Chromatography Forum

So, I have been running taurine standards (NH2CH2CH2SO2OH) on a C18 SPP 2.7 um 3x50 mm column (Its technically an amino acid). I get weird wavy baselines and split peaks. The huge peak is the derivitizing agent unreacted.

The derivitazation method is summarized in the link:

Mobile Phase used was 10mM pH=9 phosphate buffer/ MeCN at 2 ml/min with gradient run starting from 90:10 v/v buffer/MeCN going to 50:50. .

So the first day I did the runs I get good chromatograms :

Then I ran it the second day (don't mind the changes in retention times. The mobile phase ratios, flow rate, were tweaked to try to see if something worked. These are the ugly chromatograms I got:




Is this weird baseline behavior and every thing else thats ugly something that anyone came across. I am giving up. Thanks so much guys.

-Mike

I'm not sure what column you're using, but a lot of silica-based columns can't tolerate pH 9. This might explain the decreasing retention and the degrading peak quality.

Thanks for the reply.
I do add a pH=6 phosphate buffer with the carbonate pH=9 buffer, so I'm not sure that's the problem. Maybe because solution I inject has some unreacted bubbles of derivatizing reagent floating around? Is there any way to get away with derivatization by using a different column. I have a C18 on silica.

the taurine derivative shows up as the 2nd peak after the solvent peak...The small one

Thanks for the reply.
I do add a pH=6 phosphate buffer with the carbonate pH=9 buffer, so I'm not sure that's the problem. Maybe because solution I inject has some unreacted bubbles of derivatizing reagent floating around? Is there any way to get away with derivatization by using a different column. I have a C18 on silica.

Ok, your first picture says pH 9 phosphate buffer, not carbonate. What is the *actual* pH and make-up of your mobile phase? What column are you using?

So mobile phase A is 100% MeCN, mobile phase B is 0.01 M pH=6.08 phosphate buffer. The gradient runs from 90:10 phosphate buffer : MeCN all the way to 50:50.

For derivitization, a 0.01 M carbonate buffer with pH=8.99 is added, then the phosphate buffer from above is added at the end. The resulting solution for injection contains around 72% phosphate buffer and 28 % carbonate buffer (each of equal molarities).

The column used is Brownlee SPP 2.7 μm C18 3.0 x 50 mm. It is an FX-15 UHPLC from Perkin Elmer. Thank you once again.

Oh duhh... That adds up to 100%..

Ok, in more detail... for derivitazation, 1 ml taurine/water solution is mixed with 2 ml carbonate buffer (0.01 M pH=8.99), 0.5ml DMSO, 0.1 ml of 1-fluoro-2,4-dinitrobenzene. Then after 15 min. I add 6.5 ml of phosphate buffer (0.01 M pH=6.08)

You might have solubility issues with your buffer at 90% acetonitrile. To check, mix 100mL of your buffer and 900mL acetonitrile (or some other 1:9 ratio), let it sit, and look for cloudiness or a precipitate. If you see a precipitate, you might be slowly precipitating the buffer into the column and the frit at the head of the column, which will cause peak splitting and decreased retention. Can you use methanol instead of acetonitrile? Most buffers are much more soluble in methanol than acetonitrile.

I'm wondering. If I am putting acetic acid and HCl into my phosphate buffer can there be a reaction with the buffer and acetonitrile? I think I'm am ruining the buffer by adding a weak acid (acetic acid).

When and why are you adding HCl and acetic acid to your phosphate buffer?

I have a pre-made 1 M pH 7.7 phosphate buffer. So I dilute and adjust pH to 6, then put the mobile phase into my reservoir.

Dilute your buffer with phosphoric acid, or make your own buffer using phosphoric acid and Na2HPO4 or K2HPO4. I can see acidifying with HCl to get from 7.7 to 6, but why acetic acid? Why are you adding that?

Also, it is immaterial what you are adding to the phosphate buffer in relation to acetonitrile. If your phosphate buffer precipitates in a solution of 90% acetonitrile and 10% buffer, you have a problem.