790 Bull. Korean Chem. Soc. 2012, Vol. 33, No. 3 Jihye Kim et al. http://dx.doi.org/10.5012/bkcs.2012.33.3.790 Isotachophoretically Assisted On-Line Complexation of Trace Metal Ions in a Highly Saline Matrix for Capillary Electrophoresis † Jihye Kim, Kihwan Choi, Sunyoung Cho, Asif Riaz, and Doo Soo Chung * Department of Chemistry, Seoul National University, Seoul 151-747, Korea. * E-mail: dschung@snu.ac.kr Received October 26, Accepted November 30, 2011 Trace metal ions such as Cd 2+ , Ni 2+ , and Zn 2+ in a highly saline sample were subjected to on-line complexation with 4-(2-thiazolylazo) resorcinol (TAR) dissolved in a background electrolyte (BGE) under transient isotachophoresis (TITP) conditions. A long plug of the saline sample, containing the trace metal ions but devoid of TAR, was injected into a coated capillary filled with a BGE composed of 150 mM 2-(cyclohexylamino) ethanesulfonic acid (CHES) and 110 mM triethylamine (TEA) at pH 9.7. Since the electrophoretic mobility of TAR fell between the mobilities of the anionic leading electrolyte (Cl - in the sample) and the anionic terminating background electrolyte (CHES - ), a highly concentrated zone of TAR from the BGE was formed at the rear of the sample matrix and then the metal cations toward the cathode were swept by isotachophoretically assisted on-line complexation (IAOC) between the metal ions and the isotachophoretically stacked TAR. As a result, anionic metal-TAR complexes were formed efficiently, which satisfy the TITP conditions between Cl - and CHES - . The enrichment factors of metal ions including Cd 2+ were up to 780-fold compared to a conventional CZE mode using absorbance detection. The detection limits were 17 nM, 15 nM, and 27 nM for Ni 2+ , Zn 2+ , and Cd 2+ in a 250 mM NaCl matrix, respectively. Our method was successfully applied to the analysis of urine samples without desalting. Key Words : Transient isotachophoresis, Salt, Trace metals, Cadmium, 4-(2-Thiazolylazo) resorcinol Introduction For the determination of trace metal ions in capillary electrophoresis (CE), chromophoric complexation has typic- ally been used since most metal ions are neither naturally UV/Vis absorbing nor fluorescent. 1,2 Previously, we have reported transient isotachophoresis (TITP) of trace metal ions such as Ni 2+ , Fe 2+ , and Zn 2+ complexed with 4-(2- pyridylazo)resorcinol (PAR) in a high salt matrix. 3,4 Metal- PAR complexes were prepared off-line and subjected to TITP stacking, utilizing the sample matrix chloride as the leading electrolyte (LE) to achieve limits of detection (LOD) in the low/sub ppb range. However, the method was not applicable to some heavy metal ions such as Cd 2+ due to its weak complexation with PAR. 5,6 4-(2-Thiazolylazo)resorcinol (TAR), having a thiazolylazo moiety instead of the pyridylazo moiety, shows strong complexation with metal ions such as Cd 2+ , Zn 2+ , and Ni 2+ and has been used for CE of those metal ions. 6-8 When the trace metal ions were enriched first by field enhanced sample injection and then on-line complexed with preloaded TAR, LOD's were reduced to the low/sub ppb range. 8 This scheme required that samples should be dissolved in a dilute matrix and thus entailed difficulty for use with physiological samples which are usually saline. In this report, we demonstrate isotachophoretically assisted on-line complexation (IAOC) for the determination of trace metals including Cd 2+ in a highly saline matrix. A long plug of a highly saline sample devoid of the complexing agent TAR was directly injected to obtain a 330-fold enrichment for Ni-TAR, 780-fold for Zn-TAR, and 420-fold for Cd-TAR achieving LODs in the low nanomolar range. Furthermore, this IAOC method was applied to the analysis of real biological samples such as human urine without desalting. Experimental Reagents. 2-(Cyclohexylamino)ethanesulfonic acid (CHES) and triethylamine (TEA) were purchased from Sigma (St. Louis, MO, USA). Highly pure sodium chloride (99.999%), nickel(II) chloride hexahydrate, zinc nitrate hexahydrate, cadmium nitrate tetrahydrate, and TAR were from Aldrich (Milwaukee, WI, USA). 2,7-Dichloro-fluorescein (DCF) was from Merck (Darmstadt, Germany). Fluorinated carbon poly- mer neutral (FC-PN) which is a 50 g/L aqueous solution of FC-430 (3M, St. Paul, MN, USA) was purchased from J&W Scientific (Folsom, CA, USA). All reagents were used as received. A reference material for trace metals in urine (ME 28351) was obtained from Promochem (Wesel, Germany). The background electrolyte (BGE) for CE was a pH 9.7 buffer composed of 150 mM CHES and 110 mM TEA containing 0.02 vol % FC-PN and 0.2 mM TAR. A 1 mM stock solution of TAR was prepared in deionized water with a few drops of 1 M NaOH (subject to complete dissolution) Abbreviations: CHES: 2-(Cyclohexylamino)ethanesulfonic acid, DCF: 2,7-Dichlorofluorescein, PAR: 4-(2-Pyridylazo)resorcinol, FC-PN: Fluorinated carbon polymer neutral, TAR: 4-(2-Thiazolylazo) resorci- nol, TEA: Triethylamine, TITP: Transient isotachophoresis, IAOC; Isotachophoretically assisted on-line complexation † This paper is to commemorate Professor Kook Joe Shin's honourable retirement.