17_/Work procedure 1/5 Study group: Year of study: Objective: Based on construction of calibration curves for absorbance and transmittance establish the unknown concentration of a solution using absorption of light. Tasks: 1. Measure the absorbance and transmittance of solutions with different known concentrations using the spectrophotometer Spekol.. Draw two calibration curves expressing the dependence of absorbance and transmittance on the concentration of solutions. 3. Measure the absorbance and transmittance for a solution of unknown concentration. 4. Establish 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 wheel adjuster at the top right labeled "") to the % position at the lower scale (transmittance T (or ϑ) = %), which equals to absorbance (extinction) A (or E ) = on the upper scale. 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 the cuvette in front of the light slit. e) Line the needle of the potentiometer (use the adjuster at the top right labeled "1") to position 1 % at the lower scale (transmittance T, or ϑ = 1 %), which equals to the absorbance (extinction) A = (or E = ) at the upper scale. f) Repeat the calibration (items a e) once again. 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 corresponding scales. 9. Write the measured values into Table 1. 1. Repeat the calibration (item 5). Repeat the measurement (items 7 9) for the solution with the next lowest concentration. 11. Repeat the calibration (item 5). Repeat the measurement (items 7 9) for the solution of the unknown concentration. 31.1.17
17_/Work procedure /5 Study group: Year of study: 1. After the measurement, rinse the cuvettes with distilled water and let them drip. 13. Draw graphs calibration curves of both dependencies (transmittance dependence on solution concentration and absorbance dependence on solution concentration). Use only the known concentrations of solutions. Both calibration curves have to be smooth lines. 14. Read the unknown concentration from both calibration curves according to the measured values of transmittance and absorbance. Draw your reading in each graph. Compare the obtained values. 15. Fill all entries in the "Result processing" (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 of light, c is the molar concentration and x is the total thickness of absorbing layer where I is the incoming light intensity, I is the outgoing light intensity (after passing through the absorbing 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. 31.1.17
17_/Measurement sheet 3/5 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 rel = % Object of measurement: Characteristics of examined solutions: Measurement results: Table 1: Measured values of absorbance and transmittance for different solution concentrations. Measurement no. 1.. 3. 4. 5. 6. Concentration [mmol l 1 ] unknown Absorbance Transmittance [%] 1 Date and teacher's signature: 31.1.17
17_/Results processing 4/5 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: rel = % Object of measurement: Characteristics of examined solutions: 1 Measurement results: Table 1: Measured values of absorbance and transmittance for different solution concentrations. 3 Measurement no. 1.. 3. 4. 5. 6. Concentration [mmol l 1 ] unknown Absorbance Transmittance [%] 1 The unknown concentration of the solution determined according to Graph 1 and Graph (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 absorbance, transmittance, and reflectance of light and their mutual relationship. What form (functional dependency) have theoretical calibration curves of absorbance and transmittance? 3 Explain the Lambert law for radiation absorption in general. Physical principle of spectrophotometry. Evaluation of measurement conditions, microclimatic factors and possible sources of measurement errors. 31.1.17
17_/Results processing 5/5 Study group: Year of study: Comparison of concentration values obtained using both calibration curves (graphs) of absorbance and transmittance for the solution with the unknown concentration. Explain possible differences. Conclusion: Results summary and biophysical interpretation. Possible application of spectrophotometry in medicine. References: Cite references in the correct form. Obligatory protocol attachments are two hand drawn / computer processed graphs: transmittance dependence on solution concentration and absorbance dependence on solution concentration; both with drawn values for examined solution with unknown concentration (max x 5 points). * Bonus points can be given if the graphs are prepared in both versions by hand and by computer 4 (max x points). 1 Score: Date and teacher's signature: Overall max. 36 Bonus max. 6 31.1.17