ATOMIC ABSORTION SPECTROSCOPY: rev. 4/2011 ANALYSIS OF COPPER IN FOOD AND VITAMINS

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1 1 ATOMIC ABSORTION SPECTROSCOPY: rev. 4/2011 ANALYSIS OF COPPER IN FOOD AND VITAMINS Buck Scientific Atomic Absorption Spectrophotometer, Model 200 Atomic absorption spectroscopy (AAS) has for many years been one of the chemist s favorite analytical tools for quantitatively measuring very small amounts (trace amounts) of metallic elements in samples as varied as alloys, rocks and soils, foods and drinks, surface waters, biological fluids, and reagent chemicals. Although the sensitivity of this technique varies somewhat from metallic element to metallic element as well as with the method of excitation (flame vs. graphite furnace for example), this technique is generally capable of accurately measuring a metallic element s concentration in a solution in the 1 to 100 ppm range. (ppm = part per million, 1 ppm = 1mg/L = 1 µg/ml) Some metals can even be measured in the ppb, parts per billion range. Because of its ability to measure the concentration of over 60 of the metallic elements at extremely low concentrations, atomic absorption spectroscopy has many, many analytical applications including impurity analysis of substances, forensic analysis, biomedical tissue, environmental samples and chemical research. Copper is important in the function of some enzymes. It is found naturally in extremely small amounts in some foods, milk for example. But it is frequently added to foods in small amounts. The USDA s Recommended Daily Allowance ( RDA ) is just 2 mg of Cu. Because copper in very small quantities is important to our health, it is the element that we have chosen to analyze today. The basic principle is that a solution (generally aqueous) containing the metallic element is aspirated in the form of an aerosol into a high temperature flame. The flame evaporates the solvent and decomposes the compound containing the element to create gaseous state atoms of the element. This technique is often called Flame Atomic Absorption Spectroscopy or FAAS. Alternatively, an atomic vapor can be produced by rapid electrothermal heating of a graphite rod or tube on which a drop of the sample has been placed. This is accomplished using a graphite furnace. This experiment uses an acetylene flame as the vaporization method. A beam of monochromatic light with a wavelength at which only the element of interest can absorb, passes through the flame. The atoms of the element in the flame absorb some of the light. The amount of light absorbed is directly dependent upon the concentration of the element in the solution being vaporized in the flame. A beam of electromagnetic radiation characteristic of a particular element can be passed through the atomic vapor and monitored by a photomultiplier tube (PMT) detector. If the sample contains that particular element, its atoms will selectively absorb some of the electromagnetic radiation, thereby attenuating the beam and causing the detector signal to decrease. This absorbance is proportional to the concentration of that element in the vapor and hence in the original sample.

2 2 There are many experimental variables in atomic absorption. Among them are (1) the quantity of material reaching the flame, (2) the flame temperature, (3) the fuel/oxidant ratio, and (4) burner height. (1) The quantity of material reaching the flame is dependent on 4 interrelated factors: (a) The rate of aspiration of the material into the flame, (b) the viscosity of the analyte solution, (c) the solvent, and (d) the surface tension of the analyte solution. (2) The flame temperature is primarily determined by the choice of fuel and oxidant. Converse s atomic absorption spectrometers are set up to operate on an acetylene/air mixture, hence this experimental variable is largely beyond our control with our systems. (3) Furthermore, since the acetylene/air mixture is the most commonly used combination, we are not really hindered by our systems relative to most atomic absorption systems. (4) The fuel/oxidant ratio affects the flame temperature to a more limited extent. More importantly, it determines whether the flame is a reducing or an oxidizing flame. Normally the reference literature tells you which type of flame is best for that analyte. (5) There is also an optimum portion of the flame for analysis of the analyte. This variable is controlled by the burner height relative to the optical path. The optical path should be just above the tip of the inner blue cone of the flame. Another experimental consideration is the choice of calibration standards. The calibration standard should be in a matrix similar to that of the sample solution, For example, if sera are to be analyzed, then the standard solutions should be made to simulate serum. Ideally, if standards cannot be prepared in a matrix similar to that of the unknowns, then the technique of standard additions should be used. For most elements the range of analysis is between 1 and 50 ppm. Above the concentration of 50 ppm the absorbance is not linear relative to concentration for most elements. Each element has an optimum range within which the absorbanceconcentration relationship is linear (Beer s Law). However, curves are not unusual, and if you get a curve for your Beer s Law calibration graph, work with it. In spite of the previous statement, when analyzing by the standard addition method, the data is always treated as a straight line. The strongest absorption wavelength is usually used for the analysis of an element. One of the challenges you will encounter today is to get the copper out of a solid substance and into an aqueous solution. You will be working with three different types of foods : a vitamin, cereal, and Ultralight Slimfast. You may experience various degrees of success, but you should find this laboratory experience very educational.

3 3 EXPERIMENTAL PROCEDURE I. SAMPLE PREPARATION 1. VITAMIN: Place a mulit-vitamin tablet in a 125mL Erlenmeyer flask and add 25 ml of 6 M HCL. Place this on a hot plate and bring it to a boil. Set the hot plate on High. Keep a stirring rod in the flask. As soon as the mixture begins to boil, reduce the heat to a setting of about 4 on the hot plate and continue to heat it for 15 additional minutes. ADD ADDITIONAL HCl if necessary to keep the mixture wet and the volume at about 25 ml. Filter the resultant mixture through filter paper into a 25 ml volumetric flask. Rinse the beaker and filter paper several times with distilled water but be careful not to go past the line on the volumetric flask. Dilute to the mark with distilled water and thoroughly mix. 2 & 3. Cereal (or Ultralight Slimfast) in a weighing boat and transfer the cereal (or Slimfast) to a 125mL Erlenmeyer flask. Add 25 ml 6 M HCl, bring to a boil and follow the instructions above for the vitamin. After 15 minutes of heating, filter this solution into a 50 ml volumetric flask, then rinse and dilute to the mark with distilled water and thoroughly mix. Be careful not to go over the mark on the volumetric flask. Q- Why do you think we prepare the vitamin in a 250mL volumetric flask but the cereal and Slimfast in a 50 ml volumetric flask? 4. Copper unknown. Dilute to 100 ml with distilled water. II. STANDARDS You will be provided a ppm standard for copper. DO NOT WASTE THIS STANDARD. 1. Using pipets and volumetric flasks, prepare ml of each of the following standards from the ppm standard. Do your calculations before coming to the lab. Cu: 10.0, 8.0, 6.0, 4.0, and 2.0 ppm standards 2. Standard Addition Method Vitamin. You are also going to analyze your vitamin and your copper unknown by the standard addition method. Pipet ml portions of the vitamin solution into 5 different 30 ml beakers. With a 100 µl syringe add the appropriate amount of 1000 ppm Cu standard. Beaker vitamin soln (ml) 1000 pm standard (µl) ppm Cu added

4 Standard Addition Method Copper Unknown. Pipet ml portions of the unknown solution into 5 different 30 ml beakers. With a 100 µl syringe add the following amounts of 1000 ppm Cu standard to create these solutions: Beaker vitamin soln (ml) 1000 pm standard (µl) ppm Cu added III. YOUR INSTRUCTOR WILL ASSIST YOU IN TURNING ON AND LIGHTING THE INSTRUMENT. THE PROPER WAVELENGTH CAN BE SELECTED EITHER BEFORE OR AFTER LIGHTING THE FLAME. 1. Make sure the proper lamp is in position, turned on to an appropriate current, and allowed to warm up at least 10 minutes. Find the wavelength for analysis using the monochrometer dial and maximizing the energy needle (minimizing the absorbance). You may need to adjust the PMT voltage to keep all readings on scale. 2. Adjust to zero absorbance while aspirating distilled water as your blank. This constitutes a data point on the calibration curve. Then aspirate each Cu standard and the vitamin and cereal samples. Remember that you will analyze Cu in the vitamin and the copper unknown by both the Beer s Law method and the Standard Addition method. DATA TREATMENT 1. Prepare a typical Beer s Law calibration curve. If your data follows a curve, treat your calibration graph as a curve instead of a straight line.

5 5 2. From the calibration graph determine the concentrations of the copper in each of your samples including the unknowns. Remember to account for any dilutions you may be required to do in order to keep the absorbances on scale. 3. Also determine the concentrations of Cu in the vitamin and copper unknown by the Standard Addition method. See the graph below. 4. Calculate the # mg of Cu in your vitamin tablet by both methods. Remember that the total volume of the vitamin solutions was ml. Calculate a % error for each method based upon the amount of Cu on the product label. 5. Calculate the # mg of Cu in a typical bowl of cereal. First calculate the # mg in your 8 g sample. Then convert that to mg Cu per g of cereal. A serving = 1.00 oz. (1.00 oz = g). Calculate a % error based upon the product label. The RDA for Cu is 2 mg. 6. Do the same for the Slimfast. One serving of slimfast = 1.16 oz. Q-Which method gives the best results: Is there a significant difference? Which method should give the best results and why? Q-What would you have to change in the procedures in order to do the cereal and Slimfast by both methods? 1000 ppm = 1000 mg/l = 1000 µg/ml = 1 mg/ml 1 ppm = 1 µg/ml

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