Peak area

Definition

In chromatographic analysis, a peak represents the detection signal produced as a compound exits the column. The peak area is the integral of the signal under the peak (the sum of all signal values across the width of the peak). Peak area is directly proportional to the amount of compound in the sample; a larger amount of compound produces a larger peak area. This relationship is exploited to quantify how much of a target compound (such as a peptide) is present in a sample. A standard is prepared containing a known amount of the target peptide; its peak area is measured, producing a calibration curve that relates peak area to the amount of peptide. Unknown samples are then analyzed, their peak areas are measured, and the amount of compound is calculated from the calibration curve. For peptide purity assessment, the peak area of the target peptide is divided by the sum of all peak areas (which represents the total amount of all compounds), yielding the percentage purity. For example, if the target peptide peak area is 95 units and the total of all peaks is 100 units, the sample is 95% pure.

Peak area calculations are performed by the chromatography instrument's software, which automatically integrates the signal under each peak. The software must be configured correctly to properly baseline the chromatogram (subtract the background signal) and integrate peaks accurately. Manual integration is sometimes necessary if peaks are poorly resolved or if the automatic integration is incorrect. Proper integration is critical because errors in integration directly translate to errors in quantification.

For regulatory purposes, peak area must be measured using validated chromatographic methods with known accuracy and precision. Regulatory agencies require that laboratories demonstrate method validation, including accuracy (whether measured values match true values), precision (whether repeated measurements are consistent), and linearity (whether the relationship between concentration and peak area is linear across the expected range). For peptides, the most important regulatory requirement is that the target peptide has at least 95% peak area purity, indicating that the sample is at least 95% pure.