1. Beer-Lambert law
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2. Beer–Lambert law, also known as Beer's law or the Lambert–Beer law is an empirical relationship that relates the absorption of light to the properties of the material through which the light is traveling.
Schematic of the experimental setup needed to record a spectrum
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3. A(λ) = e(λ) l c
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4. Beer's Law states that
The absorbance, A(λ), of a species at a particular wavelength of electromagnetic radiation, λ, is proportional to the concentration, c, of the absorbing species
and to the length of the path, l, of the electromagnetic radiation through the sample containing the absorbing species. 
A(λ) = e(λ) l c
The proportionality constant e(λ) is called the absorptivity of the species at the wavelength, λ. [  e(λ) is called the molar absorptivity if the concentration is measured in moles/liter. ]
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5. It is common to use the energy carried by the radiation per unit area per unit time, which is called the intensity, I,
I, use to measure of the "amount" of electromagnetic radiation impinging on a surface. 
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6. For a partially transparent sample, we can consider the fraction of the intensity that is permitted to pass through the sample as a measure of the transmittance of the sample. 
We define the percent transmittance, %T , of a sample in terms of the intensity of the light incident on the sample, I0 , and the light transmitted through the sample, It
T = (It /I0 ) X %
A = log I0 /It
e l c = log I0 /It
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7. A completely transparent sample will have It = I0, and its percent transmittance will be, appropriately, 100. 
A sample which permits no radiation of a particular wavelength to pass through it will have It  = 0, and a corresponding percent transmittance of  0
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8. Some wavelengths are particularly useful in the study of Biomolecules
e(1/Mm.cm)
λmax(nm)
Chromophore
5.6
280
Tryptophane
1.4
274
Tyrosine
0.2
257
Phenylalanine
14.9
260
Adenosine
6.6
DNA
7.4
RNA
40.9
Bovine serum albumine
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9. (Type of spectrometer)
Spectrophotometer
Colorimeter
(Type of spectrometer)
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10. Absorption spectra are measured
Characterization
Absorption spectra are measured
The type of radiation is electromagnatic radiation generated by a lamp (tungesten halogen lamp)( nm).
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11. Design of spectrophotometer
Beam of light generated from lamp.
Spectrophotometers use a monochromator to analyze the spectrum
Samples are usually prepared in cuvettes; ( glass, plastic, or quartz).
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12. There are three major classes of spectrophotometers according to the beam;
a. A single beam spectrophotometer measures the absolute light intensity.
b. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths.
c. Dual beamed: two scources generate individual beams
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13. Type of cuvettes are used in spectrophotometer
Disadvantage
Advantage
Type of cuvettes
May absorb ultraviolet radiation
Useful in the visible part of the spectrum
Glass
1.May absorb ultraviolet radiation.
2. May be damaged by exposure to organic solvents
Plastic
Fragile & expensive
Don’t absorb ultraviolet radiation
Quartz
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14. Types of Detector (Photo detector)
Photomultiplier tubes
It converts light intensity into an electric signal
Photodiode
Absorption of light by a photodiode produces a current proportion to the number of photons
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15. Modern spectrophotometer
Most modern spectrophotometer are usually connected to a computer which
facilitates storage
Presentation and analysis of spectroscopic data
Function:
The spectrophotometer measures quantitatively the fraction of light that passes through a given solution.
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