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natural compound identification
Posted: Thu Nov 07, 2013 5:37 am
by LEKHA SOMAN
hi,
I have run hplc for unknown plant compound using water:ACN (60%:40%), and I am getting lot of peak but when I am repeating it for its repeatability I am not getting the same RT and intensity.
Re: natural compound identification
Posted: Thu Nov 07, 2013 6:19 am
by Gerhard Kratz
Maybe a buffer is missing while not all compounds are neutral in your sample. But please give more Details, what column, column dimensions etc.!
Re: natural compound identification
Posted: Fri Nov 08, 2013 6:08 am
by LEKHA SOMAN
Sir,
I have used C18 COLUMN and its dimention is 250* 20mm,10micro.
Re: natural compound identification
Posted: Fri Nov 08, 2013 7:31 am
by Gerhard Kratz
RP-HPLC analysis of phenolic compounds and ... - ResearchGate
www.researchgate.net/...HPLC_analysis...plant_extr...
von L Baldriµnovµ - RP-HPLC analysis of phenolic compounds and flavonoids in beverages and plant extracts using a. CoulArray detector. Methods were developed for the analysis ...
Please have a look on the above mentioned publication. They have used 5mmol Ammonium Acetate as buffer, adjusted to pH3 for example with formic acid. Why you used 10µm particles in your column?
Re: natural compound identification
Posted: Sun Nov 10, 2013 6:09 am
by Arne
There may be ghost peaks in your subsequent runs, i.e., peaks from a previous injection that take a long time to elute. Also, make sure you let the system run for a while before your first injection.
Re: natural compound identification
Posted: Sun Nov 10, 2013 8:03 pm
by artirm
Sir,
I have used C18 COLUMN and its dimention is 250* 20mm,10micro.
Do you have any specific reason to use a preparative column for analytical purposes?
Re: natural compound identification
Posted: Mon Nov 11, 2013 7:49 am
by Chris0000
Hello
A variation of the retention time can be caused by different issues
For example bad check-valves, compounds of the last run remaining on the column....
Lots of peaks is normal for natural compounds - are you doing some sample preparation?
best regards
Chris
Re: natural compound identification
Posted: Mon Nov 11, 2013 6:46 pm
by James_Ball
hi,
I have run hplc for unknown plant compound using water:ACN (60%:40%), and I am getting lot of peak but when I am repeating it for its repeatability I am not getting the same RT and intensity.
You may simply want to add a wash out step to the end of your run where you raise the organic composition of your mobile phase to 20%water:80%ACN. In many plant samples there will be oils and fats that will need more organic to elute them from the column. I analyze samples of broccoli seed extracts and we have to do this to remove the oil from the column or we get interfering peaks on following injections.