Visible and Ultraviolet Spectroscopy
An obvious difference between certain compounds is their color,
In this respect the human eye is functioning as a spectrometer
analyzing the light reflected from the surface of a solid or passing
through a liquid,
Wavelength is defined on the left below,as the distance between
adjacent peaks (or troughs),and may be designated in meters,
centimeters or nanometers (10-9 meters),Frequency is the number
of wave cycles that travel past a fixed point per unit of time,and is
usually given in cycles per second,or hertz (Hz),
Visible wavelengths cover a range from approximately 400 to 800 nm,
Violet,400 - 420 nm
Indigo,420 - 440 nm
Blue,440 - 490 nm
Green,490 - 570 nm
Yellow,570 - 585 nm
Orange,585 - 620 nm
Red,620 - 780 nm
“ROY G BIV”
Complementary colors are diametrically opposite each other,
Thus,absorption of 420-430 nm light renders a substance yellow,
and absorption of 500-520 nm light makes it red,Green is unique
in that it can be created by absoption close to 400 nm as well as
absorption near 800 nm.
Color wheel
“extensively conjugated pi-electrons”
The Electromagnetic Spectrum
Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer electrons,
There are three types of electronic transition which can be considered;
Transitions involving p,s,and n electrons,
Transitions involving charge-transfer electrons
Transitions involving d and f electrons (not covered in this Unit)
When an atom or molecule absorbs energy,electrons are promoted from their ground state to
an excited state,In a molecule,the atoms can rotate and vibrate with respect to each other,
These vibrations and rotations also have discrete energy levels,which can be considered as
being packed on top of each electronic level
UV-Visible Absorption Spectra
The energies noted above are sufficient to promote or excite a molecular electron to
a higher energy orbital,Consequently,absorption spectroscopy carried out in this
region is sometimes called "electronic spectroscopy",
Molar absorptivity
Molar Absorptivity,e = A/ c l
A= absorbance,
c = sample concentration in moles/liter
l = length of light path through the sample in cm.
Molar absoptivities may be very large for strongly absorbing chromophores
(>10,000) and very small if absorption is weak (10 to 100),The magnitude of e
reflects both the size of the chromophore and the probability that light of a given
wavelength will be absorbed when it strikes the chromophore,A general equation
stating this relationship may be written as follows,
e = 0.87*1020 R * a
(R is the transition probability (0 to 1) & a is the chromophore area in cm2)
The Importance of Conjugation
Empirical Rules for Absorption Wavelengths
of Conjugated Systems---Woodward-Fieser Rules for
Calculating the lmax of Conjugated Dienes and Polyenes
P
The amount of radiation absorbed may be measured in a number of ways,
Transmittance,T = P / P0,% Transmittance,%T = 100 T
Absorbance,A = lg P0 / P
A = lg 1 / T
A = lg100 / %T
A = 2 - lg%T
The Beer-Lambert Law
A = e b c
A is absorbance (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 with units of cm.
c is the concentration of the compound in solution,expressed in mol L-1
The reason why we prefer to express the law with this equation is because
absorbance is directly proportional to the other parameters,as long as the law is obeyed
P a t h l e n g t h / c m 0 0,2 0,4 0,6 0,8 1,0
%T 1 0 0 50 25 1 2,5 6,2 5 3,1 2 5
A b s o r b a n c e 0 0,3 0,6 0,9 1,2 1,5
The linear relationship between concentration and absorbance is both simple and
straightforward,which is why we prefer to express the Beer-Lambert law using
absorbance as a measure of the absorption rather than %T.
Note that the Law is not obeyed at high concentrations,This deviation from the Law is not dealt with here.
Question,What is the significance of the molar absorbtivity,e?
In words,this relationship can be stated as "e is a measure of the amount of light absorbed
per unit concentration",Molar absorbtivity is a constant for a particular substance.
e = A / bc
Let us take a compound with a very high value of molar absorbtivity,say 100,000 L mol-1 cm-1,
which is in a solution in a 1 cm pathlength cuvette and gives an absorbance of 1.
e = 1 / 1* c
Therefore,c = 1 / 100,000 = 1 * 10-5 mol L-1
Now let us take a compound with a very low value of e,say 20 L mol-1 cm-1,which is in
solution in a 1 cm pathlength cuvette and gives an absorbance of 1.
e = 1 / 1 * c
Therefore,c = 1 / 20 = 0.05 mol L-1
The answer is now obvious - a compound with a high molar absorbtivity is very effective
at absorbing light (of the appropriate wavelength),and hence low concentrations of a
compound with a high molar absorbtivity can be easily detected
UV visible
spectrophotometer
M o d el
W a ve l e n gt h r an ge
L i n e w i d t h ( ba ndwi dt h)
W a ve l e n gt h ac cu r ac y
W a ve l e n gt h r e p e at ab i l i t y
Pho t om e t r i c r an ge
Sa m p l e vol um e
O t he r s
Homoannular
(cisoid)
Heteroannular
(transoid)
Parent l=253 nm l=214 nm=217 (acyclic)
Increments for:
Double bond extending conjugation 30 30
Alkyl substituent or ring residue 5 5
Exocyclic double bond 5 5
Polar groupings:
-OC(O)CH3 0 0
-OR 6 6
-Cl,-Br 5 5
-NR2 60 60
-SR 30 30
Example 1:
Transoid,217 nm
Alkyl groups or ring residues,3 x 5 = 15 nm
Calculated,232 nm
Observed,234 nm
Cisoid,253 nm
Alkyl groups or ring residues,2 x 5 = 10 nm
Calculated,263 nm
Observed,256 nm
Example 2:
Transoid,214 nm
Alkyl groups ring residues,3 x 5 = 15 nm
Exocyclic double bond,5 nm
Calculated,234 nm
Observed,235 nm
Example3:
Cisoid,253 nm
Alkyl groups / ring residues,4 x 5 = 20 nm
Exocyclic double bond,5 nm
Calculated,278 nm
Observed,275 nm
Example 4:
Base values:
R = alkyl or ring residue l = 246 nm
R = H l = 250 nm
R = OH,OR l = 230 nm
Increment for each substituent:
-Alkyl or ring residue o,m 3
p 10
-OH,-OR o,m 7
p 25
-O- o,m 11
p 20
-Cl o,m 0
p 10
-Br o,m 2
p 15
-NH2 o,m 13
p 58
-NHC(O)CH3 o,m 20
p 45
-NHCH3 p 73
-N(CH3)2 o,m 20
p 85
Parent chromophore,230 nm
p-OH,25 nm
m-OH,2 x 7 = 14 nm
Calculated,269 nm
Observed,270 nm
Parent chromophore,246 nm
o-Ring residue,3 nm
m-Br,2 nm
Calculated,251 nm
Observed,253 nm
Protein absorbances come from 3 sources
Peptide Bond
Aromatic Amino Acids -- most useful
Phe e250 = 400 symmetry forbidden p --> p*
Tyr e274 = 1400 p --> p*
Trp e280 = 4500 at least 3 different transitions,A280 is one of the most commonly
used methods to measure protein concentration (aside from colorimetric
methods such as the Lowry or Bradford dye binding assays),but this method is
obviously very sensitive to differences in amino acid composition,
These transitions can change with pH; especially Tyr
Prosthetic Groups
Nucleotides --> e.g,FMN,NAD
Heme
Cu
Retinal etc..
Estimating Protein Concentration
1,Generally measure A280 and assume an average composition of Tyr
and Trp,Approximately 1 mg/ml ---> 1.0 A
2,Measure absorbance of peptide groups at l = 230 nm:
1 mg/ml ---> 3.0 A
This is not commonly used because many other groups absorb in this wavelength region,
Nucleic Acids:
Nucleotide spectra are complicated to analyze quantitatively because there are many
non-bonded electrons,Expect several different p --> p* and n --> p* transitions at
each region between 200 nm and 300 nm
All nucleotides have lmax near 260 nm which is not affected by sugar phos,Configuration
==> can measure nucleic acids at 260 nm to estimate concentration,e260 =~ 1 x 104 M-1
===> very sensitive and can measure concentrations down to approx,3 μg/ml
Hyperchromism -- A260 is lower for dsDNA than for ssDNA or for individual nucleotides,
Results from stacking of bases in the double helical conformation; quantitative explanation is
very complicated,(see Figure above)
Single-Beam UV-Vis Spectrophotometer
Introduction
Single-Beam spectrophotometers are often sufficient for making quantitative absorption
measurements in the UV-Vis spectral region,The concentration of an analyte in solution
can be determined by measuring the absorbance at a single wavelength and applying the
Beer-Lambert Law,
In either type of single-beam instrument,the instrument is calibrated with a reference
cell containing only solvent to determine the Po value necessary for an absorbance
measurement,
Pictures of single beam UV-Vis spectrophotometers (Spectronic 20 and 20D)
Dual-Beam uv-vis Spectrophotometer
Introduction
In single-beam uv-vis absorption spectroscopy,obtaining a spectrum requires manually
measuring the transmittance (see the Beer-Lambert Law) of the sample and solvent at
each wavelength,The double-beam design greatly simplifies this process by measuring
the transmittance of the sample and solvent simultaneously,The detection electronics
can then manipulate the measurements to give the absorbance.
Schematic of a dual-beam uv-vis spectrophotometer
Pictures of Lambda 3A and 4B dual-beam uv-vis spectrophotometers
Pictures of a Hitachi dual-beam UV- spectrophotometer
Picture of the sample compartment
Array-Detector Spectrophotometer
Introduction
Array-detector spectrophotometers allow rapid recording of absorption spectra,Dispersing the
source light after it passes through a sample allows the use of an array detector to simultaneously
record the transmitted light power at multiple wavelengths,There are a large number of
applications where absorbance spectra must be recorded very quickly,Some examples include
HPLC detection,process monitoring,and measurement of reaction kinetics.
Instrumentation
These spectrometers use photodiode arrays (PDAs) or charge-coupled devices (CCDs) as the detector,The
spectral range of these array detectors is typically 200 to 1000 nm,The light source is a continuum source
such as a tungsten lamp,All wavelengths pass through the sample,The light is dispersed by a diffraction
grating after the sample and the separated wavelengths fall on different pixels of the array detector,The
resolution depends on the grating,spectrometer design,and pixel size,and is usually fixed for a given
instrument,Besides allowing rapid spectral recording,these instruments are relatively small and robust,
Portable spectrometers have been developed that use optical fibers to deliver light to and from a sample,
These instruments use only a single light beam,so a reference spectrum is recorded and stored in memory to
produce transmittance or absorbance spectra after recording the sample spectrum.
Nanodrop spectrophotometer
ELISA reader
Introduction
Luminescence is the emission of light by a substance,It occurs when an electron returns to the
electronic ground state from an excited state and loses it's excess energy as a photon.
Luminescence spectroscopy is a collective name given to three related spectroscopic techniques,
They are,
Molecular fluorescence spectroscopy
Molecular phosphorescence spectroscopy
Chemiluminescence spectroscopy
Fluorescence and phosphorescence (photoluminescence)
The electronic states of most organic molecules can be divided into singlet states and triplet
states;
Singlet state,All electrons in the molecule are spin-paired
Triplet state,One set of electron spins is unpaired
Chemiluminescence
Chemiluminescence occurs when a chemical reaction produces an electronically excited species
which emits a photon in order to reach the ground state,These sort of reactions can be
encountered in biological systems; the effect is then known as bioluminescence,The number of
chemical reactions which produce chemiluminescence is small,However,some of the
compounds which do react to produce this phenomenon are environmentally significant.
A good example of chemiluminescence is the determination of nitric oxide:
NO + O3?NO2* + O2
NO2*?NO2 + hv (l = 600 - 2800 nm)
An obvious difference between certain compounds is their color,
In this respect the human eye is functioning as a spectrometer
analyzing the light reflected from the surface of a solid or passing
through a liquid,
Wavelength is defined on the left below,as the distance between
adjacent peaks (or troughs),and may be designated in meters,
centimeters or nanometers (10-9 meters),Frequency is the number
of wave cycles that travel past a fixed point per unit of time,and is
usually given in cycles per second,or hertz (Hz),
Visible wavelengths cover a range from approximately 400 to 800 nm,
Violet,400 - 420 nm
Indigo,420 - 440 nm
Blue,440 - 490 nm
Green,490 - 570 nm
Yellow,570 - 585 nm
Orange,585 - 620 nm
Red,620 - 780 nm
“ROY G BIV”
Complementary colors are diametrically opposite each other,
Thus,absorption of 420-430 nm light renders a substance yellow,
and absorption of 500-520 nm light makes it red,Green is unique
in that it can be created by absoption close to 400 nm as well as
absorption near 800 nm.
Color wheel
“extensively conjugated pi-electrons”
The Electromagnetic Spectrum
Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer electrons,
There are three types of electronic transition which can be considered;
Transitions involving p,s,and n electrons,
Transitions involving charge-transfer electrons
Transitions involving d and f electrons (not covered in this Unit)
When an atom or molecule absorbs energy,electrons are promoted from their ground state to
an excited state,In a molecule,the atoms can rotate and vibrate with respect to each other,
These vibrations and rotations also have discrete energy levels,which can be considered as
being packed on top of each electronic level
UV-Visible Absorption Spectra
The energies noted above are sufficient to promote or excite a molecular electron to
a higher energy orbital,Consequently,absorption spectroscopy carried out in this
region is sometimes called "electronic spectroscopy",
Molar absorptivity
Molar Absorptivity,e = A/ c l
A= absorbance,
c = sample concentration in moles/liter
l = length of light path through the sample in cm.
Molar absoptivities may be very large for strongly absorbing chromophores
(>10,000) and very small if absorption is weak (10 to 100),The magnitude of e
reflects both the size of the chromophore and the probability that light of a given
wavelength will be absorbed when it strikes the chromophore,A general equation
stating this relationship may be written as follows,
e = 0.87*1020 R * a
(R is the transition probability (0 to 1) & a is the chromophore area in cm2)
The Importance of Conjugation
Empirical Rules for Absorption Wavelengths
of Conjugated Systems---Woodward-Fieser Rules for
Calculating the lmax of Conjugated Dienes and Polyenes
P
The amount of radiation absorbed may be measured in a number of ways,
Transmittance,T = P / P0,% Transmittance,%T = 100 T
Absorbance,A = lg P0 / P
A = lg 1 / T
A = lg100 / %T
A = 2 - lg%T
The Beer-Lambert Law
A = e b c
A is absorbance (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 with units of cm.
c is the concentration of the compound in solution,expressed in mol L-1
The reason why we prefer to express the law with this equation is because
absorbance is directly proportional to the other parameters,as long as the law is obeyed
P a t h l e n g t h / c m 0 0,2 0,4 0,6 0,8 1,0
%T 1 0 0 50 25 1 2,5 6,2 5 3,1 2 5
A b s o r b a n c e 0 0,3 0,6 0,9 1,2 1,5
The linear relationship between concentration and absorbance is both simple and
straightforward,which is why we prefer to express the Beer-Lambert law using
absorbance as a measure of the absorption rather than %T.
Note that the Law is not obeyed at high concentrations,This deviation from the Law is not dealt with here.
Question,What is the significance of the molar absorbtivity,e?
In words,this relationship can be stated as "e is a measure of the amount of light absorbed
per unit concentration",Molar absorbtivity is a constant for a particular substance.
e = A / bc
Let us take a compound with a very high value of molar absorbtivity,say 100,000 L mol-1 cm-1,
which is in a solution in a 1 cm pathlength cuvette and gives an absorbance of 1.
e = 1 / 1* c
Therefore,c = 1 / 100,000 = 1 * 10-5 mol L-1
Now let us take a compound with a very low value of e,say 20 L mol-1 cm-1,which is in
solution in a 1 cm pathlength cuvette and gives an absorbance of 1.
e = 1 / 1 * c
Therefore,c = 1 / 20 = 0.05 mol L-1
The answer is now obvious - a compound with a high molar absorbtivity is very effective
at absorbing light (of the appropriate wavelength),and hence low concentrations of a
compound with a high molar absorbtivity can be easily detected
UV visible
spectrophotometer
M o d el
W a ve l e n gt h r an ge
L i n e w i d t h ( ba ndwi dt h)
W a ve l e n gt h ac cu r ac y
W a ve l e n gt h r e p e at ab i l i t y
Pho t om e t r i c r an ge
Sa m p l e vol um e
O t he r s
Homoannular
(cisoid)
Heteroannular
(transoid)
Parent l=253 nm l=214 nm=217 (acyclic)
Increments for:
Double bond extending conjugation 30 30
Alkyl substituent or ring residue 5 5
Exocyclic double bond 5 5
Polar groupings:
-OC(O)CH3 0 0
-OR 6 6
-Cl,-Br 5 5
-NR2 60 60
-SR 30 30
Example 1:
Transoid,217 nm
Alkyl groups or ring residues,3 x 5 = 15 nm
Calculated,232 nm
Observed,234 nm
Cisoid,253 nm
Alkyl groups or ring residues,2 x 5 = 10 nm
Calculated,263 nm
Observed,256 nm
Example 2:
Transoid,214 nm
Alkyl groups ring residues,3 x 5 = 15 nm
Exocyclic double bond,5 nm
Calculated,234 nm
Observed,235 nm
Example3:
Cisoid,253 nm
Alkyl groups / ring residues,4 x 5 = 20 nm
Exocyclic double bond,5 nm
Calculated,278 nm
Observed,275 nm
Example 4:
Base values:
R = alkyl or ring residue l = 246 nm
R = H l = 250 nm
R = OH,OR l = 230 nm
Increment for each substituent:
-Alkyl or ring residue o,m 3
p 10
-OH,-OR o,m 7
p 25
-O- o,m 11
p 20
-Cl o,m 0
p 10
-Br o,m 2
p 15
-NH2 o,m 13
p 58
-NHC(O)CH3 o,m 20
p 45
-NHCH3 p 73
-N(CH3)2 o,m 20
p 85
Parent chromophore,230 nm
p-OH,25 nm
m-OH,2 x 7 = 14 nm
Calculated,269 nm
Observed,270 nm
Parent chromophore,246 nm
o-Ring residue,3 nm
m-Br,2 nm
Calculated,251 nm
Observed,253 nm
Protein absorbances come from 3 sources
Peptide Bond
Aromatic Amino Acids -- most useful
Phe e250 = 400 symmetry forbidden p --> p*
Tyr e274 = 1400 p --> p*
Trp e280 = 4500 at least 3 different transitions,A280 is one of the most commonly
used methods to measure protein concentration (aside from colorimetric
methods such as the Lowry or Bradford dye binding assays),but this method is
obviously very sensitive to differences in amino acid composition,
These transitions can change with pH; especially Tyr
Prosthetic Groups
Nucleotides --> e.g,FMN,NAD
Heme
Cu
Retinal etc..
Estimating Protein Concentration
1,Generally measure A280 and assume an average composition of Tyr
and Trp,Approximately 1 mg/ml ---> 1.0 A
2,Measure absorbance of peptide groups at l = 230 nm:
1 mg/ml ---> 3.0 A
This is not commonly used because many other groups absorb in this wavelength region,
Nucleic Acids:
Nucleotide spectra are complicated to analyze quantitatively because there are many
non-bonded electrons,Expect several different p --> p* and n --> p* transitions at
each region between 200 nm and 300 nm
All nucleotides have lmax near 260 nm which is not affected by sugar phos,Configuration
==> can measure nucleic acids at 260 nm to estimate concentration,e260 =~ 1 x 104 M-1
===> very sensitive and can measure concentrations down to approx,3 μg/ml
Hyperchromism -- A260 is lower for dsDNA than for ssDNA or for individual nucleotides,
Results from stacking of bases in the double helical conformation; quantitative explanation is
very complicated,(see Figure above)
Single-Beam UV-Vis Spectrophotometer
Introduction
Single-Beam spectrophotometers are often sufficient for making quantitative absorption
measurements in the UV-Vis spectral region,The concentration of an analyte in solution
can be determined by measuring the absorbance at a single wavelength and applying the
Beer-Lambert Law,
In either type of single-beam instrument,the instrument is calibrated with a reference
cell containing only solvent to determine the Po value necessary for an absorbance
measurement,
Pictures of single beam UV-Vis spectrophotometers (Spectronic 20 and 20D)
Dual-Beam uv-vis Spectrophotometer
Introduction
In single-beam uv-vis absorption spectroscopy,obtaining a spectrum requires manually
measuring the transmittance (see the Beer-Lambert Law) of the sample and solvent at
each wavelength,The double-beam design greatly simplifies this process by measuring
the transmittance of the sample and solvent simultaneously,The detection electronics
can then manipulate the measurements to give the absorbance.
Schematic of a dual-beam uv-vis spectrophotometer
Pictures of Lambda 3A and 4B dual-beam uv-vis spectrophotometers
Pictures of a Hitachi dual-beam UV- spectrophotometer
Picture of the sample compartment
Array-Detector Spectrophotometer
Introduction
Array-detector spectrophotometers allow rapid recording of absorption spectra,Dispersing the
source light after it passes through a sample allows the use of an array detector to simultaneously
record the transmitted light power at multiple wavelengths,There are a large number of
applications where absorbance spectra must be recorded very quickly,Some examples include
HPLC detection,process monitoring,and measurement of reaction kinetics.
Instrumentation
These spectrometers use photodiode arrays (PDAs) or charge-coupled devices (CCDs) as the detector,The
spectral range of these array detectors is typically 200 to 1000 nm,The light source is a continuum source
such as a tungsten lamp,All wavelengths pass through the sample,The light is dispersed by a diffraction
grating after the sample and the separated wavelengths fall on different pixels of the array detector,The
resolution depends on the grating,spectrometer design,and pixel size,and is usually fixed for a given
instrument,Besides allowing rapid spectral recording,these instruments are relatively small and robust,
Portable spectrometers have been developed that use optical fibers to deliver light to and from a sample,
These instruments use only a single light beam,so a reference spectrum is recorded and stored in memory to
produce transmittance or absorbance spectra after recording the sample spectrum.
Nanodrop spectrophotometer
ELISA reader
Introduction
Luminescence is the emission of light by a substance,It occurs when an electron returns to the
electronic ground state from an excited state and loses it's excess energy as a photon.
Luminescence spectroscopy is a collective name given to three related spectroscopic techniques,
They are,
Molecular fluorescence spectroscopy
Molecular phosphorescence spectroscopy
Chemiluminescence spectroscopy
Fluorescence and phosphorescence (photoluminescence)
The electronic states of most organic molecules can be divided into singlet states and triplet
states;
Singlet state,All electrons in the molecule are spin-paired
Triplet state,One set of electron spins is unpaired
Chemiluminescence
Chemiluminescence occurs when a chemical reaction produces an electronically excited species
which emits a photon in order to reach the ground state,These sort of reactions can be
encountered in biological systems; the effect is then known as bioluminescence,The number of
chemical reactions which produce chemiluminescence is small,However,some of the
compounds which do react to produce this phenomenon are environmentally significant.
A good example of chemiluminescence is the determination of nitric oxide:
NO + O3?NO2* + O2
NO2*?NO2 + hv (l = 600 - 2800 nm)
