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Ethanolamine-HCl solution storage
Babcock_Hall:
T. Miron and M. Wilchek, A spectrophotometric assay for soluble and immobilized n-hydroxysuccinimide esters, Anal. Biochem., 1982, 126, 433–435.
I recall obtaining some information from this paper in an attempt to follow a model reaction between an amine and a succinimide ester.
Biotech78:
--- Quote from: Babcock_Hall on November 13, 2023, 10:02:18 AM ---As long as the pH is adjusted correctly, I cannot see a problem. BTW, there is some literature on following NHS coupling reactions: DOI: 10.1039/c5ay00042d. My situation was a little different; I tried to use spectrophotometry, based on the absorbance of the side-product (which as a pKa of 6) with mixed success.
--- End quote ---
I think you are right. I was talking to the millipore tech support scientist the other day and he said it could be dissolved CO2 lowering the pH. I checked the pH of stored buffer and for some reason, the pH was rather high (9.2ish) rather than what thought I had adjusted it to (8.5). Don't know it was the pH meter which needed recalibration or what. I will make the buffer again and check.
Babcock_Hall:
Borek is far more knowledgeable about pH than I am. However in my experience the pH 10 buffer standard is not indefinitely stable, perhaps because of carbon dioxide. My preliminary thinking is that a different analytical technique from SPR might be worth pursuing; the HILIC method that I cited above has a detection limit of 1 mg/L: Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC). One problem with spectrophotometry is that many other substances absorb where the anionic form of N-hydroxysuccinimide does. I had been running a reaction in an organic solvent and attempted to extract the chromophore to monitor its progress. I think such an idea has merit, but it needed some fine tuning.
Biotech78:
--- Quote from: Babcock_Hall on November 14, 2023, 10:02:04 AM ---Borek is far more knowledgeable about pH than I am. However in my experience the pH 10 buffer standard is not indefinitely stable, perhaps because of carbon dioxide. My preliminary thinking is that a different analytical technique from SPR might be worth pursuing; the HILIC method that I cited above has a detection limit of 1 mg/L: Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC). One problem with spectrophotometry is that many other substances absorb where the anionic form of N-hydroxysuccinimide does.
--- End quote ---
My primary aim is not the quantification of NHS. EDC/NHS coupling and then ethanolamine deactivation of "remaining" activated esters (that did not couple with the ligand) are the steps I am doing to prepare the SPR chip. My target analyte is a protein which will bind to the ligand I had already coupled to the activated carboxyl groups at the SPR chip surface.
Babcock_Hall:
I may not be following. Will the reaction of ethanolamine with an activated ester produce NHS?
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