Definition :Definition :

• A spectrophotometer is an instrument that

measure the amount of light absorbed or

transmitted by the sample.

Purpose :Purpose :

• Spectrophotometer is used to:1)measure the concentration of the

solution.

2)identify organic compounds by determining the absorption maximum.

Principle: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.

SingleSingle andand doubledouble beamsbeams ::

• 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 :Components of single beam :

1- Light source1- Light source..

• For ultraviolet absorption spectrophotometer • for visible absorption spectrophotometer

For ultraviolet absorption For ultraviolet absorption spectrophotometer we use 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 for visible absorption spectrophotometer we useuse

• #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.

2-2- Prism or diffraction grating :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 :3- slit : monochromators used for selecting one wave

length .

4- cell (cuvette):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):5- Detector (photometer):

a device used to convert the radiant energy to

electrical signal.

6- Read out device (Digital galvanometer) :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:Theoretical work of spectrophotometer:

Used laws:Used laws:

&&

Where

TT: transmittance of solution. II: intensity of transmitted light . (Watt/cm2)

IIoo: intensity of incident light . (watt/cm2)𝛆𝛆 : Coefficient absorptivity. (L/mole.cm) 𝐜𝐜 : Concentration of solution. (Mole/L): 𝐋: 𝐋Thickness of cuvette. (cm) AA: the absorption of light by the sample

Beer's Law:Beer's Law:• Beer's Law explains the relationship between

absorbance, at a given wavelength and concentration.

A = C L𝛆

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 Double Beam Spectrophotometer It is used to measure the ratio of light intensities

on two separate light paths.

Advantages :Advantages :

1)1) Wide applicability. Wide applicability.

2) High sensitivity.High sensitivity.

3)3) Good accuracy.Good accuracy.

4)4) Ease and convenience.Ease and convenience.

Disadvantages :Disadvantages :

1. Error in reading due to1. Error in reading due to

»the change in temperature

» nature of solution.

22. It is subject to false wavelength settingIt is subject to false wavelength setting

33. an error in wavelength setting of ±0.3 nm an error in wavelength setting of ±0.3 nm

results in an error.results in an error.

4. The equipment is generally expensive.4. The equipment is generally expensive.

Applications in our life Applications in our life

• Clinical• Food and Drink• Industrial / Pharmaceutical• Life Sciences

Mind mappingMind mapping

The EndThe End

‘‘ ‘‘best wishesbest wishes ’’ ’’