Part 5-Instrumentation: Introduction to Spectroscopy for Chemical Analysis

1. Part 5-Instrumentation: Introduction to Spectroscopy for Chemical Analysis KR LSU

2. The Spectrophotometer- Instruments

3. IMPORTANT: Absorption spectrophotometer

4. (a) (b)

5. FFYI: Double-beam spectrophotometer (better than single beam see previous page): Light passes alternately through the reference and sample cuvettes. A chopper is a mirror that rotates in and out of the light path diverting the light between the reference and sample cuvettes. Routine procedure is to first record a baseline spectrum with two reference cuvettes. The absorbance of the reference is then subtracted from the absorbance of the sample to obtain the "true" absorbance at each wavelength.

6. FYI: ….Or diode array spectrophotometer

7. Light Sources

8. FYI: Sources of radiation objects Any object that is heated emits radiation. Emission from real objects such as a tungsten filament light bulb emulate blackbody radiation (the emission is a continuous spectral distribution). Visible and infrared lamps as light sources approach blackbody radiators. The radiation from an object's surface expressed as power per unit area is the excitance (emittance), M. M = T4  Where  is the Stefan-Boltzmann constant ~ 5.7 X 10-8 W/(m2K4), T = Temperature (K) Spectral distribution of blackbody radiation

9. IMPORTANT: Lamps for absorption spectrometers Typically they are inexpensive and stable. i) Visible and Near Infrared: Tungsten Lamp, Xenon lamp ii) Ultraviolet and Visible: Quartz Halogen Lamp, Xenon lamp iii) Ultraviolet: Deuterium Arc Lamp iv) Infrared: nichrome wire , silicon carbide (globar) Examples:

10. FYI: Lasers provide ~ single  Very bright sources for spectroscopy Properties of Lasers: ·  Monochromatic (only one wavelength) ·  Collimated (emit in one direction) ·  Polarized (only one electric field vector) ·  Coherent (electric/magnetic fields in phase) ·  Expensive hHigh maintenance, but some He-Ne, and many solid state lasers may be less expensive Laser operate on the principle of trapping a large number of physical objects to a new state in a cavity, and simultaneous release of these objects to a new or old state with emission

11. Monochromators and other devices for separation of radiation objects

12. Slits +Monochromators Slits are constructed by machining a sharp edge onto two metal pieces. These lie in a plane and the spacing between them, the slit width, can be adjusted. The smaller the slit width, the better the spectral resolution. (example) Filters: Filters are used to pass on only desired wavelengths of light. A filter could be colored glass. Most likely they are also based on constructive or destructive interference of light waves. (example) Prisms Separation of wavelengths on some commercial instruments.     Prisms were used in older instruments, Quartz or salt crystals.

13. FYI: selection of colors II)       Monochromators separate wavelengths of light; they consist of both entrance & exit slits, mirrors, and diffraction grating or refraction lens/prisms, and filters. Grating–monochromators Polychromatic light is collimated (focused) into a beam of parallel rays by a concave mirror (monochromatic-one wavelength; polychromatic-many wavelengths). Rays strike the reflection grating (see next figure) and different wavelengths are diffracted (separated) at different angles. Diffracted light is focussed by a second concave mirror so that only one wavelength passes through the exit slit at a time. Grating Equation: nl = d(sinq – sinf) n = diffraction order (1…n) ; d = groove spacing;  = angle of reflection;  = angle of incidence

14. Components of a Grating Spectrophotometer i diffraction grating is ruled with a series of closely spaced parallel grooves separated by distance d. These are often constructed from aluminum metal and coated with a non oxidative coating applied. When light is reflected from the grating, each groove behaves as a source of light. When adjacent rays are in phase, they reinforce each other. When adjacent rays are out of phase, the partially or completely cancel each other. Thus can be aligned to allow only certain wavelengths to pass through.

16. Wavelength selector (monochromator) passes a narrow bandwidth of radiation (if more narrow , higher resolution!!)

17. EXPERIMENTAL PARAMETERS : how to get the most from measurements with absorption spectroscopy C Choice of the Wavelength and Bandwidth –

18. Effects of monochromator’s slit: 0.25nm,1.0nm, 2.0nm, 4.0nm

19. INTERFEROMETERS: from time and length observables to frequency/energy observables 1) allows for signal averaging 2) allows all wavelengths to be monitored simultaneously 3) mathematical process that converts data obtained in the time domain to be converted into the frequency domain.

20. ·        Allows all wavelengths to simultaneously reach the detector ·        Radiation from source reaches beam splitter, where half of the radiation hits the moving mirror and half hits the fixed mirror. ·        The beams reflect and re-combine, the emerging radiation for a wavelength exhibits constructive or destructive interference. ·        With constant mirror velocity, the wavelength modulates in a regular sinusoidal manner. ·        Both the sampling rate of radiation reaching the detector and the mirror velocity is modulated by a helium-neon laser. ·        The resulting detector signal typically is stored as a time domain spectrum (interferogram). Converted to a spectrum in the frequency domain using the mathematical process of Fourier Transform. Infra-Red spectroscopy, NMR spectroscopy: FT is a standard technique

21. Detectors

22. -detectors • The phototube is used frequently as a detector in UV-Vis spectrometers. • The cathode consists of a photo-emissive surface. • Electrons are ejected from the cathode proportional to the radiant power (photons) striking its surface. • The emitted electrons are attracted to the anode. • The accompanying voltage is fed to an amplifier and converted to a signal.

23. The Photo Multiplier Tube, (PMT) is similar to the photo tube, but is a vast improvement. • In addition to the cathode and anode, the PMT has dynodes, which produce a cascade effect on the electron emission production. • Each photon causes a ~ 107 additional electrons to be produced. • The PMT possesses high sensitivity, good S/N ratio, and excellent dynamic range. • PMTs are highly sensitive to visible and UV excitations at extremely low power conditions, (very low concentrations of analyte). • Intense light sources (such as daylight or stray light) can destroy and damage PMTs.

24. FYI: PHOTO DIODE ARRAY: diode

25. PDAs are a series of silicon photo diodes, with each having a storage capacitor, and a switch that are combined in a integrated circuit on a silicon chip. • The number of sensors (silicon photodiodes) in a PDA range from 64 to 4096. • The slit width of the instrument allows the radiation to be dispersed over the entire array, allowing the spectral information to be accumulated simultaneously. • PDAs are not as sensitive nor have the same S/N ratio as the PMT, but one gains the advantage of gathering multi-channel information (all of spectrum collected simultaneously). • Advantage of the PDA is recording the entire spectrum in a fraction of the time required for a conventional scanning spectrometer to scan one wavelength at a time. • An example of PDA use is in atomic emission spectroscopy (AES), UV-vis spectrophotometry, fluorescence spectrometry, Raman spectrometry.

26. Eliminating noise: signal averaging

27. Stray Light, Electrical Noise, Cell Positioning- • Stray light can cause problems: • Stray light arises from two major sources: • 1) Misdirected rays coming from the monochromator. • 2) Light coming from outside the instrument such as the sample compartment lid not closed properly. • Other concerns; • the correct choice of the sample cell (does it require glass or quartz?) • the alignment of the sample cell (and/or the sample cell holder) • dust & fingerprints on the cell

28. Part 5- From VUV to IR-Introduction to Spectroscopy for Chemical Analysis KR LSU

29. MOLECULAR SPECTROSCOPY: IR absorption, UV/VIS electronic absorption and emission

30. Atomic vs. Molecular • What is same what is different

32. IMPORTANT: Most atomic spectra are discrete:

33. IMPORTANT - MOLECULES: Molecular spectra are broader because of the close electronic and vibrational energies

34. FYI: MOLECULES: also electronic molecular orbitals and….. One must take into account all molecular energies from different “degrees of freedom” : translational (motion), electronic and here vibrational and rotational motion of nucleiHere are some of the degrees of freedom of H20 Harmonic oscillator models

36. "What Happens When a Molecule Absorbs Light?" What happens when the absorption process takes place for molecules and compounds? Molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe the distribution of electrons in an atom. As example; :CO:localized (Lewis) vs delocalized (MO) In an electronic transition, an electron from one molecular orbital moves to another orbital, with a concomitant increase or decrease in the energy of a molecule.

37. Molecular Spectroscopy The VIS-visible absorption methods rely on complexes or compounds forming a color and it must be easily distinguishable from other species present. The UV methods may be less specific in that typically most compounds absorb UV radiation; thus the results maybe limited to only quantitative detection (information). That is, how much is there. We have a solution containing different proteins, which absorb certain wavelengths of light (ie 254 nm). But if we require each protein's identification a more specific technique must be chosen. On the other hand the IR methods depend upon vibrational and rotational absorptions. They can give both quantitative,(how much is present) and qualitative, (compound identification) information.

38. IMPORTANT - MOLECULES: Absorption of photon: electrons gain energy (ground to excited state)

39. IMPORTANT Emission of photon: electrons lost energy (excited to ground state)

40. EXAMPLES : ABSORPTION IR and UV., atoms and molecules

41. SSinglet state(S) – when the electron in the excited state is still paired with the ground-state electron. The spins of the two electrons remains opposed. Triplet state(T) – when the electron in the excited state becomes unpaired with the ground-state electron. The spins of the two electrons are now parallel. Electronic absorption bands are broad due to the large number of vibrational and rotational states present at each electronic state. We have discussed the instrumental procedure, components and design or UV-vis spectroscopy. The visible adsorption methods rely on complexes or compounds forming a color and it must be easily distinguishable from other species present. The UV methods may be less specific in that typically most compounds absorb UV radiation; thus the results maybe limited to only quantitative detection (information). That is, how much is there. For example: We have a solution containing different proteins, which absorb certain wavelengths of light (ie 254 nm). But if we require each protein's identification a more specific technique must be chosen. On the other hand the IR methods depend upon vibrational and rotational absorptions. They can give both quantitative,(how much is present) and qualitative, (compound identification) information.

42. Example At a given wavelength, a solution of a colored compound has a molar absorptivity of 104 M-1cm-1. Calculate for a 1 cm cell containing a 0.050 mM solution of this compound, at that wavelength: (a) the absorbance, and (b) the transmittance. A = ebc = (10000M-1cm-1)(1 cm)(0.050 mM) = 0.50 A = –log10 T ‹ T = 10–A = 10-0.50 = 0.32 or %T = 32% Example At a given wavelength, a cuvet filled with a sample solution has a transmittance of 63.1%. A reference cuvet filled with the solvent has a transmittance of 94.7% at that same wavelength. What is the corresponding absorbance of the sample? A = –log10 T so Asample = –log(0.631) = 0.20 Aref = –log(0.947) = 0.02 Acorrected = Asample – Aref = 0.20 – 0.02 = 0.18 Example A 0.267 g quantity of a compound with a molecular weight of 337.69 g/mol was dissolved in 100 mL of ethanol. Then 2.000 mL was withdrawn and diluted to 100 mL. The spectrum of this solution exhibited a maximum absorbance of 0.728 at 438 nm in a 2.000-cm cell. What ise?

43. Constructing a Calibration Curve - This method is used in other chemical analyses not just spectrophotometric ones. A calibration curve is a graph showing how the experimentally measured property depends on the known concentrations of the standards. We prefer calibration procedures with a linear response, in which the corrected analyte signal is proportional to the quantity of analyte. Procedure for Constructing a Calibration Curve Step #1. Prepare known samples of analyte, covering a convenient range of concentration, and measure the response of the analytical procedure to these standards. Step #2. Subtract the average response of the three blank samples from each measured responses to obtain the corrected response. The blank measures the response of the procedure when no analyte is present. Step #3. Make of graph of corrected response vs. quantity of analyte analyzed.

44. the following information is obtained during absorbance measurements at 427 nm. standards corrected blank Absorbance conc(M) Abs readings readings readings 0.1 0.89 0.008 0.898 0.05 0 .47 0.01 0.48 0.01 0.079 0.0089 0.0879 0.005 0.04 0.009 0.049 0 0

45. W What is the fate of the absorbed electronic energy associated with UV-vis spectrometry? Sometimes it results in the emission of another photon of light (Luminescence) Excess energy is dissipated by the excited molecule through; ·collisions with other molecules (solvent) ·vibrations ·rotations ·heat ·produce a photon and relax back to the ground state. This emission process is termed luminescence. • What kinds of molecules typically exhibit luminescence? The emission spectrum typically resembles the mirror image of the absorption spectrum, but is shifted to longer wavelengths. • Highly degree of conjugation (multiple double bonds) • Aromatic molecules • Molecules with atoms, which have unpaired nonbonding valence electrons • Molecules with molecular rigidity (polycyclic) • Metal chelates

46. (a)

48. What can happen when a molecule absorbs light and an electron is promoted from the ground state, So, to a vibrationally and rotationally excited level of the excited electronic state S1? vibrational relaxation is a radiationless (does not produce photon) transfer of energy to other molecules (typically the solvent) by collisions – manifested as heat internal conversion is a radiationless transition between states with the same spin quantum numbers (e.g., S1  S0) intersystem crossing is a radiationless transition between states with different spin quantum numbers (e.g., T1  S0) fluorescence is a radiational transition between states with the same spin quantum numbers (e.g., S1  S0) phosphorescence is a radiational transition between states with different spin quantum numbers (e.g., T1  S0)

50. phosphorescence is a radiational transition between states with different spin quantum numbers (e.g., T1  S0) In general, fluorescence and phosphorescence are observed at a lower energy than that of the absorbed radiation (the excitation energy). lem > lab