Spectrophotometry

INSTRUMENTAL OF SPECTROPHOTOMETRY
A Spectrophotometry is a instrument to determine or calculate transmitant or absorbtion of a

sample as a function of wavelength , measurement of a series of samples at a single length can also
be done.
Instrument can be classified as :
1. Single beam
2. Double beam
SINGLE BEAM INSTRUMENT
Single-beam instruments can be used for quantitative measurements by measuring absorbance at a

single wavelength. Single-beam instrument has several advantages, namely simple, cheap price,
and reducing the existing costs is a real advantage. Some instruments produce single-beam
instruments for measuring ultraviolet light and visible light. The lowest wavelength is 190 to 210
nm and the highest is 800 to 1000 nm (Skoog, DA, 1996).
DOUBLE BEAM INSTRUMENT
Double-beam is made for use at wavelengths of 190 to 750 nm. Double-beam instrument which
has two rays formed by pieces of V-shaped mirrors called light-breakers. The first ray passes
through the blank solution and the second ray simultaneously passes through the sample, matching
the photodetector that comes out explaining the comparison electronically determined and shown
by the reader (Skoog, DA, 1996).
THE DIFFERENCE OF SINGLE BEAM AND
DOUBLE BEAM
The difference between the two types of spectrophotometers is only in the provision of light, where in the single-
beam, the light only passes through one direction so that the value obtained is only the absorbance value of the
solution entered.
Double-beam spectrophotometer, blank values can be directly measured together with the desired solution in one
same process. The principle is that there is a chopper that will split the beam in two, where one passes through the
blank (also called a reference beam) and the other passes through the solution (also called the sample beam).
LIGHT SOURCE
Light source
The light source on the spectrophotometer must have a stable radiation beam and high intensity.
The light source on a UV-Vis spectrophotometer is of two kinds:
a. Tungsten Lamp (Wolfram)
This lamp is used to measure samples in the visible area. The shape of this lamp is similar to
ordinary incandescent light bulbs. Has a wavelength between 350-2200 nm. The radiation spectrum
is in the form of curved lines. Generally have 1000 hours of usage.
b. Deuterium lamp
This lamp is used at a wavelength of 190-380 nm. The radiation energy spectrum is straight, and is
used to measure samples located in the UV region. Has 500 hours of usage.
MONOCHROMATOR
Monochromator
Monochromator is a device that will break polychromatic light into a single light (monochromatic) with certain wavelength components. Monochromator
parts, namely:
a. Prism
Prism will disperse electromagnetic radiation as much as possible so that it can get a good resolution of polychromatic radiation.
b. Grating (diffraction grating)
Diffraction grating gives more advantages to the spectroscopic process. The light dispersion will be spread evenly, with the same dispersion, the results of
the dispersion will be better. In addition diffraction grating can be used in the entire spectrum range.
c. Optical gap
This gap is used to direct the monochromatic light expected from the radiation source. If the gap is in the right position, then the radiation will be rotated
through the prism, so that the expected wavelength is obtained.
d. Filter
Serves to absorb the complementary colors so that the light transmitted is a colored light that matches the selected wavelength.
CELL
Usually Spectophotometry involve a solution and hence most sample containers are cells for
putting liquid into spectrophotometer light beams
DETECTOR
The detector will capture the light which is transmitted by the solution. The light is then converted into an
electrical signal by the amplifier and in the recorder and displayed in the form of numbers on the reader
(computer).
There are several types of detectors on a spectrophotometer:
1 . Phototube : 150-1000 λ range , use : UV electric current
2. Photomultiplier : 150-1000 λ range , use : UV / Vis electric current
3. Solid state : 350-3000 λ range
4. Thermocouple : 600 - 20,000 λ range , use : IR electric current
5. Thermistor : 600 - 20,000 λ range , use : IR electrical resistance

APPLICATION OF SPECTROPHOTOMETRY
• Detection of concentration of substances

• Detection of impurities
• Structure elucidation of organic compounds
• Monitoring dissolved oxygen content in freshwater and marine ecosystems
• Characterization of proteins
• Detection of functional groups
• Respiratory gas analysis in hospitals
• Molecular weight determination of compounds
• The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in
biological preparations.

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INSTRUMENTAL OF SPECTROPHOTOMETRY

A Spectrophotometry is a instrument to determine or calculate transmitant or absorbtion of a

Single-beam instruments can be used for quantitative measurements by measuring absorbance at a

There are several types of detectors on a spectrophotometer:

1 . Phototube : 150-1000 λ range , use : UV electric current

2. Photomultiplier : 150-1000 λ range , use : UV / Vis electric current

3. Solid state : 350-3000 λ range

4. Thermocouple : 600 - 20,000 λ range , use : IR electric current

5. Thermistor : 600 - 20,000 λ range , use : IR electrical resistance

• Detection of concentration of substances

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