Colorimeters

1. Colorimeters

2. Definition A colorimeter is an instrument which compares the amount of light getting through a solution with the amount which can get through a sample of pure solvent. Substances absorb light for a variety of reasons. Pigments absorb light at different wavelengths. A cloudy solution will simply scatter/block the passage of light (sometimes a colorimeter is used to monitor the growth of a bacterial or yeast culture). The % transmission or the % absorbance is recorded (you can use either). It is possible to change the color of the light that is used by using filters in the simplest equipment or an "optical wedge" .

3. How the Colorimeter Works Light from a LED light source passes through a Cuvette containing a solution sample, Some of the incoming light is absorbed by the solution. As a result, light of a lower intensity strikes a photodiode.

4. Construction The essential parts of a colorimeter are: a light source, which is usually an ordinary filament lamp an aperture which can be adjusted a detector which measures the light which has passed through the solution a set of filters in different colors filters are used to select the wavelength of light which the solution absorbs the most. Solutions are usually placed in glass or plastic cuvettes.

5. Transmittance

6. Transmittance “T” The amount of light that passes through a solution is known as transmittance T. Transmittance can be expressed as the ratio of the intensity of the transmitted light It to the initial intensity of the light beam Io ? The transmittance formula is: T = It /Io The Colorimeter produces an output voltage which varies in a linear way with transmittance, allowing a computer, calculator, or handheld to monitor transmittance data for a solution.

7. Absorbance “A” The reciprocal of transmittance of the sample varies logarithmically (base ten) with the product of three factors: e, the molar absorptivity of the solution, b the cell or cuvette width, and C the molar concentration A = log(1/T) = e b C The relationship between these two variables is inverse and logarithmic (base 10). It can be expressed as A = log(1/T)

8. T & A

9. T & A

10. Relation between Absorbance and concentration ? Beer’s law

11. Beer’s law Mathematical statement of Beer’s law For a given solution contained in a cuvette with a constant cell width, the Absorbance is proportional to the concentration: This equation shows absorbance to be related directly to concentration and represents a mathematical statement of Beer’s law. b is cuvette path length C is concentration of absorbing substance e is absorptivity (~ substance) In many chemical and biological experiments this law is assumed for making calibration line for determining concentration.

12. To obtain a Beer’s law curve, several standards (solutions of known concentration) are prepared and their absorbance values are determined using a Colorimeter. A graph of absorbance vs. concentration is then plotted. A solution of unknown concentration is placed in the colorimeter and its absorbance measured. When the absorbance of this solution is interpolated on the Beer’s law curve, its concentration is determined on the horizontal axis. Alternatively, its concentration may be found using the slope of the Beer’s law curve.

13. Concentration of unknown solution Cu Cu= Cs Au/As Cu is unknown concentration Au is unknown absorbance Cs standard concentration As is standard absorbance

14. Ranges of A and T for the colorimeterduring calibration For best results our laboratory testing of the colorimeter indicates that absorbance or transmittance values should fall with these ranges: Transmittance (T) 0.28 - 0.90 Absorbance (A) 0.050 - 0.550

15. To get sufficiently reliable results calibration solutions should be used which lie in the flat part of the curve dT% = 1% (i.e. 1% accuracy of the transmission).

16. Determining of the wavelength You can select three LED light colors: red (635 nm), green (565 nm) or blue (470 nm). There are several ways you can decide which of three wavelengths to use: Method 1. Look at the color of the solution. (Remember that the color of solution is the color of light which is not absorbed). use a different color of light that will be absorbed For example: with a blue CuSO4 solution, use the red LED (635 nm).

17. Method 2. Place a cuvette containing the solution in the colorimeter. And check to see which of three wavelengths yields the highest absorbance (low transmittance). Method 3. Directions for most colorimetry experiments express a recommended wavelength. Use the closest of the three wavelengths on the colorimeter. Even if the LED wavelength is somewhat different, a Beer's law curve can usually be obtained at almost any wavelength around the recommended wavelength.

18. Basic Colorimeter Schematic

20. Calibration

23. Example

24. Calculations

25. Automatic CAL….each time before use After powering up the Colorimeter and waiting for a 5 minute warm up, press to select a wavelength. Open the lid, insert a cuvette filled about 3/4 full of distilled water, and close the lid. Make sure that one clear side of the cuvette is lined up with the arrow at the top of the cuvette slot. Press the CAL button and hold it until the red LED begins to flash and then release the CAL button. When the LED stops flashing, the calibration is complete.

29. Maintenance Calibration Adjustment Replacement of burned-out lamp and photo detector Electronic problems are rare