Absorption Spectroscopy

• A spectrophotometer consists of two

instruments, namely a spectrometer for
producing light of any selected color
(wavelength), and a photometer for
measuring the intensity of light.
 BEER LAMBERT LAW

Light
I0 I

Glass cell filled with

concentration of solution (C)

As the cell thickness increases, the intensity of I

(transmitted intensity of light ) decreases.
 
T- Transmittance
I
T= I0 - original light intensity
I0
I- transmitted light intensity
 
I
% Transmittance = 100 x
I0
1
Absorbance (A) or optical density (OD) = Log T
I0
= Log = 2 - Log%T
I
I
Log is proportional to C (concentration of solution) and is
I0
also proportional to L (length of light path
through the solution).
A = ECL
E = Molar Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution
• Chromophores are components of
molecules which absorb light
• The type of functional groups that absorb
ultraviolet light can be conjugated species,
such as alkenes, aromatics, etc
• many metal-ligand complexes also absorb
UV/visible light.
 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
Two ways to determine concentration
from absorption

– if extinction coeff (()) and path length

are known
• Use Beer’s Law to find C
– might try at a number of different wavelengths as
a self-check

– develop calibration
• Check linearity vs concentration
Deviations from the beer Lambert's law

• Radiation reflected by the sample holder,

solution etc.
• Radiation should not provide a range of
energy levels
• Too high or too low solute concentration
• Unwanted radiation which could not be
removed by the monochromator (stray
light).
 Spectrophotometer
Instrument Design
• Light source
• tungsten-visible range (400-700 nm),
• hydrogen or deuterium for UV range (200-400 nm).
• Monochromator or filter
• Sample holder
– Quartz vs plastic
• Detector
 A monochromator:

• - a device used “to vary the wavelength of

radiation continuously over a considerable
range”
• Mechanical in construction: use “slits, lenses,
mirrors, windows, and gratings or prisms.”
– Glass prisms for visible region
– Quartz or silica prisms for UV region
 Detectors
• Convert light intensity into an electrical
signal
• Photomultiplier tubes
– With a cascade of electrons, amplify the
signal, more sensitive
• Photodiode arrays
– Cheaper and composed of silicon crystals
arranged in linear array
 Fluorescence
• Fluorescence is an absorption process
which occurs in a small group of
molecules. These molecules relax by
emission of radiation when excited, rather
than by vibration, as most molecules do.
• Fluorescencent emission is a fast process,
taking only 10-6-10-9 seconds to occur.
• Flurophores are generally aromatic rings
• Intrinsic fluors
– Tryptophan, tyrosine, phenylalanine
(frequency is low)
– NAD (generally weak fluorescence spectra)
– Bases of DNA (generally weak fluorescence
spectra)
Radiative and Non Radiative Transitions
• Because vibrational levels of the ground and
excited states of most molecules overlap, most
molecules relax by non-radiative transitions
(energy from the absorbed photon is dissipated
as vibrational relaxations)
• The conversion of electronic energy to
vibrational energy is helped if the molecule is
"loose and floppy", because it can reorient itself
in ways which aid the internal transfer of energy.
• Some chromophores are quite rigid and
inflexible molecules and have a limited
number of vibrational energy levels of
excited and ground state which do not
often overlap. These molecules relax by
radiative transition (energy dissipated by
emission of a photon) known as
fluorescence
 Radiative Transitions
• Gives rise to 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.
• It could be three types
– fluorescence
– phosphorescence
– Chemiluminescence
 Phosphorescence

• A molecule in the excited triplet state may not always

use intersystem crossing to return to the ground state. It
could lose energy by emission of a photon.
• A triplet/singlet transition is much less probable than a
singlet/singlet transition. The lifetime of the excited triplet
state can be up to several seconds, in comparison with
nanosecond average lifetime of an excited singlet state.
• Emission from triplet/singlet transitions can continue
after initial irradiation. Internal conversion and other
radiationless transfers of energy compete so
successfully with phosphorescence that it is usually seen
only at low temperatures or in highly viscous media.
• Fluorophores have a characteristic
emission and absorption spectrum
Stokes Shift
– is the energy difference between the
maximum of absorbance peak and the
maximum of emission peak
Stokes Shift is 25 nm
Fluorescence Intensity

Fluorescein
molecule
495 nm 520 nm

Wavelength
• Two further processes diminish or quench
the amount of light energy emitted from
the sample
• All forms of quenching results in the non-
radiative loss of energy
– Internal quenching (structural rearrangement)
– External quenching (interaction of the excited
molecule with another molecule)
• Quantum Yield
– A measure to quantitate fluorescence
– Q=Number of photons emitted/number of
photons absorbed
– Under a given set of conditions, Q will usually
have a fixed value for a particular fluor.
 Fluorescence Spectrophotometry
Spectroflurometer

• Source of ultraviolet radiation

• Sample compartment (do not touch! skin
oil does fluoresce)
– Cuvettes are made from borosilicate or quartz
glass
• Wavelength selector to choose excitation
radiation
• Wavelength selector to choose emission
radiation
• Detector
 Two common lamp sources
• High-pressure xenon arc lamp
- surprisingly, a continuous source
- approximates a blackbody arc, from
300 to 1300 nm
• Low-pressure mercury vapor lamp
- line source
- a few useful lines: 254, 313, 546 nm
- used in overhead lighting
 LASER (Light Amplification by the
Stimulated Emission of Radiation) sources

• More expensive
• Advantages:
- can measure samples in very small quantities (very
intense)
- provides highly specific monochromatic excitation
- very stable as a radiation source
 Optical Detectors

• Create a current proportional to light

intensity.
• Types
– Photomultiplier Tube (PMT)
– Photodiode array
– Charge Coupled device (CCD)
 Three major differences between
fluorescence and absorption spectroscopy

• Detection is at a 90 angle to the

incident radiation
• Two wavelength selectors are used
instead of one
• A more powerful light source is used
 Wavelengths Used
• Fluorescence is used primarily in the
Ultraviolet range.
• It is used mostly in the lower part of the UV
spectrum because if the energy of the light
is to high it will destroy many molecules.
 Conclusion
• Most sensitive spectroscopy because the
emission signal is measured above a low
background level.
• 1000x more sensitive than absorption
spectroscopy*
• allows experiments to be performed with
low concentration samples*
 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.
• Luminol in an alkaline solution with hydrogen peroxide
in the presence of iron or copper, produces
chemoluminescence. The luminol reaction is

luminol + H2O2 → 3-APA[◊] → 3-APA + light

luminol + H2O2 → 3-APA[◊] → 3-APA +
light

Luciferin + ATP → oxyluciferin+AMP+ PP+

CO2+light
• Why is chemiluminescence spectroscopy
a highly selective, sensitive and simple
technique?

• Hints: How common are chemiluminescent

reactions? Is the emitted radiation
measured against a noisy background?
Examples for the use of
flurosence and
chemiluminescence in
biological systems
 Examples of Assays with
Fluorescence
Bacterial Viability Assays
Cell Proliferation Assays
RNA Quantitation
DNA Quantitation
Enzyme Assays
Protein Quantitation
Reporter Gene Assays
 Bacterial Viability
• This assay utilizes mixtures of SYTO® 9 green fluorescent nucleic
acid stain and the red fluorescent nucleic acid stain, propidium
iodide.
• These stains differ both in their spectral characteristics and in their
ability to penetrate healthy bacterial cells. When used alone, the
SYTO 9 stain labels bacteria with both intact and damaged
membranes.
• In contrast, propidium iodide penetrates only bacteria with
damaged membranes, competing with the SYTO 9 stain for nucleic
acid binding sites when both dyes are present. When mixed in
recommended proportions, SYTO 9 stain and propidium iodide
produce green fluorescent staining of bacteria with intact cell
membranes and red fluorescent staining of bacteria with damaged
membranes. The background remains virtually nonfluorescent.
Consequently, the ratio of green to red fluorescence intensities
provides a quantitative index of bacterial viability
 Quantitation of DNA- Hoechst
33258
• Quantitation of DNA is an important step for many practices in
molecular biology. Common techniques that use DNA, such as
sequencing, cDNA synthesis and cloning, RNA transcription,
transfection, nucleic acid labeling (e.g. random prime labeling), etc.,
all benefit from a defined template concentration. Failure to produce
results from these techniques can sometimes be attributed to an
incorrect estimate of the DNA template used.
The concentration of a nucleic acid is most commonly measured by
UV absorbance at 260 nm (A260). Absorbance methods are limited
in sensitivity, however, due to a high level of background
interference.
Hoechst 33258, a bisbenzimide DNA intercalator, provides a
fluorometric alternative that is more sensitive than UV absorbance
methods.
 Luminescence

ATP Determinations
Cell Proliferation
Chemiluminescence Labeling
Cytotoxicity
DNA Quantitation
Enzyme Assays
Immunoassays - Alkaline Phosphatase
Mycoplasma Detection
Reporter Gene Assays
RNA Quantitation
 Reporter Gene Assay

The activity of promoter to drive the expression of geneX can be

detected by chemiluminescence (in this example luciferase was
used). If there is a mutation in the promoter of the gene that you are
studying you can clone the gene in this plasmid and compare the
expression of the gene with unmutated version)
 Enzymatic Activity
• A reliable method for ATP detection is useful for
studying enzymes that produce or degrade ATP
• The ATP-dependent oxidation of luciferin by
luciferase produces light measured by the
Luminometer. When ATP is the limiting factor in
the luciferin oxidation reaction, the amount of
light produced is proportional to the ATP
concentration of the sample.
 Cell Proliferation
• Luminescent Cell Viability Assay kits provides a
convenient, rapid, and sensitive procedure for
determining the number of viable cells in a culture based
on quantitation of ATP, which signals the presence of
metabolically active cells.
• Luciferase enzyme requires ATP in order to generate
light. Metabolically active cells produce ATP as energy
for respiration and other vital processes. After an equal
volume of Reagent is added to the cell culture,
luminescence is measured.
• Light signal is proportional to the amount of ATP present
which correlates with the number of viable cells present.
 Protein quantitation by
chemiluminescence

• Companies devise different chemiluminescence kits for different applications.

 Principle of the ProLabel Protein quantitation
Assay

• The ProLabel assay is based on enzyme fragment

complementation (2, 3).  The ProLabel tag encodes an
inactive enzyme fragment, which is expressed as an N-
or C-terminal tag fused to your protein of interest (Figure
1). When the ProLabel fusion protein is combined with
Enzyme Acceptor (EA), supplied in the Detection Kit, the
ProLabel tag and the Enzyme Acceptor combine to form
a complete, active enzyme that cleaves the
chemiluminescent substrate. The resulting signal can be
detected and quantified with any standard luminometer.
The assay provides a low threshold of detection as well
as an excellent dynamic range, allowing you to easily
detect changes in protein expression levels. 
• For FISH assays alternative to using
oligos directly labeled with fluroscent dyes
is to prepare probes(complementary oligos
to your gene of interest) using nucleotides
labeled with biotin or digoxygenin and then
use fluorescently labeled streptavidin and
fluorescently labeled anti dig antibodies.
• This Aneuploid Screen test shows a female fetus with trisomy-21.
The nucleus on the left has been hybridized to probes for
chromosomes 13 (green), and 21 (red) and clearly has three red
signals. The nucleus on the right has been hybridized to probes for
chromosomes 18 (aqua), X (green), and Y (red). Since it has two
green signals and no red signal it is female.
• Infra-red spectrophotometry - is used to
determine the presence of distinct
functional groups in organic molecules. IR
radiation causes them to vibrate at various
frequencies, allowing us to identify them.
– Mainly qualitative analysis
 NMR Spectroscopy

• NMR Spectroscopy is used to determine the carbon-

hydrogen framework of a molecule and works with even
the most complex molecules.
• The principle of NMR is based upon the spin of atomic
nuclei in an external magnetic field. Many nuclei do not
have the ability to spin in a magnetic field, but proton
(1H) and an isotope of carbon (13C) can spin in a
magnetic field, so they are the most widely used
elements in NMR. When no magnetic field is present, the
nuclear spins of magnetic nuclei are oriented randomly.
• Once a strong magnetic field is introduced to the nuclei,
they reorient their spins so their magnetic fields are
either parallel or antiparallel to the alignment of the
applied force.