Chromatography Forum

Hello,

I am currently trying to separate some steroids for quantification but my peaks are too broad and thus the LOD is not good enough for what I need.

I am using a 100mm PFP column with a gradient of methanol (65-85%) and water. The flow rate is 0.4 ml/min.

At the moment I am getting peaks about a minute wide. rate from 0.2-0.4ml/min already, have played around with column temperate and also the pH of the mobile phase but none of these have helped.

I am using an Agilent 1200 HPLC system with a BIOQ Triple quad MS.

If anyone could give me any ideas on how to improve my chromatography I would be really grateful.

Thanks,

Amy

Besides 100 mm, what are the other dimensions of your column? Particle size? What are the retention times of your peaks?

Hi,

The column is 100x2mm and is 3 µm. The retention times are currenty 3mins and 5mins.

Amy

You’ll need to describe the gradient profile, as well as sample solvent and injection volume.
Also, have you seen this method somewhere and are trying to adopt it, or are you developing a method from scratch?
Finally, are your analytes ionisable or neutral?

Best Regards

In addition to gradient profile (statrting conditions, time when gradient begins, dwell volume of system, time when gradient ends, how much time is alloted for re-equilibration, etc.), what is the concentration of the steroid standard that you are injecting?

and what about the picture which is worth more than a thousand words?

The gradient is:

0mins - 65% MEOH
2 - 85%
8 - 85%
9 - 65%
12 - 65%

The system gets three mins or so to reequilibrate back to the starting 65%.

I am pretty much developing the method from scratch, the analytes are ionisable.

I am currently injecting standards of 20ng/mL.

You didn’t mention the nature of the sample solvent. What is it?
Also the injection volume. How many µL do you inject?
And then most importantly: You write you’ve played around with pH but it didn’t make a difference. But how did you play around with the pH? Please share the pH values you’ve tried and how you achieved them (i.e. buffers and concentrations).
More pH talk: Since your analytes are ionisable, they’re most probably ionized, at present (I assume the mobile phase is currently water/methanol). So, you’ll need to suppress their ionization so that they are completely neutral. That goal should dictate the pH you’ll have to choose for further optimization of your brand new method

Best Regards

The sample solvent is methanol (with water). I make it up to mirror the % methanol of the mobile phase at the beginning. I am currently injecting 10µL.

I played around with the pH by increasing the % formic acid in the mobile phase. At 0.1% the pH was 4 and at 1 the pH was 2, the results were worse at the lower pH.

Best wishes,

Amy

Try some buffer instead of formic acid. Could be 0.05 M Sodium acetate at pH 4.7.
Please note that acetate absorbs light at shorter wavelengths (i.e. < 230 nm).
If a buffer doesn’t help, it might be your column that isn’t up to the job. Have you tried another column by the way?

Best Regards

I just went back and read the initial post. It appeared to me that you used MS detection. In that case prepare ammonium acetate buffer rather than sodium.

Best Regards

For the steroids that we work with, acetonitrile gives much better peak shape compared to methanol. A little bit of methanol (~10%?) seems to help ionization a bit. For steroids that respond better in positive mode (androgens), we use 0.2% formic acid in both the aqueous and organic parts of the mobile phase (gradient). For steroids that make negative ions, we use 2mM ethanolamine (ph~8). Just make sure your column is OK to go to that pH.

The peak shape will affect sensitivity to a certain extent, but you should also concentrate on optimizing the MS detector. Have you done any tuning with these compounds to determine optimal parameters on your detector?