I have PAH peaks broadening on C-18 column 2,1 mm x 250 mm,
MF is ACN/H2O 50:50 on start to ACN 100 % at the 30 min.
Dilution solvent is chloroform.
Concentration is 0.01 mg/ml

Your sample is dissolved in too strong a solvent. Ideally, the dilution solvent should be the same as or weaker than your initial mobile phase. If you *must* use something stronger, then try cutting the injection volume down and see if things don't improve.

-- Tom Jupille / LC Resources

If you are extracting the PAH's from a matrix with a chlorinated solvent as EPA 610 you need to solvent exchange to ACN as instructed in that method. All dilutions are then made in ACN (as T. Jupille).

In addition to the usual 'solvent strength' comments on this topic, I think a related issue is the SOLUBILITY of the injected solvent.

I've done quite a bit of work on this subject to use ethyl acetate GC extracts (water extracted with ethyl acetate) on HPLC. An example that demonstrates my point. We are unable to inject more than about 20 uL of ethyl acetate into a typical 4.6 x 150 RP column with 90/10 H2O/ACN without seeing early peaks broaden, and sometimes split. If the extract is exchanged to methanol, we can inject much more--sometimes 100 uL, depending on the analyte's retention time--without problems.

At the high water concentration, the ethyl acetate does not mix with the mobile phase, and the 'strong' ethyl acetate inhibits absorption to the column. Eventually, the analytes are absorbed, but by then your separation is compromised.

I've had some success using the injector mixing program on our HP's to increase the injection by a few more uLs.

In Mira's case, chloroform is a similar example, clearly insoluble in water/ACN.

If the mobile phase and injection solvent are not miscible, this broadening/splitting problem will occur. And, it is easy to check visually in a test tube.

May I just ask for an approximation of the sentence:

...and the 'strong' ethyl acetate inhibits absorption to the column.
Eventually, the analytes are absorbed, but by then your separation is compromised."

Do you mean competitive adsorption, or rather competitive replacement when dealing with RPLC, between the solute/matrix molecules/solvent molecules for the binding sites of stationary phase?

Best greetings,
Maciej Turowski
KIT, Japan

For our samples, I don't think the matrix matters, because standards show the same effect. Probably any early eluting peak would have the same problem.

The injection solvent eventually mixes, and the compounds adsorb to the column packing, but it takes so long the peak shape is bad. Instead of going on the column quickly, as a plug, the analyte is 'smeared' on the column because of the insolubility of the injection solvent.

In my example, 20 uL EtAc will go into solution fast enough there is no effect, but more than 20 uL will not. 5 uL of CHCl3 might work, but 10 or more won't.

This isn't a very clear chemistry answer, but I find it a way to visualize the problem.

I want to separete and determine PAHs in river water by HPLC. I need a good method for extraction of them from water before injection to instrument.

Best Regard
Shariaty

I want to separete and determine PAHs in river water by HPLC. I need a good method for extraction of them from water before injection to instrument.

Best Regard
Shariaty