Chromatography Forum

Hi,

After trying almost everything to get lower tailing of one of my peaks, I just skipped the column and injected with just a pressure regulator installed (1 meter of red PEEK capillary).

The peak that comes out has a tailing of 3.5... It improves just slightly if I lower the injection volume. Do I have retention in the LC system or does anyone have an explaination what's going on?

The method uses:
phosphate buffer pH 7.4 / ACN
fluorescence detection
sample is a flourescent dye

Hi Mattias,
there certainly is some retention on PEEK. Maybe you should change the pH of your sample solution. However, I do not know if you have any playground for that. On the other hand, think about reducing the volume you inject or shorten the tubing-maybe your sample is simply sticking on the tubing wall due to a different flow profile (you know: velocity of the solution near the tubing wall etc.) BTW, what is the volume of your red PEEK tubing (length, ID)?
Regards

Presumably your peak with the PEEK capillary is very much narrower than the one with the column, if it isn´t you have an enormous, extra-PEEK volume somewhere.
Red PEEK usually has a 0.1 mm ID so there should be about no laminar flow (which causes tailing in larger ID capillaries, though knitting can prevent that also.... discussed some time ago).
But, since there is some interaction between any two substances that can contact one should expect some tailing evn in a teflon tube.

There might also be a few other possibilities.

If you are using different tubing id's in your system, from injector to column, and column to detector, (and if a manual injector is used also the loop), you may sometime see an influence on your flow profile, especially at lower flowrates, and if the pump(s) are not suited for the purpose.

It may be also be due to shrinkage of the tubing. Especially, red and natural peek tubing are sensitive to overtightening, resulting in a restricted peek tubing. Combined with the previous, it may add to the problem.

If I were you, I'd cut all tubings, and reinject the sample without the column.

Finally, 1 m of red tubing (0.13 mm id) correspond to an extra-column volume of 13 microliter.

Hmm... Considering all the answers above, it appears that fewer people than I thought are ever measuring the system bandspreading.

When injection an analyte into a system without column, you will get fairly asymmetrical peaks. This is the nature of the beast, and has to do with the fact that the "plate count" is very low. In the extreme case, you can even get split peaks, but this is not likely to happen on a standard HPLC instrument with reasonable samples.

Bottom line: what you see is completely normal and expected. Compare it to the picture on page 349 of my book on "HPLC Columns".

An alternative in terms of dispersion in open tubes...as it was extensively studied by Ruzica & Hansen for the development of flow injection analysis (FIA).

"Flow Injection Analysis," J. Ruzica and E. H. Hansen, Analytica Chemica Acta, 99, 1978, pp. 37-76.

Ruzica, J., Hansen, E.H., 1988. Flow injection analysis. Willy, New York.

Thank you very much for your answers! I had an idealised picture of a gaussian peak coming out, but I was obviously wrong.

Since I rarely do flow injection analysis I have never thought of these effects.

Despite the tailing, the flow injection analysis works perfect with this molecule (very specific fluorescence). Why ever use "stand-alone" fluorometers...

Mattias, maybe you should ask this "Why ever...." separatly, I am sure you will feel like being on a roller coaster.

Mattias,

Not ignoring the excellent advice given above, did you try injecting something simple into your PEEK tube, using the same mobile phase eg something like toluene? It is possible that if you injecting some strongly adsorbing compound that it is sticking to something in the injector for example, and exacerbating the tailing that might occur.

HW Mueller> Now I got curious... Is it controversial in any way to use FIA instead of spectrophoto- or fluorometers? If so, maybe you are right about starting a new thread.

Well as a starter: I would have asked the question the other way around: Why do FIA when you can get much more sensitivity by just leaving it there in the cell for an optimum time, take a spectrum at leasure.... (if the substance is stable enough).

If sensitivity is not an issue, I think the automation possible with FIA is a great advantage. And in my case, the reports look much nicer from the chromatography system