By Anonymous on Wednesday, April 23, 2003 - 01:49 pm:
I have a method that I am transfering into our lab from a sister company. The method uses ion-pairing reagent in the mobile phase. The peak we are getting for our runs has a big hump on the side of it. Lowering injection volume does not help the peak shape. We have very little experience with ion pairing methods - can anyone make any suggestions on what the problem is? Are ion-pairing methods sensitive to different systems.
Thanks in advance for your thoughts
By Uwe Neue on Wednesday, April 23, 2003 - 07:04 pm:
A lot of things are possible. If more than one peak has a hump, the column is dead. It may be that the column has not yet been equilibrated with the ion-pair reagent. It takes a century to do so. Your sample may be dissolved in something that may be incompatible with the mobile phase...
Please be more specific! The more specific you are the higher is the likelyhood that somebody can help!
By C.Sychov on Thursday, April 24, 2003 - 04:31 am:
Two things may happen
1) Primitive)) you tried 2 or more ion-pairing agents when using the only column, so you peak can split.
2) Your analyte has at least 2 (or more) ionic centers, so its peak is splited. I can recommend to do something to speed up exchange procecces -- for ex. to use higher temperature. Or NOT to use ion-pair HPLC.
Constantine
By Wanda on Thursday, April 24, 2003 - 10:40 am:
1. I have found that low vs high pressure systems can cause this phenomenon.
2. The quality of the agent may be depositing crap on the column that happen to come off at that organic strength.
3. the column is shot.
4. wrong membrane used to filter MP (outside pH or organic type)
Lots of things can cause the "shoulder blob". Try looking at my suggestions in order.
By Anonymous on Thursday, April 24, 2003 - 03:21 pm:
Constantine:
Why would you get two peaks if you use a mixture of ion pair reagents?
Why would you get two peaks for a doubly charged peak? Will I get three peaks for a peak with triple charge?
By C.Sychov on Friday, April 25, 2003 - 12:35 am:
You will if the speed of ligand exchange is rather slow. Better to say, if the particular form of your analute (particular ion pair) can live in solution for the charact. time about secon of longer (correspondingly, speed constant being below 10^0 sec).
Constantine
By Anonymous on Sunday, April 27, 2003 - 12:05 pm:
Constantine
You have not answered my second question: if you get two peaks for a doubly charged peak, do I get three peaks for three charges?
Why do you think that ion-pairs live that long? Why do you think that ion-pairs exist at all?
By C.Sychov on Monday, April 28, 2003 - 01:19 am:
Dear Anonymous!
I`ve answered) I said "You will" :) .. if .. and explained in which case)))
I don`t THINK :) its theory)) this model may be calculated? this topic being mostly studied on the example of dymamic chiral chromatography (search it in J.Chrom. A for ex.)
You dont believe in ion-pairs? :)
So, than it would be religious discussion rather than scientific :)
Constantine
By Anonymous on Monday, April 28, 2003 - 03:34 pm:
Constantine
1. I have worked with mixed ion-pair reagents, and I did not get two peaks.
2. The published theories of "ion-pair chromatography" in RP LC do not assume that ion-pairs are formed. They rather deal with a slightly complicated version of an ion-exchange process. The religion is the ion-pair in ion-pair chromatography.