natural compound identification

[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.

[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.!

[LEKHA SOMAN]

Sir,
I have used C18 COLUMN and its dimention is 250* 20mm,10micro.

[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?

[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.

[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?

[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

[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.