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

Glass cell filled with concentration of solution (C)

IILight

0

As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.

 

T- Transmittance

T = I0 - original light intensity

I- transmitted light intensity

 

% Transmittance = 100 x

Absorbance (A) or optical density (OD) = 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

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).

SpectrophotometerInstrument 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

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Flu

ores

cen

ce I

nte

nsit

y

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 SpectrophotometrySpectroflurometer

• 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 AssaysCell Proliferation Assays RNA QuantitationDNA QuantitationEnzyme AssaysProtein QuantitationReporter 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.