Spectrophotometry

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  • The presence and concentration of various substances dissolved in a water sample is commonly analyzed by passing different types of light (visible, infrared, or UV) through the sample. Light can either be transmitted or absorbed by the dissolved substances.
  • Spectrophotometry

    1. 1. 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
    2. 2. • 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)
    3. 3. SPECTROSCOPY / SPECTROCHEMICAL ANALYSIS.The study how the chemical compound interacts withdifferent wavelenghts in a given region of electromagneticradiationSpectrophotometry : Quantitative measurement of thereflection or transmission properties of a material as afunction of wavelength.; Involves the use of aspectrophotometer.SPECTROPHOTOMETER : The combination of two devices, aspectrometer and a photometer.•A device that is used to measure intensity of light as a functionof the wavelength of light.• An instrument that measures the amount of light of a specifiedwavelength that passes through (is transmitted through) a sample(medium)
    4. 4. BLOCK DIAGRAM OF SPECTROPHOTOMETER
    5. 5. The Spectrophotometer Single-beam Double-beam
    6. 6. SPECTROPHOTOMETE Spectrometer: for producing light of any selected wavelength or color Photometer: used for measuring the intensity of light. Rx i t s l i t The two devices are placed at either side of a cuvette filled with a liquid E E n tr a n c e s lit D e te c to r Red I0 I R eadout d e v ic e I0= radiant power arriving at the cuvette a = absorptivity of the sample (extinction coefficient) P r is m L = length of the path through the sample C I = radiant power leaving the cuvette V io le tc = concentration vof the absorbing substance C u e tteL ig h t s o u r c e M o n o c h ro m a to r
    7. 7. 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).
    8. 8. 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 – 650si V Red 651 – 780 Near Infrared > 780
    9. 9. SPECTROPHOTOMETRY COLORIMETRY• A photometer (a device for • The measurement of color measuring light intensity) • Any technique used to evaluate an• Measure intensity as a unknown color in reference to function of the color, or more known colors specifically, the wavelength of light • It determines color based on the red,• Tungsten or xenon flashlamp blue, and green components of light absorbed by the object or sample, as the source of white light• Tungsten lamp for • Colored light beam through an measurements in visible optical filter, which transmits only region(360-900nm) one particular color / band of• Hydrogen /deuterium lamp wavelengths of light to the for UV region(200-380nm) photodectector
    10. 10. 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 LAW
    11. 11.  Spectrometer produces the light of desired wavelength and itpasses through the tube and reaches photometer that measures itsintensity. Then the photometer produces a voltage signal to a displaydevice, usually a galvanometer. As the amount of light absorbed by the liquid changes; thesignal also changes. The concentration of a substance in solution can be measured bycalculating the amount of absorption of light at the appropriatewavelength or a particular colour Reading of Spectrophotometer: (Number)- Absorbance that isdirectly proportional to the color intensity, and also theconcentration of the species responsible for the color.
    12. 12. • 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.
    13. 13. Terms:/ParametersTransmittance : 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 sampleAbsorbance units: A unit of light absorbance determined by the decrease in the amount of light in a light beamAbsorbance spectrum: A graph of a sample’s absorbance at different wavelengthsLambdamax: The wavelength that gives the highest absorbance value for a sample
    14. 14. Absorption: The Beer-Lambert Law August Beer (1825-1863): Added absorption co-efficient and related to conc. in solution. Pierre Bouguer Johan Lambert Astronomer: Light Mathematician, first to is diminished as it prove that π is irrational. passes through the No absorption coefficient atmosphere.A = − log( I1 / I 0 ) = εcl €: Extinction coefficient c: Concentration l : Path length
    15. 15. 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.BEERS 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
    16. 16. BEER–LAMBERT’S LAW (Beer–Lambert–Bouguer law) ….contdLAMBERTS LAWo 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
    17. 17. Beer – Lambert LawStates that the Absorbance (O.D) of a solution isdirectly proportional to the concentration of theabsorbing species in the solution and the pathlength.The fraction of the incident light absorbed by a solution at a given wavelength isrelated to a. thickness of the absorbing layer (path length) and b. concentration of the absorbing species
    18. 18. 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, α, and the distance the light travels through the material (i.e. the path length), ℓ.• The ABSORPTION COEFFICIENT: (α ) = 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 ; ℓ : . thickness of the absorbing layer (path length) and cross section of light absorption by a single particle;
    19. 19.   T- Transmittance I T= I0 - Original light intensity I0 I- Transmitted light intensity  I% Transmittance (T)= 100 x I0 1Absorbance (A) = Log T(OPTICAL DENSITY) I0 = Log I = KCL I0Log I is proportional to : C (concentration ofsolution) and L (length of light path through thesolution).
    20. 20. By definition of the Beer - Lambert Law. α = εc A=α ℓ A = ECL  A = Transmission/Transmissivity ; expressed in termsof Absorbance (numerical number only)- (OPTICALDENSITY)E = Molar Extinction Coefficient of the absorber (ε)-Extinction Coefficient of a solution containing 1gmolecule of solute per 1 liter of solutionC = concentration of solution ( C; moles per unitvol) L= length of light path through the solution (ℓ ; )
    21. 21. 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)
    22. 22. As Concentration (C) increases, light Absorption (A) increases,LINEARLY .· As Concentration (C) increases, light Transmission (T) decreases,EXPONENTIALLY
    23. 23. COMPONENTS OF SPECTROPHOTOMETER1. Light source(UV and visible)2. Optical system/Wavelength selector (Monochromator)3. Sample containers4. Detector5. Output: Signal processor and readout
    24. 24. SPECTROPHOTOMETER E x it s lit E n tr a n c e s lit D e te c to r Red I0 I R eadout d e v ic e P r is m V io le t C u v e tteI0= radiant power arriving at the cuvette a = absorptivity of the sample (extinction coefficient) L = length of the path through the sample CI = radiant power leaving the cuvette c = concentration of the absorbing substanceL ig h t s o u r c e M o n o c h ro m a to r
    25. 25. 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.•
    26. 26. COMPONENTS OF SPECTROPHOTOMETER 1. LIGHT SOURCE• 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
    27. 27. OPTICAL SYSTEM/WAVELENGTH SELECTOR MONOCHROMATOR• Optical device• Disperses a beam of light into its E x it s lit component wavelengths E n t r a n c e s lit D e te c to r• Allows only a narrow band of wavelengths to Red I0 I pass R eadout• Blocks all other d e v ic e wavelengths1. An entrance slit2.I =A collimating lens P r is m a = absorptivity of the sample (extinction coefficient) 0 radiant power arriving at the cuvette C u v e tte (concave) L = length of the patht through the sample C V io le I = radiant power leaving the cuvette c = concentration of the absorbing substance3. A dispersing device (usually a prism or a grating) L ig h t s o u rc e M o n o c h ro m a to r4. A focusing lens5. An exit slit
    28. 28. 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 selectfor 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
    29. 29. CUVETTES ( SAMPLE CONTAINERS)• The containers for the sample- usually plastic or quartz:• Reference solution must be transparent to the radiation which willpass through them.• Quartz or fused crystalline silica cuvettes for UV spectroscopy .• Glass cuvettes for Visible Spectrophotometer• NaCl and KBr Crystals for IR wavelengths
    30. 30. Cell Types I AND II Open-topped rectangular standard cell (a)
    31. 31. • The photomultiplier tube (In UV-Vis spectroscopy)Consists of : Detectors• 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)
    32. 32. Photomultiplier Detector
    33. 33. 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.
    34. 34. 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
    35. 35. Measuring the Absorbance
    36. 36. MEASURING THE CONCENTRATION USING STANDARD
    37. 37. DIFFERENT TYPES OF SPECTROPHOTOMETERSClassification Based on: Different measurement techniques Differ with respect to thespecies to be analysed (such as molecular or atomic spectroscopy) The sources of intensity variation: Type of radiation-matterinteraction to be monitored (such as absorption, emission, ordiffraction) The region of the electromagnetic spectrum (The wavelengthsthey work with )used in the analysis· Based on the absorption or emission of radiation, in theultraviolet (UV), visible (Vis), infrared (IR), and radio (nuclearmagnetic resonance, NMR) frequency ranges are most commonlyencountered
    38. 38. TYPES AND APPLICATIONS OF SPECTROPHOTOMETER• Primarily used for QUANTITATIVE Analysis of Known Compounds
    39. 39. Tissue absorptionMajor tissue absorbers include: Hemoglobin, lipids (beta carotene), melanin, water,proteins, blood components, body fluidsOxy and deoxy hemoglobin have distinct spectra. Optical measurements can provideinformation on tissue oxygenation, oxygen consumption, blood hemodynamics
    40. 40. APPLICATIONS 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
    41. 41. TYPES AND APPLICATION OF SPECTROSCOPY…contd Types of SpectroscopyAbsorption Spectroscopy : The power of a beam of light measured before and afterinteraction with a sample is compared. Specific absorption techniques tend to be referred to by thewavelength of radiation measured such as ultraviolet, infrared ormicrowave absorption spectroscopy Absorption occurs when the energy of the photons matches theenergy difference between two states of the material. The absorption of ultraviolet radiation by molecules isdependent upon the electronic structure of the molecule. So theultraviolet spectrum is called electronic spectrum
    42. 42. 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
    43. 43. 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 : UV absorption spectroscopy in UV/Vis spectroscopy. spectroscopy Applications- Estimation of : Niacin, Pyridoxine, Vitamin B12, Metal Determination (Fe), Fat-quality Determination (TBA) and Enzyme Activity (glucose oxidase)
    44. 44. 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)
    45. 45. 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
    46. 46. 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.
    47. 47. 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.
    48. 48. 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.
    49. 49. 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.
    50. 50. Mass Spectrometry The Components of a Mass Spectrometer 1. Ion Source 2. Analyser 3. Detector 4. Data
    51. 51. Mass Spectrometry Provides Information on1. Molecular Mass2. Molecular Structure (fragmentation)3. Elemental composition
    52. 52. MS ApplicationsNon-biomedical• Pollutant Analysis• Trace Metal Analysis• Explosive Analysis• Illegal Drug Detection• Alcohol Analysis• Organic Chemical Analysis• Inorganic Chemical AnalysisBiomedical• Proteomic Analysis• DNA sequencing• DNA fingerprinting for Forensic Applications• Biomolecule structure analysis• Polysaccharide Analysis• Metabolomic Analysis and Pharmacological Applications
    53. 53. 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 separationGC-MS Excellent in separation, identification and quantitation!
    54. 54. 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
    55. 55. 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.
    56. 56. APPLICATIONS OF NMR IN MEDICINEBRAIN  Distinguishing gray matter & white matter  Imaging posterior fossae, brain stem, spinal cord  Detect demyelinating lesions, tumors, hemorrhages, infarctionsABDOMEN 1. Metabolic liver disease 2. Measures liver iron over load in hemochromatosis 3. Focal areas of inflammation in chronic active hepatisisKIDNEYS  Distinguishing renal cortex & medulla  To evaluate transplanted kidneyPELVIS  Differentiates between BPH & prostatic carcinoma  Detects bladder tumoursHEARTo Tomographic images of heart muscle, chambers, valvular structureso Discrimination between infarcted, ischemic & normal myocardium o

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