Hello. I have 2 basic compounds to run on HPLC. These 2 compounds have 1 aromatic ring, an amine function, a ketone function, another ring (non aromatic.They have near structures and I can separate them in HPLC with a C18 column and 75% phosphate buffer (pH 3) with heptanesulfonic acid salt of Na, 25% ACN. But it work when they are in relative proportions. In reality one of them will be 130 mg/mL and the other one 1 mg/mL. But when the first one is too concentrated, it is like he wasn't retained in the column so I can't study the less concentrated. And above all I have to proportion the degradation products of the less concentrated product. Solid extraction isn't possible, so have some idea of what can I do?

P.S.: sorry for my english, I'm french.

Bonjour Anonymous, I guess your English is better than my French. For your separation problem my recommendation is to try another separation mode. HILIC, Hydrophilic Interaction seams to be better for your compounds than reversed phase mode. You can run with Acetonitrile/Water, so you have similar conditions as with RP! And you don't need heptane sulfonic acid to get sharp peaks. If you can e-mail me your address I can forward to you some information and applications on HILIC! Salut. Gerhard

From your description it appears that you are doing an isocratic separation. You may get better separation if you use a gradient separation. Start the separation with less acetonitrile (perhaps 5 or 10%) and elute with a gradient.

Do not inject too much sample. 130 mg/ml is very concentrated and this large sample could cause your main peak to broaden. use the least amount of sample possible.

Or, you might try separating these compounds as free amines rather than protonated amines. Therefore, try the separation at higher pH using a column that will tolerate high pH. (Waters and other companies have columns that are stable at high pH).

Do you need the heptane sulfonic acid? You describe a fairly large molecule that could be retained without ion pairing.

Sounds like overload. What is the sample dissolved in? Have you tried to do the separation without ion-pair reagents? Which one elutes first (it sounds as if it is the one present in the smaller concentration)?
On other suggestions, I agree with Bill.

There are a couple of other options not mentioned above. First, the concentration of the ion pair reagent effects capacity as does the amount of organic solvent. Increasing the concentration of the heptanesulfonate will increase loading capacity. Decreasing the amount of solvent present will also increase loading capacity. Furthermore, choosing a more strongly adsorbed ion pair reagent can also be used to increase capacity. For example, octanesulfonate can be substituted to increase capacity. In addition, perfluorinated carboxylic acids generally are more strongly adsorbed and will most likely give higher capacity as well.

Last but not least, if you want the maximum capacity, cation exchange is your best bet.