Farees mufti Stage Analytical Techniques in Biotechniology

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  • 1. Analytical techniques in Biotechnology
    • Topic Of Presentation:
    • UV-Visible
    • By:
    • Farees Ud Din Mufti
    • &
    • Shoaib Nawaz
    • &
    • Sana Ullah
  • 2.
    • “ The study of molecular structure and dynamics through the absorption, emission and scattering of light is called Spectroscopy”.
    • Ultra Violet
    • Ultra Violet light is found in sunlight and is emitted by electrical arcs and specialized lights such as black lights.
    • They are the electromagnetic radiations which have a shorter wavelength.
  • 3.
    • ULTRA VIOLET VISIBLE
    • As the name indicates Ultra means "beyond” and Violet a color.
    • Violet being the color of the shortest wavelengths is visible light.
    • Thus visible light have shortest wavelengths.
    • This means it uses light in the visible and adjacent (near-UV and near infrared (NIR) ranges.
    • The absorption in the visible range directly affects the perceived color of the chemicals involved.
  • 4.
    • UV-VISIBLE SPECTROSCOPY
    • The study of measurement of spectrum obtained by interaction of UV visible part of EMR (Electron Magnetic Resonance) with matter is called UV visible spectroscopy.
    • Generally, 200-400 nm (UV-rays)
    • 400-800 nm (UV-Visible rays)
  • 5.
    • BASIC PRINCIPLE
    • Electron around the nucleus revolve in orbits or shells, different e* occupy different orbits depending on their energy level, sometimes e* gives energy.
    • So move from ground state to excited state or i.e. high energy state.
    • Where they become unstable, and so try to come back.
  • 6.
    • EXPLANATION
    • UV-Visible refers to absorption spectroscopy in the ultraviolet-visible spectral region.
    • This absorption directly affects the perceived color of the chemicals involved.
    • This technique is complementary to flourescence spectroscopy, in that flourescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.
  • 7.
    • Now once either electrons move from higher to lower or from lower to higher some sort of changes occur in spectral wavelengths.
    • HYPSOCHROMIC SHIFT
    • Hypsochromic shift is a change of spectral band position in the absorption, reflectance, transmittance, or emission spectrum of a molecule to a shorter wavelngth (higher frequency).
    • Because the blue color in the visible spectrum has a lower wavelength than most other colors, this effect is also commonly called a blue shift.
     “ hypsochromic” shift “ bathochromic” shift
  • 8.
    • Bathochromic shift
    • Such shift is a change of spectral band position in the absorption, reflectance, transmittance, or emission spectrum of a molecule to a longer wavelength (lower frequency).
    • Because the red color in the visible spectrum has a higher wavelength than most other colors, this effect is also commonly called a red shift, although this usage is considered informal, and has no relation to Doppler shift or other wavelength-independent meanings of red shift.
    • This usage is often found in the scientific literature.
  • 9.
    • 1. Lambert’s Law
    • “ According to Lambert’s law, decrease in intensity of light is proportional to intensity of light is incident light and thickness of medium”.
    • Mathematically,
    • A = ab
    • Where,
    • A=absorbance
    • a=constant
    • b=thickness of medium
    cuvette source slit detector
  • 10.
    • 2. BEER's LAW
    • It states that “ Absorbance is directly proportion to concentration of the solution”.
    • Linear absorbance with increased concentration--directly proportional.
    • Makes UV useful for quantitative analysis and in HPLC detectors
    • If A is absorbance & C is concentration than,
    • A directly proportional to C
    • A = KC (K=constant)
  • 11.
    • INSTRUMENTATION
    • Here is a double-beam UV-Vis. spectrophotometer; Instruments for measuring the absorption of U.V. are made up of the following;
    • Sources (UV and visible)
    • 1. Tungten lamp (350-2500 nm)
    • 2. Deuterium (200-400 nm)
    • 3. Xenon Arc lamps (200-1000 nm)
    • Wavelength selector (monochromator)
    • Sample containers of
    • Visible; can be plastic or glass
    • UV; you must use quartz
    • Detector
    • Signal processor and readout
  • 12.
    • Sources of visible radiation
    • The tungsten filament lamp is commonly employed as a source of visible light.
    • This type of lamp is used in the wavelength range of 350 - 2500 nm.
    • The energy emitted by a tungsten filament lamp is proportional to the fourth power of the operating voltage.
    • This means that for the energy output to be stable, the voltage to the lamp must be very stable indeed. Electronic voltage regulators or constant-voltage transformers are used to ensure this stability.
  • 13.
    • Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope" which also contains the tungsten filament.
    • The iodine reacts with gaseous tungsten, formed by sublimation, producing the volatile compound WI2.
    • When molecules of WI2 hit the filament they decompose, redepositing tungsten back on the filament.
    • The lifetime of a tungsten/halogen lamp is approximately double that of an ordinary tungsten filament lamp.
    • Tungsten/halogen lamps are very efficient, and their output extends well into the ultra-violet.
    • They are used in many modern spectrophotometers.
  • 14.
    • Factors affecting UV-visible
    • 1. Solvent:
    • The effect on the absorption spectrum of a compound when diluted in a solvent will vary depending on the chemical structures involved.
    • Generally speaking, non-polar solvents and non-polar molecules show least effect.
    • However, polar molecules exhibit quite dramatic differences when interacted with a polar solvent.
    • Interaction between solute and solvent leads to absorption band broadening and a consequent reduction in structural resolution and ε max.
    • Ionic forms may also be created in acidic or basic conditions.
    • Thus care must be taken to avoid an interaction between the solute and the solvent.
  • 15.
    • 2. Surface reflections
    • Fresh white snow reflects about 85 percent of UV radiation while other bright surfaces such as sand, concrete, and water, reflect less.
    • If skiing on a spring day at the end of March, may only be 4, but due to reflection from the snow, the skier may experience.
  • 16.
    • APPLICATIONS OF UV-visible
    • Flourescent Excitation
    • Flourescent excitation - the leading method of quality control Using UV technology to perform a fluorescent inspection means that even minute hairline cracks can be reliably identified.
    • The UV handheld lamp developed by us is the most powerful small UV unit on the world market.
  • 17.
    • 2. Surface Sterilization
    • Surface Sterilization - benefiting production, efficiency and the environment.
    • For many branches of industry, sterilizing the packaging used for food, cosmetics and drugs is expensive and requires a considerable amount of chemicals.
    • UV technology offers an efficient alternative in this area. UVC light sterilizes the surface of packaging within fractions of a second, and is scientifically proven to kill up to 99.999% of bacteria.
    • Chemical substances such as hydrogen peroxide therefore no longer need to be used.
    • We offer environmentally-friendly UV sterilization which meets the requirements of state-of-the-art production processes.
  • 18.
    • 3. Stimulating Natural Sunlight
    • Stimulating Natural Sunlight - to speed up the aging process.
    • Leading automobile manufacturers exploit the substantial advantages offered by this method when examining how the aging process affects vehicle inks and plastic components.
    • Sunlight simulation equipment can be used to immediately demonstrate the effects of prolonged exposure to natural sunlight.
    • A variety of specific parameters, such as sunlight, moisture and temperature, can also be tested in climate chambers.
  • 19.
    • It is used in determination of purity of chemicals.
    • UV can be used for the determination of unknown compounds also.
    • UV spectrophotometer can be applied for the analysis of different chemicals and pharmaceuticals.
    • It helps in medicines while determining percentage purity of different pharmaceuticals.
  • 20.
    • Thank you.