Fluorescence detection - influence of solvent

[Veronika R. Meyer]

For educational purposes I am interested in an example of a HPLC separation with distinct influence of eluent impurities on fluorescence. Does anybody have knowledge of a (quantitative) analysis that was affected by impurities or oxygen in the mobile phase? I.e. a less pure solvent gave poor results whereas the same solvent of better quality worked fine? "Affected" could mean that the fluorescence response factor of a certain analyte depends on the solvent purity or the occurence of additional peaks.
Veronika R. Meyer

[Uwe Neue]

I don't have an example, and it might be tough to find one. Usually, one would do a calibration curve with the same mobile phase as the sample is run in, and then the suppression effect cancels (or better, it would show up only by comparing calibration curves obtained in different mobile phases, which is not a common thing to do).

[HW Mueller]

Yesterday I almost started typing something that would have been essentially the same as that what Uwe said. Then I thought that someone might have bodged something and have an example by accident. Also, I wanted to look at my stuff to at least find an example of different fluorescence yields due to different mobile phases. Unfortunatly, nothing was dug up within reasonable time, though I am certain that I have examples. Now, photochemistry books are full of examples of quenching, certainly you can find a compound that is quenched by a second one that you put in your mobile phase. Usually, polar solvents also quench fluorescence better than non-polar solvents, so increasing the H20 % can lower fluorescence (that´s what I looked for this morning).
One more thing: A potential quencher which is separated sufficiently from your fluoreszing analyte will not be able to quench (a "dirt" peak separated from analyte can not interfere with the fluorescence of the analyte).

[Kostas Petritis]

Veronika,

I will try to give you an example but first of all let me tell you that fluorescence quencing has been used a lot for indirect fluorescence detection and below I give some relevant references. Now if you want a hypothetical example (I do not have a real one) let's say that you are trying to analyse L-tryptophan by fluorescence detection. Now if your mobile phase contains Cu(II) as an impurity that would affect the quantitation of Tryptophan as the fluorescence of the complex Cu(L-Trp) is quenced. In my above example I assume a non constant concentration of Cu (i.e. I guess that if the concentration of Cu would be the same all the time then you would have affected your sensitivity but maybe you could still be quantitative -although difficult to validate-).

Is this the kind of example you wanted?

Title: Indirect detection of halide ions via fluorescence quenching of quinine sulfate in microcolumn ion chromatography
Author(s): Takeuchi T, Sumida J
Source: ANALYTICAL SCIENCES 20 (6): 983-985 JUN 2004

Title: Fluorescence quenching as an indirect detection method for nitrated explosives
Author(s): Goodpaster JV, McGuffin VL
Source: ANALYTICAL CHEMISTRY 73 (9): 2004-2011 MAY 1 2001

[tom jupille]

Also, ff you want to go back a quarter-century:

Bakalyar, Bradley, & Honganen, J. Chromatog., 158 (1978) 277.

or, if you have a copy, a very nice figure from that paper is reprinted in Dolan and Snyders' 1989 Troubleshooting LC Systems book on page 144.

[shirishpatel]

YA absolutely right what you are facing can be observed .

It can be due to flowcell of the FLD detector , with the use of the buffer and all flowcell performance slowly demises and your reponses also changes .

i prefer that you could inject impurity standard everyday for analysis . so there can not be confusion of change of responses .Using RRF with the method of FLD is going to give your surely a wrong results

[AdrianF]

I have done work with polyaromatic hydrocarbons eg Fluoranthene, the fluorescence is strongly influenced by oxygen. The only way I could get consistent results was to constantly degas the mobile phase with high purity helium. Flourescence was significantly less without degassing.