Spectroscopy Principles and Techniques PDF

Okay, here is the converted text from the images into a structured markdown format. # BCH 331 **SPECTROSCOPY** Spectroscopy is the study of the absorption and emission of light and other radiation by matter. It involves the splitting of light (or more precisely electromagnetic radiation) into its...

Okay, here is the converted text from the images into a structured markdown format. # BCH 331 **SPECTROSCOPY** Spectroscopy is the study of the absorption and emission of light and other radiation by matter. It involves the splitting of light (or more precisely electromagnetic radiation) into its constituent wavelengths (a spectrum), which is done much in the same way as a prism splits light into a rainbow of colors. Recently, the definition of spectroscopy has been expanded to also include the study of the interactions between particles such as electrons, protons, and ions, as well as their interaction with other particles as a function of their collision energy. Spectrometry is the measurement of the interactions between light and matter, and the reactions and measurements of radiation intensity and wavelength. In other words, spectrometry is a method of studying and measuring a specific spectrum, and it is widely used for the spectroscopic analysis of sample materials. **Differences between spectrometry and spectroscopy** Spectroscopy is the science of studying the interaction between matter and radiated energy. It's the study of absorption characteristics of matter, or absorption behavior of matter, when subjected to electromagnetic radiation. Spectroscopy doesn't generate any results, it's simply the theoretical approach to science. On the other hand, spectrometry is the method used to acquire a quantitative measurement of the spectrum. It's the practical application where results are generated, helping in the quantification of, for example, absorbance, optical density, or transmittance. **Spectrometers** A spectrometer is any instrument that's used to measure the wavelength and frequency of light, and allows us to identify and analyze the atoms in a sample we place within it. In their simplest form, spectrometers act like a sophisticated form of diffraction, somewhat akin to the play of light that occurs when white light hits the tiny pits of a DVD or other compact disk. Light is passed from a source (which has been made incandescent through heating) to a diffraction grating (much like an artificial Fraunhofer line) and onto a mirror. As the light emitted by the original source is characteristic of its atomic composure, diffracting and mirroring first disperses, then reflects, the wavelength into a format that we can detect and quantify. The basic principle shared by all spectroscopic techniques is to shine a beam of electromagnetic radiation onto a sample, and observe how it responds to such a stimulus. The response is usually recorded as a function of radiation wavelength. A plot of the response as a function of wavelength is referred to as a spectrum. It is possible for a ray of light to be absorbed by some material and simply pass through others without being affected. When a molecule absorbs light, energy is transferred from the ray of light to the molecule. If the frequency of the electronic and magnetic fields of a ray of light match the frequency at which molecules will vibrate, then light will be absorbed. If the frequency does not match, then the light will pass straight through unaltered. Inert molecules whether solid or liquid, appear colored due to the way they modify light illuminating the object. Thus, different objects absorb some wavelengths and reflect others. For example, if a white light passes through a yellow solution, it absorbs all colors except yellow. **Spectroscopic techniques** 1. Absorption spectroscopy 2. Astronomical spectroscopy 3. Atomic absorption spectroscopy 4. Circular dichroism spectroscopy 5. Electrochemical impedance spectroscopy (EIS) 6. Electron spin resonance (ESR) spectroscopy 7. Emission spectroscopy 8. Energy dispersive spectroscopy 9. Fluorescence spectroscopy 10. Fourier-transform infrared (FTIR) spectroscopy 11. Gamma-ray spectroscopy 12. Infrared (IR) spectroscopy/Vibrational spectroscopy 13. Magnetic resonance spectroscopy 14. Mass spectroscopy 15. Molecular spectroscopy 16. Mossbauer spectroscopy 17. Nuclear magnetic resonance (NMR) spectroscopy 18. Photoelectron spectroscopy 19. Raman spectroscopy 20. UV spectroscopy 21. Ultraviolet and visible (UV/Vis) spectroscopy 22. X-ray photoelectron spectroscopy **Spectrophotometry** Spectrophotometry is a measurement of how much a substance absorbs or transmits light. Spectrophotometry is widely used for quantitative analysis in various areas of science and engineering. In biochemistry for example, it is used to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or tissues for clinical diagnosis. There are several variants in spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry which are used for routine measurements in hospitals, petrochemical industry, food industry, water quality (both purity monitoring and purification), control labs, chemical and biological plants etc. Depending on the range of the wavelength of light source, a spectrophotometer can be classified basically into two different types: UV-Visible spectrophotometer: uses light over the ultraviolet range *(185-400nm)* and visible range *(400-700nm)* of electromagnetic radiation spectrum. IR spectrophotometer: uses light over the infrared range *(700-1300nm)* of electromagnetic radiation spectrum. Figure 1 is a diagram illustrating the basic structure of a spectrophotometer. It depicts a light source emitting light through a collimator (lens). The light then passes through a monochromator (prism or grating) and interacts with a sample solution in a cuvette. Finally, the light reaches a detector (photocell), which displays the results on a digital display or meter. **Fig 1: Basic structure of spectrophotometer** A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. It in general, consists of two devices; a spectrometer and a photometer. A spectrometer is a device that produces, disperses and measures light. It produces a desired range of wavelength of light. First a collimator (lens) transmits a straight beam of light (photons) that passes through a monochromator (prism) to split into several component wavelengths (spectrum). Then a wavelength selector (slit) transmits only the desired wavelengths. A photometer is a photo detector that measures the intensity of light. After the desired range of wavelength of light passes through the solution of a sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends a signal to a galvanometer or a digital display. **Principle of Spectrophotometer** The spectrophotometer technique is used to measure light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then on the display device. 1. In the spectrophotometer, a prism (or) grating is used to split the incident beam into different wavelengths. 2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the test solution. The range of the wavelengths of the incident light can be as low as 1 to 2nm. 3. The spectrophotometer is useful for measuring the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength. **Instrumentation of Spectrophotometer** The essential components of spectrophotometer instrumentation include: 4. A table and cheap radiant energy source * Materials that can be excited to high energy states by a high voltage electric discharge (or) by electrical heating serve as excellent radiant energy sources. 5. A monochromator, to break the polychromatic radiation into component wavelength (or) bands of wavelengths. * A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands. **Prisms:** * A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent. * Two types of Prisms are usually employed in commercial instruments. Namely, 600 cornu quartz prism and 300 Littrow Prism. **Grating:** * Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible, and infrared regions. 3. Transport vessels (cuvettes), to hold the sample * Samples to be studied in the ultraviolet (or) visible region are usually glasses (or) solutions and are put in cells known as "CUVETTES". * Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz. 4. A Photosensitive detector and an associated readout system * Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. * Radiation detectors generate electronic signals which are proportional to the transmitter light. * These signals need to be translated into a form that is easy to interpret. * This is accomplished by using amplifiers, Ammeters, Potentiometers, and Potentiometric recorders. **Applications** Some of the major applications of spectrophotometers include the following: * Detection of concentration of substances * Detection of impurities * Structure elucidation of organic compounds * Monitoring dissolved oxygen content in freshwater and marine ecosystems * Characterization of proteins * Detection of functional groups * Respiratory gas analysis in hospitals * Molecular weight determination of compounds * The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations. A spectrophotometer produces a variety of wavelengths because different compounds absorb best at different wavelengths. Once the intensity of light is known after it is made to pass through the cuvette, it could be related to the transmittance *(T)*. Transmittance is the fraction of light that passes through the sample. This can be calculated using the equation; $T = I/I_0$ Transmittance, $T = I/I_0$ $I$ is the light intensity after the beam of light passes through the cuvette and $I_0$ is the light intensity before the beam of light passes through the cuvette. Transmittance is related to absorption by the expression; Absorbance, $A = -log(T) = -log(I/I_0)$ Transmittance is how much of the light that passed through the sample reached the spectrophotometer. Absorbance is how much of the light has been absorbed by one of the chemicals in the solute. The concentration of the sample can be found by using Beer-Lambert Law (also known as Beer’s Law). The law states that there is a linear relationship between the absorbance and the concentration of a sample. It is expressed by the equation; $A = \epsilon c l = log(I_0/I)$ where, $c$= concentration (mol/litre) $l$ = length of light path through the cell (cm) $\epsilon$ = molar absorption coefficient (L mol$^{-1}$ cm$^{-1}$) **Procedure** **Part 1: Preparing the Samples** 1. Turn on the spectrophotometer. * Most spectrophotometers need to warm up before they can give an accurate reading. Turn on the machine and let it sit for at least 15 minutes before running any samples. * Use the warm-up time to prepare your samples. 2. Clean the cuvettes or test tubes. * If you are using cuvettes or reusable test tubes, make sure they are properly cleaned before use. Rinse each cuvette thoroughly with deionized water. * Take care with cuvettes as they can be quite expensive, particularly if they are made from glass or quartz. Quartz cuvettes are designed for use in UV-visible spectrophotometry. * When handling the cuvette, avoid touching the sides the light will pass through (generally, the clear sides of the container). If you accidentally touch these sides, wipe the cuvette down with a kimwipe (which are formulated to prevent scratching the glass). 3. Load the proper volume of the sample into the cuvette. * Some cuvettes have a maximum volume of 1 milliliter (mL) while test tubes may have a maximum volume of 5 mL. As long as the laser producing the light is passing through the liquid and not an empty part of the container, you will get an accurate reading. * If you are using a pipette to load your samples, use a new tip for each sample to prevent cross-contamination. 4. Prepare a control solution known as a blank. * The control solution has only the chemical solvent in which the solute to be analyzed is dissolved in. For example, if you had salt dissolved in water, your blank would be just water. If you dye the water red, the blank must also contain red water. The blank is the same volume as the solution to be analyzed and kept in the same kind of container. 5. Wipe the outside of the cuvette. * Before placing the cuvette into the spectrophotometer you want to make sure it is as clean as possible to avoid interference from dirt or dust particles. Using a lint-free cloth, remove any water droplets or dust that may be on the outside of the cuvette. **Part 2: Running the Experiment** 1. Choose and set the wavelength of light to analyze the sample with. * Use a single wavelength of light (monochromatic color) to make the testing more effective. The color of the light chosen should be one known to be absorbed by one of the chemicals thought to be in the test solute. Set the desired wavelength according to the specifications of your spectrophotometer. * In a classroom lab, the wavelength will likely be given to you. * Because the sample will reflect all light of the same color as it appears, the experimental wavelength will always be a different color than that of the sample. * Objects appear as certain colors because they reflect light of particular wavelengths and absorb all other colors. Grass is green because the chlorophyll in it reflects green light and absorbs everything else. 2. Calibrate the machine with the blank. * Place the blank into the cuvette holder and shut the lid. On an analog spectrophotometer, there will be a screen with a needle that moves based on the intensity of light detection. * When the blank is in, you should see the needle move to the right. Record this value in case you need it for later. With the blank still in the machine, move the needle to zero using the adjustment knob. * Digital spectrophotometers can be calibrated in the same way; they will just have a digital readout. Set the blank to 0 using the adjustment knobs. * When you remove the blank, the calibration will still be in place. When measuring the rest of your samples, the absorbance from the blank will automatically be subtracted out. * Be sure to use a single blank per session so that each sample is calibrated to the same blank. For instance, if you blank the spectrophotometer, then analyze only some of samples and blank it again, the remaining samples would be inaccurate. You would need to start over. 3. Remove the blank and test the calibration. * With the blank removed the needle should stay at 0 (zero) or the digital readout should continue to read 0. Place the blank back into the machine and ensure the needle or readout doesn't change. If the machine is properly calibrated with your blank, everything should stay at 0. * If the needle or readout is not 0, repeat the calibration steps with the blank. * If you continue to have problems, seek assistance or have the machine looked at for maintenance. 4. Measure the absorbance of your experimental sample. * Remove the blank and place the experimental sample into the machine. Slide the cuvette into the designated groove and ensure it stands upright. Wait about 10 seconds until the needle is steady or until the digital numbers stop changing. Record the values of % transmittance and/or absorbance. * The absorbance is also known as the optical density (OD). * The more light that is transmitted, the less light the sample absorbs. Generally, you want to record the absorbance values which will usually be given as a decimal, for example, 0.43. * If you get an outlying result (such as 0.900 when the rest are around 0.400), dilute the sample and measure the absorbance again. * Repeat the reading for each individual sample at least 3 times and average them together. This ensures a more accurate readout. 5. Repeat the test with successive wavelengths of light. * Your sample may have multiple unknown compounds that will vary in their absorbance depending on wavelength. To eliminate uncertainty, repeat your readings at 25 nm intervals across the spectrum. This will allow you to detect other chemicals suspected to be in the solute. **Part 3: Analyzing the Absorbance Data** 1. Calculate the transmittance and absorbance of the sample. * Many modern spectrophotometers have an output of transmittance and absorbance, but if you recorded intensity, you can calculate these values. * The transmittance $(T)$ is found by dividing the intensity of the light that passed through the sample solution with the amount that passed through the blank. It is normally expressed as a decimal or percentage. $T = I/I_0$. * The absorbance $(A)$ is expressed as the negative of the base-10 logarithm (exponent) of the transmittance value: $A = -log_{10}T$. 2. Plot the absorbance values versus the wavelengths on a graph. * The absorbance value is plotted on the vertical y-axis against the wavelength of light used for a given test plotted on the horizontal x-axis. Plotting the maximum absorbance values for each wavelength of light tested, produces the sample's absorbance spectrum and identifies the compounds making up the test substance and their proportions. * An absorbance spectrum usually has peaks at certain wavelengths that can allow you to identify specific compounds. 3. Compare your absorbance spectrum plot to known plots of specific compounds. * Compounds have unique absorbance spectrum and will always produce a peak at the same wavelength every time they are measured. By comparing your plots of unknown compounds to those of known compounds, you can identify the solutes that compose your solution. * You can also use this method to identify contaminants in your sample. If you are expecting 1 clear peak at a specific wavelength and you get 2 peaks at separate wavelengths, you know something is not right in your sample. **Atomic absorption spectroscopy** Atomic absorption is an analytical technique utilizing the principle of spectroscopy for the quantitative determination of chemical elements. **Principle of Atomic absorption spectroscopy** * Atomic absorption spectroscopy utilizes the principle that free electrons generated in an atomizer absorb radiation of different wavelengths. * The free electrons absorb UV or visible light, causing the electrons to transfer to higher energy orbits. * During this process, the absorption spectrum is released, which is detected by the photodetectors. * The absorption spectrum formed allows the quantification of free electrons in the gaseous state of the matter. * The amount of photon (radiation) absorbed results in an absorption spectrum which can then be measured in terms of absorbance. * The absorbance of a sample is dependent on the concentration of molecules in the sample. **Steps of Atomic absorption spectroscopy** * The liquid sample is mixed with a particular volume of spirit which is added to a flask which is then vaporized into a gas by a fuel-rich acetylene-nitrous oxide flame. * A lamp is set with the necessary wavelength as a light source. * The gas formed from the liquid sample is then passed through a detector that detects the absorbance of the atoms in the gas. * A similar process is performed for the detection of absorbance of solvent bank and standard solution. * A graph is plotted for the absorbance against the concentration of the molecules in the sample. Uses of Atomic absorption spectroscopy * Atomic absorption spectroscopy can be used for the quantitative and qualitative determination of metallic elements in biological systems. * This also helps in the detection of metals as an impurity in alloys and other mixtures. * Atomic absorption spectroscopy has been utilized for the purification of environmental samples like water and soil. * Detection of metals in pharmaceutical products and oil products can also be done by this method. **Atomic Emission Spectroscopy** Atomic emission spectroscopy is a method of chemical analysis that uses the intensity of light emitted from a flame, plasma, arc, or spark at a particular wavelength to determine the quantity of an element in a sample. It measures the wavelengths of photons emitted by atoms or molecules while transitioning from a high energy level to a lower energy state. The wavelength of the atomic spectral line in the emission spectrum gives the identity of the element while the intensity of the emitted light is proportional to the number of atoms of the element. The sample may be excited by various methods. **Principle of AES** * When electrons or compounds are heated either on a flame or by an electric heater, they emit energy in the form of light. * The light emitted from the compound is passed into a spectrometer then disperses the light into separate wavelengths. * Each element forms a different atomic spectrum that indicates that an atom can radiate only a certain amount of energy. * Each element emits a set of discrete wavelengths that is characteristic to it based on its electronic structure, and from these wavelengths, the elemental composition of the sample can be determined. **Steps of AES** * The solution containing the sample is heated either in a flame or with an electric heater. * The solvent evaporates first, whereas the finely divided solid particle remains in the center of the flame along with other molecules and ions. * This causes the excitation of electrons which produces the radiation of a specific wavelength. * The radiation is passed through the spectrometer where the monochromator disperses the light into different wavelengths. * Detectors detect the wavelengths in the spectroscope. * A graph of wavelength against the concentration is plotted to determine the concentration of the sample. **Uses of AES** * Emission spectroscopy has wide applications in agricultural and environmental analysis along with industrial analysis for the detection of metals and alloys. * It can also be used in the determination of lead in petrol. * This has been applied for the determination of an equilibrium constant of ion exchange resins.