Analytes to be prepared in 85% ACN with 15% deionised water. pH 5 ammonium acetate. Mobile phase buffered same way. I tried amm. formate at same pH. I tried formic acid liquid buffering medium but split peaks.
The running mobile phase equilibrating the column is 95% Amm acetate pH5. This is an amm. salt and I would expect precipitatation if 95% mobile phase prepared sample and running an 85% ACN mobile phase that is buffered because the analytes will become less soluble. This is for a HILIC setup and I am trying to set up a mini-gradient within the isocratic mode to allow the water layer to not be so pushed towards the material chemistry. So more polar interactions in the beginning to try to retain some analyte before the others. Then after 7 minutes the other analytes can become retained later in the spectrum. Moving the analytes past the To marker.
Thank you for the experience you provide. I have tried optimising other parameters in HPLC approximately and this is priority for me, on the agenda now before I try higher temperature.
I buffered the mobile phase with acetic acid of same molarity concentration at 10mM since this would be a contributing factor for the HILIC water layer to dissolve the buffer salts. I am using sonication after degassing once.
Perhaps. High concentrations of ACN are used to precipitate proteins. So the answer depends on your extract. Just in case I would use Methanol instead of ACN.
I am analysing small molecules. Gradient mode would take longer than 5 minutes and my analytes need slightly better resolution and preparing 2 mobile phases would indicate if gradient elution may be worth a try if the 10% increase in ACN shows better resolution. However the column would not be equilibrated with the 85% ACN so that may cause lack of reproducibility.
You've done a really nice job so far, but you missed one very easy step. Take your sample and pipet 10 uL into a mL or so of the eluant (simulate your injection) in a clear vial and see if you get a precipitate.
I like the concept of how you've set up your RPLC run (HILIC runs typically start with very high solvent concentrations and drop down). You want to minimize your injection to eluant volume as much as possible to avoid the perturbation from the high %solvent sample to the low %solvent eluant, because at the 95% water even the small amount of ACN that you are injecting will cause some perturbations.
Keep up the good work!
Yes! I agree with Mark Krause. The best way is to take 10 micro liters of the sample in a vial; add about 1 mL of the mobile phase and actually observe if the solution becomes turbid.
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