UV Spectroscopy.pptx FINAL YEAR SEVENTH SEM

UV VISIBLE SPECTROSCOPY PRINCIPLE, INSTRUMENTATION, APPLICATIONS

1. UV -VIS Spectroscopy Kad Dhanashree R. Modern College of Pharmacy (for ladies) Moshi , Pune spectrophotometer

2. Abstract Spectroscopy is the study of interaction of electromagnetic radiation with matter. Basic principle of spectroscopy is the Beer-Lambert’s law. In UV-VIS Spectroscopy, a continuum range of wavelengths from 200nm to 800nm is used. Spectrophotometer device is used in UV-VIS Spectroscopy. See figure1. Components of spectrophotometer Sources of light( 200nm to 800nm). Monochromator. Sample solution in cuvette. Detectors. Readout devices. Types of spectrophotometer Single and double beam instruments. Applications of UV-VIS Spectroscopy Qualitative &Quantitative analyses. FIG 1 .Spectophotometer.

3. INTRODUCTION Spectroscopy is the branch of science which deals with interaction of electromagnetic radiation with materials. In other words it is an analytical method for qualitative and quantitative analysis by use of light. The Lambert Beer law in 1852 made the basis for the quantitative evaluation of absorption measurements.

4. 1.SPECTROSCOPY When an Electromagnetic radiation is incident on a matter, phenomena like reflection, transmission, absorption ,are occurring. Spectroscopy is the study of interaction of electromagnetic radiation with matter based on the Bohr-Einstein frequency relationship E=hv , here h is the proportionality constant called Planck’s constant (6.626 x 10-34 J s) and v is frequency.

5. Measurement of radiation intensity as a function of wavelength is described by spectroscopy, as shown in figure 2. All forms of spectroscopy use part of the electromagnetic radiation to give us information about the materials. FIG.2 .Spectroscopy graph.

6. 1.1 SPECTRUM The spectrum is formed by electromagnetic waves and the wavelength is varies. See figure 3. When a narrow beam of light is allowed to pass through a prism/grating, it is dispersed into seven colors from red to violet and the band is called Spectrum. . See figure 4. FIG.3.Electromagnatic spectrum . FIG.4.Glase prism dispersion

7. 1.2 PRINCIPLE Basic principle of spectroscopy is the Beer-Lambert’s law . 1.2.1 BEER LAW Beer's law stated that absorbance is proportional to the concentrations of the material sample. 1.2.2 LAMBERT LAW Lambert's law stated that absorbance of a material is directly proportional to its thickness (path length).

8. The modern derivation of the Beer–Lambert law combines the two laws and correlates the absorbance to both the concentrations and the thickness of the material. FIG 5. Beer–Lambert law.

9. 2. UV-VISIBLE SPECTROSCOPY Ultraviolet–visible spectrum can be generated when ultraviolet light and visible light(200-800nm) are absorbed by materials. The spectrum can be used to analyze the composition and the structure of the materials. For a particular wavelength in the ultraviolet–visible ranges, the absorption degree is proportional to the components of the materials. Therefore, the characteristics of the materials are quantitatively reflected by the spectrum, which changes with the wave-length. Ultraviolet–visible spectrum consists of an absorption spectrum. An absorption spectrum gives information about the molar absorptivity, concentration of the sample, optical path length.

10. 2.1 INSTRUMENTATION 2.1.1 SOURCE of LIGHT. 2.1.2 MONOCHROMATOR. 2.1.3 SMPLE SOLIOTION in CUVETTE. 2.1.4 PHOTO DETECTOR. 2.1.5 READOUT DEVICE. FIG 6 .Components of spectrophotometer.

11. The monochromator unit consists of : Entrance slit: defines narrow beam of radiation from source. Collimating mirror : (polished surface) collimates the lights. Diffraction grating or Prism (make of quartz): disperses the light into specific wavelength. Focusing mirror: captures the dispersed light & sharpens the same to the sample via exit slit Exit slit: allows the corrected wavelength of light to the sample .

12. Advantages of UV-Vis spectroscopy Versatility : UV-Vis spectroscopy can be used to study a wide range of samples, including liquids, gases, and solids. It can also be used to study samples in different states, such as solutions, suspensions, and emulsions. Non‑destructive : The procedure is nondestructive, allowing the sample to be reused or processed or analysed further. Rapid : Rapid measurements allow for simple integration into experimental methods. Sensitivity : UV-Vis spectroscopy is a highly sensitive technique that can detect even small concentrations of a substance in a sample. Easy to use : The instruments are simple to operate and require minimal training prior to use. Minimal processing : In general, data analysis needs minimum processing, hence requiring minimal user training. Quantitative analysis:  UV-Vis spectroscopy can be used to determine the concentration of a substance in a sample, which makes it useful for quantitative analysis. Non-destructive:  UV-Vis spectroscopy does not damage or alter the sample being studied, making it a non-destructive technique. High-throughput:  UV-Vis spectroscopy can be used to analyze multiple samples simultaneously, which makes it useful for high-throughput applications. Low cost:  UV-Vis spectroscopy equipment is relatively low cost and easy to operate and maintain. Widely available:  UV-Vis spectroscopy is widely available in research and industrial laboratories, making it easily accessible for a wide range of applications. High-resolution:  UV-Vis spectroscopy can be used to study samples at high-resolution, which allows for detailed analysis of the sample. Wide range of applications:  UV-Vis spectroscopy has a wide range of applications, including in analytical chemistry, biochemistry, materials science, and in industries such as Pharmaceuticals, food, water and environment analysis, etc.

13. Major disadvantages of uv-vis spectroscopy are: Not linear for high concentration. Does not work with compounds that do not absorb light at this wavelength region. Generates significant heat and requires external cooling.

14. 2. Pharmaceutical analysis In the pharmaceutical sector, UV-Vis spectroscopy is one of the most prevalent applications. Specifically, analysing UV-Vis spectra with mathematical derivatives enables the resolution of overlapping absorbance peaks in the original spectra to identify individual pharmaceutical drugs. By applying the first mathematical derivative to the absorbance spectra, benzocaine , a local anaesthetic , and chlortetracycline, an antibiotic, can be recognised simultaneously in commercial veterinary powder formulations. By constructing a calibration function for each component, simultaneous quantification of both chemicals was feasible over a concentration range of micrograms per millilitre . 3. Bacterial culture UV-Vis spectroscopy is utilised frequently in bacterial cultivation. To assess cell concentration and monitor growth, 600 nm OD readings are routinely and rapidly acquired at a wavelength of 600 nm. 600 nm is widely employed and recommended because to the optical properties of bacterial growth conditions in which they are cultivated and to avoid injuring the cells when they are needed for further investigation. Applications of UV-Vis spectroscopy UV-Vis has been adapted to numerous situations and purposes such as but not limited: 1. DNA and RNA analysis The rapid verification of the purity and concentration of RNA and DNA is a ubiquitous application. Table 1 provides a summary of the wavelengths employed in their analysis and what they represent. When preparing DNA or RNA samples for downstream applications such as sequencing, it is sometimes crucial to ensure that neither sample is contaminated with the other or with protein or chemicals derived from the isolation procedure.

15. 4. Beverage analysis Another common application of UV-Vis spectroscopy is the determination of specific chemicals in beverages. Caffeine content must be under specified legal limits which can be measured with UV light. Certain types of colourful compounds, such as the anthocyanin present in blueberries, raspberries, blackberries, and cherries, can be easily identified using UV-Vis absorbance by matching their known peak absorbance wavelengths in wine.  Other applications Analytical chemistry:  UV-Vis spectroscopy is used to determine the concentration of a substance in a sample, to identify unknown compounds, and to monitor chemical reactions. Biochemistry : UV-Vis spectroscopy is used to study the structure and function of biomolecules , such as proteins, nucleic acids, and pigments. Environmental science:  UV-Vis spectroscopy is used to monitor the quality of water and air, to detect pollutants and to study the photochemistry of atmospheric gases. Food industry:  UV-Vis spectroscopy is used to measure the concentration of food ingredients, to monitor the quality of food products, and to detect contaminants. Materials science:  UV-Vis spectroscopy is used to study the electronic and optical properties of materials, such as semiconductors, dyes, and pigments. Organic chemistry:  UV-Vis spectroscopy is used to study the electronic structure of organic molecules, to identify functional groups and to study the mechanism of chemical reactions. Medical research:  UV-Vis spectroscopy is used to study the properties of blood, to monitor the level of glucose in blood, and to study the photochemistry of biological systems. Forensics : UV-Vis spectroscopy is used to analyze trace evidence, such as fibers and paint, to identify the source of a sample. Industrial process control:  UV-Vis spectroscopy is used to monitor the progress of chemical reactions in industrial processes, to optimize conditions and to control the quality of the final product.