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For the quantification of non-volatile substances, HPLC is very well suited. But in order to be able to determine the content, comparative values are necessary. These are obtained by measuring a standard whose material is known. The retention time and the peak area can then be referred to the data of the substances from the sample.
In many cases, such pure substances which are suitable as standard exist, but this can be difficult, for example, with natural products. They may be mixtures of isomers or racemates. Therefore, purity and, if necessary, content must be examined.
If there is a need for a standard mix, the individual components must also be exactly identified. There are two possibilities for calibration: one can summarize the surfaces of all peaks and correlate the weight of the substances with the sum. The other possibility only considers one, usually the largest peak for calibration. This is possible as long as it is exactly the same substance; Other batches can lead to deviations and thus to incorrect results. In the case of a batch change, it is necessary to include corrective factors to make the results comparable.
In summary, quantitative results can only be achieved with the use of a defined standard which are considered to be reliable and reproducible. Such standards are either commercially available or have to be quantified themselves.