Niğde University, Faculty of Science and Arts, Chemistry Department, Niğde, Turkey.
Talanta. 2011 Sep 15;85(3):1472-82. doi: 10.1016/j.talanta.2011.06.026. Epub 2011 Jun 17.
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.
通过酰胺键形成将腐殖酸（HA）固定到氨丙基硅（APS）上来获得的材料的固定相特性进行了研究。从元素分析、FTIR、热重分析、pH 值零电荷测量、电位滴定和接触角测量等方面对材料进行了表征。通过元素分析结果确定了结合到 APS 上的 HA 的量，发现为 170mgHA/gAPS。在不同 pH 值的水介质中研究了材料的稳定性，在 pH=8 时释放的 HA 量不超过总固定化 HA 的 2%。在 HPLC 系统中，使用一些低分子量极性化合物（即一些核苷和碱基）作为测试溶质，研究了经过良好表征的材料的固定相特性。研究了一些实验变量（如柱条件、流动相组成和温度）对研究化合物的色谱行为的影响。还研究了不同 pH 值的铵溶液对物种保留性能的作用。保留因子（k'）与有机改性剂体积百分比呈 U 形曲线，这被评估为固定相的 RPLC/HILIC 混合模式行为的指示。通过几何方法分析了 RPLC 和 HILIC 模式之间的正交性，发现为 48.5%。在每种研究模式下都实现了研究化合物组的基线分离，并且根据应用的 HPLC 模式观察到化合物洗脱顺序的一些差异。在 RPLC 和 HILIC 模式下记录的色谱图与在相似条件下在 APS 上记录的色谱图进行了比较，从而详细评估了 HA 固定化过程的影响/重要性。根据获得的结果，固定化 HA 被表示为 HPLC 分离的有用固定相。
