The stationary phase characteristics of the material obtained through immobilization of humic acid (HA) to aminopropyl silica (APS) via amide-bond formation were investigated. The material was characterized in terms of elemental analysis, FTIR, thermogravimetric analyses, pH point of zero charge measurements, potentiometric titrations, and contact angle measurements. Amount of HA bonded to APS was determined from the elemental analysis results, and found as 170 mgHA/gAPS. Stability of the material was studied in aqueous media at different pH values, and amount of HA released at pH=8 did not exceed 2% of the total immobilized HA. Stationary phase characteristics of the well-characterized material were investigated in an HPLC system by using some low-molecular weight polar compounds (i.e. some nucleosides and nucleobases) as test solutes. Effect of some experimental variables such as column conditioning, composition of mobile phase, and temperature on the chromatographic behavior of the studied compounds was studied. Role of ammonium solutions at different pH values on retentive properties of the species was also studied. Retention factors (k') versus volume percentage of organic modifier exhibited a U-curve, which was evaluated as an indication for RPLC/HILIC mixed-mode behavior of the stationary phase. Orthogonality between RPLC and HILIC modes was analyzed through geometric approach, and found as 48.5%. Base-line separation for the studied groups of compounds was achieved under each studied mode, and some differentiations were observed in elution order of the compounds depending on the HPLC mode applied. Chromatograms recorded under RPLC and HILIC modes were compared with those recorded on APS under similar conditions, and thus the influence/importance of HA immobilization process was evaluated in detail. In light of the obtained results, immobilized HA is represented as a useful stationary phase for HPLC separations.
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