A spectrophotometer is an important scientific device that divides complicated light into spectral lines. The spectrometer's measurement range normally comprises the visible light region with wavelengths ranging from 380 to 780 nm and the ultraviolet light region with wavelengths ranging from 200 to 380 nm. Because different light sources have varied emission spectra, different luminous bodies can be utilised as the instrument's light source. A tungsten lamp's emission spectrum: light of 380-780nm wavelength emitted by a tungsten lamp is refracted by a prism to produce a continuous chromatogram consisting of red, orange, yellow, green, blue, indigo, and violet; this chromatogram can be utilised as visible light. The spectrophotometer's light source. The spectral range of the spectrometer.

The visible light area of the spectrometer has a wavelength range of 400 to 760 nm, and the ultraviolet light region has a wavelength range of 200 to 400 nm. Because different light sources have varied emission spectra, different luminous bodies can be utilised as the instrument's light source.

The tungsten lamp emission spectrum: a prism is used to refract the light spectrum of 400760nm wavelength emitted by the tungsten lamp light source, resulting in a continuous chromatogram composed of red-orange, yellow-green, blue indigo, and purple; this chromatogram can be used as a visible light spectrophotometer light source.

The absorption spectrum of the substance

If a solution of a certain substance is placed between the light source and the prism, the spectrum displayed on the screen is no longer the spectrum of the light source, and several dark lines appear, indicating the light source emission spectrum of certain wavelengths. The solution absorbs and vanishes. This spectrum is known as the solution's absorption spectrum once it has been absorbed by the solution. various substances have various absorption spectra. As a result, the chemicals in the solution can be identified using the absorption spectrum.

Use of spectrometer

Nucleic acid quantification

The spectrophotometer's most commonly utilised function is nucleic acid quantification. The spectrometer can measure the concentration of oligonucleotides, single-stranded and double-stranded DNA, and RNA in buffer. The absorption wavelength of nucleic acid's maximum absorption peak is 260 nm. Because each nucleic acid's molecular makeup differs, so does its conversion factor. To quantify various forms of nucleic acids, the correct coefficients must be chosen ahead of time. 1OD absorbance, for example, is similar to 50g/ml dsDNA, 37g/ml ssDNA, 40g/ml RNA, and 30g/ml Olig.

After the test, the absorbance value is translated by the aforementioned coefficient to obtain the matching sample concentration. Select the correct programme before the test, enter the volume of the original solution and the diluent, and then test the blank solution and sample solution. However, the endeavour was not without its challenges. The most difficult problem for experimenters may be unstable readings. The larger the shift in absorbance, the greater the sensitivity of the device.

Direct quantification of protein (UV method)

This method involves directly testing the protein at 280nm. If you use the Warburg formula, the photometer will display the sample concentration immediately, or you can use the associated conversion technique to convert the absorbance value to the sample concentration. The protein determination procedure is straightforward: first test the blank solution, then test the protein directly. Because the buffer contains certain impurities, it is usually essential to remove the 320nm "background" information and turn this function on. The absorbance value of A280, like that of the test nucleic acid, must be larger than 0.1A, and the optimal linear range is between 1.0 and 1.5. The direct protein quantification approach is best suited for assessing purer, single-component proteins. The UV direct quantification approach is faster and easier to use than the colorimetric method; nevertheless, it is sensitive to interference from parallel compounds such as DNA; it also has low sensitivity and requires a higher concentration of protein.

Bacterial cell density

A laboratory spectrometer can be used to determine the growth density and period of bacteria. The OD600 method is the industry standard for monitoring the development of microorganisms in liquid cultures. As the blank solution, use the culture solution without bacteria, and then quantify the culture solution with bacteria after the culture. The OD value of the bacterial solution may appear negative at times during the experiment. The color-developing media is utilised, which means that once the bacteria have been grown for a while, they react with the medium and generate a colour change reaction. Furthermore, the analysed samples cannot be centrifuged, and the bacteria must be kept in suspension.