SPECTROSCOPY

Light interacting with matter as an analytical tool

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)

Spectroscopic Techniques and Chemistry they Probe

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

Spectroscopic Techniques and Common Uses

UV-vis Atomic Absorption FT-IR Raman FT-NMR X-Ray Spectroscopy X-ray Crystallography UV-vis region UV-vis region IR/Microwave IR/UV Radio waves X-rays X-rays Quantitative analysis/Beers Law Quantitative analysis Beers Law Functional Group Analysis Functional Group Analysis/quant Structure determination Elemental Analysis 3-D structure Anaylysis

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

Quantitative Spectroscopy
Beers Law Al1 = el1 bc e is molar absorptivity (unique for a given compound at l1) b is path length c concentration

Beers Law
source slit cuvette detector

A = -logT = log(P0/P) = ebc T = Psolution /Psolvent = P/P0 Works for monochromatic light Compound x has a unique e at different wavelengths

Characteristics of Beers 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

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

Typical Beers Law Plot

1.2 1 0.8 0.6 0.4 0.2 0 0.0 y = 0.02x

20.0

40.0

60.0

concentration (uM)

UV-Vis Spectroscopy
UV- organic molecules
Outer electron bonding transitions conjugation

Visible metal/ligands in solution

d-orbital transitions

Instrumentation

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 Beers Lawtype analyses The most common detector for an HPLC

Molecules have quantized energy levels: ex. electronic energy levels. hv

energy

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.

Broad spectra
Overlapping vibrational and rotational peaks Solvent effects

Molecular Orbital Theory

Fig 18-10

 2p

2p

2s

2s

Ethane

C C

hv

H C C H H H H H

(a high energy transition)

m ax

= 135 nm

Absorptions having max < 200 nm are difficult to observe because everything (including quartz glass and air) absorbs in this spectral region.

C C

hv

= hv =hc/

Example: ethylene absorbs at longer wavelengths: max = 165 nm = 10,000

C O

n
hv

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

C C

> >
H

135 nm 165 nm 183 nm weak

C C

C O

n >

C O

150 nm > n 188 nm > n 279 nm weak >

180 nm

C O

A
279 nm

Conjugated systems:
C C

LUMO

HOMO

Preferred transition is between Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).

Note: Additional conjugation (double bonds) lowers the HOMOLUMO energy gap: Example: 1,3 butadiene: max = 217 nm ; = 21,000 1,3,5-hexatriene max = 258 nm ; = 35,000

Similar structures have similar UV spectra:

O O O

m ax

= 238, 305 nm

m ax

= 240, 311 nm

m ax

= 173, 192 nm

Lycopene:

m ax

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

m ax

Metal ion transitions

E Degenerate D-orbitals of naked Co

D-orbitals of hydrated Co2+ Octahedral Configuration

Octahedral Geometry
H2O H2O H2O Co2+ H2O H2O H2O

Instrumentation
Fixed wavelength instruments Scanning instruments Diode Array Instruments

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

Scanning Instrument
Scanning Instrument monochromator

Tungsten Filament (vis)

slit

slit

Photomultiplier tube cuvette

Deuterium lamp Filament (UV)

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

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

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

Single beam vs. double beam

Source flicker

Diode array Instrument

mirror Tungsten Filament (vis) slit slit Diode array detector 328 individual detectors

cuvette Deuterium lamp Filament (UV)

monochromator

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

Diode array

HPLC-UV
HPLC Pump

Mobile phase

Sample loop

6-port valve

HPLC column UV detector

syringe Solvent waste