I am working in glycerol oxidation and I am using HPLC for analysis but I have found that the peak of glyceraldehyde overlapped with another peak which is unknown for me. I need to assess quantitatively the amount of glyceraldehyde in my reaction.
1. you can develop a new HPLC method
2. try to make spiking of your reaction sample with glyceraldehyde reference to calculate relative retention time (RRT) of glyceraldehyde.
3. you can determine glyceraldehyde by use LC/MS/MS with MRM method to check if any traces of glyceralgehyde exist or not
4. you can use LC/MS/MS with MRM method to quantitiative of your product
You an try changing using gradient elution. It should get rid of the overlapping peak if properly done. Or like Naser have said, use coupled methods to analyse the compound.
thanks so much for answering me , actually I have changed all the parameter during calibration of glycerol derivatives and I got the best method for separation because as you know the products which is expected to obtain from glycerol oxidation all almost same function such as glyceric acid , oxalic acid , tartonic acid, glycolic acid dihydroxyacetone , and glyceraldehyde . and when i make the reaction the peak which is attributed to glyceraldehyde was overlapped with new peak which is also unknown for me. and i saw this when diluted the sample i mean in low concentration .other wise i saw it one peak in high concentration.
to Mr.Saleem Nasir, the method which you talked about it it is not available in our department so if you have any further suggestion i will be thankful
Probably you can separate the overlapping peaks by derivatization of glyceraldehyde with 2,4-DNPH. Due to change in polarity the retention time may vary and separation may occur. Hope it works.
ok try to do PDA test of purity of this peak at different concentration
could please Mr.Naser explain me more about PDA because I have never used it before, thanks alot
ok ... photodiode array detector can scanning your sample at all lambda 190 to 700 nm and can give you more details about your peak like
1. purity of your peak if you used 3D scanning
2. give you if any un-separated peak exist behind your peak
3. give you lambda max of your peak
you can use PDA also to get good evidance if the shoulder peak is real peak or not by 3D scannig
but you should prepare different concentration from your sample then check your sequence by PDA to get good indication about your purity and maxiumum wave length of your peak
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