Title of Invention	"A NOVEL DEVICE FOR THE QUANTIFICATION OF LEAD AND COPPER ALONE OR SIMULTANEOUSLY IN AQUEOUS/NON-AQUEOUS & BIOLOGICAL SAMPLES."
Abstract	Various toxic metals namely arsenic, lead, mercury, cadmium, zinc, copper and other environmental pollutants have been described as a source of poisoning in humans and animals. The major source of contamination is either through environmental exposure or through dietary intake of contaminated foods and water. In view of their significance in human life, it is important to estimate the level of these metal ions in environmental, industrial, food and biological samples. The present invention relates to a cost-effective device and process for the quantification of lead and copper alone and or simultaneously in aqueous, non-aqueous and biological samples, wherein anodic stripping voltammetry technique is used for the quantification at low ppb (µg/1) level using a special type of sensing electrode.

Title of Invention

"A NOVEL DEVICE FOR THE QUANTIFICATION OF LEAD AND COPPER ALONE OR SIMULTANEOUSLY IN AQUEOUS/NON-AQUEOUS & BIOLOGICAL SAMPLES."

Abstract

Various toxic metals namely arsenic, lead, mercury, cadmium, zinc, copper and other environmental pollutants have been described as a source of poisoning in humans and animals. The major source of contamination is either through environmental exposure or through dietary intake of contaminated foods and water. In view of their significance in human life, it is important to estimate the level of these metal ions in environmental, industrial, food and biological samples. The present invention relates to a cost-effective device and process for the quantification of lead and copper alone and or simultaneously in aqueous, non-aqueous and biological samples, wherein anodic stripping voltammetry technique is used for the quantification at low ppb (µg/1) level using a special type of sensing electrode.

Full Text	The present invention relates to a novel device for the quantification of lead and copper in aqueous/ non-aqueous & biological samples and a process thereof. The present invention particularly relates to a device and method for the quantification of lead and copper alone and or simultaneously in aqueous/ non-aqueous & biological samples by electrochemical technique. Various toxic metals namely arsenic, lead, mercury, cadmium, zinc, copper and other environmental pollutants have been described as a source of poisoning in humans and animals. The major source of contamination is either through environmental exposure or through dietary intake of contaminated foods and water. Therefore monitoring/quantification of the toxicants in water and biological samples assumes great significance in the health surveillance and safety of humans. Lead poisoning is an environmental disease, but it is also a disease of lifestyle. Lead is one of the best-studied toxic metal, and as a result we know more about the adverse health effects of lead than virtually any other chemical. The health problem caused by lead has been well documented over a wide range of exposures in every continent. Lead poisoning continues to be one of the most prevalent occupational and environmental illnesses affecting adults. It has also adverse neurological effects, with decrease in IQ and reading ability in young and adults, and affects virtually every system in the body. Blood lead levels as low as 10 jig/dl cause adverse health effects in children. Copper is an essential metal ion which is also toxic and the detection of copper in food, water as well as in blood is very important. Copper affects the stability of DMA, alters the fluidity of plasma membranes, inhibits various enzymes and diminishes or enhances mitochondria respiration etc. Acute poisoning causes several types of diseases and affects the liver, central nervous system, kidneys and heart and may lead to death. Wilson's disease is the prototypical disease caused by copper toxicosis, affecting first the liver and later the central nervous system, eyes and kidneys. Copper is also an essential element and its deficiency causes ischemic heart disease, references may be made as 1] E. J. Underwood, "Trace elements in Human and Animal Nutrition", 4th Ed., Academic press, NY, USA, 1979 and 2] B. Venugopal, T.D. Luckey, "Metal toxicity in mammals", Vol. 1, Plenum press, NY, USA, 1977). In view of their significance in human life, it is important to estimate the level of these metal ions in environmental, food and biological samples. However handling of a large number of samples (water, food items, blood, urine etc.) obtained during field surveys and their processing for chemical determinations through sophisticated laboratory equipment and infrastructure facilities are usually time consuming and very costly, requires excessive investments on spares, chemicals and manpower. Therefore there is an urgent need for the development of quick, reliable, sensitive and cost effective technologies for the quantification of lead and copper for low ppb(^.g/t) level. The well known and reliable analytical methods for the quantification of lead and copper are atomic emission spectroscopy with inductively coupled plasma excitation, Atomic Absorption Spectrophotometry or Neutron activation analysis or x-ray fluorescence. These methods are having the following drawbacks. a. Analysis with a highly sophisticated instrument of very high cost and need a well- trained personnel and equipped laboratory. b. These techniques are not possible in field conditions c. Portable device is not possible. Apart from the above conventional techniques, various electrochemical methods are also reported for the quantification of lead and copper. Reference may be made to 1] A.M. Bond "Modern Polarographic Methods in Analytical Chemistry", Marcel Deckker, New York, 1980; 2] J. Wang, "Striping analysis-Principals, Instrumentation and Applications", VCH, Deerfield Beach ., FL 1985. Above references reported mainly the polarographic techniques for the estimation of the several metal ions including lead and copper. These techniques were having its own limitations because use of Hg hanging electrodes which were itself very toxic and also detection limit was quite low. Latter Stripping Voltammetric techniques were introduced with solid stationary metal/modified electrodes with relatively low detection limit. Reference may be made to 1] Differential pulse anodic stripping voltammetric determination of Pb(ll) with N-P-chlorophenylicinnamohydroxamic acid modified carbon paste electrode, Degefa, Tesfaye Hailu, Chandravanshi et al, Electroanalysis, 119170, 1305, 1999; 2] Determination of lead(ll) and cadmium (II) in hard and soft wheat by derivative potentiometric stripping analysis, F.Lo Coco, P. Monotti, V. Fiecchi and L. Ceccon, Analytica Chimica Acta, 409, 1-2, 93, 2000; 3] Anodic and Cathodic stripping voltammetry in the simultaneous determination of toxic metals in environmental samples, Clinio Lacatelli, Electroanalysis, 9, 13, 1997; 4] Lead Analyzer, US Patent Nos. 4,201,646, 4,374,041 and 4,090,926, ESA Analytical Ltd., 14 Cromwell Mews, St. Road, Stives, Huntingdon, Cambridgeshire PE 17 4HJ England However, none is capable to detect lead and copper ions at low ppb level (ug/l) with good reproducibility, mainly because of the limitations of the electrodes and electrolytes. All the above techniques used a stationary sensing electrodes in acidic electrolyte. The major Drawbacks associated with the earlier works are: 1) Stationary (non-rotating) working electrode is used, which is unable to overcome the problems of hydrogen evolution at negative potential and also poisoning of the electrodes. 2) High acid concentration is used mainly for biological and complex matrix samples, this causes the large gas evolution during the metal deposition as well and may cause damage to the electrodes and device parts on long use. 3) Some times the metal ions are not reducible in the matrix and therefore it requires more negative potential and special type of working electrodes, which gives irreproducible results with stationary electrodes. 4) It is hazardous to use high acid concentration, which is common in the conventional methods. 5) Precise and reproducible estimations of higher concentration of lead/copper may not possible. In our co-pending patent application no. 774/Del/2000, we have described and claimed a novel device for the quantification of arsenic in water and a process thereof, wherein the novel device for the quantification of arsenic is based on the anodic stripping voltammetry. The device consisted a three electrodes configuration of a novel side tip gold working electrode along with platinum counter/auxiliary electrode and conventional reference electrode. The novel gold electrode of rotating type and of specific construction was used for the low ppb (\|j.g/l) estimation of arsenic in acidified waster sample. The gold electrode was constructed by embedding the known size of gold metal circular/triangular/elliptical thin plate at the side surface (towards bottom) of the inert material hollow cylinder and taking a wire connection from the embedded gold plate in order to make the electrical contact. The above gold electrode was attached to a rotating assembly and all the three electrodes were fitted in an electrochemical cell having typical composition of electrolyte such as 4% (v/v) mineral acid and low ppb of copper ions for the quantification of arsenic as low as 5 ppb. The main object of the present invention is to provide a device for the quantification of lead and copper alone and simultaneously in various samples such as aqueous/ non-aqueous & biological samples. Another object of the present invention is to provide a process for the quantification of lead and copper, using the novel device of the present invention. Yet another object of the present invention is to provide a novel electrochemical device and process for the quantification of lead and copper. Still another object of the present invention is to provide a simple, easy and safe technique (non-hazardous) for the quantification of lead and copper. Another object of the present invention is to provide a sensing electrode for lead and copper, such as platinum, gold or any noble metal or carbon paste or graphite/glassy carbon tip embedded at the side of a hollow cylinder of inert material. Yet another object of the present invention is to develop a portable device useful for sensing lead and copper alone and simultaneously in various samples. In the present invention the basic principle of anodic stripping voltammetry with three electrodes configuration has been used to provide a novel electrochemical device and process for the quantification of low ppb (^.g/l) level of lead and copper in aqueous/ non-aqueous & biological samples. In the present invention the three electrodes configuration essentially consists of a metal or carbon tipped sensing electrode, an inert metal counter/auxiliary electrode and a conventional reference electrode. The inventive step of the novel device of the present invention resides in the rotating sensing electrode constructed by embedding the known size of noble metal or carbon paste or graphite/glassy carbon circular/triangular/elliptical thin plate or rod at the side surface or bottom of the hollow cylinder made up of inert material and aqueous or non-aqueous electrolytic solution, which is either acidic 0.01N to 1N or neutral electrolyte of soluble salts 0.01M to 1 M. A wire connection from the embedded metal or carbon plate/rod is taken in order to make the electrical contact. The electrode top is equipped with a screw type connection to attach with a rotator assembly/ prime mover. The above sensing electrode is attached to the rotating assembly and all the three electrodes are fitted in an electrochemical cell having acidic or neutral electrolyte for the quantification of lead and copper as low as 5 ppb. In the present invention the sample may be either aqueous or non-aqueous and verity of the samples may be used like natural water, petroleum, biological etc, while in co-pending patent application no. 774/Del/2000, it was limited to aqueous (water) samples only. In the present invention the nature of the electrolyte may be either acidic or neutral, while in our pending patent application no, 774/Del/2000, it must be acidic. Also in the present invention in the sensing electrode the metal or carbon tip may be present either at the side or at the bottom of the rotating electrode body, while in our pending patent application no. 774/Del/2000, essentially it must be present at the side of the electrode body. Therefore, the novelty of the present invention lies in the device capable of quantification of lead and copper alone and simultaneously in various samples at low ppb levels. The non-obvious inventive step reside in the metal/carbon tipped rotating sensing electrode. In figure-1 of the drawings accompanying this specification the schematic diagram of the novel device of the present invention is shown. Wherein the various parts are, electrolytic container (1), electrolyte (2), inert material cylindrical substrate (3), sensing tip (4), inert auxiliary electrode (5), reference electrode (6), rotating assembly/prime mover (7), variable voltage source (8), current measuring device (9) and connecting wires (10). Accordingly, the present invention provides a novel device for the quantification of lead and copper alone or simultaneously in aqueous/ non-aqueous & biological samples, which comprises a three electrode electrochemical cell (1) essentially containing acidic/neutral electrolyte (2), wherein the sensing electrode consist of an inert material cylindrical substrate (3) having a metal/carbon tip (4) embedded onto the side surface (towards bottom) or at the bottom of the said substrate (3), the said electrode embedded substrate being connected by known means to a rotating assembly/prime mover (7) capable of providing a variable speed in the range of 100 to 2000 rpm, the said metal/carbon tip (4) being provided with an electrical connection by means of a connecting wire (10) to a negative terminal of a known variable voltage source (8), the positive terminal of the said variable voltage source (8) being connected to an inert auxiliary electrode (5) and a standard reference electrode (6), the said sensing, auxiliary and reference electrodes being connected to a known current measuring device (9) in series to the said variable voltage source (8). In an embodiment of the present invention the electrolyte in concentration range of 0.01 M to 1 M used may be such as HCI, H2SO4, HNO3, HCIO4, other acids, KCI, NaCI or any other soluble salts. In still another embodiment of the present invention the inert substrate of the rotatable sensing electrode may be such as Teflon, glass, plastics. In yet another embodiment of the present invention the metal/carbon sensing tip used may be made up of nobel metal such as gold or platinum or carbon paste or graphite/glassy carbon. In yet another embodiment of the present invention the metal/carbon sensing tip used may be such as circular, elliptical, triangular of the surface area in range of 1mm2 to 10 mm2. In another embodiment of the present invention the prime mover used may be such as variable speed electric motor. In yet another embodiment of the present invention the auxiliary electrode used may be such as platinum, gold, glassy carbon, titanium, stainless steel. In still another embodiment of the present invention the reference electrode used may be such as any commercially available reference electrode. In another embodiment of the present invention the variable voltage source used may be such as any commercially available potentiostat capable of providing sweeping potential in the range of minus 1.0 V to plus 0.6 V in differential pulse mode. In still another embodiment of the present invention the current measuring device used may be such as analog or digital capable of measuring current in the range of 50 nA to 25 ^A. Accordingly, the present invention provides a process for the quantification of lead and copper alone and or simultaneously in aqueous/ non-aqueous & biological samples using the device as described above, which comprises placing a sample in the electrochemical cell containing acid of concentration in the range of 0.01 N to 1N or neutral electrolyte of suitable soluble salt of concentration in the range of 0.01M to 1M, applying negative potential to the sensing electrode in the range of minus 1.0 V to minus 0.2 V for a period in the range of 1 second to 5 minutes with the rotation speed in the range of 100 to 2000 rpm, allowing to stand/rest for a period in the range of 1 second to 120 seconds followed by scanning in differential pulse mode from minus 0.6 V to plus 0.6 V at a scanning rate in the range of 1 mV/s to 500 mV/s and measuring the maximum current, estimating lead and copper alone or simultaneously either by the predetermined standard slope or by standard addition method by adding atieast two known concentrations of the respective metal ions. In the present invention the basic principle for the quantification of lead and copper ions are based on the anodic stripping voltammetry. This method can be classified in two steps, first is the quantitative deposition of the metal ions over the electrode in form of elemental metal and second is the oxidation of deposited metal back to the solution from the electrode. Lead or copper ions are commonly present in the sample in +2 oxidation states (Pb"1"2, Cu+2). The deposition of the Pb^/Cu*2 in its elemental state can be done either in neutral solution using some salt solution as an electrolyte or some specific pH solution using acids or buffer. The complex mediums specially biological fluids are very complicated and creates a lot of interference for the detection of lead/copper ions. Therefore, for complex matrix, an acidic electrolyte is preferred to overcome the matrix effect. While we have also demonstrated a simple salt electrolyte for estimation of lead/copper for the complex matrix viz. blood. The estimation can be divided in two steps: In the first step of the process i.e. deposition, a negative potential is applied for a certain time period (depending on matrix and concentration) to the sensing electrode. The strength of the electrolyte can be optimized according to the need. During the deposition of the metal ions in the acidic medium, evolution of the Cb gas and HZ gas may take place at auxiliary and sensing electrode respectively and both the gases evolve at the electrodes in the form of fine bubbles. In the case of some other electrolytes, some gases may also evolve during the deposition. These gases interfere during the deposition of the metal ions, therefore, must be removed immediately after their formation. In order to get the least interference by the gas bubbles formed during the deposition, the sensing electrode is used with the rotation arrangement. Side tip electrode is used with high speed rotation in order to remove the gases from the electrode. In the second step of the process, the deposited metals are allowed to oxidise by applying a sweeping potential in differential pulse mode with low scan rate followed by a equilibrium potential (the sweeping potential range must cover the oxdidation potential of the deposited metal). The equilibration time is important for the removal of the impurities, which may be deposited over the electrode along with the target metal ions. The estimation of the oxidation current peak gives an estimation of the corresponding metal ions in the solution, with the help of a calibration plot. The oxidation peak current vs. lead or copper concentration plot (calibration curve) showed a linear relation. The lead and copper alone and simuitaneously estimated up to 5 ppb and above in various aqueous and non-aqueous samples including biological samples. The present invention provides a novel device useful for quantification of (ead and copper electrochemicaily alone or simultaneously, which comprises a special type of sensing electrode along with a metal auxiliary and a reference electrode placed in a suitable container for holding the fluid in which metal ion is to be quantified. All the three electrodes being connected to a voltage source which can apply the potential to the sensing electrode in differential pulse mode. In the present invention the sample may be either aqueous or non-aqueous. A verity of the samples may be used like natural water, petroleum, biological materials etc, while in co-pending patent application no. 774/Del/2000, arsenic detection is limited to aqueous (water) samples only. Moreover the nature of the electrolyte may be either acidic or neutral, while in our pending patent application no. 774/Del/2000, it must be acidic. The sensing electrode is used here either may be metal or carbon tip at the side or at the bottom of the rotating electrode body, while in our pending patent application no. 774/Del/2000, it must be present at the side of the electrode body. The invention is further illustrated with the help of the following examples and therefore should not be construed to limit the scope of the present invention in any manner whatsoever. Example 1 Quantification of lead in water Gold bottom disk rotating electrode (BDRE) (2 mm disk diameter) situated at the bottom of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Tap water sample (5 ml) was taken and KCI salt was added to make it to 0.5M concentration. Sample was placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied to the gold electrode for 60s for the deposition of any lead present in the sample at the electrode. After deposition, another 30s equilibration time was given at the -0.5 V to remove the impurities (impurities for which the oxidation potential is below -0.5V) deposited along with the lead at the surface of the BDRE at the rotation of 1000 rpm. A sweeping potential (in differential pulse mode) was applied in the range of -0.5 V to 0.0 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V was obtained as shown in curve A of Fig. 2(a) of the drawing accompanying this specification. In the same solution four different known concentrations of lead were added to get the total concentration of 10 ppb, 20 ppb, 30 ppb and 40 ppb and experiment was repeated under the same conditions. The lead oxidation peak for each addition was recorded as shown as curves B, C, D and E of Fig. 2(a) of the drawing. The peak current vs. lead concentration was plotted for preparing the calibration curve as shown in Fig. 2(b) of the drawing. A linear nernstian plot was observed in the range of 10 ppb to 40 ppb of lead as shown in Fig. 2(b) of the drawing. The tap water sample contains 21.47 ppb lead as estimated with the help of calibration curve. When the experiment was repeated in 0.1 M HCI as electrolyte, same results were obtained with ±5% deviation. Example 2 Quantification of lead in Rat's blood with two point calibration: Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Rat's blood (0.5 ml) was taken and diluted to 5 ml with 0.1N HCI. Sample was shaken for five minutes and placed in the cell. A deposition potential of-0.6 V vs. Ag/AgCI was applied to the gold electrode for 60s to deposit lead if present in the sample at the surface of SDRE at the rotation of 1000 rpm. After deposition, another 30 s equilibration time was given at the -0.4 V to remove the impurities (impurities for which the oxidation potential is below -0.4V) deposited along with the lead. A sweeping potential (in differential pulse mode) was applied in the range of -0.4 V to 0.0 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V was obtained as shown in curve A of Fig. 3(a) of the drawing accompanying this specification. In the same solution two different known concentrations of lead were added to get the total concentration of 10 ppb and 20 ppb and experiment was repeated under the same conditions. The lead oxidation peak for each addition was recorded as shown as curves B and C of Fig. 3(a) of the drawing. The peak current vs. lead concentration was plotted for preparing the calibration curve as shown in Fig. 3(b) of the drawing. A linear nernstian plot was observed as shown in Fig. 3(b) of the drawing. The blood sample contains 23.58 x 10 = 235.8 ppb (x 10 because of dilution) or 23.58 ^.g/dl lead as estimated with the help of calibration curve. When the experiment was repeated in 0.5 M KCI as electrolyte, same results were obtained with ±5% deviation. Example 3 Quantification of lead in human blood with two point calibration: Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Human blood (1 ml) was taken and diluted to 5 ml with 0.5 M KCI solution. Sample was shaken for five minutes and placed in the cell. A deposition potential of -0.6 V vs. Ag/AgCI was applied to the gold electrode for 60 s to deposit lead if present in the sample at the surface of SDRE. After deposition, another 30 s equilibration time was given at the -0.4 V to remove the impurities (impurities for which the oxidation potential is below -0.4V) deposited along with the lead. A sweeping potential (in differential pulse mode) was applied in the range of -0.4 V to 0.0 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V was obtained as shown in curve A of Fig. 4(a) of the drawing accompanying this specification. In the same solution two different known concentrations of lead were added to get the total concentration of 20 ppb and 40 ppb and experiment was repeated under the same conditions. The lead oxidation peak for each addition was recorded as shown as curves B and C of Fig. 4(a) of the drawing. The peak current vs. lead concentration was plotted for preparing the calibration curve as shown in Fig. 4(b) of the drawing. A linear nernstian plot was observed as shown in Fig. 4(b) of the drawing. The blood sample contains 24.26 x 5 = 121.30 ppb (x 10 because of the sample was diluted 5 times) or 12.13 ug/dl lead as estimated with the help of calibration curve. When the experiment was repeated in 0.1 N HCI as electrolyte, same results were obtained with ±5% deviation. Example 4 Quantification of copper in water Gold bottom disk rotating electrode (BDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Tap water (5 ml) was taken and KCI salt was added to make it to 0.5M concentration. Sample was placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied for 60 s to deposit copper if present in the sample at the surface of BDRE at the rotation of 1000 rpm. After deposition, another 30 s equilibration time was given at the 0.0 V to remove the impurities (impurities for which the oxidation potential is below 0.0V) deposited along with the copper. A sweeping potential (in differential pulse mode) was applied in the range of 0.0 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at 0.3 V ± 0.05 V was obtained as shown in curve A of Fig. 5(a) of the drawing accompanying this specification. In the same solution four different known concentrations of copper were added to get the total concentration of 10 ppb, 20 ppb, 40 ppb and 50 ppb and experiment was repeated under similar condition. The copper oxidation peak for each addition was recorded as shown as curves B, C, D and E of Fig. 5(a) of the drawing. The peak current vs. copper concentration was plotted for preparing the calibration curve as shown in Fig. 5(b) of the drawing. A linear nernstian plot was observed in the range of 10 ppb to 50 ppb of copper as shown in Fig. 5(b) of the drawing. The unknown sample was containing 5.17 ppb copper as estimated with the help of calibration curve. When the experiment was repeated in 0.1 N HCI as electrolyte, same results were obtained with ±5% deviation. Example 5 Quantification of copper in blood with two point calibration: Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 \|jA range which was interfaced with a computer. Human blood sample (1 ml) was taken and diluted to 5 ml with 0.5 M KCI solution. Sample was shaken for five minutes and placed in the cell. A deposition potential of -0.6 V vs Ag/AgCI was applied to the gold electrode for 60 s to deposit copper if present in the sample at the surface of SDRE at the rotation of 1000 rpm. After deposition, another 30 s equilibration time was given at the 0.0 V to remove the impurities (impurities for which the oxidation potential is below 0.0V) deposited along with the copper. A sweeping potential (in differential pulse mode) was applied in the range of 0.0 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at 0.3 V ± 0.05 V was obtained as shown in curve A of Fig. 6(a) of the drawing accompanying this specification. In the same solution two different known concentrations of lead were added to get the total concentration of 100 ppb and 200 ppb and experiment was repeated under the same conditions. The copper oxidation peak for each addition was recorded as shown as curves B and C of Fig. 6(a) of the drawing. The peak current vs. copper concentration was plotted for preparing the calibration curve as shown in Fig. 6(b) of the drawing. A linear nernstian plot was observed as shown in Fig. 6(b) of the drawing. The blood sample contains 200.0 x 5 = 1.0 ppm (x 10 because of the sample was diluted 5 times) copper as estimated with the help of calibration curve. When the experiment was repeated in 0.1 N HCI as electrolyte, same results were obtained with ±5% deviation. Example 6 Quantification of lead and copper simultaneously in water with two point calibration: Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Tap water sample (5 ml) was taken and KCI salt was added to make it to 0.5M concentration. Sample was placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied to the gold electrode for 60s for the deposition of any lead or copper present in the sample at the electrode. After deposition, another 30s equilibration time was given at the -0.5 V to remove the impurities (impurities for which the oxidation potential is below-0.5V) deposited along with the lead/copper at the surface of the SDRE at the rotation of 1000 rpm. A sweeping potential (in differential pulse mode) was applied in the range of-0.5 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V for lead and 0.3 V ± 0.05 V for copper was obtained similar to the case of alone estimation. In the same solution two different known concentrations of lead and copper was added respectively and experiment was repeated under the same conditions. The peak current vs. lead and copper concentration were plotted separately for preparing the calibration curve similar to the earlier experiments, and concentration of each were found same within the ±5% deviation. When the experiment was repeated in 0.1 M HCI as electrolyte, same results were obtained with ±5% deviation. Example 7 Quantification of lead and copper simultaneously in human blood with two point calibration: Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Human blood (1 ml) was taken and diluted to 5 ml with 0.5 M KCI solution. Sample was shaken for five minutes and placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied to the gold electrode for 60s for the deposition of any lead or copper present in the sample. After deposition, another 30s equilibration time was given at the -0.5 V to remove the impurities (impurities for which the oxidation potential is below -0.5V) deposited along with the lead/copper at the surface of the SDRE at the rotation of 1000 rpm. A sweeping potential (in differential pulse mode) was applied in the range of -0.5 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V for lead and 0.3 V ± 0.05 V for copper was obtained similar to the case of alone estimation. In the same solution two different known concentrations of lead and copper was added respectively and experiment was repeated under the same conditions. The peak current vs. lead and copper concentration were plotted separately for preparing the calibration curve similar to the earlier experiments, and concentration of each were found same within the ±5% deviation. When the experiment was repeated in 0.1 M HCI as electrolyte, same results were obtained with ±5% deviation. The main advantages of the present invention are: 1. A quick, reliable and reproducible estimation of copper and lead in various samples from 5 ppb and above without any interference of other chemicals or ions. 2. A non-hazardous, easy and cost effective technology. 3. A simultaneous estimation of lead and copper in the same sample without any interference. We claim 1. A novel device for the quantification of lead and copper alone or simultaneously in aqueous/ non-aqueous & biological samples, which comprises a three electrode electrochemical cell (1) essentially containing acidic/neutral electrolyte (2), wherein the sensing electrode consists of an inert material cylindrical substrate (3) having a metal/carbon tip (4) embedded onto the side surface (towards bottom) or at the bottom of the said substrate (3), the said electrode embedded substrate being connected by known means to a rotating assembly/prime mover (7) capable of providing a variable speed in the range of 100 to 2000 rpm, the said metal/carbon tip (4) being provided with an electrical connection by means of a connecting wire (10) to a negative terminal of a known variable voltage source (8), the positive terminal of the said variable voltage source (8) being connected to an inert metal auxiliary electrode (5) and a standard reference electrode (6), the said sensing, auxiliary and reference electrodes being connected to a known current measuring device (9) in series to the said variable voltage source (8). 2. A novel device as claimed in claim 1, wherein the electrolyte in concentration range of 0.01 M to 1 M used rarj1 tojuch as HCI, H2SO4, HNO3, HCIO4, other acids, KCI, NaCI or any other soluble salts. 3. A novel device as claimed in claims 1-2, wherein the inert substrate of the rotatable sensing electrode is such as Teflon, glass, plastics. 4. A novel device as claimed in claims 1-3, wherein the sensing tip used is made up of noble metal such as gold, platinum or any noble metal or carbon paste or graphite/glassy carbon. 5. A novel device as claimed in claims 1-4, wherein the metal/carbon sensing tip used is such as circular, elliptical, triangular of the surface area in range of 1mm2 to 10 mm2. 6. A novel device as claimed in claims 1-5, wherein the prime mover used is such as variable speed electric motor. 7. A novel device as claimed in claims 1-6, wherein the auxiliary electrode used is such as platinum, gold, glassy carbon, titanium, stainless steel. 8. A novel device as claimed in claims 1-7, wherein the reference electrode used is such as any commercially available reference electrode. 9. A novel device as claimed in claims 1-8, wherein the variable voltage source used is such as any commercially available potentiostat capable of providing sweeping potential in the range of minus 1.0 V to plus 0.6 V in differential pulse mode. 10. A novel device as claimed in claims 1-9, wherein the current measuring device used is such as analog or digital capable of measuring current in the range of 50 nA to 25 uA. 11. A process for the quantification of lead and copper alone and or simultaneously in aqueous/ non-aqueous & biological samples using the novel device as claimed in claims 1-10, which comprises placing a sample in the electrochemical cell containing acid of concentration in the range of 0.01 N to 1N or neutral electrolyte of suitable soluble salt of concentration in the range of 0.01 M to 1M, applying negative potential to the sensing electrode in the range of minus 1.0 V to minus 0.2 V for a period in the range of 1 second to 5 minutes with the rotation speed in the range of 100 to 2000 rpm, allowing to stand/rest for a period in the range of 1 second to 120 seconds followed by scanning in differential pulse mode from minus 0.6 V to plus 0.6 V at a scanning rate in the range of 1 mV/s to 500 mV/s and measuring the maximum current, estimating lead and copper alone or simultaneously either by the predetermined standard slope or by standard addition method by adding at least two known concentrations of the respective metal ions. 12. A novel device for the quantification of lead and copper in aqueous/ non-aqueous & biological samples substantially as herein described with reference to the examples and drawings accompanying this specification. 13. A process for the quantification of lead and copper in aqueous/ non-aqueous & biological samples substantially as herein described with reference to the examples and drawings accompanying this specification.

Full Text

The present invention relates to a novel device for the quantification of lead and copper in aqueous/ non-aqueous & biological samples and a process thereof.
The present invention particularly relates to a device and method for the quantification of lead and copper alone and or simultaneously in aqueous/ non-aqueous & biological samples by electrochemical technique.
Various toxic metals namely arsenic, lead, mercury, cadmium, zinc, copper and other environmental pollutants have been described as a source of poisoning in humans and animals. The major source of contamination is either through environmental exposure or through dietary intake of contaminated foods and water. Therefore monitoring/quantification of the toxicants in water and biological samples assumes great significance in the health surveillance and safety of humans.
Lead poisoning is an environmental disease, but it is also a disease of lifestyle. Lead is one of the best-studied toxic metal, and as a result we know more about the adverse health effects of lead than virtually any other chemical. The health problem caused by lead has been well documented over a wide range of exposures in every continent. Lead poisoning continues to be one of the most prevalent occupational and environmental illnesses affecting adults. It has also adverse neurological effects, with decrease in IQ and reading ability in young and adults, and affects virtually every system in the body. Blood lead levels as low as 10 jig/dl cause adverse health effects in children.
Copper is an essential metal ion which is also toxic and the detection of copper in food, water as well as in blood is very important. Copper affects the stability of DMA, alters the fluidity of plasma membranes, inhibits various enzymes and diminishes or enhances mitochondria respiration etc. Acute poisoning causes several types of diseases and affects the liver, central nervous system, kidneys and heart and may lead to death. Wilson's disease is the prototypical disease caused by copper toxicosis, affecting first the liver and later the central nervous system, eyes and kidneys. Copper is also an essential element and its deficiency causes ischemic heart disease, references may be made as 1] E. J. Underwood, "Trace elements in Human and Animal Nutrition", 4th Ed., Academic press, NY, USA, 1979 and 2] B. Venugopal, T.D. Luckey, "Metal toxicity in mammals", Vol. 1, Plenum press, NY, USA, 1977).
In view of their significance in human life, it is important to estimate the level of these metal ions in environmental, food and biological samples. However handling of a large number of samples (water, food items, blood, urine etc.) obtained during field surveys and their processing for chemical determinations through sophisticated laboratory equipment and infrastructure facilities are usually time consuming and very costly, requires excessive investments on spares, chemicals and manpower.

Therefore there is an urgent need for the development of quick, reliable, sensitive and cost effective technologies for the quantification of lead and copper for low ppb(^.g/t) level.
The well known and reliable analytical methods for the quantification of lead and copper are atomic emission spectroscopy with inductively coupled plasma excitation, Atomic Absorption Spectrophotometry or Neutron activation analysis or x-ray fluorescence. These methods are having the following drawbacks.
a. Analysis with a highly sophisticated instrument of very high cost and need a well-
trained personnel and equipped laboratory.
b. These techniques are not possible in field conditions
c. Portable device is not possible.
Apart from the above conventional techniques, various electrochemical methods are also reported for the quantification of lead and copper. Reference may be made to 1] A.M. Bond "Modern Polarographic Methods in Analytical Chemistry", Marcel Deckker, New York, 1980; 2] J. Wang, "Striping analysis-Principals, Instrumentation and Applications", VCH, Deerfield Beach ., FL 1985. Above references reported mainly the polarographic techniques for the estimation of the several metal ions including lead and copper. These techniques were having its own limitations because use of Hg hanging electrodes which were itself very toxic and also detection limit was quite low. Latter Stripping Voltammetric techniques were introduced with solid stationary metal/modified electrodes with relatively low detection limit. Reference may be made to 1] Differential pulse anodic stripping voltammetric determination of Pb(ll) with N-P-chlorophenylicinnamohydroxamic acid modified carbon paste electrode, Degefa, Tesfaye Hailu, Chandravanshi et al, Electroanalysis, 119170, 1305, 1999; 2] Determination of lead(ll) and cadmium (II) in hard and soft wheat by derivative potentiometric stripping analysis, F.Lo Coco, P. Monotti, V. Fiecchi and L. Ceccon, Analytica Chimica Acta, 409, 1-2, 93, 2000; 3] Anodic and Cathodic stripping voltammetry in the simultaneous determination of toxic metals in environmental samples, Clinio Lacatelli, Electroanalysis, 9, 13, 1997; 4] Lead Analyzer, US Patent Nos. 4,201,646, 4,374,041 and 4,090,926, ESA Analytical Ltd., 14 Cromwell Mews, St. Road, Stives, Huntingdon, Cambridgeshire PE 17 4HJ England However, none is capable to detect lead and copper ions at low ppb level (ug/l) with good reproducibility, mainly because of the limitations of the electrodes and electrolytes. All the above techniques used a stationary sensing electrodes in acidic electrolyte.
The major Drawbacks associated with the earlier works are:
1) Stationary (non-rotating) working electrode is used, which is unable to overcome the problems of hydrogen evolution at negative potential and also poisoning of the electrodes.

2) High acid concentration is used mainly for biological and complex matrix samples, this
causes the large gas evolution during the metal deposition as well and may cause
damage to the electrodes and device parts on long use.
3) Some times the metal ions are not reducible in the matrix and therefore it requires more
negative potential and special type of working electrodes, which gives irreproducible
results with stationary electrodes.
4) It is hazardous to use high acid concentration, which is common in the conventional
methods.
5) Precise and reproducible estimations of higher concentration of lead/copper may not
possible.
In our co-pending patent application no. 774/Del/2000, we have described and claimed a novel device for the quantification of arsenic in water and a process thereof, wherein the novel device for the quantification of arsenic is based on the anodic stripping voltammetry. The device consisted a three electrodes configuration of a novel side tip gold working electrode along with platinum counter/auxiliary electrode and conventional reference electrode. The novel gold electrode of rotating type and of specific construction was used for the low ppb (|j.g/l) estimation of arsenic in acidified waster sample. The gold electrode was constructed by embedding the known size of gold metal circular/triangular/elliptical thin plate at the side surface (towards bottom) of the inert material hollow cylinder and taking a wire connection from the embedded gold plate in order to make the electrical contact. The above gold electrode was attached to a rotating assembly and all the three electrodes were fitted in an electrochemical cell having typical composition of electrolyte such as 4% (v/v) mineral acid and low ppb of copper ions for the quantification of arsenic as low as 5 ppb.
The main object of the present invention is to provide a device for the quantification of lead and copper alone and simultaneously in various samples such as aqueous/ non-aqueous & biological samples.
Another object of the present invention is to provide a process for the quantification of lead and copper, using the novel device of the present invention.
Yet another object of the present invention is to provide a novel electrochemical device and process for the quantification of lead and copper.
Still another object of the present invention is to provide a simple, easy and safe technique (non-hazardous) for the quantification of lead and copper.

Another object of the present invention is to provide a sensing electrode for lead and copper, such as platinum, gold or any noble metal or carbon paste or graphite/glassy carbon tip embedded at the side of a hollow cylinder of inert material.
Yet another object of the present invention is to develop a portable device useful for sensing lead and copper alone and simultaneously in various samples.
In the present invention the basic principle of anodic stripping voltammetry with three electrodes configuration has been used to provide a novel electrochemical device and process for the quantification of low ppb (^.g/l) level of lead and copper in aqueous/ non-aqueous & biological samples. In the present invention the three electrodes configuration essentially consists of a metal or carbon tipped sensing electrode, an inert metal counter/auxiliary electrode and a conventional reference electrode. The inventive step of the novel device of the present invention resides in the rotating sensing electrode constructed by embedding the known size of noble metal or carbon paste or graphite/glassy carbon circular/triangular/elliptical thin plate or rod at the side surface or bottom of the hollow cylinder made up of inert material and aqueous or non-aqueous electrolytic solution, which is either acidic 0.01N to 1N or neutral electrolyte of soluble salts 0.01M to 1 M. A wire connection from the embedded metal or carbon plate/rod is taken in order to make the electrical contact. The electrode top is equipped with a screw type connection to attach with a rotator assembly/ prime mover. The above sensing electrode is attached to the rotating assembly and all the three electrodes are fitted in an electrochemical cell having acidic or neutral electrolyte for the quantification of lead and copper as low as 5 ppb. In the present invention the sample may be either aqueous or non-aqueous and verity of the samples may be used like natural water, petroleum, biological etc, while in co-pending patent application no. 774/Del/2000, it was limited to aqueous (water) samples only. In the present invention the nature of the electrolyte may be either acidic or neutral, while in our pending patent application no, 774/Del/2000, it must be acidic. Also in the present invention in the sensing electrode the metal or carbon tip may be present either at the side or at the bottom of the rotating electrode body, while in our pending patent application no. 774/Del/2000, essentially it must be present at the side of the electrode body. Therefore, the novelty of the present invention lies in the device capable of quantification of lead and copper alone and simultaneously in various samples at low ppb levels. The non-obvious inventive step reside in the metal/carbon tipped rotating sensing electrode.

In figure-1 of the drawings accompanying this specification the schematic diagram of the novel device of the present invention is shown. Wherein the various parts are, electrolytic container (1), electrolyte (2), inert material cylindrical substrate (3), sensing tip (4), inert auxiliary electrode (5), reference electrode (6), rotating assembly/prime mover (7), variable voltage source (8), current measuring device (9) and connecting wires (10).
Accordingly, the present invention provides a novel device for the quantification of lead and copper alone or simultaneously in aqueous/ non-aqueous & biological samples, which comprises a three electrode electrochemical cell (1) essentially containing acidic/neutral electrolyte (2), wherein the sensing electrode consist of an inert material cylindrical substrate (3) having a metal/carbon tip (4) embedded onto the side surface (towards bottom) or at the bottom of the said substrate (3), the said electrode embedded substrate being connected by known means to a rotating assembly/prime mover (7) capable of providing a variable speed in the range of 100 to 2000 rpm, the said metal/carbon tip (4) being provided with an electrical connection by means of a connecting wire (10) to a negative terminal of a known variable voltage source (8), the positive terminal of the said variable voltage source (8) being connected to an inert auxiliary electrode (5) and a standard reference electrode (6), the said sensing, auxiliary and reference electrodes being connected to a known current measuring device (9) in series to the said variable voltage source (8).
In an embodiment of the present invention the electrolyte in concentration range of 0.01 M to 1 M used may be such as HCI, H2SO4, HNO3, HCIO4, other acids, KCI, NaCI or any other soluble salts.
In still another embodiment of the present invention the inert substrate of the rotatable sensing electrode may be such as Teflon, glass, plastics.
In yet another embodiment of the present invention the metal/carbon sensing tip used may be made up of nobel metal such as gold or platinum or carbon paste or graphite/glassy carbon.
In yet another embodiment of the present invention the metal/carbon sensing tip used may be such as circular, elliptical, triangular of the surface area in range of 1mm2 to 10 mm2.
In another embodiment of the present invention the prime mover used may be such as variable speed electric motor.

In yet another embodiment of the present invention the auxiliary electrode used may be such as platinum, gold, glassy carbon, titanium, stainless steel.
In still another embodiment of the present invention the reference electrode used may be such as any commercially available reference electrode.
In another embodiment of the present invention the variable voltage source used may be such as any commercially available potentiostat capable of providing sweeping potential in the range of minus 1.0 V to plus 0.6 V in differential pulse mode.
In still another embodiment of the present invention the current measuring device used may be such as analog or digital capable of measuring current in the range of 50 nA to 25 ^A.
Accordingly, the present invention provides a process for the quantification of lead and copper alone and or simultaneously in aqueous/ non-aqueous & biological samples using the device as described above, which comprises placing a sample in the electrochemical cell containing acid of concentration in the range of 0.01 N to 1N or neutral electrolyte of suitable soluble salt of concentration in the range of 0.01M to 1M, applying negative potential to the sensing electrode in the range of minus 1.0 V to minus 0.2 V for a period in the range of 1 second to 5 minutes with the rotation speed in the range of 100 to 2000 rpm, allowing to stand/rest for a period in the range of 1 second to 120 seconds followed by scanning in differential pulse mode from minus 0.6 V to plus 0.6 V at a scanning rate in the range of 1 mV/s to 500 mV/s and measuring the maximum current, estimating lead and copper alone or simultaneously either by the predetermined standard slope or by standard addition method by adding atieast two known concentrations of the respective metal ions.
In the present invention the basic principle for the quantification of lead and copper ions are based on the anodic stripping voltammetry. This method can be classified in two steps, first is the quantitative deposition of the metal ions over the electrode in form of elemental metal and second is the oxidation of deposited metal back to the solution from the electrode. Lead or copper ions are commonly present in the sample in +2 oxidation states (Pb"1"2, Cu+2). The deposition of the Pb^/Cu*2 in its elemental state can be done either in neutral solution using some salt solution as an electrolyte or some specific pH solution using acids or buffer. The complex mediums specially biological fluids are very complicated and creates a lot of interference for the detection of lead/copper ions. Therefore, for complex matrix, an acidic electrolyte is preferred to overcome the matrix effect. While we have also demonstrated a simple salt electrolyte for estimation of lead/copper for the complex matrix viz. blood. The estimation can be divided in two steps:

In the first step of the process i.e. deposition, a negative potential is applied for a certain time period (depending on matrix and concentration) to the sensing electrode. The strength of the electrolyte can be optimized according to the need. During the deposition of the metal ions in the acidic medium, evolution of the Cb gas and HZ gas may take place at auxiliary and sensing electrode respectively and both the gases evolve at the electrodes in the form of fine bubbles. In the case of some other electrolytes, some gases may also evolve during the deposition. These gases interfere during the deposition of the metal ions, therefore, must be removed immediately after their formation. In order to get the least interference by the gas bubbles formed during the deposition, the sensing electrode is used with the rotation arrangement. Side tip electrode is used with high speed rotation in order to remove the gases from the electrode.
In the second step of the process, the deposited metals are allowed to oxidise by applying a sweeping potential in differential pulse mode with low scan rate followed by a equilibrium potential (the sweeping potential range must cover the oxdidation potential of the deposited metal). The equilibration time is important for the removal of the impurities, which may be deposited over the electrode along with the target metal ions. The estimation of the oxidation current peak gives an estimation of the corresponding metal ions in the solution, with the help of a calibration plot. The oxidation peak current vs. lead or copper concentration plot (calibration curve) showed a linear relation. The lead and copper alone and simuitaneously estimated up to 5 ppb and above in various aqueous and non-aqueous samples including biological samples.
The present invention provides a novel device useful for quantification of (ead and copper electrochemicaily alone or simultaneously, which comprises a special type of sensing electrode along with a metal auxiliary and a reference electrode placed in a suitable container for holding the fluid in which metal ion is to be quantified. All the three electrodes being connected to a voltage source which can apply the potential to the sensing electrode in differential pulse mode. In the present invention the sample may be either aqueous or non-aqueous. A verity of the samples may be used like natural water, petroleum, biological materials etc, while in co-pending patent application no. 774/Del/2000, arsenic detection is limited to aqueous (water) samples only. Moreover the nature of the electrolyte may be either acidic or neutral, while in our pending patent application no. 774/Del/2000, it must be acidic. The sensing electrode is used here either may be metal or carbon tip at the side or at the bottom of the rotating electrode body, while in our pending patent application no. 774/Del/2000, it must be present at the side of the electrode body.
The invention is further illustrated with the help of the following examples and therefore should not be construed to limit the scope of the present invention in any manner whatsoever.
Example 1
Quantification of lead in water
Gold bottom disk rotating electrode (BDRE) (2 mm disk diameter) situated at the bottom of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer.
Tap water sample (5 ml) was taken and KCI salt was added to make it to 0.5M concentration. Sample was placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied to the gold electrode for 60s for the deposition of any lead present in the sample at the electrode. After deposition, another 30s equilibration time was given at the -0.5 V to remove the impurities (impurities for which the oxidation potential is below -0.5V) deposited along with the lead at the surface of the BDRE at the rotation of 1000 rpm. A sweeping potential (in differential pulse mode) was applied in the range of -0.5 V to 0.0 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V was obtained as shown in curve A of Fig. 2(a) of the drawing accompanying this specification. In the same solution four different known concentrations of lead were added to get the total concentration of 10 ppb, 20 ppb, 30 ppb and 40 ppb and experiment was repeated under the same conditions. The lead oxidation peak for each addition was recorded as shown as curves B, C, D and E of Fig. 2(a) of the drawing. The peak current vs. lead concentration was plotted for preparing the calibration curve as shown in Fig. 2(b) of the drawing. A linear nernstian plot was observed in the range of 10 ppb to 40 ppb of lead as shown in Fig. 2(b) of the drawing. The tap water sample contains 21.47 ppb lead as estimated with the help of calibration curve. When the experiment was repeated in 0.1 M HCI as electrolyte, same results were obtained with ±5% deviation.
Example 2 Quantification of lead in Rat's blood with two point calibration:
Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device

capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer. Rat's blood (0.5 ml) was taken and diluted to 5 ml with 0.1N HCI. Sample was shaken for five minutes and placed in the cell. A deposition potential of-0.6 V vs. Ag/AgCI was applied to the gold electrode for 60s to deposit lead if present in the sample at the surface of SDRE at the rotation of 1000 rpm. After deposition, another 30 s equilibration time was given at the -0.4 V to remove the impurities (impurities for which the oxidation potential is below -0.4V) deposited along with the lead. A sweeping potential (in differential pulse mode) was applied in the range of -0.4 V to 0.0 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V was obtained as shown in curve A of Fig. 3(a) of the drawing accompanying this specification. In the same solution two different known concentrations of lead were added to get the total concentration of 10 ppb and 20 ppb and experiment was repeated under the same conditions. The lead oxidation peak for each addition was recorded as shown as curves B and C of Fig. 3(a) of the drawing. The peak current vs. lead concentration was plotted for preparing the calibration curve as shown in Fig. 3(b) of the drawing. A linear nernstian plot was observed as shown in Fig. 3(b) of the drawing. The blood sample contains 23.58 x 10 = 235.8 ppb (x 10 because of dilution) or 23.58 ^.g/dl lead as estimated with the help of calibration curve. When the experiment was repeated in 0.5 M KCI as electrolyte, same results were obtained with ±5% deviation.
Example 3
Quantification of lead in human blood with two point calibration:
Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer.
Human blood (1 ml) was taken and diluted to 5 ml with 0.5 M KCI solution. Sample was shaken for five minutes and placed in the cell. A deposition potential of -0.6 V vs. Ag/AgCI was applied to the gold electrode for 60 s to deposit lead if present in the sample at the surface of SDRE. After deposition, another 30 s equilibration time was given at the -0.4 V to remove the impurities (impurities for which the oxidation potential is below -0.4V) deposited
along with the lead. A sweeping potential (in differential pulse mode) was applied in the range of -0.4 V to 0.0 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V was obtained as shown in curve A of Fig. 4(a) of the drawing accompanying this specification. In the same solution two different known concentrations of lead were added to get the total concentration of 20 ppb and 40 ppb and experiment was repeated under the same conditions. The lead oxidation peak for each addition was recorded as shown as curves B and C of Fig. 4(a) of the drawing. The peak current vs. lead concentration was plotted for preparing the calibration curve as shown in Fig. 4(b) of the drawing. A linear nernstian plot was observed as shown in Fig. 4(b) of the drawing. The blood sample contains 24.26 x 5 = 121.30 ppb (x 10 because of the sample was diluted 5 times) or 12.13 ug/dl lead as estimated with the help of calibration curve. When the experiment was repeated in 0.1 N HCI as electrolyte, same results were obtained with ±5% deviation.
Example 4
Quantification of copper in water
Gold bottom disk rotating electrode (BDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer.
Tap water (5 ml) was taken and KCI salt was added to make it to 0.5M concentration. Sample was placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied for 60 s to deposit copper if present in the sample at the surface of BDRE at the rotation of 1000 rpm. After deposition, another 30 s equilibration time was given at the 0.0 V to remove the impurities (impurities for which the oxidation potential is below 0.0V) deposited along with the copper. A sweeping potential (in differential pulse mode) was applied in the range of 0.0 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at 0.3 V ± 0.05 V was obtained as shown in curve A of Fig. 5(a) of the drawing accompanying this specification. In the same solution four different known concentrations of copper were added to get the total concentration of 10 ppb, 20 ppb, 40 ppb and 50 ppb and experiment was repeated under similar condition. The
copper oxidation peak for each addition was recorded as shown as curves B, C, D and E of
Fig. 5(a) of the drawing. The peak current vs. copper concentration was plotted for preparing the calibration curve as shown in Fig. 5(b) of the drawing. A linear nernstian plot was observed in the range of 10 ppb to 50 ppb of copper as shown in Fig. 5(b) of the drawing. The unknown sample was containing 5.17 ppb copper as estimated with the help of calibration curve. When the experiment was repeated in 0.1 N HCI as electrolyte, same results were obtained with ±5% deviation.
Example 5 Quantification of copper in blood with two point calibration:
Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 |jA range which was interfaced with a computer.
Human blood sample (1 ml) was taken and diluted to 5 ml with 0.5 M KCI solution. Sample was shaken for five minutes and placed in the cell. A deposition potential of -0.6 V vs Ag/AgCI was applied to the gold electrode for 60 s to deposit copper if present in the sample at the surface of SDRE at the rotation of 1000 rpm. After deposition, another 30 s equilibration time was given at the 0.0 V to remove the impurities (impurities for which the oxidation potential is below 0.0V) deposited along with the copper. A sweeping potential (in differential pulse mode) was applied in the range of 0.0 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at 0.3 V ± 0.05 V was obtained as shown in curve A of Fig. 6(a) of the drawing accompanying this specification. In the same solution two different known concentrations of lead were added to get the total concentration of 100 ppb and 200 ppb and experiment was repeated under the same conditions. The copper oxidation peak for each addition was recorded as shown as curves B and C of Fig. 6(a) of the drawing. The peak current vs. copper concentration was plotted for preparing the calibration curve as shown in Fig. 6(b) of the drawing. A linear nernstian plot was observed as shown in Fig. 6(b) of the drawing. The blood sample contains 200.0 x 5 = 1.0 ppm (x 10 because of the sample was diluted 5 times) copper as estimated with the help of calibration curve. When the experiment was repeated in 0.1 N HCI as electrolyte, same results were obtained with ±5% deviation.
Example 6 Quantification of lead and copper simultaneously in water with two point calibration:
Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer.
Tap water sample (5 ml) was taken and KCI salt was added to make it to 0.5M concentration. Sample was placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied to the gold electrode for 60s for the deposition of any lead or copper present in the sample at the electrode. After deposition, another 30s equilibration time was given at the -0.5 V to remove the impurities (impurities for which the oxidation potential is below-0.5V) deposited along with the lead/copper at the surface of the SDRE at the rotation of 1000 rpm. A sweeping potential (in differential pulse mode) was applied in the range of-0.5 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V for lead and 0.3 V ± 0.05 V for copper was obtained similar to the case of alone estimation. In the same solution two different known concentrations of lead and copper was added respectively and experiment was repeated under the same conditions. The peak current vs. lead and copper concentration were plotted separately for preparing the calibration curve similar to the earlier experiments, and concentration of each were found same within the ±5% deviation. When the experiment was repeated in 0.1 M HCI as electrolyte, same results were obtained with ±5% deviation.
Example 7
Quantification of lead and copper simultaneously in human blood with two point calibration:
Gold side disk rotating electrode (SDRE) (2 mm disk diameter) situated on the side of a Teflon rod (4mm diameter and 4 cm in length) having screw type connection was used along with a platinum auxiliary and Ag/AgCI reference electrodes. An electrochemical cell of 5 ml sample capacity equipped with three electrodes along with sensing electrode rotating assembly was used. All the three electrodes were connected with a Potentiostat, device
capable of applying sweeping potential in differential pulse mode in the range of -1.0 V to + 0.6 V and measuring current in 50 nA to 25 uA range which was interfaced with a computer.
Human blood (1 ml) was taken and diluted to 5 ml with 0.5 M KCI solution. Sample was shaken for five minutes and placed in the cell and a -0.6 V potential vs. Ag/AgCI was applied to the gold electrode for 60s for the deposition of any lead or copper present in the sample. After deposition, another 30s equilibration time was given at the -0.5 V to remove the impurities (impurities for which the oxidation potential is below -0.5V) deposited along with the lead/copper at the surface of the SDRE at the rotation of 1000 rpm. A sweeping potential (in differential pulse mode) was applied in the range of -0.5 V to 0.5 V vs. Ag/AgCI with 20 mV/s scan rate to determine the oxidation current. Current vs. potential plot was recorded and peak at -0.2 V ± 0.05 V for lead and 0.3 V ± 0.05 V for copper was obtained similar to the case of alone estimation. In the same solution two different known concentrations of lead and copper was added respectively and experiment was repeated under the same conditions. The peak current vs. lead and copper concentration were plotted separately for preparing the calibration curve similar to the earlier experiments, and concentration of each were found same within the ±5% deviation. When the experiment was repeated in 0.1 M HCI as electrolyte, same results were obtained with ±5% deviation.
The main advantages of the present invention are:
1. A quick, reliable and reproducible estimation of copper and lead in various samples from
5 ppb and above without any interference of other chemicals or ions.
2. A non-hazardous, easy and cost effective technology.
3. A simultaneous estimation of lead and copper in the same sample without any
interference.

We claim
1. A novel device for the quantification of lead and copper alone or simultaneously in aqueous/ non-aqueous & biological samples, which comprises a three electrode electrochemical cell (1) essentially containing acidic/neutral electrolyte (2), wherein the sensing electrode consists of an inert material cylindrical substrate (3) having a metal/carbon tip (4) embedded onto the side surface (towards bottom) or at the bottom of the said substrate (3), the said electrode embedded substrate being connected by known means to a rotating assembly/prime mover (7) capable of providing a variable speed in the range of 100 to 2000 rpm, the said metal/carbon tip (4) being provided with an electrical connection by means of a connecting wire (10) to a negative terminal of a known variable voltage source (8), the positive terminal of the said variable voltage source (8) being connected to an inert metal auxiliary electrode (5) and a standard reference electrode (6), the said sensing, auxiliary and reference electrodes being connected to a known current measuring device (9) in series to the said variable voltage source (8).
2. A novel device as claimed in claim 1, wherein the electrolyte in concentration range of 0.01 M to 1 M used rarj1 tojuch as HCI, H2SO4, HNO3, HCIO4, other acids, KCI, NaCI or any other soluble salts.
3. A novel device as claimed in claims 1-2, wherein the inert substrate of the rotatable
sensing electrode is such as Teflon, glass, plastics.
4. A novel device as claimed in claims 1-3, wherein the sensing tip used is made up of
noble metal such as gold, platinum or any noble metal or carbon paste or graphite/glassy
carbon.
5. A novel device as claimed in claims 1-4, wherein the metal/carbon sensing tip used is
such as circular, elliptical, triangular of the surface area in range of 1mm2 to 10 mm2.
6. A novel device as claimed in claims 1-5, wherein the prime mover used is such as
variable speed electric motor.
7. A novel device as claimed in claims 1-6, wherein the auxiliary electrode used is such as
platinum, gold, glassy carbon, titanium, stainless steel.

8. A novel device as claimed in claims 1-7, wherein the reference electrode used is such as
any commercially available reference electrode.
9. A novel device as claimed in claims 1-8, wherein the variable voltage source used is
such as any commercially available potentiostat capable of providing sweeping potential
in the range of minus 1.0 V to plus 0.6 V in differential pulse mode.
10. A novel device as claimed in claims 1-9, wherein the current measuring device used is
such as analog or digital capable of measuring current in the range of 50 nA to 25 uA.
11. A process for the quantification of lead and copper alone and or simultaneously in
aqueous/ non-aqueous & biological samples using the novel device as claimed in claims
1-10, which comprises placing a sample in the electrochemical cell containing acid of
concentration in the range of 0.01 N to 1N or neutral electrolyte of suitable soluble salt of
concentration in the range of 0.01 M to 1M, applying negative potential to the sensing
electrode in the range of minus 1.0 V to minus 0.2 V for a period in the range of 1 second
to 5 minutes with the rotation speed in the range of 100 to 2000 rpm, allowing to
stand/rest for a period in the range of 1 second to 120 seconds followed by scanning in
differential pulse mode from minus 0.6 V to plus 0.6 V at a scanning rate in the range of
1 mV/s to 500 mV/s and measuring the maximum current, estimating lead and copper
alone or simultaneously either by the predetermined standard slope or by standard addition
method by adding at least two known concentrations of the respective metal ions.

12. A novel device for the quantification of lead and copper in aqueous/ non-aqueous &
biological samples substantially as herein described with reference to the examples and
drawings accompanying this specification.
13. A process for the quantification of lead and copper in aqueous/ non-aqueous &
biological samples substantially as herein described with reference to the examples and
drawings accompanying this specification.

Documents:

13-del-2002-abstract.pdf

13-del-2002-claims.pdf

13-del-2002-correspondence-others.pdf

13-del-2002-correspondence-po.pdf

13-del-2002-description (complete).pdf

13-del-2002-drawings.pdf

13-del-2002-form-1.pdf

13-del-2002-form-18.pdf

13-del-2002-form-2.pdf

13-del-2002-form-3.pdf

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Patent Number

217509

Indian Patent Application Number

13/DEL/2002

PG Journal Number

15/2008

Publication Date

11-Apr-2008

Grant Date

26-Mar-2008

Date of Filing

10-Jan-2002

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	RAJIV PRAKASH	INDUSTRIAL TOXICOLOGY RESEARCH CENTRE,M.G.MARG, LUCKNOW-226001,INDIA.
2	RAMESH CHANDRA SRIVASTAVA	INDUSTRIAL TOXICOLOGY RESEARCH CENTRE,M.G.MARG, LUCKNOW-226001,INDIA
3	PRAHLAD KISHORE SETH	INDUSTRIAL TOXICOLOGY RESEARCH CENTRE,M.G.MARG, LUCKNOW-226001,INDIA

PCT International Classification Number

G01N7/08

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA