Chromatography Forum

my colleague run 8-OH quinoline through luna C18 150x4.6 at 1ml/min, room temperature, MP 15% MeOH and 85% Buffer( NaH2PO4 6g/L pH 3.5) to give tailing 0.7;
he claimed the tailing became 1.1 when he changed the buffer pH to 2.8.
Is this posssile?
If yes, what's the mechanism?
Thanks

Hi ym3142,

This is certainly possible and not at all surprising. At pH 3.5 the phosphate has no buffering capacity and that’s why your colleague expeariances bad fronting – the compound “decidesâ€

Donka, thanks

Hi, Donka,
Thank again for last reply but would you mind explaining that the 8-OH quinoline was fully protonated at both pH 3.5 and 2.8 and both samples were prepared in the corresponding mobile phase how the BUffer(capacity) plays the role?
Thanks and have a nice weekend,

sorry, I found it's Danko.

Hi again,

I’ll try, but would you mind telling me what the pKa for 8-OH quinoline is?

Best Regards

hi, Danko,
I did not find the pka for 8-OH quinoline but phenol is 10 and quinoline is 4.9. so what happens if pka was (assumed) 4.5 and my buffer pH=3.5?
Thanks

hi,
I think the pka of your compound will be around 4.5 to 5.0. So why dont you try with an acetate buffer since it will have its buffering range between 3.76 to 5.76.
without knowing the exact pKa of the 8 -OH quinoline it will be difficult to explain the mechanism

Shan

shan,
thank you for your interesting. please assume what ever pka to fit the Buffer"capacity" theory as sugggested by Danko [quote]This is certainly possible and not at all surprising. At pH 3.5 the phosphate has no buffering capacity and that’s why your colleague expeariances bad fronting – the compound “decidesâ€

Hi ym3142,

Let’s assume that the analyte is a weak acid (pKa about 4 or something like that), in order to illustrate the situation I referred to. At pH 3.5 some of it will be protonated and some of it will be deprotonated (at pH 4 the ratio will be halv and halv). The deprotonated part will act as a base.
If the phosphate (the mobile phase) is adjusted to pH 3.5, it has no capacity to prevent the analyte to act as a base (too little phosphoric acid in the mixture), which means; pH elevation upon injection of the sample. Higher pH usually means shorter retention time (on a reverse phase column) due to ionized sites i.e. the molecule exhibits polar properties.
Because of the limited amount of the analyte, compared to the mobile phase, this change of pH will only be effective in short period of time (until more phosphoric acid arrives with the flow).
So we have short term pH raise hence shorter retention time, but only for a fraction of the compound and then pH drop, which means the rest of the analyte is retained according to its polarity at pH 3.5. Chromatographically, the peak will be fronting.
Nothing of this will happen, if the pH of the mobile phase is within the buffer capacity of phosphate or whatever the buffer is.

Hope it sounds logical.

Best Regards

Danko, Thanks.