COLORIMETER Principle, Procedure & Steps for B.Sc. Biotech/Botany Sem-2
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A colorimeter is a photometric device used to establish the concentration of a solution by measuring its absorbance of light at specific wavelengths, adhering to Beer’s and Lambert’s laws. The instrument employs an optical system, filters, and a photocell to produce results that indicate the relationship between light absorbance and concentration. It finds applications in various fields including food processing, pharmaceuticals, and water purity assessment, though it has limitations in measuring colorless substances and requires careful operational precautions.
COLORIMETER Principle, Procedure & Steps for B.Sc. Biotech/Botany Sem-2
- 1. WHAT IS COLORIMETER ? Colorimeter is works on principle of photometry A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light. Principle Light from a broad spectrum LED is focused through an unique optical system a filter test tube to the photocell. Output from the photocell is fed into an amplifier and the output of the amplifier to the Digital Panel Meter (DPM). A set of filters, enabling a wide range of range of analytical tests, are mounted on a turret so that the required filter can easily be selected. The operation of the unit is very simple, the user needs to adjust display 0.00 OD or 100% T with blank and then take the reading of the identified standard / sample with Auto Zero facility. The microprocessor based unit when opted for, provides multiple facilities within the same compact size with Auto Zero facility It is a photometric technique which states that when a beam of incident light of intensity Io passes through a solution, the following occur: • A part of it is reflected which is denoted as Ir • A part of it is absorbed which is denoted as Ia • Rest of the light is transmitted and is denoted as It Therefore, Io = Ir + Ia + It To determine Ia the measurement of Io and It is sufficient therefore, Ir is eliminated. The amount of light reflected is kept constant to measure Io and It. Beer’s & Lambert’s Law
- 2. • The amount of light absorbed or transmitted by coloured is in accordance with the Beer’s & Lambert’s Law. • Beer’s law : It states that the intensity of the colour is directly proportional to the concentration of coloured particle in the solution. • Lambert’s Law :It states that the amount of the light absorbed by a coloured solution depends on the length of the column or the depth of the liquid through which light passes. • The Beer & Lambert Law combines these two laws. Beer’s law: According to this law the amount of light absorbed is proportional to the solute concentration present in solution. Log10 Io/It = asc where, as is absorbency index c is the concentration of solution Lambert’s law: According to this law the amount of light absorbed is proportional to the length as well as thickness of the solution taken for analysis. A = log10 Io/It = asb Where, A is the test absorbance of test as is the standard absorbance b is the length / thickness of the solution
- 3. FUNCTION OF A COLORIMETER Color is the combination of wavelengths of varying strength to produce a sum light frequency. For example, the color white is the equal presence of all wavelengths across the visible light spectrum. The basic function of a colorimeter is to determine what quality of color is emitted from solution. In colorimetric determinations A specific reagents are used which react with the specific component and form a colored complex. The concentration of the colored complex is directly proportional to the concentration of the component in the specimen. That colour density absorbed specific spectum of light and rest of light get transmitted from speciment. That transmitted light is detected by colorimeter detector. According to following formula, Optical density is calculated. O.D. = 2 – log %T O.D. is directly proportional to concentration of substance. FUNCTION OF EACH COMPONANT Light source Two kinds of lamp. 1. Halogen Deuterium • for measurement in the ultraviolet range 200 – 900 nm 2. Tungsten lamp • for measurement in the visible 400 – 760 nm and near-infrared ranges
- 4. Instrumentation of Colorimeter • Light Source: The source of light should produce energy with enough intensity to cover the entire visible spectrum (380-780 nm). Commonly, Tungsten lamps are used as a light source for measurement in the visible spectrum and near-infrared ranges. Halogen deuterium is suitable for measurement in the UV range (200-900 nm). • Slit: It reduces unwanted or stray light by allowing a light beam to pass through. • Condensing lens: Parallel beam of light emerges from condensing lens after the light passes through slit incidents on it. • Monochromator: It filters the monochromatic light from polychromatic light, which absorbs unwanted light wavelengths and permits only monochromatic light. These are of three types: prism, grating, and glass. • Prism: It facilitates the refraction of light when it passes from one medium to another. • Glass: It selectively transmits light in certain ranges of wavelengths. • Gratings: These are made of graphite, which separates light in different wavelengths.
- 5. • Cuvette (Sample cell): The monochromatic light from the filter passes through the colored sample solution placed in the cuvette. Their sizes range from square, and rectangle to round and have a fixed diameter of 1cm. These are of three types based on the substances these are made of: Glass, Quartz, and Plastic cuvette. • Glass cuvettes are cheap and absorb light of 340 nm wavelength. • Quartz cuvettes facilitate entry of both lights of UV and visible ranges. • Plastic cuvettes are cheaper, easily scratched, and have shorter lifespans. • Photocell (Photodetector): These photosensitive devices measure light intensity by converting light energy into electrical energy. • Galvanometer: The electrical signal generated in a photocell is detected and measured by a galvanometer. It displays optical density (OD) and percentage transmission.
- 6. Types of Colorimeters Several types of colorimeters are as follows: 1. Densitometers: Determine the density of a material. 2. Spectrophotometers: Measure the spectral reflectance and transmittance of a surface. 3. Tristimulus colorimeter: Employed to measure the tristimulus values of a color. Colorimeter Operating Procedure 1. Switch the device on by rotating the Power Switch knob in a clockwise direction (toward the right). 15 minutes of warming up time for the colorimeter is required to stabilize the light source and the detector. 2. After the warm-up period, turn the Wavelength Control knob to the appropriate wavelength. 3. Press the MODE control key until the light next to “Transmittance” turns on to switch the display mode to transmittance. 4. Use the Zero Control knob to set the display’s T-factor to 0.0%. Make this adjustment while ensuring the sample chamber is empty and the cover is securely closed. 5. Place the blank solution in a cuvette until it reaches the top of the triangle on the side of the cuvette. To get rid of any fluids or fingerprints on the cuvette’s exterior, wipe it with a Kimwipe. Both will obstruct the light’s ability to travel and result in inaccurate readings. 6. Place the tube gently but completely into the cuvette chamber, with the vertical guide line facing in the direction of your right. The guideline on the cuvette should now be lined up with the guideline on the sample chamber by rotating the cuvette 90 degrees in a clockwise orientation. This method protects the cuvette against scratches in the light- transmitting portions. Erroneous measurements can result from scratches on the cuvette. Close the compartment’s cover.
- 7. 7. Set the display to 100.0% using the Transmittance/Absorbance control knob. 8. Press the MODE control key and switch the Status Indicator light to read Absorbance. The display should indicate 0.0 if the Transmittance calibration was done correctly. No further adjusting is necessary. Use the Transmittance / Absorbance control knob to set the display to 0.0 if it does not already show that value. Switch the display back to Transmittance using the MODE key. 9. Reverse the previous process to remove the cuvette from the compartment by rotating it 90 degrees counter-clockwise before doing so. You should put the solution whose absorbance you want to test in another cuvette. Similar to before, place it inside the chamber. a. Directly from the digital display, read the %T value. b. Select Absorbance using the MODE key, then take the A value directly from the digital display. Select Transmittance once more. 10. Reverse the process you used to insert the cuvette to remove it from the sample compartment. Close the compartment’s cover. Applications of Colorimeter • It is employed in the printing industries to evaluate the caliber of printing ink and paper. • These are used in the food and food processing industries. • It is frequently used in laboratories and hospitals to determine the biochemical composition of samples like blood, urine, cerebral spinal fluid, plasma, serum, etc. • It is used in the textile and paint industries. • Diamond dealers also examine the visual characteristics of priceless stones using a colorimeter. • The instrument is also employed in cosmetology to measure the UV protection level of skin-care products. • They are used to evaluate the water’s purity and screen for the presence of chemicals like cyanide, iron, fluorine, chlorine, molybdenum, etc.
- 8. • They are employed to evaluate the color contrast and brightness of screens on mobile devices, computers, and televisions to give people the greatest viewing experience. • A colorimeter is also employed in the pharmaceutical sector to spot inferior goods and medications. • Blood samples are tested using a colorimeter to determine the amount of hemoglobin present. • Disadvantages of Colorimeter • The procedure of determining the concentration of colorless substances becomes laborious. • Colorimeter does not function in the ultraviolet or infrared spectrum since it only measures wavelength absorbance in the visible range of light (400nm to 700nm). • A spectrum range must be set rather than a specific wavelength to measure the absorbance. • Measurements might be challenging on surfaces that reflect light. • Precautions • It is best to keep the instrument’s external environment stable while measuring. For instance, it’s best to avoid flashing ambient light while measuring. • Avoid allowing liquids, powders, or solid foreign particles to enter the measuring caliber or within the device. Avoid making contact with the instrument and colliding with it. • It should not be utilized in humid environments or around water mist. To prevent damage to the instrument, it should be stored in a dry, cool atmosphere. • A clean set of cuvettes should be used. Fill the cuvettes to the top of the triangular mark or two-thirds full. For the blank solution, always use the same cuvette; for the sample, always use the other cuvette. • For heat to dissipate and wires and sockets to be easily accessible, we need ample space (at least three inches) surrounding the unit.