Here I am conjugating FITC dye with penta ethyle tetra amine, but I am suffering with the separation problem of unconjugated dye, can anyone help to solve this problem?
I had a similar problem and used an MPLC to separate FITC from my product.
However, I had to use a very shallow gradient (chloroform-methanol; normal phase silica gel) to achieve a suitable separation.
How about using an HPLC system with built-in fluorescence detector.
Usually, this works well for us when separating conjugates from free fluorescent dyes.
Of course, when such an instrument is not available or your HPLC-columns are for analytical quantities only, flash column chromatography using silica and dichloromethane/methanol as solvent should also work.
From my experience, size-exclusion gel filtration using Sephadex G-25 most of the time does not provide enough resolution to clearly separate conjugates from free dyes (your conjugate is max. double in size compared to FITC).
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