acetic acid buffer

[sglrio]

i used hplc to detect metabolite products of dye decolorization. i used solvent acetonitrile and acetic acid buffer 30 mM pH 4.5. after i inject my sample i always find peak of acetic acid with high intensity. i just wonder, how to remove presence of acetic acid in peak? thank you.

[tom jupille]

Use 30 mM acetate in both your diluent and your mobile phase. If you *still* see a peak for acetic acid, that means that the excess is, in fact, part of your sample.

[sglrio]

thank you. i forgot to tell something. my sample also contains acetic acid buffer. when i inject only water, acetic acid did not appear. just right now i inject dye in water (not in acetic acid buffer), acetic acid peak appears again.

by the way, i little bit dont understand about addition acetic acid in diluent and in mobile phase. what's the meaning of diluent? thank you so much for your respond.

[HPLCaddict]

when i inject only water, acetic acid did not appear. just right now i inject dye in water (not in acetic acid buffer), acetic acid peak appears again.

When there's really no acetic acid in your sample solvent, there are two possibilities: Either the peak you're seeing IS acetic acid and it is comming from your dye sample (and not the solvent). Or it is NOT acetic acid but some other component of your dye sample.
Just to be sure, that peak is positive (upwards), right?

If possible, you should use your mobile phase as solvent for your sample. This way, you minimize the possibility of seeing extra peaks that do not originate from the sample.

[sglrio]

hello.. i change the wavelength and acetic acid peak has already dissappear. however, i still confuse about how to choose wavelength. my initial sample has maximum absorbance at 595 nm. so when i inject sample in that absorbance, the initial sample peak is appear. but my treated sample (metabolite product from my initial sample) can not detected. so what should i do? i should get the new absorbance to detect other compounds? sorry, too many question. it's the first time i use hplc. thank you so much..

[HPLCaddict]

Generally speaking, you should set the wavelength so you can detect all analytes of interest with satisfying sensitivity.
Do you want to see the initial substance AND its metabolite? Do you want to see only the metabolite? Do you want to see only the initial substance?
If you want to analyse only one of these two, you should set the detection wavelength to its absorption maximum. This way, the other peak might get very small or even dissapear completely, but who cares - you're not interested in it, right?
If you want to analyse both simultaneously, you'll have to find a wavelength where both show enough absorption. If that's not possible you can use different wavelengths for both analytes - either in one run, if your equipment (detector) is capable of it (i.e. simultanous measurement of two wavelengths or changing of the wavelength between the two peaks), or you'll have to split the analysis in two runs with different wavelengths.

[sglrio]

actually i have to get all compounds (initial and its products) and i got difficulties about it. yeah, i think i should repeat them. i use fraction collector to collect the samples.

[HPLCaddict]

Ah, so you're doing preparative chromatography. Bad luck - reruns are a bit more of work
I'd suggest to try a low wavelength, something like 210nm - should increase the chance to see everything. But be careful, you'll see more junk, also. You should develop the method on an analytical system first before going to preparative work...

[prepcolumn]

hello.. i change the wavelength and acetic acid peak has already dissappear. however, i still confuse about how to choose wavelength. my initial sample has maximum absorbance at 595 nm. so when i inject sample in that absorbance, the initial sample peak is appear. but my treated sample (metabolite product from my initial sample) can not detected. so what should i do? i should get the new absorbance to detect other compounds? sorry, too many question. it's the first time i use hplc. thank you so much..

hello
595 nm is the visible range, that's why you may easily see your initial peak (which is a dye). As far as i understand your metabolite is decolorized which means your metabolite has less conjugation then initial product. This causes the wavelenth shift to lower (below 350-400 nm). In that case you should select two wavelenghts to detect both compounds.

for acetic acid case; maybe acetic acid forms during the chemical transition. Not to detect acetic acid you may select a wavelenth higher than 220-230nm if possible

good luck