1. A spectrophotometer measures the light that passes through a liquid sample and gives readings in percent transmittance and absorbance. 2. As concentration of an absorbing substance increases, less light is transmitted through the sample according to Beer-Lambert's law. 3. Key aspects of a spectrophotometer include the light source, monochromator, sample cells, detector, and associated electronics for amplification and readout. Spectrophotometers are used for a variety of applications including quantitative analysis and molecular structure determination.

1. UV - VISIBLE SPECTROPHOTOMETER HARIS P

2. Spectrophotometer • Measures the light that passes through a liquid sample • Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)

3. The Electromagnetic Spectrum  = c /  E = h 

4. Absorbance and Complementary Colors

5. I o I Cell with Pathlength, b, containing solution light source detector blank where I o = I concentration 2 concentration 1 b with sample I < I o The process of light being absorbed by a solution As concentration increased, less light was transmitted (more light absorbed).

6. The law states that the amount of light absorbed by a solution (colored) is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material (path length). Absorbance is also called optical density A = abc where a – molar absorptivity, b – pathlength, and c – molar concentration BEER - LAMBERT'S LAW

7. Some terminology I – intensity where I o is initial intensity T – transmission or %T = 100 x T (absorption: Abs = 1 – T or %Abs = 100 - %T) T = I/ I o A – absorbance A = - log T = -log I/ I o

8. The blank contains all substances expect the analyte. Is used to set the absorbance to zero: A blank = 0 This removes any absorption of light due to these substances and the cell. All measured absorbance is due to analyte. The Blank

9. Conventional Spectrophotometer 1. A stable and cheap energy source. 2. A monochromator to break the polychromatic radiation into component and wavelength/bands of wave length. 3. Transparent vessels (cuvettes) to hold the sample. 4. A photo sensitive detector and associated amplifier and recorder

10. Conventional Spectrophotometer Optical system of a split-beam spectrophotometer

11. LIGHT SOURCES UV Spectrophotometer 1. Hydrogen Gas Lamp 2. Mercury Lamp Visible Spectrophotometer 1. Tungsten Lamp IR Spectrophotometer 1. Carborundum (SIC)

12. Light Source Deuterium Arc Lamp UV Region Wavelength Range : 190~420nm Tungsten Lamp Wavelength Range : Part of the UV and the whole of the Visible range ( 약 350 ~ 2,500nm) Xenon Lamp Wavelength Range : 190~800nm

13. Monochromator Accepts polychromatic input light from a lamp and outputs monochromatic light Components : Entrance slit, Dispersion device, Exit slit. The resolving element are of two kinds namely, prisms and diffraction gratings. Simple glass prisms are used for visible range. For UV region silica, fused silica or quartz prism is used. Fluorite is used in vaccum UV range. Gratings are often used in the monochromators of spectrophotometers operating in UV, visible and infra red regions. Their resolving power is far superior to that of prisms & they yield a linear resolution of spectrum.

14. Dispersion Devices Non-linear dispersion Temperature sensitive Linear Dispersion Different orders

15. CELLS UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass IR Spectrophotometer NaCl

16. Cell Types I Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume

17. Cell Types II Micro cell (a) for very small volumes and flow-through cell (b) for automated applications

18. Detection Devices Most detectors depend on the photoelectric effect, where incident light photons) liberates electrons from a metal or other material surface. Important requirements for a detector (1)high sensitivity to allow the detection of low levels of radiant energy, (2)short response time, (3)long term stability, and (4)an electronic signal which is easily amplified for typical read out apparatus, Ultraviolet and visible radiation detectors are photocells, phototubes and photo multiplier tubes.

19. Photomultiplier Tube Detector Anode High sensitivity at low light levels Cathode material determines spectral sensitivity Good signal/noise Shock sensitive

20. Amplification and Readout Radiation detectors generate electronic signals which are proportional to the transmitter light. These signals need to be translated to a form that is easy to interpret. This is accomplished by using amplifiers, ammeters, potentiometers, and potentiometric recorders.

21. 1. Qualitative Analysis 2. Quantitative Analysis 3. Molecular weight determination 4. Study of cis-trans Isomerism 5. Other Physiochemical studies 6. Control of Purification 7. Difference Spectroscopy 8. Turbidimetry Applications of UV - Visible Spectroscopy

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