Poor retention and resolution

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

13 posts Page 1 of 1
Dear everyone,

I have been trying to determine aromatic hydrocarbon types in middle distillates of petroleum products with HPLC-RID, heptane as mobile phase and an amino column, according to EN 12916.

However, after a few injections of the standard, my column exhibits poor retention and resolution for the 7 compounds. I replaced the column with a new one (different manufacturer) and obtained the same pattern.

System backpressure and baseline signal are stable. After receiving the column, I have equilibrated it directly with heptane for approximately 1 hour, and stored it in heptane.

Any suggestions concerning what may be happening and possible ways to troubleshoot?
Heptane as a mobile phase should be fairly benign as far as column lifetime for an amino bonded phase (anything aqueous would be a different matter) so my guess is that there is some junk in your samples that is sticking tightly to the column. A guard cartridge (changed frequently!) or a regular flush with a more polar solvent (yes, it will take a long time for the RI baseline to settle down) might help. Solid phase extraction sample cleanup might be another solution.
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374
What is the column shipping solvent? If it isn't miscible with heptane it would better to equilibrate the column with i-PrOH.
Best regards,
Dmitriy A. Perlow
What about accumulating water from sample and mobile phase on the colum?

Maybe try to dry your heptane with molecular sieve or filter through a column dried bare silica.

also test if the problem correlates with the volume mobile phase pumped or the no of injections.

Maybe you can reactivate your columns by pumping dried i-PrOH and then back to dried heptane.
Thanks everyone,

residual water was indeed the case. Once I dried the mobile phase with sodium sulfate, everything went smoothly!

Does anyone know how to eliminate the drift in the RI detector? This seems to be the next problem with this analysis...
It is easy and hard at the same time. Check if all variables are persistent through the time: mobile phase, stationary phase etc. For example plug out the column and connect pump directly with detector, check if drift still exists.
Best regards,
Dmitriy A. Perlow
Hi stdev,

RIDs are tricky. What we used to do at Merck was to place as much of the capillary tubing from the column outlet leading into the column heater as we could and thermally jacket the capillary as well. RIDs can be very touchy when it comes to thermal gradients. Don't know what temp your separation is at, but you can also consider placing a heating magnetic stirrer under the eluent reservoir. I've not done this with RID, but with UV detection of short-chain organic acids (temp of separation 60 degrees Celsius, Bio-Rad HPX-87C column, also kind of touchy with thermal gradients).

Best Wishes!

stdev wrote:

Does anyone know how to eliminate the drift in the RI detector? This seems to be the next problem with this analysis...

RI detector is very temperature sensitive. So you need to make sure that:

1.RI optical cell temperature is at least the same as column temperature (I'd recommend 5 degrees higher than column)
2.RI detector is not close to source of air (air con, other LC system, draught, lab door, window etc)
3.Reference cell is always flushed with fresh mobile phase before any run (even if LC is dedicated for the same merhod all the time)
4.Capillary between column and detector is as short as possible
5.Method is fully optimized - all compounds are flushed out from column after run.


Tomasz Kubowicz
Thanks everyone!
Dear Everyone!
I've been having trouble in separating an acidic herbicide mesotrione and it's metabolites AMBA (2-amino-4-methylsulfonyl benzoic acid) and MNBA (4-methylsulfonyl-2-nitrobentoic acid) in one run.
The peak shape of the two metabolites are seem to be ok, in various separation methods, but for mesotrione i cannot produce an acceptable peak.

Has anyone tried this?
I've tried RP, HILIC, and polar organic modes, and i also tried a carbon column, still no result.
I'd appreciate the help.
Thank you,
Acidic? What's the ionizable group in the mesotrione molecule?

Please specify what the starting mobile phase was in HILIC, what solvent the sample was in when you injected it, and which HILIC column you used.
PolyLC Inc.
(410) 992-5400
According to literature, mesotrione mainly exists in it's enolic tautomer form with a pKa around 3. It's two main metabolites are true carboxylic acids.
I used a Phenomenex Kinetex HILIC column (100*3 mm, 2.6u) with a gradient of 95% ACN:0.1% AcOH in water to 50:50.
The solvent was acetonitrile. However in case of AMBA and MNBA i could reach satisfying retention and peak shape, for mesotrione i couldn't.
Today i could produce an acceptable peak for mesotrione with a Zorbax Eclipse Plus C18 column, and a general gradient of 10% ACN to 95% ACN, with 0.1% AcOH.
Thank you for your reply, i'd be still interested in any better solution, or some method with which i could measure these three compounds in a single run.
In place of the acetic acid, try adding 20 mM ammonium formate (prepared from a stock solution in the pH range 3.5-4.5) to both the mobile phases and the sample solvent. That's 20 mM overall, not just in the aqueous portion of the solutions. Use the starting mobile phase as your sample solvent.
PolyLC Inc.

(410) 992-5400

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