Principle of Spectrophotometer:
A spectrophotometer is an analytical instrument used to measure the absorption or transmission of light by a substance across a specific wavelength range. It operates based on the principle that different substances absorb light at different wavelengths, allowing for the quantitative analysis of various compounds and biochemical parameters.
The basic components of a spectrophotometer include a light source, monochromator, sample holder, photodetector, and display or output device. The spectrophotometer emits light of a specific wavelength, which passes through the sample. The photodetector measures the intensity of light transmitted through or absorbed by the sample, and the results are displayed or recorded for further analysis.
Spectrophotometry: Spectrophotometry measures the absorption or transmission of light by a sample across a range of wavelengths. It quantifies the amount of light absorbed by a substance at specific wavelengths and provides information about the concentration or quantity of the absorbing species in the sample.
Instrument Example: UV-Vis Spectrophotometer (e.g., Thermo Scientific Evolution™ 201 UV-Vis Spectrophotometer)
Use of Spectrophotometer in Biochemical Estimation:
Spectrophotometry is widely employed in biochemical estimation to determine the concentration of specific compounds or biochemical parameters in a sample. The followings are some commonly measured parameters and associated methods:
Protein Concentration: The Bradford method is frequently used to estimate protein concentration. It involves the reaction of protein molecules with a dye (e.g., Coomassie Brilliant Blue) that undergoes a color change upon binding to proteins. The intensity of the resulting color is directly proportional to the protein concentration and can be measured using a spectrophotometer at a specific wavelength (usually 595 nm).
Nucleic Acid Concentration: UV spectrophotometry is commonly used to determine the concentration of nucleic acids (DNA and RNA). Nucleic acids absorb ultraviolet (UV) light at specific wavelengths due to the presence of aromatic bases. The absorbance at 260 nm is measured, and a known extinction coefficient is used to calculate the concentration.
Enzyme Activity: Spectrophotometry is frequently employed to measure enzyme activity based on the change in absorbance resulting from a reaction catalyzed by the enzyme. The reaction produces a product that exhibits a measurable change in absorbance at a specific wavelength. The rate of change in absorbance is proportional to the enzyme activity and can be used to calculate enzyme kinetics.
Enzyme Substrate Concentration: In enzyme-substrate assays, the spectrophotometer is used to monitor the changes in absorbance resulting from the conversion of the substrate to the product. The reaction progress is measured at a wavelength specific to the product or substrate, and the initial rate of the reaction is used to determine the substrate concentration.
Enzyme Inhibition: Spectrophotometry is also employed to study enzyme inhibition by measuring the changes in absorbance resulting from the interaction between an inhibitor and the enzyme. The decrease in enzyme activity is reflected in the change of absorbance at a specific wavelength.
In all these applications, a spectrophotometer provides a quantitative measurement of the absorbance or transmission of light, allowing for the estimation of various biochemical parameters. The concentration of the target compound or parameter is calculated using standard calibration curves or known extinction coefficients, enabling researchers to analyze and compare different samples for research, diagnostics, or quality control purposes.
Fluorescence: Fluorescence is a phenomenon where certain molecules absorb light at a specific wavelength and emit light at a longer wavelength. It involves the absorption of light energy, followed by the emission of light with lower energy (longer wavelength). Fluorescence spectroscopy measures the intensity and characteristics of the emitted light, which can provide information about the chemical structure and environment of fluorescent molecules.
Instrument Example: Fluorimeter or Fluorescence Spectrophotometer (e.g., Horiba Fluorolog®-3 Fluorescence Spectrophotometer)
Colorimetry: Colorimetry involves the measurement of the intensity and characteristics of visible light absorbed or transmitted by a sample. It is primarily used to quantify the concentration of colored compounds or to determine the color properties of substances. The colorimetric analysis relies on the perception of color by the human eye and typically uses standardized color scales or comparisons to known standards.
Instrument Example: Colorimeter (e.g., Hach DR 900 Colorimeter)
These instruments represent commonly used examples for each technique, and various manufacturers offer a range of models with different features and capabilities.
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