i am trying to develope a metod for separation of acidic compounds such as ferulic, caffeic, coumaric and hydroxbenzoic acid, i'm using hypersil bds c18 column, and literature says that mobile phase should be something about 80:20 water:methanol with addition of acetic acid to ph 2.5, i've tried various variations of mobile phase composition also with acn and buffers, and there is a separation but the peaks are very broad (flow 1.5 ml/min) they are fronting (big assymetry) and the left side is distorted, the temperature is 38 C, and ph allways about 2,5. What is wrong and what should i try? change column (aquasil c18, nucleosil c18, nova-pak c18), but the esters of hydroxybenzoic acid are runnin very well. i am wating for you advice maybe some of you tried to do sth like that. thanks

Try injecting less, or make the sample in a weaker solvent.

We use acetic acid-water and ACN or methanol also, with no issues. I agree with Anonymous above. In general, when peak shapes are poor, try injecting less and note whether stuff is better, worse, or the same, that's a good start.

If the left side of the peak is distorted (fronting) the problem is usually the injection conditions. As above, injecting less is likely to help. Most likely, the sample is made up in a "strong" solvent. Usually, this is an excess of organic solvent, but in the case of acidic samples it could also be the pH of the sample.

Since the esters of the hydroxybenzoic acid are running well, and the rest does not, I bet it is the pH of the sample. Add some acetic acid to the sample, and the problem should disappear.

Please check this out as alternative approach:
We have several columns for analysis of organic acids-good peak shape and symmetry, high efficiency
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http://allsep.com/makeChr.php?chr=Chr_031 for benzoic, maleic, fumaric and phtalic acid
http://allsep.com/makeChr.php?chr=Chr_030 for succinic, malic, maleic and fumaric acids
http://allsep.com/makeCmp.php?cmp=Cmp_035 for hydroxybenzoic acids

http://allsep.com/makeCmp.php?cmp=Cmp_045 for formyl-pyrrol carboxylic acid

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mail@allsep.com

hi, i was trying to inject samples disolved in methanol, acidic methanol, or acidic mobile phase, to acidify solvents i used acetic acid, final concentration 2,5%, i am injectin 20 microliters and the absorbance i at 0,1 units level. i also though that the ph of sample is wrong but i have checked it and eliminated this problem, so my question is still opened

Injecting the sample in a more polar solvent might help. If this does not eliminate the fronting, then we are looking at the wrong area.

What happens if you switch your temperature control off and run this at room temperature? If the peaks are fine, then the fronting could come from the temperature control, or lack thereof. The mobile phase needs to be preequilibrated to the column temperature. Standard column heating systems provide a pre-column tuning for this equilibration. Check your care and use manual for information!

There is no need to buy a new column from Allsep. This separation should run well on any one of the columns that you have tried.

chromatograms of samples in 100% methanol are also bad, and the problem exists in room temperature and in higher temp, there is only minor difference,nearly the same, what should i check next?

Yes, I expect chromatograms from samples in 100% methanol to be bad, due to the fact that the sample solvent is too strong. Water is more polar than methanol. Dissolve the sample in water with some acetic acid!

Since the problem is not there at room temperature, but does occur when the sample is dissolved in methanol, we are back to the issue of the sample solvent.

Hi, finaly this separation is succesfull, using 80:20 3% acetic acid in water:methanol 38 degrees flow 1,5, i dissolved samples in mobile phase and it is OK, there is only one thing left, the peaks are slightly tailing. Thank you Uwe for your help.