Chromatography Forum

Hi I am noticing some odd things when using Tetrabutylammonium Hydrogen Sulfate when separating a phosphonate.

At 5mM at pH 7.1, I am noticing high peak tailing (1.5). I tried using a 10mM concentration at pH 7.1 (Both buffered with 5 mM Ammonium Phosphate). I am seeing the peak with short equilibration/saturation of column, but gradually over time, the peak becomes very broad with high tailing and low sensitivity.

So, how does the concentration of IPReagent affect peak sensitivity? I was under the impression that, generally, smaller IPReagents require a higher concentration than if I were to use a bigger one. Could it be the high ionic strength buffer than is causing a problem? What is the best way to buffer in IPC?

Would there be a better IPReagent in this particular case? Maybe something that isn't so strong? n-pentylamine or diethylamine?

Thanks

Cannot explain why your peaks become broader with time, but it appears as your are using quite a lot of IP reagent and not very much buffer (compared to what I am used to). How much acetonitrile do you use?

You are eluting your compound mainly with the buffer, and if you have too low conc I guess stange things may happen. I have also noticed that the column itself is quite important. Standard C18 columns works best. Columns with polar groups (like "Aqua" or nitrile columns) give very strange chromatography together with IP reagents. Core-shell columns like the Kinetex C18 also give very strange peak shape.

Not very much help, and I think there are others on the forum that can give more scientific explainations .

biphosphonates (especially zoledronic acid) are tough to handle with traditional IP chromatography. It has been years but as far as i remember i could not achieve to get the peak below 1.6 tailing with ion pair reagents and again if my mind is right i gave 1.8 tailing as system suitability..

what is the typical amount of buffer concentration that should be present?

interestingly, injections under these conditions yield no detection!

what's going on?!