98% vs 99% peptide purity: what the difference does and does not tell you

What the percentage actually measures

GenScript describes peptide purity as usually determined by reversed-phase HPLC or UHPLC, and as the percentage of the target peptide compared with impurities that absorb at 220 nm. [1] Bachem says purity is typically assessed by UV detection at 210 to 220 nm, with the main peak area relative to the total area of all peaks reflecting purity. [3]

Two consequences follow. First, purity is a ratio of peak areas, not a weight of material. Water and counter-ions do not appear in it; Bachem describes net peptide content separately, as the percentage of peptide relative to non-peptidic material, mostly counter-ions and moisture. [3] Second, anything that does not absorb at the detection wavelength, or that elutes under the main peak, is invisible to the figure.

The arithmetic: impurity per weighed sample

The table uses fictional numbers: a 10 mg weighed sample with 80% net peptide content, so about 8 mg of peptidic material. It assumes impurities respond to UV like the main peptide, which real impurities may not, so treat the mass column as an approximation.

• The step from 98% to 99% halves the visible impurity. The step from 95% to 98% removes more than half of what remains.
• Net peptide content often matters more for how much target peptide you weigh. Moving from 70% to 80% content changes peptidic mass by 1 mg in 10 mg, far more than the 0.08 mg between 98% and 99% purity.
• GenScript notes that lot-to-lot variability becomes greater the lower the purity. [1]

Why the same lot can read 98% or 99%

An HPLC purity value belongs to its method: column, gradient, run time, wavelength and how peaks are integrated. A steeper gradient or shorter run can merge a close impurity into the main peak and raise the reported purity. Integration settings decide where small peaks start and end.

ICH Q2(R2) says specificity in a separation method can be shown by the resolution of the two components that elute closest to each other, that representative chromatograms should be used to demonstrate it, and that where one procedure does not discriminate enough, a combination of two or more is recommended. [4] That is the standard a pharmaceutical method is held to. A research certificate rarely shows its validation, which is why the chromatogram and method details matter.

Why a single percentage is weak evidence

A number on a product page is a claim about some lot, at some time, by some method. Without the chromatogram you cannot see whether the main peak is sharp and well separated, whether there are shoulders, or how many small peaks were integrated. Without the method you cannot compare two certificates. Without the lot number you cannot tell whether it describes your material.

GenScript says it provides a certificate, mass spectrometry and HPLC data for each peptide, and uses ESI mass spectrometry to confirm molecular weight. [2] Thermo Fisher notes that HPLC-UV is sufficient to estimate yield, but that a mass spectrometer is required to look at impurities. [5] A purity percentage with no identity result does not show that the main peak is the peptide you ordered.

Do you need 99%? How suppliers frame grades

GenScript's published purity guide groups peptides into tiers at 75% or more, 85% or more, 95% or more and 98% or more, and lists typical research applications for each. For example, it lists quantitative receptor-ligand interaction studies and NMR studies at 95% or more, and crystallography and structure-activity relationship studies at 98% or more. [1] Its tiers stop at 98%; it does not define a separate 99% grade.

Choose the tier from your method's sensitivity to impurities, then ask for documentation that supports the tier. A lot-matched 98% certificate with chromatogram, method and mass spectrometry identity tells you more than an unsupported 99%.

• Ask for the integrated chromatogram and peak table for your lot.
• Ask for the column, gradient, run time and wavelength.
• Ask for an identity result by mass spectrometry for the same lot.
• Ask for net peptide content if you will weigh the material for a quantitative method.
• Record "purity method not stated" rather than accepting a bare percentage as equivalent to a documented one.