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Spectrophotometer introducing
A spectrophotometer is an instrument that measures light absorption by a sample to determine the concentration of solutes. It operates by diffracting light into a spectrum and detecting intensities to produce absorption graphs, following the Beer-Lambert law. Various components, including light sources, monochromators, detectors, and cuvettes, contribute to its functionality, and it can be classified into single beam, double beam, and split beam types for different measurement applications.
Spectrophotometer introducing
- 2. DEFINITION A spectrophotometer isan instrument that measures the amount of light absorbed by a 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 in a cuvette placed in the spectrophotometer .
- 3. The spectrophotometer techniqueis to measures light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then the display device .
- 4. PURPOSE (USES) A spectrophotometeroptically determines the absorbance or transmission of characteristic wavelengths of radiant energy (light) by a chemical species in solution. Each molecule absorbs light at certain wavelengths in a unique spectral pattern because of the number and arrangement of its characteristic functional groups, such as double bonds between carbon atoms. According to the Beer-Lambert law, the amount of light absorbed at these wavelengths is directly proportional to the concentration of the chemical species. Spectrophotometers are used to identify organic compounds by determining the absorption maxima (which for most compounds and groups of compounds have very distinct fingerprints (that's what the absorption curves and peaks are called). If one is working in the range of 380 to 700 nm, the spectrophotometers can also be used for color determination within this spectral range.
- 5. -In the Figurebelow the red part of the spectrum has been almost completely absorbed by CuSO4 and blue light has been transmitted. Thus, CuSO4 absorbs little blue light and therefore appears blue. -We will get better sensitivity by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum. But to do this, we have to isolate the red wavelengths
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- 7. 1)Light source The functionof the light source is to provide a sufficient of light which is suitable for marking a measurement. The light source typically yields a high output of polychromatic light over a wide range of the spectrum.
- 8. I) Tungsten Lamp TungstenHalogen Lamp, it is the most common light source used in spectrophotometer. This lamp consists of a tungsten filament enclosed in a glass envelope, with a wavelength range of about 330 to 900 nm, are used for the visible region. They are generally useful for measuring moderately dilute solutions in which the change in color intensity varies significantly with changes in concentration . It has long life about 1200h.
- 9. II) Hydrogen /Deuterium Lamps For the ultraviolet region, hydrogen or deuterium lamps are frequently used. their range is approximately 200 to 450 nm. Deuterium lamps are generally more stable and has long life about 500h.This lamp generates continuous or discontinuous spectral.
- 10. III) Xenon flashlamps Xenon flash lamps have several advantages as the following : 1)Their range between ( 190nm - 1000 nm) 2) Emit both UV and visible wavelengths 3) Long life 4) Do not heat up the instrument 5) Reduce warm up time
- 11. 2) Dispersion devices *Monochromator Acceptspolychromatic input light from a lamp and outputs monochromatic light. Monochromator consists of three parts: I) Entrance slit II) Exit slit III) Dispersion device
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- 13. Dispersion devices : Dispersiondevices causes a different wavelength of light to be dispersion at different angles monochromators used for function. *Types of dispersion devices : 1)Prism Prism is used to isolate different wavelength .If a parallel beam of radiation falls on a prism , the radiation of two different wavelength will be bent through different angles. Prism may be made of glass or quartz. The glass prisms are suitable for radiation essentially in the visible range whereas the quartz prism can cover the ultraviolet spectrum also. It is found that the dispersion given by glass is about three times that of quartz.
- 14. 2)Filter Filters separate differentparts of the electromagnetic spectrum by absorbing or reflecting certain wavelengths and transmitting other wavelengths. *Absorption filters are glass substrates containing absorbing species that absorb certain wavelength. A typical example is a cut on color filter, which blocks short wavelength light such as an excitation source, and transmits longer wavelength light such as fluorescence that reaches a detector. *Interference filters are made of multiple dielectric thin films on a substrate. They use interference to selectively transmit or reflect a certain range of wavelengths. A typical example is a Bandpass interference filter that transmits a narrow range of wavelengths, and can isolate a single emission line from a discharge lamp.
- 16. 3) Diffraction gratings Diffractiongrating is an optical component with a regular pattern, which splits (diffracts) light into several beams travelling in different directions. 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. The diffraction grating disperses the light into a linear spectrum of its component wavelengths, which is then directed, in whole or in part along the light path of the instrument.
- 17. Combinations of lenses,slits, and mirrors. Variable slits also permit adjustments in the total radiant energy reaching the detector. The Ebert and Czerny-Turner monochromators and their variations are combinations of prisms or gratings and focusing devices . Ebert and Czerny-Turner Monochromator.
- 18. *Optical Materials 1)Mirrors Type ofrays Mirror material X-rays – Ultraviolet(UV) Aluminum Visible Aluminum Near infrared Gold Infrared (IR) Copper or Gold
- 19. 2)Lenses Rays Material X-rays UltravioletFused silica , Sapphire Visible Glass Infrared Glass
- 20. 4)Absorption cells(Cuvettes) A cuvetteis a kind of cell (usually a small square tube) sealed at one end, made of Plastic, glass or optical grade quartz and designed to hold samples for spectroscopic experiments. Cuvette should be as clear as possible, without impurities that might affect a spectroscopic reading. Like a test-tube, a cuvette may be open to the atmosphere on top or have a glass or Teflon cap to seal it shut.
- 21. Cuvettes are chosenfor transparency in the spectral wavelengths of interest. For measurements in the visible region, cuvettes of optical glass are sufficient; however, optical glass absorbs light below 350 nm , and more expensive quartz or fused silica must be used for these wavelengths. The sample cuvettes are placed in a darkened analysis chamber; some chambers have rotating carousels that can hold several cuvettes.
- 22. 5)Detectors Any photosensitive devicecan be used as a detector of radiant energy .The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking Them .
- 23. *Types of detectors 1)Silicon PIN Photodiodes Photovoltaic V-Series Blue enhanced for spectral range from 350nm to 1100nm; designed for low-noise, D.C. to medium bandwidth applications. Active areas range from .31mm² to 100mm². Applications include: low light level measurements, particle counting, chemical and analytical measurement and detection.
- 24. 2)Gallium Nitride (GaN)UV Detectors This family of Gallium Nitride (GaN) UV Detectors are Schottky processed fully passivated U.V. photodiodes. Spectral range from 200 nm to 365 nm and is ideal for UVA or UVB sensing applications and is packaged with a quartz window.
- 25. 6)Display devices The datafrom a detector are displayed by a readout device, such as an analog meter, a light beam reflected on a scale, or a digital display , Or liquid crystal display(LCD) .The output can also be transmitted to a computer or printer.
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- 27. TYPES OF SPECTROPHOTOMETER Thereare two classes of spectrophotometers: 1)Single beam The single beam spectrophotometer was the first invented, and all the light passes through the sample. In this case, to measure the intensity of the incident light, the sample must be removed so all the light can pass through. This type is cheaper because there are less parts and the system is less complicated.
- 28. The advantages ofthe single beam design are low cost, high throughput, and hence high Sensitivity , because the optical system is simple is that an appreciable amount of Time elapses between taking the reference (I) and Making the sample measurement (Io) so that there can be problems with drift. This was certainly true of Early designs but modern instruments have better electronics and more stable lamps, so stability with single beam instruments is now more than Adequate for the vast majority of application.
- 30. 2)Double beam The doublebeam instrument design aims to eliminate drift by measuring blank and sample virtually simultaneously. A "chopper" alternately transmits and reflects the light beam so that it travels down the blank and the sample optical paths to a single detector. The chopper causes the light beam to switch paths at about 50 Hz causing the detector to see a "saw tooth" signal of Io and I which are processed in the electronics to give either transmittance or absorbance as output. To measure a spectrum with a double beam instrument the two cuvettes, both containing solvent are place in the sample and reference positions and a "balance" measurement is made. This is the difference between the two optical paths and is subtracted from all subsequent measurement. The sample is then placed in the sample cuvette and the spectrum is measured. I and Io are measured virtually simultaneously as described above.
- 31. The advantage ofthe double beam design Is high stability because reference and sample are measured virtually at the same moment in time. The disadvantages are higher cost, lower sensitivity because throughput of light is poorer because of the more complex optics and lower reliability because of the greater complexity.
- 32. 3)Split Beam The splitbeam spectrophotometer is similar to the double beam spectrophotometer but it uses a beam splitter instead of a chopper to send light along the blank and sample paths simultaneously to two separate but identical detectors. Thus blank and sample measurements can be made at the same moment in time. Spectra are measured in the same way as with a double beam spectrophotometer. is good stability, though not as good as a double beam instrument because two detectors can drift independently, and good noise, though not as good as a single beam instrument because the light is split so that less than 100% passes through the sample.
- 33. SPECTROPHOTOMETERS VS. COLORIMETERS Understanding thetypes of instruments available for measuring color is important when choosing the instrument to purchase or use for your application . The "colorimeter" and "spectrophotometer" cause some confusion . both types of instruments provide data obtained over the same range of visible wavelengths (about400- 700nm) but may treat this data differently. Spectrophotometers and colorimeters are instruments that measure color intensities of solutions by applying a light source to the solution.
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