1. Cairo University Faculty Of Engineering chemical engineering department
SPECTROPHOTOMETER

2. Definition
A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. Spectrophotometer techniques are used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed in the spectrophotometer .

3. The spectrophotometer technique is to measures light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then the display device .

4. Purpose (Uses)
1)Measure the concentration of the solution A spectrophotometer optically determines the absorbance or transmission of characteristic wavelengths of radiant energy (light) by a chemical species in solution. Each molecule absorbs light at certain wavelengths in a unique spectral pattern because of the number and arrangement of its characteristic functional groups, such as double bonds between carbon atoms. According to the Beer-Lambert law, the amount of light absorbed at these wavelengths is directly proportional to the concentration of the chemical species.

5. 2) Identify organic compounds by determining the absorption maximum.
Spectrophotometers are used to identify organic compounds by determining the absorption maxima (which for most compounds and groups of compounds have very distinct fingerprints (that's what the absorption curves and peaks are called).
3) Used for color determination within the spectral range
If one is working in the range of 380 to 700 nm,
the spectrophotometers can also be used for color
determination within this spectral range

6. Example
-In the Figure below the red part of the spectrum has been almost completely absorbed by CuSO4 and blue light has been transmitted. Thus, CuSO4 absorbs little blue light and therefore appears blue.
-We will get better sensitivity by directing red light through the solution because CuSO4 absorbs strongest
at the red end of the visible spectrum. But to do this, we
have to isolate the red wavelengths

7. Internal Components

8. 1)Light source
The function of the light source is to provide a
sufficient of light which is suitable for marking a
measurement. The light source typically yields a
high output of polychromatic light over a wide
range of the spectrum.

9. I) Tungsten Lamp
Tungsten Halogen Lamp, it is the most common light source
used in spectrophotometer. This lamp consists of a tungsten
filament enclosed in a glass envelope, with a wavelength
range of about 330 to 900 nm, are used for the visible region.
They are generally useful for measuring moderately dilute
solutions in which the change in color intensity varies
significantly with changes in concentration . It has long life
about 1200h.

10. II) Hydrogen / Deuterium Lamps
For the ultraviolet region, hydrogen or deuterium
lamps are frequently used.
their range is approximately 200 to 450 nm.
Deuterium lamps are generally more stable and has
long life about 500h.This lamp generates continuous
or discontinuous spectral.

11. III) Xenon flash lamps Xenon flash lamps have several advantages as the following : 1)Their range between ( 190nm nm) 2) Emit both UV and visible wavelengths 3) Long life 4) Do not heat up the instrument 5) Reduce warm up time

12. 2) Dispersion devices *Monochromator Accepts polychromatic input light from a lamp and outputs monochromatic light. Monochromator consists of three parts: I) Entrance slit II) Exit slit III) Dispersion device

13. Monochromator

14. Dispersion devices :
Dispersion devices causes a different wavelength of light
to be dispersion at different angles monochromators used
for function.
*Types of dispersion devices :
1)Prism
Prism is used to isolate different wavelength .If a parallel beam of radiation falls on a prism , the radiation of two different wavelength will be bent through different angles.
Prism may be made of glass or quartz. The glass prisms are suitable for radiation essentially in the visible range whereas the quartz prism can cover the ultraviolet spectrum also.
It is found that the dispersion given by
glass is about three
times that of quartz.

15. Filters separate different parts of the electromagnetic
spectrum by absorbing or reflecting certain wavelengths
and transmitting other wavelengths.
*Absorption filters are glass substrates containing absorbing
species that absorb certain wavelength. A typical example is a cut
on color filter, which blocks short wavelength light such as an
excitation source, and transmits longer wavelength light such as
fluorescence that reaches a detector.
*Interference filters are made of multiple dielectric thin films
on a substrate. They use interference to selectively transmit or
reflect a certain range of wavelengths.
A typical example is a Bandpass interference filter that
transmits a narrow range of wavelengths, and can isolate
a single emission line from a discharge lamp.

17. 3) Diffraction gratings Diffraction grating is an optical component with a regular pattern, which splits (diffracts) light into several beams travelling in different directions. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as a dispersive element. The diffraction grating disperses the light into a linear spectrum of its component wavelengths, which is then directed, in whole or in part along the light path of the instrument.

19. 3)Focusing devices
Combinations of lenses, slits, and mirrors. Variable
slits also permit adjustments in the total radiant energy
reaching the detector. The Ebert and Czerny-Turner
monochromators and their variations are combinations of
prisms or gratings and focusing devices .
Ebert and Czerny-Turner
Monochromator.

20. *Optical Materials
1)Mirrors
Type of rays Mirror material
X-rays – Ultraviolet(UV) Aluminum
Visible Aluminum
Near infrared Gold
Infrared (IR) Copper or Gold

21. 2)Lenses
Rays Material
X-rays Ultraviolet Fused silica , Sapphire
Visible Glass
Infrared Glass

22. 4)Absorption cells(Cuvettes) A cuvette is a kind of cell (usually a small square tube) sealed at one end, made of Plastic, glass or optical grade quartz and designed to hold samples for spectroscopic experiments. Cuvette should be as clear as possible, without impurities that might affect a spectroscopic reading. Like a test-tube, a cuvette may be open to the atmosphere on top or have a glass or Teflon cap to seal it shut.

23. Cuvettes are chosen for transparency in the spectral wavelengths of interest. For measurements in the visible region, cuvettes of optical glass are sufficient; however, optical glass absorbs light below 350 nm , and more expensive quartz or fused silica must be used for these wavelengths. The sample cuvettes are placed in a darkened analysis chamber; some chambers have rotating carousels that can hold several cuvettes.

24. 5)Detectors Any photosensitive device can be used as a detector of radiant energy .The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking Them .

25. *Types of detectors 1) Silicon PIN Photodiodes Photovoltaic V-Series Blue enhanced for spectral range from 350nm to 1100nm; designed for low-noise, D.C. to medium bandwidth applications. Active areas range from .31mm² to 100mm². Applications include: low light level measurements, particle counting, chemical and analytical measurement and detection.

26. 2)Gallium Nitride (GaN) UV Detectors This family of Gallium Nitride (GaN) UV Detectors are Schottky processed fully passivated U.V. photodiodes. Spectral range from 200 nm to 365 nm and is ideal for UVA or UVB sensing applications and is packaged with a quartz window.

27. 6)Display devices The data from a detector are displayed by a readout device, such as an analog meter, a light beam reflected on a scale, or a digital display , Or liquid crystal display(LCD) .The output can also be transmitted to a computer or printer.

28. Theoretical work of spectrophotometer
First we but the sample into a Cuvette then the light source generates light at a specific wave length or wave lengths , the light passes through the dispersion devices that separate the light into its components wavelengths .
Slits then isolate the wave lengths needed for measurement with a * Bandpass filter to improve its purity . Next , the light passes through the sample ,and a portion of radiant energy absorbed . The remaining light is transmitted to the Photometer ,which converts light energy to electrical energy can be registered on a meter or digital readout.
The amount of light absorbed depends on the nature of the concentration of the sample .
*Bandpass filter is a device that passes frequencies within a
certain range and rejects frequencies outside that range.”

30. Used Laws :

33. Types of spectrophotometer
There are two classes of spectrophotometers: 1)Single beam The single beam spectrophotometer was the first invented, and all the light passes through the sample. In this case, to measure the intensity of the incident light, the sample must be removed so all the light can pass through. This type is cheaper because there are less parts and the system is less complicated.

34. The advantages of the single beam design are low cost, high throughput, and hence high Sensitivity , because the optical system is simple. The disadvantage is that an appreciable amount of Time elapses between taking the reference (I) and Making the sample measurement (Io) so that there can be problems with drift. This was certainly true of Early designs but modern instruments have better electronics and more stable lamps, so stability with single beam instruments is now more than adequate for the vast majority of application.

36. 2)Double beam
The double beam instrument design aims to eliminate drift by measuring blank and sample virtually simultaneously. A "chopper" alternately transmits and reflects the light beam so that it travels down the blank and the sample optical paths to a single detector. The chopper causes the light beam to switch paths at about 50 Hz causing the detector to see a "saw tooth" signal of Io and I which are processed in the electronics to give either transmittance or absorbance as output.
To measure a spectrum with a double beam instrument the two cuvettes, both containing solvent are place in the sample and reference positions and a "balance" measurement is made. This is the difference between the two optical paths and is subtracted from all subsequent measurement. The sample is then placed in the sample cuvette and the spectrum is measured. I and Io are measured virtually simultaneously as described above.

37. The advantage of the double beam design
Is high stability because reference and sample are measured virtually at the same moment in time. The disadvantages are higher cost, lower sensitivity because throughput of light is poorer because of the more complex optics and lower reliability because of the greater complexity.

39. 3)Split Beam
The split beam spectrophotometer is similar to the double beam spectrophotometer but it uses a beam splitter instead of a chopper to send light along the blank and sample paths simultaneously to two separate but identical detectors. Thus blank and sample measurements can be made at the same moment in time. Spectra are measured in the same way as with a double beam spectrophotometer.
The advantage of this design is good stability, though not as good as a double beam instrument because two detectors can drift independently, and good noise, though not as good as a single beam instrument because the light is split so that less than 100% passes through the sample.

40. Spectrophotometers vs. colorimeters
Understanding the types of instruments available for measuring color is important when choosing the instrument to purchase or use for your application . The "colorimeter" and "spectrophotometer" cause some confusion . both types of instruments provide data obtained over the same range of visible wavelengths (about nm) but may treat this data differently.
Spectrophotometers and colorimeters are instruments that measure color intensities of solutions by applying a light source to the solution.

42. Prepared by Ahmed Mostafa Ahmed Ashraf Amir Ehab Islam Hamdy
Peter Wageeh