Hello to all,
I am encountering the peak separation problem in my HPLC experiment. i have attached the chromatogram with this post.
Column - 25 cm X 4.6mm, 5u particle size
Solvent A - 100% water with 0.05% HFBA
Solvent B - 60% acetonitrile +40% water with 0.05% HFBA
Elution condition- Binary gradient
5min-0% solvent B
6min- 10% solvent B
30min - 22% solvent B
31min - 100% solvent B
41min - 100% solvent B
42 min - 0% solvent B (100% solvent A)
52 min - 0% solvent B (100% solvent A)
(I hope this kind of gradient elution is correct for this column)
As you see the chromatogram, the separation is not good between large peak with 8.9min retention time and small peak with 10.08 min ( .
I am interested to quantify these peaks including peaks at 17min and 19 min . Where as the peak at 14.9 min is ref.std
In an recent experiment i tried with increased con. of HFBA (0.1%) in solvent B but with no success (if it is required i can also attach the chromatogram).
Is there any one got better idea to trouble shoot this problem , I will appreciate the future comments.