Analytical Communications, June 1996, Vol33 (I zyxw 93-1 96) zyx 193 z Determination of Calcium and Magnesium in Sea-water Using a Dynamically Coated Porous Graphitic Carbon Column With a Selective Metallochromic Ligand as a Component of the Mobile Phase zyxwvu Brett Paull, Peter A. Fagan and Paul R. Haddad Department of Chemistry, University of Tasmania, GPO zyxwvuts Box 252 C, Hobart, TAS 7001, Australia A simple liquid chromatographic procedure for the separation and detection of magnesium and calcium in saline samples has been developed. A selective colour-formingmetallochromic ligand, o-cresolphthalein complexone, was used both to dynamically coat a pH tolerant porous graphitic carbon reverse phase column and also as a component of the mobile phase. The metals were separated by a chelating ion exchange mechanism at pH 10.5 and eluted as coloured chelates, which were detected using direct spectrophotometry at 572 nm. The procedure was applied to the determination of magnesium and calcium in a sea-water sample and the results compared to those obtained using AAS. The spectrophotometric detection of metal ions following their chromatographic separation, using colour-forming metallo- chromic ligands as post-column reagents (PCR) is now a well established analytical technique. The extensive choice of suitable reagents available allows both sensitive and often highly selective detection of almost all metals. However, a drawback to the above technique is that post-column reaction detection requires the addition of one or more reagent pumps to the conventional HPLC system, in order to deliver the PCR into the eluent flow prior to detection. Also, long reaction coils are often required to facilitate adequate mixing of the eluent and PCR. An alternative to the above technique is to include a colour- forming reagent within the actual mobile phase itself. This eliminates the need for both additional pumping systems and reagent mixing coils and also reduces the number of solutions which need to be prepared. Metal ions are detected using direct spectrophotometry as they elute from the analytical column in the form of coloured metal chelates. Previous work by Zenkil and Toei2-7 has successfully applied this technique to a number of metals in various samples using ion chromatography (IC). Examples of metallochromic ligands used include chloro- sulfonazo III,1 o-cresolphthalein c omple~one,~~ arsenazo 1115-6 and Xylenol Orange.7 Of the above metallochromic ligands, o-cresolphthalein complexone is the most sensitive and selective for the alkaline earth metals, particularly calcium and magnesium. Toei in- cluded o-cresolphthalein complexone24 as a component of the mobile phase for the separation and detection of alkaline earth metals in a range of sample types, including clinical samples, milk samples and sea-water samples. Ion chromatography was used to separate the metals present in the above samples and thus control of the mobile phase pH was an important factor, as this had an effect upon retention times. The acceptable working conditions were determined to be between pH 3 and 6. However, at these pH values o-cresolphthalein complexone remained in a colourless uncomplexed form and the post- column addition of ammonia was required to raise the mobile phase pH to 10.2, at which the coloured metal complexes were formed and could be detected at 572 nm. Owing to the need to adjust the pH of the mobile phase after it has passed through the column, Toei’s method offered little advantage, in terms of the instrumentation and the number of solutions required, over the post-column reaction methods initially described. This paper details the preliminary results of an investigation into the use of a dynamically coated pH tolerant porous graphitic carbon column for the separation of calcium and magnesium, and their subsequent detection as coloured o- cresolphthalein complexes in saline natural water samples. The graphitic carbon column allowed a mobile phase containing the above colour-forming ligand to be used at pH 10.5, thus removing the need for the addition of any post-column buffering reagents. Calcium and magnesium were separated on the above column by a dynamic chelating ion exchange mechanism and formed coloured complexes with the o- cresolphthalein ligand within the mobile phase as they passed through the column. The eluting coloured chelates were detected spectrophotometrically at 572 nm. An investigation into the effects of flow rate, temperature, ligand concentration and percentage methanol, upon both the retention times and sensitivity was carried out. The linearity and reproducibility of the method has been evaluated and the method applied to the analysis of calcium and magnesium in sea-water. The results were compared to those achieved using AAS. Experimental Instrumentation A Waters model 600 programmable pump (Waters, Milford, MA, USA) was used to deliver the mobile phase. Sample injection was carried out using a Waters model U6K injector. The analytical column was a Hypercarb porous graphitic carbon reverse phase column (100 zyxw X 4.6 mm id), combined with a guard column (10 X 4.6 mm id), supplied by Shandon HPLC (Runcorn, Cheshire, UK). A Waters model 486 UV/VIS spectrophotometric detector was used to monitor the eluting complexes at 572 nm. This was interfaced to a Maxima 820 data station (Waters). A Varian SpectrAA. 800 Atomic Absorption Spectropho- tometer (Varian, Victoria, Australia) was used to provide comparative results for the analysis of sea-water samples. Reagents o-Cresolphthalein complexone zyx (3.3’-bis[NJV-bis(caboxy- methyl)aminomethyl]-o-cresolphthalein) was obtained from Published on 01 January 1996. Downloaded by CSIRO Library Services on 11/06/2013 09:15:31. View Article Online / Journal Homepage / Table of Contents for this issue