Chromatography Forum

i ame working on two projects with HILIC seperation on Waters BEH amide column. both methods use a gradiënt form 30% water to 70 % water. eluens contains 50mM ammoniumbicarbonate pH10.
analytes are oxalate and citrate in method 1 and pyridoxalphosphate and thiaminephosphate in method 2.
samples are disolved in water/acetonitrile 30/70, injectionvolume is 0,5 ul.
all the compounds tail. on the same instrument a method is runnning for steroids in RP that gives symetrical peaks.
does anybody have an idea what is cousing this tailing an how to avoid this?

Hi Aldijkhuizen,

BEH amide is a non-endcapped phase--the solvent you're using seems to me to be appropriate. At this pH, all silanols will be negatively charged as well as all four analytes in these two methods.

Lessening the concentration of ammonium carbonate or switching to ammonium acetate at ca. 10 mM (basic pH) may help with the organic acids as well as for the B6 and B1.

http://www.waters.com/webassets/cms/lib ... A64081.pdf

Best Wishes!

Hi Again, Aldijkhuizen,

Waters does not seem to recommend the use of ammonium carbonate--please refer to:

http://wwwp1.waters.com/webassets/cms/s ... 001371.pdf

and Table 2, pg. 5.

I can 't find recomamdation against ammoniumbicarbonate. I tested and it worked better then acetate.

Tailing is an indication that there is still an interaction between your 'targets of interest' and the stationary phase or column. Thus, it may be easier to protonate those targets so they don't see the column and interact and therefore use an acidic mobile phase (0.1% formic acid).

Itried acidic. gave terrible peaks.

If that is the case try a neutral pH (~7) with Ammonium Formate. Adjust your mobile phase composition with a higher concentration of Acetonitrile (Methanol and IPA too). Check the internet. If this still does not work you might have to try 'ion pairing' with TEA.

But this means you will have to dedicate your column! TEA can't be flushed out.

Hi Aldijkhuizen,

For fun, why not try out the applications notes on pgs. 34 and 39 of:

http://www.waters.com/webassets/cms/lib ... A64081.pdf

perhaps the issues with tailing will be mitigated? Apologies for misreading your eluent composition from above.

I agree with Matt. Look at Waters has done. Why reinvent the wheel?

Bruce Neagle

I tried waters method. It was a total disaster for my compounds. Horible peaks of 2 minutes width. 50 mM ammoniumbicarbonate pH10 gave acaptable width but still some tailing.anybody an idea how to decrease tailing?

Tailing is an indication that your molecules are not 'happy' with the chromatographic conditions. They actually prefer the stationary phase and will stick to it.

Try a 'higher' composition of ACN (even 50%!) from your current conditions using 10% increments until you are pleased with both the peak shapes and the baseline separation. You may even have to use a stronger solvent like IPA instead of ACN. Good luck!

Hi Again, Aldijkhuizen,

It seems strange to me that Waters would publish a completely poor method or two that would not "work out of the box," so-to-speak.

There's no doubt that you want to have basic conditions for these separations and with this phase (amino).

Are you certain that the strong eluent and weak eluent in your gradient program are not inadvertently reversed on the instrument? Remember for HILIC, water is the strong eluent and the organic component (ACN) is the weak one. This could cause tailing, I think.

Otherwise, the variables you have to work with are temperature and salt type and/or concentration to eliminate tailing.

Remember also that HILIC takes a Long Time (twice as long as RP) to equilibrate, that may also make a difference.

Best Wishes, again. I'll keep thinking about the trouble(s) to see if I've missed anything. The next step would be to change phases I'd think.

Mac-mod has a nice guide for that:

http://mac-mod.com/pdf/product-bulletin ... 0Guide.pdf

Just to think about:
Did you change also the strong and weak needle wash according to the HILIC mode of operation? So make sure your weak wash solvent contains not more water than your initial eluent.

Also make sure your re-equilibration is enough, I would go to at least 10, better 20 column volumes of initial mobile phase + the dwell volume of your system.
Dividing this by your flow rate will give the time you need for equlibration.

I checked a and b solvent..
Because i ame working on agilent infinity 2 this strong/weak wash issue does not apply.
My methos is a gradient not isocratic.
Is it worth trying IPA instead of acetonitril?
Lower buffer concentration gave worse results, higher concentration was not posible. Maybey when switching to IPA higher concentration is posible?

IPA is a possibility (but it depends on your sample matrix). I have never used IPA in a UPLC or HILIC. IPA is protic and closer to Methanol. Use 5% initially. Remember with HILIC, the higher the water content the retention time will decrease and the peak will run toward the injection front (the opposite of reverse phase).