I ran a non-trageted HPLC, because I have no standards to compare with. Can I know the compounds in a certain retention time and wavelength? How?
Depending on the conditions of separation you can say something about the polarity of the compound and something on the basis of its UV spectra. That's all :)
Regards
G
Not really! Many classes of chemicals may have the same UV maximum (PDA/DAD scan), but the chromatographic retention time will be different for each compound. In addition, the detector response for each compound will be different so without the 'pure' (>99%) reference standard you can't say anything.
If you don't have first information about the sample there is no way you can identify the analyte in question without reference. Library data base May help to some degree. No reference no qualitative analysis in HPLC. Retention time will not suffice the need. LC-MS hyphenated technique will certainly help.
How will I be able to analyze my data ? Shall I compare peaks of same retention time?
You wasted your time! If you have an MS/MS detector you might be able to use 'collision induced daughters' (CID) for an identification. But this won't allow quantification since there is NO information on the detector response.
You will have to have an organic chemist make a ~99% pure reference standard for quantification About 500 mg will do.
Dear Aya, Multiple compound can have same retention time and UV spectra. Therefore, I suggest that if you are getting a single good peak, then subject it to mass by direct infusion. Alternatively, if you are getting multiple peak, then you should collect the peak coming out of the detector end concentrate it and subject to mass. Your last option is to subject sample for LCMS.
I agree that LC/MS is the best option. However, some writers pointed out some questions that may be addressed and help you.
First, do you have the whole UV/Vis spectrum for each acquisition time window?
Second, are your peaks pure substances or mixtures? Can you change chromatographic conditions and your peaks stay the same? The whole UV/Vis spectrum stays the same along each chromatographic peak? The latter condition would be a good indication of peak purity.
Third, if you have any kind of standards that - you suspect - may be the same substance of a chosen peak, you can collect its spectrum in a chromatographic run with the same conditions and check not only retention time, but also the overlapping of UV/Vis spectra from your standard and the analyte peak. If they are the same pure substance, their spectra must be identical.
However, your peak may have contaminants from the original sample and differ. And even if your peaks superimpose each other, you wouldn't be 100% sure without another independent confirmation, although you would have a good indication. This rule applies to mass spectra as well, but mass spectra TOGETHER with retention time and UV/Vis overlapping spectra would probably suffice for any journal.
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