NIKHIL.K.POTDUKHE
Outline of UV spectrophotometer Outline of Recombinant DNA technology Application of UV spectroscopy in recombinant DNA technology References
Lambert law: “When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of the light”. Beer law: “The intensity of beam of monochromatic light decreases exponentially with the increase in concentration of the absorbing substance arithmetically”.
A = abC or A = ξbC A: absorbance b : Sample Path length C : Sample Concentration a : Absorbance Constant ξ : Molecular Absorbance Constant
Deviations in absorptivity coefficients • At high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity Scattering of light • due to particulates in the sample Fluorescence or Phosphorescence of the sample Changes in refractive index at high analysis concentration Shifts in chemical equilibria as a function of concentration
The recorder assembly The spectrometer itself – this houses the lamps, mirrors, prisms and detector. The spectrometer splits the beam of radiation into two and passes one through a sample and one through a reference solution (that is always made up of the solvent in which you have dissolved the sample). The detector measures the difference between the sample and reference readings and communicates this to the recorder. The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes. The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are normally designed to allow the radiation to pass through the sample over a distance of 1cm
Cont….. Spectrometric instruments have a common set of general features. Here we look at specific features for the UV/Visible experiment. Sources: D2 lamp, W filament (halogen lamp), and Xe arc lamp. Wavelength Selectors: Filters and Monochromators. Sample Containers: Fused silica, quartz, and glass. Detectors: Phototube, PMT, photodiode, photodiode array,CCD array
Strike a low voltage DC arc in a lamp filled with D2. Gives continuum emission from 160 to 400 nm.
Reflecting focusing assembly Lamp Condenser Lens A heated W filament, gives off blackbody radiation. Add a small amount of a halogen gas. Sublimated W reacts with halogen to form tungsten halide; does not deposit on quartz cover (no blackening) but does redeposit on filament (extends life).
Tube filled with Xe (or sometimes a mixture of Hg and Xe), invented in 1940, commercialized in 1961 by Osram. Pass a low voltage DC current to excite Xe. The broad spectral output closely resembles natural daylight, and is often used in 150 Watt Xe lamp projection systems (e.g. 15 kW IMAX systems)
• A filter is device that allows the required wavelength to pass A filter is device that allows the required wavelength to pass but absorb of other wavelength wholly or partially . but absorb of other wavelength wholly or partially . • Filters are of two type - Filters are of two type - Absorption filter – It work by selective absorption of unwanted – It work by selective absorption of unwanted Absorption filter light. Absorption filter is solid sheet of glass colored by light. Absorption filter is solid sheet of glass colored by pigment which is dispersed in glass. pigment which is dispersed in glass. Interference filter - It work by reflection of unwanted - It work by reflection of unwanted light. A Interference filter semitransparent metal film is deposited on a plate of glass . Then it is coated with dielectric material . When ray of light incident on it part of light reflects back whereas remaining light is trans mitted
A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input. Accepts polychromatic input light from a lamp and outputs monochromatic light. Components : Entrance slit, Dispersion device, Exit slit Entrance slit provides narrow source light to avoid overlapping of monochromatic light. Exit slit select narrow band of dispersed spectrum for observation of detector.
• Diffraction grating is an optical component with a regular pattern, which splits (diffracts) light into several beams traveling in different directions. • Grating consist of large number of parallel lines ruled on highly polished surface such as alumina. • The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as a dispersive element.
• Prism is a transparent optical element with flat, polished surfaces that refract light. • The prism disperse the light radiation in to individual colors or wavelength. • Prisms are typically made out of glass, but can be made from any material that is transparent to the wavelengths for which they are designed. • The resolution depends upon the size and refractive index. • Prisms have higher dispersion in the UV region.
Successful spectroscopy requires that all materials in the beam path other than the analyte should be as transparent to the radiation as possible. Also, the geometries of all components in the system should be such as to maximize the signal and minimize the scattered light. Polystyrene 340-800 nm Methacrylate 280-800 nm Glass 350-1000 nm Suprasil Quartz 160-2500 nm • Keep the cuvette clean. •Don’t clean with paper products (Kim-wipe); use optical paper. •Store dry. •Don’t get finger prints on them. •Store carefully and gently.
Detectors : • Phototube • Photomultiplier tube • Photodiode • Channeltron
Phototube: Detector is composed of : Photo cathode- It is coated with elements of 1. high atomic volume like Potassium or silver oxide. Collector anode 2. Photo cathode liberates electron towards anode when light incident on it
Photomultiplier tube: It is most sensitive of all detector . In this detector multiplication of photoelectron by secondary emission of electron achieved by using photo diode and series of anode (dynodes) Up to 10 dynodes are used , maintained at 75 – 100 V higher than preceding one It can detect very week signal. light dynodes anode electrons photocathode voltage divider network high voltage
Photodiode: A photodiode is formed by sandwiching an undoped layer of Si between a heavily doped p-layer and a heavily doped n-layer. Photons whose wavelength is between 400 nm and 1100 nm can be absorbed in the intrinsic layer, producing an electron-hole pair. The bias potential sweeps these carriers to the opposite regions, producing a current in the external circuit. Photodiodes are more sensitive than phototubes , but far less sensitive than PMT’s, since they only generate ~1 electron-hole pair per photon. On the other hand they are about the size of a transistor and require no high voltage support.
A continuous dynode chain is built into a single unit. Excellent and widely used electron multiplier. If the front end is a photo emissive surface then you have a compact “PMT”. Channeltrons require high vacuum to operate.
Mechanism:
Cont…. A beam of light from a visible and/or UV light source is separated into its component wavelengths by a prism or diffraction grating. Each monochromatic (single wavelength) beam in turn is split into two equal intensity beams by a half-mirrored device. One beam, the sample beam , passes through a small transparent container (cuvette) containing a solution of the compound being studied in a transparent solvent. The other beam, the reference (colored blue), passes through an identical cuvette containing only the solvent. The intensities of these light beams are then measured by electronic detectors and compared. The ultraviolet (UV) region scanned is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm.
Single beam spectrophotometer: This consist of tungsten lamp as source of light . This light radiation focused on slit by using concave mirror. This light passes through simple absorption filter where the only required wavelength of light passed through it and passed through it and falls on the sample cell where the solution to be analyzed is present . The sample or standard solution absorbs apart of the radiation and rest is transmitted . The intensity of transmitted light is determined by photovoltaic cell.
Double beam spectrophotometer: It is similar to that of single beam instrument . Here the light beam after passing through filter is split into sample beam and reference beam by using beam splitter . These beam pass through sample and reference solution and fall onto detector separately The final read out is in absorbance or transmittance , obtained after electronic manipulation of 2 detectors.
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