During my time of pandemic shelter-in-place, like many others I turned to online streaming for distraction. In my search for programs, I found intense fascination with baking competitions. Captivated how the same list of ingredients used could form such different results.
Some of the most interesting variations were found during bread week. I watched in wonder how the various kneading and proving times greatly influenced the pore structure of the bread, and the judging experience. I learned how underproving yielded small pores, making a dense and undesirable bread. Properly proved breads showed a larger pore structure, a lighter bread, and more desirable experience with the judges.
As I reflected upon these episodes, my thoughts returned to chromatography. How pore structure relates to chromatographic separations, and I started to find a crossover with my favorite baking shows and column chemistry.
Most simply, we appreciate that columns with a small pore structure can be an issue when working with larger molecular weight samples, such as proteins, mRNA and larger oligonucleotides. We chromatographically experience the challenge of a too small pore structure as carryover, excessive peak tailing, and in very challenging situations, losing the ability to detect the analyte of interest.
When working with a wide pore column, our large molecular weight samples will have a different experience. Using mRNA as an example, on a conventional silica column with pore sizes 200Å or less, it can be difficult for the sample to elute from the column. If it does, the peak is often tailing and demonstrating poor resolution for its impurities. Running the same mRNA sample on a column with a wide pore structure, such as the supermacroporous DNAPac RP (average pore size greater than 1000Å), customers are able to characterize their larger samples, separate out impurities, and, for further characterization, couple to a mass spectrometer. Simply put, because the sample is not caught up in the pore structure it has room to separate, the chromatography yields a better qualitative and quantitative result.
But as they say on these baking shows, it is not just how it looks, but also about the flavor. I correlate this to the overall performance of the column — will it be robust, offer a long lifetime? Or will it be like a bread with a soggy bottom, and just be a disappointment in the end? Scientists running the DNAPac RP, can take advantage of the column’s polymer backbone, which is able to handle the simultaneous oligonucleotide method requirements of elevated column temperatures and higher pH. Silica columns, regardless of their ability to handle high pH, start to become brittle at higher temperatures (> 60°C). This is a challenge as many oligonucleotide separations perform better at higher temperatures. So how can a chromatographer find the right balance of resolution and column lifetime? The polymeric DNAPac RP columns show temperature stability up to 110°C, and users experience double the column lifetime for their separations compared to conventional silica material. Couple this with the high resolving power of the wide pore material, now that’s something you can really sink your teeth into!
For more information on how Thermo Scientific polymeric wide pore columns can help your large protein or oligonucleotide separations, please visit www.thermofisher.com/bioLC.