Recommended tools and devices: spectrophotometer Spekol, cuvettes, set of chromatic solutions varying in concentration, distilled water

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1 16_Spectrophotometry.xlsx/Work procedure Study group: Year of study: Objective: Establish the unknown concentration of a solutionusing absorption of light. Tasks: 1. Measure the absorbance and transmittance of solutions with different known concentrations using the spectrophotometer Spekol.. Draw calibration curves expressing the dependence of absorbance and transmittance to the concentration of solutions. 3. Measure the absorbance and transmittance for a solution of unknown concentration. 4. State the unknown concentration of the solution from both calibration curves due to measured absorbance and measured transmittance. Compare the results. 5. Explain the obtained results and possible differences. Recommended tools and devices: spectrophotometer Spekol, cuvettes, set of chromatic solutions varying in concentration, distilled water Procedure: 1. Work alone.. Turn the light source of spectrophotometer on. 3. Adjust the screw of monochromator in order to set the appropriate wavelength λ (if yellow colour solutions are used, select λ = 49 nm). 4. Check the cleanness of the cuvettes. Do not touch them on glossy surface, only on the rough ones. If needed, clean the glossy surface. 5. Calibrate the spectrophotometer: a) Switch the light shutter (on the bottom left) to the position "". This closes the slit of the monochromatic light. b) Line the needle of the potentiometer (use the adjuster at the right top labeled "") to the % position at the lower scale (transmittance T (resp. ϑ) = %), which equals to absorbance (extinction) A (or E ) =. c) Switch the light shutter to the position "I". This opens the slit of the monochromatic light. d) Fill the cuvette with distilled water and put it into the moveable holder. Shift the holder with cuvette in front of the light slit. e) Line the needle of the potentiometer (use the adjuster at the right top labeled "1") to position 1% at the lower scale (transmittance T, respectively. ϑ = 1%) which equals to the absorbance (extinction) A = (or E = ). f) Repeat the calibration (items a e) once more. 6. Fill next cuvette with the solution of the lowest available concentration. 7. Put the cuvette into the moveable holder and shift the holder (with the cuvette) in front of the light slit. 8. Read the absorbance and transmittance values from the scales. 9. Write the measured values into Table Repeat the measurement (items 7 9) for the solution with the next lowest concentration. Before each measurement, repeat the calibration (item 5). 11. Repeat the measurement (items 7 9) for the solution of the unknown concentration. Before the measurement repeat the calibration (item 5). 1. After the measurement, rinse the cuvettes with distilled water and let them drip /5

2 16_Spectrophotometry.xlsx/Work procedure Study group: Year of study: 13. Draw graphs calibration curves of both dependencies (solution concentration dependence of transmittance and of absorbance). Use only the known concentrations of solutions. 14. Read the unknown concentration from both calibration curves according the measured values of transmittance and absorbance. Compare the obtained values. 15. Fill all entries in the "Results" (to verify the equivalence of the established values of transmittance and absorbance, you can use Figure 1). 16. Explain the results and discuss possible differences. Figure 1: Comparison of transmittance and absorbance. Applied equations: Lambert Beer's law: 1 where ε is the molar absorption coefficient for a given wavelength, c is a molar concentration and x is a total where I is the entering light intensity, I is the light intensity after passing through the layer % 1 % Note Orange painted cells have to be filled in BEFORE your arrival on practical training. Green painted cells have to be filled during the measurement, or later during protocol processing. Parts of the procedure denoted in italics designate your homework processing measurement results at home /5

3 16_Spectrophotometry.xlsx/Measurement sheet Study group: Year of study: Date of the measurement: I have studied the theory for practical training from: Time of the measurement: (specify a complete bibliographical reference including appropriate chapter and page designation) Used devices and tools: Microclimatic conditions of measurement: atmospheric pressure p = mmhg = kpa air temperature t = C relative humidity H rel = % Object of measurement: Characteristics of examined solutions: Measurement results: Table 1: Measured values of absorbance and transmittance for differenct solution concentrations. Measurement no Concentration [mmol l 1 ] unknown Absorbance Transmittance [%] 1 Date and teacher's signature: /5

4 16_Spectrophotometry.xlsx/Results processing Study group: Year of study: Date of the measurement: Used devices and tools: Time of the measurement: 1 1 Microclimatic conditions of measurement: 1 atmospheric pressure p = kpa room air temperature t = C relative air humidity: H rel = % Object of measurement: Characteristics of examined solutions: 1 Measurement results: Table 1: Measured values of absorbance and transmittance for differenct solution concentrations. 3 Measurement no Concentration [mmol l 1 ] unknown Absorbance Transmittance [%] 1 The unknown concentration of the solution determined according to Chart 1 and Chart (in protocol attachment) from the absorbance curve (c A ) and transmittance curve (c T ): c A = mmol l 1 c T = mmol l 1 Discussion: Physical principle of spectrophotometry. 3 Evaluation of measurement conditions, microclimatic factors and possible sourcesof measurement errors. Comparisson of particular collected values of examined solution concentration /5

5 16_Spectrophotometry.xlsx/Results processing Conclusion: Results summary and biophysical interpretation. Possible application of spectrophotometry inmedicine. 3 References: Cite references in the correct form. NOTE: Obligatory protocol attachments are two hand drawn graphs of dependence of transmittance (absorbance) to solution concentration. *Optional protocol attachment is a computer processed graph of the dependencies stated above with labeled unknown concentration. 1 4 Score: Date and teacher's signature: Overall max. 33 Bonus max /5