My target compound contains several quaternary ammonium centers (positive charge), various hydroxy groups, and a couple of benzyl groups. I ran a 0.6% solution of the componud on a C-18 column with 50:50 ACN:water at 1mL/min for 30 min with a photodiode array detector. The resulting chromatogram showed only a small peak (0.02AU) at 1 min when viewed at 254nm. At 200nm the absorbance increased to 1.2AU. If I assume that my compound has a high absorption at 254nm because of the benzyl groups (we're running UV), it would seem that my target compound never eluted. I've had some suggestions to add an ion pairing reagent, but I also have heard that this can really complicate things and to only use it as a last resort. Is ion-pairing chromatography my only option? Does anyone have any suggestions/ideas regarding different columns, eluents, or if my assumptions about no retention might be incorrect. Any comments are greatly appreciated.

Dora;

I could recommend a couple of things regarding the problem you describe. Quaternary amines can be highly retained on C18 columns due to the presence of multiple alkyl groups. You mention the compound has several of them and if they are rather large (Propyl or larger), it is quite likely your compound is not eluting with the mobile phase employed. Try using 80%ACN/H2O or perhaps a less retentive column, such as a C4 or C8 one.

Adding an ion-pair reagent is likely to improve peak shape and separation of impurities. if you try this, I would recommend 0.05% TFA (trifluoroacetic acid) in your mobile phase. You have a PDA detector and therefore you can tell if the UV scan of your signal resembles that of the compound you inject.

Good luck;

Benjamin

Dora,

Your problem is most likely due to the high affinity of quaternary compounds to silica silanols. If you are determined to use C18 silica, make sure that you work at the minimum possible pH. You don't really need to use an ion pair reagent as this will only increase the concentration of solvent required for elution and prolong column equilibration. Also, some electrolyte in the mobile phase is also helpful in order to stabilize the electrical double layer when separating such compounds. The other option is to use cation exchange as a separation mechanism. Again, silica silanols may present problems so better results are achievable with polymer based phases.

If my memory doesnīt fail me the absorption of phenyls is quite weak there. That is, your compound could be eluting.
This is a another nice example of how difficult it is to do chromatography without having any data, here UV, on the substance.
If you can not predict the UV spectrum, canīt you purify the stuff by some unequivocal means, and then inject it without a column to get a spectrum (careful not so simple), etc.? Or collect it and do another type of identification?
Again, it could be instructive to be informed of your problem solving.

Incidentally, why would one use ion pairing if the compound itself is well retained?

Some buffers are needed to get rid of residual silanol effects. We also have seen some hydrophilic interaction when working with high percentage organic on C18, that is, RT increases with increasing organic percentage.