CHE130L Analytical Chemistry Laboratory I. OBJECTIVES II. III.

Introduction

MALAYAN COLLEGES LAGUNA

EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS

Upon completion of the experiment, the student should be able to: operate and analyze output from a UV-VIS spectrophotometer; determine the wavelength of maximum absorption by plotting the absorption curves of some substances; construct Beers law plot for a given analyte; and construct calibration curves to determine the concentration of analyte in an unknown sample.

A. LABORATORY EQUIPMENT / INSTRUMENTS Apparatus 100-mL volumetric flasks 50-mL volumetric flasks 50-mL beakers 10-mL pipet Rubber aspirator Spectrophotometer B. CHEMICALS AND REAGENTS Chemicals/Materials 0.02M KMnO4 solution 0.04M CuSO4 3.0M NH3 solution Distilled water in wash bottle Unknown samples DISCUSSION OF FUNDAMENTALS Quantity 5 5 2 1 1 1

Over the course of years, the nature of light is explained as a property of two, or a duality. Since light is emitted and absorbed into tiny packets known as photons, and exhibits properties of both waves and particles, this property is referred to as the wave-particle duality of light. This will then tell that like everything else, light is also a particle. Since light is a type of EMR (or electromagnetic radiation) that is in a form of a transverse wave, it contains energy (since waves do not flow unless Experiment 7: Spectrophotometric Methods Page | 1

CHE130L Analytical Chemistry Laboratory

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EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS work is exerted, and therefore, energy is present). By the virtue of collision theory, we can assume a particle/molecule in an open space, and is not affected by any other factors other than environmental factors (such as gravity and the like). When particles collide with each other, they can transfer the energy that they have, and to another, and so on ad infinitum, gone from kinetic to potential. So, if light strikes a molecule, by the lights particle nature, it can transfer the energy that it contains by letting the molecule absorb the lights photons, as stated earlier, defined as tiny packets that are present in light. With that, the energy of the molecule increases. Because of the energy that it contains, by the concept of hybridization, the energy of the electrons that the outer shell contains will step up a block (i.e. from s to p block, etc.) or as what we call an excited state. The excited state of a molecule will preferably want to be more stable, and in doing so, the electron will now go back to its lower (or neutral) state. By virtue of law of conservation of energy, the energy that is absorbed by the electron must be equal to the energy that the electron emits again to go back to its initial state (or going back to stability). Since the energy goes up, it affects the wavelength of the wave and by the EMR spectra, the transition of electrons are occurring in the UV (ultra-violet) and the visible (light) region (since the EMR spectra uses wavelengths to differ types of one another). This transition will then be affected by the following factors: the specific types of groups, bonds and functional groups that the molecule contains. This is possibly due to the fact that the molecules differ in bond length, angle and the like, as they differ in their composition. Ultimately, it will make the wavelength and intensity of absorption differ for each type of molecule. A method in analytical chemistry is used to possibly use this property of molecules when exposed to light. This method is called spectrophotometry. Spectrophotometry differs in which EMR spectra they particularly scan in. Examples would be IR spectrophotometer, UV-Vis spectrophotometer and the like. A spectrophotometer has a standard light source, depending on what type it is. With this, we are going to focus on the spectrophotometer that uses the visible spectra of light, which is the UV-Vis spectrophotometer. From a standard source of light, the solution will then absorb EMR from it; therefore the solvent is the matrix of the sample. By all means, the solvent must be the blank of the solution. With this, the concentration of the solution must be related to the absorption of light. By virtue of proportionality, when light strikes the solution, some of the light is transmitted (or passes through) and the others are absorbed by the absorbing species. Therefore, light generally goes by a ratio of the transmitted light and the original light, defining transmittance as a percentage of the transmitted light and the original emitted light. Absorbance is related to transmittance in a non-linear fashion. Since absorbance and transmittance undergoes a logarithmic relationship (as 1/T=A, making a one sided hyperbola), and inversely proportional if considered. With this, we can tell that the absorbance is measured by how pH is related to the hydronium ion concentration, that is A=-log (T). Experiment 7: Spectrophotometric Methods Page | 2

CHE130L Analytical Chemistry Laboratory IV.

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EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS Transmittance shows a non-linear relationship to the concentration of the solution, since it does not undergo a direct relationship with the solution, as the transmitted light passes through the solution itself. With that, absorbance is computed to get the relationship among the light given off and the concentration, which is proposed by Beer-Lambert law, or Beers Law. It is depicted by the formula A=bC, wherein is a proportionality constant that is defined by several factors which include the wavelength of light being used, the identity of the solution, its solvent and temperature, among the like. With this, it will form a linear relationship among the concentration C to the absorbance A, and by running it on solutions of known concentrations, the formula can extensively be used to pave the way of determining unknown concentrations of similar solutions by appropriate methods. METHODOLOGY A. Preparation of KMnO4 solution From the stock solution of 0.02M KMnO4 solution, 2.0 mL was pipetted and was diluted to 50.0 mL

From the diluted solution, aliquots of different volumes (1.00, 2.50, 5.00, 7.50, and 10.00) were pipetted to different flasks. They were all diluted to the mark and labeled. B. Preparation of CuSO4 solution From the stock 0.04M CuSO4 solution, aliquots of different volumes (1.00, 2.50, 5.00, 7.50, and 10.00) were pipetted to different 50-mL volumetric flasks.

3.0M NH3 solution was added to each to develop a royal blue color. The Cu(NH3)42+ complex was formed. All were diluted to mark and labeled.

A blank solution consisting of 10 mL 3.0 NH3 and 40 mL distilled water was prepared. Experiment 7: Spectrophotometric Methods Page | 3

CHE130L Analytical Chemistry Laboratory V.

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EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS

C. Plotting the absorption curve The wavelength of the spectrophotometer was set to 400nm and was calibrated using the prepared blank solutions.

In decreasing concentrations, the absorptions were read, with increasing increment of 20nm on the wavelength until 700nm.

DESCRIPTION OF THE APPARATUS/ SET-UP Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer that measures light intensity. The spectrophotometer provided was a Genesys 20 UV-Vis spectrophotometer, an easy-to-use device made for teaching in the labs. It is able to measure a wide range of analytes. It gives direct measurements of absorbance and transmittance as they are the data most required for the test the instrument is used. There is also a concentration mode for quantitative analysis.

Figure 1. Genesys 20 UV-Vis spectrophotometer

Experiment 7: Spectrophotometric Methods

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CHE130L Analytical Chemistry Laboratory VI. DATA SHEET

MALAYAN COLLEGES LAGUNA

EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS

10 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 10 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 1.43 1.472 1.106 0.954 0.594 0.374 0.427 0.491 0.437 0.267 0.098 0.429 0.308 0.281 -0.1 -0.033 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 1.412 1.414 1.093 0.942 0.564 0.308 0.304 0.304 0.247 0.133 0.023 0.404 0.29 0.267 -0.1 -0.04 -0.002 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

7.5 1.412 1.449 1.098 0.942 0.56 0.293 0.272 0.257 0.199 0.102 0.009 0.4 0.287 0.265 -0.1 -0.041 -0.002 7.5 1.43 1.472 1.109 0.953 0.585 0.345 0.369 0.402 0.347 0.205 0.065 0.419 0.3 0.275 -0.1 -0.036 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

5 1.412 1.448 1.098 0.939 0.554 0.276 0.241 0.21 0.152 0.068 -0.009 0.394 0.282 0.26 -0.1 -0.043 0 5 1.43 1.474 1.115 0.957 0.579 0.319 0.311 0.311 0.254 0.142 0.034 0.414 0.298 0.274 -0.1 -0.034 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

2.5 1.412 1.446 1.098 0.938 0.548 0.261 0.21 0.164 0.105 0.035 -0.027 0.389 0.278 0.256 -0.1 -0.046 -0.001 2.5 1.43 1.465 1.108 0.949 0.566 0.295 0.271 0.253 0.196 0.099 0.008 0.402 0.288 0.264 -0.1 -0.042 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

1 1.412 1.444 1.096 0.935 0.544 0.251 0.193 0.139 0.079 0.017 -0.037 0.386 0.276 0.255 -0.1 -0.047 -0.002 1 1.43 1.459 1.106 0.944 0.553 0.262 0.207 0.158 0.099 0.031 -0.028 0.391 0.279 0.257 -0.1 -0.046
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Experiment 7: Spectrophotometric Methods

CHE130L Analytical Chemistry Laboratory

MALAYAN COLLEGES LAGUNA

EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS

700 20 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

0.007

700 15

0.005

700 10

0.009

700 5

0.002

700 2

1.432 1.482 1.115 0.954 0.577 0.315 0.308 0.33 0.353 0.373 0.376 0.822 0.707 0.661 0.2 0.283 0.282

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

1.432 1.474 1.112 0.951 0.57 0.299 0.278 0.279 0.282 0.282 0.272 0.271 0.599 0.56 0.108 0.203 0.213

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

1.432 1.467 1.11 0.949 0.564 0.285 0.25 0.231 0.214 0.194 0.171 0.606 0.494 0.461 0.016 0.124 0.145

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 5 blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

1.432 1.457 1.104 0.942 0.553 0.266 0.216 0.177 0.137 0.098 0.061 0.492 0.38 0.352 -0.085 0.034 0.068

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 2

1.432 1.455 1.103 0.942 0.551 0.258 0.2 0.149 0.098 0.048 0.004 0.433 0.323 0.299 -0.1 -0.01 0.031

20 blank 1.432 400 1.515 420 1.132 440 0.97 460 0.606 480 0.375 500 0.419 520 0.519 540 0.622 560 0.717 580 0.77 600 1.236 620 1.114 640 1.042 660 0.544 680 0.584 700 0.538

15 blank 1.432 400 1.491 420 1.123 440 0.963 460 0.592 480 0.344 500 0.36 520 0.417 540 0.476 560 0.529 580 0.554 600 1.009 620 0.891 640 0.834 660 0.357 680 0.421 700 0.4

10 blank 1.432 400 1.498 420 1.131 440 0.969 460 0.591 480 0.328 500 0.318 520 0.338 540 0.359 560 0.377 580 0.378 600 0.824 620 0.709 640 0.663 660 0.203 680 0.287 700 0.287

1.432 1.476 1.12 0.957 0.574 0.293 0.255 0.236 0.217 0.197 0.174 0.609 0.497 0.464 0.019 0.126 0.148

blank 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

1.432 1.466 1.113 0.951 0.562 0.273 0.22 0.178 0.136 0.095 0.057 0.487 0.376 0.349 -0.087 0.033 0.068
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Experiment 7: Spectrophotometric Methods

CHE130L Analytical Chemistry Laboratory VII. SAMPLE COMPUTATIONS CuSO4: A = 1.3902 x 10-3 B =70.389 r = 0.99968 y = 70.389 + 1.3902 x 10-3 VIII. RESULTS AND DISCUSSIONS

MALAYAN COLLEGES LAGUNA

EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS

Unknown: 0.538 = 10.389x + 1.3902 x10-3 x = 1.623 x 10-3 M

Spectrophotometry is the measurement of the reflection properties of a material in terms of wavelength. It involves the use of a spectrophotometer, a device that measures the intensity of light that passes through the specified medium. As a consequence of light absorption the beam of light that emerges from the sample has a diminished intensity symbolized by I. Fewer Figure 2. Spectrophotometer photons leave the sample than entered it. The ratio I/I0 is the fraction of light that actually passes the sample and is called the transmittance, t. This quantity is generally expressed as a percentage. As an example, a certain solution held in a particular curvette may have a 10% transmittance at 450 nm wavelength. This statement means that when light of 450 nm wavelength (a shade of blue) passes the tube only 1/10 of the 450 nm photons remain in the beam; the rest are absorbed by the sample. This behavior makes no implication about the transmittance at some other wavelength of light. Indeed the same sample might have a transmittance of 100% at 500 nm indicating that a beam of 500 nm light (a kind of green) passes through the sample tube without any detectable absorption of light.

Experiment 7: Spectrophotometric Methods

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CHE130L Analytical Chemistry Laboratory

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EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS The transmittance of a solution containing a light absorbing material, the analyte, is related to experimental conditions by Beer's Law. -log I/I0 = -log T = A = abC In this equation T is the transmittance, expressed as a decimal (10% transmittance corresponds to t = 0.10) and A is called the absorbance. C is the concentration of the analyte, b is the length of the light path through the absorbing solution and a is the absorptivity, a number which depends both on the nature of the light absorbing substance and the wavelength of light. When b is expressed in cm and C in mol/L units a has units of L mol-1cm-1 and is termed the molar 2 absorptivity or the molar extinction coefficient and may be symbolized as . In other words, Beer's Law is sometimes written as A = bC. It can be seen that the absorbance varies linearly with the concentration and path length. This is where the Beers law equation was yielded. Beers law states that: where is the molar absorptivity, is the path length and is the analyte concentration. The experiment required the measurement of different concentration of four solutions, two of known starting molarities and two unknowns. This is not true all the times for Beers Law is also limited by chemical and instrumental factors. Such causes include scattering of lights in the sample, fluorescence or phosphorescence of the sample, shifts in chemical equilibria, stray lights, and others.

Figure 3. Different concentrations of KMnO4

Figure 4. Different concentrations of CuSO4

Experiment 7: Spectrophotometric Methods

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CHE130L Analytical Chemistry Laboratory IX. X. REFERENCES

MALAYAN COLLEGES LAGUNA

EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS

But graphing the results, the resulting graph still shows how Beers Law basically works, thus showing the success of the experiment:

SUMMARY AND CONCLUSIONS This experiment concluded the assorted concepts of spectrophotometry. Potassium permanganate is prepared by diluting it two times, with the second time getting different aliquots from the first dilution. It is diluted in distilled in water, which is used as the blank of the solution. Copper sulfate is prepared from a standard stock solution, and by different aliquots. It is diluted with ammonium + water, which will be used as a blank. Ammonia is used to form the copper-ammono complex, which is characterized by a stronger color of royal blue, from an initial color of light blue. All of the solutions are labeled accordingly. By setting the blank to 0 absorbance, each of the solutions are set to 400 nm and getting the individual absorbance by 20 nm increments to 700 nm. By doing the same preparation to the unknown samples of both solutions, this process is also repeated. The absorbance vs. wavelength of each of the solutions is graphed, along with the Beers law graph (absorbance vs. concentration). By computation (interpolation or other methods appropriate), the concentration of the unknown can be determined accordingly.

Experiment 7: Spectrophotometric Methods

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CHE130L Analytical Chemistry Laboratory

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EXPERIMENT NO. 7 SPECTROPHOTOMETRIC METHODS Christian, Gary D. 2004. Analytical chemistry (6th ed.). John Wiley and Sons Inc. Hage, David S. and James D. Carr. 2011. Analytical chemistry and quantitative analysis. New Jersey: Pearson Prentice Hall. Harris, Daniel C. 2003. Quantitative chemical analysis. (6th ed). New York: W. H. Freeman and Company. Madamba, Lilia S.P. 1995. Chemistry 32 Laboratory Instruction Manual (3rd rev). Los Baos: Analytical and Environmental Chemistry Division, Institute of Chemistry, University of the Philippines Los Baos. Skoog, Douglas et. al. 2004. Fundamentals of Analytical Chemistry (8th ed.). Singapore: Thomson Learning.

Experiment 7: Spectrophotometric Methods

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