Chromatography Forum

I am now working on As speciation, using a C18 column (5um, 15cm). We have 7 species in one run but no matter what I did, changed pH, or different ion-paring reagent, or other modifier, I can't seperate them all at one time. Do you think a smaller partical size column (or/and longer column) would certainly help?

Agilent recently offered an ion-X column for As speciation. I haven't read the article below. Talk to them, and maybe they'll run a sample or suggest the best option for you.

http://www.chem.agilent.com/scripts/Lit ... 989-5505EN

Bruce Hamilton

Try complexing with EDTA and then use an ion-X column instead of a C18.

J. Anal. At. Spectrom., 2004, 19, 973-978
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Selective arsenic speciation analysis of human urine reference materials using gradient elution ion-exchange HPLC-ICP-MS

Jens J. Sloth, Erik H. Larsen and KÃ¥re Julshamn

Iceman, to answer your original question: unless you are reasonably close to acceptable resolution, merely using a smaller particle or longer column will not help much.

Resolution is approximately proportional to the square root of the plate number. If you double the plate number (e.g., by making the column twice as long) you will increase your resolution by 40% (the square root of 2 is about 1.4).

As suggested by the previous posts, a different separation mechanism with different (better) selectivity is probably a better approach.

Dear all, thank you so much for your help!

Bruce: We have noticed the 'Agilent column' as well, we also read the paper. But the application note from Agilent using this column didn't try to sepearte MMA(III) and DMA(III). Using an C18 column with ion-paring reagent at higher pH, we are able to obtain the same good results as Agilent provided.

Jdh199: Thank you I also read this paper too, The resolution of those As species are excellent. However, there's no application of MMA(III) and DMA(III). Even the published papers dealing with MMA(III) DMA(III) were not satisfactory. I wish I could use gradient too, but our HPLC pumps are only isocratic. It's a pity.

Tom: Yes we were very close to separate all the species only two peaks were merged. But we do have 2 C18 columns and maybe I will simply try to couple these two columns and see if we can get better resolution.

I was recently sent a method a friend of mine had developed for analysing trace metals in API by HPLC.
It involves a pre-column complexation with DCC.

If you would like a copy of the method (written for Pt but it worked for most catalytical metals in developments provided good complex formation) to use as a basis drop me an e-mail and I'll forward it to you.

Cheers, Pat.

I was recently sent a method a friend of mine had developed for analysing trace metals in API by HPLC.
It involves a pre-column complexation with DCC.

If you would like a copy of the method (written for Pt but it worked for most catalytical metals in developments provided good complex formation) to use as a basis drop me an e-mail and I'll forward it to you.

Cheers, Pat.

That's great, Pat. My email address: mannheim@gmail.com
Thank you!

We use a method similar to the paper I quoted. Ours is not a gradient per se, instead it switches from 100% A to 100% B, then back to 100% A over a 12 minute run. Do you have the set up to do that? A a simple switching valve with two isocratic pumps could do it.

Here's an example chromatogram:



ps... FYI, the new Agilent column retails at $2500 CDN for the column, plus $550 CDN for the guard column I think I'll stick to our current one at less than half that price.

ps... FYI, the new Agilent column retails at $2500 CDN for the column, plus $550 CDN for the guard column I think I'll stick to our current one at less than half that price.

Ouch!. For that price I'd expect their technical support to run several samples for the original poster to clearly show that the column would perform the desired separation, before any purchase..

In fact, I'd almost expect them to include somebody to run the chromatograph with any purchased As speciation column .

I'm very keen to encourage column manufacturers to provide technical support for columns, and the more requests they get for evaluations, the more they might provide such support to ensure customers buy the appropriate column. If a column does the job well, price may become less of an issue for some customers.

Bruce Hamilton

have you tried already a 25 or 30 cm 5u column?

if they can't give the "N power" to separate your analytes then none of the smaller particle columns won't probably either at least according to the theoretical values and the "pope plots",

like it was suggested above you will need to find a better suited chemical appraoch.

if you are going to try and couple; and you can convince your boss to do it, try maybe to do an isocratic 2D approach proposed by BISCHOFF.
as their are trying to push their new "poplc" they might help you in the method development side.

that sounds interesting, but I don't really know what BISCHOFF is?

that sounds interesting, but I don't really know what BISCHOFF is?

http://bischoff-chrom.de/?url=poplc&lang=en

Arsenic species cover the range in properties including anions, cations, betaines etc so the best selectivity is offered by ion exchange. I have been working on a dual IC-MS method that separates ASIII, ASV, MMA, DMA and Arsenobetain using anion exchange and mixed mode anion/cation exchange so that all of these species are retained and can be detected using ESI-MS detection in one analytical run. All of these species are also well separated from sodium, chloride and other common matrix ions. Copies of a poster and an oral presentation are available from Dionex.