[Babcock_Hall]

I am not concerned what are the products of reaction between ethanolamine and esters

What I am trying to do is to question (in a constructive way) your choice of method.  What evidence from the literature is there that SPR can detect the success of the reaction that blocks the surplus esters?  I suggest considering one of two methods found in the literature as alternatives.  BTW I coupled alkaline phosphatase to a Sepharose 4B activated as the 6-aminohexanoic acid N-hydroxysuccinimidyl ester long ago.  I used Tris to quench, but I did not think about trying to follow the quenching reaction to completion; so I admire your efforts in that regard.

[Biotech78]

I am not concerned what are the products of reaction between ethanolamine and esters

What I am trying to do is to question (in a constructive way) your choice of method.  What evidence from the literature is there that SPR can detect the success of the reaction that blocks the surplus esters?  I suggest considering one of two methods found in the literature as alternatives.  BTW I coupled alkaline phosphatase to a Sepharose 4B activated as the 6-aminohexanoic acid N-hydroxysuccinimidyl ester long ago.  I used Tris to quench, but I did not think about trying to follow the quenching reaction to completion; so I admire your efforts in that regard.

You may have a point. SPR may not be ideal to detect the success of the reaction we are talking here but it can nonetheless indicate how successful the reaction was. If ethanolamine does react with the esters on SPR chip, there will be an RU shift compared to baseline. If not, all the ethanolamine will be washed away in the wash step. Aim of my experiment is SPR itself. Ester-ethanolamine reaction is just a "preparative" step and because it is for SPR, I have to do in "on chip". All I can say is that is all I can do in the given circumstances.

[Babcock_Hall]

Last night I went to the library and into the journal stacks (which may not be available much longer) to re-read the article (Miron and Wilchek 1982 Analytical Biochemistry 126(2):433-435) on using spectrophotometry to follow coupling reactions involving NHS esters.  When I consulted this article before, I was running a reaction in an organic solvent and attempting to extract N-hydroxysuccinimide into the aqueous layer of a micro-extraction.  One thing that limited the usefulness of this technique in my hands was that there was a large value of the absorbance at time zero.  This might have been due to the reagent itself or to an impurity in the starting materials.

I am not trying to beat a dead horse, but if there were some way to perform UV spectrophotometry in your application (I know almost nothing about SPR chips), the spectrophotometric method might have more than one use.  One of their applications was to measure how much active ester was found on commercial gel designed for coupling to macromolecules, and I can imagine that this might be useful in your work as well.