Chromatography Forum

Hello to all,
I'm trying to develop a method for impurities determination. I'm using a mobile phase with 50% ACN - 50% H20 and a solvent with 30% ACN. The injection volume is high, 300 μl because the low concentration of my product. When i run in HPLC the ref solution of 1% (dilution 1ml of test solution to 100 ml with solvent) then an appropriate peak displays in my chromatogram. When i run a spiked sol 1% (dilution of 1 ml of test solution to 100 ml with placebo) then the peak of interest is poor with very low area. I noticed that the baseline with placebo is higher so the baseline covers my peak. If i use lower injection volume then the peak improve but i cant use lower injection volume because of my products low concentration. Can you help me how can i fix my baseline without modify the injection volume? Or if there is any other solution i can try?
Thnx in advance

I noticed that the baseline with placebo is higher so the baseline covers my peak.

Does that mean that your analyte peak is lost in the tail of a placebo peak? If you could post a link to a chromatograms (analyte only, placebo only, and spiked placebo), it would be easier to advise. Instructions for linking are here: viewtopic.php?f=1&t=2617

If it is a case of mass (of the placebo) overload, then changing the injection volume won't help; what you would need to do is to improve the resolution between the interfering placebo peak and your analyte.

If it is a case of volume overload, you may be able to improve things by using a weaker diluent (100% water if possible). Your current diluent is already weaker than your mobile phase, which is a good thing, so I don't know how much additional improvement is there. All you can do is try.

Thank you for the fast response Tom. Well today i tried lower injection volume and i got the ideal chrom with 50μl inj volume. The area of the peak is far too low so i must try something else. My products formula includes high amounts of NaCl so i think thats the reason why at 2mins appears a huge peak in my chrom (overloading the column) which affects my analyte at 9mins. I'm thinking of trying to start my method with 80-20 H2O-ACN so get rid of the salt and then change to 50-50 for elute my analyte. What do you think of this idea?
Thank you again

I'm thinking of trying to start my method with 80-20 H2O-ACN so get rid of the salt and then change to 50-50 for elute my analyte.

I generally try to avoid "step" changes (differences in mixing characteristics can make them difficult to transfer), but in this case I think it is worth a try. The other thing to try might be to use less ACN in the diluent and see if that lets you increase the volume again.

We'll I tried changes in solvent,even 100% water, with no improvement. I have good results using as mobile phase 20% ACN for at least 5mins and change to 50% ACN. So now I just have to make some small changes to optimize the gradient to display the rest impurities peaks in an acceptable time.

Glad to hear it's working out!