Uracil is a naturally occurring pyrimidine derivative. Uracil is used for drug delivery and as a pharmaceutical. Uridine is a product of uracil and sugar ribose. Uridine is one of components of ribonucleic acid. Uracil and uridine are slightly basic in nature. Both compounds are hydrophilic and uracil is very often is used as a void marker in reversed-phase chromatography. Uracil and uridine are separated based on their polar properties on a Primesep N HILIC column. Uracil and uridine are monitored by UV.

Uracil and uridine are separated based on their polarity. The mechanism of retention is pure HILIC mode. Retention time is controlled by varying the amount of ACN. Buffer concentration and pH will affect ionization and polarity of the stationary phase, and thus indirectly affect retention of neutral compounds. Column is compatible with all general detection techniques.

Introduction

Hydrogen-bonding interaction offers unique selectivity based on number of “interaction points” available for hydrogen bonding. One of the useful characteristics to determine retention patterns in hydrogen-bonding mode is the molecular polar surface area (PSA). This calculated parameter is usually used for prediction of drug transport properties, but we successfully applied it to hydrogen-bonding interactions. Polar surface area is defined as a sum of surfaces of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.  Since those polar  atoms can participate in hydrogen-bonding interaction, estimation of elution order can often be made based on PSA. While PSA is a good indicator of elution time, it must be noted that polar surface area does not account for the accessibility of hydrogen-interaction sites. Not every polar surface participates in intermolecular hydrogen interactions with the stationary phase.

Proximity of “interaction points” to each other within one molecule also needs to be considered since molecules can form an intramolecular hydrogen-bonding, which competes with intermolecular interaction between analyte and stationary phase. This reduces retention time in hydrogen-bonding mode.  Such structural factors provides unique selectivity among similarly structural (isomers, homologs, degradation products, precursors) molecules.

Since SHARC 1 column is a mixed-mode column, pKa is another useful parameter in method development for these columns. SHARC columns operate in non-aqueous mobile phase, but some effect of charge interaction of stationary phase and ionizable molecules still exists and contributes to the retention profile.

Nucleobases, like xanthines, have a lot of interaction points due to the presence of multiple nitrogens and oxygens. Nucleobases in most cases elute according the number of interaction points and PSA value. Thymidine (3 interaction points, PS value of 104), uridine (4 interaction points, PS value of 124 ), adenosine (4 interaction points, PS value of 139), guanosine (5 interaction points, PS value of 159 ), and cytidine (4 interaction points, PS value of 139) were separated based on hydrogen-bonding properties. Some abnormality was observed in case of cytidine and it was attributed to better accessibility of groups contributing to hydrogen-bonding.

Application Notes: Nucleosides are glycosylamines consisting of nucleobase linked to ribose or deoxyribose sugar and are building blocks for DNA and RNA. These compounds are very polar and contain groups available for hydrogen bonding interaction. Thymidine, uridine, adenosine, guanosine and cytidine were separated using a hydrogen-bonding method. There is a strong correlation between the retention time and mobile phase composition. The strength of hydrogen-bonding interaction increases as the number of hydroxyls in the analytes increase. Additionally the rder of elution for compounds depends on the ratio of the mobile phases: acetonitrile and methanol. Our method is compatible with LC/MS and preparative chromatography.

Application Columns: SHARC 1, 3.2x100 mm, 5 um, 100A, To learn more about SHARC 1 columns click here. To order this column click here. To see more chromatographic separations check our web site.

Application Compounds: Thymidine, uridine, adenosine, guanosine and cytidine

Th