Symmetrical peak question

[diggatee]

Hello board!

This is my first post on here, I looked on the FAQ's but did not see a specific answer for my problem, so here goes...

I just switched over to an R & D position from a QC lab, and this is my first method development problem: I'm developing a method for Glycine using reversed phase HPLC and an ion pairing mobile phase with 5% Methanol. My extraction solvent is simply a low pH water. The standard and sample peaks elute cleanly and without interferences, but the peak of interest isn't exactly symmetrical. It is not really tailing with a curve, it's more like a triangle with straight lines that don't point straight up. USP tailing factor of ~1.8. The pH's of the mobile phase and extraction solvent are similar, and the tailing factor comes down with lower concentration, but I don't want to go much lower than where it currently is.
Quantitation is fine, but this is my first new method and I would like it to be perfect.

Thanks

[Kostas Petritis]

If you can disclose the information, what is the ion pairing agent you use and at what concentration? Also as it is a little bit unclear, when you say: "The standard and sample peaks elute cleanly and without interferences, but the peak of interest isn't exactly symmetrical." is your peak of interest Glycine?

Have you tried to increase your ion pairing reagent and/or further decrease the Methanol concentration and at the same time decrease the lenght of your ion-pairing reagent (if you get too much retention)? What is the injected concentration of your Glycine (and what are the dimension of your column)?

[diggatee]

Kostas,

My ion pairing agent is HSA at 2.0 g/L, and my peak of interest is Glycine. I would rather not go to a higher concentration of HSA, I've found that too much salt on the system can gunk it up.
After reading your reply, I tried bleeding my mobile phase with some pure water to decrease the methanol content, but it had no effect (at least at the concentration I tried). I'm not sure what you meant by "decrease the length of my ion pairing reagent", but my retention time is only 3.5 minutes, so I'd rather not shift it back any further. Concentration of Standard is ~1.0 mg/ml and column is 4.6 x 150 mm Luna C18(2).

I think those are all the specifics of my method, but I was thinking there might be more general ideas for future reference, possibly any little tricks people have found that I might try?

Thank you all in advance for your time.

[Peter Apps]

Could you post a chromatogram ?

What is your injection volume ? With such a short retention time, and not running a gradient (?) the peak shape might be very strongly influenced by the injection.

What is the pH of your sample solvent, and are standards dissolved in the same solvent ? Is this pH the same as the pH of the mobile phase ?

Peter

[diggatee]

Peter,

Thank you for your time. Unfortunately I do not have the knowledge of how to post a chromatogram on here. My injection volume is 10 microliters, and I am not running a gradient, although I do have a wash phase at the end. I will try adjusting the injection size today to see what effect it has on the shape. The pH's of my mobile phase and extraction solvent are both 2.3 and the samples and standards are dissolved in the same extraction solvent.
I had seen an article in a magazine about the effect of pKa on peak shape, and it suggested that if my mobile phase was too basic it would produce the shape I am seeing. With that being said, I don't want to go any lower than pH 2 for this run.
A previous method for Glycine used a mobile phase of 0.1% TFA, and the peak was more symmetrical, however various other business practices dictated that I not use that mobile phase. I checked the pH of that phase, and it was roughly the same as my new one. That's when I started pulling my hair out and posted here.

Thanks again to anyone taking the time to read my tales, and thanks to those who have responded. Hope everyone is doing well today.

[tom jupille]

Based on everything you have said, it sounds like overload is the most likely problem. How much glycine are you injecting (mass) and how are you detecting?

[diggatee]

Tom,

The original method that I based developed this from used Glycine at 2mg/ml concentration. When I performed a linearity check I went from 8 mg/ml to 0.1 mg/ml and noticed that tailing and symmetry factors went down with lower standard concentration.

Unfortunately Glycine doesn't have a big spectrum and I'm viewing at 205 nm to get a good peak. I moved concentration to 1 mg/ml and peak size is only about 0.04 AU. Can I be overloading with such a small peak?

As I said before, quantitation is fine, but I'm developing this method for my former colleagues in the QC lab, and I know they will perform it and say "Look at this crappy peak" and then tease me about it. I'm thinking the method is okay as it is, I just wanted to avoid the heckling.

Thanks everyone so much for all your help, I truly appreciate it.

[Kostas Petritis]

Diggatee,

From my experience with ion pairing reagents and amino acids you seem like you are indeed overloading your column. The use of longer ion-pairing reagents provide a little bit higher loading capacities due to higher amounts of ion pairing reagent that are adsorbed dynamically in the column (i.e. 0.1% HFBA mobile phase will be much more easy to overload than 0.5 mM PDFOA).

Also remember that a small peak in UV does really mean nothing as it strongly depends on the molecule chromophore...

You might want to have a look in the publications below:

Petritis et al. J. Chromatogr. A 833 (1999), 147-155.
Petritis et al. J. Chromatogr. A 961 (2002) 9-21.

[Albany-12303]

You have a rather high conc of Glycine that you are injecting and the RT is ony a few minutes. My guess is that the analyte is not being fully PICed.

Can you add the same conc of PIC reagent to your diluent? In my experience, that would help.

Oh, and increasing the column temp (to 35 or 40 deg) may also help, but this can be a hit/miss thing.

Regards