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-vis 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.
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)
(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-vis 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.
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)
