Chromatography Forum

Hi, I'm relatively new to HPLC.

How would you deal with this kind of problem? Where would you start?

I'm having trouble with the peaks I'm getting. They seem to overlay, and looked likes a two-headed peak wanting to separate. (see image 1 in this link: https://drive.google.com/file/d/1uFiJCC ... sp=sharing)

Now, I know I can work on the resolution of the 2nd and the 3rd peaks. But based on my previous runs, the peaks are single and nice in the next image. (see image 2: https://drive.google.com/file/d/1Y1vQRd ... sp=sharing)

Here's some details:

Shimadzu HPLC-20AD
Mobile Phase - 25% Methanol, 75% Phosphate Buffer
Total flow - 1.5 mL/min
column - Chromolith® SpeedROD RP-18 endcapped 50-4.6 HPLC column

I used a mixed standard of NDS isomers in the chromatograms presented.

Thank you very much.

Hi Marino,

This issue looks like your isomers/analytes are partially ionized. I know you mentioned a buffer solution for your mobile phase, now is the mobile phase buffered to what pH? Additionally, what are your target analytes moieties? Are any of them acids or bases? These types of split peaks can be due to some of your analyte retaining as a neutral molecule, while the other portion is eluting ionized (simple explanation). This will distribute your analytes in a larger range where some may experience more time as a neutral molecule (better retention for hydrophobic stationary phases), and some spend more time charged (better retained in water).
Another potential issue may be your lamp's lifetime, but I'm not certain about this suggestion-- it wouldn't hurt checking the hours though. My Shimadzu lamp is typically good for 2000 hours, however the lamps generally start going bad after two years with or without use.

Tyler Smith.

What are you analyzing? You can use mixed-mode chromatography if any of your compounds ionizable and can get a much better separation, efficiency and peak shape. Contact me if you want to discuss your options

If conditions are the same for the two chromatograms, it is very likely that the column is irreversibly damaged.

When all the peaks in the chromatogram show the same symptom (in this case, partial splitting) it is likely that the problem occurs while the peaks are still together (i.e., at or prior to the head of the column).
If this were a conventional column, I would suspect a void space at the head of the column. This isn't supposed to happen with monolithic columns*, so the next most likely source is excessive pre-column void volume (possibly an improperly assembled end fitting?).
If the column has seen a fair amount of use, the quickest solution would be to simply replace it, being careful to push the tubing all the way down into the end fitting before you tighten the fitting.
If you want to be systematic about it, loosen and tighten all the pre-column fittings (everything between the outlet of the injector and the inlet of the column) and re-test. If the peaks now look OK you have fixed the problem and learned a lesson. If the peak shape problem persists, that indicates a bad column which should be replaced.
* yeah, it's not supposed to happen, but it does!

Hi all. Thank you very much for your ideas. I changed the pre-column filter and made sure that no space in between the tubing and the column. And this is my chromatogram now.

https://drive.google.com/file/d/114oV7v ... sp=sharing

Thanks again.

And this is my chromatogram now.

The peak splitting has disappeared. However, the peak tailing is higher than it was on the "ideal chromatogram", the retention times are reduced 2-fold, and the resolution is lowered.

I would suspect a void space at the head of the column. This isn't supposed to happen with monolithic columns*
* yeah, it's not supposed to happen, but it does!

It is supposed that the void space is formed between the monolith and the wall of its PEEK housing.