Principle of UV Visible Spectroscopy - Explanation, FAQs

Light source:- A constant source capable of emitting light across a wide variety of wavelengths is required for this light-based technique. In both the UV and visible ranges, a single xenon lamp is widely utilized as a high-intensity light source. Xenon lamps, on the other hand, have higher costs and are less stable than tungsten and halogen lamps. A tungsten or halogen lamp is usually used for visible light, whereas a deuterium lamp is commonly used for UV light in devices with two lamps. The light source of the device must switch during measurement since two distinct light sources are required to scan both the UV and visible wavelengths. This switchover usually happens between 300 and 350 nm, when the light emission from both light sources is similar and the transition can be smoother.

*Wavelength selection:- From the wide range of wavelengths radiated by the light source, certain wavelengths relevant to the sample type and analyte for detection must be chosen for sample inspection. The methods used are described below;

(i) Monochromators- Light is separated into a small band of wavelengths by a monochromator. It is most commonly based on diffraction gratings that can be rotated to select the desired wavelength of light by changing the incoming and reflected angles.

(ii) Absorption filters- Colored glass or plastic absorption filters are often used to absorb specific wavelengths of light.

(iii) Interference filters- Also known as dichroic filters, are made up of numerous layers of dielectric material, with interference occurring between the thin layers of materials. These filters can be used as a wavelength selector by removing unwanted wavelengths through destructive interference.

(iii) Cutoff filters- It allows light to pass through either below (short pass) or above (long pass) a given wavelength. Interference filters are routinely used to achieve this.

(iv) Bandpass filters- Type of filter that allows a wide range of wavelengths to pass through and are made by combining short pass and long pass filters.

*Sample analysis:- The light then passes through a sample, regardless of whatever wavelength selector is utilised in the spectrophotometer. Measurement of a reference sample, is referred to as a blank sample. In a cuvette sometimes the same solvent is filled which is used to create the sample. When the sample is used for measurement, the reference is an aqueous buffered solution that does not include the substance of interest. The sterile culture media would be used as a reference while analysing bacterial cultures. The device then uses the reference sample signal to assist in obtaining the genuine absorbance values of the analytes.

*Detection:- After the light has gone through the sample, a detector converts the light into an electronic signal that can be analyzed. Detectors are often made of photoelectric coatings or semiconductors.

UV-Visible spectroscopy is commonly employed in analytical chemistry, particularly for quantitative study of a single analyte. UV-Visible spectroscopy, for example, can be used to do quantitative analysis of transition metal ions. Furthermore, UV-Visible spectroscopy can be used to do quantitative examination of conjugated organic molecules. It should also be noted that in some circumstances, this sort of spectroscopy can be used on solid and gaseous analytes.

The visible spectrum ranges from 400 to 700 nm, while the UV ranges from 100 to 400 nm. Deep UV refers to the wavelength range of 100–200 nm.

The Photomultiplier tube is a common UV-Vis spectroscopy detector. A photo emissive cathode (a cathode that emits electrons when hit by radiation photons), multiple dynodes (a device that emits several electrons for each impacting electron), and an anode make up the device.