HPLC
High-performance liquid chromatography. It tells you what share of the detected material is the main peptide. It does not tell you how much peptide is in the vial.
High-performance liquid chromatography (HPLC) pushes a dissolved sample through a packed column under pressure. Different molecules stick to the column packing to different degrees, so they come out at different times. A detector at the end records each one as a peak on a chart called a chromatogram. The time a peak appears is its retention time; the area under the peak reflects how much of that substance was detected.
For peptides the usual setup is reversed-phase HPLC: a column coated with oily C18 chains, a mobile phase that moves from water to acetonitrile, and a small amount of trifluoroacetic acid (TFA) to sharpen the peaks. Detection is by ultraviolet light at around 214 to 220 nm, where the peptide bond absorbs.
What "98% purity" means
Purity by HPLC is usually area percent: the area of the main peak divided by the area of all peaks the detector saw. A result of 98 percent means 98 percent of the UV-absorbing material that came off the column was in the main peak. The other 2 percent is related substances: truncated chains, chains with a missing or extra amino acid, oxidised or deamidated forms, and other by-products of synthesis or storage.
| Question | Does HPLC purity answer it? |
|---|---|
| What share of the detected peptide material is the target? | Yes |
| How many milligrams of peptide are in the vial? | No. That is peptide content, a separate test |
| Is the main peak actually the right molecule? | Not on its own. Identity needs mass spectrometry or a reference standard |
| Is there water, salt or counter-ion in the powder? | No. These are invisible at the UV wavelength used |
| Is there endotoxin or bacterial contamination? | No. Needs an endotoxin test and a sterility test |
How to read a chromatogram
A useful report shows the chromatogram, not just the number. Look for one tall main peak and small side peaks. Check that the retention time of the main peak matches a reference standard, if one was run. Note the date and batch number, and whether they match the vial. A purity figure from a different batch, or a chromatogram with no batch at all, does not describe the vial in your hand.
Two peaks can also overlap. An impurity that comes off the column at nearly the same time as the main peptide can hide inside the main peak and inflate the purity figure. Pairing HPLC with mass spectrometry, as LC-MS, helps catch this, because each peak then carries a mass.
Purity versus content
This is the most common misreading. A vial labelled 10 mg with 99 percent HPLC purity does not hold 9.9 mg of peptide. A freeze-dried peptide also contains water and counter-ions such as acetate or TFA left over from synthesis and purification, and none of those show on the UV trace. The salt forms page explains why a milligram of salt is not a milligram of peptide.
When you add a vial to the app's vial tracker, you can attach its batch and source, so the purity report and the doses drawn from it stay together.
Related terms: purity testing, certificate of analysis, mass spectrometry, peptide salt forms. Back to the glossary.
Questions people ask
What does HPLC purity mean?
The share of the UV-detected material in a sample that sits in the main peak, usually reported as area percent. The rest is related impurities.
Does 99 percent purity mean 99 percent of the vial is peptide?
No. HPLC purity does not measure water, salts or counter-ions, so the peptide content of the powder can be noticeably lower than the purity figure.
Can HPLC confirm a peptide is what the label says?
Not on its own. It needs a matching reference standard or mass spectrometry to confirm identity.
Why is TFA used in HPLC?
Trifluoroacetic acid is added to the mobile phase to sharpen peptide peaks. It is also why peptides purified this way often end up as TFA salts.
Sources
- HPLC analysis and purification of peptides (Mant et al.), Methods in Molecular Biology, 2007
- The role of counter-ions in peptides: an overview (Sikora et al.), Pharmaceuticals, 2020
- Mass spectrometry-based proteomics (Aebersold and Mann), Nature, 2003
