Chromatography Forum

Hello to all,

I have tried precolumn derivatization by using 50 µl of 500 µg/ml FMOC-CI solution and 50 µl 0.2 M borate buffer, pH 8. The samples were kept at 50 C for 20 min. in hot air oven. The chromatograms of blank solutions (without analyte) are different each other.
How can I solve this problem?

Thanks

Hi ebruu

more information is required.
maybe your column is not equilibrated enough?
what is your chromatography method? column type? and such...

how many blanks have you done

Hi,

Blank solution preparation: To 50 µl acetonitrile, 50 µl 0,2 M borate buffer solution, pH : 8, was added, followed by addition of 50µl 500 µg/ml FMOC-CI solution resulting mixture is vortex for 10 s. The derivatization was performed at 50 C for 20 min in hot air oven.

Chromatographic seperation : I have used for chromatographic separation Hypersil MOS C8 column (5µm partical size, 200 mm x 4.6 mm i.d.)
Mobile phase is consist of methanol : water ( 20:80, v:v) and flow rate is set at 1 ml/dk
Prior to analyse, the column is flushed with the mobile phase for one hour and the baseline is observed stabile.

I have sent two blank chromatograms, both of two blank chromatograms are the same day.

May be not stable in hot air oven temperature?

Thanks

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What is ml/dk? What are you trying to derivatize? If memory doesn´t fail me this is usually done at room temp. Also where is unreacted FMOC-Cl? Where does FMOC-OH and FMOC-NH2 come? Most of your peaks, if not all, could be system peaks.

1, what is the void time?
2, the two blank from the same or different vial?
3, the sample solvent seems to be strong. how much is the injection?
4, could the reaction inconsistent?

1-Void time is 0,9 (why is it necessary?)
2- I have injected from different vial
3- Injection volume is 20 µl
4- Although I have preparated the blanks according to the same procedure, the resulting blank chromatograms are very different.

I do know why void is 0,9(hour? minute? second?).

Try inject from the same vial. IF c-grams are still diff, then you have LC issue. IF same, your have prep issue.
good luck,

If the flow is 1ml/min the to (tm) would be expected considerably above 0.9min.
Incidentally, I was not talking about a blank, but injection of reagent FMOC-CL.

Try to ramp the mobile phase to a high percent of methanol after your peak comes out to wash the FMOC from the column before the next injection. Then re-equilibrate the column for several minutes at 20:80 methanol:water before the next injection. You can also try to cleanup the sample prior to injection. It would help if we know what you are trying to derivatize.