I had peaks that were tailing quite a bit so I added TEA in my mobile phase to reduce this effect and now my peaks are fronting? What should I try next? The analytes are dissolved in mobile phase and I am injecting ~ 3.5ug on a 4.6 X 250 column so I do not think that I am overloading.

Questions: column type; analyte; pH of buffer; is the tailing a new phenomenon; ???

Column is a Ultrasphere C-18, basic drug, pH 4.7 The tailing has isn't new always had some, the fronting is.

Is the fronting terrible, or is it minor? Some columns are packed to give some fronting, so that the tailing does not look so bad.

I have three analytes, all used to tail pretty significantly so I added TEA to reduce it. At what point do you define fronting as minor or terrible? Looking at my standards - the first peak's tailing factor (TF) ranges from 0.82 - 1.2 (split about 50:50 around 1), the second peak's TF ranges from 0.87 - 0.97 and the third peak TF looks fine. I tried to look up the USP guidelines on fronting to see if they are still with in system suitibility requirements but I couldn't find anything. I know that the tailing factor has to be <=2 but shouldn't fronting have to be above some value?

I have "heard" (vendor seminar) that pH 3 will protonate silanols sites and reduce tailing. pH 4.7 leaves some of these silanols unprotanated.

I have not had a chance to "test" this out with my applications.

In reversed-phase liquid chromatography, interaction between weak acids and weak bases with residual silanol groups can cause tailing. The poor peak shape can be controlled with the proper pH or with the addition of a modifier such as triethylamine to prevent weak base tailing. If all peaks in the chromatogram appear tailed, the peak shape has resulted from a problem with deterioration of the column or because of extra-column effects.

Basic substances typically show fronting on badly deactivated phases that show silanol-activity as these substances show strong interactions with negatively charged silanols. Therefore I would recomend using a better deactivated phase that can be supplied by most HPLC column manufacturers. Have a look at Waters Symmetryshield, Phenomenex Luna, Macherey-Nagel Nucleodur Gravity, Merck Purospher etc. If You need to get more retention of your compounds there are silica columns that are stable in the pH range well above pH 10 where your substances don`t carry positive charges.

Our SOP's (for compliance with GLP's) specify that tailing factor (T) should be between 0.5 and 2, where T is defined as

T = (w_0.05)/2f

where w_0.05 = width at 5% height and

f = width of front half of peak at 5% height

Thanks to All! I found this site while searching for an answer to my question. I wasn't sure how things would turn out but I would say that it has been pretty darn helpful.

So here is my next question... my standards vs my internal standard give a beautiful line even with the fronting. According to MG's comment on their SOP for GLP compliance the range of my fronting is acceptable. Would most of you recommend to use this method (with the fronting) or not? And to help me continue with my ongoing HPLC education could you share the reasoning behind your answer.

Thanks - LP

I think you should run a System Suitability test. The purpose of the system suitability test is to ensure that the complete testing system(including instrument, reagents, columns, analysts) is suitable for the intended application.