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.

Xanthines are good candidates to be separated by hydrogen bonding with SHARC1. They have a lot of hydrogen bonding interaction points. The theory of PSA and accessibility of groups explains retention time and order of elution very well. Caffeine (PSA-61.8), 3-Methylxanthine (PSA-83,6), 1-Methylxanthine (PSA-83.6) and xanthine (PSA-94.4) eluted according to the PSA number. In caffeine, three of the nitrogens have substituted methyls, which make these groups unavailable for hydrogen interaction. 3-methyl-, and 1-methylxanthine only have one substitution and thus are retained longer. Xanthine which has no substitution on nitrogen retained the longest.

Application Notes: Xanthines are polar neutral compounds which are hard to retain and separate by traditional reversed-phase chromatography. However a hydrogen bonding method makes separation possible due to an observable correlation between the number of hydrogens available for interaction and retention time. Molecules with no hydrogens available for interactions retain less, and compound with multiple hydrogen donors retain the most. Retention time can be controlled by changing ratio of ACN:MeOH. Other protic and aprotic solvents can be used to control retention time and selectivity of separation.

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:  Caffeine, 3-methylxanthine, 1-methylxanthine, and xanthine