1,10-Phenanthroline Determination of Total Iron Edward Cieplechowicz 100406700 Experiment Done on: Jan 23rd, 2012 Submitted on:

Jan 30th, 2012 Unknown Number: 3 Marking Lab: Andrew Frank

Purpose

The purpose of this experiment is to determine the concentration of an u nknown solution using molecular absorption spectroscopy. The samples will be te sted using a microplate reader. Theory In this experiment this week spectroscopy was used to determine an unkno wn concentration of iron using Beers Law. Beers Law is used to determine the amoun t of absorbance of a compound or chemical. The equation for Beers law is: . In t he equation the A is the amount of absorbance that occurred in the test. is the extinction coefficient which is different for every compound or chemical. The co efficient tells one the molar absorptivity of said compound or chemical. C is th e equation is the concentration of the sample inside the cells in the microplate and finally the path is L in the equation. The path length is the length is the depth of the cell and it was 1cm. The equation of Beers law also shows us that t he coefficient, concentration and path length are directly proportional to the a bsorbance. (Dr.Smith, Jan 12th, 2012) In the spectroscopy done this week a microplate reader was used. This is a spectroscopy machines that emits a specific wavelength of light (508nm for th is experiment) through each of the cells recording the absorbances. An advantage of using a microplate reader is that small volumes of sample are used to get an absorbance as well as multiple samples can be down at a time. The microplates th at were being used had 96 individual cells that could hold samples. This allowed for multiple trails to be tested simultaneously. The reaction that occurred this week in the lab was of the reduction of Fe3+ which is shown below. 2 Fe+3 + 2 NH2OH.HCl + 2 OH2 Fe2+ + N2 + 4 H2O + H+ + ClThen when the Iron is reduced it is reacted with 1,10-phenanthroline to form its bright orange complex which is shown below. Fe2+ + 3 o-Phen Fe(o-Phen)32+ (http://www.chem.uky.edu/courses/che226/Labs/050-Fe_Absorption.pdf) In the experiment when making the stock solution H2SO4 is added before t he solution is filled to the make. This is to lower the pH of the solution. The Iron 2+ complex likes to form in more acidic solutions as well they are more sol uble in more acidic solutions. The structure of H2SO4 is shown below. (http://themedicalbiochemistrypage.org/heme-porphyrin.html) (http://www.lenntech.com/periodic/water/iron/iron-and-water.htm)

Next in the experiment was the reduction of Fe3+ and that was done with the Hydr oxylamine hydrochloride. In this reaction the Hydroxylamine hydrochloride is the reduction agent due to the fact the irons charge drops from 3+ to 2+. The full reaction with the both Iron and Hydroxylamine hydrochloride is shown below as we ll as the structure of hydroxylamine hydrochloride. 2 Fe+3 + 2 NH2OH.HCl + 2 OH2 Fe2+ + N2 + 4 H2O + H+ + ClThe half reaction below shows what is happening strictly to the iron in this sta ge of the experiment. Fe3+ + 1e Fe2+ The next part of the experiment is the Iron complex that forms. This hap pens when the reduced iron reacts with 1,10-phenanthroline. This is a 3 to 1 rea ction as indicated by the balance chemical reaction equation. Fe2+ + 3 o-Phen Fe(o-Phen)32+ Three 1,10-phenanthroline react with the carbon making 2 bonds one at each nitr ogen in the phenanthroline structure. These bonds would be 90o to one another ma king a octahedral complex. The most absorbance with the highest intensity will o ccur at the wavelength of 508nm. (http://www.chem.uky.edu/courses/che226/Labs/050-Fe_Absorption.pdf) Finally the sodium acetate is added to the solution as a buffer. This wi ll make sure that the pH is in the proper region for the complex to be stable. S ince the sodium acetate is a much weaker acid it will not change the pH much but it will make it closer to neutral. (http://www.csudh.edu/oliver/che230/labmanual/iron.htm) Procedure The first thing that was done in this weeks lab was the preparation of th e standard solution. 0.0175g 0.0001g of ferrous ammonium sulphate hexahydrate wa s massed out on the analytical balance. Then once massed the transfer quantitati vely to a 250 ml volumetric flask and add about 100 ml of deionized water. And s wirl until complete dissolve. Next add 3.75 ml of 3M H2SO4 using a graduated cyl inder. Once the acid is added fill the volumetric flask to the mark with deioniz ed water. Next iron standards will be prepared with a unknown and blank. Into a la belled 5 ml volumetric flask and 2 ml of deionized water. This is the blank and will be treated like the other samples. The blank will have all the reagents lis ted except Fe2+. Now grab 5 more 5 mL volumetric flaks and fill then with the ir on standard respectively with theses volumes; 50 L, 250 L, 500 L, 1mL and 1.75 mL. Next receive an unknown iron sample and dilute to the mark of that 10 vo lumetric flask. Once diluted and mixed well transfer 1 mL of the unknown solutio n using a micropipette to a separate 5 mL volumetric flask and label it as the u nknown flask. Next add the following volumes of the chemicals in this order to e ach of the seven 5 mL volumetric flasks. First add 50 L of 100g/L Hydroxylamine H ydrochloride, 500 L of 1g/L 1/10-phenanthroline and finally 400 L of 1.2M sodium a cetate. So when those are added in the correct order and volume fill the flask t o the 5 mL mark with deionized water and mix really well. Once done mixing allow the solutions to react for 10 min and mix again. Transfer the solutions into th eir own 20 mL vials and then transfer 350 L of each to its proper cell in the mic roplate using a micropipette. Repeat this 2 more times but the blank does not have to be made again. O nce all the solutions are made and in the microplate they can be put into the mi croplate reader and tested for their absorptions at 508nm. To us the microplate reader first click on the microplate reading softwa re. Click advance reads on the left hand side drop down menu and then click setu p. Set the wavelength to 508nm. Label the placement of the blanks and samples on the grid. Click start and then record the absorbances observed. (https://mylearningspace.wlu.ca/d2l/lms/content/viewer/main_frame.d2l?ou=41072&t Id=269120) Observations

In each of the preparation there were 3 trails in each. This gave nine trials fo r each concentration as well as the unknown. There was one blank and 2 trails fo r the blanks to see how blank they actually were. Table 1: Preparation one absorbances. Concentration Trail 1 Trail 2 Trail 3 1 (50 L) 0.0207 0.0225 0.0206 2 (250 L) 0.1170 0.1141 0.1141 3 (500 L) 0.1945 0.1919 0.1894 4 (1mL) 0.3999 0.3991 0.4169 5 (1.75mL) 0.6560 0.6853 0.6578 Unknown 0.0307 0.0294 0.0260

Table 2: Preparation 2 absorbances Concentration Trail 1 Trail 2 Trail 3 1 (50 L) 0.0216 0.0232 0.0203 2 (250 L) 0.1025 0.1091 0.1124 3 (500 L) 0.2015 0.2321 0.3142 4 (1mL) 0.4063 0.5309 0.5478 5 (1.75mL) 0.7014 0.7715 0.7735 Unknown 0.0282 0.0711 0.0598 Table 3:Preperation 3 absorbances. Concentration Trail 1 Trail 2 Trail 3 1 (50 L) 0.0508 0.0334 0.0630 2 (250 L) 0.1067 0.1541 0.1541 3 (500 L) 0.3109 0.3132 0.3176 4 (1mL) 0.4234 0.4208 0.4473 5 (1.75mL) 0.8569 0.7921 0.8441 Unknown 0.0319 0.0295 0.0350 Table 4: Blank trials Blank Trial Number 1 0.0003 2 0.0013 Absorbance

This shows us that the blanks where indeed very blank with absorbances being extr ememly close to zero.

Results Table 5: Concentrations of the diluted standard samples 1-5. Dilution of Standard Concentration (mM) Standard 0.1785 1 (50 L) 3.57 x 10-5 2 (250 L) 1.785 x 10-4 3 (500 L) 3.57 x 10-4 4 (1mL) 7.14 x 10-4 5 (1.75mL) 1.249 x 10-3 Table 6: Average concentration that have been Q-tested Dilution of Standard Average Absorbances 1 (50 L) 0.0306 2 (250 L) 0.1205 or 0.1108 3 (500 L) 0.2517 4 (1mL) 0.4436 5 (1.75mL) 0.7487

Unknown 0.03796 Table 7: Standard Deviation for absorbance Dilution of Standard Standard Deviation of Absorbances 1 (50 L) 0.0157 2 (250 L) 0.01974 3 (500 L) 0.06038 4 (1mL) 0.05636 5 (1.75mL) 0.0766 Unknown 0.01603 Sample calculations Standard concentration in mM Mass = 0.0175g MM = 392.14 g/mol

Volume = 250 mL = 0.250 L

Standard Dilution C1 = standard concentration in mM v1 = Standard volume in liters v2 = Dilut ion volume in liters C2 = New concentration in mM mM Q-test Q = Gap/Range 0.0207, 0.0225, 0.0206, 0.0216, 0.0232, 0.0203, 0.0508, 0.0334, 0.0630 Outliner = 0.0630 =0.0630-0.0508 / 0.0630-0.0206 =0.0123 / 0.0423 =0.6360 Q accepted = 0..437 Q accepted > Q calculated There the value is not rejected Average of Concentration 1 0.0306 Standard Deviation of Concentration 1

Finding unknown concentration of sample Y is absorbance and x is the concentration = x Multiply x5 to get undiluted concentration and x = 0.0001731 mM 1.731x10-5 mol/L MM = 55.85 1.731x10-5 mol/L x 55.85 g/mol = 9.667x10-6 g 9.667x10-6g = 0.9667x10-3mg mg/L = ppm 0.9667x10-3 ppm Molar absorptivity Rearrange for and you get L mol-1 cmDiscussion In this weeks experiment Beers Law was used to determine the concentration of an unknown Fe2+ solution. With the standardized Beers Law plot the observed a

bsorbance is needed for the unknown and then solve for x and a diluted concentra tion of was received and then multiplied by 5 to receive the undiluted concent ration of 0.0001731 mM. This concentration of iron converted to ppm is 0.9667x10 -3 ppm. The calculations of the Molar absorptivity were much less than the expec ted value in the text that was given. The text said that there would be a absor ptivity of around 11,100 mol/L. the calculated was 2192.9 mol/L. this is a diffe rence of about 8900 mol/L. This could affect by 3 things that are in beers law. T he path length, the absorbance and the concentration of the solution affect the molar absorptivity. These parameters could be adjusted to get the desired 11,100 mol/L absorptivity. One source of error was the buoyancy correction of the analytical balanc e. This affects the fourth decimal place on the read out. How this works is ever ything something gets put on the balance the air gets displace changing the read ing. Another source of error was that there could have been imperfections in the micr oplates and that could have caused an increase in absorbance. This would affect the calculated concentration and the calculated molar absorptivity. Questions 1. The experiment could be changes to differentiate between Fe2+ and Fe3+ b y reacting a chemical that not only react with just Fe2+ like 1,10-phenanthrolin e. In this experiment that only reacted with Fe2+ but if there was any Fe3+ in s olution it would not show up. So one could also test for Fe3+ ion complexes as w ell as using absorption for the same standard. 2. The difference between measurement uncertainties is due to the precision of the device or instrument doing the work. An example is buoyancy correction o n an analytical balance. The total uncertainties are due to everything that is h appening this includes measurement uncertainties. This includes human error, non -human error and random error than can occur. For these reasons measurement unce rtainties will be much smaller than the total uncertainty. The absorbances for the same solutions were all very close to one another in some situations only differing from 0.0001 in absorbance but in some differing by up to 1400 in absorbance. The instrument differences was much smaller than the dif ferences of averaged concentrations which were bigger than the ones of the same sample and that is to be expected due the fact that making 2 solutions with the exact same convention is difficult Conclusion In conclusion to this experiment a linear trend was observed in the Beers Law plots with the diluted samples. With that standardized data and unknown conc entration was determined as well as a value for molar absorptivity. The values a re 0.9667x10-3 ppm and 2192.9 mol/L respectively.

Bibliography 1. 1,10-Phenanthroline Dermination of total Iron https://mylearningspace.wlu.ca/d2l/lms/content/viewer/main_frame.d2l?ou=41072&tI d=269120, accessed January 26th,2012. 2. D.S.Smith. Lecture:Absorbance and some stats. CH262. January 12th,2012 3. Colorimetric Fe Analysis, http://www.csudh.edu/oliver/che230/labmanual/i ron.htm Accessed January 26th,2012 4. EXPERIMENT 5,Molecular Absorption Spectroscopy: Determination of Iron W ith 1,10-Phenanthroline,http://www.chem.uky.edu/courses/che226/Labs/050-Fe_Absor ption.pdf accessed January 26th, 2012. 5. http://themedicalbiochemistrypage.org/heme-porphyrin.html accessed Janua

ry 26th,2012 6. Water Treatment Solutions, Lenntech, http://www.lenntech.com/periodic/wa ter/iron/iron-and-water.htm Accessed January 26th,2012.