Chromatography Forum

I have a combination of a very basic compound (oxymetazoline) along with a series of neutral steriods which I am currently seperating all of them beautifully with a Ace 3 C18 column using a 15ul injection. Mobile phase A is 100% water with 0.1% TFA and mobile phase B is 100%ACN with 0.1%TFA. I need to raise the injection volume to at least 40ul to meet DL and QL S/N requirements for oxy, however, the tailing in the response factor standard for oxy jumps to ~1.7-1.8.
Someone sugested to add a minimal amount of TEA to the mobile phase, but I'm curious of the chemistry. The TFA is there as an ion-pair reagent, but does the addition of the TEA negate its effect? SHould the TEA only be added to mobile phase A?

TEA was traditionally added to counteract tailing of basic compounds caused by interactions with "activated" silanols. It is much less widely used with today's higher purity columns. Since your tailing seems to be caused by overload rather than secondary interactions, I'm not sure that the TEA would help all that much. Furthermore, the addition of TEA will very likely change the selectivity, so that the method will have to be reworked.

Adding TEA to both the A and B solvents is a good idea, but not necessarily at the same concentration (the distribution coefficient of the TEA between the mobile and stationary phases will change as a function of organic content).

Bottom line is that addition of TEA will most likely mean reworking the whole method. A tailing factor of 1.8, while not good, is not catastrophic, particularly with a large peak. If this were my problem, and I still had good resolution, I'd choose to live with the tailing.

thanks Tom. I'm going to reduce the concentration of the response factor standard and see if the tailing is concentration dependant. I don't believe it is due to the fact that the steriods which elute ~ 8 minutes later have a tailing of 1.0 regardless of injection volume (information that I forgot to provide earlier). They also have a much higher molar absortivity than oxymetazline, thus their response is ~15x greater than the less hydrophobic oxymetazoline peak.

Is there a column on the market which has SiO- at a pH near 2? Could it be that there is a strong pH incompatibility at the injection? Or is the base tailing due to decomposition on the column?
(The tail probably disappeared in the noise at the lower inj. amount).