Introduction to Physical Basis: LIGHT: Absorption , Electromagnetic spectrum, Wavelength, Colour Principle of Spectrophotometry Components of a Spectrophotometer Working of Spectrophotometer

ter Types of Spectrophotometer Applications of Spectrophotometry

Optical methods based on interactions of light with matter (fluid/ biological sample)
Basic Principles Absorption Scattering
Multiple scattering/Diffusion Single scattering

Fluorescence Microscopy Optical Coherence Tomography Photodynamic therapy

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

August Beer (1825-1863): Added absorption co-efficient and related to conc. in solution.

Pierre Bouguer Astronomer: Light is diminished as it passes through the atmosphere.

Johan Lambert Mathematician, first to prove that is irrational. No absorption coefficient

A log( I1 / I 0 ) cl
: Extinction coefficient c: Concentration

l : Path length

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)

SPECTROSCOPY / SPECTROCHEMICAL ANALYSIS.

The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic radiation Spectrophotometry : 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)
.

BLOCK DIAGRAM OF SPECTROPHOTOMETER

Single-beam

Double-beam

SPECTROPHOTOMETER
Exit slit Entrance slit Red I0 I Readout device Detector

I0= radiant power arriving at the Prism Icuvette = radiant power leaving the
cuvette

a = absorptivity of the sample (extinction L = length of the path through the sample coefficient) Cuvette Violetc = concentration of the absorbing substance C

Light source

Monochromator

 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).

COLOR
Ultraviolet Violet

WAVELENGTH ( in nm)
< 380 380 435 436 480 481 490 491 500 501 560 561 580 581 595 596 650 651 780 > 780

Visible Light

Blue Greenish-blue Bluish-green Green Yellowish-green Yellow Orange Red Near Infrared

SPECTROPHOTOMETRY

COLORIMETRY

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)

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

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 Lamberts 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 sample Absorbance: 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 samples absorbance at different wavelengths Lambdamax: The wavelength that gives the highest absorbance value for a sample

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

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 Law

States 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

Transmittance
Defined as the ratio of the intensity of light emerging from the solution (I) to that of incident light entering (Io)
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, , and the distance the light travels through the material (i.e. the path length), .
The ABSORPTION COEFFICIENT: ( ) = Molar absorptivity (extinction coefficient) of the absorber, (c) X the concentration (c) of absorbing species in the material

I0 : intensity(power) of the incident light I : intensity(power) of the transmitted light ; : . thickness of the absorbing layer (path length) and cross section of light absorption by a single particle;

T- Transmittance

T=

I I0

I0 - Original light intensity

I- Transmitted light intensity I I0

% Transmittance (T)=

100 x

Absorbance (A) = Log

(OPTICAL DENSITY)

1 T
I0

= Log

= KCL

I0 Log is proportional to : C (concentration of solution) and LI(length of light path through the solution).

By definition of the Beer - Lambert Law.

= c
A=

A = ECL
A = Transmission/Transmissivity ; expressed in terms of Absorbance (numerical number only)- (OPTICAL DENSITY) E= Molar Extinction Coefficient of the absorber ()Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution C = concentration of solution ( C; moles per unit vol) 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 Transmission (T) decreases: EXPONENTIALLY (INVERSLY)

1. 2.

3.

4.
5.

Light source(UV and visible) Optical system/Wavelength selector (Monochromator) Sample containers Detector Output: Signal processor and readout

Exit slit SPECTROPHOTOMETER Entrance slit Red I0 I Readout device Detector

Prism

I0= radiant power arriving at the cuvette I = radiant power leaving the cuvette

Violet = absorptivityCuvette (extinction coefficient) a of the sample

L = length of the path through the sample C c = concentration of the absorbing substance

Light source

Monochromator

Conventional Single Beam Spectrophotometer

Schematic of a conventional single-beam spectrophotometer

Split Beam Spectrophotometer

Optical system of a split-beam 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.

1. LIGHT SOURCE(UV AND VISIBLE)

LIGHT SOURCE
Deuterium LampsContinuous spectrum in the ultraviolet region is produced by electrical excitation of deuterium at low pressure. (160nm375nm) Tungsten Filament Lampsthe 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

2. OPTICAL SYSTEM/WAVELENGTH SELECTOR (MONOCHROMATOR)

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

Exit slit Entrance slit Red I0 I Readout device Detector

1. An entrance slit 2.I = radiant power arriving at the cuvette A collimating lens Prism a = absorptivity of the sample (extinction coefficient) 0 Cuvette Violet L = length of the path through the sample C (concave) I = radiant power leaving the cuvette c = concentration of the absorbing substance 3. A dispersing device (usually a prism or a grating) Light source Monochromator 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

3.

SAMPLE CONTAINERS ( CUVETTES)

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 Visible Spectrophotometer NaCl and KBr Crystals for IR wavelengths
Exit slit Entrance slit Red I0 I Readout device Detector

Prism

Violet

Cuvette

Light source

Monochromator

Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume

Micro cell (a) for very small volumes and flow-through cell (b) for automated applications

4. DETECTOR

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 (UVVis); Thermocouple (IR); Thermister (IR)

Photomultiplier Detector

5. OUTPUT: SIGNAL PROCESSOR AND READOUT (DISPLAY DEVICE)

 Consist

of a movingcoil 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.

MOVING COIL METER

DISPLAY DEVICE

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

Avoid very high or low absorbencies when drawing a standard curve.

Plot the Absorbance vs. Concentration to get a straight line

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

 Primarily

used for QUANTITATIVE Analysis of Known Compounds

Tissue absorption

Major tissue absorbers include: Hemoglobin, lipids (beta carotene), melanin, water, proteins, blood components, body fluids Oxy and deoxy hemoglobin have distinct spectra. Optical measurements can provide information on tissue oxygenation, oxygen consumption, blood hemodynamics

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 SPECTROSCOPYcontd Types 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 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

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

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 : UV absorption spectroscopy in UV/Vis spectroscopy. Applications- Estimation of : Niacin, Pyridoxine, Vitamin B12, Metal Determination (Fe), Fat-quality Determination (TBA) and Enzyme Activity (glucose oxidase)

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 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

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.

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.

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, an EUV photon, or an ultraviolet photon.

Unique among the various techniques Mass spectrometry: Highly sensitive detection and identification technique, allowing determination of molecular structures, and thus of a samples 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.

Mass Spectrometry
The Components of a Mass Spectrometer

1. 2. 3. 4.

Ion Source Analyser Detector Data

Mass Spectrometry

Provides Information on
1. 2. 3. Molecular Mass Molecular Structure (fragmentation) Elemental composition

Non-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 Disease Diagnosis Eg:

Mass Spectrometry
Hyphenated techniques; GC-MS
GC (Gas Chromatograph)

Excellent in separation and quantitation Poor in identification

MS (Mass Spectrometer)
Excellent in identification and quantitation Poor in separation

Excellent in separation, identification and quantitation!

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.

 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.

APPLICATIONS OF NMR IN MEDICINE

BRAIN
Distinguishing gray matter & white matter

Imaging posterior fossae, brain stem, spinal cord Detect demyelinating lesions, tumors, hemorrhages, infarctions ABDOMEN 1. Metabolic liver disease 2. Measures liver iron over load in hemochromatosis 3. Focal areas of inflammation in chronic active hepatisis KIDNEYS Distinguishing renal cortex & medulla To evaluate transplanted kidney PELVIS Differentiates between BPH & prostatic carcinoma Detects bladder tumours HEART o Tomographic images of heart muscle, chambers, valvular structures o Discrimination between infarcted, ischemic & normal myocardium