Chromatography Forum

Hi
Can anyone suggest some idea for my problem?
I use phosphate buffer as my mobile phase and column is Discovery C18.Most of the times i get a hump at the RT of Main peak,Sometimes not.
We suspect that water causes that problem.Anybody else has experienced the same? and how can I overcome this?..

"The devil is in the details".

Isocratic or gradient?
100% buffer or is there an organic solvent in there?
What injection volume?
What is the diluent?
How are you detecting (if UV, what wavelength)?
What buffer concentration and pH?
Does the hump show up with blank injections?

Isocratic or gradient?
Gradient
100% buffer or is there an organic solvent in there?
Mobile A is 100% buffer; Mobile B is 50:50 Mobile A : ACN
What injection volume?
20 µl
What is the diluent?
Mobile A
How are you detecting (if UV, what wavelength)?
UV-254 nm
What buffer concentration and pH?
0.025M phosphate buffer and pH of both mobile phases 3.0
Does the hump show up with blank injections?
Yes,then in standard injection the main peak elutes on the hump

Since it is a gradient please post your gradient conditions too.
What exactly is a "hump"? Are you saying it's very broad?
If it is...sounds maybe like something that is strongly retained on the column and taking more than one gradient run to elute.

From your description, it looks to me the buffer might be the source of problem. To confirm it, adjust the equilibration time to 50% shorter and 100% longer to see if a smaller and a bigger humps can be observed respectively. It will help to use the highest quality of both phosphate salt and water for the buffer. Also you might use background subtraction to get acceptable result.

A very broad hump in gradient HPLC can result from two things:
- build-up on the column of contamination in the weak solvent
- RI mismatch between the solvents.

As XL suggested, if the size of the hump varies with equilibration time, then it is contamination. If it does not vary with equilibration time, then it is probably a refractive index effect (RI is a non-linear function of composition, which is why the baseline shift can appear as a "hump". If the hump is very broad, your best approach is to simply ignore it and let the data system deal with it (in the vicinity of the peak, the data system should treat it the same way as it would treat drift).

All UV detectors show some response to RI, but iff the hump is excessively large, try to run the same method on a different instrument of the same model (if available). If both systems show about the same amount of hump, then it's an optics design issue, and there's not a lot you can do about it chromatographically. If one of the systems looks worse than the other, then it is a service issue; if the flow cell is poorly aligned with the optical axis of the detector, proportionally more light enters the cell "off axis", and RI sensitivity is exacerbated.

Thanks Tom..You may be right.We will try to solve as per your suggestion..