Two abbreviations appear again and again on peptide certificates: HPLC and LC-MS. They sound alike and are often run on similar instruments, yet they answer different questions. This guide explains each and how they fit together.
HPLC in brief
HPLC stands for high-performance liquid chromatography. A dissolved sample is pushed through a column packed with fine particles. Different molecules travel through at different speeds, so they leave the column at different times. A detector at the end records a signal over time and draws a chromatogram, a line with peaks.
For peptides the usual set-up is reversed-phase HPLC (RP-HPLC) with a UV detector, since peptide bonds absorb ultraviolet light. Reports may write this as HPLC-UV or RP-HPLC with UV detection.
What an HPLC result tells you
The main peak is the compound the lab is looking at, and smaller peaks are other substances that absorb at the detector’s wavelength. Purity is calculated as the area of the main peak divided by the total area of all peaks, shown as a percentage. It is a relative figure under one set of conditions. It tells you how clean the sample looks to that detector. It does not, by itself, prove which compound produced the main peak, and anything that does not absorb at the chosen wavelength will not show up.
LC-MS in brief
LC-MS joins liquid chromatography to a mass spectrometer. The chromatography stage separates the sample as before. The mass spectrometer then measures the mass-to-charge ratio of the molecules coming off the column. Because every compound has a characteristic molecular mass, the result can be compared with the expected mass of the compound named on the label.
What an LC-MS result tells you
LC-MS speaks mainly to identity. A match between the measured and expected mass supports the claim that the compound is what it says it is. Depending on how it is run, it can also detect impurities that differ in mass from the main compound, such as truncated or modified sequences. Some set-ups are used semi-quantitatively; others are used only to confirm mass.
Why they complement each other
HPLC-UV asks, “How pure is the main peak?” LC-MS asks, “What is the main peak?” A report with both gives a more complete picture than either alone. If only one is shown, read it with its limits in mind: a strong purity figure without identity data leaves the identity question open, and a mass match without a purity figure leaves the purity question open.
Other items on a report
Neither method measures endotoxin; that uses a separate assay described in our endotoxin testing explainer. A quantity line, when printed, comes from a calibrated measurement and has its own tolerance.
Examples in our library
Our NAD+ 1000 certificate lists LC-MS identity confirmation from Freedom Diagnostics. Many other entries in the COA library list HPLC-UV or RP-HPLC purity from laboratories such as Janoshik and Chromate. Each entry shows the method, the batch number and the date, so you can see exactly what was measured.
Common questions
Is LC-MS better than HPLC? Neither is better; they answer different questions, so many laboratories run both.
Why do two laboratories report slightly different purity figures? Columns, gradients, wavelengths and integration settings all influence the area-based figure.
Does a method line guarantee a result is accurate? It describes how the number was obtained, which helps you judge what it can and cannot show.
Reading tips
- Find the method line first.
- Decide whether the report speaks to purity, identity or both.
- Check that the batch number matches your vial.
- Remember that a percentage is only as meaningful as the method behind it.
For a complete walk-through of a report, see how to read a certificate of analysis, and for our approach as a whole see Lab Testing & Quality.
All products are sold for laboratory research use only.
