Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol‐modified CO2 and water as the polar additive

The separation of four water soluble nucleobases (thymine, uracil, adenine, and cytosine) via supercritical fluid chromatography with a CO₂‐based mobile phase containing an alcohol modifier and additive is described. Methanol, ethanol, 1‐propanol, and 1‐butanol were examined in conjunction with water as a neutral additive. Packed column stationary phases included silica bonded diol, cyanopropyl, and 2‐ethyl pyridine. Thymine and uracil eluted with good peak shapes without additive, while adenine and cytosine yielded late eluting, severely tailing peaks. The addition of up to 5% water to each of the five alcohols gave rise to much sharper peaks that eluted under gradient conditions in less than 10 min with no baseline noise. Results with water under identical chromatographic conditions were compared with formic acid and ammonium acetate as additives. Water proved to be much superior to formic acid, and it was comparable to ammonium acetate. The role of water was speculated to not only enhance the solvating power of the binary mobile phase for water soluble analytes, but the common elution pattern exhibited by each of the three stationary phases suggested that water had altered the surface chemistry of the packed phase.