Chromatography Forum

Dear members,
We have different peak-shape while using USP method.
The mobile phase is a mixture of 1400 mL of water, 400 mL of acetonitrile and 2.0 mL of triethylamine in a 2000-mL volumetric flask (adjusted with 0.2 N NaOH or dilute glacial acetic acid to a pH of 5.9 +/- 0.1), and diluted with water to volume. We make an adjustment to flow rate and wavelength detection, it is set at 1.5 mL/minute and 230 nm respectively. (see: USP29-NF24 Page 2036)



What should we do to repair the fronting peak-shape of the second peak (tailing factor 0.5)? The first is normal, the tailing factor is about 1.2.

Regards,
Siswanto Tanuatmojo

What is the compound?

Trimethoprim (front) and Sulfamethoxazole.

If the method allows it, reduce the injection volume by a half.

Peter

Hi Syx
I doubt it very much, that reducing the injection volume will help, unless this means reduction of the load. It is not “strong solvent effectâ€

Reducing the injection volume by a half will reduce the load by a half - not so ?

Peter

Of course Peter. And this would be the â€

Have you tried different pH?

At pH 5.9 trimetoprim will be fully charged (pKa=7.12) but sulfamethoxazole (pKa=5.7) will only be partially charged.

Maybe you should try to analyse it fully charged (decrease pH) or uncharged (increase pH). Secondly one could always try to increase the concentration of triethylamine.

CSV- I guess Syx is restricted due to following a USP method. However, I am surprised that the pH of this method is 5.9 because this is outside the buffer range of acetate (pKa = 4.75). But I suppose it's only just outside... Nevertheless this could be the cause of the problem.

This looks like a mass overload to me as there is no effect on the first peak. Of course reducing the injection volume will also reduce the injected mass. Reducing the mass will also reduce the "load" on the buffer. So doing this may solve your problem but not tell you exactly what is going on.

I would remark that simple overloading usually gives rise to tailing peaks in RP-HPLC, although fronting peaks are commonly shown in GLC.

to me , maybe the MP and the column do not have the best match.

Syx, hopefully you don´t let USP prevent you from finding a better (correcter, optimum) pH situation? Peter´s contribution at the water cooler is very relevant here.

The pH specification is strange. Victor has pointed this out already. I suspect that this is a case where the pH has been adjusted after the addition of the organic solvent. in this case, the buffering capacity may not be entirely unreasonable, and this could improve the peak shape.

Also, fronting peaks are no unusual, if there is no buffering capacity in the "buffer".

I would adjust the pH to the specified pH after the addition of the acetonitrile and see how things look then.

I'm sorry that I did not tell you that we always adjust the pH before organic solvent addition. What I was wrote here is USP procedure and we did not apply it exactly.

I would remark that simple overloading usually gives rise to tailing peaks in RP-HPLC, although fronting peaks are commonly shown in GLC.

Depends on what is being "overloaded".

Tailing will arise when the equilibrium distribution skews toward the mobile phase as loading increases (I agree that's the most common case in LC, because there is only a limited amount of stationary phase surface available).

Fronting will arise when the equilibrium distribution skews toward the stationary phase as loading increases. That can happen in LC if the analyte has limited solubility in the mobile phase, if the amount of analyte overwhelms the buffer capacity, or if the analyte molecules tend to aggregate.

Out of those possibilities, I would agree with what seems to be the consensus: the most likely culprit is the pH being outside the working range of the buffer(s) used.

Siswanto,

What I would suggest then is to follow the procedure (as I read it) and adjust the pH AFTER the addition of the organic solvent. The peak shape will then be fine again.

Don't forget, some of the USP procedures have been written a long, long time ago, when the issues of pH adjustment had not yet been discussed in the literature.

Let us know if the problem goes away!