GOVERNMENT COLLEGE UNIVERSITY LAHORE Assignment of Basic Biotechnology bmitted To: Sir Tayyab \ Submitted By: Ammarah Riaz Roll 0478-BS-CHEM-21 AL Semester: 2nd Topic of Assignment: Spectrophotometer Scanned with CamScannerTable of contents A) Indicator. 8) Spectrophotometer.. 2. Two different types of spectrophotometer. A) _Ultraviolent spectrophotometer. 8) Visible light spectrum spectrophotometer.. Method of interaction with light wave: 4, Parts of a spectrophotometer A) Light source. a) Incandescent Lamy b) Laser Source: 8) Focusing lens or mirror C) Prisms and gratings. D) Cuvettes.. £) Photodetectors. F) Digital display or mete! 5, How a spectrophotometer works?... a) Beers law or Beers Lambert La i. Absorption Spectrum. 6) Function of a spectrophotometer. » 7). Overview of quantitive of spectrophotometer... 8).Application: 9}. Reference... Scanned with CamScannerSpectrophotometer “A spectrophotometer is an instrument that measures the amount of light absorbed by the sample.” + Spectrophotometer techniques are used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution ina cuvette placed in spectrophotometer 1) Methods to detect molecules: a) Indicator(Quickest method) Indicator solutions change colours when a molecule of interest is present. Allows scientist to detect colourless molecules in a solution. Examples: Bradford protein reagent b) Spectrophotometer ‘An instrument that measures the amount of light that passes through (is transmitted through) a sample. > Uses a type of light to detect molecules in a solution. > Light is atype of energy, and the energy is reported as wavelengths, in nanometers (nim). 2. Two different types of Spectrophotometer: 1.Ultraviolet (UV) Spectrophotometers. + Uses ultraviolet light of wave lengths from 200 nm to 350 nm. 2.Visible (VIS) Light Spectrum Spectrophotometers. + Uses i ite i sible light (white light) of wave lengths from 350 nm to 700 nm. Method of i ‘“*cthod of interaction with light waves: 2 Scanned with CamScannerSpectrophotometer shines a beam of light on a sample. The molecules in the sample interact with the light waves in of 3 ways: * Absorb the energy «Reflect the energy ‘© Transmit the energy between and through the atoms and molecules of the sample For example, consider blue molecules, all the wavelengths of light are absorbed, except for the blue ones. The blue wavelengths are transmitted or reflected off the molecules. If these blue wavelengths hit a detector (such as in the spectrophotometer or the nerve cells in ‘your eye), they appear blue. Molecules are whatever colour of light that they do not absorb. Like, green molecules appear green because they absorb most wavelengths of visible light, except the green wavelengths. 4. Parts of a spectrophotometer: © Light source Collimator(lens) % Prism or grating % Wavelength selector(sit) wwe —Nanaunas hs cr a voce hn Scanned with CamScanner+ Cuvette + Detector Digital display or meter Outer parts | eT Ngee. kne ee O- | eeaaA eee aia ee dea aeae eles ‘Three different types of the device available are 1. The Single Beam Spectrophotometer Lf tm ght Entrance Monochromator Exit. © Cuvet De a a a Jetector Meter ouble-beam:- ace Spectrophotometer: ee, — O-b-m--L4 f-5 , — wasn Lbs . tggee Monesvomnon ght Tat Beto Meter Scanned with CamScannerSa suttiviont amount of tight which is suitable for making a + Wop mroasuroetett, over a wide © Thetighe source Qpioally yields a high output of polychnomatic i range ofthe specter, Types of tight sources use it spectrophotonneters include: Incandescent lamps and tasers + Incandescent Lamp Hap: The most common source of visible and near (2 wavelength 320 to 2500 nm) in the ultraviolet region Thi “Ss oF varios frequencies into an extremely intense focused, and nearly Light. Through selection of erent materials, emnlitad by the laser ane obtained, s Scanned with CamScannerS or mirror; + Slits, and mirrors which relays and focuses light through the 8. Focusing te a mbinations of lenses instrument, C. Prisms and Gratings: ‘+ Prisms separates white light into a continuous spectrum by refraction with shorter ‘wavelengths being bent or refracted more than longer ones. * Diffraction gratings are prepared by depositing a thin layer of aluminium-copper alloy oon the surface of a fat glass plate, then ruling many small parallel grooves into the metal coating Diffraction gratings are prepared by depositing a thin layer of alurninium-copper alloy on the surface of a flat glass plate, then ruling many smalll parallel grooves into the metal coating. D. Cuvettes: a small vessel used to hold oT 8 Tiquid sample to be analysed in the light path ofa speetrophotometer, * May be round, square or rectangular and are constructed from glass, silica (optical grade quartz) or plastic, * Quartz or fused crystalline silica cuvettes for UY. spectroscopy, * Glass cuvettes for Visible Spectrophotometer * NaCl and KBr Crystals for IR wavelengths, * Cuvettes are expensive and fragil Sic Properly and carefully MICexcept for “disposable” plastic ones).Use them * Do not scratch rf ; vettes; do ae are "OF store them in wire racks or clean with by rushes of Scanned with CamScanner+ Wash cuvet immediately ater use, E. Photodetectors: ‘These are devices that convert light into an electric signal that is proportional to the ‘number of photons striking its photosensitive surface. F. Digital display or meter: ‘+ Using the computer, output from calibrator is digitally stored © digital signals from blanks are subtracted from calibrators and unknowns, and ‘she concentration of unknowns is automatically calculated 5. How a spectrophotometer works: ‘The spectrophotometer measures the amount of light transmitted through the sample (Transmittance). For ths, first light hits the prism or grating, itis split into the different colours. ‘The wavelength knob rotates the prism/grating, directing different colours of light toward the sample. ‘The wavelength of light produced by the lamp. ‘The molecules in the sample either absorb or transmit the light energy of one wavelength or another. The detector measures the amount of light being transmitted by the sample and reports that value directly (% transmittance) or converts it to the amount of light absorbed in absorbance units (au) using Beers Law. Scanned with CamScanner© The concentration of an unknown sample can be determined by comparing the absorbance data to standards of known concentration. * The data generated with the set of known standards is called a standard curve. Beers Law or Beers Lambert Seer Law or Beers Lambert law: A=2-l0g10% ‘Spectrophotometer measure transmittance but transmittance is not linear when plotted against concentration. 0 Concentration ‘Therefore, transmittance is converted to absorbance A= -logl0 (transmittance) ‘Absorbance vs concentration is linear. < ° Poncentration Absorption Spectrum After collecting data for your concentration an absorption spectrum graph is created. 8 Scanned with CamScanner* These can be used when attempting to identify unknown substances ‘The absorbance spectrum isa graph ofa sample's absorbance at different wavelengths, Wersterat ott my Example of bsarpton spectrum of Chlorophyta & b, and Cxotenods 6. The function of a spectro} hotometer ~~ mora spectrophotometer ‘The spectrophotometer can measure the Smount of absorbance or lack of absorbance of ‘lifferent coloured light fora given molecule. ‘The relationship of concentration ina solution: * The concentration of molecules solution affects the solution’s absorbance, Remember isa ratio - when we change one number — it affects the ratio * Hfthere are more molecules in one solution than in another, than there are ‘more molecules to absorb the light, 9 Scanned with CamScannerB, Plot a standard curve with absorbance on the X axis and analytic concentration on the ¥ axis C. Measure the absorbance of the ‘unknown(s) . Determine the concentration of material of interest in the unknowns based on the standard curve “ypicalyinvoves a STANOARO CURVE, _aescrbance (Y ais) concentration (or amount (Kas Linear Range + Ifthere is too much or too little analytic, spectrophotometer cannot read the absorbance accurately 8. Applications of a spectrophotometer ‘+ Determines the presence and concentrations of samples # Determines the purity of a sample. ‘© Look at the change of samples overtime. Reference The principles of use of a spectrophotometer and its application in the ‘measurement of dental shades{2003]. Spectrophotometry FUNDAMENTALS (Chapters 17, 19,20), Dr. G. Van Biesen, ‘Win2011 10 Scanned with CamScanner