Internal Calibration Standard Curve for Amine Concentration Determination

By: Dr. Teeradet Supap

This post, we explain how the concentration of an unknown amine is determined using the internal standard curve method which is known to help reduce run to run/day to day inconsistency, thus improving the analysis accuracy.

From the graphical abstract;

Step 1 is used to prepare standard samples containing different concentrations of the amine of interest whose solutions are spiked with the same concentration of an internal standard compound. Selection of the internal standard must be done carefully so that the chosen standard has no or little interference to the amine in the sample. This means the standard should be structurally similar to that of the amine to ensure the compatibility and inertness when present in the sample. The standard must also chromatographically separate completely from the amine peak and all other species that are also present in the sample. This is essential for the accurate determination of the internal standard and amine responses used to generate the calibration curve.

Step 2 involves a use of a chromatographic instrument such as Gas Chromatograph (GC) or Liquid Chromatograph (LC) to analyze all the standard samples made from Step 1. The instrument separates the internal standard and the amine whose responses are displayed as peaks in the chromatogram. Since, the peak size measured as peak area/height, is directly proportional to the compound concentration. The internal standard peak area remains the same in all samples while that of amine increases with an increase of the amine concentration.

Step 3 uses the amine and the internal standard peak area ratios and their corresponding concentration ratios to generate the calibration curve. The curve also provides the equation that mathematically expresses the relationship of the peak area and concentration ratios useful for the concentration determination of any unknown amines. This area ratios are used to compensate for the above-mentioned run to run/day to day inconsistency that occurs due to drifts of the instrument run conditions.

In Step 4, the unknown amine is prepared similarly to those of the standard samples with the identical concentration of internal standard being added. The unknown sample is subsequently analyzed to obtain the peak area ratio of the amine and the internal standard. The exact concentration of the unknown amine is finally obtained by calculation using the calibration curve equation generated from step 3.