak.
- Main peak. The compound you ordered. Purity is this peak's area as a share of all peak areas.
- Shoulder or close neighbour. Often a related impurity, such as a truncated or oxidised sequence.
- Early small peaks. Other impurities. A clean trace has few, and each is small.
- Baseline. Should be flat and quiet. A wandering baseline makes area figures less reliable.
What HPLC can and can't show
HPLC is good at separating synthesis by-products that differ from the target, such as deletion sequences missing one amino acid, truncated chains, and oxidized or deamidated forms. Their combined area is what pulls a purity figure below 100%.
It has real limits, though:
- It doesn't identify peaks on its own. Retention time isn't unique to one compound. Matching the time of a reference standard is supporting evidence, not proof of identity.
- Impurities can hide. A compound that leaves the column at the same moment as the main peak is counted as part of it. Method studies show that changing the column or mobile phase can turn a hidden impurity into a visible shoulder.
- It only sees what absorbs. Water and most salts don't appear as peaks, so they don't lower the purity number.
- The result belongs to the method. The column, gradient, wavelength and integration settings all affect the figure.
How mass spectrometry works
A mass spectrometer turns molecules into charged ions, sorts them by mass-to-charge ratio, written m/z, and counts them. For peptides, the two common ways of making ions are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
MALDI mostly produces ions carrying a single charge, so the main peak sits close to the peptide's mass plus one proton. Electrospray often adds several protons at once. A peptide with a mass of 1,500 Da might appear near m/z 1,501 with one charge, 751 with two and 501 with three. Software combines this series back into one neutral mass, a step called deconvolution.
Two more details matter when you compare numbers. The monoisotopic mass uses only the most common isotope of each element, while the average mass weights all isotopes by natural abundance. For a peptide of this size they differ by about one unit, and by more for larger ones, so a report should say which it used. It should also state the tolerance it accepted, because instruments differ widely in precision.
Reading a mass spectrum
The horizontal axis is m/z and the vertical axis is signal intensity. On a good identity result, the tallest peak, or the deconvoluted mass, lines up with the expected value for the sequence, as in the example below. The report line might read “expected 1,532.8 Da, observed 1,532.9 Da”.
Smaller peaks are common and not automatically a problem. Some are the same molecule carrying a different charge. Others sit at a recognizable offset from the main mass:
- About +22: a sodium ion in place of a proton, usually a feature of the measurement rather than the sample.
- +16: often oxidation, most commonly of methionine.
- About +1: possible deamidation, though this is hard to tell apart from the natural isotope peaks on a lower-resolution instrument.
- Minus the mass of one amino acid: a deletion sequence left over from synthesis.
What mass spec can and can't show
A matching mass is strong evidence that the main compound is the intended molecule. It can't settle everything:
- Same-mass variants look identical. A scrambled sequence, leucine swapped for isoleucine, or a D-form amino acid in place of the usual L-form gives the same mass. Confirming sequence order needs tandem mass spectrometry (MS/MS) or other methods.
- Peak height isn't quantity. Different molecules ionize with different efficiency, so a spectrum on its own is not a purity measurement.
Some labs couple the two techniques, called LC-MS, so that the liquid leaving the HPLC column flows straight into a mass spectrometer. That can put a tentative mass on impurity peaks as well as the main one. Practice varies, and many research COAs report the two tests separately.
The practical rule is to look for both. A complete report pairs an HPLC trace and peak table with a mass result that shows observed and expected values side by side.
Questions
Does a 99% HPLC result mean the vial is 99% peptide?
No. It means the main peak made up about 99% of the peak area the detector counted. Water and counter-ions don't appear as peaks, so the powder can contain noticeably less peptide by weight than the purity figure suggests.
Why might two labs report different purity for the same batch?
Purity depends on the method: the column, solvent gradient, detection wavelength and how peaks are integrated. A method with better separation can reveal an impurity that another method hides under the main peak. Small differences are normal, while large ones are worth asking about.
Is a mass spec result enough on its own?
No. It confirms that the main compound has the expected mass, but it doesn't measure how much of the sample is that compound. It also can't distinguish molecules with the same mass, such as a scrambled sequence.
What is the difference between ESI and MALDI?
Both are ways of turning peptides into ions for mass spectrometry. MALDI mostly produces singly charged ions, while electrospray often produces several charge states that software converts back into one mass. Either can give a valid identity result.
This guide is about laboratory documentation and handling. It contains no dosing or medical advice, and nothing here describes use in people or animals.