SPECTROPHOTOMETRIC DETERMINATION OF TRACE IRON AS A 1,10-PHENANTHROLINE COMPLEX BACKGROUND

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1 #5. Determination of Trace Iron 41 EXPERIMET 5. SPECTROPHOTOMETRIC DETERMIATIO OF TRACE IRO AS A 1,10-PHEATHROLIE COMPLEX BACKGROUD 1,10-phenanthroline (phen) forms an intensely red complex with iron(ii) that may be exploited to determine iron concentrations in the range of parts per million. The reaction is 3 phen + Fe 2+ <==============> Fe(phen) 3 2+ The reagent has the structure shown below. Other reagents which are used for the determination of iron are shown also. C C 1,10-Phenanthroline Bipyridine 2,4,6,-Tripyridyl-s- triazine The molar absorptivity (a in Beer's law) of the complex is 8650 L/mol-cm at 522 nm, the wavelength of maximum absorption. The complex forms rapidly is stable over the ph range 3 to 9, and may be used to determine iron(ii) concentrations in the range of 0.5 to 8 ppm. Iron(III), if present, must be reduced to iron(ii) to produce the colored species. A suitable reagent for this purpose is hydroxylamine hydrochloride, H 2 OH.HCl. The concentration of iron in the sample could be calculated from Beer's law if the molar absorptivity and solution thickness were known. It is preferable, however, to prepare one or more standards and compare absorbance readings of the sample and standard solutions, since this technique minimizes the effects of instrument and solution variation.

2 42 #5. Determination of Trace Iron Although spectrophotometric methods are normally accurate to about 1%, higher accuracy and precision can be obtained with more sophisticated instruments. In most cases an accuracy of 1% at the level of milligrams per liter is sufficient. The standard in this experiment, FeSO 4 (H 4 ) 2 SO 4 6H 2 O, though not a primary standard, is available with a purity greater than 99%, which is adequate. PROCEDURE Reagent List: Unknown Sample - do not dry ferrous ammonium sulfate sulfuric acid (H 2 SO 4 ) - conc. 18 M hydroxylamine hydrochloride - 10% solution needed 0- phenanthroline 0.25% soln. sodium acetate - 10% solution needed cobalt chloride HCl conc. 12M Due to the availability of Spectronic 20 spectrophotometers, this experiment demands that you share an instrument with a partner. One set of standard solutions per group is acceptable. Report your partners name to the instructor. Collect an unknown sample which is contaminated with Fe from your laboratory instructor. Preparation of Standard Iron Solution Weigh to the nearest 0.1 mg, enough FeSO 4 (H 4 ) 2 SO 4 6H 2 O to prepare 250 ml of a solution M in iron. Transfer the salt to a 250-mL volumetric flask, dissolve it in distilled water, add 8 ml of 3 M H 2 SO 4, dilute to volume with distilled water, and mix. Pipet 10 ml of this solution into 100-mL volumetric flask, add 4 ml of 3 M H 2 SO 4, and dilute to volume. 1 1 The iron concentration of this standard solution should be known to within about 0.5%. Do not exceed 4 ml of H 2 SO 4, or the buffering capacity of the sodium acetate, to be added later, may be exceeded.

3 #5. Determination of Trace Iron 43 AALYTICAL PROCEDURE Preparation of Solutions for Measurement Obtain the total mass of your sample (mass maybe used as a check) and then place in a 500-mL volumetric flask. Dissolve in distilled water, add 20 ml of 3 M H 2 SO 4 and then dilute to volume with distilled water. Pipet 5 ml portions of this stock solution into two 50-mL volumetric flasks. Standard Solutions Into a set of four 50-mL flasks, pipet 5, 10, 15, and 20 ml of the standard iron solution. 2 Prepare a blank by adding 0.4 ml (about 10 drops) of 3 M H 2 SO 4 to another 50-mL volumetric flask. 3 Add to each of the seven flasks, in order, 1 ml of 10% hydroxylamine hydrochloride solution, 10 ml of 0.25% phenanthroline solution, and 4 ml of 10% sodium acetate solution, 4 mixing after each reagent is added. Dilute to volume. Measurement of Absorption Spectrum To obtain an absorption spectrum of the iron-phenanthroline complex, first measure and record % transmittance values of the most concentrated standard solution at 20-nm intervals over the spectral range of 400 to 640 nm. (The procedure is given in later pages). Convert the %T readings to absorbance, and plot absorbance (vertical axis) against wavelength (horizontal axis) in your laboratory notebook. From this graph select the wavelength to be used for measurement of the unknown solutions. Check the selected value with your laboratory instructor before proceeding. Measurement of Standards and Samples Using matched tubes, read the % transmittance (%T) of each solution several times at the wavelength of maximum absorbance. Set the %T reading to 100 with the blank solution between each group of six sample and standard readings. All solutions may be discarded in the sink upon completion of the experimental work. Report the total weight in milligrams of iron in your sample. 2 A 5-mL pipet should be used for this purpose. 3 A 100-mL volumetric flask may be used if convenient; in that case use 0.8 ml of 3 M H 2 SO 4. 4 The sodium acetate plus sulfuric acid gives an acetic acid-sodium acetate buffer in the ph region of about 4.5 to 5. At a ph much below 4 bipyridine exists predominantly in the protonated form, and complex formation with the iron may not be complete.

4 44 #5. Determination of Trace Iron CALCULATIOS Prepare a calibration curve by plotting the absorbance (calculated from the % transmittance) of the standard solutions (vertical axis) against the concentration of iron(ii) in the unknown solutions and calculate the mg of iron in the original sample. Also calculate the mg of iron by the least squares procedure described below. Here is a example SIMILAR to the calculations you must perform: By use of the foregoing procedure standard absorbance values were obtained as follows: Std 1: 0.120, 0.125, 0.130; Std 2: 0.248, 0.255, 0.252; Std 3: 0.382, 0.385, 0.384; Std 4: 0.504, 0.502, The sample readings were 0.337, 0.335, for Sample 1, and 0.341, 0.336, and for Sample 2. If the weight of ferrous ammonium sulfate taken was g, how many milligrams of iron did the unknown contain? The Graphical Method First calculate the concentration of iron in each of the standard solutions: mg Fe in stock soln = (at. wt. Fe)(0.204)(1000 mg/g) (mol. wt. FeSO 4.(H 4 ) 2 SO 4.6H 2 O)(250 ml) = mg/ml in 250-mL Flask (0.116) (V 10 ) (100.0 ml) = mg/ml in 100-mL flask (0.0116) (V 10 ) (50.0 ml) = mg/ml = 2.32 µg/ml in Std 2 For Stds 1, 3, and 4 the corresponding values are 1.16, 3.48, and 4.65 µg/ml. ext plot a calibration curve using the average absorbance values, as shown in the accompanying figure. Finally, read the concentration of iron in the two unknown solutions from the graph (3.09 µg Fe/mL in this example). In this example the two sample solutions gave essentially identical absorbance readings.

5 #5. Determination of Trace Iron Absorbance µg Fe/mL Figure IV-1. Calibration curve for the iron(ii) phenanthroline complex, based on data given in example. From the concentration of iron in the unknown solution the total iron in the original sample is calculated as follows: For Unknown Solution 1; (3.09 µg/ml)(50.0 ml) = 154 µg in the 50-mL flask. Since this amount of iron came from a 10-mL aliquot of a 100-mL sample, the total iron in the sample is 154 µg ml V 10 = 1540 µg, or 1.54 mg

6 46 #5. Determination of Trace Iron THE SPECTROIC 20 SPECTROPHOTOMETER. OPERATIG PROCEDURE AD PROCEDURE FOR MATCHIG SAMPLE CELLS The Spectronic 20 Operating Instructions for Bausch and Lomb Spectronic Turn on the instrument by rotating the amplifier control clockwise. (Knob - front - lower left ) 2. Set the wavelength control (knob - top panel right side ) to the desired wavelength. With the amplifier control, set the meter needle to zero on the percent-transmittance scale. The setting corresponds to infinity on the absorbance (optical density on older instruments) scale. 3. Dry the outside of the matched sample tubes with a lintless towel or tissue. Insert a tube containing a blank 1 into the sample compartment. Position the tube in the instrument with the aid of the index mark. Close the cover of the compartment. 4. Rotate the light control ( knob - front - lower right ) until the meter reads 100 on the percent-transmittance scale (zero on the absorbance scale). 5. Remove the blank tube and recheck the zero reading. Replace the blank tube with one containing a standard or sample. Read the absorbance directly and record it. The cover of the sample compartment should be closed for all readings. Variations of ±1% in the readings are normal. 1 A blank solution is one that contains all the reagents for an analysis but not the material under test.

7 #5. Determination of Trace Iron If readings are to be taken at another wavelength, remove the sample tube and insert the blank tube. Turn the light control counterclockwise before changing the wavelength setting; otherwise, increased sensitivity of the photodetector at the new wavelength may result in a signal of sufficient magnitude to damage the meter. Set the new wavelength. 7. To record multiple wavelength readings or to record a spectrum, repeat steps 4, 5, and 6 until readings at all desired wavelengths have been taken. Readjust the light control whenever the wavelength setting is changed. The zero percent-transmittance setting (dark current) also should be checked periodically and readjusted as needed with the amplifier control. Readings taken at 10- to 20-nm intervals are sufficient to outline an absorption spectrum except at absorption peaks or shoulders, where additional points may be needed to characterize the curve more completely. ear the ends of the spectral range of the instrument (below about 350 and above 650 nm) a 100% transmittance reading may be impossible to obtain with the blank. SAMPLE-CELL MATCHIG Sample Cells (Cuvettes) in Spectrophotometry Several different types of sample cells are used in spectrophotometry. Less expensive instruments are designed to use test tubes for liquid samples. To ensure that the solution path length is the same for sample and standard solutions, a matched set of tubes is needed; the use of a single tube for all measurements, although possible, is inconvenient. Tubes are matched by placing a solution of intermediate absorbance in each and comparing absorbance readings. One tube is picked arbitrarily as a reference, and others are selected that give the same reading within 1%. Tubes should be matched in a separate operation before any spectrophotometric experiments are begun. Follow the procedure outlined below. PROCEDURE Selection of Matched Tubes for Bausch and Lomb Spectronic 20 Spectrophotometer Obtain a supply of 13- by 100-mm test tubes that are clean, dry, and free of scratches. You will need at least 3 testtubes which are matched.

8 48 #5. Determination of Trace Iron Half-fill each tube with a solution of approx. 2% CoCl 2.6H 2 O. The solution contains about 2 g of CoCl 2.6H 2 O in 100 ml of 0.3 M HCl. 2 Take the tubes to a Spectronic 20. Set the wavelength to 510 nm on the spectrophotometer. (See operating instructions for Bausch and Lomb Spectronic 20 at the beginning of this section.) With the amplifier control, set the instrument to read zero. Place a vertical index mark near the top of one of the tubes, and insert the tube into the sample compartment. Adjust the light control so that the meter reads 90% transmittance. Using this tube to check periodically the 90% reading, insert the other tubes and record their transmittance. If the percent transmittance is within 1% of the initial tube, place an index mark so that the tube can be inserted in the same position every time. If it is not within 1%, rotate the tube to see whether it can be brought into range. In future measurements insert each tube in the same position relative to its index mark. Once you have obtained at least 3 tubes that match proceed with the section Measurement of Absorption Spectrum. To compensate for variations between instruments, use the same one for both tube matching and experimental work. 2 This solution is recommended because it is stable, has a broad absorption band at about the center of the visible region, and transmits about 50% in a 1-cm cell.

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