1. Spectrophotometry

2. Instrumental Analysis
Analytical Chemistry
Quantitative
Gravimetric Analysis
Volumetric Analysis
Instrumental Analysis
Qualitative

3. Spectrophotometry
Spectrophotometry is one of the most useful tools available to the biochemist.
It offers a high degree of precision, sensitivity, and accuracy. In addition, it is inexpensive and applicable to the measurement of a variety of substances.

4. Principles of Spectrophotometry
There is interaction between electromagnetic radiation (light) and matter
A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light.

5. Principles of Spectrophotometry
The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, The signal changes as the amount of light absorbed by the liquid changes.

6. Electromagnetic Radiation (EMR)
Energy
10-200nm vacuum UV region
nm near UV
nm visible region

7. Electromagnetic Spectrum
Type of Transition
Wavelength Range
Frequency Range (Hz)
Type of Radiation
nuclear
<1 pm
gamma-rays
inner electron
1 nm-1 pm
X-rays
outer electron
400 nm-1 nm
ultraviolet
750 nm-400 nm
4-7.5x1014
visible
outer electron molecular vibrations
2.5 µm-750 nm
1x1014-4x1014
near-infrared
molecular vibrations
25 µm-2.5 µm
infrared
molecular rotations, electron spin flips*
1 mm-25 µm 3x
microwave
nuclear spin flips*
>1 mm
<3x1011
radio waves

9. Ultra-Violet/Visible Spectrophotometry
UV/VIS Spectrophotometry is used to determine the absorption or transmission of UV/VIS light (180 to 820 nm) by a sample.
It can also be used to measure concentrations of absorbing materials based on developed calibration curves of the material.

10. Spectrophotometer
Quantitative
Device which measures the amount of light absorbed by a substance.
The amount of light absorbed is directly related to the concentration of the analyte in the solution (A= a b c ) Beer’s law.
Each substance has maximum absorption of light at certain wavelength (λmax)
Qualitative

11. Components of spectrometry
Lamp: light source(s)
Monochromator: a means of isolating a particular wavelength band of the light source, provide Monochromatic light which is light in which all photons have the same wavelength
Cuvette or cell: a sample holder
Detector: a device to measure light intensity.

12. Single Beam Spectrometer
Instrument
Single Beam Spectrometer
Prism
Diffraction grating
Optical filtration
Light source
Tungsten (visible)
Deuterium or hydrogen (UV)
Cell
Glass (visible)
Quartz( UV)

13. The light source shines onto or through the sample.
The sample transmits or reflects light.
The detector detects how much light was reflected from or transmitted through the sample.
The amplifier then converts how much light the sample transmitted or reflected into a number.

14. A schematic showing that a single-color light is selected from continuous spectrum light source by a monochromator. The monochromator usually includes a grating which separates different color light at different angle. A specific color (angle) light passes through a slit and exits the monochromator.

15. Types:
A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample.
A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted.

16. Single Beam Spectrometer

17. Double Beam Spectrometer

18. Experiment

19. Objective: Determine the absorbance spectrum of a sample solution.
Determine the wavelength of maximum absorbance.

20. Approach:
Measure the intensity of transmitted light for various wavelengths of light.
For each wavelength, calculate the absorbance of the solution.
Construct the absorbance spectrum by plotting A vs λ.
Determine λmax by locating the wavelength at which the absorbance is greatest.

21. absorbtion spectrum
The spectrum itself is a plot of absorbance vs wavelength and is characterized by the wavelength (λmax) at which the absorbance is the greatest.

23. The value of λmax is important for several reasons
The value of λmax is important for several reasons. This wavelength is characteristic of each compound and provides information on the electronic structure of the analyte. In order to obtain the highest sensitivity and to minimize deviations from Beer's Law (see subsequent pages on this topic), analytical measurements are made using light with a wavelength of λmax.

24. Double Beam Spectrometer