Quantitative Analysis by Spectrophotometric Methods Essay
Abstract In this experiment, the absorbance of KMnO4 was measured by spectrophotometric method to determine the molar concentration and the molar extinction coefficient of KMnO4. In part 1, in order to determine the maximum absorbance wavelength of KMnO4, we measured the absorbance of the sample solution which contains KMnO4 at the wavelengths between 330nm and 660nm, and plotted the ? and A points; the ? max was 530nm. In part 2, the effect of concentration on the absorbance was examined. We prepared five differently concentrated (but, same path length) solutions, and measured the absorbance of them at the ? ax(530nm) discovered in part 1; According to the results, higher concentrated solution had higher absorbance value. The extinction coefficient(? ) could be calculated from the results determined in part 2 and Beer’s Law; ? = 1.
7 x 103. In part 3, the absorbance of the KMnO4 solution of unknown concentration was measured, and using Beer’s law and dilution equation, the initial concentration of the unknown was determined; The concentration of the solution (unknown # : 15) was calculated to be 3. 3 x 10-3M. Introduction Our eyes are sensitive to light which lies in a very small region of the electromagnetic spectrum labeled “visible light”.This “visible light” corresponds to a wavelength range of 400 – 700 nanometers (nm) and a color range of violet through red. The human eye is not capable of “seeing” radiation with wavelengths outside the visible spectrum.
The visible colors from shortest to longest wavelength are: violet, blue, green, yellow, orange, and red. Ultraviolet radiation has a shorter wavelength than the visible violet light. Infrared radiation has a longer wavelength than visible red light. The white light is a mixture of the colors of the visible spectrum. Black is a total absence of light.Figure 5.
1 The electromagnetic spectrum. Although visible light acts as a wave in some respects, it also displays properties characteristic of particles. The particle-like properties of visible light are exhibited through small, energy-bearing entities known as photons. The energy of a photon is: E photon = hc / ? (1) where h = Planck’s constant, 6. 626 x 10-34 J/s, c = speed of light, 3.
00 x 108 m/s, and ? = wavelength of light. Light is energy, and when energy is absorbed by a chemical it results in a change in energy levels of the chemical.Molecules normally exist in discrete energy levels. Vibrational energy levels exist because molecular bonds vibrate at specific frequencies.
Electronic energy levels exist because electrons in molecules can be excited to discrete, higher energy orbitals. The energy (E) of light depends on its wavelength. Longer wavelengths (infrared) have less energy than shorter wavelengths (ultraviolet). A molecule will absorb energy (light) when the energy (or wavelength) exactly matches the energy difference between the two energy states of the molecule.In absorption, light — sunlight which is white light — strikes an object and part of the light may be absorbed by the object.
The light we see coming from that object is the light which was not absorbed by the object. We see the “not-absorbed” light as the color of the object. If no light is absorbed, the object appears to be colorless. A spectrophotometer is employed to measure the amount of light that a sample absorbs.
The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons.When a photon encounters a molecule, there is a chance the molecule will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam.
The ratio of transmitted light intensity(I) to the incident light intensity(I0) is the transmittance, T: T = I / I0 (2) The amount of light a sample absorbs is affected by its concentration. If there are samples of same substances but different concentrations, the amount of the absorbed light will be different.In higher concentrated solution, more absorbing molecules are present in the path of the light, and the chance the light strikes the molecules will increase. So, more light will be absorbed by the sample; less light will be transmitted.
In contrast, in lower concentrated solution, less light will be absorbed, and more light will be transmitted. The concentration is represented by the symbol C and is typically measured in mole/L. Another factor that affects the amount of light a sample absorbs is the path length which is the length of sample that the light passes through.The path length is represented by the symbol l and is typically measured in centimeters. When the light travels through longer distance, it will strike more absorbing molecules, so more light will be absorbed, and less light will be transmitted. The relationship of two factors (path length & concentration) can be combined to yield a general equation called Beer’s Law.
– log10T = A = ? lC (3) The quantity ? is the molar absorptivity; in older literature it is sometimes called the molar extinction coefficient.It is the measure of how strongly a substance absorbs light at a particular wavelength; a larger extinction coefficient means that substance absorbs more light. The units of ? are usually in M-1cm-1 or L mol-1cm-1. A is the absorbance of light by a sample, and in this experiment, it was measured directly by a spectrophotometer.
Experimental 1. Maximum Absorbance Wavelength: For the first part of this experiment, we determined the wavelength at which a selected substance (KMnO4) would absorb best. We obtained 10ml of 0. 0040M KMnO4, and using a graduated 1.
0-ml pipette and a pipetting bulb, transferred 1. 00ml of it into a 25. 0-ml volumetric flask. We filled the flask half full with deionized water, and added 1.
0ml of 3. 0M H2SO4. After mixing the contents, we filled the flask completely with deionized water and mixed them again. After the sample solution was prepared, we calibrated a spectrophotometer using a blank solution; in this experiment, deionized water was used for the blank solution because H2SO4 is transparent in the visible region. For measuring the absorbance of KMnO4, first we selected a wavelength, and placed a cuvette containing blank solution, then replaced it with the other cuvette containing the prepared KMnO4 solution.
The absorbance was measured at the wavelengths between 360nm and 660nm, and each time a new wavelength was selected, the spectrophotometer was recalibrated. After all the absorbance values were measured, we plotted the absorbance data versus the wavelength to determine ? max which was used for the remainder of the experiment. 2. Standard Absorbance Curve: For the second part of this experiment, we examined the effect of varying the concentration on the absorbance. Five differently concentrated KMnO4 solutions were prepared for this part.
For each 25. 00ml sample, 1. 00ml, 0. 80ml, 0. 60ml, 0.
40ml, and 0. 0ml of 0. 0040M KMnO4, 1. 0ml of 3.
0M H2SO4, and deionized water were added. The concentration of each solution was calculated using the dilution equation, M1V1 = M2V2. After a sample was prepared, we set the spectrophotometer to the ? max discovered in first part, calibrated using the blank, and then examined the absorbance of the sample. After all the findings were examined, we made a plot of the absorbance versus concentration.
The molar extinction coefficient could be calculated using the Beer’s Law and the measured results. The inside diameter of the cuvette (path length) was measured with calipers. 3.Concentration of an Unknown: In the last part, we determined the concentration of unknown solution using the results (the path length and the extinction coefficient) determined in the second part.
We obtained a solution of KMnO4 of unknown concentration. We followed the same procedure as for the second part to dilute the unknown solution; added KMnO4, H2SO4, and deionized water to make 25. 00ml sample, and calibrated the instrument with the blank solution at the ? max, and then measured the absorbance. The concentration of the unknown before it was diluted could be determined using the dilution equation in a reverse way.Results & Discussion 1.
Maximum Absorbance Wavelength: In part 1, we determined the sample’s absorbance spectrum to find the wavelength (? max) at which KMnO4 absorbs best. The absorbance data measured in this part is shown in Table 1, and Figure 1 is the absorbance spectrum constructed by plotting A vs ?. Table 1: Data for Maximum Absorbance Wavelength Wavelength360380400420440460480500 Absorbance0. 1450.
0770. 0210. 0080. 0170. 0510. 1320.
253 Wavelength520540560580600620640660 Absorbance0. 3620. 3600. 2460. 1130.
0400. 0300. 0220. 018 Figure 1: Absorbance Spectrum of KMnO4According to the graph, ? max of KMnO4 is about 530nm, and it means that at the wavelength 530nm, KMnO4 absorbs light best. 2. Standard Absorbance Curve: In part 2, we examined the effect of varying the concentration on the absorbance.
We prepared five differently concentrated solutions (the higher concentrated solution appeared darker pink-violet), and using dilution equation, the concentrations were calculated. Figure 2: Color of permanganate in different concentrated solution The calculated concentration and the measured absorbance of each solution are summarized in Table 2.Table 2: Data for Standard Absorbance Curve Volume (ml)Concentration (M)Absorbance 1. 001.
6 x 10-40. 320 0. 801. 3 x 10-40. 252 0.
609. 6 x 10-50. 185 0. 406. 4 x 10-50.
122 0. 203. 2 x 10-50. 057 As shown in Table 2, when concentration decreased, the absorbance value also decreased. A plot of the absorbance verses concentration (Figure 4) resulted a straight line, and according to the equation (3), the Beer’s Law, we knew that the slope(2036.
563) of the line equals ? l. The path length(l) of the cuvette was measured to be 1. 18cm, and the extinction coefficient(? ) at 530nm was calculated to be 1. x 103; The molar extinction coefficient varies with the wavelength of light used in the measurement. Figure 3: A plot of Absorbance vs.
Concentration 3. Concentration of an Unknown: The concentration of the unknown (#15) solution was calculated using Beer’s Law and dilution equation. Table 3 shows the data gathered. Table 3: Data and Calculation for Unknown KMnO4 Concentration TrialVol. of KMnO4 AbsorbanceCon.
after dilution (M)Con. before dilution (M) 11. 0 ml0. 2511.
3 x 10-43. 3 x 10-3 20. 8 ml0. 2121.
1 x 10-43. 4 x 10-3 30. 6 ml0. 1557. 7 x 10-43.
3 x 10-3 The mean concentration before dilution was calculated to 3. x 10-3M. In part 1, the maximum wavelength of KMnO4 was found as 530nm. When MnO4- is dissolved in water, it appears pink-violet, and as shown in color wheel below, it absorbs primarily yellow-green light.
(The color a substance appears to be is directly across the wheel from the color of light that substance has absorbed. ) The range of the wavelength of green-yellow light is between 495nm ~ 590nm, so the maximum wavelength measured (530nm) is reasonable. Figure 4: The Color Wheel The absorbance spectrum shows how the absorbance of light depends upon the wavelength of the light.The spectrum itself is a plot of absorbance vs. wavelength and is characterized by the wavelength (? max) at which the absorbance is the greatest.
The value of ? max is important for several reasons. This wavelength is characteristic of each compound and provides information on the electronic structure of the analyte. In order to obtain the highest sensitivity and to minimize deviations from Beer’s Law, analytical measurements are made using light with a wavelength of ? max. In part 2, we observed that the absorbance was lower in less concentrated solution.Before the experiment was done, we expected that in lower concentrated solution, because fewer number of absorbing molecules would exist, so less light would be absorbed; the expectation was correct. As I mentioned above, the solution appears pink-violet because permanganate absorbs primarily green-yellow light.
The pink-violet color was darker in higher concentrated solution as in figure 2, and it can be explained that more green-yellow light was absorbed, so the violet color appeared darker. In part 3, we measured the absorbance of diluted unknown KMnO4 solution to determine the concentration of it.I got the sample of unknown number 15. We’d already known the value of the path length and the extinction coefficient, so we could calculate the concentration using the Beer’s Law. The solution was diluted to 25ml, so, in order to determine the initial concentration of the unknown, we used the dilution equation in a reverse way.
The absorbance of the unknown was similar to the absorbance of the known sample, so I expected that the concentration would be similar; the known concentration was 0. 0040M, and the unknown concentration determined to be 0. 0033M.