Question

Explain how to find the concnetration of an unknown when the when five reference have a mixture of five different compounds. by internal standard method

Answer 1

AOAC international methods are specific for each compound and chemical reactions. So, without the prior information about the compound/ molecule/ chemical species, it won’t be possible to mentioned any specific AOAC or any other standard method/ protocol.

I assume that the sample contains an unknown compound (for instance, molecule X) and the reference has a mixture of 5 different standard compounds (for instance, A, B, C, D and E).

The concentration of unknown compound can be worked out as follow-

1. Identify the compound X.

It can be done by analyzing the absorption spectra of compound X using spectrophotometer or HPLC.

Compare the spectral properties (absorption spectra) of compound X with A, B, C, D and E.

The molecule in the standard with same absorption spectrum as that of X identifies it. For example, if the absorption spectra of X matches with that of C in the standard, the unknown compound X is C.

2. The standard solution labels the concentration of each compound.

Prepare aliquots of different concentration (say, 0.00, 0.02, 0.04, 0.08, 0.10 units, etc.) of compounds C.

3. Take the OD of the aliquots of compound C at the specified wavelength (using spectrophotometer or HPLC, whichever is the best suited one- cost, availability and protocol). Prepare the standard graph- usually done using the software coupled with the instrument itself.

4. Take the OD of the X (confirmed to be C).

Calculate the concentration of C in the unknown sample by using the standard graph for compound C.