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Falling Victim to One of LC's Classic Blunders: Mismatching Your Diluent and Mobile Phase

30 Dec 2019


An early lesson most of us learn in liquid chromatography is this: Always match your diluent to your mobile phase.  Once the exams are done, if you learned this in a college course, or your manager has walked away, you start flexing this “requirement” a little to see how matched they really need to be.  I get it! You don’t want to have to blow off all your organic, because that takes time.  Or if you’re scouting various columns, you just want to use the same sample you used in HILIC and reversed-phase tests.

In some cases, you can get away with this possibly because your analyte has plenty of affinity for your stationary phase, and that extra bit of strong solvent isn’t enough to ruin your separation.  While in other cases your analytes elute well passed the dead volume of the column, so your peak shape is still good.  In these instances, it can be OK to bend the “rules” a little.  Sometimes though, you have an analyte so sensitive, that even 5% residual acetonitrile in your diluent is enough to ruin your chromatography altogether.  I present to you this case study on acrylamide.


Acrylamide is a small polar molecule (show image).  It is akin to Iocane powder.   It’s odorless, tasteless, dissolves instantly in liquid, and is [not] among the more deadly poisons known to man.  Though as a side note it is thought to be a probable carcinogen with repeated prolonged exposure.  In reality, acrylamide is soluble in water and methanol and is very soluble in acetonitrile. Additionally, it is difficult to retain on most LC columns.  Earlier this year, we developed a product specifically for acrylamide analyses and have developed applications in various matrices.

Early on in method development, we noticed that acrylamide was very sensitive to residual organic solvent leftover from the sample preparation procedure.  In the published EN 16618 method, the final extract is in a water/methanol eluate.  The step before analysis is a lengthy blow down; a step many, including myself, are tempted to short change.  In the QuEChERS method, the final sample diluent is acetonitrile (MeCN), which is even more detrimental to chromatographic analysis and requires complete solvent replacement.

To better understand the sensitivity of acrylamide to organic diluent, we deliberately performed an extraction of acrylamide from potato chips/crisps and added organic solvent in known volumes to see how this changed the chromatography from an ideal sample prep to one that might be more “lazy.”  In our method, we use a 100% aqueous mobile phase with 0.001% formic acid, meaning that our diluent should also be 100% aqueous.   As more organic was added to the diluent, retention time decreased and peak width broadened.  While 10% methanol in the diluent still gave decent retention and chromatography, 20% or more methanol resulted in poor peak shape and retention.  In the case where there is even 5% residual MeCN, massive peak broadening occurred and when more MeCN was present, the acrylamide peak split and then eventually eluted with the matrix contaminant peak.


While acrylamide qualifies as an extreme example, this data shows the necessity of ensuring your diluent matches your starting mobile phase.  We also noticed that having strong organic in your autosampler rinsing routine could result in a similar effect on your chromatography.  So in 2020, and beyond, next time you see poor chromatography with early eluting or poorly retained peaks, consider checking your diluent composition.  Your solution might be as simple as making sure your diluent better matches your mobile phase.

Happy New Year from all of us here at Restek!