1. Course on Analytical Methods

2. Electronic Spectroscopy Ultraviolet and visible spectroscopy Scope Some applications Some features of measurements Identification of organic species Quantification of Inorganic species Colorimetric analysis The origin of the analytical signal Excitation of atom or molecule by ultra violet or visible radiation 190-900 nm

4. PHOTON IN PHOTON OUT The essential features Count the number of photons (intensity) Energy analysis Analyze other effects (polarizations)

5. Where in the spectrum are these transitions?

6. X-ray: core electron excitation UV: valance electronic excitation IR: molecular vibrations Radio waves: Nuclear spin states (in a magnetic field) Electronic Excitation by UV/Vis Spectroscopy :

7. Ultraviolet (UV) Spectroscopy – Use and Analysis Of all the forms of radiation that go to make up the electromagnetic spectrum UV is probably the most familiar to the general public (after the radiation associated with visible light which is, for the most part, taken for granted). UV radiation is widely known as something to be aware of in hot weather in having a satisfactory effect of tanning the skin but which also has the capacity to damage skin cells to the extent that skin cancer is a direct consequence of overexposure to UV radiation. This damage is associated with the high energy of UV radiation which is directly related to its high frequency and its low wavelength (see the equations below). c =  E = h  E = (hc)/ E  1/ E = energy; c = speed of light; = wavelength;  = frequency; h = Planck’s constant

8. Ultraviolet (UV) Spectroscopy – Use and Analysis If, having passed through the material, the beam is diffracted by passing through a prism it will produce a light spectrum that has gaps in it (caused by the absorption of radiation by the transparent material through which is passed). When continuous wave radiation is passed through a prism a diffraction pattern is produced (called a spectrum) made up of all the wavelengths associated with the incident radiation. When continuous wave radiation passes through a transparent material (solid or liquid) some of the radiation might be absorbed by that material. This slide is part automatically animated – if animation does not occur click left hand mouse button. Radiation source Diffraction prism Spectrum Transparent material that absorbs some radiation Spectrum with ‘gaps’ in it The effect of absorption of radiation on the transparent material is to change is from a low energy state (called the ground state) to a higher energy state (called the excited state). The difference between all the spectroscopic techniques is that they use different wavelength radiation that has different associated energy which can cause different modes of excitation in a molecule. For instance, with infra red spectroscopy the low energy radiation simply causes bonds to bend and stretch when a molecule absorbs the radiation. With high energy UV radiation the absorption of energy causes transition of bonding electrons from a low energy orbital to a higher energy orbital. The energy of the ‘missing’ parts of the spectrum corresponds exactly to the energy difference between the orbitals involved in the transition.

9. Ultraviolet (UV) Spectroscopy – Use and Analysis   ** ** n Occupied Energy Levels Unoccupied Energy Levels The bonding orbitals with which you are familiar are the  -bonding orbitals typified by simple alkanes. These are low energy (that is, stable). Next (in terms of increasing energy) are the  -bonding orbitals present in all functional groups that contain double and triple bonds (e.g. carbonyl groups and alkenes). Higher energy still are the non-bonding orbitals present on atoms that have lone pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds). All of the above 3 kinds of orbitals may be occupied in the ground state. Two other sort of orbitals, called antibonding orbitals, can only be occupied by an electron in an excited state (having absorbed UV for instance). These are the  * and  * orbitals (the * denotes antibonding). Although you are not too familiar with the concept of an antibonding orbital just remember the following – whilst electron density in a bonding orbital is a stabilising influence it is a destabilising influence (bond weakening) in an antibonding orbital. Antibonding orbitals are unoccupied in the ground state UV A transition of an electron from occupied to an unoccupied energy level can be caused by UV radiation. Not all transitions are allowed but the definition of which are and which are not are beyond the scope of this tutorial. For the time being be aware that commonly seen transitions are  to  * which correctly implies that UV is useful with compounds containing double bonds. A schematic of the transition of an electron from  to  * is shown on the left. Increasing energy

10. Ultraviolet (UV) Spectroscopy – The Instrumentation The instrumentation used to run a UV is shown below. It involves two lamps (one for visible light and one for UV light) and a series of mirrors and prisms as well as an appropriate detector. The spectrometer effectively varies the wavelength of the light directed through a sample from high wavelength (low energy) to low wavelength (high energy). As it does so any chemical dissolved in a sample cell through which the light is passing may undergo electronic transitions from the ground state to the excited state when the incident radiation energy is exactly the same as the energy difference between these two states. A recorder is then used to record, on a suitable scale, the absorption of energy that occurs at each of the wavelengths through which the spectrometer scans. The recorder assembly The spectrometer itself – this houses the lamps, mirrors, prisms and detector. The spectrometer splits the beam of radiation into two and passes one through a sample and one through a reference solution (that is always made up of the solvent in which you have dissolved the sample). The detector measures the difference between the sample and reference readings and communicates this to the recorder. The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes. The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are normally designed to allow the radiation to pass through the sample over a distance of 1cm.

11. Ultraviolet (UV) Spectroscopy – The Output The output from a UV scanning spectrometer is not the most informative looking piece of data!! It looks like a series of broad humps of varying height. An example is shown below. Decreasing wavelength in nm Increasing absorbance * *Absorbance has no units – it is actually the logarithm of the ratio of light intensity incident on the sample divided by the light intensity leaving the sample. There are two particular strengths of UV (i) it is very sensitive (ii) it is very useful in determining the quantity of a known compound in a solution of unknown concentration. It is not so useful in determining structure although it has been used in this way in the past. The concentration of a sample is related to the absorbance according to the Beer Lambert Law which is described above. A = absorbance; c = concentration in moles l -1 ; l = pathlength in cm ;  = molar absorptivity (also known as extinction coefficient) which has units of moles -1 L cm -1. Beer Lambert Law A = .c.l

12. Ultraviolet (UV) Spectroscopy – Analysing the Output Handling samples of known concentration If you know the structure of your compound X and you wish to acquire UV data you would do the following. Prepare a known concentration solution of your sample. Run a UV spectrum (typically from 500 down to 220 nm). From the spectrum read off the wavelength values for each of the maxima of the spectra (see left) Read off the absorbance values of each of the maxima (see left). Then using the known concentration (in moles L -1 ) and the known pathlength (1 cm) calculate the molar absorptivity (  ) for each of the maxima. Finally quote the data as follows (for instance for the largest peak in the spectrum to the left and assuming a concentration of 0.0001 moles L -1 ). max = 487nm A= 0.75  = 0.75 /(0.001 x 1.0) = 7500 moles -1 L cm -1 Determining concentration of samples with known molar absorptivity (  ). Having used the calculation in the yellow box to work out the molar absorptivity of a compound you can now use UV to determine the concentration of compound X in other samples (provided that these sample only contain pure X). Simply run the UV of the unknown and take the absorbance reading at the maxima for which you have a known value of . In the case above this is at the peak with the highest wavelength (see above). Having found the absorbance value and knowing  and l you can calculate c. This is the basis of your calculation in Experiment 4 of CH199 and also the principle used in many experiments to determine the concentration of a known compound in a particular test sample – for instance monitoring of drug metabolites in the urine of drug takers; monitoring biomolecules produced in the body during particular disease states Beer Lambert Law A = .c.l

14. UV / visible Spectroscopy / nm Abs / nm Abs

15. UV / visible Spectroscopy

16. Electronic transitions involve the promotion of electrons from an occupied orbital to an unoccupied orbital. Energy differences of 125 - 650 kJ/mole.

17. UV / visible Spectroscopy Beer-Lambert Law A = log(I O /I) =  cl

18. UV / visible Spectroscopy A = log(I O /I) =  cl –A = Absorbance (optical density) –I O = Intensity of light on the sample cell –I = Intensity of light leaving the sample cell –c = molar concentration of solute –l = length of sample cell (cm) –  = molar absorptivity (molar extinction coefficient)

19. UV / visible Spectroscopy The Beer-Lambert Law is rigorously obeyed when a single species is present at relatively low concentrations.

20. UV / visible Spectroscopy The Beer-Lambert Law is not obeyed: –High concentrations –Solute and solvent form complexes –Thermal equilibria exist between the ground state and the excited state –Fluorescent compounds are present in solution

21. UV / visible Spectroscopy The size of the absorbing system and the probability that the transition will take place control the absorptivity (  ). Values above 10 4 are termed high intensity absorptions. Values below 1000 indicate low intensity absorptions which are forbidden transitions.

22. UV / visible Spectroscopy Organic Spectroscopy Transitions between MOLECULAR ORBITALS

23. UV / visible Spectroscopy Highest occupied molecular orbital HOMO Lowest unoccupied molecular orbital LUMO

24. UV / visible Spectroscopy

25. Not all transitions are observed There are restrictions called Selection Rules This results in Forbidden Transitions

26. UV / visible Spectroscopy The characteristic energy of a transition and the wavelength of radiation absorbed are properties of a group of atoms rather than of electrons themselves. The group of atoms producing such an absorption is called a CHROMOPHORE

27. UV / visible Spectroscopy

29. It is often difficult to extract a great deal of information from a UV spectrum by itself. Generally you can only pick out conjugated systems.

30. UV / visible Spectroscopy

31. ALWAYS use in conjunction with nmr and infrared spectra.

32. UV / visible Spectroscopy As structural changes occur in a chromophore it is difficult to predict exact energy and intensity changes. Use empirical rules. Woodward-Fieser Rules for dienes Woodward’s Rules for enones

33. UV / visible Spectroscopy 1. Bathochromic shift (red shift) –lower energy, longer wavelength –CONJUGATION. 2. Hypsochromic shift (blue shift) –higher energy, shorter wavelength. 3. Hyperchromic effect –increase in intensity 4. Hypochromic effect –decrease in intensity

34. Spectroscopic Techniques and Chemistry they Probe UV-visUV-vis regionbonding electrons Atomic AbsorptionUV-vis regionatomic transitions (val. e-) FT-IRIR/Microwavevibrations, rotations RamanIR/UVvibrations FT-NMRRadio wavesnuclear spin states X-Ray SpectroscopyX-raysinner electrons, elemental X-ray CrystallographyX-rays3-D structure

35. Spectroscopic Techniques and Common Uses UV-visUV-vis region Quantitative analysis/Beer’s Law Atomic AbsorptionUV-vis region Quantitative analysis Beer’s Law FT-IRIR/MicrowaveFunctional Group Analysis RamanIR/UV Functional Group Analysis/quant FT-NMRRadio wavesStructure determination X-Ray SpectroscopyX-raysElemental Analysis X-ray CrystallographyX-rays3-D structure Anaylysis

36. Different Spectroscopies UV-vis – electronic states of valence e/d-orbital transitions for solvated transition metals Fluorescence – emission of UV/vis by certain molecules FT-IR – vibrational transitions of molecules FT-NMR – nuclear spin transitions X-Ray Spectroscopy – electronic transitions of core electrons

37. Quantitative Spectroscopy Beer’s Law A l1 = e l1 bc e is molar absorptivity (unique for a given compound at l 1 ) b is path length c concentration

38. Beer’s Law A = -logT = log(P 0 /P) = ebc T = P solution /P solvent = P/P 0 Works for monochromatic light Compound x has a unique e at different wavelengths cuvette source slit detector

39. Characteristics of Beer’s Law Plots One wavelength Good plots have a range of absorbances from 0.010 to 1.000 Absorbances over 1.000 are not that valid and should be avoided 2 orders of magnitude

40. Standard Practice Prepare standards of known concentration Measure absorbance at max Plot A vs. concentration Obtain slope Use slope (and intercept) to determine the concentration of the analyte in the unknown

41. Typical Beer’s Law Plot

42. UV-Vis Spectroscopy UV- organic molecules –Outer electron bonding transitions –conjugation Visible – metal/ligands in solution –d-orbital transitions Instrumentation

43. Characteristics of UV-Vis spectra of Organic Molecules Absorb mostly in UV unless highly conjugated Spectra are broad, usually to broad for qualitative identification purposes Excellent for quantitative Beer’s Law-type analyses The most common detector for an HPLC

44. Molecules have quantized energy levels: ex. electronic energy levels. energy hv energy }  = hv Q: Where do these quantized energy levels come from? A: The electronic configurations associated with bonding. Each electronic energy level (configuration) has associated with it the many vibrational energy levels we examined with IR.

45. Broad spectra Overlapping vibrational and rotational peaks Solvent effects

46. Molecular Orbital Theory Fig 18-10

47. 2s       2p n

48. max = 135 nm (a high energy transition) Absorptions having max < 200 nm are difficult to observe because everything (including quartz glass and air) absorbs in this spectral region. Ethane

49. Example: ethylene absorbs at longer wavelengths: max = 165 nm  = 10,000  = hv =hc/

50. The n to pi* transition is at even lower wavelengths but is not as strong as pi to pi* transitions. It is said to be “forbidden.” Example: Acetone: n  max = 188 nm ;  = 1860 n  max = 279 nm ;  = 15

51.  135 nm  165 nm n  183 nmweak  150 nm n  188 nm n  279 nmweak A 180 nm 279 nm

52. Conjugated systems: Preferred transition is between Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO). Note: Additional conjugation (double bonds) lowers the HOMO- LUMO energy gap: Example: 1,3 butadiene: max = 217 nm ;  = 21,000 1,3,5-hexatriene max = 258 nm ;  = 35,000

53. Similar structures have similar UV spectra: max = 238, 305 nm max = 240, 311 nm max = 173, 192 nm

54. max = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm max (Actual) = 474.

55. Metal ion transitions Degenerate D-orbitals of naked Co D-orbitals of hydrated Co 2+ Octahedral Configuration EE

56. Co 2+ H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O Octahedral Geometry

57. Instrumentation Fixed wavelength instruments Scanning instruments Diode Array Instruments

58. Fixed Wavelength Instrument LED serve as source Pseudo-monochromatic light source No monochrometer necessary/ wavelength selection occurs by turning on the appropriate LED 4 LEDs to choose from photodyode sample beam of light LEDs

59. Scanning Instrument cuvette Tungsten Filament (vis) slit Photomultiplier tube monochromator Deuterium lamp Filament (UV) slit Scanning Instrument

60. sources Tungten lamp (350-2500 nm) Deuterium (200-400 nm) Xenon Arc lamps (200-1000 nm)

61. Monochromator Braggs law, nl = d(sin i + sin r) Angular dispersion, d r/ d = n / d(cos r) Resolution, R = /  nN, resolution is extended by concave mirrors to refocus the divergent beam at the exit slit

62. Sample holder Visible; can be plastic or glass UV; you must use quartz

63. Single beam vs. double beam Source flicker

64. Diode array Instrument cuvette Tungsten Filament (vis) slit Diode array detector 328 individual detectors monochromator Deuterium lamp Filament (UV) slit mirror

65. Advantages/disadvantages Scanning instrument –High spectral resolution (63000), /  –Long data acquisition time (several minutes) –Low throughput Diode array –Fast acquisition time (a couple of seconds), compatible with on-line separations –High throughput (no slits) –Low resolution (2 nm)

66. HPLC-UV Mobile phase HPLC Pump syringe 6-port valve Sample loop HPLC column UV detector Solvent waste

67. UV / visible Spectroscopy The radiation which is absorbed has an energy which exactly matches the energy difference between the ground state and the excited state. These absorptions correspond to electronic transitions.

68. Many organic molecules have chromophores that absorb UV UV absorbance is about 1000 x easier to detect per mole than NMR Still used in following reactions where the chromophore changes. Useful because timescale is so fast, and sensitivity so high. Kinetics, esp. in biochemistry, enzymology. Most quantitative Analytical chemistry in organic chemistry is conducted using HPLC with UV detectors One wavelength may not be the best for all compound in a mixture. Affects quantitative interpretation of HPLC peak heights Why should we learn this stuff? After all, nobody solves structures with UV any longer!

69. Uses for UV another aspect Knowing UV can help you know when to be skeptical of quant results. Need to calibrate response factors Assessing purity of a major peak in HPLC is improved by “diode array” data, taking UV spectra at time points across a peak. Any differences could suggest a unresolved component. “Peak Homogeneity” is key for purity analysis. Sensitivity makes HPLC sensitive e.g. validation of cleaning procedure for a production vessel But you would need to know what compounds could and could not be detected by UV detector! (Structure!!!) One of the best ways for identifying the presence of acidic or basic groups, due to big shifts in for a chromophore containing a phenol, carboxylic acid, etc. “bathochromic” shift “hypsochromic” shift

70. The UV Absorption process –    * and    * transitions: high-energy, accessible in vacuum UV ( max <150 nm). Not usually observed in molecular UV-Vis. –n   * and    * transitions: non-bonding electrons (lone pairs), wavelength ( max) in the 150-250 nm region. –n   * and    * transitions: most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with max = 200-600 nm. –Any of these require that incoming photons match in energy the gap corrresponding to a transition from ground to excited state. –Energies correspond to a 1-photon of 300 nm light are ca. 95 kcal/mol

71. What are the nature of these absorptions? Example for a simple enone π π n π π n π* π π n  -  *; max =218  =11,000 n-  *; max =320  =100 h 170nm photon Example:    * transitions responsible for ethylene UV absorption at ~170 nm calculated with ZINDO semi-empirical excited- states methods (Gaussian 03W): LUMO  g antibonding molecular orbital HOMO  u bonding molecular orbital

72. How Do UV spectrometers work? Rotates, to achieve scan Matched quartz cuvettes Sample in solution at ca. 10-5 M. System protects PM tube from stray light D2 lamp-UV Tungsten lamp-Vis Double Beam makes it a difference technique

73. Experimental details What compounds show UV spectra? Generally think of any unsaturated compounds as good candidates. Conjugated double bonds are strong absorbers Just heteroatoms are not enough but C=O are reliable Most compounds have “end absorbance” at lower frequency. Unfortunately solvent cutoffs preclude observation. You will find molar absorbtivities  in Lcm/mol, tabulated. Transition metal complexes, inorganics Solvent must be UV grade (great sensitivity to impurities with double bonds) The NIST databases have UV spectra for many compounds

74. An Electronic Spectrum Absorbance Wavelength,, generally in nanometers (nm) 0.0 400800 1.0 200 UV Visible Make solution of concentration low enough that A≤ 1 (Ensures Linear Beer’s law behavior) Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent. Can extract the  value if conc. (M) and b (cm) are known UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV

75. Solvents for UV (showing high energy cutoffs) Water205 CH3C  N 210 C6H12210 Ether210 EtOH210 Hexane 210 MeOH210 Dioxane 220 THF220 CH2Cl2 235 CHCl3245 CCl4265 benzene 280 Acetone 300

76. Organic compounds (many of them) have UV spectra One thing is clear Uvs can be very non- specific Its hard to interpret except at a cursory level, and to say that the spectrum is consistent with the structure Each band can be a superposition of many transitions Generally we don’t assign the particular transitions. From Skoog and West et al. Ch 14

77. The Quantitative Picture Transmittance: T = P/P 0 P 0 (power in) P (power out) Absorbance: A = -log10 T = log10 P0/P B(path through sample) The Beer-Lambert Law (a.k.a. Beer’s Law): A = ebc Where the absorbance A has no units, since A = log10 P0 / P e is the molar absorbtivity with units of L mol-1 cm-1 b is the path length of the sample in cm c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)

78. Beer-Lambert Law Linear absorbance with increased concentration-- directly proportional Makes UV useful for quantitative analysis and in HPLC detectors Above a certain concentration the linearity curves down, loses direct proportionality--Due to molecular associations at higher concentrations. Must demonstrate linearity in validating response in an analytical procedure

79. Polyenes, and Unsaturated Carbonyl groups; an Empirical triumph R.B. Woodward, L.F. Fieser and others Predict max for π  * in extended conjugation systems to within ca. 2-3 nm. Homoannular, base 253 nm Acyclic, base 217 nm heteroannular, base 214 nm Attached groupincrement, nm Extend conjugation+30 Addn exocyclic DB+5 Alkyl +5 O-Acyl 0 S-alkyl +30 O-alkyl +6 NR2 +60 Cl, Br +5

80. Interpretation of UV-Visible Spectra Transition metal complexes; d, f electrons. Lanthanide complexes – sharp lines caused by “screening” of the f electrons by other orbitals One advantage of this is the use of holmium oxide filters (sharp lines) for wavelength calibration of UV spectrometers See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14

81. Quantitative analysis Great for non-aqueous titrations Example here gives detn of endpoint for bromcresol green Binding studies Form I to form II Isosbestic points Single clear point, can exclude intermediate state, exclude light scattering and Beer’s law applies

82. More Complex Electronic Processes Fluorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of the same multiplicity Phosphorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of different multiplicity Singlet state: spins are paired, no net angular momentum (and no net magnetic field) Triplet state: spins are unpaired, net angular momentum (and net magnetic field)