Spectrophotometry .pdf

Biotechnology SPECTROPHOTOMETRY Ronald P. Cabral,M.D.,DPPS Clinical Laboratory Scientist Asst. Professor II LNU College of Medicine & College of Dentistry Special lecturer University of the East College of Dentistry  Fundamentals of Sp...

Biotechnology SPECTROPHOTOMETRY Ronald P. Cabral,M.D.,DPPS Clinical Laboratory Scientist Asst. Professor II LNU College of Medicine & College of Dentistry Special lecturer University of the East College of Dentistry  Fundamentals of Spectrophotometry Properties of Light Types of Light – The Electromagnetic Spectrum  Fundamentals of Spectrophotometry Properties of Light Types of Light – The Electromagnetic Spectrum Note again, energy (E) of light increase as frequency (ν) increases or wavelength (λ) decreases  Fundamentals of Spectrophotometry Absorption of Light 1.) Colors of Visible Light Many Types of Chemicals Absorb Various Forms of Light The Color of Light Absorbed and Observed passing through the Compound are Complimentary Therefore: The concentration of a substance in directly proportional to the amount of light absorbed.  DNA Analysis (After extraction) Nucleic acid (DNA/RNA) Qualitative analysis Quantitative analysis Size-Quality Purity- Yield Agarose gel electrophoresis Spectrophotometry  Common Absorbing Biochemicals O The bases of nucleic N acids NH N N NH2 H NH2 O Guanine H3C N N NH NH2 N N N O H N H Thymine Adenine N O H Cytosine Nucleic Acid Absorption Properties Base λmax (nm) ε (mM-1cm-1) Guanine 275 8.0 Adenine 260 12.9 Cytosine 265 5.8 Thymine 258 8.0 Relationship between %Transmittance and light path length and concentration 100 100 80 80 60 60 %T %T 40 40 20 20 0 length 0 concentration Transmittance = IT/I0 = e-αcl where α is an extinction constant, c is concentration and l is light path length  Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design An instrument used to make absorbance or transmittance measurements is known as a spectrophotometer  Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design Light Source: provides the light to be passed through the sample - Tungsten Lamp: visible light (320-2500 nm) - based on black body radiation: Low pressure (vacuum) heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid Tungsten Filament filament - Deuterium Lamp: ultraviolet Light (160-375 nm) D2or H2 Gas In presence of arc, some of the electrical energy is absorbed by Filament D2 (or H2) which results in the disassociation of the gas and 40V Electric Arc release Sealed Quartz Tube of light Electrode D2 + Eelect ! D *2 ! D’ + D’’ + hν (light produced) Excited state  Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design Wavelength Selector (monochromator): used to select a given wavelength of light from the light source - Prism: - Filter:  Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design Sample Cell: sample container of fixed length (b). - Usually round or square cuvet - Made of material that does not absorb light in the wavelength range of interest 1. Glass – visible region 2. Quartz – ultraviolet 3. NaCl, KBr – Infrared region