Area percent purity (revision 10)

Area percent purity is the integrated area of the principal peak in a chromatogram expressed as a percentage of the total integrated area of all peaks. It is the figure almost always meant when a research-peptide certificate reports "purity", and it is reported without further qualification often enough that its limitations are worth stating plainly.^[1]

The measure has one defining property from which everything else follows: it is relative to what was detected, not to what was weighed. A species that does not elute, does not absorb at the detection wavelength, or co-elutes with the principal peak contributes nothing to the denominator and therefore cannot reduce the figure. Water, counterion and inorganic residue are invisible to it in the ordinary case, which is why a vial can honestly carry a certificate reading 99.2% and contain substantially less than 99.2% peptide by mass.^[1]

Area percent is nevertheless the right measure for the question it answers, which is how much of the material that eluted was the intended species relative to everything else that eluted. Read as a purity ratio it is informative; read as a mass fraction it is simply a different quantity, and that substitution is the commonest analytical error in this field.

A sample is dissolved, injected onto a reverse-phase column, and eluted under a gradient of increasing organic modifier. A detector — most often ultraviolet absorbance at 214 nm, where the peptide bond absorbs — produces a signal against time. Peaks are integrated, and the principal peak's area is divided by the sum of all integrated areas.

Four choices in that description change the answer, and none of them is usually stated on a certificate:

• The gradient. A shallow gradient resolves species that a steep one runs together. Two laboratories can obtain two honest and different figures for one vial by choosing differently here.
• The detection wavelength. At 214 nm the peptide bond dominates and most peptide-related impurities are seen. At 280 nm only aromatic residues respond, and a related substance without one is invisible.
• The integration rule. Valley-to-valley and drop-line integration assign the area under an unresolved shoulder differently, and the difference lands entirely on the principal peak.
• The integration threshold. Peaks below the threshold are not counted at all, so raising it raises the reported purity without any change to the material.^[1]