Dear Sir/Madam,

I am a postgraduate in NUS of Singapore.During these days, I am utilizing LC(Rp)--Flourescence detector(FD) to develope a quantitative method for my basic compound (containing -NH). However, there are some problems troubling me.

1. When I just changed the buffer from (0.06M)Potassium dihydrogen Phosphate/Phosphatic Acid to (0.06M)Ammonium Acetate/Acetic Acid, the signal of FD lowered almost 7 times (peak height). I want to konw if Buffer of Ammonium Acetate is not suitable for FD or not. What is the reason?

the following is my chromatography condition:
Column: Hypersil-C18(ODS ), 250*4.6mm;
Mobile Phase:Acetonitrile-Buffer(400:600), containing 0.05M Triethylamine, PH=3;
Excitation Wavelength:202nm
Emission Wavelength:300nm

2. Observing the strength of signal, I want to know if the peak height is the first choice or peak area. Why?

3. When I added ion-pair reagent (hexanesulfonic sodium) to the mobile phase, the signal of my basic compound did almost not change in strength, retention time and peak shape. If the IP is not useful for some compounds because of their stereo structure. What is the reason?

Many thanks,

Without knowing more details about the structure of you analyte, we can make some generalizations:

1. Buffer effects. Buffers are themselves weak acids/bases and as such are only effective within 1 to 1.5 pH units of their own pKa. This means that acetate (pKa approx 4.7) is not a good buffer at pH 3. (If the reasons for this are unclear, find a faculty member and review the physical chemistry of buffers and pH). As a corrolary, it is not unusual for spectral characteristics to change as a function of pH if the charged groups are coupled to the chromophore or fluorophore; this is why indicators work (again, if this is unclear, find a faculty member who will discuss the molecular basis of UV absorbance and fluorescence with you). If you change to a different buffer that doesn't control pH well, a change in spectral characteristics is not unexpected.

2. Peak are is more directly related to concentration (the peak height relationship assumes that width is constant). However, height measurements can be more reproducible if:
- flow rate varies (think of the analyte staying in the flow cell too long -- the area will be wrong but the height may not change much).
- near the detection limits (it's easier for the data system to estimate the average baseline and the top of the peak than it is to identify the exact beginning and end when signal-to-noise ratio is poor)
- when peaks are inadequately resolved (fewer assumptions need to be made by the data system).

3. There is a very good explanation of "ion-pair" chromatography by Bruce Freeman in the "Cationic Ion Pair Reagent" thread here. You may not have added enough of the sulfonate to significantly affect retention.

1) Your exitation wavelength is 202nm. I suspect that a good bunch of photons that would normally be exciting your molecule and producing fluorescence are getting absorbed by your acetate buffer. The net result - less fluoresence.

Also, even if you start with 0.06 M acetate, your final acetate concentration will be very high because you have had to titrate your TEA and have added enough acetic acid to overcome the buffering region of the acetate (at pH 5.7 to 3.7). This higher acetate concentration (now >100 mM) will exaserbate the above effect even further. Consider dropping your TEA and acetate concentration, consider formic acid (pKa = 3.1)or stick to phosphate.

3) agree with above that you may not have added enough IP, particularly if there is a high concentration of other anions in solution that will compete to ion-pair with your molecule (whether you want them to or not). You may be swamping out the sulfonate IP effect with your high ionic strength.

Let me know if this helps?