Chromatography Forum

Update, I got peaks by ~doubling the tubing length from my Rx coil to the detector. Peak looks Gaussian but small.
I am using the 547 buffer recipe of 19.1g borate, 100 mg OPA, and 100 ul of 1:1 ACN/ mercaptol ethanol.
I am following a paper from Waters who's recipe is 19.1 g borate, 800mg OPA and 2 ml mercapto ethanol (no idea if it is a 1:1 mix with ACN).
I'll try the Waters recipe tomorrow.

Bleach?? The old stuff I used was an Acros product of 5%, while Clorox is 8.25%.
None of the supply houses I checked has small bottles of 8.25%.
They have either 4-5%, or 10-15% . Argh!
Am I chasing my tail?
Should I just borrow a small amount from the cleaning crew?

Bleach?? The old stuff I used was an Acros product of 5%, while Clorox is 8.25%.
None of the supply houses I checked has small bottles of 8.25%.
They have either 4-5%, or 10-15% . Argh!
Am I chasing my tail?
Should I just borrow a small amount from the cleaning crew?

That is what we usually do, just get some bleach from the microbiology department.

Update, I got peaks by ~doubling the tubing length from my Rx coil to the detector. Peak looks Gaussian but small.
I am using the 547 buffer recipe of 19.1g borate, 100 mg OPA, and 100 ul of 1:1 ACN/ mercaptol ethanol.
I am following a paper from Waters who's recipe is 19.1 g borate, 800mg OPA and 2 ml mercapto ethanol (no idea if it is a 1:1 mix with ACN).
I'll try the Waters recipe tomorrow.

I used that Waters mixture about 20 years ago when I had the Waters equipment. It will work but the excessive OPA and mercaptoethanol will stain all of your tubing dark blue and you will need to wash the cell often with straight Acetone to keep it clean.

I switched to the Hamilton PRP X400 column from the Waters column and never had any problems. We tried the Pickering column and while it worked great, it was not compatible at all with Methanol, and if you didn't get the Methanol washed out after doing Carbamates you would ruin a $1500 column.

Look up the Hamilton procedure and they have trouble shooting ideas which say that if you have AMPA but no Glyphosate you need more bleach. Too much bleach and you will begin to lose both, so it is a balancing act. Also if the reactor is too hot, you will not get peaks, 36C is what we use.

James, using the Hamilton column do you use the carbamate buffer ? 100mg OPA and 100ul 1:1 mercaptain/ ACN?

Sorry I missed the reply. We have used the carbamate buffer with success, and also a more concentrated buffer which is about 5x more borate. I always use the same OPA and mercapto as the carbamates.

The big thing I did was make sure the eluant going into the detector had a pH of at least 10, to make sure the reaction would work. If it is low just add some potassium hydroxide to the OPA reactant until you have the proper pH at the detector entrance.

Yup, did it,killed my column. Getting results, but my glyphosate peak is ~2 min wide @ baseline, AMPA~3.5 min!

Question for James, can I use Waters post column chemistries with the PRP-X400 column?

Yup, did it,killed my column. Getting results, but my glyphosate peak is ~2 min wide @ baseline, AMPA~3.5 min!

Question for James, can I use Waters post column chemistries with the PRP-X400 column?

It shouldn't be a problem. Just look at the flow ratio to try to keep the same concentration in the final mixture. If the Hamilton column uses half the flow rate as the Water then reduce the reactant flows by half, just as an example.

Thanks