Transcript SPECTROSCOPY - University of Massachusetts Boston

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
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
Spectroscopic Techniques and Common Uses
UV-vis
UV-vis region
Quantitative
analysis/Beer’s Law
Atomic Absorption
UV-vis region
Quantitative analysis
Beer’s Law
FT-IR
IR/Microwave
Functional Group Analysis
Functional Group
Analysis/quant
Raman
IR/UV
FT-NMR
Radio waves
X-Ray Spectroscopy
X-rays
Elemental Analysis
X-ray Crystallography
X-rays
3-D structure Anaylysis
Structure determination
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
• Beer’s Law
Al1 = el1bc
e is molar absorptivity (unique for a
given compound at l1)
b is path length
c concentration
Beer’s Law
slit
cuvette
source
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
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
Standard Practice
• Prepare standards of known
concentration
• Measure absorbance at lmax
• Plot A vs. concentration
• Obtain slope
• Use slope (and intercept) to determine
the concentration of the analyte in the
unknown
A
Typical Beer’s Law Plot
1.2
1
0.8
0.6
0.4
0.2
0
y = 0.02x
0.0
20.0
40.0
concentration (uM)
60.0
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 Beer’s Lawtype analyses
• The most common detector for an
HPLC
Molecules have quantized energy levels:
ex. electronic energy levels.
energy
energy
hv
}
= 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
s*
p*
2p
n
2p
p
s
s*
2s
2s
s
Ethane
C C
s*
s*
hv
s
s
s
H
C C
H
H
HH
H
s*
lmax = 135 nm (a high energy transition)
Absorptions having lmax < 200 nm are difficult to observe because
everything (including quartz glass and air) absorbs in this spectral
region.
C C
s*
p*
s*
p*
= hv
=hc/l
hv
p
p
s
s
p
p*
Example: ethylene absorbs at longer wavelengths:
lmax = 165 nm = 10,000
C O
s*
p*
s*
p*
n
hv
n
p
p
s
s
n
p*
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s* lmax = 188 nm ; = 1860
np* lmax = 279 nm ; = 15
C C
ss*
135 nm
C C
pp*
165 nm
ns*
183 nm
weak
pp*
ns*
np*
150 nm
188 nm
279 nm
weak
H
C O
C O
180 nm
C O
A
279 nm
l
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:
lmax = 217 nm ; = 21,000
1,3,5-hexatriene
lmax = 258 nm ; = 35,000
Similar structures have similar UV spectra:
O
O
O
lmax = 238, 305 nm
lmax = 240, 311 nm
lmax = 173, 192 nm
Lycopene:
lmax = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm
lmax(Actual) = 474.
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
Deuterium lamp
Filament (UV)
Photomultiplier
tube
cuvette
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/dl = n / d(cos r)
• Resolution, R = l/l=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
Diode array detector
328 individual detectors
Tungsten
Filament (vis)
slit
slit
cuvette
Deuterium lamp
Filament (UV)
monochromator
Advantages/disadvantages
• Scanning instrument
– High spectral resolution (63000), l/l
– 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)
HPLC-UV
HPLC
Pump
Mobile
phase
Sample
loop
6-port
valve
HPLC
column
UV
detector
syringe
Solvent
waste