What HPLC purity does and doesn't tell you

What HPLC measures

HPLC separates the parts of a sample by pushing it through a column under pressure, then records how much material comes out at each point in time. Different molecules move through the column at different speeds, so a mixture separates into distinct groups as it comes out, each one called a peak. How big a peak is shows how much of that ingredient was in the sample.

A purity result is a ratio: the size of the peak for the target compound, divided by the combined size of every peak the lab's detector picked up, written as a percentage. "99.2% purity" means that one peak made up 99.2% of everything detected.

Four things a purity percentage does not establish

It does not confirm identity. HPLC identifies a peak by comparing how long it took to come off the column against a known reference sample run the same way. Two different molecules can, in rare cases, take almost the same amount of time. Purity testing assumes it already knows which peak is the target; it doesn't independently prove that on its own. That's what mass spectrometry is for. See identity testing by mass spectrometry.

It does not confirm the exact amount or total weight. A 99% purity reading means 99% of what the detector saw was the target compound. It says nothing about how many milligrams are actually in the vial.

It does not confirm sterility. Purity and sterility are measured by completely different methods. HPLC has nothing to say about whether the sample carries live bacteria. See endotoxin and sterility: two different questions.

It does not confirm the endotoxin level. Same reasoning: a separate test measures endotoxin, and a high purity result carries no information about it either way.

Why the method used matters

HPLC purity figures are usually reported one of two ways, and the number they produce isn't the same kind of measurement:

• UV/area-percent purity reports what share of the light-absorbing material in the sample is the target peak. This is what's most commonly quoted, and what "HPLC purity" usually means unless stated otherwise.
• Mass-based purity accounts for things that don't show up the same way, like water content or leftover salts, and can report a different, often lower, number for the exact same sample.

Two certificates reporting different purity numbers for material that's otherwise identical aren't necessarily disagreeing. They may just be reporting two different kinds of measurement. See why purity percentages are not comparable across labs for how the method and reporting choice change what a number means.