[coquim]

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?

thanks a lot and have fun!!

[Arkcon]

That is a problem with ion pair reagents, and why some people do anything to avoid using them.  They don't really wash free, with light to moderate washing, so they remain, and interfere with the chromatography of future runs on the same column even if they're not in the mobile phase.

On the other hand, with vigorous washing, you can strip off the ion pair reagent, and then, it will take a substantial equilibration to get it back to the way it was before.

OK, some practical tips.  To use your time effectively, start equilibrating your column while you're doing other things at the start of the day.  If you can, program the system to start before you even come in that day.  You have to go home and sleep, the system doesn't have to be idle while you do that.

A good rule of thumb, is to do test injections.  Inject a standard, twice, and get the same retention time, before you start making injections you intend to acquire data from.  If the retention time is shifting, or there are contamination peaks, or shapes are changing, equilibrate more, or better yest, re inject, until the system stabilizes.

[coquim]

thanks Arkcon! i imagine that! f...!! @#$%&!!!
i thought yesterday before to do that and i went home thinking about this would be probably happen, but of course i wasn´t sure 
by any chance, do you know how should i wash the column? in wich way?
i think may be the estabilization take it about 2 or 3 hours? don´t you think?

[JGK]

For a trouble shooting guide to ion pairing check out

http://chromatographyonline.findpharma.com/lcgc/Column%3A+LC+Troubleshooting/Ion-Pairing---Blessing-or-Curse/ArticleStandard/Article/detail/494775?contextCategoryId=9678

I would also recommend adding this link to your favourites, the troubleshooting articles are well worth reading and consulting

http://chromatographyonline.findpharma.com/Column%3A+LC+Troubleshooting

[coquim]

thanks a lot, i´m gonna read this right now!... by the way...mobile phase has been running for at least tree hours and i have seen changes, but i cannot reproduce yesterday´s retention time...could i make it some day? 

[coquim]

excellent reading, JGK thanks!
i have a doubt by the way, is acceptable to use the same IPC marked column for example with Tetrabuthyl ammonium and in other circunstances use it with hexane sulfonic acid or may i use differents columns with differents IPC regeants?
Thanks again!

[JGK]

My experience with IPC is that the columns are best used with the same Ion pair reagent throughout their life.

Similarly, Columns used with IP reagents seldom work the same on subsequent analyses and often fail to return to their normal state. Consequently, columns which may be re-used should be clearly identified if they have been used for IPC.

Luckily, my only contact with IPC these days is using trifluoroacetic acid (TFA) which is easily removed from the columns.

[coquim]

mmmmm!...
i´ve just bought Tfa regeant...it´s good to know that i can clen well this regeant from column...could i use this column after washing and use it from non ip regeants? i know what you´ve said about to identify those col. but if tfa easily removed, may be i could use that col with other kind of "normal" regeants.
  is not recomended, isn´t it?

Thanks a lot!

[JGK]

mmmmm!...
i´ve just bought Tfa regeant...it´s good to know that i can clen well this regeant from column...could i use this column after washing and use it from non ip regeants? i know what you´ve said about to identify those col. but if tfa easily removed, may be i could use that col with other kind of "normal" regeants.
  is not recomended, isn´t it?

Thanks a lot!

I have used columns with TFA and subsequently non IP assays.

[coquim]

i imagine you use it after, without alter the selectivity...
thanks!!!