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Determining Wavelength of Light for Absorbance Measurement

A step-by-step guide on how to determine the wavelength of light to use for measuring absorbance, including preparing standard solutions and creating a calibration line.

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Determining Wavelength of Light for Absorbance Measurement

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  1. STEP 1 – DETERMINE THE WAVELENGTH OF LIGHT TO USE If a solution appears red, it is primarily absorbing its complimentary color, green EX8-1 (of 8)

  2. STEP 1 – DETERMINE THE WAVELENGTH OF LIGHT TO USE ABSORBANCE SPECTRUM – A graph of the absorbance of a solution at different wavelengths EX8-2 (of 8)

  3. STEP 1 – DETERMINE THE WAVELENGTH OF LIGHT TO USE WAVELENGTH OF PEAK ABSORBANCE – The wavelength of light most strongly absorbed EX8-3 (of 8)

  4. STEP 2 – PREPARE STANDARD SOLUTIONS Use a series of Red Dye #40 solutions of known concentrations in order to measure their absorbance's at the peak wavelength to plot absorbance vs. concentration Absorbance Concentration of Red Dye #40 EX8-4 (of 8)

  5. STEP 2 – PREPARE STANDARD SOLUTIONS STANDARD SOLUTIONS – Solutions of known concentration EX8-5 (of 8)

  6. STEP 3 – MAKE A CALIBRATION LINE A@560.0 = mx + b m (slope) = 44.32 b (Y-intercept) = 0.001 correlation = 0.997 C: .0000g/L .0030g/L .0060g/L .0075g/L .0090g/L A: 0.000 0.241 0.478 0.722 0.961 y = mx + b A@560 = (44.32 L/g)C + 0.001 m = Δy ____ Δx = Δ Absorbance _____________________ Δ Concentration = no units ____________ g/L = L/g EX8-6 (of 8)

  7. STEP 4 – MEASURE ABSORBANCE OF YOUR SAMPLE If your diluted Gatorade sample has an absorbance of 1.352, find its concentration A@560.0 = mx + b m (slope) = 44.32 b (Y-intercept) = 0.001 correlation = 0.997 A@560 = (44.32 L/g)C + 0.001 A@560 = (44.32 L/g)c + 0.001 1.352 = (44.32 L/g)c + 0.001 1.351 = (44.32 L/g)c 1.351 = c ____________ 44.32 L/g 0.030 g/L = c EX8-7 (of 8)

  8. STEP 4 – MEASURE ABSORBANCE OF YOUR SAMPLE Calculate the extinction coefficient at this wavelength, with units A = (44.32 L/g)c + 0.001 A = ɛlc + b m m = ɛl m = ɛ ___ b = 44 L/gcm = 44.32 L/g _____________ 1.00 cm EX8-8 (of 8)

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