Chromatography Forum

Hello All; I am trying to develop a metohd for determination of Water soluble vitamins in Flour (Nicotininc acid, Thiamine, Folic A and Riboflavine). my LC method is quite fine but my SPE metohd is not so well.

I am using Sep Pak C18 flushing 10 ml of MeOH and 10 ml of H2O; load my flour extract and then I elute with 10 ml of a 50:50 mix of MeOH:Buffer (pH4.5); but I am loossing Thiamine and Nicotinic Acid; I has tried eluting at different pH's but allways the same problem. I has tried conditioning the cartirdge with buffer at low pH's too (4 and 5) but I can not retain these vitamins; obviosly I am loosing them when I charge so I need some help; I think I must to regulate the pH of my extract before load but I do not know how.
When I going to low pH nicotininc acid should stay unoinized and then should fix to the cartridge but theory fail (probably it must be protonated).

Any body has experience with this analysis? any advice?
I am working with a method I found in the internet about determination of vitaqmins in tarhana but after test many modifications the best resul I found was the one I described above but still with terrible recovery.

Before I forget it; I am extracting with a phosphate buffer at pH of 4.

Thanks for everything.

Oscar.

Hi Oscar,

You might like to shortly describe your LC method as well. If it works fine, it might contain valuable information that can be of help in the SPE context.

Best Regards

You are likely to get better results with phosphate pH 2. If this still does not retain the vitamins, I would use an Oasis MCX (mixed-mode cation exchanger) at the same pH. With the mixed-mode packing, you get retention from both the hydrophobic interaction and from ion exchange. For these two compounds, the hydrophobicity may be too weak.

Thank you Danko and Uwe.

My chromatographic method is as follow:
T (min) A(%) B(%)
0 98 2
5 98 2
18 75 25 (gradient)
23 75 25
A= KH2PO4 0.1MpH7, B= MeOH
Teperature= 30C
Column Atalntis T3, 5umx4.6mmx150mm. I have quite good retention because Nicitininc acid have 4min. of RT and Thiamine have 7min.

Uwe; do you think I have to flush PO4 pH2 before load my sample? and reach the same pH for extract?
My problem is that I dojust have a few MCX not enough to do run many test but what I really have is Sep Pak, the Lab got a lot of them.

any additional comment will be appreciated.

Regards.

Oscar.

The reason for the adequate retention with the LC method is probably the fact that the Atlantis column is designed for retention of hydrophilic, as well as, hydrophobic compounds. So you might achieve better results if you emulate the LC method – stationary phase wise. I think Uwe is the right man to suggest which type SPE is similar to the Atlantis stationary phase.
I also agree that the first thing to do (before experimenting with another SPE type) is, trying much lower pH (ca. 2.2 - phosphate).

Best Regards

With phosphate pH 2 in your sample, I don't think you need to prewash the Sep-Pak with anything different from what you are doing now.

If the analytes are not retained, I think you will need to consider the ion-exchanger. If they are nicely retained, you may also consider changing the pH in your LC method. I will look up some HPLC methods for these things.