HPLC Difficulty - Please Help

sykkea · Joined: 27 Mar 2009 Posts: 3

Hey, all. I took a look at the troubleshooting guide posted here, and to my knowledge, it's not addressing the difficulty I'm having. It's a bit of a strange/long story, so... here goes nothing:

For research, I've been using a 50mM solution of triethylamine at pH of about 6.6 for the aqueous portion (95%; other 5% is HPLC-grade MeOH). It gives me the retention times I need, the best separation out of any other mobile phase I've messed with... no snags.

Usually, I make 500mL of it at a time. My overseeing professor and I decided to make more, just so save us some extra time. We ran through the calculations in order to make a 2L batch and did so. This is where it gets strange. Whenever I made 500mL, it had an initial pH of around 3.7 that I had to adjust to what we wanted — 6.6 — with 1M NaOH dropwise. HOWEVER!! When I make 2L of it, it's suddenly sitting at an initial pH of 6.79.

I took it to the professor, who acknowledged that it was indeed strange, but told us to go leave it. I received an email the next day saying that when she ran it, the retention times were not what they should be. I went in today to try it again.

I used freshly cleaned equipment to make the new batch, just in case the other was due to some weird contaminant. No such luck, this one's sitting at an initial pH of 6.74. I am SO CONFUSED! Professor once again told me to leave it, and she's going to email me on Monday with results... but I am not optimistic.

Does anyone, anyone have any sort of insight on this? I'm seriously just... baffled.

Chris · Guest

How can a 50 mM solution of NEt3 in water have an initial pH of either 3.7 or 6.8? It should be basic, after all.
How exactly do you prepare the buffer?

Guest · Posted: Fri Mar 27, 2009 8:59 pm Post subject:

I could have sworn it would be basic, too. But... I quit trying to beg an explanation when I saw that the first couple of runs worked. I promise you, that's what the pH meter was reading. And yes, we do calibrate it before use.

I'm simply following instructions my professor gave me. 13.765g of solid triethylamine measured out, dissolved in 2L of DI H2O. That's all. Supposedly, after that, it's 1M NaOH until the desired pH.

I suppose if it went basic like I originally thought it would have, I'd have to correct it with HCL dropwise...

sykkea · Joined: 27 Mar 2009 Posts: 3

Chris · Guest

Triethylamine is a liquid at RT...

sykkea · Joined: 27 Mar 2009 Posts: 3

If the lab were still open, I would go in and grab the stock bottle to tell you exactly what it is. But it is a solid.

... I know I'm left sounding like just another idiot, and for that, I apologize. Perhaps I should just take my leave and wait to see what Monday brings.

Rod · Joined: 22 Nov 2006 Posts: 182

may be what you use is Et3N*HCl?
because Et3N is liquid - no doubts.

In any case, it does not explain different pH at different scale.

Such kind of things happen to everyone from time to time. Honestly, I believe there is no miracle behind this story. There is a mistake somewhere, e.g.:
1)different batches of reagents (are you sure there is no 2nd bottle looking like that one next to it? Sometimes content of a bottle does not correspond to a label on it.
2) do you check pH of water you use every time?
3) mistake in the course of pH calibration (eg pH standards are not good anymore)