Power point presentation on Spectrophotometry and Atomic Absorption Spec.pptx

This power point presentation is prepared by scientist B. Hassan of the department of chemical pathology, school of medical laboratory science, Usmanu Danfodiyo University Sokoto

1. SPECTROPHOTOMETRY AND ATOMIC ABSORPTION SPECTROPHOTOMETRY PRINCIPLE AND ITS BIOCHEMICAL APPLICATIONS (MLC 902: DIAGNOSTIC CLINICAL CHEMISTRY) PRESENTATION BY HASSAN, Abubakar Bello (Adm NO.: 24311226001) SUBMITTED TO Dr. JELANI ISMAILA DEPARTMENT OF CHEMICAL PATHOLOGY SCHOOL OF MEDICAL LABORATORY SCIENCE USMANU DANFODIYO UNIVERSITY, SOKOTO August , 2025 8/19/25 1

2. OUTLINE Introduction Spectrophotometry Principle of spectrophotometry Components of spectrophotometer Types of spectrophotometry Biochemical applications of spectrophotometry Limitation of spectrophotometry Conclusion References 8/19/25 2

3. INTRODUCTION Every compound that is present in the nature has a property to absorb, transmit or reflect light (electromagnetic radiation) at a certain wavelength This property of the compounds helps to measure quantitatively by using spectrophotometric techniques Spectrophotometry is a technique which deals with the measurement of the interaction of light with materials When light falls on a material that can be reflected, transmitted, scattered, or absorbed and at the same time the material on which light has fallen can emit absorbed light with different frequency (Germer et al ., 2014). 8/19/25 3

4. SPECTROPHOTOMETRY Spectrophotometry on the other hand, is a method use to measure how much a chemical substance absorbs light (at one or more wavelengths) by measuring the intensity of light as the beam of light passes through a sample solution This depends on the concentration of that chemical substance and the basic principle is that each compound absorbs light over a certain range of wavelength while Spectrophotometer is an instrument that measures the intensity of light after it passes through a sample solution Depending on the range of wave length of the light source, spectrophotometers can be classified into two different types UV range spectrophotometer: Uses light over the ultraviolet range, and wavelength ranges between 185 - 400 nm Visible range spectrophotometer : Uses a tungsten light range and wavelength ranges between 400 - 700 nm ( Zwinkels , 2015 ) 8/19/25 4

5. SPECTROPHOTOMETRY CONT’D Different types of spectrophotometry is well known and widely used technique to identify and quantify compounds in the field of research as well as in the industrial and clinical laboratories Figure 1. Spectrophotometer device ( Zwinkels , 2015 ) 8/19/25 5

6. PRINCIPLE OF SPECTROPHOTOMETRY The techniques is based on the estimation of light absorbing nature of the substances in the solution to be measured The basic principle of spectrophotometry revolves around 2 Laws i.e the Beer-Lamberts’s laws which relate the absorption of the light to the properties of the materials through which the light is travelling Beer’s Law: August Beer states that the absorbance of a solution is directly proportional to the concentration of a solution (A œ c ) or transmittance of a solution decreases exponentially with increase innthe concentration of the solution. Lambert’s Law: Johann Eienrich Lambert states that the aborbance of a solutions is directly proportional to the thickness of the optical path (light path) i.e (A œ b ) The two Laws combined to form the Beer-Lambert’s law, which forms the basis of the principle of operation for all spectrophotometric measurements Beer-Lambert’s Law states that, the absorbance of a solution is directly proportional to the concentrstions of the solution and the thickness of the path through which light travel or pass (Ochei and Kolhatkar, 2007) 8/19/25 6

7. COMPONENTS OF SPECTROPHOTOMETER The spectrophotometer consist of two devices: Spectrometer which produces a desired range of wavelength of light, they include light source, collimator (lens), monochromator, wavelength selector (slit) Photometer which detects light absorbed by the sample as the light from the slit passess through the solution and then send sigal to the galvanometer or digital display Figure 2 . Basic instrumentation of spectrophotometer ( Upadhyay et al ., 2009 ) . 8/19/25 7

8. COMPONENTS OF SPECTROPHOTOMETER CONT’D LIGHT SOURCE: is the source of illumination for the intrumets that produces a desired range of wavelength and it has two light sources namely Tungsten lamp emits visible light at 400-700nm Deuterium lamp which emits light in the near ultraviolet radiation region 200-380nm MONOCHROMATOR: is a device that can break polychromatic radiation in to components wavlengths through fefraction by a quart prism of diffraction by grating and it consist of the following units Entrance slit Collimating lens (Polished surface) Dispersing system (Prism or grating) Focusing lens or mirror Exil stilt WAVELENGTH SELECTOR (SLIT): This allow a narrow beam of selected monochromatic to pass through the same solutiom, hight transmission and it limit the radiation to be absorbed by a sample ( Upadhyay et al ., 2009 ) 8/19/25 8

9. COMPONENTS OF SPECTROPHOTOMETER CONT’D SAMPLE CONTAINERS (CUVETTES): A cuvette is a small vessel used to hold sample/solution to be analyzed in the light path of a spectrophotometer It may be round, square or rectengular and it is constructed from silica (quartz) or plastic, square or rectengular cuvettes have a plane-parallel optical surface and a constant light path Most cuvettes have a 1.0cm light path and must be kept constant to have absorbance proportional to concentration PHOTOSENSITIVE DETECTORS: The detector convert the transmitted radiants energy into equivalent amount of electrical energy, it is highly sensitive and fast response over a considerable range of wavelength The electrical signal produced by the detector must be directly proportional to the transmitted intensity READ-OUT DEVICES: Electrical signals from the detectors are displayed on the meter or read out system which provide a visual numerical display of absorbance or converted values of concentrations ( Upadhyay et al ., 2009 ) 8/19/25 9

10. TYPES OF SPECTROPHOTOMETRY UV-visible spectrophotometry Infrared spectrophotometry Fourier transform infrared spectroscopy (FTIR) Raman spectroscopy Spectro-fluorometry ( Rand , 1972 ) 8/19/25 10

11. BIOCHEMICAL APPLICATIONS OF SPECTROPHOTOMETRY Qualitative and Quantitative determinations of enzymes assays, proteins and hormones Detection of impurities: used in the identification of pure analytes Pharmaceutical analysis: used in determination of drug concentration Quantification and identification of organic compounds D etermination of micromolar concentrations of substances in blood, urine and other body fluids Identification of compounds: used in various chemical compounds and functional groups in organic molecules IR-S I nvolved in the characterization of nano particles particularly in the study of physicochemical characteristics of drug nanocarriers FTIR has been used for the characterizing the unpredictability in fuel stability of various biodiesel and antioxidant samples FTIR with attenuated total reflectance (ATR) accessory was used in forensic analysis to get biochemical information on postmortem interval estimation based on pericardial fluids of rabbit ( Rojas and Canopavo n, 2005 ) 8/19/25 11

12. BIOCHEMICAL APPLICATIONS OF SPECTROPHOTOMETRY CONT’D Molecular diagnosis of cervical cancer: Raman spectroscopy is powerful tool in biochemical investigation, particularly in identifying and characterizing the structure of biomolecules, cells, and tissues, this approach is very helpful for the scientists to diagnose the malignant neoplasm which leads to the cervical cancer In agriculture, food and biosystems: Raman spectroscopy is often used for early detection of plant diseases In forensic toxicology Spectro-fluorometry is used in identification of 3-D structure of protein In food: fluorescence spectroscopy plays an important role in the determination of numerous food components, adulterants, additives and contaminants The more common applications of spectrofluorometer include qualitative analysis and quantitative analysis ( Rojas and Canopavo n, 2005 ) 8/19/25 12

13. LIMITATIONS OF SPECTROPHOTOMETRY Sensitivity and Selectivity: Spectrophotometry has lover sensitivity compared to some other analytical techniques, making it less effective for detecting very low concentrations of analytes It can be challenging to differentiate between compounds that absorb light at similar wavelengths especially in complex mixtures, this lack of selectivity can lead to inaccurate measurements Interference: Excipients in formulations can interfere with the measurements by absorbing light at the same wavelength as the analyte leading to the inaccurate results Coloured subtances, complexing reagents and oxidizing/reducing agents can also interfere with spectrophotometric measurements Instrumental factors: Misalignments of components or noise within the instrument can also contribute to inaccuracies Light scattering: Suspended solids or bubbles in liquid/solution samples can scatter light there by affecting measurements Stray light: Imperfect wavelength selectors can allow unwanted wavelength to pass through leading to errors Deviation from Beer-Lambert’s Law: At very high concentration or when analyte’s behavior deviates from ideal behaviour 8/19/25 13

14. CONCLUSION Spectrophotometry measures how a chemical substances absorbs light by measuring the intesity of light as the beam passes throug a sample solution and the basic principle is that each compound absorbs or transmits light over a certain range of wavelength. Therefore, the spectrophotometric techniques are very useful in elementary analysis in almost all fields by providing reliable information about elements to be analyzed. 8/19/25 14

15. SOME CITED REFERENCES Germer , T . A . , Zwinkels , J . C, Tsai , B . K. (2014). Theoretical concepts in spectrophotometric measurements. Experimental Methods in the Physical Sciences ; 46 :11-66 . Rand , R . N. (1972). The role of spectrophotometric standards chemistry laboratory. Journal of Research of the Notional Bureau of Standards-A . Physics and Chemistry; 76 ( 5 ):499-508 Rojas , F . S . , CanoPavón , J . M. (2005). Spectrophotometry : Biochemical Applications. Encyclopedia of Analytical Science. 2nd ed. Amsterdam, Netherlands: Elsevier . The Lancet publishers, Elsevier ; P p . 366-72 Upadhyay , A . , Upadhyay , K . , Nath , N. (2009). Biophysical Chemistry (Principals and Techniques). Girgaon, Mumbai, India: Himalaya Pub House Zwinkels , J. (2015). In: Luo R, editor. Light, Electromagnetic Spectrum. Encyclopedia of Color Science and Technology. New York: Springer Science and Business Media ; Pp 204 8/19/25 15

16. OUTLINE Introduction Atomic Absorption Spectrophotometry Principle of Atomic Absorption spectrophotometry Components of AAS Types of AAS Biochemical applications of AAS Limitation of AAS Conclusion References 8/19/25 16

17. INTRODUCTION Atomic absorption Spectrophotometry ( AAS) is an analytical method used for the qualitative and quantitative determination of chemical elements, especially in trace element analysis The process is based upon the absorption radiant energy usually in ultraviolet (UV) and visible region (V) of electromagnetic radiation The process begins with atomization, which is a process that dissociates elements into atoms by heat of several thousand degrees but less than 3000°C When gaseous solution is employed, emission and absorption analysis is obtained, at this temperature, only a small fraction of mono-atomic particles is excited to a higher electronic state (excited state) which emits radiation and revert back to ground state almost immediately because it has a short life span of between 10-8 to 10-9 seconds (Rechard and Jack, 1993) 8/19/25 17

18. ATOMIC ABSORPTION SPECTROPHOTOMETRY Atomic Absorption S pectrometry (AAS) is an analytical technique that measures the concentrations of elements. It is so sensitive that it can measure down to parts per billion of a gram ( μ g/L) in a sample On the other hand, Atomic Absorption Spectrophotometry is an analytical techniques used to determine the concentration of specific elements in a sample by measuring the absorption of the electromagnetic radiation by free gaseous atoms, it is a highly sensitive methods particularly useful for the quantifying trace elements Figure 3. Atomic Absorption Spectroscopy ( Beaty and Kerber, 1993 ) 8/19/25 18

19. PRINCIPLE OF ATOMIC ABSORPTION SPECTROPHOTOMETRY The working principles of atomic absorption spectroscopy are largely based on the Louis deBroglie theory which states the quantized energy of electrons. Further, deBroglie theory was elaborated by Wolfgang Pauli and therefore, the improved deBroglie theory states that no two electrons can share the same four quantum numbers Atoms have valence electrons present in the outermost orbit of the atom, and therefore atoms can be excited when irradiated that creates an absorption spectrum When an atom is excited, the valence electron moves up an energy level. The energies of the various stationary states, or restricted orbits, can then be determined by these emission lines The resonance line is then defined as the specific radiation absorbed to reach the excited state Figure 4. Atomic Absorption Process (Butcher, 2005) 8/19/25 19

20. COMPONENTS OF AAS LIGHT SOURCE: The light source is usually a hollow-cathode lamp of the element that is being measured, lasers are intense enough to excite atoms to higher energy levels and they allow atomic absorption and atomic fluorescence measurements in a single instrument ATOMIZER: Atomic absorption spectroscopy requires the analyte atoms to be in the gas phase. Ions or atoms in a sample must undergo desolvation and vaporization in a high-temperature source such as a flame or graphite furnace SPECTROMETER: It is used to separate the different wavelengths of light before they pass to the detector, this spectrometer used in AAS can be either singlebeam or double-beam LIGHT S EPARATION A ND D ETECTION: In atomic absorption spectroscopy (AAS), spectrometers use monochromators and detectors for uv and visible light ( Haswell, 1991 ). 8/19/25 20

21. TYPES OF AAS FLAME AAS: The techniques requires a liquid to be asprited, aeronized and mixed with combastible gases such as acetylene and air or acetylene and nitrous oxide and the mixture is ignited in a flame whose temperature ranges from 2100 to 2800℃ When metals are exposed to heat, they absorb light that emit from the source and each metals absorb light at a characteristic wavelength Advantage: Short analysis, good precision, easy to used and cheap Limitation: Lack of sensitivity, dynamic range, requires flammable gases and most not contain excessive amount of dissolved solids GRAPHITE FURNANCE AAS: Uses a graphite coated furnance to vaporized the sample which are deposited in a small graphite coated tibe which can then be heated to vaporize and atomize the analyes and the graphite tube are heated using a high current power supply at 3000℃ Advantage: Sample size (as low as 0.5µL), very little or no sample prepartion is needed, high sensitivity due to eaired sample is atomized at one time and free atoms remain in the optical path length , ultra trace analysis is possible and reduced sample volume 8/19/25 21

22. BIOCHEMICAL APPLICATIONS OF AAS Qualitative and quantitative analysis: AAS found to be a very good tool especially for the determination of food quality Determination of metallic elements in biological system: AAS can be used in the estimation of metals in body fluids like in serum, whole blood, apart from urine and in tissues for toxicological investigation in clinical studies Determination of metallic element in food industry, raw materials and impurities like lead which is toxic Determination of Ca, Mg, Na, K in serum. AAS is a sensitive and highly selective spectrometric technique for the determination of many elements at trace and ultra-trace levels Determination of lead in petrol: Tetra ethyl lead ((C2H5) 4 Pb) and tetramethyl lead ((CH 3 ) 4 Pb) are two anti knocking agents used in petrol Mining: By using AAS, the amoutn of materials such as gold in rock can be determined to see whether it is worth mining the rocks to extract the gold Phar maceutical analysis: AAS is used in some pharmaceuticals manufacturing processes ( García and Báez , 2012 ) 8/19/25 22

23. LIMITATIONS OF AAS Spectral interferences: Include absortion by other closely absorbing atomic species, absorption by molecular species close to the spectral line of element of interest and background emission Chemical interference: Sample contain species which forms a thermally stable compound Matrix interference: When a sample is more viscous or has different surface tension than the standard resulting in differences in sample uptake rate Ioniation interference: Excess energy of the flame can lead to excitation of ground state atom to ionic state by loss of electrons there by resulting in depletion of ground state atoms Require high level of operator skills Standard additions of calibration is required more frequently Expensive e.g graphite tube (Skoog et al ., 2016). 8/19/25 23

24. CONCLUSION Atomic Absorption Spectrophotometry is a simple techniques that can give both quantitative and qualitative results that are adquate for clincal analysis, experiments and demonstrations. As these techniques are applicable and useful in areas of biological/medical, agricultural, environmental analysis, pharmaceuticals and food industry. T he techniques produce accurate and less expensive compare to other analytical techniques. 8/19/25 24

25. SOME CITED REFERENCES Beaty, R. D., and Kerber, J. D. (1993). 2nd edition, Concepts , Instrumentation and Techniques in Atomic Absorption Spectrophotometry Norwalk, CT: The Perkin-Elmer Corporation, Pp 96. Richard , D. , B and Jack , D. K. (1993) . 2nd Edition, Concepts, Instrumentation and Techniques in Atomic Absorption Spectrophotometry , The Perkin-Elmer Corporation , Pp 267 Butcher , D . J. (2005). Atomic Absorption Spectrometry : Interferences and Background Correction. Encyclopedia of Analytical Science. 2nd ed . Amsterdam, Netherlands: Western Carolina University, Cullowhee, NC, USA, Elsevier ; P p . 157-163. Haswell, S. J. (1991). Analytical Spectroscopy Library-Volume 5. Atomic Absorption Spectrometry: Theory, Design and Application. Elsevier , Pp 196. García , R. , and Báez , A. P. (2012) . Atomic Absorption Spectrometry (AAS), Atomic Absorption Spectroscopy, Dr. Muhammad Akhyar Farrukh (Ed.), InTech, Available from: http://www.intechopen.com/books/atomic-absorption-spectroscopy/atomic absorption-spectrometry-aas Skoog, D. A., James, H.E., and Crouch, S. R. (2016). Introduction: Atomic absorption and flame spectrophotometry. Principles of instrumentation and method of analysis, Illinois, 6th edition , Pp 978 8/19/25 25

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