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Ultraviolet-visible (UV-Vis) spectroscopy is a widely used technique in many areas of science ranging from bacterial culturing, drug identification and nucleic acid purity checks and quantitation, to quality control in the beverage industry and chemical research. This article will describe how UV-Vis spectroscopy works, how to analyze the output data, the technique's strengths and limitations and some of its applications.
What is UV-Vis spectroscopy?
UV-Vis spectroscopy is an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. This property is influenced by the sample composition, potentially providing information on what is in the sample and at what concentration. Since this spectroscopy technique relies on the use of light, let’s first consider the properties of light.
Light has a certain amount of energy which is inversely proportional to its wavelength. Thus, shorter wavelengths of light carry more energy and longer wavelengths carry less energy. A specific amount of energy is needed to promote electrons in a substance to a higher energy state which we can detect as absorption. Electrons in different bonding environments in a substance require a different specific amount of energy to promote the electrons to a higher energy state. This is why the absorption of light occurs for different wavelengths in different substances. Humans are able to see a spectrum of visible light, from approximately 380 nm, which we see as violet, to 780 nm, which we see as red.1 UV light has wavelengths shorter than that of visible light to approximately 100 nm.
How does a UV-Vis spectrophotometer work?
Light source : -
As a light-based technique, a steady source able to emit light across a wide range of wavelengths is essential. A single xenon lamp is commonly used as a high intensity light source for both UV and visible ranges. Xenon lamps are, however, associated with higher costs and are less stable in comparison to tungsten and halogen lamps.
Wavelength selection : -
In the next step, certain wavelengths of light suited to the sample type and analyte for detection must be selected for sample examination from the broad wavelengths emitted by the light source. Available methods for this include
Monochromators - A monochromator separates light into a narrow band of wavelengths. It is most often based on diffraction gratings that can be rotated to choose incoming and reflected angles to select the desired wavelength of light. The diffraction grating's groove frequency is often measured as the number of grooves per mm. A higher groove frequency provides a better optical resolution but a narrower usable wavelength range. A lower groove frequency provides a larger usable wavelength range but a worse optical resolution. 300 to 2000 grooves per mm is usable for UV-Vis spectroscopy purposes but a minimum of 1200 grooves per mm is typical.
Absorption filters - Absorption filters are commonly made of colored glass or plastic designed to absorb particular wavelengths of light.
Sample analysis
Whichever wavelength selector is used in the spectrophotometer, the light then passes through a sample. For all analyses, measuring a reference sample, often referred to as the "blank sample", such as a cuvette filled with a similar solvent used to prepare the sample, is imperative. If an aqueous buffered solution containing the sample is used for measurements, then the aqueous buffered solution without the substance of interest is used as the reference. When examining bacterial cultures, the sterile culture media would be used as the reference. The reference sample signal is then later used automatically by the instrument to help obtain the true absorbance values of the analytes.
Detection
After the light has passed through the sample, a detector is used to convert the light into a readable electronic signal. Generally, detectors are based on photoelectric coatings or semiconductors.
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1 окт 2024
