Chromatography Forum

HI everybody!, i got a serious problem here....yesterday, after i trying a lot to separate vit b-15, vit b-6 and orphenadrine i made it! but today when i try to reproduce and quantificate oh no!!! sh....!!!f...!! %&@#!!! nothing is correct, the second and third peak have disappear, instead a clean second and thrid peak i´ve got a second and auful doble peak ( like a castle ).
the mobile phase is Tetrabuthylammonium 3,4g and H2KPo4 6,8 g ph=2,9
86% of this, 8% ACN and 6% MeOH...the only thing that i did yesterday was to wash the column with 80%h2O and 20% MeOh about 1,30Hs. I´m thinking that the column was marked with this mobile phase and after i washed it, i´ve lost this estabilization...
I know that i musn´t use the column marked with IPC to something else...is it correct to wash this marked columns in this way? what should i do in order to prevent this?...i want to avoid this problems of course, so how can i reproduce this method?

Hello Coquim.

I would like to know what kind of column are you using?

These sound to me like a dewetting phenomenom.
I use to analyze NO3 and NO2 by RP using PIC, and I use to have the same problem; and this was because the column.

Oscar.

ion-pair chromatography needs the ion-pair reagent adsorbed on the column. If you wash it off, you need to put it back on.

With other words, if you want to do ion pair chromatography, you need to keep the ion-pair reagent in the mobile phase. So you need to go back and start all over.

I think that you are using a wrong approach. Here is why:
1. Vitamin B6/pyridoxine is basic and hydrophilic in nature; you need to use HFBA or similar acidic ion-pairing reagent (not TBA-Chloride). If you are using Tetrabuthylammonium it is not serving as IP reagent for B6. At higher pH 6.8 pyridine ring is not ionized but it is still very hydrophilic due to the presence of hydroxyl groups

2. Vitamin B15/Pangamic acid is acidic and hydrophilic, but if you use lower pH you can retain it by weak RP interaction

3. Orphenadrine is hydrophobic and basic and there is no problem to retain it.

You can try to use one of the sulphonates as IP reagent but this will increase retention of orphenadrine even more, you will have huge gap between peaks unless you use sharp gradient.
If you are flexible with column choice I would use either mixed-mode or HILIC. Please contact me by email and I will send you application for retention of ascorbic acid (behaving similar to your B15), B6 vitamin and hydrophobic basic molecule (similar to your orphenadrine)

Thanks to everyone!
Oscar: i´m using C18 4,6 x 15mm from shimadzu.
Uwe Neue: you are right, and i really thought about this but i didn´t think that washing fully the column i put the regeant out from column, in fact i thought the union fron TBA with non polar stationary phase was strong enough to keep inside of...
SIELC_Tech: You are very kind, and of course you are right, but, unfortunely, in the mean time, i´m not flexible at all, i have only 3 columns c18, and i don´t have enough regeants...by the way, i have a question about Heptane sulfonic acid and tba for example, are those compatible in order to use the same column? ( because both are ip regeant) or may i use different columns with these? if would be possible to use both with the same column ( i don´t so but...) how can i wash the column? and how is the correct procedure to wash the most columns once you use it with ipc regeant??
thanks again!!

Regeneration of columns after usage of ion-pair reagent (previously discussed):

http://www.sepsci.com/chromforum/viewto ... ing+column

Regards

There have been examples in the past where very hydrophobic ion pairing reagents have been used to "permanently" modify reverse phase columns and then use them without incuding ion pairing reagent in the mobile phase. The coating/modification was done at very high organic or organic only mobile phases (i.e. 100% ACN) and then you needed to operate it at highly aqueous mobile phases after washing it extensively with that mobile phase. Of course such methods didn't survive the course of time as coating was of course highly dependant on ion pairing reagent concentration and temperature so it was pretty difficult to make two identical columns.

In your case, TBA is not hydrophobic enough to try something like this so as indicated by Uwe, you need to include it at all times...

thank you so much for your answers people

i clearly see that´s difficult to reproduce the same result with these kind of method (IPC).

You can actually be reproducible with IPC methods (otherwise nobody would use them or being able to validate them). Only the variation I mentioned is more challenging...