Chromatography Forum

Hello,
Any neuro/catecholamine people out there. Need some serious help here, relatively new (2 yrs.) to HPLC. We are using BAS reverse phase HPLC to detect dopamine concentration for a dialysis exp. Does anyone know of a way to increase the retention time for dopamine, separating it from DOPAC and the mobile front in the process. Currently, dopamine peaks at just about 3 minutes on the downslope of DOPAC. To further complicate things, we have ascorbate (.25mM) and perchloric acid in the sample which swamps the dopamine curve. Adding ascorbate oxidase does not do anything in the context of the perchloric acid. I think my best bet is to increase dopamine retention time relative to mobile front if possible. Here is the current setup that works great with sample containing no ascorbate, again dopamine peaks at about 3 minutes on the downslope of DOPAC.
BAS microbore column
1X100 mm
3 mm ODS
C18
100 microliters/min. rate

Mobile phase pH = 3.4, 1 liter
containing 14.5 mM sodium phosphate
30 mM Sodium Citrate
10mM Diethylamine HCl
2.2 mM 1-octanesulfonic acid
108 microliters Disodium EDTA
40 ml acetonitrile
10 ml tetrahydrofuran

We already tried increasing the sample pH with sodium hydrochloride to prevent breakdown of the ascorbate oxidase but doesn't work, and other suggestions, help. Thanks a lot in advance, and hope to hear from someone. This is a great resource.

Dopamine and the silanols of your ODS coulum will become more attracted to each other at lower pH. I would suggest lowering the pH to the 2.5 to 3 range. An ODS coulmn should be able to handle that without falling apart on you.

Also, I do not know exactly how much you are looking to change ... but if it is a lot, then what I would really look at is chaning the mobile phase to something like 0.025 M KH2PO4 with 0.3 mM heptasulfonic acid in water and adjusting to a pH of 3. Using this you are essentially playing with ion exchange on a reverse phase column (as you are currently doing) .... but the mobile phase is easier to make. Additionally, the amount od organic in the mobile phase you are currently using is causing you to loose retention time of dopamine. So, run at 100% water until you get the dopamine off and then slowly switch to organic to get the late eluters/column wash...

You could also try a stronger ion paring reagent such as sodium laurel sulfate, but it will require more organic to move it off the column.

Here are bunch of application for dopamine. With Primesep mixed mode columns you can achieve any retention (from void to 60 minutes) just by playing with mobile phase composition:
http://www.sielc.com/compound_108.html

Regards,

Vlad

okay, this sounds promising, thanks shaun. I think I'm gonna have to spend the next couple of days troubleshooting this by testing mobile phases. Vlad, I'll look into mobile phases before thinking about columns for now, but the website looks like something for me to consider in the future. Any other suggestions from others are welcome cause I'll be working on this for a while.

Cheers,
Mark

Here are the conditions we use here at Dionex for catecholamines with electrochemical detection: http://www1.dionex.com/en-us/acclaim_li ... 27727.html

Below is a catecholamine separation done on Unison UK-C18:
http://www.imtakt.com/TecInfo/TI174E.pdf