Assembly and Application of New Calcium Ion –Selective Electrode for Determination of Calcium Ions .

: A new calcium ion-selective electrode was prepared from calcium glyxolate complex doped with PVC and plasticizer .The electrode was characterized by its stable Nernstian response 29.97mV/decade. with a linear range of ( 10 -5 - 10-1 ) M , detection limit of 10-5 M and response time of ( 20 - 60 sec.) within the electrode lifetime which lasted four months. Suitable pH range for electrode response was (5 - 9) and little temperature effect was found on the efficiency of the electrode. High selectivity of the electrode to calcium ions in the presence of many cation was found which clarified from the lower potentiometric selectivity coefficients . Most of the anions are severely interfered as a result of precipitation or stable complex formations with calcium ions and these interferents were exploited in the determination of the anions or the calcium ions. Encouraging and promising results were obtained using this new calcium ISE in the determination of calcium in drinking water and human blood serum coincidental with the spectrophotometric methods.


Introduction :
The first calcium ion-selective electrode was fabricated by Ross (1) in 1967 for clinical purposes .The assembly of the electrode was based on liquid ion-exchanger and neutral carrier (2) .The best development of calcium ISES was the incorporation of ion exchanger or neutral carrier in PVC matrix (3,4) .These PVC matrices membrane ISEs were simply designed , considerably economic in sensor materials and offer longer life times which led to more and wide applications (5) especially in clinical analysis (6)(7)(8).
Recently , a new calcium ISEs was fabricated from calcium complex with zincon in PVC instead of ion-exchangers or neutral carriers (9) .The electrode found wide applications in the determination of calcium ions using different techniques .After the success of using calcium complex with zincon doped in PVC to prepare an electroactive membrane for calcium determination , the present work is concentrated on the homemade of new calcium ionselective electrode from the complex with Glyoxal bis ( 2 -hydroxyanil , GBHA) doped in PVC with certain plasticiser and solvent .

Experimental:
All chemicals used in this investigation are of analytical-reagent grade .Double distilled water was used for the preparation of solutions.Most measurements were carried out at room temperature .Preparation of calcium glyoxalate complex (10) : 100ml of 0.01M of alcoholic solution of glyoxal (GBHA) was added to 200ml of 0.04 M of alcoholic solution of NaOH with continuous stirring at room temp.until pale yellow colour was observed.The whole mixture was gradually added with stirring to 300 ml of 0.01 M CaCl 2 in a waterbath at 60 ْ◌ C .The mixture got red colour which was an indication of complex formation.The mixture was left at this temperature until smooth red precipitate occurred , then kept at 5 ْ◌ C for two hours until complete coagulation of the precipitate.The precipitate was filtered , washed and dried at 115 ْ◌ C and then reserved in a desiccator containing silica gel .

Identification of Ca-GBHA Complex: IR Identification :
IR spectra of GBHA and Ca-GBHA complex were measured in KBr discs using Philips PU 9706 IR spectrophometer in the range of ( 4000-600 ) cm-1 on the same chart .

UV-ViS analysis of glyoxal and its complex with Ca :
Small amounts of GBHA and Ca-GBHA complex were dissolved in ethanol to form 0.01M for each and the UV-Vis spectra were taken using 6405 JENWAY UV-Vis spectrophotometer in a range of (200-800) nm against ethanol as a blank.Absorption bands spectrum were obtained for GBHA at certain wavelength which were shifted into longer wavelengths for Ca-GBHA complex.Determination of the percentage of calcium in Ca-GBHA complex : 0.02 g of Ca-GBHA was ashed in a Muffle furnace at 500 ْ◌C for one hour.The residue was dissolved in sufficient volume of HNO3 (1:1) and completed to 50 ml with distilled water.A flame emission photometer (Jenway, UK) was calibrated by series calcium standard solutions and the concentration of calcium in the Ca-GBHA complex was picked up from the calibration curve.

Preparation of the master membranes of Ca-ISEs:
The master membrane of the Ion-Selective electrode was prepared according to the technique of Craggs et al (11), which involved mixing 0.04g of Ca-GBHA with 0.36g of Tributylphosphate (as plasticiser) with stirring until homogeneous mixture was observed.A mixture of 0.17g of PVC (BreonIII EP , BP Chemicals International Ltd.) was dissolved in 7 ml of THF and added to the above mixture with continuous stirring .The whole mixture was transferred into a cylindrical glass casting fixed on a smooth glass plate.The glass cylinder was covered with filter paper pad and held by a suitable weights and left for 72 hours for gradual evaporation of the solvent.The membrane was prised out from inside edges of the ring , wrapped with tissue paper and stored in a plastic container and kept in a refrigerator at 4 ْ◌C.

Assembly of Ca-ISEs:
A disc of 8 mm diameter and 0.4 mm thickness from each master membrane was cut by suitable porer and held to polythane tube by adhesive of PVC in THF .The polythane tube was filled with a mixture of 0.1M CaCl2 and 0.1 M KCl saturated with AgCl to form the internal reference solution.Ag/AgCl electrode was inserted inside the internal solution and connected to a convenient cable to the ion-meter .The electrode was put in 0.1 M calcium ion solution for 24 hours before application .

Calibrations of the assembled Ca-ISE:
A series of standard solutions ranged from 10-6 -10 -1M were prepared from calcium chloride di-hydrated salt.
The calibrations were performed for all the prepared electrodes and the recorded potentials were plot versus log [ Ca2+] and from the calibration curves many important informations were obtained like slopes, linear ranges , detection limits which illustrate the efficiencies of the electrodes.Calibrations were also accomplished at different periods to find out the active lifetimes of the electrodes.

pH effect on the responses of the electrodes:
Tris-Buffer (Tris (Hydroxy methylamino methane ) was used to prepare buffer solutions with pHs in the range of 2.3 -11.7 applying hydrochloric acid (10% and 25%) to lower pH and concentrated ammonia to raise it.The potentials of the prepared solutions were measured by Ca-ISEs relative to saturated calomel reference electrode and plotted VS. pH values.

Effect of temp. on the response of Ca-ISEs No. 1 :
The effect of temperature on the response of Ca-ISE number (1) was studied by potential measurements for a series of calcium standard solutions at different temperatures ( 25 , 35 and 45 ْ◌ C) .The slopes determined by Ca-ISE No. (1) were not mostly affected by temperature changes.But the electrode membrane were intensively affected by high temperatures ( above 60 ْ◌C) at which the membranes decomposed and solubility of the components especially the Ca-GBHA complex and the plasticizer (12) .

Interference studies of cations on calcium ion determination by Ca-ISE No. ( 1 ) :
Mixed solution method( 13) was used to study the effect of a number of cations on the response and selectivity of the new Ca-ISE number (1) .The method involved constant activity of the interferent ion while variation the calcium ion activity in the range of 10-5 -10-1 M. The following ions ; Na+ , K + NH4 + , Ag + , Ba 2+ , Mg 2+ , Ni 2+ , Co 2+ , Zn 2+ , Al 3+ , Cr3+ and Fe 3+ were examined as interferents in the determination of calcium ions put and selectivity coefficient ,K,Ca,M .The potentiometric selectivity coefficients were calculated from the following relation : aCa2+ = K Pot,Ca,M x aM Zca/ZM Where aCa2+ = the activity of calcium ions which were practically determined from the calibration curve , aM = the activity of the interferent cation and ZCa and ZM are charges of Ca 2+ and interferent cation respectively .

Applications of the Ca-ISE No.(1) : Determination of Calcium in drinking water:
Direct potentiometric method , calibration curve method and potentiometric titration method were applied for the determination of calcium ion in drinking water using the new Ca-ISE No.(1) relative to reference calomel electrode.
The classical titration method of calcium ions with EDTA in the presence of murexide metad -ion indicator was used to determine calcium ions for comparison at pH = 12 (1M NaOH ) .
Flame atomic emission method was also used to determine calcium ions in drinking water for comparison which was considered the reference method.

Potentiometric titration determination of some anions with calcium ions:
Since most of anions are severely interfered in the determination of calcium ions as a result of precipitations and complex formations , this technique was utilized in the determination of oxalate , sulphate , carbonate , phosphate and fluoride ions , using different concentrations .A clear , suitable and practical potencial shifts were observed at equivalence points.Chloride and Nitrate ions don't interfere , thus , calcium chloride and calcium nitrate solutions were used as standards for calibration of Ca-ISEs.

Determination of calciumin human blood serum:
Three samples each of 5 ml of blood were taken from three persons and transferred into centrifuge tubes and left for half an hour at room temperature .The tubes were transferred into a centrifuge and switched on in a speed of 300 rpm for ten minutes to get blood serum.
The blood serum was transferred into clean and dry tubes and firmly tied up and kept at -20 ْ◌C until biochemical tests.The calibration curve method was set up to determine calcium concentration in blood serum using the new Ca-ISE No (1).
A spectrophotometric method( 14) was also used for comparison depending on the complex formation of ortho-cresolphthaline with calcium at pH 10.7 and at wavelength of 570 nm.

Results and Discussions :
(1) Identifcation of the glyoxal and its complex with calcium ion: IR Spectra of glyoxal and its complex with calcium ions : Table (2)depicts IR spectra of the functional groups of glyoxal and its calcium complex.It is clear from the table the absorption band at 1604 cm-1 of C=N group in glyoxal has shifted into lower wave number 1590 cm-1 in the complex due to participations of the imine group C=N in the coordination sites with calcium ion.
The disappearance of the absorption band at 3379 cm-1 due to OH phenol groups in the complex is an indication of engagements of both OH groups in the formation of the complex , after displacement of acidic hydrogen ion(15).The appearance of broad peak at 3417 cm-1 in the complex represents the absorption band of water molecules only in the complex which offers it octahedral form (16). Table (2) : IR spectra of functional groups in glyoxal and its complex with calcium.

Group
Glyoxal cm -1 Glyoxal complex cm - Three absorption bands were obtained in glyoxal at 216 , 298 and 465 nm which were shifted into longer wavelengths in the complex at 253 , 320 and 510 nm table ( 3) .
These shiftings were due to the engagement of imine and hydroxyl groups in coordination with calcium ions (17) .Therefore , the opportunities of formation of inter and intra molecular hydrogen bondings in glyoxal were lowered or diminished in the complex which assist the transferring of electrons at lower energies and higher wavelengths which is another indication of complex formation .

Table ( 3) UV-Vis absorption bands of glyoxal and its calcium complex in ethamal .
Glyoxal Flame atomic emission technique was applied to determine calcium in calcium glyoxalate complex using calibration curve method.The percentage was found to be 12.63% with a standard deviation of  0.22 .The theoretical percentage of calcium was calculated and found 12.73% which insured the combination of two water molecules with complex to form the octahedral structure ( 16) of the complex in order to approach this percentage .Fig( 1) exhibits the proposed structures of glyoxal and its complex with calcium.
(2) :Calibration of the four calcium Ion-selective electrodes: The electrodes were calibrated using a series of standard calcium solutions in concentrations ranged ( 10-6 -10-1 ) M , relative to calomel reference electrode.Table ( 4) includes information obtained from the calibration curves of these electrodes .The linear range of the response of the electrodes were ( 10-5 -10 -1 ) M with a detection limit of 10-5 M and response time in the range of ( 20-60 ) sec.(Fig .2) .
The response time became longer for dilute solutions and multi use of the electrodes (18) .The calibrations of the electrodes were carried out at different periods .The electrode lifetime approached its end when its slope became very low and far from Nernstian and its linear range has been shortened to ( 10-3 -10 -1 ) M.
The response mechanism of the electrodes depend on the ionic conductivity of calcium ions within the membrane and their exchange with the corresponding calcium ions in the external solutions that generates a potential directly proportional to the log activity or concentration of the calcium ions (19).
It is clear from table(4) that the Ca-GHBA , Ca-ISE No.( 1) is superior to the electrode No.2 assembled from Ca Bis -TMBPP ion exchanger from the higher slopes values and longer lifetime .It was anticipated that improvement may be attained to ISE No.(1) by addition either the ion exchanger or Iodine , but shorter lifetimes and lower slopes were obtained (Table 4) .Therefore , there is no advantages from addition ion exchanger or iodine to Ca-GHBA complex and most of the research was confined on Ca-ISE No. (

1). (3) : pH effect on the response of the new Ca-ISE No. (1) .
The best pH for the linear response of the new Ca-ISEs No.(1) was found in the range of (5)(6)(7)(8)(9) where the electrode response was suitable and steady for most calcium ion concentrations, (Fig. 3) .
The range was diverged with more dilutions of concentrations where the response was affected by the extreme acidic and basic media At pH above 9 , the decrease in electrode response was due to formation of Ca(OH) + which is not responded by the electrode and the formation of Ca (OH)2 precipitate (20).
At pH less than 5 , the elevation in electrode response was due to increase in hydrogen ions which penetrate inside the membrane causing positive increase in potential as a result of response of the electrode to hydrogen ions in addition to calcium ions (20).
This range of pH is near to the pH range of Ca-ISEs fabricated from calcium and zincom complex ( 5-10) (9).

(4) : Interference studies of cations on the response of the new Ca-ISE No(1) :
It was found in this research that there is no interference of the cations: Na+ , K+ , Mg 2+ , Ba 2+ , NH4+ , Ni 2+ , Co 2+ and Fe 3+ on the response of the new Ca-ISE No.(1) at low concentrations (10-5 -10-3 ) M .The electrode has recorded rather little effect of these cations (Table 5).However , the selectivity of the electrode was relativity high which was proved from the very low values of selectivity coefficients in the range of ( 1.6 x 10 -3 -1.6 x 10-2 ) .(table 5) .The lower interferences exhibited in the presence of sodium , potassium and magnesium ions would enable the electrode to be used in the biological researches and studies (6).The calcium concentration levels found in drinking water were within the acceptable World Health Organization specialists (21) .Therefore , the new Ca-ISE assembled from calcium glyoxalate complex offered high efficiency in determination of calcium in aqueous solutions.

Determination of some anions by Potentiometric titration with standard calcium ion solutions:
Since most anions showed an intense interferences with calcium ions , these reactions were utilized in the determination The concentrations of these anions by potentiometric titration with standard calcium solutions using the new ISE.No.(1) (Fig 4).Different concentrations of anions solutions were used and the results were compiled in table (7) .
A sudden shift in potentials at equivalence points have been found for oxalate , carbonate , phosphate , sulphate and fluorides.However , there was no clear precipitate of calcium sulphate because of the high solubility of CaSO4 (22) ( 0.15g/l = 150 mg/l) and KSP CaSO4 = 2.3 x 10-4 at 25 ْ◌C .Therefore , there was a suitable opportunity to form ion pairs Ca2+, So42-instead of precipitate.Table (7) exhibits the results of potentiometric titrations of these anions with their relative errors which were attributed to experimental errors.Table (8) contains the concentrations of free calcium ions in three samples of human blood serum determined by Ca-ISE No.1 using calibration curve technique which were matching the results obtained by spectrophotometric method (14) .The first and third samples showed normal average levels of calcium concentrations in human body while the second sample showed shortage in calcium levels which should be cured (14).  1) may be due to the little interferences of cations in the blood that should be characterized by a special blank.

Conclusion:
A new Ca-ISEs assembled from calcium glyoxalate complex offered high efficiency in determination of calcium ions in drinking water and blood serum .Little interferences were found by many cations but most of anions were severely interfered which were exploited for determination either the anions or calcium ions.