Simplified LC Method Development for Nucleotides

Guest Author: Christina Dyczewski, Executive Account Manager

Earlier this year I dusted off my traveling satchel and ventured out into the field to visit customers. I was excited to be face-to-face with people I have been working with over the last few years. These trips give me the amazing opportunity to listen to customers’ requests, trials, and any pitfalls they experience in regard to LC method development and column selection. However, something that really stood out to me was the high number of requests I received surrounding LC method for nucleotides, the building blocks of nucleic acids. This request came from a lot of customers working with metabolites, although we have seen this as a trending topic for peptide and oligonucleotide synthetic chemists.

There are many selectivity options for method development for nucleotides, however for this discussion, we are going to stick with a good old C18.  With that said, a “Plain Jane” C18 by itself will not do the trick—it is not retentive enough for such polar analytes.  I have seen methods that utilize a phosphate buffer/ammonium acetate mix1, as this yields great peak shape.  I have also seen ion pairing agents, such as TBAHS2, used to decrease tailing and improve peak shape.  Even though using an exotic mix of modifiers or using an ion pairing agent can do the job of yielding adequate peak shape and separation, it is not the most appealing route. Using such modifiers can be too harsh for columns, ultimately leading to decreased column lifetimes and extended column equilibration and cleaning time.  Also, such conditions are not so nice to your LC/MS.

Since we want to stay away from ion pairing agents and non-mass spec friendly modifiers, the next step would be to find a C18 column that has some affinity for retaining polar compounds.  This lack of polar selectivity is the reason why nucleotides aren’t able to retain as well on a regular C18 column.  Using a column with a different selectivity factor can dramatically improve your separation, since selectivity is a significant variable in the resolution equation:


I have included a couple of examples of this improved peak shape for nucleotides in the adenosine family.  The Luna C18(2) is a standard C18 phase (image 1), while the Luna Omega is a Polar C18 with a polar modified surface (image 2).  The only difference is the pH was slightly altered.  You can see that the resolution and separation is dramatically improved with the Luna Omega Polar C18.

Image 1: Adenine family using Luna C18(2) (formate)


Image 2: Adenosine Nucleotides Luna Omega 3 μm Polar C18


Another factor to consider when wanting to improve separation is core-shell (or solid core, as it is sometimes referred to) verses fully porous columns.  As more and more core-shell columns enter the market, it appears that they are becoming the column of choice when it comes to method development.  Core-shell medias allow for faster run times, as well as improved resolution.  The reason for this is its optimal linear velocity being higher than fully porous medias, as well as the core-shell morphology itself3.  With so many different particle sizes becoming available, they are no longer limited to being used on a UHPLC system.

I realize that with so many different options, it might be overwhelming to choose just one column.  I invite you to reach out to your local Account Manager, or contact us on live chat, to further discuss your options.  And if you have a different but tricky separation that you are having trouble with, please do not hesitate to mention that as well.  We enjoy providing solutions for our customers and discussing matters in detail.

Don’t be afraid to schedule a visit or a phone call. Who knows… maybe I will even see you on my next trip!


  1. Yiping Ren1, Jingshun Zhang2, Xiaodan Song3, Xiaochun Chen1, and Duo Li2 (April 2011). Simultaneous Determination of 5′-Monophosphate Nucleotides in Infant Formulas by HPLC–MS.
  2. Shukla, Arpit (2017). How to Separate Nucleotides Using Ion-paired Reverse Phase HPLC [Web log post]. Retrieved November 22, 2017, from
  3. RichardHayesa,AdhamAhmeda,TonyEdgeb and HaifeiZhanga (May 2014). Core–shell particles: Preparation, fundamentals and applications in high performance liquid chromatography.
Simplified LC Method Development for Nucleotides
Article Name
Simplified LC Method Development for Nucleotides
This article discusses the different ways to develop a simpler LC method when working with nucleotides and here are many selectivity options.

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