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reflectance

Spectrophotometry. scattering. reflectance. Emmision . Absorbtion. transmitance. EMR. refractance. raman. Characteristics of Light Wavelength = distance between two peaks in a wave l (lambda) is the symbol Meters = m is the unit

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reflectance

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  1. Spectrophotometry scattering reflectance Emmision Absorbtion transmitance EMR refractance raman

  2. Characteristics of Light • Wavelength = distance between two peaks in a wave • l (lambda) is the symbol • Meters = m is the unit • Frequency = number of complete waves passing a given point per second • n (nu) is the symbol • Hertz = Hz = s-1 is the unit • Amplitude = A = measure of the intensity of the wave, “brightness” • l and n are inversely proportional • If one increases, the other decreases

  3. Complementary Colors Color Absorbed violet violet-blue blue blue-green green yellow-green yellow orange red purple Color Observed green-yellow yellow orange red purple violet violet-blue blue blue-green green l of Max. Absorption 380-420 420-440 440-470 470-500 500-520 520-550 550-580 580-620 620-680 680-780 UV : 10-400 nm UV-C : 100- 280 UV-B : 280- 315 UV-A : 315- 400

  4. 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

  5. This spectrum of a sunscreen lotion is in the ultraviolet region. UV-B is the shorter  or higher energy and thus more harmful to skin.

  6. Comparison of atomic and molecular spectra

  7. All spectrometric methods use a range of wavelengths Instead of monochromatic Range called bandwidth . A narrower bandwidth represents better performance

  8. BLANK TYPES : RGT/SAMPLE/DW / AIR The Beer-Lambert Law

  9. Photodiode Array Spectrophotometer

  10. Double Beam Instrument P0() P() E Detector & readout Wavelength selector Source sample Cells Quartz: 180 – 3500 nm Si glass: 380 – 2000 nm W – 350 -2200 nm D2 – 160 – 380 nm

  11. SPECTROPHOTOMETER •

  12. SOURCE MONOCHROMATOR SAMPLE HOLDER DETECTOR Tungsten Lamp Tungsten evaporate ,black surface lamp Tungsten (W) Filament l: 350-2200 nm (VIS/near-IR) Intensity nonvariable in constant voltage Deuterium Arc Lamp & Mercury Lamp continues wave D2 + electrical energy  D2*  D2 + hn D2 lamp to cover entire UV region (200-400nm) SILICA CARBIDE (Carborundum) – IR SORCE LIGHT Light Amplification by Stimulated Emission of Radiation Lasers highly monochromatic (bandwidths of 0.01 nm or less) Lasers 100 - 10,000 nm , monochromatic, high power ( LINEAR WAVE)

  13. MONOCHROMATOR Infrared monochromatic Ray Red Orange SLIT Yellow Polychromatic Green PRISM Ray Blue Violet Ultraviolet Polychromatic Ray Monochromatic Ray SOURCE SAMPLE HOLDER DETECTOR MC’s Filter (used to reject/select broad bands of radiation,excitation and emmision wavelength. filter is colored glass) Prism (used in older instruments . Tilt prism to allow various l’s to exit from acontinuous source Grating (Czerny-Turner grating monochromator) • grooved, reflective surface(300/2000-cm) each groove is a “light source” • different l’s of light are bent at different angles. • Position slit to allow only one wavelength to pass after grating has spread the spectrum via diffraction. Each made with specificline spacing(d) - silica (SiO2) coated Aluminum

  14. MONOCHROMATOR d Grating Filters SOURCE SAMPLE HOLDER DETECTOR n = d(sin I - sin r) I = constant; therefore   r r I

  15. Cuvettes Flat surface best – better reproducibility Cleaning: H2SO4 or Hcl+Ethilicalchohol+Water in 1 :3 : 4 ratio respectively. (glass adsorb uv spectrum) SAMPLE HOLDER SOURCE MONOCHROMATOR DETECTOR Take 1gm of Potassium dichromate, add a little distilled water and very slowly add approximately 100ml of Conc.H2SO4. Keep the cells in this acidic solution for a maximum period of 12hrs. Wash the cells thoroughly with distilled water before use. Quartz 160-2500 Methacrylate 280-800Glass 350-1000Plastic( polystyrene) Visible regionNaCl or KBr IRPolyethylene Far IR Standard : L=1 cm , V= 3 ml Microplates are used when volumes are below 200 L

  16. DETECTOR h e- -V SOURCE MONOCHROMATOR SAMPLE HOLDER Detector converts incident light to an electrical signal that we can measure and process Phototube l: UV/VIS amplifier Photosensitive, negatively charged electrode ( Photoemissive cathode ). Emits electrons when struck by UV / VIS light. Electrons move through vacuum to anode (+) and produce current proportional to intensity. Detector response is a function of l therefore must blank at after each l change. hn

  17. Photo Multiplier Tube - PMT l: UV/VIS -HV+ V h e- e- e- e- hn e- e- e- e- e- e- e- e- e- -HV -HV+ 2V Similar to Phototube YET more sensitive Electrons amplified via collection of positively charged dynodes Cascade of e-’s where >106 electrons are collected for each photon striking the cathode e-

  18. Photodiodes DETECTOR Si Si Si Si Si Si hn Al Si P Si Si Si Si Si Si Si Si Si n-type Si p-type Si Photodiodes I SOURCE MONOCHROMATOR SAMPLE HOLDER

  19. Photodiode Array l: UV/VIS white light Sample Source Grating Polychromator Photodiode Array SOURCE MONOCHROMATOR SAMPLE HOLDER DETECTOR Collect entire spectrum simultaneously . Collection of diodes for each wavelength. Grating reflects dispersed radiation to all diodes. Faster yet less sensitive than phototube Infrared methods uses thermal transducers e.g. Thermocouples consist of a pair of junctions formed when two pieces of a metal that are different are fused at the ends

  20. Quality control of spectrophotometer 1-Wavelenght accuracy 2- linearity 3- photometric accuracy 1- Observation 2- Intensity of light source 2-1 mecury :313-365-405-436-546nm 2-2 deuterium : 486-656 nm Holmium oxide: narrow SBW (less than8nm)blank:perch.A 3- Filter λmax:279,287,333,360,418,536 1-Wavelenght accuracy Didymium oxide: broad SBW (more than 8nm)blank:air max 530 & 585 nm potassium dichromat(257,350nm) paranitrophenol(401nm) 4- Chemical solvents ammonium cobalt sulphate(512nm) cyanomethemoglobin(540nm)

  21. 2- linearity (paranitrophenol-450),(ammonium cobalt sulphate- 512) 1 (cooper sulphate- 650),(cyanomethemoglobin-540) (green food color-630),(nickel sulphate-550) didymium oxide in 550nm against air 2 0(air) ---- 0.096 , 0.25(air) ---- 0.346 0.5(air) ---- 0.596 , 0.75(air) ---- 0.846 3- photometric accuracy 3-1 ammonium cobalt sulphate 400nm----0.012,450mn-----0.077 500nm----0.163,550nm-----0.077 3-2 potassium dichromat 350nm----0.536±0.005(blank : 0.01N H2SO4)

  22. CHECKING THE PERFORMANCE OF THE SPECTROPHOTOMETER 1. Preparation of Blank: Measure accurately 10 ml of “Anal R” quality HCl of 36-46% concentration. Add it slowly to 400 ml distilled water filled in one litre volumetric flask. Make it up to mark with distilled water and mix it up well. 2. Preparation of sample Weigh exactly 22.2 gm of “Anal R” quality Cobalt Chloride(COCl2 ).Transfer into one litre volumetric flask. Dissolve it in 1% Hydrochloric Acid (HCl) and make it up to the mark. 3- measure the Transmittance/absorbance at wavelength 480 to 540 nm at 5nm interval. 4- Graph should be in increasing order up to 510-515nm wavelength and then in decreasing order

  23. Thank you

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