Electronic Excitation by UV/Vis Spectroscopy :

Transcription

1 SPECTROSCOPY Light interacting with matter as an analytical tool III Pharm.D Department of Pharmaceutical Analysis SRM College Of Pharmacy,Katankulathur

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

3 Spectroscopic Techniques and Chemistry they Probe UV-vis UV-vis region bonding electrons Atomic Absorption UV-vis region atomic transitions (val. e-) FT-IR IR/Microwave vibrations, rotations Raman IR/UV vibrations FT-NMR Radio waves nuclear spin states X-Ray Spectroscopy X-rays inner electrons, elemental X-ray Crystallography X-rays 3-D structure

4 UV-vis Spectroscopic Techniques and Common Uses Atomic Absorption UV-vis region UV-vis region Quantitative analysis/beer s Law Quantitative analysis Beer s Law FT-IR IR/Microwave Functional Group Analysis Raman IR/UV Functional Group Analysis/quant FT-NMR Radio waves Structure determination X-Ray Spectroscopy X-rays Elemental Analysis X-ray Crystallography X-rays 3-D structure Anaylysis

5 Different Spectroscopies UV-vis electronic states of valence e/dorbital 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

6 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

7 Beer s Law source slit cuvette detector 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

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

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

10 Typical Beer s Law Plot A y = 0.02x concentration (um)

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

12 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 Lawtype analyses The most common detector for an HPLC

13 Molecules have quantized energy levels: ex. electronic energy levels. hv energy energy } ΔΕ = 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.

14 Broad spectra Overlapping vibrational and rotational peaks Solvent effects

15 Molecular Orbital Theory Fig 18-10

16 σ π 2p π n 2p σ σ 2s 2s σ

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

18 ΔΕ= hv =hc/λ Example: ethylene absorbs at longer wavelengths: λ max = 165 nm ε= 10,000

19 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 σ λ max = 188 nm ; ε= 1860 n π λ = 279 nm ; ε= 15

20 λ σ > σ π > π 135 nm 165 nm n > σ 183 nm weak π > π 150 nm n > σ 188 nm n > π 279 nm weak 180 nm A 279 nm

21 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: λ max = 217 nm ; ε= 21,000 1,3,5-hexatriene λ max = 258 nm ; ε= 35,000

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

23 λ max = (8) + 11*( *11) = 476 nm λ max (Actual) = 474.

24 Metal ion transitions ΔE Degenerate D-orbitals of naked Co D-orbitals of hydrated Co 2+ Octahedral Configuration

25 Octahedral Geometry H 2 O H 2 O H 2 O Co 2+ H 2 O H 2 O H 2 O

26 Instrumentation Fixed wavelength instruments Scanning instruments Diode Array Instruments

27 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 sample beam of light LEDs photodyode

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

29 sources Tungten lamp ( nm) Deuterium ( nm) Xenon Arc lamps ( nm)

30 Monochromator Braggs law, nl = d(sin i + sin r) Angular dispersion, dr/dλ = n / d(cos r) Resolution, R = λ/δλ = nn, resolution is extended by concave mirrors to refocus the divergent beam at the exit slit

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

32 Single beam vs. double beam Source flicker

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

34 Advantages/disadvantages Scanning instrument High spectral resolution (63000), λ/δλ 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)

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