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.
You can try gel filtration with low resolution matrix like G-25 or G-50 (desalting column- use very thin and long column). protect the column from light to avoid the bleaching of the FITC-conjugated compound .
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).
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes