Hi everyone
Can i identify Peaks of complicated crud extract by congruent retention times compared with standards without using other methods ?
Thank you in advance
For proper identification it is absolutely necessary to use combination of different techniques and methods: HPLC retention of standard compounds, UV spectra, different columns and mobile phases, hydrolysis and MS/MS fragmentation. By well-known compounds and matrices usually retention and UV spectra are sufficient.
Best regards
Not really. Similar compounds can have the same retention times and same wavelength maxima.
Depends completely on your detector. If you have a MS/MS then the answer is probably. If you have RI or UV then the answer is absolutely no. HPLC is a relatively low resolution chromatographic technique (as compared, for example, to HRGC) and as Bruce points out you get a lot of peak overlap.
For crude extract, you better go with HPTLC technique, you can not only compare the Rf value but also can get the spectra of that particular component from the same plate and you can quantify the compound in your extract by densitometric analysis. And regarding similar compounds which have almost similar Rf value, you can go for 2D chromatogram development process for better separation.
HPLC analysis can be used for identification of compounds using Reference compounds by comparing their Rt, this identification is confirmed by Spiking technique) spiking the standard reference to the extract to indicate the accurate Rt. of peak in the extract.
For proper identification it is absolutely necessary to use combination of different techniques and methods: HPLC retention of standard compounds, UV spectra, different columns and mobile phases, hydrolysis and MS/MS fragmentation. By well-known compounds and matrices usually retention and UV spectra are sufficient.
Best regards
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts