際際滷

際際滷Share a Scribd company logo

Beers law

C
Claire Balase

This document describes using Beer's Law and a colorimeter to determine the molar extinction coefficients of three common food dyes - erythrosin B, erioglaucine, and sunset yellow. Standard curves of absorbance versus concentration were prepared for each dye. The slope of each standard curve plot equals the molar extinction coefficient. The calculated coefficients from this experiment matched well with literature values, demonstrating the accuracy of using Beer's Law and a colorimeter for this type of analysis.

1 of 5
Downloaded 76 times
Beer's Law and Molar Extinction Coefficient Background and Objectives Colorimeters (and spectrophotometers) measure absorbance of light of a specific wavelength by a solution. Absorbance values can be used to determine the concentration of a chemical or biological molecule in a solution using the Beer-Lambert Law (also known as Beers Law). Beers Law states that absorbance of a sample depends on the molar concentration ( ), light path length in centimeters ( ), and molar extinction coefficient ( ) for the dissolved substance at the specified wavelength (了)1 . Beer-Lambert Law: An example of a Beers Law plot (concentration versus absorbance) is shown below. The slope of the graph (absorbance over concentration, / ) equals the molar absorptivity coefficient, 竜 x . The objective of this lab is to calculate the molar extinction coefficients of three different dyes from their Beers Law plot. 1 Path length (distance that light travels through the solution) is determined by the cuvette that the sample is placed in. Most colorimeters and spectrophotometers, including the one in this kit, use cuvettes with a path length of 1 cm. Molar extinction coefficient is a measure of how strongly a substance absorbs light at a particular wavelength, and is usually represented by the unit M-1 cm-1 or L mol-1 cm-1 .
Food dyes are used to color a variety of food products such as sweets, cereal and sports drinks and are often used in high school and undergraduate labs2 . The 3 dyes used in this lab were chosen as they absorb in the range of the colorimeter LED wavelengths. Erythrosin B Erioglaucine Sunset Yellow 2 For example: Sigman and Wheeler 2004, J. Chemical Education 81 (10): 1475-1478; Henary and Russell, 2007, J. Chemical Education 84 (3) 480-482.
Materials The following list of materials is required for this lab. More details can be found in the Appendix (online). Assembled Educational Colorimeter kit from Lab 1 Powdered food dyes erythrosin B, erioglaucine and sunset yellow Analytical scale 3 x 250 mL volumetric flasks 15 x test tubes (>5 mL) 1 mL fixed volume pipette 16 x cuvettes Water Methods This lab uses the Educational Colorimeter Plotting program. Before starting the lab, download the software and review the operation of this program (details online and in your Users Manual). Step 1: Prepare 1 mM stock of dyes Erythrosin B (FW: 879.86): e.g. 0.218 g in 250 mL distilled water Erioglaucine (FW: 792.85): e.g. 0.198 g in 250 mL distilled water Sunset Yellow (FW:452.37): e.g. 0.113 g in 250 mL distilled water Step 2: Preparation of standard curve 1. Dilute the 1 mM stock solutions as shown in Table 1 using a 250 mL volumetric flask. Label these flasks working stock; 2. For each of the 3 dyes, prepare a series of standard curve dilutions as shown in Table 2 using the test tubes. Label tubes #1-5 for each dye; Step 3: Measure absorbance with the colorimeter and plot data 1. Launch the colorimeter plotting program. Calibrate the device with a cuvette containing water as described in Lab 1. 2. Starting with erythrosin B, measure the absorbance for each standard curve solution with the appropriate color channel3 , and enter the corresponding concentration in the program; 3. Once all the samples are measured, click on the Plot button. Repeat measurements for erioglaucine and sunset yellow. Record values for the slope in Table 3. 3 To determine which color channel to use, measure absorbance at all 3 wavelengths as described in Lab 1.
Table 1: Preparation of working solutions Dye Volume of 1 mM stock Concentration of working stock Color channel/ wavelength Erythrosin B 1 mL in 250 mL 4.00 袖M Green/528 nm Erioglaucine 2.5 mL in 250 mL 10.00 袖M Red/625 nm Sunset Yellow 10 mL in 250 mL 40.00 袖M Blue/470 nm Table 2: Preparation of standard curves Tube # Volume of working stock Erythrosin B Erioglaucine Sunset Yellow 1 1 mL + 4 mL H2O 0.8 袖M 2 袖M 8 袖M 2 2 mL + 3 mL H2O 1.6 袖M 4 袖M 16 袖M 3 3 mL + 2 mL H2O 2.4 袖M 6 袖M 24 袖M 4 4 mL + 1 mL H2O 3.2 袖M 8 袖M 32 袖M 5 5 mL + 0 mL H2O 4 袖M 10 袖M 40 袖M Table 3: Molar extinction coefficient Plotted Slope (袖M vs. Abs) Molar extinction coefficient (M-1 cm-1 ) Reported value (Sigma spec sheets) Erythrosin B 0.056 56,000 at 528 nm 82,500 (524-528 nm) Erioglaucine 0.098 98,000 at 625 nm 80,000 (627-637 nm) Sunset Yellow 0.020 20,000 at 470 nm 20,000 (479-485 nm)
Sample Data Fig 1: Image of cuvettes with 3 different food dye standard curves Fig 2: Sample data - Erioglaucine standard curve

More Related Content

Beers law

  • 1. Beer's Law and Molar Extinction Coefficient Background and Objectives Colorimeters (and spectrophotometers) measure absorbance of light of a specific wavelength by a solution. Absorbance values can be used to determine the concentration of a chemical or biological molecule in a solution using the Beer-Lambert Law (also known as Beers Law). Beers Law states that absorbance of a sample depends on the molar concentration ( ), light path length in centimeters ( ), and molar extinction coefficient ( ) for the dissolved substance at the specified wavelength (了)1 . Beer-Lambert Law: An example of a Beers Law plot (concentration versus absorbance) is shown below. The slope of the graph (absorbance over concentration, / ) equals the molar absorptivity coefficient, 竜 x . The objective of this lab is to calculate the molar extinction coefficients of three different dyes from their Beers Law plot. 1 Path length (distance that light travels through the solution) is determined by the cuvette that the sample is placed in. Most colorimeters and spectrophotometers, including the one in this kit, use cuvettes with a path length of 1 cm. Molar extinction coefficient is a measure of how strongly a substance absorbs light at a particular wavelength, and is usually represented by the unit M-1 cm-1 or L mol-1 cm-1 .
  • 2. Food dyes are used to color a variety of food products such as sweets, cereal and sports drinks and are often used in high school and undergraduate labs2 . The 3 dyes used in this lab were chosen as they absorb in the range of the colorimeter LED wavelengths. Erythrosin B Erioglaucine Sunset Yellow 2 For example: Sigman and Wheeler 2004, J. Chemical Education 81 (10): 1475-1478; Henary and Russell, 2007, J. Chemical Education 84 (3) 480-482.
  • 3. Materials The following list of materials is required for this lab. More details can be found in the Appendix (online). Assembled Educational Colorimeter kit from Lab 1 Powdered food dyes erythrosin B, erioglaucine and sunset yellow Analytical scale 3 x 250 mL volumetric flasks 15 x test tubes (>5 mL) 1 mL fixed volume pipette 16 x cuvettes Water Methods This lab uses the Educational Colorimeter Plotting program. Before starting the lab, download the software and review the operation of this program (details online and in your Users Manual). Step 1: Prepare 1 mM stock of dyes Erythrosin B (FW: 879.86): e.g. 0.218 g in 250 mL distilled water Erioglaucine (FW: 792.85): e.g. 0.198 g in 250 mL distilled water Sunset Yellow (FW:452.37): e.g. 0.113 g in 250 mL distilled water Step 2: Preparation of standard curve 1. Dilute the 1 mM stock solutions as shown in Table 1 using a 250 mL volumetric flask. Label these flasks working stock; 2. For each of the 3 dyes, prepare a series of standard curve dilutions as shown in Table 2 using the test tubes. Label tubes #1-5 for each dye; Step 3: Measure absorbance with the colorimeter and plot data 1. Launch the colorimeter plotting program. Calibrate the device with a cuvette containing water as described in Lab 1. 2. Starting with erythrosin B, measure the absorbance for each standard curve solution with the appropriate color channel3 , and enter the corresponding concentration in the program; 3. Once all the samples are measured, click on the Plot button. Repeat measurements for erioglaucine and sunset yellow. Record values for the slope in Table 3. 3 To determine which color channel to use, measure absorbance at all 3 wavelengths as described in Lab 1.
  • 4. Table 1: Preparation of working solutions Dye Volume of 1 mM stock Concentration of working stock Color channel/ wavelength Erythrosin B 1 mL in 250 mL 4.00 袖M Green/528 nm Erioglaucine 2.5 mL in 250 mL 10.00 袖M Red/625 nm Sunset Yellow 10 mL in 250 mL 40.00 袖M Blue/470 nm Table 2: Preparation of standard curves Tube # Volume of working stock Erythrosin B Erioglaucine Sunset Yellow 1 1 mL + 4 mL H2O 0.8 袖M 2 袖M 8 袖M 2 2 mL + 3 mL H2O 1.6 袖M 4 袖M 16 袖M 3 3 mL + 2 mL H2O 2.4 袖M 6 袖M 24 袖M 4 4 mL + 1 mL H2O 3.2 袖M 8 袖M 32 袖M 5 5 mL + 0 mL H2O 4 袖M 10 袖M 40 袖M Table 3: Molar extinction coefficient Plotted Slope (袖M vs. Abs) Molar extinction coefficient (M-1 cm-1 ) Reported value (Sigma spec sheets) Erythrosin B 0.056 56,000 at 528 nm 82,500 (524-528 nm) Erioglaucine 0.098 98,000 at 625 nm 80,000 (627-637 nm) Sunset Yellow 0.020 20,000 at 470 nm 20,000 (479-485 nm)
  • 5. Sample Data Fig 1: Image of cuvettes with 3 different food dye standard curves Fig 2: Sample data - Erioglaucine standard curve