High!
I have to set up an method for low molecular weight carboxylic acids (C2-C5) using HPLC. Iīve looked around and found the BioRad Aminex HPX-87H column to be suited best (alternatives: Waters KC811 expensive, Dionex not usefull in our lab)
Could anyone provide me with doīs and dontīs for this column or are there better alternatives??

Thanks for any help

tomtom

tomtom - there is still RP chromatography on endcapped C18 columns. I have seen some applications using buffer at pH 2.4-3.0 as solvent (my girlfriend works with GromSil ODS HD, but Waters Atlantis column has a similar application). Also polar embedded columns should be suitable.
In the end you have homologues that differ drastically in their polarity.

Dear TomTom, I've found a publication which might be helpful. Separation of carboxylic acids on a weakly cation-exchange resin by ion-exclusion chromatography, J. of Chromatogr., Vol. 850 pages 187-196 (1999). Even that publication is from the last century, that application still works. Good luck. Gerhard

I have done about a zillion of this separation. Get ES Industries aqueous column Aquasep, use 0.02% phosphoric acid and acetonitrile. Start at 0% acetonitrile and ramp up till last acid comes off. UV at 205 nm. detection limit for acetic is less than 1 ppm with 10 uL injection! a recent J. Chromatogr publication describes this very separation. I don't have the citation but it is under my name.

some of the polar embedded columns contain basic sites and therefore are a disaster for this separation,see JChromatogr. 868(2000)41.

tomtom

I'm curious what you meant by "Dionex not useful in our lab". Perhaps you tried using an ICE AS6 for this application. Certainly, this column wouldn't be a good choice since its considerably more hydrophobic than conventional gel based material such as the BioRad HPX-87H column (and hence not a great choice for acids as hydrophobic as valeric acid) but the ICE AS1 column is essentially equivalent to the BioRad HPX-87H column and it works well for this application.

Regarding do's and don't's, a crucial point is to take care of divalent cations. Such a column has a high-capacity so it will take a while for the problem to manifest but the resin shrinks in the alkaline earth form and eventually shrinkage will damage chromatographic performance (and the problem is not always fully reversible). If you believe your samples may contain significant amounts of these metal ions, it's advisable to either remove them using readily available sample prep tools or periodically regenerate the column using relatively concentrated acid (i.e. 0.25 molar sulfuric acid). Also, the valeric acid in particular will benefit from the addition of some acetonitrile to the eluent. A gel based material takes quite a while to reach equilibrium with solvent so be patient. 5% acetonitrile is a good starting point.

He probably meant that Dionex would not sell to him if he does not have a Dionex instrument.

I've always been fascinated by that business model. I was trying to develop a method for simple sugars and I knew that the Dionex Carbopac was the best column out there. They would not sell me the column because I did not have a Dionex LC.

To this day I still cannot believe it.

Dear all!
Thankīs for your input.
First of all I have to correct for some misunderstanding: we have two Dionex HPLC in our lab which are in daily use with inorganic anions and cations, so we wonīt change the configuration - thatīs what I meant with "not usefull"

Divalent cations will be a problem, the matrix is anaerobic sludge from a waste water treatment plant (Ca~50-200mg/l, Mg~10-100mg/l),the questions is if it is better to use a silica based column?

tomtom

the cations will certianly not be a problem with the reversed phase column I recommended. They may be a problem on an ion exclusion column. I suspect there will be soluble humic material in this sample and it may bind forever on polymer or reversed phase. This application may call for a guard column. I think that column life my be the most significant issue to resolve. This would likely be a nice capillary electrophoresis method as it would be more immune to the junk(putting it kindly) in your sample. Depending on what ever else is in there you may be able to do the whole separation on a reversed phase guard column. These acids are miles apart. Don't forget to acidify sample.

If the levels are approx 0.01% or greater you might do well with GC after acidification of sample. Frequent liner changes would be essential.

Dear Bill
my preferences now are for the Aquasep-column, in addition the price is significant lower.
Humic acids: I have no information about quantities of humic acids in anaerobic sludge but I suggest they will be inside.
Sample preparation will be a great part of the analytical procedure since proteins, fats and oligosaccharides are in solution and should not precipitate on the analytical column, a guard column will be used.
What column length (guard column??) would you recommend for the mentioned analytes?

tomtom

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Dionex not very useful .... the small print says that you invalidate your warranty if you use non-dionex columns in a dionex IC. How crap is that!.

If all you had to worry about was C2 to C5 straight chain acids, I am fairly positive that the separation could be done only on an AquaSep guard column. but, who knows what may in your sample that will absorb by UV. So, perhaps you will need greater resolution, or worse there will be so much UV absorbing junk around that the method will fail by UV detection.

In that case, an Dionex AS 10 guard column will probably do the separation just fine, and conductivity detection, with all its problems, will be more selective. It is my favorite place to start and many times it works just fine and at a lower cost than a full sized column. I have not kept up with newer Dionex columns so there may be a better choice now days. Also, Old Waters 510 pumps will pump dilute sodium hydroxide for at least 10 years, as will Perkin Elmer pumps. So, the eluent can be pumped on either of these systems and probably others as well.

I am glad these are your samples to handle and not mine. The smell must be something. Typically such a system will be run until the bugs canabilize themselves, so there may not be significant "digestable" material around, just humus.

I have bought Dionex stuff since the company started and I was not fond of their marketing philosophy either. But, in all fairness, they have improved greatly. So, let us encourage this trend. I am sure all their scientists feel about these things as we do. So, let us not discourage their participation in the forum.

probrably this is not the better place to write this but,GC is a much better choice.
I am performing 600 of this analyses in cattle's rumen in a 5 minutes run.the only problem is that you need a FFAP column but a packed 6 ft SS column with H3PO4 and chromosorb can do the job very well.

Thanx to all, I learned much from your input

tomtom

I want to detect piperine from a poly herbal medicine. could any one can send me the easiest HPLC method (If have paper plz send)

We routinely do small organic acids by HPLC and GC. When dealing with aqueous solvents on HPLC we use a Prevail organic acids from Alltech with 0.08% formic acid or 25mM KH2PO4 pH 2.5 with varying amounts of ACN - detection 210nm. If using an organic solvent we tend to use GC on a FFAP column which is quicker and more sensitive.
John