effect of diluent solvent pH

[Kaoula]

Hi All:

we have anlysed folic acid using mobile phase at pH 4.5 The diluent solvent is NaOH 1N and mobile phase we have obtained 2 peaks of folic acid instead 1 peak

when we have used NaOH 0.1 N instead 1N we have obtained 1 peaks only
please I need your advice????!!!!!

[JA]

this is probably just a case of insufficient buffer capacity in your mobile phase. Some of the other posters on this board will likely answer your question in better technical detail, but assuming they're not at work and you need an answer now:

For ionisable compounds it is possible to observe peaks at separate RTs for the ionised and neutral forms. You control the degree of ionisation with the mobile phase pH and for best reproducibility keep the pH 2 or more units away from the pKa of the compound.

When you dissolve your sample in 1N NaOH the carboxylic functions are fully dissociated (ionised). When the sample is injected into the pH 4.5 mobile phase some of the analyte may be neutralised, but evidently not all (the two peaks). With 0.1N NaOH the strength of your pH 4.5 buffer is sufficient to fully neutralise the injected analyte and one peak results.

There's couple of options to try:

- Use a lower concentration of base in your diluent.
- Try dissolving the sample in water alone; folic acid should have some solubility
- Use smaller injection volumes
- Use higher concentration of buffer in your mobile phase

HTH

[Bill Tindall]

Well said JA. And I would add that if many samples were injected from the 1 M NaOH the first part of the column has probably dissolved. This hole could also result in a split peak (two peaks).

If the separation is done at pH 4.5, the combination of sample buffer capacity, eluent buffer capacity and sample size must result in the pH of the sample plug being near 4.5 as it hits the head of the column. 1M NaOH has a huge buffer capacity and almost any imaginable sample size will still be at pH 12 or greater when it hits the column.

[Kaoula]

thank you very match -JA and Bill Tindall - for your advice

please notify me if it exist a refference explain the effect of diluent solvent pH

Kaoula

[Bill Tindall]

I don't know of a reference. Maybe someone from LCR will know. Let me make some reasonable assumptions. Suppose the flow in the separation is 1 ml/min and the injection volume is 10 microliters. If the injection gets diluted over a 10 second interval that would result in a dilution of the injection of 10/1 . More dilution will occur as the injection band spreads down the column, but what is important is the dilution as the injection band hits the head of the column. I think that a good assumption for this initial dilution will be in the range of 5/1 to 50/1. 5/1 to 20/1 is my best guess, but that is just a reasonable guess. I suspect that someone has calculated it but I am unaware of where to find the calculation.

If your injection of 1 M NaOH is diluted even 20/1 it will overwhelm a .1M eluant buffer. So, your analyte will reach the column in a solution of very high pH. This high pH will neutralize (ionize)even weak acids, as well as dissolve silica.

[Uwe Neue]

I got some stuff from our seminars on preparative chromatography, where one is dealing with a very similar situation. You get two peak for one compound, if the mobile phase buffer is too weak to change the ionization of all the compound injected, and you get a single clean peak, if you work out a way to convert the compound into a single state. If you contact me, I can send you the publication, or you can look it up yourself: Chromatographia Supplement Vol 57 (2003), pages S 121-127.