Determination of Trace Amounts of Copper by Phenanthroline Derivate Reagents and Use of Monolithic Column
Simona Procházková 1
Radoslav Halko 1
Michael Murkovic 2
1 Comenius University, Faculty of Natural Sciences, Department of Analytical Chemistry, Iľkovičova 6, 842 15 Bratislava IV, Slovakia 2 Graz University of Technology, Department of Food Chemistry and Technology, Petersgasse 12/2, A-8010 Graz, Austria
|Section:||Utilization of instrumental methods in the analysis of biologically important substances|
Copper is a nutrient required for many biochemical and physiological functions. In humans this trace element is third in abundance, after Fe(III) and Zn(II), among the essential heavy metals. In the body, copper shifts between the cuprous Cu(I) and the cupric Cu(II) forms and though the majority of the body’s copper is in the Cu(II) form it is usually in the Cu(I) form inside of cells.
Determination of copper in urine is important in the diagnosis of an inborn error of metabolism, Wilson’s disease, and various kinds of anemia. Copper accumulates in the liver, brain, kidney, and cornea, and urinary excretion of copper is increased. Especially in the diagnosis of Wilson’s disease, urine copper determination should be performed because serum copper determination is not sufficient for this purpose in the diagnosis of this disease. The measurement of copper in the urine is better, because the excretion of copper in the urine is higher than 1.5 μmol per 24 h. The excretion of copper in the urine of those affected by Wilson’s disease is several fold higher, usually 5–7 μmol per 24 h .
Our work consists of two part: 1) Spectrophotometric study of condition of chelate copper(I)–neocuproine and chelate copper(I)–bathocuproine 2) Determination of trace amounts of copper in human urine by RP-LC after pre-column derivatization and use of High Resolution RP-18e column.
Phenanthroline derivate reagents (2,9-dimethyl-1,10-phenanthroline, neocuproine, and 2,9-dimethyl-4,7-diphenyl-1,10-phenantroline, bathocuproine) are diamine chelators which form 2:1 complexes with Cu(I) at pH 4.0 - 7.0. In the presence of a reducing agent (ascorbic acid), the strong complex stabilizes the copper in its reduced form of Cu(I) in a tetrahedral binding geometry , while the induced conformational changes of the complex result in the appearance of a strong absorption band in the 450 to 550 nm wavelength range. As the molecular mass of the chelating reagent increases the absorbance band intensity also increases, as can be confirmed.
Our research was based on the abovementioned available facts. The various factors were monitored such as different molar ratios, pH and stability of the copper(I)–neocuproine and of the copper(I)–bathocuproine chelates. The ligands were used without further purification.
 X. Ding, H. Xie, Y. J. Kang, J. Nutr. Biochem. 22 (2011) 301–310.