Hello to all,

I am encountering the peak separation problem in my HPLC experiment. i have attached the chromatogram with this post.


Experimental conditions:
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.

Thanks
Rajeev

change the acetonitrile concentration and/or gradient rate should help

and adjust solution pH with HCl could be another option

Please indicate the type of column you are using (manufacturer and bonded phase, e.g., Zorbax ODS).

next, change the program so that the acetonitrile % changes at a slower rate. For example, change the gradient from 0 % B at time = 0 to 15 % B at 30 minutes.

consider a high efficiency column (4.6 X 150 mm, 3 um)

consider a different reverse phase column (with different selectivity). You will have to research this topic to understand it better.