Chromatography Forum

Hi,
I have a few questions regarding alterations to the mobile phase. I use HPLC-ECD to analyse amines etc including dopamine and its metabolites, noraldrenaline, serotonin and its metabolites, two internal standards (all in all 10 compounds). I could do this fine, then I changed column to a microbore (from 4.6x150 with 5um to 1x50 with 2.5um) and I have lost all my resolution. My mobile phase was 75mM NaH2PO4, 1.7mM octane sulfonic acid, 25uM EDTA pH 3 ; 90-10 v/v acetonitrile.
I have read the following:
Increase buffer concentration = decreased retention times (RT) of all compounds
Increased Octane sulfonate concentration = increased RT of amines
Increased pH = decreased RT of metabolites
Increased organic = decrease RT of all
Increased EDTA = decreased baseline (I have otherwise also)

Are these assumptions correct. I will be beginning microdialysis soon and not only do I need resolution but my sensitivity needs to be exceptional and I don't want to blindly test 20 different Mobile Phases when I could be more refined in my approach.
Appreciate the help

I suspect that the problem is not with the mobile phase. From 4.6 mm to 1 mm diameter columns is a pretty drastic step, and 1 mm columns are not compatible with all HPLCs (you need low volume injectors, connections and detector cells etc).

Check all your plumbing before you start playing with mobile phase.

Good luck Peter

^ what he said...

Extra column volume will hurt your separations a lot more than you're used to when you run a column that small. You will also have less capacity. If your flow cell path length and/or volume have not been optimized for this setup, you may suffer a net loss in sensitivity too.

In other words, I would look into going back to the old column and trying to increase the sample size (vol. and/or conc.) until you see unacceptable resolution or fronting on the larger peaks.

P.S. Octane sulfonic acid at pH3?

Agree with all posts.

I'd also recommend a longer column length.
150 (5um) is more comparable to 75 (2.5um)

Once you check that the system volume in your LC
has been optimized - you can try 2 x 75 mm (2.5um).

Thanks for the tips,
I spose I should give you an idea of my setup as well. I rarely inject more than 10ul at a time so I don't think it is injection volume that is an issue. I use an ESA 5014B microdialysis cell (-150mV / +250mV) with an ESA coulochem III detector and an agilent 1100 pump/autosampler. The whole system is only 6 months old. In terms of capacity I inject from 10-100pg at a time (femtomole range).

I aim to test microdialysate fluid which has very low sample volume and concentration (low femtomoles) so do you think it would still be my system or that given the drastic change in condition I should keep tinkering with the mobile phase composition.
Thanks alot for all your help too.

Edit: I based the method of a paper (Rossi et al. 2005 Neurochem Res) and they used Octane sulfonic acid at pH3 - would this be a problem?

I rarely inject more than 10ul at a time so I don't think it is injection volume that is an issue.

I beg to differ. 10 μL may seem small to you, but it's huge for that small column.

A good estimate of column void volume (volume of mobile phase inside the column) is Vm ≈ 0.5 x L x dia^2. That means that the void volume for your 50 x 1 mm reversed-phase column is only about 25 μL. To put that in perspective, your original 150 x 4.6 mm column had a void volume of about 1500 μL. Your 10 μL injection on the small column is equivalent to a 600 μL injection on your original column.

Edit: I based the method of a paper (Rossi et al. 2005 Neurochem Res) and they used Octane sulfonic acid at pH3 - would this be a problem?

I can't find a handy reference, but I am under the impression that chain length is roughly proportional to the effective buffering range of the alkyl sulfonate - eg C5 sulfonic acid might be more appropriate for lower pH while a C8 or C10 would be more appropriate for a pH much closer to 7.

Thanks for the input

Tom - I only thought 10ul wasn't that much as all the dialysis papers I read using microbore inject this or more but I will try to lower my injection volume; what would you suggest as an appropriate injection?. Also the 'empty column volume' as said in the user manual states 0.1mL (2.5mL for a my old column) although, I take it this is somewhat different to column void volume.

DR- I used to use heptane sulfonic acid but after seeing octane more I changed over. I have just (over the weekend) performed a mobile phase test using multiple concentrations (0.6, 1.7, 2.1, 2.5mM) and as expected all my amine retentions increased with increasing concentration so it seems to be working.

I really appreciate the help and to let you know I have run 7 different MP's now and my resolution is getting better, hopefully soon I can maximise the use of my new microbore column

For reversed-phase columns, a good rule of thumb is that the column void volume (volume of mobile phase contained in the column) is
Vm ≈ 0.5 * L * dc^2 where L is column length and dc is column internal diameter.

Another useful rule of thumb is that the injection volume should be no more than 15% of the volume (baseline width) of the narrowest peak. This assumes that the sample is dissolved in mobile phase (or equivalent solvent strength). If the sample is dissolved in something stronger, then you have to inject less; if dissolved in something weaker, then you can inject more.

I agree with Tom
I think you should try. Your column is lower diameter, your retention time may decrease, the intensity may increase so the volume of injection may decrease.