April 17, 2024, 11:56:55 PM
Forum Rules: Read This Before Posting


Topic: Ethanolamine-HCl solution storage  (Read 1836 times)

0 Members and 1 Guest are viewing this topic.

Offline Biotech78

  • Regular Member
  • ***
  • Posts: 21
  • Mole Snacks: +0/-0
Ethanolamine-HCl solution storage
« on: November 10, 2023, 01:43:44 PM »
Hi
Anyone knows the recommended conditions for storage of ethanolamine-hcl solution. I need to use it in an SPR experiment to inactivate the remaining NHS-esters after EDC/NHS coupling step.
However, I have observed that ethanolamine-hcl may not be working properly. It does not cause any (net) RU shift. I suspect I am not handling the solution properly. Plus my analytes are binding to control channels. Dissociation is virtually impossible for both test and control channels indicating proteins (analytes) are binding to the unreacted activated COOH groups on the sensor surface
How do I store the solution? Refrigerated? Frozen? or need to wrap parafilm around falcon/bottle cap? protection from light? what am I doing wrong?
So far what I know is that in solid form its hygroscopic, the solution shouldn't be. I saw one paper recommended make solution and then store in aliquots on -20. Light sensitivity not reported.
I make solution in a 50mL falcon, adjust ph with NaOH (8.5) and store at 4, no wrapping no light protection

Offline rolnor

  • Chemist
  • Sr. Member
  • *
  • Posts: 2212
  • Mole Snacks: +149/-10
Re: Ethanolamine-HCl solution storage
« Reply #1 on: November 10, 2023, 03:21:39 PM »
It should be stable if it is a tight stopper on the flask, just glass is fine.
« Last Edit: November 10, 2023, 03:38:17 PM by rolnor »

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 5608
  • Mole Snacks: +321/-22
Re: Ethanolamine-HCl solution storage
« Reply #2 on: November 12, 2023, 12:06:18 PM »
Can you explain the term "RU shift?"

Offline Biotech78

  • Regular Member
  • ***
  • Posts: 21
  • Mole Snacks: +0/-0
Re: Ethanolamine-HCl solution storage
« Reply #3 on: November 12, 2023, 02:42:18 PM »
Since this is for an SPR experiment, RU stands for resonance unit. 1RU shift is equal to a critical angle shift of 10-4 deg

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 5608
  • Mole Snacks: +321/-22
Re: Ethanolamine-HCl solution storage
« Reply #4 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.
« Last Edit: November 13, 2023, 11:43:55 AM by Babcock_Hall »

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 5608
  • Mole Snacks: +321/-22
Re: Ethanolamine-HCl solution storage
« Reply #5 on: November 13, 2023, 02:48:17 PM »
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.

Offline Biotech78

  • Regular Member
  • ***
  • Posts: 21
  • Mole Snacks: +0/-0
Re: Ethanolamine-HCl solution storage
« Reply #6 on: November 13, 2023, 08:19:17 PM »
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.
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.

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 5608
  • Mole Snacks: +321/-22
Re: Ethanolamine-HCl solution storage
« Reply #7 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.  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.
« Last Edit: November 14, 2023, 10:52:31 AM by Babcock_Hall »

Offline Biotech78

  • Regular Member
  • ***
  • Posts: 21
  • Mole Snacks: +0/-0
Re: Ethanolamine-HCl solution storage
« Reply #8 on: November 14, 2023, 10:29:31 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.
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.

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 5608
  • Mole Snacks: +321/-22
Re: Ethanolamine-HCl solution storage
« Reply #9 on: November 14, 2023, 10:57:56 AM »
I may not be following.  Will the reaction of ethanolamine with an activated ester produce NHS?

Offline rolnor

  • Chemist
  • Sr. Member
  • *
  • Posts: 2212
  • Mole Snacks: +149/-10
Re: Ethanolamine-HCl solution storage
« Reply #10 on: November 14, 2023, 11:36:10 AM »
no it will quench any remaining suc-ester

Offline rolnor

  • Chemist
  • Sr. Member
  • *
  • Posts: 2212
  • Mole Snacks: +149/-10
Re: Ethanolamine-HCl solution storage
« Reply #11 on: November 14, 2023, 11:40:51 AM »
If the pH of the ethanolamine solution is stable it is ok I think the problem is coming from elswhere

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 5608
  • Mole Snacks: +321/-22
Re: Ethanolamine-HCl solution storage
« Reply #12 on: November 14, 2023, 01:34:33 PM »

Offline Biotech78

  • Regular Member
  • ***
  • Posts: 21
  • Mole Snacks: +0/-0
Re: Ethanolamine-HCl solution storage
« Reply #13 on: November 14, 2023, 03:45:15 PM »
If the pH of the ethanolamine solution is stable it is ok I think the problem is coming from elswhere
I am still not able to figure out where the problem is. pH is okay. I recalibrated the pH meter and the buffer pH was around 8.43 (vs expected 8.5) which is within the expected error range. Could it be that the problem is at EDC/NHS step? Not enough esters to react with ethanolamine?

Offline Biotech78

  • Regular Member
  • ***
  • Posts: 21
  • Mole Snacks: +0/-0
Re: Ethanolamine-HCl solution storage
« Reply #14 on: November 14, 2023, 04:16:03 PM »
no it will quench any remaining suc-ester
https://www.sciencedirect.com/topics/medicine-and-dentistry/n-hydroxysuccinimide#:~:text=The%20NHS%20ester%20end%20of,hydrolysis%20rate%20of%20the%20ester.
If ethanolamine reacts with a succinimide-ester, N-hydroxysuccinimide will be a by-product.
I have a COOH group on my chip surface, I add EDC and NHS which leads to the formation amine reactive NHS esters. I add a ligand, say protein or any other molecule with an amine group, it reacts with esters and forms a stable amide bond. Now not all the esters would react with my ligand so if I bring in another protein, it's amine groups will also react with the remaining esters and form an amide bond. This is what we dont want. We want this last protein (analyte) to bind to the ligand only. So we use any primary amine to "block" or "inactivate" the "surplus" esters. This is where ethanolamine comes in. Ethanolamine will block the unreacted esters so my analyte will not have any esters to react with and will only bind to the ligand.
I am not concerned what are the products of reaction between ethanolamine and esters

Sponsored Links