Chromatography Forum

Dear Sirs
I'm trying to separate B3 and B6 in a phenyl column, Microsorb phenyl, the reason is we detect cafeine, sorbic and benzoic acid in this column and the juices we produce have also this two vitamins.
My conditions are
Temperature 20º
Acetic acid 0.1%:80
ACN with acetic 0.1%:20
1 ml / min
I have good results for sorbic and Benzoic, but pics of B3( 4.3min ) and B6(10.9 min) have problems with tailing and the sensibility for both are not good enough for me.
I decided to change phosphoric instead of acetic acid, and the results have surprised me. Pics are better, sensibility is better for the two vitamins and also the tailing, but they co-elute B3 (2.1 min) and B6 (2.2) so by now I could separate it.
I know that phosphoric is more acid (pH= 2.4) than acetic (pH=3.7) but I don't think this is the reason, maybe I'm wrong.
I don't know why phosphoric has produced this effect.
Anyone could give me some idea to solve the problem I mean:
Obtain good results with conditions with acetic acid or
Separate vitamins in conditions with phosphoric acid.
I hope you could understand my explanation.

Thanks in advance.

P.S. I have the chromatograms scanned so I could send to anyone, I don't know if I could load on the message or how to do it.

It is hard to clarify your problem. For separation of Nicotinamide (B3) and Pyridoxine HCL (B6) by HPLC, we are using ion-paired RP HPLC. The basic characteristic of two these vitamins may be the cause of tailing peak. Reduce the pH of mobile phase or add triethylamine is the ways to reduce the interaction between silanol activity of column with these vitamin so it reduce the tailing factor.

P.S. I have the chromatograms scanned so I could send to anyone, I don't know if I could load on the message or how to do it.

Instructions on posting chromatograms are here:
viewtopic.php?f=1&t=2617

Dear Sir
I would try with triethyl amine, I thought of but I did'nt because it could affect to sorbic and benzoic, but better answer is just to prove it.
I'll try with the conditions with acetic, but I like more the results with phosphoric because the shape of the pics are better.
Thanks in advance.

Andrew

Hello again
I've been following the instructions for the image so I hope I have done it in the correct way, here it is:


You will see the chromatograms number 3 and 4, which are the conditions with acetic and phosphoric, number one and two are the vitamins alone with phosphoric. C is caffeine, S and B, sorbic and Benzoic and the vitamins B3 and B6.
I hope this will show my problem better.

Thanks

Ok, sorry to everybody, I think now you can see it.
thanks

Pyridoxine is notorius for tailing. Both vitamins are weak bases (pKa ~4-5) so reducing the pH with phosphoric (how much?) increases their ionization which decreases retention. Try reducing the %ACN when using H3PO4 to see if you can get baseline separation with the other compound eluting within a reasonable amount of time.

useful link:
http://web.squ.edu.om/med-Lib/MED_CD/E_ ... -table.pdf

Hi Andrew,

The attached application note may be of some help.

http://dlibrary.dionex.com/Public/View. ... tionID=110

Although you probably don't have the same system, the principle should be achievable.

Hope this helps

Thanks to Carl and Anthony,
I'll prove both thinks, in order of course, because I was trying some injections with TEA with really bad results in both cases, with phosphoric and acetic. With tea I obtain that the two vitamins co-elute at 2 min more or less and the pics of the other substances were bad than without TEA.
The table you have send me is really useful, and I already got it and you have confirmed what I suspected.
Thanks for your time, as soon as I could try the methods I'll tell you about the results.

Andreu Pulido

Dear all
Finally I've achieved a reasonable separation between B3 and B6, my conditions are
0.2% TFA:ACN 80:20, 1 ml/min, 25ºC in a phenyl column, sorbic and benzoic and Caffeine had better separation
so, I'm trying with 0.8 ml/min, because retention times for B3 and B6 are at 2 min, so I would like to reduce a little beat more. In my product I have the same separation, so by now I just have to quantify them.
Thanks to all your suggestions, they had been really useful and so I've learned a lot with all the proves.
I'll try to embed a Chromatogram as soon as I can.
Thanks to everybody.