Peak Height or Peak Area? – Which is the right choice for quantitative chromatographic calculations

Peak Height or Peak Area? – Which is the right choice for quantitative chromatographic calculations

A typical chromatogram comprises of several peaks varying in size. The height of each peak is in proportion to the amount of the particular component present in the sample mixture injected into the chromatograph.

  Ideal Chromatographic  Peaks

The chromatogram shown above is an ideal one showing symmetrical shaped peaks rising above a stable horizontal baseline. It can be seen that the peaks are well separated from one another and there are no overlap or shoulder bands. In such situations it is immaterial whether you use peak height or peak area in quantitative calculations.

Real-life sample chromatograms may not be that simple and will more than often show deviations resulting in non-Gaussian peaks due to several factors some of which are listed below:

  • Presence of interfering compounds or impurities in the sample mixture
  • Changes in operational parameters such as column temperature, flow rate of carrier stream, injection volume, etc.
  • Leakages due to septum coring or worn out ferrules, O-rings or joint fittings.
  • Detector response variation due to nature of compound
  • Residual impurities present from earlier analysis.

Due to such reasons the chromatograms can deviate from ideal peak shapes and distortions in chromatograms become apparent as shown below

                                       Distorted Chromatographic Peaks

In such cases complications arise due to unsymmetrical peak responses and unstable baselines which are not uniformly flat. Peak height based calculations will lead to errors in quantitative estimations. Closely appearing peaks cannot be integrated in a reproducible manner because neighbouring peaks or overlaps influence peak height and area integration.The same is true for peaks arising from sloping or noisy baselines.

Peak integration is a mathematical operation performed by the chromatographic software to measure the area under a peak. The area measurement is based on integration which hypothetically divides the region below the peak into several rectangles which are summed up to give the total area under the peak.

In order to define this area the software permits either manual or automatic marking of the start and end points of the peak. The baseline is then drawn between the start and end points to define the area calculation. It is important that same algorithm is used for area calculation of standard as well as sample peaks

Asymmetrical peaks often result due to peak tailing. It is difficult to reproducibly mark the endpoint of such peaks. This leads to errors in reporting of areas resulting in lowering of precision and accuracy of quantitative measurements.

Peak areas are used for most quantitative chromatographic estimations. Peak heights can vary due to distortion of the shapes such as the broadening or fronting and tailing. However, in such situations areas are not affected and show high reproducibility.

On the other hand for very small peaks resulting from trace amounts of impurities peak height calculations may be a better option as errors in small variations in making start and end point of peaks become negligible.

The answer to the choice is based on your judgement of the quality of the chromatogram. However, in a majority of situations it would be advisable to make use of peak area measurements to get the highest levels of accuracy and precision.

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  1. Dear Sir,
    I need to know how calculate done the area percentage of individual peak, can you help

    1. Manual calculation is superseded in today’s instruments. The software provides you the area under a peak in terms of digital counts.It also takes into account the best fit and takes care of the underlying parameters so you are saved a lot of calculation and personal judgements.

  2. hi sir,

    I need to know about points that you will look at when reviewing the chromatograms for a batch, for Assay results. Focus on Data Integrity.

    What manual integrations are prohibited/not allowed?how it can be detected from empower/chromaleon?

    1. Modern software takes care of best fits in calculations and you do not have to bother yourself with graphic details. Manual integrations can introduce errors arising from personal bias.

  3. Why need a signal to noise ratio in HPLC .
    And which purpose use this test.

    1. Signal to noise is very important for establishing the Limit od Detection and Limit of Quantification for any method. Usually, S/N of 3 is acceptable for LOD and 10 for LOQ.

  4. Hi Dr.
    I was wondering what is the limit of the peak area? if it less than 1000 does the area seems valid?

    1. Hi the area by itself is not god enough to decide. You need to look at the signal to noise ratio and repeatability of the response. So a peak area of 1000 will be good if the noise has an area of 100 (S/N – 10) but is noise is more it will not be.

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