Unexpected Details

When we think about setting up a chromatography experiment, we probably think about details such as making sure our glassware is clean, using low-binding and/or high recovery sample vials for small quantity protein samples, lowering the temperature of the autosampler to protect the sample, and double-checking method settings in the software. One thing we may not think about is the pipette used to prepare mobile phases! I had thought about glass versus plastic solvent bottles before, I hadn’t thought about the pipette or graduated cylinder.

The example below is a standard mixture of oligonucleotides on Agilent’s AdvanceBio Oligonucleotide column. The mobile phase for this method contains triethylamine (TEA), which serves as an ion pairing reagent so that polar oligos can be retained on the reverse phase column. The only difference between these two chromatograms is the pipette used to add TEA to the mobile phase – in the top chromatogram a disposable plastic pipette was used but in the bottom chromatogram a glass graduated cylinder was used. Clearly this detail matters!

Adding a smaller quantity of ion pairing agent to the mobile phase will result in lower retention of the sample – exactly what we see when the TEA was added with plastic. Interactions between the TEA and the plastic likely resulted in incomplete transfer of the pipetted material.

This example is a good reminder to think carefully about everything that contacts your sample AND your mobile phase.

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