OBJECTVES: EXPERMENT #3 A Beer's Law Study To operate a Spectronic 20 To convert from percent transmission to absorbance units To plot absorbance versus wavelength and find max To plot absorbance versus concentration and demonstrate Beer's Law To find the slope of the Beer's Law plot at max Colorimetry or colorimetric analysis follows the variation in color intensity (absorbance) of a solution as the absorbing species changes in concentration. A solution has a color because the solute or solvent absorbs part of the visible region of the electromagnetic spectrum: the nonabsorbed part of the spectrum passes through the solution. The color passing thorough is complimentary to the color absorbed. (The concept of complimentary colors may be explained in more detail in your textbook.) The absorption of any type of electromagnetic radiation follows Beer's Law. This states that the absorbance is directly proportional to the concentration of the absorbing species. This is described mathematically by equation (1), A = bc (1) where A is the absorbance, is the molar absorptivity or molar extinction coefficient, b is the length of the path (sample) through which the radiation travels, and C is the molar concentration. Unless otherwise specified the standard value for b is 1.00 cm, and the unit of is M -1 cm -1 ; A has no units. A plot of A (vertical axis) versus C (horizontal axis) is a straight line passing through the origin whose slope is b (see Figure below). By measuring the absorbances for a series of solutions of known concentrations, the straight line can be determined. From the measured absorbance of an unknown solution, the concentration of the absorbing species can be determined as shown by the dotted line in Figure. While the human eye can detect differences in color intensity reasonably well, quantitative measurements are made with a spectrophotometer. A schematic of a typical spectrophotometer is shown on below in Figure. A beam of light from a source is focused onto a device (monochromator) that disperses the beam into its various wavelengths and selects one of these wavelengths to pass through the sample. A detector measures the intensity of this monochromatic radiation and compares it with the intensity of the radiation striking the sample. This data is then displayed on an analog or digital readout. Figure : Linear Relationship Between Absorbance and Concentration -- Beer's Law Light Source Monochromator Sample Cell Detector Display Figure : Schematic Representation of a Spectrophotometer 23 P a g e
EXPERMENT #3 A BEER'S LAW STUDY The instrument does not actually measure absorbance. The electronic components compare the intensity of the radiation striking the sample, o, to the intensity of radiation passing through the sample, t. The transmittance, T, is defined according to equation (2), and the per cent transmittance is shown in equation (3). t T = (2) t %T = x 100 (3) o The absorbance, A, is defined by equation (4). o A = log (4) t Manipulating the above equations results in the relationship between absorbance and transmittance, shown in equation (5). A = 2.00 - log(%t) (5) While Beer's law measurements can be made at any wavelength, error is kept to a minimum and sensitivity at a maximum by using a wavelength at which the absorbance is a maximum. n Part of this experiment a plot of absorbance versus wavelength will be constructed to find the wavelength of maximum absorbance. Using this wavelength, a plot of absorbance versus concentration (a calibration curve) will be constructed in Part. n Part the calibration curve determined in Part will be used to determine the concentration of an unknown solution. (f your calculator has the ability to perform a linear regression analysis and you would like to learn how to use this function, consult your instructor. The slope calculations become trivial.) o NOTE: The material you will be working with is an acid-base indicator that has a well defined color at a ph of 6.86. The indicator and the dilution solutions (a ph 6.86 phosphate buffer solution) are non-hazardous and can be disposed by flushing down the sink. The indicator is the same color as the complex ion Cu(NH3)4 2+ and will subsequently be referred to as copper() ion. 24 P a g e
EXPERMENT #3 A BEER'S LAW STUDY PROCEDURE: (Work in pairs.) Spectronic 20 Operating nstructions 1. Turn power control switch clockwise until you hear the click. Allow instrument to warm up for a least 10 minutes. 2. Select a wavelength. 3. With no sample present and the cover to the sample-holder closed, turn zero-adjust to place the analog or digital display to zero per cent transmittance. 4. Fill cuvette to half with a blank solution (dilution solution in this experiment), place in sample holder, align the mark on cuvette with mark on sample-holder, and close the cover. Adjust the light control knob to place the display at 100% transmittance. 5. Fill the cuvette to half with sample, carefully wipe the outside of the cuvette with a Kimwipe, place in sample holder, align the mark on cuvette with mark on sample-holder, and close the cover. Read the percent transmittance from the display, then calculate absorbance. 6. Remember, when you change wavelengths, steps 3 and 4 must be repeated. t is also a good idea to repeat these steps when working at a single wavelength for a prolonged period of time. 7. The following WEB site contains useful information on the operation of a Spectronic 20. http://genchem.chem.wisc.edu/labdocs/ cell chamber wavelength selector on/off and zero adjust 100% adjust 25 P a g e
EXPERMENT #3 A BEER'S LAW STUDY Part 1. Locate the stock copper() ion solution and record its concentration on your data sheet. 2. Obtain 500 ml of dilution solution in a clean dry flask or beaker. 3. Prepare a series of 10 reference solutions of known concentrations as follows. For reference solution 1, dispense exactly 1.00 ml of the copper() stock solution, using a 1.00 ml volumetric pipet, into a 50 ml volumetric flask. Fill each flask to the calibration line of with the dilution solution. 4. Repeat this procedure using the proper volumetric pipet (or pipet combination) to dispense 2.00. 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00, and 10.00 ml of the copper() solution into a 50 ml volumetric flask. Fill each flask to the calibration line with the dilution solution. Clearly label each flask. 5. For measuring transmittance you only need one cuvette. Rinse the cuvette with several small portions of the solution to be measured and discard the rinsings. Then put about an inch and a half of solution in the cuvette and record the transmittance on your data sheet. Remember to wipe the cuvette clean with a Kimwipe each time you take a measurement. When placing the cuvette in the holder, line up the white marking on the cuvette with the raised plastic indicator on the sample holder. Part 6. Using the solution prepared from 5.00 ml of the copper() stock solution measure the transmittance over the range from 360-700 nm in 10 nm intervals. At each wavelength adjust the 0% and 100% transmittance as described in the Spectronic 20 Operating nstructions. Calculate the absorbance for each transmittance as described in equation (5). Plot absorbance versus wavelength on graph paper and choose the wavelength of maximum absorbance. Part will be performed at this wavelength. PART 7. At the wavelength determined in step 6 measure the transmittance for the remaining reference solutions. Remember to set the 0% and 100% scales whenever you change wavelength. Make a Beer's Law plot and determine its slope. Part V 8. Obtain an unknown, measure its transmittance, and determine its concentration from the calibration curve in Part. 9. Clean-up: All solutions can be flushed down the sink. Thoroughly rinse the each pipet with deionized water and replace in the pipet rack. 26 P a g e
NAME Section Date Data and Observations: A Beer's Law Study Concentration of stock copper() ion solution: Unknown Solution: Reference Solution ml of Cu() Stock Solution Concentration of Reference Solution %T Absorbance, A at max 1 2 3 4 5 6 7 8 9 10 Unknown Sample calculation of reference solution concentration: 27 P a g e
Determination of max Plot of Absorbance versus Wavelength for Cu() Solution Wavelength %T A Wavelength %T A 360 530 370 540 380 550 390 560 400 570 410 580 420 590 430 600 440 610 450 620 460 630 470 640 480 650 490 660 500 670 510 680 520 690 700 (Record %T to the nearest tenth and circle max in the above table.) 28 P a g e
NAME Section Date Coordinates of the two points selected: Slope of Beer's Law Plot: y 1 : x 1 : ` y 2 : x 2: Calculated Slope: Molar extinction coefficient: Concentration of Unknown from Beer's Law Plot: ndicate on the Beer's Law plot how concentration was determined. 29 P a g e
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NAME Section Date ADDTONAL ASSGNMENT: A Beer's Law Study 1. A stock solution of Cu 2+ ion has a concentration of 2.50 x 10-3 M. Several milliliter portions of this solution are diluted to 50 ml in a volumetric flask. Complete the column headed concentration of diluted solution. Volume of Stock Solution, ml Concentration of Diluted Solution, M %T Absorbance, A 1.00 93.1 2.00 86.7 3.00 80.7 4.00 75.2 5.00 70.0 6.00 65.0 7.00 60.5 8.00 56.4 9.00 52.5 10.00 48.9 2. Calculate the absorbance for each diluted solution from the given %T. 3. Plot absorbance (vertical axis) versus concentration (horizontal axis) for the above data. Calculate the slope of the best straight line through all the points. Remember that (0,0) is also a point. 4. What is the concentration of an unknown solution, if its percent transmittance is 73.8? Show on the graph paper how you arrived at your answer. 31 P a g e
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