Chromatography Forum

Hi All; has been a while (around 4 months) I am not here; to much things to do lately.

I have a problem and I would like to know if some one can help me since I do not have to much experience with this kind of separation

I am trying to do an analysis of Galactose in urine samples using an Alliance 2795 with IRD using a Carobopac PA1.
My movil Phase is NaOH 20mM.

At the beginning the Rt of galactose is around 6 minutes but as the time pass the retention start to goes shorter and the peak overlaps with glucose. I read and I found this is caused because of CO2.
I regenerated the column passing 60 ml of HCl 1N follow by NaOH 200mM and after that RT got fine again, but the peak got broader, and after 1 hr Galactose started to elute earlier in the chromatogram.

I have two main question:
1. how can I remove CO2 from my MP? I was thinking to try to attach some kind of NaOH pellets filter on my bottle but if some one have any advice it would be welcome

2. How can I regenerate my column in order to get nice peaks again?

can this column only work with an IC provided with CO2 removal device or could work with a normal HPLC?

Thanks in advance for your help.

Oscar

Hello Oscar,

we use the PA1 column for glycan analysis. With our system setup we keep the mobile phase headspace pressured with nitrogen and this reduces some of the problems you are seeing. this may not be the easiest thing for you to setup, but I thought I'd mention it.

On your second problem though, I've found that the PA1 column can be regenerated with sodium acetate. We run 0.1M sodium hydroxide/1M sodium acetate as mobile phase B on our system and when the chromatography is becoming poor I run for 30 mins at 50% B to regenerate.

Good luck

Simon

Hello Sipeelie; thanks for your help; I will regenerate with HCl in order to do it deeply and then I will apply your recommendation once RT start to change; I find hard to pressurize my movil phase but is a good Idea; I will see what I can do.

additional advises will be welcome

Best Regards

Oscar