Chromatography Forum

Dear all,
I am carrying a derivatization process and analysing the product by HPLC with fluorescent detector on C8 column (Eclipse XDB- Zobrax, 150x4.6mm). I don't know the structure of the product but speculate that it is a base. Followings are the different chromatographic conditions I have run:
1. 80% MeOH-acetate buffer pH 5.5 : can't see the peak after 30 min.
2. 80% MeOH-acetate buffer pH 4: peak at 15.1min
3. 80% MeOH-phosphate buffer pH 3.5: peak at 14.5 min
4. 80% MeOH-phosphate buffer pH 3: peak at 7.3min
4. 80% MeOH-phosphate buffer pH 2.5: peak at 4.6min
I think my compound is too sensitive with the change of pH at range < 3.5, so for the robustness of the method I choose the pH 4 for the buffer. I increased % of MeOH to 90% with the hope to shorten the retention time. Surprisingly, the peak splits to 2 peaks, 1 peak at 6.9 min and 1 peak at 13.1 min; the sum of the area is similar to the area of the peak at 80% MeOH.
I also tried with acetonitrile and mixture of acetonitrile and methanol and got similar results (with pH from 2.5 to 3.5, the retention time changes about 50% with 0.5-unit difference of the pH and the peak splits when the % of organic solvent is high).
I don't know which the best pH is for my mobile phase and why my peak splits like that?
I am waiting for suggestions from all of you. Thanks in advance!

Three suggestions:
The sample could have aged or degraded.
The dual peak comes from a mismatch between the sample solvent and the mobile phase.
The retained peak was always there, but you did not see it in 80% MeOH because the retention was too large.

Dear Uwe,
Thanks for your suggestion. However, my samples are always freshly prepared so I am sure it isn't the degradation. It may due to the mismatch of the sample solvent and the mobile phase. I can't change the sample solvent because I have to inject the sample directly after derivatization process. So do you think if I keep working with low % of organic solvent (80% or less), it should be OK? Could you please suggest me what is the pKa of my compound? Why pH from 2.5-3.5 of the buffer made the big change the retention time like that? Which is the best buffer for a robust method in my case?
By the way, which buffer is the best for buffer from 3.1-3.8 because phosphate buffer only for pH <3.1 and acetate buffer for pH from 3.8 to 5.8?
Alll your suggestions are greatly apreciated!

It would help if you tell us what derivatizing reagent you used, what you know about your unknown, and whether the two peaks show the same excitation/emission behavior.

From what you told us I think that the second suggestion of Uwe is the most likely cause of your problem. It looks like you have a good method for your compound (i.e. pH 4 and 15.1 min of retention time).

As you just want to increase your throughput at this time and organic solvent or pH changes can not be performed I would rather play with the flow rate and/or the length of the column (i.e. maybe only 3-5 cm would be OK) and/or change to a C4 instead of a C8.

By the way formate buffer should do the job for the pH you want to achieve.

Dear TOT,

Please conseder 2 isomers originated from native compound or from
fluorescence-labelling compound

As Kostas has mentioned, formic acid might be a good choice for the intermediate pH range. It has a pK of 3.75, one unit less than acetic acid.

TOT,

As Uwe suggests, your problem could be related to a mismatch of your sample solvent and your mobile phase. What is your sample dissolved in? Do you have more solvent in your sample than in your mobile phase? This can sometimes cause split peaks although it more often causes peak shape distortion. What is the pH of your sample? If it's dramatically different than your mobile phase, this could also create the problem you describe.

Thanks all for suggestoins.
It was really the mismatch of my sample and mobile phase, my sample is at pH 10 and the mobile phase with buffer pH3. I am trying to increase the pH of my sample now, hopefully, it will be better.
Best regards,
TOT.