Spectrophotometer

Definition : A spectrophotometer is an instrument that measure the amount of light absorbed or transmitted by the sample.

Purpose : Spectrophotometer is used to: 1)measure the concentration of the solution. 2)identify organic compounds by determining the absorption maximum.

Principle: Spectrophotometer consists of two instruments: 1-spectrometer to produce light for any selected wave length. 2-photometer to measure the intensity of light, and the analyte is put between them.

Single and double beams : In the early days of spectroscopy, double beam spectrophotometers were popular but now it is thought that the single beam spectrophotometer is more advantageous.

Components of single beam :

1- Light source. For ultraviolet absorption spectrophotometer for visible absorption spectrophotometer

For ultraviolet absorption spectrophotometer we use # H2 lamp its wave length ranges from 190 to380 nm.

#D2 (deuterium) lamp its wave length ranges from185 to400 nm. We prefer D2lamp because of its higher stability & it emits continuous radiation.

for visible absorption spectrophotometer we use #tungsten lamp (350~2200nm) #tungsten halogen lamp (240~2500nm) We prefer tungsten halogen lamp because it has longer life, can be used at lower wave length.

2- Prism or diffraction grating : Dispersion devices causes a different wavelength of light to be dispersion at different angle Optical materials : lenses. prism diffraction grating

3- slit : monochromators used for selecting one wave length .

4- cell (cuvette): The cuvette or absorption cells, must made from material that is transparent to radiation in the spectral region of interest.

5- Detector (photometer): a device used to convert the radiant energy to electrical signal.

6- Read out device (Digital galvanometer) : The data from the detector are displayed by a readout device , such as an analog meter ,digital display or liquid crystal display. The out put can also transmitted to computer or printer .

Theoretical work of spectrophotometer:

Used laws: &

Where T: transmittance of solution. I: intensity of transmitted light . (Watt/cm2) Io: intensity of incident light . (watt/cm2) 𝛆 : Coefficient absorptivity. (L/mole.cm) 𝐜 : Concentration of solution. (Mole/L) : 𝐋Thickness of cuvette. (cm) A: the absorption of light by the sample

Beer's Law: Beer's Law explains the relationship between absorbance, at a given wavelength and concentration. A = 𝛆 C L Where: A = absorbance. 𝛆 = molar absorptivity coefficient. (L/mole.cm) L = length of the light path. (cm) C = concentration of the solute. (mole/L)

Double Beam Spectrophotometer It is used to measure the ratio of light intensities on two separate light paths.

Advantages : Wide applicability. High sensitivity. Good accuracy. Ease and convenience.

Disadvantages : 1. Error in reading due to the change in temperature nature of solution. 2. It is subject to false wavelength setting

3. an error in wavelength setting of ±0.3 nm results in an error. 4. The equipment is generally expensive.

 Applications in our life  Clinical Food and Drink Industrial / Pharmaceutical Life Sciences

Mind mapping Spectrophotometer Applications Disadvantages Advantages Single & double beams Double beam Single beam Used laws Theoretical work Components cell slit Prism Light source visible UV Principle Purpose Definition

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