1 3 DOI 10.1007/s10337-014-2678-9 Chromatographia (2014) 77:771–781 ORIGINAL HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase Hayriye Aral · Tarık Aral · K. Serdar Çelik · Berrin Ziyadanog ˘ ulları · Recep Ziyadanog ˘ ulları Received: 12 October 2013 / Revised: 26 March 2014 / Accepted: 28 March 2014 / Published online: 10 May 2014 © Springer-Verlag Berlin Heidelberg 2014 Many red chilli peppers were screened and three of them contained Sudans dyes. Keywords Amide-embedded stationary phase · Phenolic compounds · Herbicides · Anilines · Sudan dyes · Nucleotides and nucleosides Introduction Conventional C18 stationary phases are the most widely used stationary phases for reversed-phase HPLC applica- tions. The use of polar-embedded stationary phases for RPLC separations of alkaline and acidic small polar com- pounds has become very popular in the last decade [1–3]. Stationary phases with embedded amide or other polar functionalities were originally intended mainly for reversed- phase applications in water-rich mobile phases [4–7]. Recent investigations describing stationary phases con- taining polar-embedded groups have shown the superior performance of these new phases over conventional C8 and C18 phases for alkaline analytes [8, 9]. In addition, the amide phases have shown enhanced selectivity towards low-molecular-weight acids [4]. Small polar compounds are often very challenging to method development due to lack of retention on conven- tional reversed-phase columns. Although very effective in separating small polar compounds, normal-phase liquid chromatography (NPLC) methods are generally not desir- able for routine applications in the pharmaceutical indus- try due to poor reproducibility and difficulty in interfac- ing with (MS) detectors. The polar embedded stationary phases can enhance the retention of polar compounds with a mostly aqueous mobile phase in reversed-phase liquid chromatography RPLC [10]. Abstract Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chroma- tographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleo- tides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity pos- sibly through secondary interactions with the model com- pounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the sta- tionary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. H. Aral · T. Aral (*) · K. S. Çelik Department of Chemistry, Faculty of Science and Art, University of Batman, Batman, Turkey e-mail: tarik.aral@batman.edu.tr B. Ziyadanog ˘ ulları · R. Ziyadanog ˘ ulları Department of Chemistry, Faculty of Science, University of Dicle, Diyarbakir, Turkey