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- Posts: 5
- Joined: Wed May 25, 2016 1:59 pm
After my last question was answered in such a prolific way and solved my problem, I have decided to try my luck once more. In order to describe my problem in a more practical way, I have attached three chromatograms which you can find at the end of this post.
My primary aim is to establish a method with which I can isolate and quantify peaks 1, 2 and 5 clearly and without disturbance. I started method development with a long gradient, which has now changed to two isocratic steps, and a program which looks like this (blatantly copied from my earlier post):
I am using a C18 column with 3,5 µm, 4.6x150 mm dimensions and have initially - to figure out when my compounds elute in general - run a long gradient over 20 min, with an initial 4 min 0% B @ 1 ml/min to exclude that they do not bind to the column. After I noticed that the compounds elute early, I have run an isocratic program with which I can very nicely see my peaks elute from the column.
Buffer A: 0.5% Acetic acid, pH 3.9
Buffer B: 100% MeOH
Column temperature: 40°C
Injection volume: 50 µl
8% B for 7.5 min @ 1 ml / min
10% B for 12.5 min @ 1ml / min
followed by washing and equilibration
As you can see in the chromatograms, I have included two compounds (3 and 4) which are of indirect interest to me, and I mainly wanted to exclude that they collide with any of my important peaks. This has worked out nicely, however, I was not able to separate them clearly despite running two different attempts of doing so:
- Chromatogramm 1:
8% B for 7.5 min @ 1 ml / min
10% B for 12.5 min @ 1ml / min
followed by washing and equilibration - Chromatogramm 2:
8% B for 7.5 min @ 1 ml / min
9% B for 12.5 min @ 1ml / min
followed by washing and equilibration - Chromatogramm 3:
8% B for 20 min @ 1 ml / min
followed by washing and equilibration
As you can see, the problem gets even worse and I now observe a total fusion of the peak. I now have the following questions:
- Since I have to introduce two more compounds which might elute in the range of peak 1 and peak 2, how can I achieve a better separation of peaks 1 and 2, as well as 3 and 4?
- How low can the amount of MeOH in a program be? Are percentages of e.g. 3% manageable?
- If I had to switch columns to get a clearer separation of peaks, would an increase in length help?
I'd be grateful for any help!