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UV ray spectrophotometer

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The spectrophotometer technique is to measures light intensity as a function of wavelength.
• Measures the light that passes through a liquid sample
• Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)

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UV ray spectrophotometer

  1. 1. UV- Visible Spectrophotometry
  2. 2. • The spectrophotometer technique is to measures light intensity as a function of wavelength. • Measures the light that passes through a liquid sample • Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)
  3. 3. The electromagnetic spectrum
  4. 4. Energy levels of a substance in solution UV-Visible spectroscopy: Valance electronic excitation
  5. 5. The human eyes sees the complementary to which that is absorbed
  6. 6. The absorption spectrum is complementary to the transmission of light. Chlorophyll is green because it absorbs strongly in the blue (435 nm) and red (660 nm) regions of the spectrum. Thus, the "transmission window" is left around 550 nm, which corresponds to green light. The absorbance spectrum of chlorophyll
  7. 7. The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole, and the near ultraviolet region, out to 200 nm, extends this energy range to 143 kcal/mole The energies noted above are sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, absorption spectroscopy carried out in this region is sometimes called "electronic spectroscopy". A diagram showing the various kinds of electronic excitation that may occur in organic molecules is shown on the left. Of the transitions outlined, only the two lowest energy ones are achieved by the energies available in the
  8. 8. THE ABSORPTION SPECTRUM When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. An optical spectrometer records the wavelengths at which absorption occurs, together with the degree of absorption at each wavelength. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength (λ) is known as a spectrum. The significant features:  λmax (wavelength at which there is a maximum absorption)  єmax (The intensity of maximum absorption) UV-visible spectrum of isoprene showing maximum absorption at 222 nm
  9. 9. Light is a form of electromagnetic radiation. When it falls on a substance, three things can happen: • the light can be reflected by the substance • it can be absorbed by the substance • certain wavelengths can be absorbed and the remainder transmitted or reflected Since reflection of light is of minimal interest in spectrophotometry, we will ignore it and turn to the absorbance and transmittance of light.
  10. 10.  I – intensity where Io is initial intensity  T – transmittance or %T = 100 x T (T = I/ Io)  A – absorbance A = log1/ T = log Io/ I (absorption: Abs = 1 – T or %Abs = 100 - %T) Some terminology
  11. 11. ABSORBANCE LAWS BEER’S LAW The Effect of Concentration “ The intensity of a beam of monochromatic light decrease exponentially with the increase in concentration of the absorbing substance” . Arithmetically; - dI/ dc ᾱ I I= Io. eˉkc --------------------------eq (1)
  12. 12. Transmittance and concentration – Beer’s Law
  13. 13. LAMBERT’S LAW The Effect of Cell Path Length “When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of the light” mathematically; -dI/ dt ᾱ I I= Io. eˉkt --------------------------eq (2)
  14. 14. Transmittance and pathlength – the Bouguer Lambert’s Law
  15. 15. The law states that the amount of light absorbed by a solution (colored) is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material (path length). Absorbance is also called optical density the combination of eq 1 & 2 we will get A= Kct A= ℇct (K=ℇ) where ℇ – molar absorptivity, t – thickness/pathlength, and c – molar concentration
  16. 16. Molar Absorptivity, ε = A / c l (where A= absorbance, c = sample concentration in moles/liter & l = length of light path through the sample in cm.) Because the absorbance of a sample will be proportional to the number of absorbing molecules in the spectrometer light beam (e.g. their molar concentration in the sample tube), it is necessary to correct the absorbance value for this and other operational factors if the spectra of different compounds are to be compared in a meaningful way. The corrected absorption value is called "molar absorptivity", and is particularly useful when comparing the spectra of different compounds and determining the relative strength of light absorbing functions (chromophores). Molar absorptivity (ε) is defined as:
  17. 17. Deviation from Beer-Lamberts law • High concentration of analyte • Scattering of light due to particulates in sample • Fluoresecence or phosphorescence of the sample • Non-monochromatic radiation
  18. 18.  The blank contains all substances except the analyte.  Is used to set the absorbance to zero: Ablank = 0  This removes any absorption of light due to these substances and the cell.  All measured absorbance is due to analyte.
  19. 19. Nature of Shift Descriptive Term To Longer Wavelength Bathochromic To Shorter Wavelength Hypsochromic To Greater Absorbance Hyperchromic To Lower Absorbance Hypochromic Terminology for Absorption Shifts
  20. 20.  The spectrophotometer technique is to measures light intensity as a function of wavelength.  It does this by: 1. diffracting the light beam into a spectrum of wavelengths 2. direct it to an object 3. receiving the light returned from the object 4. detecting the intensities with a charge-coupled device 5. displaying the results as a graph on the detector and then the display device Spectrophotometer
  21. 21. block diagrammatic representation of UV-spectrophotometer
  22. 22. Spectrophotometer: a) Single-beam b) Double-beam [4]
  23. 23. Components of spectrophotometer  Source  Monochromator  Sample compartment  Detector  Recorder Instrumentation
  24. 24. LIGHT SOURCES Characteristics of good light source: 1. High intensity but small surface area 2. Wide spectral range 3. Stable output 4. Long life at reasonable cost UV Spectrophotometer 1.Hydrogen or Deuterium Lamp (Wavelength Range :190~420nm) 2.Mercury Lamp 3.Xenon Lamp (Wavelength Range :190~800nm) Visible Spectrophotometer 1.Tungsten Lamp (Part of the UV and the whole of the Visible; Wavelength Range :350~2500nm) UV-Vis spectrophotometer have both deuterium & tungsten lamps.
  25. 25. Accepts polychromatic input light from a lamp and outputs monochromatic light Characteristics desired of a monochromator 1. minimum absorption of light as it passes through the system 2. High degree of accuracy in wavelength selection 3. High spectral purity over a broad spectral range Components : Entrance slit, Dispersion device, Exit slit. The resolving element are of two kinds namely, Prisms Simple glass prisms are used for visible range. For UV region silica, fused silica or quartz prism is used. Fluorite is used in vaccum UV range. Gratings are often used in the monochromators of spectrophotometers operating in UV, visible and infra red regions. Their resolving power is far superior to that of prisms & they yield a linear resolution of spectrum. MONOCHROMATOR
  26. 26. UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass SAMPLE CONTAINERS / SAMPLE CELLS / CUVETTES
  27. 27. Three common types of detectors are used I. Barrier layer cell II. Photo cell detector III. Photomultiplier , Photo voltaic cells DETECTORS Convert radiant energy (photons) into an electrical signal The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking them
  28. 28. RECORDERS Display devices: The data from a detector are displayed by a readout device, such as an analog meter, a light beam reflected on a scale, or a digital display , or LCD The output can also be transmitted to a computer or printer
  29. 29. 1. Concentration measurement  Prepare samples  Make series of standard solutions of known concentrations APPLICATIONS
  30. 30. APPLICATIONS  Set spectrophotometer to the λ of maximum light absorption  Measure the absorption of the unknown, and from the standard plot, read the related concentration
  31. 31. APPLICATIONS 2. Detection of Impurities  UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material.
  32. 32. APPLICATIONS 3. Structure elucidation of organic compounds.  From the location of peaks and combination of peaks UV spectroscopy elucidate structure of organic molecules: o the presence or absence of unsaturation, o the presence of aromatic ring
  33. 33. APPLICATIONS 4. Chemical kinetics  Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed.
  34. 34. Limitations of UV-visible spectrosco
  35. 35. 1. Sample UV-vis spectroscopy works well on liquids and solutions, but if the sample is more of a suspension of solid particles in liquid, the sample will scatter the light more than absorb the light and the data will be very skewed. Most UV-vis instruments can analyze solid samples or suspensions with a diffraction apparatus (Figure), but this is not common. UV-vis instruments generally analyze liquids and solutions most efficiently.Schematic representation of the apparatus for collecting UV-vis spectra from solid materials
  36. 36. 2. Calibration and reference • A blank reference will be needed at the very beginning of the analysis of the solvent to be used (water, hexanes, etc), and if concentration analysis needs to be performed, calibration solutions need to be made accurately . • If the solutions are not made accurately enough, the actual concentration of the sample in question will not be accurately determined.
  37. 37. 3. Choice of solvent Every solvent has a UV-vis absorbance cutoff wavelength. The solvent cutoff is the wavelength below which the solvent itself absorbs all of the light. So when choosing a solvent be aware of its absorbance cutoff and where the compound under investigation is thought to absorb. If they are close, chose a different solvent. Table below provides an example of solvent cutoffs. UV absorbance cutoffs of various common solvents solvents UV absorbance cutoff (nm) Acetone 329 Benzene 278 Dimethylformamide (DMF) 267 Ethanol 205 Toluene 285 water 180
  38. 38. 4. Choice of container The material the cuvette (the sample holder) is made from will also have a UV-vis absorbance cutoff. Glass will absorb all of the light higher in energy starting at about 300 nm, so if the sample absorbs in the UV, a quartz cuvette will be more practical as the absorbance cutoff is around 160 nm for quartz Three different types of cuvettes commonly used, with different usable wavelengths. Material Wavelength range (nm) Glass 380-780 Plastic 380-780 Fused quartz Below 380
  39. 39. 5. Concentration of solution To obtain reliable data, the peak of absorbance of a given compound needs to be at least three times higher in intensity than the background noise of the instrument. Obviously using higher concentrations of the compound in solution can combat this. Also, if the sample is very small and diluting it would not give an acceptable signal, there are cuvettes that hold smaller sample sizes than the 2.5 mL of a standard cuvettes. Some cuvettes are made to hold only 100 μL, which would allow for a small sample to be analyzed without having to dilute it to a larger volume, lowering the signal to noise ratio.
  40. 40. JOB EFFECT If we continue to take measurements beyond the colour reagent limit, it is observed that the linearity of the Beer- Lambert calibration does not continue indefinitely but forms a plateau at a point which indicates that there is insufficient reagent to produce any more colour. This phenomenon is known as job effect. To extrapolate beyond the linear portion of the curve, therefore, would potentially introduce enormous errors.

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