Chromatography Forum

Hey there,



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!

Cortical wrote:

• 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?

1) My first suggestion would be to first inject the two new compounds (if you have them isolated) and see where they elute. No point in trying to improve the resolution between peaks 1 and 2 if you don't need to.

2) 3% should be manageable, but as a personal rule, I don't ask the pump to blend mobile phases in increments smaller than 5%. Your mileage may vary.

3) An increase in length would help but not as much as you think. Look up the fundamental resolution equation. The N term - plate count - is increased when you increase the column length but you'll see it's only by a square root. When you need to increase resolution, increasing N isn't the best first approach.

Some things that might help us help you: a time axis on your chromatograms as well a the current resolution between Peaks 1 and 2.