0. UV-visible spectroscopy
How They Work

1. What is Spectroscopy?
The study of molecular structure and dynamics through the absorption, emission and scattering of light.

2. What is Light?
According to Maxwell, light is an electromagnetic field characterized by a frequency f, velocity v, and wavelength λ. Light obeys the relationship
f = v / λ.

3. The Electromagnetic Spectrum
E = hn
n = c / l

4. Wave Number (cycles/cm)
Spectroscopy
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X-Ray UV
Visible IR
Microwave
200nm
400nm
800nm
WAVELENGTH(nm)

5. Transmission and Color
The human eye sees the complementary color to that which is absorbed

6. Absorbance and Complementary Colors

7. Two-Component Mixture
Example of a two-component mixture with little spectral overlap

8. Two-Component Mixture
Example of a two-component mixture with significant spectral overlap

9. Influence of 10% Random Error
Influence on the calculated concentrations
Little spectral overlap: 10% Error
Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%)

10. Absorption Spectra of Hemoglobin Derivatives

11. Light Sources UV Spectrophotometer 1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
InfraRed (IR) Spectrophotometer
1. Carborundum (SIC)

12. Dispersion Devices Non-linear dispersion Temperature sensitive
Different orders

13. Dispersion of polychromatic light with a prism
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
SLIT
PRISM
Polychromatic Ray
Monochromatic Ray
Prism - spray out the spectrum and choose the certain wavelength (l) that you want by moving the slit.

14. Photomultiplier Tube Detector
High sensitivity at
low light levels
Cathode material
determines spectral sensitivity
Good signal/noise
Shock sensitive
Anode

15. The Photodiode Detector
Wide dynamic range
Very good
signal/noise at high light levels
Solid-state device

16. Schematic Diagram of a Photodiode Array
Same characteristics
as photodiodes
Solid-state device
Fast read-out cycles

17. Conventional Spectrophotometer
Schematic of a conventional single-beam spectrophotometer

18. Conventional Spectrophotometer
Optical system of a double-beam spectrophotometer

19. Conventional Spectrophotometer
Optical system of a split-beam spectrophotometer

20. Definition of Resolution
Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths

21. Instrumental Spectral Bandwidth
The SBW is defined as the width, at half the maximum intensity, of the band of light leaving the monochromator

22. Natural Spectral Bandwidth
The NBW is the width of the sample absorption band at half the absorption maximum

23. Transmission Characteristics of Cell Materials
Note that all materials exhibit at least approximately 10% loss in transmittance at all wavelengths

24. Cells UV Spectrophotometer Quartz (crystalline silica)
 Visible Spectrophotometer
Glass
 IR Spectrophotometer
NaCl

25. Cell Types I Open-topped rectangular standard cell (a)
and apertured cell (b) for limited sample volume

26. Cell Types II
Micro cell (a) for very small volumes and flow-through cell (b) for automated applications

27. Transmittance and Concentration The Bouguer-Lambert Law

28. Transmittance and Path Length: Beer’s Law
Concentration

29. The Beer-Bouguer-Lambert Law

30. BEER LAMBERT LAW
As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.

31. R- Transmittance
R = I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution). I I0 I I0 1 T I0 I I I0

32. A  CL = KCL by definition and it is called the Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient g of solute per liter of solution
A = ECL
E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution

33. E differs from K (Specific extinction Coefficient) by a factor of molecular weight.
UNITS
  A = ECL
A = No unit (numerical number only)

34. L = Cm
C = Moles/Liter
A = KCL
A = No unit C = Gram/Liter L = Cm K=
Liter
Cm  Gram

35. STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHOD
Run the sample for spectrum
2. Obtain a monochromatic wavelength for the maximum absorption wavelength.
3. Calculate the concentration of your sample using Beer Lambert Equation: A = KCL

36. There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where becomes non-linear.

37. SPECTROMETRIC ANALYSIS USING STANDARD CURVE
Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

38. Every instrument has a useful range for a particular analyte.
Often, you must determine that range experimentally.
This is done by making a dilution series of the known solution.  
These dilutions are used to make a working curve.

39. Make a dilution series of a known quantity of analyte and measure the Absorbance. Plot concentrations v. Absorbance.

40. What concentration do you think the unknown sample is?

41. In this graph, values above A=1. 0 are not linear
In this graph, values above A=1.0 are not linear. If we use readings above A=1.0, graph isn’t accurate.

42. The best range of this spectrophotometer is A=0. 1 to A=1
The best range of this spectrophotometer is A=0.1 to A=1.0, because of lower errors. A=0.4 is best.

43. Relating Absorbance and Transmittance
Absorbance rises linearly with concentration. Absorbance is measured in units.
Transmittance decreases in a non-linear fashion.
Transmittance is measured as a %.
Absorbance = log10
(100/% transmittance)

45. Precision and Accuracy