Chromatography Forum

Hello to all,

I have been working on the specific components of collagen. People from different labs reported different excitation and different emission wavelengths for the same compound. Of course, they are using different buffer systems, but, most of them are using reversed phase column.

So, come back to the question, is selection of excitation and emission wavelengths depends on

1. what buffer we use for the assay ? (even though everybody is working on the same compound)

2. or, the nature of the reversed phase column?

Thanks in advance for the answers
Rajeev

Polarity of the medium (solvent), pH, temp.,... can have a profound effect on fluorescence. That is why fluorescence is used to study conformation, etc., of proteins. As far as the column is concerned, I doubt that the denaturing effect by different RP columns is so different that it would show up even after the protein dissolved in mobile phase.
What do you mean with "same compound"? All used an identical sample? I just can´t picture collagen as being a single compound.

HW mueller,

Thanks for your replay. I am sorry for the confusion. I have been working on different cross links of collagen such as HP, LP and pentosidine (instead i just wrote specific components of collagen).

Different labs are using different sources to identify the above cross links (cartilage, bone etc.), In my case, I am using tendons.

Here are the few refs:
Journal of chrmatography B, 703 (1997) 37-44
Analytical biochemistry 278, 99-105 (2000)
Biochimica et Biophysica Acta 1272 (1995) 53-60

As i said before, i am using tendon as a source to measure HP,LP and pentosidine
Buffer A: 0.05% HFBA in D.D water
Buffer B: 0.05% HFBA in 60% acetonitrile in water

rajeev

Yes to both. Protic solvents like water and methanol do quench fluorescence more than aprotic solvents like acetonitrile. Just one example is aflatoxin B1: strongly fluorescent under normal-phase conditions, but under reversed-phase conditions it is so weak that derivatization needs to be used. Some fluorophores are less sensitive to this effect, for example FMOC.

The column effect is more related to the changes in mobile phase needed to elute the compounds in a convenient time and resolution.

rajeev, judging from what you have told us, it appears that everybody has a different substance (not a single, homogeneous compound), and possibly different media, so different spectra are to be expected.