A spectrophotometer is an instrument that measures the amount of light transmitted through a sample. It uses either ultraviolet or visible light, and detects molecules in a solution based on which wavelengths of light are absorbed. It works by shining a light source through a sample, and measuring how much light is absorbed, reflected, or transmitted. This allows it to determine characteristics of unknown samples by comparing them to measurements taken of samples of known concentration.

1. SPECTROPHOTOMETER

2. Spectrophotometer
☐ Spectrophotometer
An instrument that measures the amount of light that
passes through (is transmitted through) a sample.

3. Spectrophotometer
Uses a type of light to detect molecules in a solution
Light is a type of energy, and the energy is reported
as wavelengths, in nanometers (nm).

4. Two different types of
Spectrophotometer:
Ultraviolet (UV) Spectrophotometers.
■ Uses ultraviolet light of wave lengths from 200 nm
to 350 nm.
Visible (VIS) Light Spectrum
Spectrophotometers.
■ Uses visible light (white light) of wave lengths from
350 nm to 700 nm.

5. The visible light spectrum
R O Y G B I V

6. Spectrophotometer
☐ Shines a beam of light on a sample.
☐ The molecules in the sample interact with the light
waves in of 3 ways:
Absorb the energy
Reflect the energy
Transmit the energy between and through the
atoms and molecules of the sample.

7. Blue molecules absorb the other
colors of visible light.
Blue molecules are blue because they reflect blue light.

8. How a spectrophotometer works:
Consider blue molecules, all the wavelengths of light
are absorbed, except for the blue ones.
The blue wavelengths are transmitted or reflected off
the molecules. If these blue wavelengths hit a detector
(such as in the spectrophotometer or the nerve cells in
your eye), they appear blue.

9. Spectrophotometer
The spectrophotometer measures the amount of light
transmitted through the sample (Transmittance).
By using an equation (Beers law), it converts the
transmittance data to an absorbance value.

10. Spectrophotometer
The concentration of an unknown sample can be
determined by comparing the absorbance data to
standards of known concentration.
The data generated with the set of known standards
is called a standard curve.

11. Parts of a spectrophotometer
• Inner parts
• Lamp
• Prism or grating that direct light of a specific wavelength.
☐

12. VIS Spec vs. UV spec
Visible spectrophotometer
■ Contains a tungsten lamp that produces white light.
Ultraviolet spectrophotometer
■ Contains a deuteriumlamp that produces light in the
UV light part of the spectrum.

13. Parts of a Spectrophotometer
☐ Outer parts:
Sample Holder
Display
Knobs or buttons used to calibrate the
spec to measure the designated molecule.
Wavelength
Selection

15. How a spectrophotometer works:
• Visible Spectrophotometer
■ White light hits the prism or grating, it is split into the colors
of the rainbow (Visible Spectrum).
■ The wavelength knob rotates the prism/grating, directing
different color of light toward the sample.

16. How a spectrophotometer works:
☐ The wavelength of light produced by the tungsten lamp
range from about 350 nm (Violet light) to 700 nm (red
light).
☐ The molecules in the sample either absorb or Transmit
the light energy of one wavelength or another.

17. How a spectrophotometer works:
☐ The detector measures the amount of light being
transmitted by the sample and reports that value
directly (% transmittance) or converts it to the amount of
light absorbed in absorbance units (au) using Beers
Law.

18. Beers Law
T=10-A
A= Absorbance
T=Optical depth

19. The function of a spectrophotometer
The spectrophotometer can
measure the amount of absorbance
or
lack of absorbance of different colored light for a given
molecule.

20. Applications of a spectrophotometer
☐ Determines the presence and concentrations of
samples.
☐ Determines the purity of a sample.
☐ Look at the change of samples over time.