analytical Spectrophotometry lecture.pptx

Chemistry lecture for students

1. Analytical Spectrophotometry Dr . M Farooque

2. Analytical spectrophotometry Analytical spectrophotometry is a technique that uses light absorption to measure the concentration of substances in a solution. It's a versatile tool that can be used in many fields, including chemistry, biology, and manufacturing.

3. Properties of light and its interaction with matter Light is a form of energy that causes the sensation of vision. Different theories on the nature of light were proposed on the basis of the fact that energy can be transferred from one point to another, either by particle motion or by wave motion. Light travels in a straight line. The speed of light is faster than sound. Light travels at a speed of 3 x 108 m/s.

4. Light, as an electromagnetic wave, interacts with matter primarily through reflection, absorption, transmission, and scattering; when light encounters a material, it can bounce off (reflect), be taken in by the material (absorb), pass through (transmit), or be deflected in various directions (scatter), depending on the properties of the light and the material it interacts with.

5. Reflection Refraction Scattering Dispesion Interference Diffraction Total internal reflection polarization Following are the properties of light

6. Reflection of light Reflection of light is when light rays bounce off a surface and change direction. This happens when light waves hit a surface that doesn't absorb the light's energy.

7. Refraction of light The refraction of light is the bending of light rays as they pass from one medium to another, thereby changing the path of the rays. Refraction occurs due to a change in the speed of the light ray or wave.

8. Scattering of light Light scattering is the way light behaves when it interacts with a medium that contains particles or the boundary between different mediums where defects or structures are present.

9. Dispersion of light Dispersion of light is the process of splitting white light into its component colors. This occurs when white light passes through a transparent medium, such as a glass prism.

10. Interference of light Interference of light is a phenomenon that occurs when two or more light waves interact with each other. It can result in the waves' combined amplitude increasing or decreasing.

11. Polarization Polarization is the process of converting non-polarized light into polarised light. The light in which particles vibrate in all various planes is known as unpolarised light. Polarization of light refers to the phenomenon in which waves of light or electromagnetic radiation are restricted to vibrate in a single direction.

12. Electromagnetic radiations electromagnetic radiation" refers to  a form of energy that travels as waves consisting of oscillating electric and magnetic fields .

13. Relation between frequency, velocity and wave number Wavelength (λ) - The wavelength of light is defined as the distance between the crests or troughs of a wave motion. Frequency (f) - Frequency is the number of occurrences of a repeating event per unit time. In the case of light, frequency refers to the number of times a wavelength is repeated per second. The unit used most often to describe frequency is Hz which means "per second" or /s.

14. Wave number (k) - The wave number also known as propagation number or angular wavenumber is defined as the number of wavelengths per unit distance the spacial wave frequency and is known as spatial frequency. Velocity of a wave (v) - The wave velocity is the speed at which the disturbance moves. Wave velocity is sometimes also called the propagation velocity or propagation speed because the disturbance propagates from one location to another.

15. All these are inter-related by the following formulae: λf = c k = 1/ λ v = λf

16. Spectrophotometry Spectrophotometry is a method 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. The basic principle is that each compound absorbs light over a certain range of wavelength.

17. Spectrophotometer is an instrument that measures the intensity of light after it passes through a sample solution. Spectrophotometer device

18. 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 wave length ranges between 185 - 400 nm.  Visible range spectrophotometer: Uses a tungsten light range and wave length ranges between 400 - 700 nm.

19. Note: Notation of wavelength is Lambda (λ) Nanometer: It is the unit of measuring wavelength in nano-meter (nm = 1*10-9 m).

20. The need for a spectrometer is to produce various wavelengths since the absorbance depends on the compatibility of components with different wavelengths. For example, the highest absorption of p-nitrophenol (acid form) occurs at approximately 320 nm, while p-nitrophenolate (basic form) takes place at 400 nm

21. absorption of p-nitrophenol in acidic and basic medium

22. Beer-Lambert Law When a monochromatic light of initial intensity Io passes through a solution in a transparent vessel, some of the light is absorbed so that the intensity of the transmitted light I is less than Io. There is some loss of light intensity from scattering by particles in the solution and reflection at the interfaces, but mainly from absorption by the solution. The relationship between I and Io depends on the path length of the absorbing medium, l, and the concentration of the absorbing solution, c. These factors are related in the laws of Lambert and Beer.

23. If material bodies are exposed to radiation, part of the incident radiation is absorbed, a part is scattered and a part is transmitted. As a result of absorption the intensity of light passing through material bodies, i.e. the intensity of transmitted light, decreases. The fraction of incident light absorbed depends on the thickness of the absorbing medium. Lambert derived a quantitative relationship between the decrease in intensity of a monochromatic light due to the passage through a homogeneous medium of thickness dx and the intensity of light I. This law is known as Lamberts law, and may be stated as The decrease in intensity of light with thickness of the absorbing medium at any point is directly proportional to the intensity of light

24. Limitations of Beer-Lambert law The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of non-linearity of the law occur in the following conditions: deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity scattering of light due to particulates in the sample fluoresecence or phosphorescence of the sample changes in refractive index at high analyte concentration shifts in chemical equilibria as a function of concentration non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band

25. Other limitations include: The electromagnetic radiation should be monochromatic. The light beam should not be scattered. The solution should be diluted

26. single beam double beam Light path One beam of light passes through the sample Two beams of light, one through the sample and one through the reference Calibration Manually switch between sample and reference cuvettes Automatic correction for light loss Accuracy Less accurate due to intensity fluctuations More accurate and reliable due to compensation for fluctuations

27. Single-Beam Spectrophotometer and How Does It Work? A single beam spectrophotometer is a device that measures how much light a substance absorbs. It uses a single beam of light to determine the concentration, purity, and chemical properties of a substance. How it works A light beam passes through a sample holder The intensity of the light is measured before and after passing through the sample The difference in light intensity is used to calculate the concentration of the sample

28. A spectrophotometer must first be calibrated by using standard solutions with the solute of known concentration in the test solution. For this, the Cuvettes are filled with the standard solutions and set in the Cuvette holder of the spectrophotometer. Single-beam spectrophotometers use a single light beam from a light source to illuminate both the sample and the reference sites. The light wavelength that is employed in this situation is chosen by the monochromator.

29. Instrumentation of single beam spectrophotometry

30. The Basics of Single-Beam Spectrophotometer Instrumentation Three separate light sources are frequently utilized to create light with various wavelengths in spectrophotometers. A tungsten lamp is the most typical type of light source used in spectrophotometers for the visible spectrum. Familiar sources of ultraviolet radiation include the deuterium lamp and the hydrogen lamp. The best sources of infrared (IR) radiation are Nernst filaments or globals. The monochromatic light that enters the Cuvette is partially reflected, partially absorbed by the solution, and partly transmitted through the solution before striking the photodetector system. The transmitted light intensity is calculated by the photodetector system and converted into electrical impulses.

31. These electrical impulses are measured by the galvanometer, which then digitally displays the results. The solution under analysis has an absorbance or optical density, which is a digital representation of the electrical impulses. More light will be absorbed with a higher absorption rate, and more light will be transmitted through a solution with a lower absorption rate. It influences the galvanometer reading and reflects the solute concentration in the solution

32. Advantages and Disadvantages Single Beam Spectrophotometer Advantages Disadvantages 1. improve efficiencey 1. It does not account for errors like voltage fluctuations that affect the outcomes 2. Convenient to use 2. Only counts the absorbance of either the sample or the reference blank at one time 3. Simplistic in its design 4. More affordable 5. High sensitivity for detection

33. Double beam spectrophotometry The wavelength range of a double-beam Uv-Vis spectrophotometer is 185 to 1000 nm. This device divides the monochromator’s light output into two beams. Both the reference beam and the sample reading beam are employed. Different areas of the two light beams are illuminated and radiated. The entire set of samples is radiated from the second part, while the first part illuminates the referencestandard.

34. It removes the inaccuracy that results from variations in the light output and the detector’s sensitivity. Reference Beam: This beam is used to measure lamp energy by passing through the reference standard. Sample Beam: To reflect sample absorption, this beam travels through the sample.

35. Instrumentation

36. A substantial amount of radiation must be absorbed by the atom in the double-beam UV-visible spectrophotometer before it can move from the ground state to an excited state and proceed to higher excited states. In the context of spectrophotometry, the term “absorption” refers to the actual physical act of absorbing light, whereas “absorbance” refers to a sample’s capacity to do so. In single-beam and double-beam spectrophotometers, it may also detect the attenuation of the instrument’s lens, which is brought on by absorption. The wavelength of the primary incident light affects the diffraction angles in this device. The apparatus has disks that aid in the formation of the diffraction angles. As a result, measurements of the biochemical components in the sample can be made more accurately.

37. lamps as sources of light In a spectrophotometer, the "lamp" used as a light source is typically a deuterium lamp for ultraviolet wavelengths and a tungsten halogen lamp for visible light wavelengths; meaning most spectrophotometers use a combination of both lamps to cover a wider spectrum of light. The desirable features for light sources are: Uniform light output over specified wavelength range Stability of response over extended time periods Long usage life span Low-cost

38. Deuterium Lamps Deuterium lamps are most common light sources for the UV region ( 160 – 375 nm). Excitation of deuterium gas at low pressure by applying a discharge results in generation of UV radiation and for applications in vacuum UV region special lamps operating down to 115 nm are available. The gas envelope and windows are made of quartz as glass absorbs in this region. Deuterium lamps exhibit superior stability in comparison to the Xenon and Mercury- Xenon lamps .Deuterium lamps also have long service life. However, before operation sufficient warm-up time is necessary and a stable power supply is necessary for maintaining a constant current and the temperature of the lamp during operation.

39. Xenon Sources Xenon sources cover the entire range UV- visible and even beyond (160 – 2000 nm). Emission is intense in the UV region which rapidly levels out in other regions. Such lamps have lower stability than deuterium lamps. Pulsed Xenon lamps provide additional benefits like less heat generation, longer lamp life due to lower duty cycle and reduced decay of sample due to intermittent exposure. Mercury-Xenon lamps contain a mixture of Xenon gas and Mercury vapour. Such lamps emit a broad spectrum and include sharp peaks in both UV and visible region corresponding to mercury’s spectral lines. Such lamps have increased utility over xenon lamps due to high output intensity, stability and longer useful life.

40. Tungsten Lamps Tungsten filament lamp has been conventionally used as a light source in the visible region covering 350 – 2200 nm. It consists of a thin coiled tungsten filament sealed inside an evacuated glass bulb. Power supply should ensure constant voltage to the lamp for a stable response. Tungsten – halogen lamps consist of a quartz envelope filled with an inert gas and a small amount of halogen such as iodine. Iodine forms tungsten iodide which is volatile in nature.The tungsten iodide molecules decompose on coming in contact with hot filament and redeposit tungsten onto the filament. Tungsten – halogen lamps bring the output closer to the UV region (240-2500 nm) and also extend useful lamp life to almost double of normal tungsten lamp

41. Monochromator A monochromator is a device that separates different wavelengths of light from a given light source. The main components typically include an entrance slit, mirrors and a light disperser. A prism or grating is often used as the light disperser. Most monochromators have an exit slit where the separated light leaves the device, but monochromators used in spectrographs have an array detector, usually a CCD, replacing the exit slit to capture data from a range of wavelengths at once, resulting in real‑time data

42. DEFINITION OF IR SPECTROSCOPY A technique that studies the interaction of infrared radiation with matter. Used to identify the functional groups and study molecular vibratios .

43. Importance Identifies unknown compounds. Analyses molecular structure. Distinguishes similar molecules.

44. What is infrared radiation? Part of the electromagnetic ( lower energy than visible light) Wavelengths range: 2.5-16 micrometer or 4000-625cm-1 in wave number.

45. How IR radiation interact with molecules? Causes bonds to vibrate( stretching or bending) Only vibrations that change the dipole moment absorb IR radiation.

46. Types of molecular vibration Stretching vibration; Bond length changes( it stretches or compresses) while bond angle remain same. Bending vibrations; Bond angle changes while bond length remains same like bending a flexible rod.

47. Symmetric vibration Asymmetric vibration Bonds stretch and contract together. Example; In the symmetric stretch, both C H bonds stretch at the same time and contract at the same time. One bond stretches while other contracts. Example; In the antisymmetric stretch, one C H bond stretches while the other contracts

48. Symmetric vibration Asymmetric vibrations

49. In-plane bending Scissoring Rocking Atoms move close and farther, like scissors. Example; both hydrogen atoms move closer and then apart, like closing and opening scissors. Atoms move side-to side together. Both hydrogen atoms sway side-to-side together, like rocking in sync.

50. Scissoring Rocking

51. Out-of plane bending Twisting Wagging Atoms rotate in opposite direction. Example; atoms rotate around the bond axis. Atoms move up and down in the same direction. Example; atoms swing up and down like a dog tail.

52. Infrared spectrum A graph showing absorptions of IR radiation at different wave numbers. X-axis: wave number (cm-1), Y-axis: % of transmittance.

53. Regions of spectrum Functional group region (4000-1500 cm-1): Identifies the functional groups (e.g., O-H, C-H, C=O). Finger print region (1500-600 cm-1): Unique for every molecule; helps to identify compounds.

54. Key features in an IR spectrum Broad peaks O-H stretches( alcohols, carboxylic acids), 3200-3600 cm-1 . Sharp peaks Triple bonds( alkyne , nitrile ), around 2100-2300cm-1. Intense peaks C=O stretches( carbonyl groups), near 1700cm-1. Weak peaks Bending vibration in finger print region. Example: C-H bending vibrations in the fingerprint region.

55. Applications of IR spectroscopy Identification of functional groups Example; O-H( alcohols), C=O( carbonyl groups) Structural analysis Analyses double/triple bonds and molecular geometry.

56. Example: Ethanol( C2H5OH) Broad peak at 3200-3600 cm-1 O-H stretch( alcohol) Sharp peak at 2900 cm-1 C-H stretch. Fingerprint region unique bending region.

57. Than k You .

58. references http://life.nthu.edu.tw/~labcjw/BioPhyChem/Spectroscopy/beerslaw.htm https://www.slideshare.net/MorshedulHaque/beer-lamberts-law https://www.slideshare.net/JALEEL/beer-lambert https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law https://pubs.acs.org/doi/pdf/10.1021/ed074p744.3 http://www.collin.edu/chemistry/Handouts%202009/Beer’s%20Law.pdf