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Is defined as measurement of intensity of light at selected wavelength Used in quantitative & qualitative analysis of various chemical & biological procedures Very accurate & sensitive method Depends on light absorbing property of a substance or derivative of substance being analysed Intensity of light passing through a solution having absorbing substances is decreased Decrease fraction of light is then detected ,measured & used to relate the light transmitted or absorbed to concentration of analyte Principle Light is an electromagnetic radiation described as photon of energy traveling in waves According to Planket,s formula E=hv As h=constant (6.62 x10 -27 ) Frequency(v) Frequency(v) is inversely proportional to wavelength so energy of electromagnetic radiation(light) is also inversely proportional to wavelength, smaller the wavelength more is frequency & more is energy & vice versa v=frequency, v= 1/

Percent Transmittence It is rate of radiant energy transmitted(T) divided by radiant energy incident on sample (I) and is % T = (Is / Ir) * 100

Absorbance When light passes through a solution having light absorbing substance , light is absorbed by this substance ,this absorbance is directly proportional to concentration of substance Both transmittance & absorbance are mathematically related to concentration of substance by formula called BEER LAW which states that concentration of a substance is directly proportional to amount of light absorbed & inversely proportional to logarithm of light transmitted and is A=abc A=absorbance a=proportionally constant defined as absorbity b=light path in centimeters (1cm) c=concentration of absorbing compounds expressed in gm/l A level of 100 % transmittance is obtained if no light is absorbed. Spectrophotometer is always set as 100% transmittance while light passing through blank or reference

INSTRUMENTATION Modern instruments use narrow wavelength range of spectrum for measurement Those that use filter for this purpose are called filter photometer Those that use prisms or grating are called spectrophotometer It is further divided into single or double beam spectrophotometer Single beam

Double beam

1. LIGHT SOURCE Tungsten or tungsten iodide lamps used for visible & near infrared light 15% of radiant energy fall in visible light. Deuterium & low pressure mercury lamps used for ultrviolet & visible light. High pressure mercury lamps used for ultraviolet to mid visible. LASER(light amplification by stimulated emission of radiation)

(550-1818nm) 2. MONOCHROMATORS Isolation of individual wavelength of light is function of monochromator. The degree of wavelength isolation is a function of device used & width of entrance & exit slits Band pass of monochromator defines the range of wavelength transmitted & is calculated as width at more then half the maximum transmittance

FILTER Thin layer of color glass Not true filter Can transmit wide range of wave-length Spectral purity of filter is described as spectral band width Commonly used glass filter has 50nm bandwidth PRISM Prism separate white light into continuous spectrum by refraction with shorter wavelength being bent or refracted more than longer wavelength as it passes through prism

GRATING Thin layer of aluminum copper alloy on surface on glass than ruling many parallel grooves into matel coating usually 10000 to 20000/ inches FIBER OPTICS

3. CUVETS Small vessels used to hold liquid samples to be analysed in light path of spectrophotometer may be round square or rectangular made of glass or silica or plastic Most has 1 cm light path It must be clean, clear, no scratching otherwise will effect absorbance No alkaline solution should be used Can be cleaned by rinsing in water. 4. PHOTO DETECTORS It convert light energy in electrical energy and is equal to number of photons striking to photosensitive surface. Photocells Photocells composed of film of light sensitive material usually selenium on a plate of iron. Over the light sensitive material is thin transparent layer of silver when exposed to light. Electron in light sensitive material are excited and released to flow to highly conductive silver.

It generates its own electromotive force which can be measured. Current produced is proportional to incident light radiations. In expensive, durable, but temperature sensitive. Phototubes Phototubes similar to above one but it requires outside voltage for operation. It has negative charge cathode and two charge anode enclosed in glass case. Photomultiplier tubes A photomultiplier tube contains a cathode, a light sensitive metal and a series of dynodes, all of which are enclosed in an evacuated glass enclosure. It detects and amplifies radial energy cathode anode (dynodes) Triggers a cascade of electrons within the photomultiplier tube. They have extremely rapid response time, are very sensitive and slow to fatigue.

5. READ OUT DEVICES Electrical energy from detector is displayed on some meter or read out meter. Now replaced by digital read out device that provide usual numeric display of absorbance or converted value of concentration. 6. CALIBERATION

Read the manufacturers instruction before handling instruments. Said the instrument to 0/0 T with the help of zero setting knob. During the zero setting light is not allowed to pass through cuvets. Match to cuvets after filling with water and take absorbance reading, record the error. Pour the blank into one of cuvets after taking water out. Set the instrument to 100 % T, for blank use, the blank setting knob. Perform the required analytical procedure using various concentration of standard solution. Take the absorbance reading of each solution. Rinse the cuvets each time with water and always stat with lowest concentration. Record the reading in tabular form, absorbance reading against the concentration of standard. Calibration curves should be at the angle of 45 degree 7. QUALITY ASSURANCE Performing atleast the following checks should validate instrument function, wavelength accuracy, stay light, and linearity. Wavelength accuracy means that the wavelength indicated on control dial is the actual wavelength of light passed by the monochromator. Stray light refer to any wavelength outside the band transmitted by the monochromator common causes of stray light are reflection of light from scratches on optical surface are dust particle anywhere in the light path and higher order spectra produce by differaction gratings result in absorbance error. Can be corrected by using cut off filters which eliminate all radiation at wavelength beyond one of interest.

Linearity Demonstrated when a change in concentration result in a strainght line calliberation curve. Colored solution must be carefully diluted and used to check linearity using the wavelength of maximal absorbance for that color. Sealed set of different colors and concentration are available commercially. Set of neutral density filters to check linearity over a range of wavelength are also commercially available