Introduction To The Principle And Application Of General Components Of Spectrophotometer

Spectrophotometer is widely used in biological, chemical, clinical and environmental research.
Spectrophotometry measures how much light a chemical can absorb by passing a beam of light through a sample in a spectrophotometer.
By measuring the detected light intensity, the method can be used to determine the concentration of solute in the sample.
The concept of Spectrophotometry
The beam sent to the sample consists of a photon beam.
When the photons meet the molecules in the sample, the molecules can absorb some of them, reduce the number of photons in the beam and reduce the intensity of the detection signal.
The transmittance is part of the light passing through the sample. It is defined as the light intensity passing through the sample at the incident light intensity.
The absorbance is opposite to the transmittance, corresponding to the amount measured by the spectrophotometer.
According to the absorbance, the concentration of the solution sample can be determined from the bill Lambert law, which shows that there is a linear relationship between the absorbance and the sample concentration. According to the beer Lambert law, absorbance is the product of absorption coefficient, which is a measure of the amount of light absorbed by solute at a given wavelength and the distance through which light passes. Sample, or travel distance, and solute concentration. In general, the aim of measuring absorbance is to measure the concentration of the sample.
Components of spectrophotometer
Each spectrophotometer consists of a light source, a collimator (lens or a focusing device for transmitting strong and straight beams), a monochromator for separating beams of different wavelengths, and a length selector for selecting the wave or groove of the desired wavelength. The wavelength of the light used in the spectrophotometer presented in this video is in the range of ultraviolet and visible light. The spectrophotometer also includes a sample holder, a photodetector and a screen displaying the results of the detector.
The most recent spectrophotometer is directly coupled to a computer, where experimental parameters can be controlled and the results displayed.
Working principle of spectrophotometer
When carrying out spectrophotometry, it is important to take appropriate precautions, such as wearing gloves, depending on the type of biological or chemical solution you use.
Turn on the device and allow the lamp and electronics to heat before measuring the UV-Vis spectrum of the sample.
Prepare a blank sample of the same solution with the same pH and similar ionic strength, but without the components to be analyzed; since the cuvette and solvent can disperse light, it is necessary to analyze the sample.
The traditional spectrophotometer sample holder is designed to hold plastic and quartz bowls. Pipette the original solution into a bowl.
After wiping off any fingerprints and splashing them on the outside of the bowl, insert the cuvette correctly into the sample holder and close the lid door.
Never forget to close the door because UV radiation from an open spectrophotometer can damage your eyes and skin.
Program the desired wavelength or wavelength range that will be transmitted to the sample. This depends on the best wavelength that the component being analyzed can absorb. The machine is then zeroed by measuring the blank sample, which subtracts the bottom absorbance due to the sample buffer.
Depending on the type of spectrophotometry experiment you are doing, it may be necessary to generate a standard curve prior to sample measurement, from which the concentration of the compound analyzed in the sample can be determined.
Allow the sample to reach the proper temperature and mix gently to avoid introducing bubbles. The sample can then be added directly to the bowl inside the machine and a reading can be made.
After the sample has been measured for absorbance, the experiment is properly calculated; for example, to determine the concentration or enzyme activity level.
Application of spectrophotometer
One of the common uses of spectrophotometer is to measure cell density. Cell density measurement can be used to produce the logarithmic growth curve of bacteria, from which the optimal time for the integration of recombinant protein can be determined.
The spectrophotometer can also be used to measure the chemical reaction rate. In this embodiment, the absorbance is used to monitor the enzymatic reaction, which disappears with time through an intermediate reaction of 452nm. The rate of this enzymatic step can be calculated by matching the data with the appropriate equation.
The introduction of micro spectrophotometer eliminates the need of sample holder. These spectrophotometers use surface tension to maintain the sample.
Microspectrophotometer is the best choice to measure the mass and concentration of small and expensive samples, such as biomolecules, including proteins and nucleic acids.
The absorbance of proteins at 280nm depends on the content of aromatic side chains found in tryptophan, tyrosine and phenylalanine, as well as the disulfide bond between the two cysteines.
The concentration of protein can be determined by its absorbance at 280nm and its absorption coefficient, which is based on the composition of amino acids.
DNA and RNA have the maximum absorbance at 260nm, from which their concentrations can be determined. The purity of nucleic acids can also be evaluated from the ratio of absorbance readings to specific wavelengths.