Chromatography Forum

Hi.

I need advice about improving peak shape on HILIC column. My analyte is pramipexole. I tried buffer of ammonium acetate with added acetic acid and pH equilibrated to 9,8 mixed with ACN (40:60). Retention is ok, but tailing of peak is 1.3 and peak area is quite small. With decrease tempretaure, peak shape is even poorer.

Please, do you have any advice how to improve peak shape?

Best regards,
Natasa

tailing of peak is 1.3

Tailing 1.3 (T = (a+b)/2a at 5 % height?) is acceptable for most applications. The range from 0.8 to 1.5 (or even to 1.8 starting from Dec. 2022 for USP and from Jan. 2023 for Eur. Ph.) is a default acceptance range in USP and Eur. Ph.

peak area is quite small

Are the amount injected and the detection conditions appropriate?

Hi Natasha,

Depending on the column, amines can be particularly difficult to get ideal peak-shapes with. This may be part of the issue you are seeing here, but we have no column information right now. Additionally, your mobile-phase prep has me slightly confused, as I don't understand how it is possible to create a mobile-phase of pH 9 (I think 9) with ammonium acetate and acetic acid?

Vmu also brings up a great point, what are the detection conditions, are you visually the molecule at an appropriate uV abs for example?

Tyler

Thank you for your answers.

I know that tailing is in acceptable range. I am trying to get even better peak shape.

My buffer contains from ammonium acetate with added acetic acid and fixed to pH 9,8 with triethylamine. I added acetic acid, because without it, I have problem with repeatability.

I just do not know, if maybe on HILIC column would change of pH of mobile faze (from 9,8 to about pH make difference in peak shape? Or it could get better on other HILIC column? Now I have Kinetex HILIC (150 mm x 4,6 mm, 2,7 um). I choose HILIC column, because of good selectivity.

Maybe you have any idea of using diffrent mobile faze that could also/better work for analyse of amine on HILIC column?

According to maximum of analyte on spectro, I think that wave lenght is ok (263 nm). Injection volume is 50 uL. I also can not change sample concentration.

Hi Natasha,

I've done some quick research on a few methods so take this with a grain of salt but most of it is good news. All of the methods I read about were done under acidic conditions, on C18 columns, and I'll link them below to see if you can mimic or use their methods. The first method I read about used an ion-pairing reagent, but for the others I could not find evidence of one being used. Here's the link to those:
Indian Journal Of Pharmaceutical Sciences: https://www.ijpsonline.com/articles/est ... iew=mobile
And another off Research Gate:
https://www.researchgate.net/publicatio ... osage_Form

I'm biased to the second method, but it is important to note that they utilize an ion-pairing reagent, which will stick to your column and may potentially be there forever. So if you do utilize their method, then you should dedicate a particular column to this separation and note that it is an ion-pairing column now.

As for your sample volume, I'd decrease the injection volume to 20 ul and see how the peak-shape is affected, additionally what flow-rate are your using?

Commercially, pramipexole is sold as the dihydrochloride salt. That means that the two ionizable nitrogens in the molecule will have chloride as their counterion when injected. The anion in your mobile phase is acetate. At some point during the sample's migration through the column, the amine residues will exchange chloride ion for acetate as their counterion. Pramipexole with chloride ion in its ion pairs will differ in polarity from pramipexole with acetate in its ion pairs, so the two alternative salt forms will migrate through a HILIC column at different rates. This counterion mismatch frequently results in peaks that tail or even two different peaks that are connected by a continuum. An example of the latter is presented in Anal. Chem. 80 (2008) 62-76. Try increasing the concentration of acetate in your sample solvent so that the counterion exchange occurs prior to injection.

I wouldn't be surprised if this molecule could be analyzed using RP-HPLC. High pH and something like an Evo C18 column might work, or a polar C18 column at neutral pH.

Is the reason for the high pH in the mobile phase that you have better separation there?

Positively charged molecules like pramipexole are very suitable for the Kinetex HILIC column where they will have great retention at ambient pH.

If you want the sharpest peak in history, I would use acetonitrile in channel A and 100 mM of ammonium acetate buffer pH 5 in channel B. And then do a buffer gradient from 10 - 50% B in (maybe) 15 min. Collect the UV-signal at 270 nm where you have good response and no background from the buffer. Remember to equilibrate well before next injection.

Maybe the separation is terrible, but the main peak will be extremely sharp