Modern Optical Spectroscopy
http://www.scientific-web.com/en/Books/ModernOpticalSpectroscopyWithExercises.html
Shu-Ping Lin, Ph.D.
Institute of Biomedical Engineering E-mail: [email protected]
Website: http://web.nchu.edu.tw/pweb/users/splin/
Introduction & the Beer-Lambert Law
Energy Absorption
The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy. The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition.
Wave Number (cycles/cm)
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
Wavelength (nm)
Spectral Distribution of Radiant Energy
Region of the Electromagnetic Spectrum
V = Wave Number (cm-1)
l = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
u = Frequency of Radiation (cycles/sec)
The energy of photon:
h (Planck's constant) = 6.62 x 10-27 (Ergsec)
V =u
C l=
E = h = hC
lu
C=
lu C = ul
Electromagnetic Radiation
Visible
Ultra violet
Radio
Gamma ray
Hz
cm
cm-1
Kcal/mol
eV
Type
Quantum Transition
Type
spectroscopy
Type
Radiation
Frequency
υ
Wavelength
λ
Wave
Number V
Energy
9.4 x 107 4.9 x 106
3.3 x 1010 3 x 10-11 1021
9.4 x 103 4.9 x 102
3.3 x 106 3 x 10-7 1017
9.4 x 101 4.9 x 100
3.3 x 104
3 x 10-5 1015
9.4 x 10-1 4.9 x 10-2
3.3 x 102
3 x 10-3 1013
9.4 x 10-3 4.9 x 10-4
3.3 x 100
3 x 10-1 1011
9.4 x 10-7 4.9 x 10-8 3.3 x 10-4
3 x 103 107
X-ray
Infrared
Micro-wave
Gamma ray emission
X-ray absorption, emission
UV absorption
IR absorption
Microwave absorption
Nuclear magnetic resonance
Nuclear
Electronic (inner shell)
Molecular vibration
Electronic (outer shell)
Molecular rotation
Magnetically induced spin states
Spectral Properties, Application and Interactions of Electromagnetic Radiation
Dispersion of Polymagnetic Light with a Prism
Prism - Spray out the spectrum and choose the certain wavelength (l) that you want by slit.
Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Electronic Spectroscopy Ultraviolet and visible
Where in the spectrum are these transitions?
Why should we learn this stuff? After all, nobody solves structures with UV any longer!
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 is very 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
Ultra Violet Spectrometry
The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule.
So the ultraviolet spectrum is called electronic spectrum.
The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and n-orbitals from the ground state to higher energy states. This is also called energy transition. These higher energy states are molecular orbitals called antibonding.
Electronic Molecular Energy Levels
The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.
Ener
gy
*
*
n
*
*
n
*
n
*
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Spectrophotometer
An instrument which can measure the absorbance of a sample at any wavelength.
Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits
Instrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions.
Emit fluorescent light as energy decreases
Ground state
Sample
90 ° C
Detector UV Light Source
Monochromator Monochromator
Antibonding
Antibonding
Nonbonding
Bonding
Bonding Energy
->
->
'
'
'
' '
n-> n
n-> '
Electron's molecular energy levels
Fluorometer
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Spectrophotometry
Key Concepts
• Lambert’s Law of Absorption
• Beer’s Law
• Beer-Lambert Law
• Absorption Cross-Sections
• Photometric quantities
• Spectrophotometer
• The Cary 50 Spectrophotometer
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Lambert‟s Law of Absorption
xeI
I
Ix
I
-=
-=
0
d
d
The intensity I0 if a beam of light decreases exponentially as it passes though a uniform absorbing medium with the linear decay constant α.
Restatement: In a uniform absorbing medium, the intensity of a beam of light decreases by the same proportion for equal path lengths traveled.
The linear decay constant α is a characteristic of the medium. It has units of reciprocal length. α is the path length over which the intensity is attenuated to 1/e.
Photo: http://www-history.mcs.st-andrews.ac.uk/history/PictDisplay/Lambert.html
α
I0
I(x)
x
Lambert described how intensity changes with distance in an absorbing medium.
xeIxI -= 0)(
leII -= 0
I
l
xeII
xII
-=
-=
0
dd
Johann Heinrich Lambert 1728-1777
The distance traveled through the medium is called the path length.
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Lambert’s Law of Absorption (base 10)
xkxeI
I -- == 100
Typically base 10 is used in photometry.
xkx IeII -- == 1000
10ln=k
k is the path length over which the intensity is attenuated to 1/10.
xk
I
I -=100
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Lambert’s Law Example If one slab of absorbing material of thickness l reduces the intensity of a beam of light
to half.
α I0
2
110
0
== - lk
I
II
l
And three slabs will reduce the intensity of a beam of light to one eight.
Then two slabs of the same absorbing material will then reduce the intensity of a beam of light to one quarter.
4
1
2
110
2
2
0
=
== - lk
I
Iα I0 I
l
α
l
8
1
2
110
3
3
0
=
== - lk
I
Iα I
l
α
l
α I0
l
Beer found that Lambert’s linear decay constant k for a solution of an absorbing substance is linearly related to its concentration c by a constant, the absorptivity ε, a characteristic of the absorbing substance.
Restatement: The linear decay constant k is linear in concentration c with a constant of proportionality ε.
(August Beer, 1825-1863)
A colored absorber has an absorptivity that is dependent on wavelength of the light ε(λ). The absorptivity is the fundamental property of a substance. This is the property that contains the observable spectroscopic information that can be linked to quantum mechanics (also see absorption cross section.)
ck =
Typical units are: k cm−1; c M (moles/liter); ε M−1cm−1
Beer’s Law
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Photometric Quantities
Transmittance (T)
Absorbance (A) (AKA optical density, O.D.)
0I
IT = usually given in percent
TI
IA loglog
0
-=
-= by convention, base 10 logs are used
In photometry we measure the intensity of light and characterize its change by and object or substance. This change is typically expresses as percent transmittance or absorbance.
Frequently when your primary interest is the light beam
Used almost exclusively when your interest concerns the properties of the material
Beer – Lambert Law
Glass cell filled with
concentration of solution (C)
IILight
0
As the cell thickness increases, the transmitted intensity of light of I decreases.
T- Transmittance
T = I0 - Original light intensity
I- Transmitted light intensity
% Transmittance = 100 x
Absorbance (A) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution).
I
I0
I
I0
I0
I
1
T
I
I0
Beer-Lambert Law
Lambert‟s and Beer‟s Laws are combined to describe the attenuation of light by a solution. It is easy to see how the two standard photometric quantities can be written in terms of this law.
lcII -= 100
lcA
TI
IA
=
-=
-= loglog
0
xcT
I
IT
-=
=
10
0
Transmittance Absorbance
http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law
#Beers Law states that absorbance is proportional
to concentration over a certain concentration range
A = absorbance = molar extinction coefficient (M-1 cm-1 or mol-1 L cm-1) c = concentration (M or mol L-1) l = path length (cm) (width of cuvette)
Beer-Lambert Law
Beer‟s law is valid at low concentrations, but breaks down at higher concentrations
For linearity, A < 1
1
Beer-Lambert Law
If your unknown has a higher concentration than your highest standard, you have to ASSUME that linearity still holds (NOT GOOD for quantitative analysis)
Unknowns should ideally fall within the standard range
25
Quantitative Analysis
A < 1 If A > 1:
Dilute the sample Use a narrower cuvette
(cuvettes are usually 1 mm, 1 cm or 10 cm)
Plot the data (A v C) to produce a calibration „curve‟
Obtain equation of straight line (y=mx) from line of „best fit‟
Use equation to calculate the concentration of the unknown(s)
26
Quantitative Analysis
Calibration curve showing absorbance as
a function of metal concentration
y = 0.9982x
R2 = 0.9996
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2
Concentration (mg L-1)
Ab
so
rban
ce (
no
un
its)
Steps in Developing a Spectrometric Analytical Method
1. Run the sample for spectrum
2. Obtain a monochromatic wavelength for the maximum absorption wavelength.
3. Calculate the concentration of your sample using Beer Lambert Equation: A = ECL
Wavelength (nm)
Absorbance
0.0
2.0
200 250 300 350 400 450
Spectrometer Reading
Slope of Standard Curve = A
C
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where becomes non-linear.
x
x
x
Spectrometric Analysis Using Standard Curve
1 2 3 4
0.4
0.8
1.2
Concentration (g/l) glucose
Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line
Homeworks
1. Calculate the Molar Extinction Coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 cm cell.
Solution C x 105 M Io I
A 2.23 100 27
B 1.90 100 32
2. The molar extinction coefficient (ε) of compound riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance
reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample?
3. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 cm quartz cell was 0.4. What is the molar extinction coefficient of compound Y?
4. Calculate the molar extinction coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the following data which were obtained in 1 cm cell.
Solution C x 105 M I0 I
A 2.0 100 30
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Cross-Sections and Absorptivity the connection to single particles and
molecules
The absorption of light by particles (and single molecules) is characterized by an absorption cross section C. In this model the particle is replaced by a perfectly absorbing sphere with a cross sectional area C. This cross section is a property of the particle and is not related to its geometric cross sectional area. The concentration of particles per unit volume is N.
=
=
=
-
3
3
cm
liter 1010ln
10ln
CN
NCk
NC
A
typical units are: C cm2; N cm−3
The cross section can be directly related to the molar absorptivity. NA is Avagadro‟s number. units are: C cm2; N cm−3; NA mole−1; ε M−1cm−1
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Efficiency
The absorption efficiency Q of a particle is the ratio of its absorption cross section C to its geometric cross section Cgeo.
Absorption efficiency is dimensionless.
geoC
CQ =
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Extension to Scattering and Extinction
Attenuation of light by absorption and scattering both obey Lambert‟s Law. Thus we can extend our treatment of absorption to scattering and extinction. (Recall that extinction is the effect of absorption + scattering.)
)cxcxA
QQQ
CCC
scaabsext
scaabsext
scaabsext
scaabsext
==
=
=
= The scattering efficiency can be much larger than unity.
Extinction paradox: Qext = 2 (Qabs = 1; Qsca = 1) for an perfectly absorbing particle very large compared to the wavelength of light.
Note:
•All of these quantities are in general wavelength dependent.
•Our discussion has not included the mechanism (cause) of absorption and scattering.
•There are many different mechanisms that cause of absorption and scattering.
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Instrumentation
Spectrometer: measures I vs λ. Simply measures the spectrum of the light (e.g. emission spectroscopy).
Spectrophotometer: measures I/I0 vs λ. Measures how the sample changes the spectrum of the light (e.g. transmission, reflection, scattering, fluorescence).
All spectrophotometers contain a spectrometer.
-meter: the detector is electronic
-graph: light intensity recorded on film
photometer: measures I/I0 without λ selection.
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The Spectrophotometer
Measures absorbance as a function of wavelength
Components: light source, monochromator, sample cell, detector, optical system.
monochromator sample cell detector
light source
slit
diffr
act
ion g
rating
balance the forces:
Computer controlled acquisition of absorption spectra
Cary 50 UV-Vis Spectrophotometer
sample
detector
light source
monochromator
Can you find the diffraction grating and the slit?
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Making a Measurement with the Cary 50
First, measure the baseline using a blank sample. This is raw I0. The blank sample is the cuvette with deionized water (everything but your nanoparticles). This corrects for any absorption due to the cuvette, water, and variations of the light intensity of the light source, monochromator, etc.
Second, measure the zero by inserting the beam block. This corrects the instrument for the detector background.
Third, measure your sample. This is the raw I. The Cary 50 automatically calculates the corrected intensities (I and I0) by subtracting the zero from each of the raw intensities.
Subsequent measurements do not require re-measuring the blank and zero, simply repeat step 3.
TA
zeroIraw
zeroIraw
I
IT
log
00
-=
-
-==
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Applications of Spectrophotometry
Spectroscopy
Chemical Analysis: trace analysis, pH, forensic, in situ
monitoring, remote monitoring, geology, astronomy, ....
Particle size
Thin film characterization
Color matching
Optics
Sample Cells (Cuvettes) UV Spectrophotometer
Quartz (crystalline silica): works in the UV region below 350 nm, can be employed across the whole UV-Visible wavelength range, 190-1,000 nm
Visible Spectrophotometer
Optical glass: employed in the region above 300nm
Moulded plastic cells: used in the visible region above 350 nm, low cost
Common size of cells is the 1cm rectangular cell, which has an optical path length of 1cm and path width of 1 cm. Typically, these cells hold 2-3 ml of sample. When sample volume is an important issue Consider
micro cells
Light Sources
UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
Uses for UV, continued
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 l for a chromophore containing a phenol, carboxylic acid, etc.
l
“bathochromic” shift “hypsochromic” shift
The UV Absorption process
• * and * transitions: high-energy, accessible in vacuum UV (lmax <150 nm). Not usually observed in molecular UV-Vis.
•n * and * transitions: non-bonding electrons (lone pairs), wavelength (lmax) 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 lmax = 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
What are the nature of these absorptions?
Example: * transitions responsible for ethylene UV absorption at ~170 nm calculated with ZINDO semi-empirical excited-states methods (Gaussian 03W):
HOMO u bonding molecular orbital LUMO g antibonding molecular orbital
h 170nm photon
Example for a simple enone
π
π
n
π
π
n
π*
π
π
n
π* π*
π*
π*
π*
π* π*
π*
-*; lmax=218 =11,000
n-*; lmax=320
=100
How Do UV spectrometers work?
Two photomultiplier inputs, differential voltage drives amplifier.
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
Rotates, to achieve scan
Diode Array Detectors
Diode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity,
The Multiplex advantage
Disadvantage, resolution is 1 nm, vs 0.1 nm for normal UV
Model from Agilent literature. Imagine replacing “cell” with a microflow cell for HPLC!
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 absorptivities in L•cm/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
An Electronic Spectrum
Abso
rbance
Wavelength, l, generally in nanometers (nm)
0.0
400 800
1.0
200
UV Visible
lmaxwith certain extinction
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.
Solvents for UV (showing high energy cutoffs)
Water 205
CH3CN 210
C6H12 210
Ether 210
EtOH 210
Hexane 210
MeOH 210
Dioxane 220
THF 220
CH2Cl2 235
CHCl3 245
CCl4 265
benzene 280
Acetone 300
Various buffers for HPLC, check before using.
Chemical Structure & UV Absorption
Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region.
Chromophoric Structure
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261
Organic compounds (many of them) have UV spectra
From Skoog and West et al. Ch 14
One thing is clear
UVs can be very non-specific
It is hard to interpret except at a cursory level, and hard 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.
An Example--Pulegone
Frequently plotted as log of molar
extinction So at 240 nm, pulegone has a molar extinction of 7.24 x 103
Antilog of 3.86
O
Can we calculate UVs? Electronic Spectra
Wavelength (nm)
Molar Absorptivity (l/mol-cm)
220 230 240 250 260 270 280 290 300
0
10049
20097
30146
40194
50243
nacindolA
Electronic Spectra
Wavelength (nm)
Molar Absorptivity (l/mol-cm)
220 230 240 250 260 270 280 290 300
0
10394
20789
31183
41578
51972
Nacetylindol
Semi-empirical (MOPAC) at AM1, then ZINDO for config. interaction level 14
Bandwidth set to 3200 cm-1
The orbitals involved
Electronic Spectra
Wavelength (nm)
Molar Absorptivity (l/mol-cm)
200 210 220 230 240 250 260 270 280 290 300
0
11097
22195
33292
44390
55487
Nacetylindol
Showing atoms whose MO‟s contribute most to the bands
The Quantitative Picture
• Transmittance:
T = P/P0
B(path through sample)
P0
(power in)
P (power out)
• Absorbance:
A = -log10 T = log10 P0/P
• The Beer-Lambert Law (a.k.a. Beer‟s Law):
A = bc Where the absorbance A has no units, since A = log10 P0 / P
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)
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.
Polyenes, and Unsaturated Carbonyl groups; an Empirical
triumph R.B. Woodward, L.F. Fieser and others
Predict lmax for π* in extended conjugation systems to within ca. 2-3 nm.
Homoannular, base 253 nm
heteroannular, base 214 nm
Acyclic, base 217 nm
Attached group increment, nm
Extend conjugation +30
Addn exocyclic DB +5
Alkyl +5
O-Acyl 0
S-alkyl +30
O-alkyl +6
NR2 +60
Cl, Br +5
Similar for Enones
O
x
b b O O
X=H 207
X=R 215
X=OH 193
X=OR 193
215 202 227 239
Base Values, add these increments…
Extnd C=C +30
Add exocyclic C=C +5
Homoannular diene +39
alkyl +10 +12 +18 +18
OH +35 +30 +50
OAcyl +6 +6 +6 +6
O-alkyl +35 +30 +17 +31
NR2
S-alkyl
Cl/Br +15/+25 +12/+30
b g d,
With solvent correction of…..
Water +8
EtOH 0
CHCl3 -1
Dioxane -5
Et2O -7
Hydrcrbn -11
Some Worked Examples
O
Base value 217 2 x alkyl subst. 10 exo DB 5 total 232 Obs. 237
Base value 214 3 x alkyl subst. 30 exo DB 5 total 234 Obs. 235
Base value 215 2 ß alkyl subst. 24 total 239 Obs. 237
Distinguish Isomers!
HO2C
HO2C
Base value 214 4 x alkyl subst. 20 exo DB 5 total 239 Obs. 238
Base value 253 4 x alkyl subst. 20 total 273 Obs. 273
Generally, extending conjugation leads to red shift
“particle in a box” QM theory; bigger box
Substituents attached to a chromophore that cause a red shift are called “auxochromes”
Strain has an effect…
lmax 253 239 256 248
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
Benzenoid aromatics
From Crewes, Rodriguez, Jaspars, Organic Structure Analysis
UV of Benzene in heptane
Group K band () B band() R band
Alkyl 208(7800) 260(220) --
-OH 211(6200) 270(1450)
-O- 236(9400) 287(2600)
-OCH3 217(6400) 269(1500)
NH2 230(8600) 280(1400)
-F 204(6200) 254(900)
-Cl 210(7500) 257(170)
-Br 210(7500) 257(170)
-I 207(7000) 258/285(610/180)
-NH3+ 203(7500) 254(160)
-C=CH2 248(15000) 282(740)
-CCH 248(17000) 278(6500
-C6H6 250(14000)
-C(=O)H 242(14000) 280(1400) 328(55)
-C(=O)R 238(13000) 276(800) 320(40)
-CO2H 226(9800) 272(850)
-CO2- 224(8700) 268(800)
-CN 224(13000) 271(1000)
-NO2 252(10000) 280(1000) 330(140)
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
Binding of a lanthanide complex to an oligonucleotide
Quantitative analysis
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)
UV Spectrometer Application
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
Flurometric Application
Thiamin (365 nm, 435 nm)
Riboflavin
Vitamin A
Vitamin C
Visible Spectrometer Application
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)