Spectrophotometer

Theory of the method:

Spectrophotometry

Measurement of concentration using a spectrophotometer

Materials:

    • Helios epsilon spectrophotometer
    • Acridine-orange solutions: 2‰, 4‰, 8‰, x‰
    • Distilled water
    • Cuvettes (5) for spectrophotometer
    • Marker

Procedure:

A. Measure the absorbance:

Fig. 1 Helios epsilon spectrophotometer

1. Switch on the spectrophotometer (wait 5 min. before to start, the apparatus will check all the functions)

2. Select the absorbance method (Mode knob)

3. Select the wavelength (two horizontal arrow knobs); the measurements will be performed from 10 to 10 nm from 440 to 540 nm

4. Place the cuvette with distilled water in the spectrophotometer

5. Set the transmittance to zero/ close and 100/ open (Zero knob)

6. Place one by one the cuvettes with acridine-orange solution (2‰, 4‰, 8‰, x‰) in the spectrophotometer and read the absorbance; it is possible to print the results

7. Repeat the steps for all of the other wavelengths

8. Place all of your data in a table (the columns: A, the rows: wavelength)

B. Calculate the unknown concentration of acridine-orange solution

9. Draw a graph A = f(λ)

10. On the same graph draw all of four curves for each acridine-orange concentration

11. Select the wavelength with maximum absorbance for all four solutions

a) Calculate the concentration using the graph method:

12. Draw a graph A = f(c)

13. By intra-, extrapolation calculate the unknown concentration of acridine-orange solution

b) Calculate the concentration using the least square theory:

14. Calculate a and b parameter for the regression line equation:

y = a + b x

A = a + b cx

where,

A = absorbance,

cx = unknown concentration

15. Using the previous formula calculate the unknown concentration using the regression software

C. State two conclusions regarding to your results.