PRINCIPLES AND APPLICATION OF SPECTROPHOTOMETRY IN DISEASE DIAGNOSIS Absorption : UV/Visible/IR • Certain molecules absorb light in a characteristic way: helps to identify and quantify biological molecules • Absorption occurs when the energy contained in a photon is absorbed by an electron resulting in a transition to an excited state • The absorption efficiency of an analyte is affected by: The nature of the analyte, number of available microstates, The solvent • Absorption spectroscopy: Bioanalytical methods; signal intensity is directly proportional to the concentration
Transcript
PRINCIPLES AND APPLICATION OF SPECTROPHOTOMETRY IN DISEASE DIAGNOSISAbsorption : UV/Visible/IR
• Certain molecules absorb light in a characteristic way: helps to identify and quantify biological molecules
• Absorption occurs when the energy contained in a photon is absorbed by an electron resulting in a transition to an excited state
• The absorption efficiency of an analyte is affected by: The nature of the analyte, number of available microstates, The solvent
• Absorption spectroscopy: Bioanalytical methods; signal intensity is directly proportional to the concentration
• Pigment Chlorophyll- which absorbs light; in the blue and red region of the visible light spectrum.
• For this reason, leaves are- green (because they reflect green).
• If Leaf is extracted in an organic solvent, the leaf extract (containing the solute chlorophyll) with a high chlorophyll content will produce: dark green colour
• A leaf extract with a low chlorophyll content will yield a pale green extract.
Spectrophotometry is• a mean of measuring how densely green
the solution is.(concentration)
The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic radiationSpectrophotometry : Quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.; Involves the use of a spectrophotometer.SPECTROPHOTOMETER : The combination of two devices, a spectrometer and a photometer.•A device that is used to measure intensity of light as a function of the wavelength of light.
• An instrument that measures the amount of light of a specified wavelength that passes through (is transmitted through) a sample (medium)
.
SPECTROSCOPY / SPECTROCHEMICAL ANALYSIS.
BLOCK DIAGRAM OF SPECTROPHOTOMETER
The Spectrophotometer Single-beam
Double-beam
I0= radiant power arriving at the
cuvette I = radiant power leaving the cuvette
L = length of the path through the sample Cc = concentration of the absorbing substance
a = absorptivity of the sample (extinction coefficient)
SPECTROPHOTOMETER
E ntrance s lit
E x it s lit
D e tecto r
C uve tte
R ed
V io le tP rism
M onochrom ato rL igh t source
I0I
R eadoutdevice
Spectrometer: for producing light of any selected wavelength or color Photometer: used for measuring the intensity of light. The two devices are placed at either side of a cuvette filled with a liquid
PRINCIPLES : Spectroscopy:
Deals with the production, measurement, and interpretation of spectra arising from the interaction of electromagnetic radiation with matter.
Electromagnetic spectrum of energy: the gamma rays (wavelengths < 0.1 nanometres) to radio waves (wavelengths > 250 millimetres.)
Spectroscopy deals with : the ultraviolet (180 to 380nm) the visible (380 to 800nm) the infrared (0.8 to 50 micrometres).
Colors & Wavelengths
COLOR WAVELENGTH (λ in nm)
Ultraviolet < 380
Violet 380 – 435
Blue 436 – 480
Greenish-blue 481 – 490
Bluish-green 491 – 500
Green 501 – 560
Yellowish-green 561 – 580
Yellow 581 – 595
Orange 596 – 650
Red 651 – 780
Near Infrared > 780
Vis
ible
Lig
ht
SPECTROPHOTOMETRY
• A photometer (a device for measuring light intensity)
• Measure intensity as a function of the color, or more specifically, the wavelength of light
• Tungsten or xenon flashlamp as the source of white light
• Tungsten lamp for measurements in visible region(360-900nm)
• Hydrogen /deuterium lamp for UV region(200-380nm)
COLORIMETRY
• The measurement of color • Any technique used to
evaluate an unknown color in reference to known colors
• It determines color based on the red, blue, and green components of light absorbed by the object or sample,
• Colored light beam through an optical filter, which transmits only one particular color / band of wavelengths of light to the photodectector
Spectroscopy and Spectrophotometry
• Light can either be transmitted or absorbed by dissolved substances
• Presence & concentration of dissolved substances is analyzed by passing light through the sample
• Spectroscopes measure electromagnetic emission
• Spectrophotometers measure electromagnetic absorption • Principle: Based on Beer Lambert’s LAWBased on Beer Lambert’s LAW
Spectrometer produces the light of desired wavelength and it passes through the tube and reaches photometer that measures its intensity.
Then the photometer produces a voltage signal to a display device, usually a galvanometer.
As the amount of light absorbed by the liquid changes; the signal also changes.
The concentration of a substance in solution can be measured by calculating the amount of absorption of light at the appropriate wavelength or a particular colour Reading of Spectrophotometer: (Number)- Absorbance that is directly proportional to the color intensity, and also the concentration of the species responsible for the color.
• To use absorbance for analytical purposes, a calibration curve must be generated by measuring the absorbance of several solutions that contain known concentrations of analyte.
If development of color is linked to the concentration of a substance in solution then: That concentration can be measured by determining the extent of absorption of light at the appropriate wavelength.
For example : Hemoglobin appears red • Hemoglobin absorbs blue and green light rays much
more effectively than red.) • Thus, The degree of absorbance of blue or green light is
proportional to the concentration of hemoglobin.
Terms:/Parameters Transmittance : The passing of light through a
sampleAbsorbance: Amount of light absorbed by a
sample (the amount of light that does not pass through or reflect off a sample)
%Transmittance: The manner in which a spectrophotometer reports the amount of light that passes through a sample
Absorbance units: A unit of light absorbance determined by the decrease in the amount of light in a light beam
Absorbance spectrum: A graph of a sample’s absorbance at different wavelengths
Lambdamax: The wavelength that gives the highest absorbance value for a sample
Absorption: The Beer-Lambert Law
Pierre Bouguer
Astronomer: Light is
diminished as it passes
through the atmosphere.
August Beer (1825-1863): Added absorption co-efficient and related to conc. in solution.
Johan Lambert
Mathematician, first to prove that is irrational. No absorption coefficient
clIIA )/log( 01
€: Extinction coefficient
c: Concentrationl : Path length
BEER–LAMBERT’S LAW (Beer–Lambert–Bouguer law)
• Relates the absorption of light to the properties of the material through which the light is travelling.
BEER'S LAW • When monochromatic light (light of a specific wavelength) passes
through a solution there is usually a quantitative relationship between the solute concentration and the intensity of the transmitted light
• The amount of light absorbed by the a medium ( solution/ sample) is proportional to the concentration of the absorbing material or solute present.
• Thus the concentration of a coloured solute in a solution may be determined in the lab by measuring the ABSORBANCY OF LIGHT AT A GIVEN WAVELENGTH
BEER–LAMBERT’S LAW (Beer–Lambert–Bouguer law)….contd
LAMBERT'S LAW o Lambert described how intensity changes
with distance in an absorbing medium. o The amount of light absorbed by the a
medium ( solution/ sample) at a given wavelength is proportional to thickness of the absorbing layer: path length of the light
Beer – Lambert LawStates that the Absorbance (O.D) of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.
The fraction of the incident light absorbed by a solution at a given wavelength is related to a. thickness of the absorbing layer (path length) and b. concentration of the absorbing species
I0 : intensity(power) of the incident light I : intensity(power) of the transmitted light ; ℓ : . thickness of the absorbing layer (path length) and cross section of light absorption by a single particle;
Transmittance
There is a logarithmic dependence between the transmission (or transmissivity), T, of light through a substance and The product of : the absorption coefficient of the substance, α, and the distance the light travels through the material (i.e. the path length), ℓ.
Defined as the ratio of the intensity of light emerging from the solution (I) to that of incident light entering (Io)
• The ABSORPTION COEFFICIENT: (α ) = Molar absorptivity (extinction coefficient) of the absorber, (c) X the concentration (c) of absorbing species in the material
T- Transmittance
T = I0 - Original light intensity
I- Transmitted light intensity
% Transmittance (T)= 100 x
Absorbance (A) = Log
(OPTICAL DENSITY)
= Log = KCL Log is proportional to : C (concentration of solution) and L (length of light path through the solution).
I
I0
I
I0
I0 I
1
T
I0 I
By definition of the Beer - Lambert Law.
α = εc
A = α ℓ
A = ECL A = Transmission/Transmissivity ; expressed in terms of Absorbance (numerical number only)- (OPTICAL DENSITY)
E = Molar Extinction Coefficient of the absorber (ε)- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution
C = concentration of solution ( C; moles per unit vol) L= length of light path through the solution (ℓ; )
IMPLICATIONS OF BEER-LAMBERTS LAW• The absorbance (A) becomes linear with the
concentration ( C; number density of absorbers)• Thus, if the path length and the Molar absorptivity ae
known; & the absorbance is measured: The concentration of the substance (or the number density of absorbers) can be obtained.
• As Concentration (C) increases, light Absorption (A) increases, LINEARLY
As Concentration (C) increases, light Absorption (A) increases, LINEARLY .· As Concentration (C) increases, light Transmission (T) decreases,EXPONENTIALLY
COMPONENTS OF SPECTROPHOTOMETER
1. Light source(UV and visible) 2. Optical system/Wavelength selector
(Monochromator) 3. Sample containers 4. Detector 5. Output: Signal processor and readout
I0= radiant power arriving at the cuvette I = radiant power leaving the cuvette
L = length of the path through the sample Cc = concentration of the absorbing substance
a = absorptivity of the sample (extinction coefficient)
SPECTROPHOTOMETERE ntrance s lit
E x it s lit
D e tecto r
C uve tte
R ed
V io le tP rism
M onochrom ato rL igh t source
I0I
R eadoutdevice
WORKING OF SPECTROPHOTOMETER
• White light radiation source that passes through a MONOCHROMATOR ( prism or a diffraction grating that separates the white light into all colors of the visible spectrum) .
• After the light is separated, it passes through a FILTER (to block out unwanted light, sometimes light of a different color) and a SLIT (to narrow the beam of light).
• Next the beam of light passes through the SAMPLE that is in the sample holder.(cuvette)
• The light passes through the sample and the unabsorbed portion (reflected) strikes a PHOTODETECTOR that produces an electrical signal which is proportional to the intensity of the light.
• The signal is then converted to A READABLE OUTPUT (absorbance )that is used in the analysis of the sample.
• Calibration curve : generated by measuring the absorbance of several solutions that contain known concentrations of analyte.
•
• Deuterium Lamps － Continuous spectrum in the ultraviolet region is produced by electrical excitation of deuterium at low pressure. (160nm- 375nm)
• Tungsten Filament Lamps － the most common source of visible and near infrared radiation ( at wavelength 320 to 2500 nm)
• Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths from 200 to 800 nm)- in UV Spectrophotometer
• Silicon Carbide (SiC) Rod : Radiation at wavelengths:1200 -40000 nm
• NiChrome wire (750 nm to 20000 nm); ZrO2 (400 nm to 20000 nm) – for IR Region:
• Laser: Used when high intensity line source is required
COMPONENTS OF SPECTROPHOTOMETER1. LIGHT SOURCE
I0= radiant power arriving at the cuvette I = radiant power leaving the cuvette
L = length of the path through the sample Cc = concentration of the absorbing substance
a = absorptivity of the sample (extinction coefficient)
E ntrance s lit
E x it s lit
D e tecto r
C uve tte
R ed
V io le tP rism
M onochrom atorL igh t source
I0I
R eadoutdevice
OPTICAL SYSTEM/WAVELENGTH SELECTOR MONOCHROMATOR
• Optical device• Disperses a beam of
light into its component wavelengths
• Allows only a narrow band of wavelengths to pass
• Blocks all other wavelengths
1. An entrance slit 2. A collimating lens
(concave)3. A dispersing device
(usually a prism or a grating)
4. A focusing lens 5. An exit slit
MONOCHROMATOR
•Czerny-Turner setup
• AS A FILTER: It will select a narrow portion of the spectrum
(the bandpass) of a given source.
• IN ANALYSIS: the monochromator will sequentially select
for the detector to record the different components
(spectrum) of any source or sample emitting light.
• Mirror collimates light (parallel rays)
• Gating disperses light ( Prisms were formerly used)
• Light coming through entrance slit is polychromatic
• Light out of exit slit is monochromatic
CUVETTES ( SAMPLE CONTAINERS)• The containers for the sample- usually plastic or quartz:• Reference solution must be transparent to the radiation which will pass through them. • Quartz or fused crystalline silica cuvettes for UV spectroscopy . • Glass cuvettes for VisibleVisible Spectrophotometer• NaCl and KBr Crystals for IR wavelengths
Open-topped rectangular standard cell (a)
Cell Types I AND II
Detectors
• The photomultiplier tube (In UV-Vis spectroscopy)Consists of : • A photoemissive cathode (a cathode which emits electrons
when struck by photons )• Several dynodes (which emit several electrons for each
electron striking them) • An anode. • Produces an electric signal proportional to the radiation
intensity• Signal is amplified and made available for direct display• A sensitivity control amplifies the signal• Examples: Phototube (UV); Photomultiplier tube (UV-Vis);
Thermocouple (IR); Thermister (IR)
Photomultiplier Detector
5. OUTPUT: SIGNAL PROCESSOR AND READOUT (DISPLAY DEVICE)
DISPLAY DEVICE (Output device)
• Consist of a moving–coil meter or a pen recorder displaying % transmission (%T).
• At present: Instrument control, operation, standardization and data processing or storage: carried out by a microcomputer or microprocessor built in or interfaced to it.
Steps in working with spectrophotomoter
When warming up the spectrophotometer, there should be no cuvettes in the
machine Preparation of samples A series of standard solutions of known concentration Set spectrophotometer to wavelength of maximum light absorption• Measure light absorbance of standards Set the % transmittance of light as 0% In the sample space, lodge a cuvette, filled with solvent and close the sample
space. Set the transmittance at 100% For comparing, fill the cuvette with sample and place it in sample space and
close the sample space. Note down the reading on the Photometer for calculations. Plot standard curve: Absorbance vs. Concentration Calculating the concentration of sample using Beer Lambert Equation:
A = ECL
Measuring the Absorbance
MEASURING THE CONCENTRATION USING STANDARD
DIFFERENT TYPES OF SPECTROPHOTOMETERS
Classification Based on: Different measurement techniques Differ with respect to the species to be analysed (such as molecular or atomic spectroscopy) The sources of intensity variation: Type of radiation-matter interaction to be monitored (such as absorption, emission, or diffraction) The region of the electromagnetic spectrum (The wavelengths they work with )used in the analysis· Based on the absorption or emission of radiation, in the ultraviolet (UV), visible (Vis), infrared (IR), and radio (nuclear magnetic resonance, NMR) frequency ranges are most commonly encountered
TYPES AND APPLICATIONS OF SPECTROPHOTOMETER
• Primarily used for QUANTITATIVE Analysis of Known Compounds
Tissue absorption
Major tissue absorbers include: Hemoglobin, lipids (beta carotene), melanin, water, proteins, blood components, body fluidsOxy and deoxy hemoglobin have distinct spectra. Optical measurements can provide information on tissue oxygenation, oxygen consumption, blood hemodynamics
APPLICATIONS OF SPECTROPHOTOMETERAPPLICATIONS OF SPECTROPHOTOMETER
Forensic sciences. Molecular biology: in measuring the growth of micro
organisms like bacteria. UV-Vis : Most Popular in Pharmaceutical, Foods and
Paints Industries, Water Laboratories In Disease diagnosis/ Pathological states (changes):
detected by the analysis of various samples.,taken from the body : are analyzed in three different areas –Chemistry, Hematology and Microbiology section
Blood (the blood plasma, and the formed elements – the blood cells )- The most common substance for analysis
TYPES AND APPLICATION OF SPECTROSCOPY…contdTypes of Spectroscopy
Absorption Spectroscopy :
The power of a beam of light measured before and after interaction with a sample is compared.
Specific absorption techniques tend to be referred to by the wavelength of radiation measured such as ultraviolet, infrared or as ultraviolet, infrared or microwave absorption spectroscopy microwave absorption spectroscopy
Absorption occurs when the energy of the photons matches the energy difference between two states of the material. The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule. So the ultraviolet spectrum is called electronic spectrum
Ultraviolet Spectroscopy
All atoms absorb in the Ultraviolet (UV) region because these photons are energetic enough to excite outer electrons.
Used in quantifying protein and DNA concentration, the ratio of protein to DNA concentration in a solution; Amino Acids (aromatic), Pantothenic Acid, Glucose Determination and Enzyme Activity (Hexokinase)
Several amino acids usually found in protein, such as tryptophan, absorb light in the 280 nm range and DNA absorbs light in the 260 nm range. (Ratio of 260/280 nm absorbance- general indicator of the relative purity of a solution)
Used as a detector for high performance liquid chromatography (HPLC). The presence of an analyte gives a response which can be assumed to be proportional to the concentration
Visible Spectroscopy
Many atoms emit or absorb visible light. In order to obtain a fine line spectrum, the atoms must be in
a gas phase. This means that the substance has to be vaporised. The spectrum is studied in absorption or emission. Often combined Often combined : UV absorption spectroscopy in UV/Vis : UV absorption spectroscopy in UV/Vis
spectroscopyspectroscopy. Applications- Estimation of : Niacin, Pyridoxine, Vitamin
B12, Metal Determination (Fe), Fat-quality Determination (TBA) and Enzyme Activity (glucose oxidase)
Infrared Spectroscopy• The IR spectral region Further subdivided into ; near-infrared
(NIR), mid-infrared (MIR), and far-infrared (FIR) based on wavelength.
• The MIR region : most familiar to the organic chemist as offers the possibility to measure different types of interatomic bond vibrations at different frequencies.
• In organic chemistry the analysis of IR absorption spectra shows types of bonds are present in the sample.
• IR-based methods: Most common clinical analytical tests, those involving serum, whole blood, and urine.; fluids that are less commonly assayed (e.g. saliva and amniotic fluid)
Near /Mid Infrared Spectroscopy
• Near Infrared Spectroscopy : NIRange, immediately beyond the visible wavelength range, -Much greater penetration depth into the sample than in the case of mid IR spectroscopy range.
• Allows large samples to be measured in each scan• Practical applications : Medical diagnosis,, pharmaceuticals/medicines,
biotechnology, genomics analysis, proteomic analysis, interatomics research, inline textile monitoring, food analysis and chemical imaging/hyperspectral imaging of intact organisms, agricultural: rapid grain analysis; insect detection
• Forensic lab application, crime detection and various military applications.
• To identify changes in biofluid metabolite concentrations reflecting site and mechanism-specific toxicity, to define novel indices of toxic insult, to evaluate control data, to monitor disease progression and response to therapeutic intervention and to track progression and regression of toxin-induced lesions over a time period
•
X-Ray Spectroscopy• When X-rays of sufficient frequency (energy) interact with a
substance, inner shell electrons in the atom are excited to outer empty orbitals, or they may be removed completely, ionizing the atom.
The inner shell "hole" will then be filled by electrons from outer orbitals.
The energy available in this de-excitation process is emitted as radiation (fluorescence) or will remove other less-bound electrons from the atom (Auger effect).
The absorption or emission frequencies (energies) are characteristic of the specific atom.
Used in chemistry and material sciences to determine elemental composition and chemical bonding.
Atomic Absorption Spectroscopy -
Uses a pre-burner nebulizer (or nebulizing chamber) to create a sample mist and a slot-shaped burner that gives a longer path length flame.
The nebulizer and flame are used to desolvate and atomize the sample, but the excitation of the analyte atoms is done by the use of lamps shining through the flame at various wavelengths for each type of analyte.
The amount of light absorbed after going through the flame determines the amount of analyte in the sample.
A graphite furnace for heating the sample to desolvate and atomize is commonly used for greater sensitivity.
Good sensitivity and selectivity: Used for trace elements in aqueous (and other liquid) samples.
Photo Emission Spectroscopy
Photoelectron spectroscopy
Refers to energy measurement of electrons emitted
from solids, gases or liquids by the photoelectric effect, in order to determine the binding energies of electrons in a substance.
Various techniques, depending on whether the ionization energy is provided by an X-ray photon or an ultraviolet photon.
Mass Spectroscopy
• Unique among the various techniques• Mass spectrometry: Highly sensitive detection and
identification technique, allowing determination of molecular structures, and thus of a sample’s composition
• Weigh atoms, molecules, cluster, nano-particle, virus, cell and etc. In general, it can only determine mass (mass-to-charge ratio (M/Z) for a particle in gas phase.)
• .For most mass spectrometers, Z is equal to 1 so that mass can be determined
• Involves the interaction of electromagnetic radiation or some form of energy with molecules.
• The molecules absorb the radiation and produce a spectrum : during absorption process or as the excited molecules return to the ground state.
MS ApplicationsNon-biomedical• Pollutant Analysis• Trace Metal Analysis• Explosive Analysis• Illegal Drug Detection• Alcohol Analysis• Organic Chemical Analysis• Inorganic Chemical Analysis Biomedical• Proteomic Analysis• DNA sequencing• DNA fingerprinting for Forensic Applications• Biomolecule structure analysis• Polysaccharide Analysis• Metabolomic Analysis and Pharmacological Applications
Mass SpectrometryHyphenated techniques; GC-MS
Excellent in separation and quantitation
Poor in identification
Excellent in identification and quantitation
Poor in separation
Excellent in separation, identification and quantitation!
GC-MS
MS (Mass Spectrometer)
GC (Gas Chromatograph)
Raman Spectroscopy • Interactions between matter and electromagnetic radiation also give rise
to scattering processes, such as elastic scattering, and inelastic scattering • It relies on inelastic scattering, or Raman scattering, of
monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range.
• The laser light interacts with molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser photons being shifted up or down.
• The shift in energy gives information about the phonon modes in the system.
• This process,it takes place with no change in frequency• for the radiation forming the beam involved. • To study vibrational, rotational, and other low-frequency modes in a
system.
Nuclear Magnetic Resonance Spectroscopy
• Analyses the magnetic properties of certain atomic nuclei to determine different electronic local environments of hydrogen, carbon, or other atoms in an organic compound or other compound
• Used to determine the structure of the compound.
