Thin layer chromatographic separation of cobalt from nickel on impregnated silica gel layers: quantitative determination by digital image analysis

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1 2012 年 10 月 Vol. 30 No. 10 October 2012 Chinese Journal of Chromatography 1081 ~ 1088 Articles DOI: / SP. J Thin layer chromatographic separation of cobalt from nickel on impregnated silica gel layers: quantitative determination by digital image analysis P A MOHAMED NAJAR, R G SONALI, M T NIMJE, K V RAMANA RAO (Jawaharlal Nehru Aluminium Research Development and Design Centre, Nagpur , India) Abstract: Thin layer chromatography ( TLC) of cobalt and nickel has been performed on silica gel layers induced with alkali mediated cellulose extract. A novel combination of 10% aqueous solutions of Tween-20 and potassium thiocyanate in 1 1 (v / v) was identified as the best mobile phase for the selective separation of Co 2 + from Ni 2 + on the impregnated Silica Gel G layers. The chromatographic characteristics of the cations were studied and the limits of detection as well as the limits of quantification for Co 2 + and Ni 2 + were determined. The quantitative estimation of the cations was achieved from the digital image analysis of respective chromatograms. The proposed quantitative method was successfully applied with % error for the determination of Co 2 + from Ni 2 + in spiked samples of bauxite, soil and rock containing common cations such as Al 3 +, Fe 2 +, Ti 4 +, Zn 2 +, Mn 2 +, Cu 2 +, Cr 6 +, Mg 2 +, etc. under the optimized chromatographic conditions. Key words: thin layer chromatography ( TLC); cobalt; nickel; quantification; digital image analysis CLC number: O658 Document code: A Article IC: (2012) Heavy metals are the most stable and significant environmental contaminants. The metal cations such as cobalt and nickel are common water pollutants and often present in industrial liquid wastes. The present choices of these elements are directly connected with their identities as rare metals of closely related chemical and physical identities as well as the levels of toxicity to natural ecosystem[1]. Although nickel and cobalt are essential trace elements for living organisms, these are toxic when ingested in higher quantities. Their existence in the natural environment causes disturbances in the growth and development of ecological units and indirectly or directly affects human health. They are also of considerable interest to allergology and allergy [ 2-4 ]. Consequently, the development of reliable and cost-effective quantitative analytical tools for these metal ions plays a significant role in environmental studies. The recent literature on quantitative thin layer chromatography ( QTLC) of organics has revealed some interesting applications of digital imaging of TLC-chromatograms for the quantitative evaluation of paracetamol and caffeine[5], food colour dyes [ 6 ], parabens in pharmaceutical preparations[7] as well as the utilization of image analysis software in quantitative thin layer chromatography[8] for organics. Very recently the pioneering work of Hess[9] illustrated the development and application of hyphenated TLC-digital image analysis ( DE-TLC) as a cost-effective alternative for TLC-optical scanning densitometry. Other significant developments were the application of field portable scanner for quantitative analysis of TLC plates based on multi-wavelength image processing[10] as well as the development and application of TLC-digital image analysis system for the determination of cichoric acid present in Echinacea purpurea ( L. ) Moench[11]. Corresponding author: P A MOHAMED NAJAR. Fax: , najarp@ hotmail. com. Foundation item: Science & Technology Wing, Ministry of Mines, Government of India is acknowledged for Received date: financial support to the project on Development of Rapid Analytical Procedures for Cobalt, Chromium and Nickel.

2 1082 色谱第 30 卷 The TLC separation of Co 2 + from Ni 2 + [ 12 ] followed by the quantitative determination by spectrophotometry [ 13, 14 ] was reported and it is observed that the majority of the chromatographic systems used for the selective separation of Co 2 + from Ni 2 + comprised multicomponent mobile phases and generally impregnated adsorbents [ 15 ] containing ion exchange gels. One of the reliable combination of mobile phase systems consisting dimethylamine with acetone as well as methanol [16] resulted the separation of Co 2 + from Ni 2 +. Unfortunately, these combinations appeared least user friendly due to the release of toxic fumes all through the homogenization process. In order to overcome these difficulties, in the present study the emphasis was on utilizing simple, cost-effective and easily reproducible chromatographic systems for the selective TLC separation of Co 2 + from Ni 2 +. This article reports the development of a complementary analytical procedure which can be utilized in the quantitative analysis of coexisting Co 2 + from Ni 2 + in a variety of geological sample matrices by hyphenated TLC-digital image analysis. It is relevant to note that only densitometry and spectrophotometry have been used in the past for the quantitative analysis of metal ions by TLC. 1 Experimental 1. 1 Materials Instrumentations Chromatographic trials were performed with glass plates (7 cm 2. 5 cm, 10 cm 10 cm, 10 cm 20 cm ) coated with suitable adsorbents. Hanna (India) ph meter model HI 8424 USA was used for all the ph measurements. Photometric measurements were carried out by PC based TLC Imaging and Analysis System ( Syngene, UK ) equipped with Chroma Scan tools Chemicals and reagents Cobalt chloride, cobalt nitrate, nickel chloride, nickel nitrate, potassium thiocyanate, potassium chloride, sodium chloride, sodium hydroxide, Silica Gel H and Silica Gel G were obtained from E. Merck, India. Dimethylglyoxime, ethyl alcohol, methyl alcohol, ammonia buffer solution and acetone were obtained from Qualigens, India. Microcrystalline cellulose and standard solutions (1 g / L) of cobalt and nickel were procured from E. Merck, Darmstadt, Germany. All other chemicals used were also of analytical grade reagents Materials used 1% reference standards were prepared in double distilled water with the respective salts. The standard solutions of cobalt and nickel (1 g / L) in alkaline medium were used as received. Test samples were prepared by spiking appropriate volumes of 1% cobalt and nickel in dilute acidic solutions of bauxite, igneous rock and soil samples. The resultant clear solution was made up with double distilled water and preserved in polyethylene flasks for chromatographic studies Preparation of test samples Bauxite The powdered bauxite sample (1 g) was weighed and transferred into a clean 500 ml beaker and made wet with a few drops of double distilled water. The sample slurry was homogenized with 80 ml acid mixture of 35% HCl-70% HNO 3-98% H 2 SO 4 (3 1 4, v / v / v). The contents in the beaker were digested on a hot plate at (100 ± 5) for 45 min. Precipitated silica in the solution was separated by filtration through a Whatman No. 40 ashless filter paper and subjected for alkali fusion. Fused silica was fumed with 6% HF and concentrated H 2 SO 4 for removal of silica and recovery of undigested metallic portions in bauxite. The undigested residue was subjected for fusion with Na 2 CO 3. The alkaline residue was dissolved in double distilled water containing dilute HCl (1 1, v / v) and mixed with the parent solution and made up to the mark in 100 ml polyethylene flask Rock and soil Rock and soil samples were prepared by similar procedure as described for bauxite. The approximate chemical compositions of some representative samples used for this study are shown in Table 1.

3 第 10 期 P A MOHAMED NAJAR, et al. : Thin layer chromatographic separation of cobalt from nickel on impregnated silica gel layers: quantitative determination by digital image analysis 1083 Table 1 Constituent Al 2 O 3 1) Fe 2 O 3 1) SiO 2 1) TiO 2 1) Chemical composition of samples used for the study Bauxite A Bauxite B Rock C Rock D Soil E Soil F LOI 1) Co 2) 0. 0l Nil Cr 2) ND Mn 2) ND ND Ni 2) ND ND ND ND ND Cd 2) Nil ND ND ND ND ND Zn 2) ND Cu 2) ND ND ND ND Mg 2) Ca 2) ND ND Chemical compositions: 1)%; 2) μg / L. ND: not reported. Nil: not present. LOI: loss on ignition Test solutions 1% aqueous solutions of chloride, nitrate and sulfate salts of cobalt and nickel were prepared in demineralized water containing a few drops of corresponding acid to limit the extent of hydrolysis. All other aqueous solutions used for the chromatographic study were also prepared in demineralized water Buffer solution Acetate buffer of ph ~ was prepared by dissolving 25 g ammonium acetate in 50 ml hot double distilled water followed by the addition of glacial acetic acid ( ~ 3 ml). The mixture was diluted to 100 ml in a standard flask. Ammonia buffer was used as received, and sodium borate buffer ( ph ~ ) was prepared by dissolving 0. 8 g NaOH and 4. 7 g boric acid in 100 ml double distilled water Stationary phase Plain cellulose microcrystalline (S 1 ), Silica Gel H (S 2 ), Silica Gel G (S 3 ), the mixtures of the cellulose microcrystalline with Silica Gel H & Silica Gel G separately (S 4 & S 5 ) in 1 1, 3 7, 7 3, 9 1 and 1 9 ( w / w), Silica Gel H and Silica Gel G impregnated with alkaline cellulose extract ( S 6 & S 7 ) as well as alumina G ( S 8 ) were used as adsorbent materials Mobile phase The solvent systems in Table 2 were used as the mobile phases Chromatography Sample purification One percent test solutions ( bauxite, rock, soil) containing Al 3 +, Fe 2 + and Ti 4 + as major components were evaporated on a hot plate to ensure the removal of acids. The dried sample was dissolved in double distilled water containing a few Solution No. M 1 M 2 M 3 M 4 M 5 M 6 acetone Table 2 1% (v / v) Tween-20 10% (v / v) formic acid 100 g / L NaCl 10 g / L KSCN 100 g / L NaOH Mobile phases and their compositions Composition M 7 dimethylamine + 10% formic acid + acetone (8 2 8, 8 3 8, v / v / v) M 8 dimethylamine + 10% formic acid + methanol g / L NaCl ( , v / v / v / v) M 9 dimethylamine + 10% formic acid + methanol (8 2 8, v / v / v) M g / L NaOH + 10% formic acid + H 2 O (1 1 8, v / v / v) M g / L NaCl + 10% formic acid (8 2, 7 3, 9 1, v / v) M g / L KSCN + 10% formic acid + acetone (8 4 4, v / v / v) M g / L KSCN + 10% formic acid g / L NaCl (8 2 8, v / v / v) M g / L KSCN g / L NaCl + acetone (4 2 2, v / v / v) M g / L KSCN + 10% formic acid + acetone g / L NaCl ( , v / v / v / v) M g / L KSCN + 10% formic acid + acetone g / L NaCl ( , v / v / v / v) M g / L KSCN + 1% Tween-20 + acetone (1 1 1, 2 2 1, v / v / v) M g / L KSCN + 1% Tween % formic acid (1 1 1, 2 2 1, v / v / v) M g / L KSCN + 1% Tween-20 (2 1, 8 2, 1 1, 9 1, 7 3, v / v) M g / L KSCN + 10% Tween-20 (2 1, 8 2, 1 1, 9 1, 7 3, v / v)

4 1084 色谱第 30 卷 drops of dilute HCl (1 1, v / v) and made up to 100 ml in a standard flask for chromatographic study. Appropriate dissolution of purified sample was made according to the requirement and nature of studies carried out Preparation of TLC plates TLC plates were prepared by mixing 1 3 ( w / v) silica gel varieties or 1 4 ( w / v) microcrystalline cellulose and double distilled water. The slurry obtained was shaken mechanically for 5 min after which it was spread over polished glass plates (3 cm 7 cm for qualitative studies; 10 cm 10 cm and 10 cm 20 cm for quantitative studies). The layer thickness on the glass plate was physically measured and maintained mm ( average) for optimization. The plates were dried at room temperature and activated at (100 ± 5) for 1 h in an electric oven. The activated plates were stored in an airtight chamber. Impregnated plates were prepared by mixing Silica Gel G or Silica Gel H with alkaline extract of microcrystalline cellulose in double distilled water under the same experimental conditions as nonimpregnated plates. The alkaline extract of cellulose was prepared by dissolution of 10 g microcrystalline cellulose in 50 ml NaOH solution (200 g / L) followed the overnight refrigeration at 0-4. The undissolved cellulose fibers were filtered through double layered Whatman No. 1 filter paper and the filtrate of alkaline extract was collected. For the preparation of silica gel slurry, 20 ml alkaline extract of cellulose was diluted to 100 ml with double distilled water and used in place of water for making silica gel slurry. The dried plates were activated at (100 ± 5) for 1 h and stored as described for unimpregnated plates Procedure Standard chromatographic procedure [ ] has been used for sample application on TLC plates, chromatogram development, visualization of the separated constituents and characterization based on the respective R F ( retardation factor) values calculated from the equation R F = R L + R T / 2, where, R L is the R F of leading front and R T is the R F of trailing front Detection and qualitative separation For the separation, 1% aqueous salt solutions containing Co 2 + and Ni 2 + were prepared separately and mixed in 1 1 ( v / v) ratio and 1 ml sample mixture was diluted with 1 ml acetate buffer. Approximately 5 μl of the synthetic sample mixture was loaded on the chromatographic plates (7 cm 2. 5 cm) coated with S 1-8. The chromatography was performed for achieving the detection of Co 2 + and Ni 2 + and for observing their migration trend from the point of sample application. Among the various chromatographic systems studied, S 4-7 and M 7-20 were found resulting better detection and tendency for differential migrations of the cations as shown in Fig. 1. From the experimental data, S 7 -M 20 (1 1) has been identified as the best chromatographic system suitable for the selective separation of Co 2 + from Ni 2 +. Co 2 + was self detected as bright blue colored spot in the presence of KSCN due to the formation of cobalt thiocyanate complex. Ni 2 + was detected as bright pink spot by spraying 0. 5% ethanolic dimethylglyoxime. The R F values recorded for the ions in their mixture were compared with that recorded for the ions in individual salt solutions. Both readings were found varying slightly from each other under the same experimental conditions. Fig. 1 TLC Separation of Co 2 + and Ni 2 + on impregnated silica gel layers Quantitative studies The quantitative determinations of Co 2 + and Ni 2 + were carried out by digital image analysis. The chromatography was performed under optimized experimental conditions ( Table 3) for the separation of Co 2 + and Ni 2 +. For the quantitative deter-

5 第 10 期 P A MOHAMED NAJAR, et al. : Thin layer chromatographic separation of cobalt from nickel on impregnated silica gel layers: quantitative determination by digital image analysis 1085 minations, a set of standard sample solutions (0. 5%, 1%, and 1. 5%) were prepared by dissolving the salts of respective chlorides in double distilled water. The salt solutions were mixed in 1 1 (v / v) and the sample mixture was diluted with double distilled water and acetate buffer ( ph ~ 6. 98) in (v / v / v) for loading on the TLC plates. Calibration curves were constructed for the determination of Co 2 + and Ni 2 +, by loading 5 μl of the solutions containing , , μg of Co 2 + and , , μg of Ni 2 + respectively on the TLC plates coated with S 7 (10 cm 10 cm) at 1. 5 cm above from the lower edge by using micropipette. The plates were developed with mobile phase M 20 (1 1) and the cations were detected ( R F ( Co 2 + ) = and R F (Ni 2 + ) = 0. 67) on separate TLC plates. Table 3 Optimized chromatographic conditions for separation of Co 2 + from Ni 2 + Item Condition Nature aqueous, acidic Concentration 0. 01% % Sample loading volume 1-5 μl Stationary phase Solid liquid ratio of silica gel and 1 3 (w / v) alkali extract Solid liquid ratio of cellulose and alkali 1 1 (w / v) Dilution of alkaline cellulose extract 2 times Plate size 10 cm 10 cm 10 cm 12 cm Slurry volume ml Layer thickness ~ 2. 5 mm Activation temperature (100 ± 2) Plate activation time (60 ± 5) min Mobile phase 100 g / L KSCN + 10% Tween-20 aqueous, 1 1 (v / v) Mobile phase volume 10 ml Mobile phase ascent 7 cm Chromogenic reagent Co 2 + Ni 2 + self detected dimethylglyoxime The digital images of the chromatograms ( Fig. 2) were recorded in dark chamber equipped with a 16 bit digital camera controlled by ChromaScan TLC image analysis software for documenting digital images of the chromatogram for quantitative assessment. Tracks I and II representing standard concentration of Ni 2 + and Co 2 + spots in the digital image have been evaluated for assessing linearity with respect to area under the curve. From the peak area corresponding to the concentration range of Ni 2 + and Co 2 + loaded on the TLC plate, the calibration curves were constructed. Fig. 2 Digital image analysis of chromatograms of Ni 2 + (Track I) and Co 2 + (Track II) 1, 2 and 3 represent the number of loading trials. Hamilton syringe was loaded with sample solution three times and two spots were loaded on the TLC plate every time Application Since the concentrations of Co 2 + and Ni 2 + in real samples such as bauxite, soil and rock were found bare minimum (Table 1) for effective TLC detection, the samples were spiked with specific quantities of standard salt solutions. The spiked samples were prepared separately by mixing 1% aqueous solutions of CoCl 2 6H 2 O and NiCl 2 6H 2 O with 1% solutions of bauxite ( A), soil ( C) and rock (E) in (v / v / v). The chromatography was performed with the spiked samples under optimized chromatographic conditions in S 7 - M 20 (1 1). The peak areas corresponding to the cations present in unknown samples were determined by the simultaneous loading of respective standards ( samples A, C, E) and sample solutions ( samples B, D and F) side by side on the same TLC plate and the chromatography was performed. From the peak area obtained from the digital image analysis of the chromatograms of standard and sample spots of Co 2 + and Ni 2 +, the recoveries of Co 2 + and Ni 2 + in the unknown samples were determined. The error E was calculated by standard statistical equation: E = A - B B 100%

6 1086 色谱第 30 卷 where, A: composition of constituent in the unknown sample; B: composition of constituent in standard sample. 2 Results and discussion The chromatographic behaviors of cobalt and nickel in different samples were examined on selective chromatographic systems comprising mainly aqueous potassium thiocyanate, Tween- 20, Silica Gel G with alkaline extract of microcrystalline cellulose. The results of these studies are summarized in Table 3 & 4 and Fig Nature of sample To study the effects of sample composition and matrix effect of inorganic cations on the separation of coexisting Co 2 + and Ni 2 +, an aliquot ( 5 μl) containing metal ions such as Al 3 +, Fe 2 +, Mn 2 +, Zn 2 +, Cd 2 +, Cu 2 +, Cr 6 + etc. in the samples of bauxite, rock and soil was spotted on the TLC plates coated with S 5. The plates were developed by the solvent system M 20 (1 1). The spots of Co 2 + and Ni 2 + in the geological samples were detected under the same experimental conditions for synthetic salt solutions. The R F values recorded for Co 2 + and Ni 2 + in spiked samples revealed that the compositional variations ( R F for Co 2 + and R F = for Ni 2 + ) do not affect the binary separation of Co 2 + and Ni 2 +. Repeated trials under the change of temperature (25 to 50 ) and the change of adsorbent moisture showed reproducible R F values ( Fig. 3 ) under the same experimental conditions. The effect of adsorbent moisture on the separation was studied with non-activated, activated and reactivated ( after sample loading ) TLC plates (S 7 ). An overall variation in R F of was noticed with Co 2 + and Ni 2 +. The important aspect of this study revealed the efficiency and selectivity of the chromatographic system S 7 - M 20 (1 1). It was observed that the presence of foreign ions in milligram levels such as Al 3 +, Fe 2 + and Ti 4 + and microgram levels of trace elements ( Table 1) did not hamper the separation of Co 2 + from Ni 2 +. The chromogenic reagents used for Co 2 + from Ni 2 + were selective and did not form colored spots of other elements. The location of Al 3 + and Fe 2 + were identified ( R F ) by spraying with 0. 1% aqueous aluminum and potassium ferrocyanide respectively. Both the spots appeared tailing from the point of sample application and diffused each other. The lower R F of both the cations indicated its zero interference in the mutual separation of Co 2 + and Ni 2 +. Though attempts were made for the detection of Ti 4 + in bauxite samples by spraying 0. 50% tiron, the spot was not detected with the system of S 7 -M 20 (1 1). The study recorded negligible variations of R F in the presence of the impurity elements, i. e. the changes were ΔR F < for both of Co 2 + and Ni 2 + ( Fig. 4), where ΔR F represents the difference between the R F of cobalt or nickel in the salt and the R F of cobalt or nickel in spiked samples. Fig. 4 Range of variation of R F of Co 2 + and Ni 2 + with respect to matrix effect Fig. 3 Effect of sample composition on R F variation of Co 2 + and Ni Effect of ph The ph values of the sample and mobile phase played a significant role in the detection and mobility of the cations. High R F value ( from ) was observed for both of Co 2 + and Ni 2 + with the systems of S 1-5 -M The differential migrations of Co 2 + and Ni 2 + were found possible only in

7 第 10 期 P A MOHAMED NAJAR, et al. : Thin layer chromatographic separation of cobalt from nickel on impregnated silica gel layers: quantitative determination by digital image analysis 1087 neutral to medium alkaline range ( ph ). The acidic ph range was not preferred for the mutual separation of the cations and in order to maintain medium alkaline ph range, the sample solution was neutralized with acetate buffer ( ph ~ 6. 98) and silica gel was impregnated with alkaline extract of microcrystalline cellulose. In order to verify the ph effect, trials were carried out without acetate buffer as well as with ammonia buffer (ph) and borate buffer (ph ) in place of acetate buffer. In the presence of ammonia buffer, Co 2 + gave tailed spots and the detection of Ni 2 + appeared sluggish Nature of chromatographic system The nature of chromatographic system was found decisive for the differential migrations of Co 2 + and Ni 2 +. The scope of varying adsorbent combinations was constricted since the priority was high on realizing simple, nontoxic and greener chromatographic system for attaining binary separation of the cations having closely related physical and chemical properties. It was observed from the R F variations that the presence of alkali and neutral nature of cellulose in silica gel system hold the potential of resulting differential migrations of Co 2 + and Ni 2 +. However, the direct addition of alkali as impregnant as well as in the mobile phase was not preferred in terms of its adverse effects such as the tailing and moderate spot colour intensity. The alkaline extract of cellulose has been found suitable for imparting the controlled presence of cellulose and alkali in silica gel for resulting highly compact ( R L of Co 2 + and Ni R T 0. 15) spots Conversely, the presence of surfactant in the mobile phase was found intensify the spot colour for resulting in high values for limit of detection and limit of quantification Limits of detection and quantification The limits of detection ( LOD) of Co 2 + and Ni 2 + were evaluated in the chromatographic system of S 7 -M 20 (1 1). Different volumes of standard sample solutions ( g / L) were loaded on TLC plates and the chromatography was performed under the optimized conditions. The method was repeated with successive lowering of the amount of cation until no spot visibility attained on the TLC plate. Also, the procedure was repeated for attaining the limit of quantitation ( LOQ) by recording the digital image of spots until no signal to noise ( peak for spot image) on the TLC plate. The minimum amount of cations which gave a visual indication on the TLC plate after development in the system of S 7 -M 20 (1 1) was considered as the LOD and which gave a measurable peak during the digital image analysis of chromatogram was considered as the LOQ. The lowest LOD goes down to 1-3 μg for both of Co 2 + and Ni 2 +. The thiocyanate and surfactant ( Tween-20 ) present in the mobile phase were attributed for the high LOD values of Co 2 + and Ni 2 + respectively. Similarly the reported LOQ values for both cations were in the range of 4-6 μg. The details of the study are summarized in Table 4. Table 4 Limits of detection of cations in samples and complex matrices Sample Cobalt nitrate 3 LOD / μg Co 2 + Ni 2 + Nickel nitrate 3 Mixture (1 1 1, v / v / v) 4 3 Mixture (1 1 1, v / v / v) + soil 7 5 Mixture (1 1 1, v / v / v) + igneous rock 4 3 Mixture (1 1 1, v / v / v) + river sediment 10 8 Mixture (1 1 1, v / v / v) + plant effluent 9 8 Mixture (1 1 1, v / v / v) + bauxite 5 4 Mixture (1 1 1, v / v / v) + leafy vegetable extract 9 15 Average of five consecutive trials under optimized chromatographic conditions Quantitative determination In order to achieve the accuracy and reproducibility of the digital image analysis of chromatograms of Co 2 + and Ni 2 +, the chromatography was performed with spiked samples of varying chemical compositions ( Table 1). The laboratory made TLC plates were prepared under optimized experimental conditions ( Table 3) for achieving consistency in R F values, spot size, colour stability and development time. The quantitative assessment of Co 2 + and Ni 2 + in the samples was determined from the digital image of respective chromatograms. The peak values obtained after the assessment of digital images of chromatograms were found proportional to the Co 2 + and Ni 2 + concentrations

8 1088 色谱第 30 卷 applied to the chromatographic plate. Subsequently, the concentrations of Co 2 + and Ni 2 + in unknown samples (samples B, D, F) were determined relative to the respective standards as shown in Fig. 5. Since the natural presence of Co 2 + and Ni 2 + in the samples was negligible, it is presumed that matrix effect was the only parameter influenced in the determination of the cations in the spiked samples. However, the reproducible data obtained for Co 2 + and Ni 2 + for both standards as well as samples confirm the reliability of TLCimage analysis method developed. The comparative quantitative analysis data obtained from the samples of unknown concentration were listed in Table 5. The error calculated after image analysis of the individual cations was within the range of tolerance up to the second decimal place. The relative standard deviation (0. 01% %) was calculated from five consecutive measurements of peak area obtained for any particular sample. The chromatograms were developed under the optimized chromatographic conditions and the digital image was recorded for spot area measurements. Fig. 5 Digital image analysis of Co 2 + and Ni 2 + in different sample matrices Table 5 Quantitative determination of Co 2 + and Ni 2 + by digital image analysis Analyte Bauxite sample Rock sample Soil sample A( known) B( unknown) Error / % C( known) D( unknown) Error / % E( known) F( unknown) Error / % Co Ni Each value was average of five consecutive scanning of five different spots of the same sample loaded on the TLC plate. 3 Conclusions The present chromatographic procedure on the separation and quantitative determination of cobalt and nickel using hyphenated TLC-digital image procedure revealed the possibility of an improved and rapid quantitative methodology for the analysis of heavy metal cations in real samples. The investigation also proved that TLC procedure can be successfully modified and hyphenated with sophisticated instrumental methods for the qualitative and quantitative determination of trace elements. Acknowledgement The authors thank Dr. J. Mukhopadhyay, Director JNARDDC for his constant encouragement, useful suggestions and permission for publishing the work. References: [1] Dojlido J R, Best G A. Chemistry of Water and Water Pollution. New York: Ellis Horwood Limited, 1993 [2] Baralkiewicz D, Karas Z, Siepak J. Chem Anal, 1997, 42: 691 [3] Delesculuse J, Dinet Y. Dermatology, 1994, 189: 56 [4] Rudzki E, Prystupa K. Contact Dermatitis, 1994, 30: 254 [5] Soponar F, C ǎt ǎlin Moţ A, Sarbu C. Chromatographia, 2009, 69(1 / 2): 151 [6] Soponar F, Cǎ tǎ lin Moţ A, Sarbu C. J Chromatogra A, 2008, 1188(2): 295 [7] Casoni D, Anamaria I T, Costel S. J Liq Chromatogr Rel Technol, 2011, 34(10 / 11): 805 [8] Johnsson R, Träff G, Sundén M, et al. J Chromatogr A, 2007, 1164: 298 [9] Hess A V I. J Chem Educ, 2007, 84(5): 842 [10] Aldridge P K, Callis J B, Burns D H. Rev Sci Instrum, 1992, 63(10): 4333 [11] Tie-xin T, Hong W. J Chromatogr Sci, 2008, 46(6): 560 [12] Mohamed A, Nasim K T, Ahmad J, et al. Analusis, 1995, 23: 243 [13] Mohammad A, Sirwal Y H. Indian J Chem Tech, 2004, 11: 726 [14] Mohamed A, Mohamed Najar P A, Iraqi E. Indian J Chem Tech, 1999, 6: 38 [15] Mohamed A, Iraqi E, Sirwal Y S. Sep Sci Tech, 2003, 38 (10): 2255 [16] Mohamed A, Sirwal Y H. Acta Chromatogr, 2003, 13: 117 [17] Stahl E. Thin-Layer Chromatography: A Laboratory Hand Book. 2nd ed. New York: Springer-Verlag, 1969: 52 [18] Touchstone J C. Practice of Thin Layer Chromatography, 3rd ed. New York: John Wiley and Sons Inc, 1992: 60 [19] Mohamed Najar P A. [ Ph. D Thesis]. India: Aligarh Muslim University, 1997 [20] Spangenberg B, Poole C F, Weins C. Quantitative Thin Layer Chromatography: A Practical Survey. New York: Springer-Verlag, 2011