Chapter 3

3.1 Materials
Ice sample were taken from Mrs. Freeze ice manufacturing plant. Concentrated ammonium hydroxide were purchased from laboratory equipment and supplies division of University of Santo Tomas. Concentrated rubeanic acid is purchased from a University of Santo Tomas supplier. The equipments used are USB 2000 Ocean Optics UV-Vis spectrophotometer and Lambda 35 Perkin Elmer UV-Vis spectrophotometer of Thomas Aquinas Research Complex (TARC), University of Santo Tomas

3.2 Preparation of Reagent

Initially we will consider concentrated ammonia as the reagent for Cu +2, A spectrum in the visible region will be scanned to show the Lambda max of the absorbed light. Then solutions of Cu+2 will be prepared for about 50-150 ppm in increments of 20 ppm. This will be the standards to read the corresponding absorbance. A calibration curve will serve as the guide for determining Cu+2 concentration in real samples (ice water from malabon area). The sensor will be characerized according to stability of signal, response time, repeatability, sensitivity and limit of detection. In preparing rubeanic acid, the necessary steps are taken in consideration: 1. 2. 3. 4. 5. 6. 7. Bring sections to water via xylene and ethanol. Place in the working rubeanic acid solution overnight at 37C. Place into 70% ethanol for 15 minutes. Place into absolute ethanol for 6 hours. Lightly counterstain with alcoholic eosin. Rinse well with absolute ethanol. Clear with xylene and mount with a resinous medium.

Copper forms a chelate with rubeanic acid, as do some other metals. Of these, cobalt and nickel may be confused with copper, so sodium acetate is added to the solution to inhibit them. Rubeanic acid is also known as ethanedithioamide, dithiooxamide or dithiooxalic diamide. It would appear that the copper is chelated between the sulphur and nitrogen

3.3 Application to real samples

Ice samples from different places in malabon area are taken and melted naturally in a sterilized container, each is placed in a cuvette for scanning using two different methods (Present and Traditional) and reagents (Ammonia and Rubeanic Acid).

3.4 Spectrophotmeteric measurement

In order to determine the concentration of copper in the unknown solutions we shall first construct a standard plot of the concentration of copper vs. the absorbance of solutions having known copper concentrations. According to Beer's Law, absorbance is directly proportional to concentration and so the resulting plot should be a straight line. The stock solution of copper was prepared by diluting 10ml of the 50-150 ppm solution. Buffer solution was prepared by dissolving ammonium chloride in the de-ionized water, PH is now checked with the reagent concentration

3.5 Testing for copper

Present Method The absorbance of copper solutions was read usingthe Ocean Optics and Perkin Elmer UV-Vis spectrophotometer within the range of 50ppm 150ppm. Stability tests will be measured for three hours and the repeatability test will be conducted within three minutes interval for an hour. Sensitivity test of the copper sensor will be tested using the Perkin Elmer UV-Vis spectrophotometer, using varying solution concentration the sensitivity of the absorbent will be read for four minutes interval in an hour. All process is repeated until all the solutions are scanned Traditional Method A drop of sample solution which is as near neutral as possible is placed on filter paper, a drop of rubeanic acid solution is added and the paper is held over ammonia. Copper is indicated by a dark green or black color. A blank should be run with distilled water. A Heavy filter paper is impregnated with the rubeanicacid reagent and allowed to dry. The test solution is acidified with acetic acid and a drop placed on the filter paper. Another method that can be used is Electrolysis. Shocking water in swimming pools gets rid of contaminants in the water, the high concentration of chlorine increase when shocked and the water may turn green if large amount of copper concentration is binded by the ionic bond positive and negative charges.