Hi there,
I was wondering how to determine the substitution value(something like ~umole/g) for the resin in solid phase peptide synthesis(Fmoc chemistry). I have seen people mentioning about measuring Fmoc absorbance but not sure of how would that be. Could anyone help me with that?
Thanks,
Chun
You generally do this by weighing in the resin, diluting it with a known volume and then cleaving the Fmoc from the resin using a base. The dibenzofulvene which forms upon Fmoc cleavage can then be quantified at 294 or 304 nm. I use a protocol based on this paper: M. Gude, J. Ryf, P. White, Lett. Pep. Sci. 2002, 203–206
I agree with Christina's answer. When I do loading determination I measure the piperidine-dibenzofulvene adduct absorbance at 301 nm using the extinction coefficient of 7800 mL/mmol*cm. There are many ways to accomplish this, just make sure you use the correct extinction coefficient that corresponds to the wavelength you are measuring. Personally, the aapptec guide has worked well for me.
https://www.peptide.com/custdocs/1198.pdf
Fmoc method (UV) for the loading of Fmoc-amino acid (or FmocNH-peptide) on the resin!
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