A colorimeter is a laboratory equipment used to measure the absorbance of light of particular wavelengths in a specific solution. The different solutions absorb light of a different wavelength equal to the concentration of the solution when exposed to light. This is the basis of colorimetry or simply Beer-Lambert’s Law.
A calorimeter is a photometric device with measured light in the visible range of the electromagnetic spectrum. A colorimeter is applicable in areas with less analytical depth because it covers only a few spectral ranges (visible range), unlike other photometric devices. The fields that use it are measuring ink color in forensic science, beverage color analysis in food industries, and yeast and bacterial culture growth in microbial laboratories.
Principle of Colorimeter
The principle of the equipment is colorimetry. Here, solution analysis occurs based on the solution’s absorbance of visible range light. Firstly, the white light beam is passed through the color filter. Then the filtered light passes through the solution through the cuvette. Here the intensity of light leaving the solution will be less than the incident light. The solution is believed to absorb the lost light. The loss of light or absorbance is directly proportional to the concentration of the solution.
Theoretically, the Beer-Lambert law gives the relationship between concentration and light absorbed. The law states that “the light transmitted through a solution changes in an inverse logarithmic relationship to the sample concentration.”
The percent transmittance (%T) readings is converted into an inverse logarithmic form which is called optical density units (OD) or absorbance (Abs), to take measurements in terms of concentration directly as well as linearly.
The formula is as follows;
OD (optical density) or Abs (absorbance) = log (100/ % T)
During colorimetric analysis, the readings obtained for the optical density or absorbance are related directly to the concentration of the solutions (C). Likewise, the optical density is directly proportional to the sample path length (L). Which means;
Abs ∝ CL
or Abs = εCL; here, ε is the molar absorptivity or extinction coefficient.
The used cuvette’s path length in the experiment is fixed and known. Likewise, the value of molar absorptivity is also fixed for a particular molecule. The absorbance is calculated using the %T. In this way concentration of the solution can be easily determined.
Parts of Colorimeter
The essential parts of the colorimeter include a light source, cuvette chamber or sample container, cuvette, filter, detector, and galvanometer.
- Light source: In the colorimeter, it produces light energy of the required intensity throughout the visible spectrum, 380-780 nm. The light source used in the laboratory equipment is a simple tungsten lamp that provides light in the visible range.
- Cuvette chamber or sample container: It is the area in the equipment where cuvettes or container is held. It is present at the topmost part of the equipment.
- Cuvette: The analysis of solutions in a colorimeter requires a container. The container must be transparent, so material like clear plastic and glass is preferred. Cuvettes are fused glass cells, either rectangular or square shaped, having exact path lengths, usually 10 or 20 mm, but larger path lengths are also available. It can be made of plastics or glass and offers precision as known path length is used during measurement. The commonly used cuvettes are 45 mm high and can hold 4 ml of solution. Test tubes can also be used while performing analysis and is a cheaper alternative to cuvettes. But test tubes lack accuracy and precision.
- Filter: The types of filters depend on the company manufacturing colorimeter. It depends on the wavelength, i.e., monochromatic (only one wavelength) or polychromatic (white light). The available options are; gelatin, interference, grating, and prisms.
- Gelatin filters: It is formed by sandwiching a thin layer of colored gelatin between two thin glass plates. These are cost-effective filters but can absorb 30-40% of all incident radiation, decreasing the detectors’ energy throughput.
- Glass filters: Another type of filter is colored glass filters with wide band passes up to 150 nm. Specific wavelengths are achieved by combing different glass filters.
- Interference filter: It comprises many reflecting but semi-transmitting films of silver separated by thin layers of transparent dielectric material. When white light passes through the dielectric layers, multiple reflections appear between the semi-transparent mirrors. Here, some energy from the light rays passes straight through the filter. It is the desired wavelength for analysis. The thickness of the dielectric layer determines the resultant wavelength of the light.
- Grating monochromator: It produces monochromatic light and consists of many parallel grooves placed closely together on a polished surface like steel, glass, or quartz. A typical grating may consist of 500-600 lines/mm, but research-based instruments may contain 1200-2000 lines/mm. When the incident light hits the grating, various wavelengths of white light deflect at different angles.
- Prism: It separates white light into its components. The required spectrum is selected by rotation of the prism. The prism used in colorimeter is made up of glass and operates at the range of 350-800 nm wavelength.
- Detector: It helps in converting the resulting transmitted light rays once it passes the sample container into an electrical signal are the detectors. It is also called photocell. Based on the material used, many types of sensors are used in colorimeters. Some commonly used detectors are selenium photocell, phototube, and silicon photocell.
- Selenium photocell: It is the simplest type of detector and does not require any power supplies for functioning.
- Phototube: It is made up of a glass bulb coated with photosensitive materials like cesium or potassium.
- Silicon photocell: It generates electrons when a photon of light strikes the semi-conductive surface of the silicon photocell.
- Galvanometer: It measures the electric signal generated by the detectors and displays the value in the display area.
- Display: It can be analog or digital. The analog appears like meters and is calibrated in absorbance along with a supplementary percent transmission scale. The digital display indicates an absorbance value identical to the resolution despite the change in the magnitude of data.
Types of Colorimeter
The colorimeter are divided into various types based on its size and the filters used in the instrument. On the basis of size colorimeters are of two types; benchtop and portable colorimeters. Whereas based on the filters used, it is of three types; tristimulus, densitometer, and spectrophotometer colorimeters.
Based on size
- Benchtop colorimeter: It slightly larger in size and require a benchtop for operating. It comes in the wavelength rage 420-660 nm. It is highly accurate and consumes only 1.5 ml of reagent. It is suitable in analysis of compounds in different laboratories.
- Portable/handheld colorimeter: It is a compact device that can be easily carried outside laboratory setting. It is useful in food analysis and water analysis at outdoor settings. The wavelength range provided by general manufacturers are 420-660 nm.
Based on filters
- Tristimulus colorimeters: This type of colorimeters uses three filters to measure the intensity of three primary colors-RGB (red, green, and blue). It is the most common type filter used in the colorimeter.
- Densitometer colorimeters: It has a single filter for measuring intensity of color of certain light. It is useful in measuring density of bacterial and yeast growth.
- Spectrophotometer colorimeters: It uses prism for breaking down white light into different spectral of color. It helps in measuring the spectral distribution of light sources.
Based on the display
- Analog colorimeter: The display area of the analog colorimeter has scale. The upper number scale denotes the transmittance, whereas the lower scale represents the absorbance. The change in the arrow head’s placement indicates the absorbance and transmittance.
- Digital colorimeter: The digital colorimeter displays on an LED screen. The absorbance and % transmittance displays in digits. The digital colorimeter is quickly replacing analog colorimeters.
Operating a Colorimeter
Steps of operating colorimeter
- Select the required filter.
- Calibrate the colorimeter.
- Fill two-thirds of the cuvette with the desired sample solution.
- Slide the lid of the cuvette chamber and place the cuvette with the sample inside it.
- Press the T button or test button to start testing the sample solution.
- Observe the absorbance in the display area.
How to calibrate a colorimeter?
- Fill two-thirds of the cuvette with distilled water.
- Slide the lid of the cuvette chamber and place the cuvette inside it.
- Press the CAL or R button in the colorimeter until the LED light flashes.
- When the LED light stops flashing, the calibration completes, and the absorbance shown in the display should be 0.00 (100% transmittance).
- Now, remove the cuvette and start analyzing test samples.
Things to consider
- Pre-heating the instrument for 5 minutes before use is necessary.
- Calibration after every filter change is a must.
- Fill the cuvette two-thirds to three-fourths for good transmittance.
- Placing the cuvette in the right way is also essential.
- Cover the cuvette with a lid before the experiment decreases the risk of spillage.
Uses of Colorimeter
The colorimeter is used in various fields of science as well as non-science for measuring the concentration of solutions or density of the solution. The following are the uses of colorimeter based on the areas:
- In the clinical laboratory, a colorimeter is used to analyze urine, plasma, serum, and cerebrospinal fluids for biochemical studies.
- Studying the growth density of bacterial and yeast cultures using a densitometer colorimeter is very helpful in the microbiology laboratory.
- The concentration of food preservatives and harmful toxins in food industries is analyzed using colorimeters.
- Quality control in various laboratories, like water quality in the water supply area quality of drugs produced by pharmaceutical industries, is analyzed using a colorimeter.
- In the textile and paint industries, to analyze different colors.
- In forensic science, a colorimeter helps in analyzing different samples.
Advantages of Colorimeter
- It is low-cost equipment.
- A colorimeter is easy to repair and maintain.
- It is a simple instrument to use.
- The handheld colorimeter is very helpful for on-site analysis.
Disadvantages of Colorimeter
- Some surfaces can reflect the light hindering the specificity of the equipment.
- It does not work in ultraviolet and infrared rays.
- The colorimeter is not applicable for colorless substances.
- Product manuals: Omega engineering (no date) Product Manuals | Omega Engineering. LaMotte. Available at: https://www.omega.com/en-us/pdf-manuals.
- A guide to colorimetry – cole-parmer (no date). Sherwood Scientific . Available at: https://archive-resources.coleparmer.com/Manual_pdfs/Sherwood/Chroma%20Colorimeters/Manuals/A%20Guide%20to%20Colorimetry.pdf.
- Colorimeter User Manual – Vernier (no date). Available at: https://www.vernier.com/manuals/col-bta/