1. UV-VIS Spectroscopy
Dr. AKM Shafiqul Islam

2. Spectrometer Experiment
Irradiance in
Irradiance out
Pathlength
Irradiance, P, is measured in W·m-2
Transmittance, T, is the fraction of original light not absorbed by the sample, or
Where po = is power of incident light, p = power of transmitted light and b = path length

3. A light source can be a lamp, laser, or even a light bulb
A light source can be a lamp, laser, or even a light bulb. A monochromator (“one color”) is used to select a particular wavelength. This is typically a grating, but can be a prism or filter. The light then passes through the sample, containing the analyte. Afterwards, the light is detected.

4. Putting this in logarithmic form

5. In 1852, Beer and Bernard each stated that a similar law holds for the dependence of concentration, c
Where k’ is a new constant

6. Combining these two laws which describes the dependence of T on both the path length and concentration
Where a is a combined constant of k and k’

7. It is more convenient to remove negative sign on right hand side, we get
The transmittance is given by
or,

8. Now, we can write

9. Absorbance and Beer’s Law
Absorbance, A, the amount light absorbed by the sample is related to transmittance:
Beer’s law relates the absorbance of a chemical to its concentration:
b is the pathlength, typically in cm, and c is the concentration of the chemical species in M
 is the molar absorptivity, the unit that tells how much light is absorbed for a given wavelength.  has units of M-1 cm-1

10. Wavelengths and Color

11. Beer’s Law Assumptions
The light being shined on the sample must be monochromatic (one color or wavelength)
The analyte must not be participate in a concentration dependent equilibrium
This isn’t a good technique for many weak acid systems, as dilution increases dissociation and HA and A- probably don’t have the same absorbance

12. To do a Spectroscopic Analysis
You need:
A continuous light source
A wavelength selector
A sample cell
A detector
The sample cell is called cuvet and can be made of many substances
Glass (good for visible)
Quartz (UV-vis)
NaCl/KBr (IR)

13. Spectroscopic Procedure
You may have a single-beam or double beam
Single-beam instrument has one sample holder, you must swap blank and sample
Double-beam instrument splits light output between two holders so you can measure blank and sample
A baseline spectrum is a spectrum of a reference solution (solvent or reagent blank)
We try to do an analysis at the λmax if we can
Sensitivity is greatest at maximum absorbance
Curve is relatively flat in case the monochromator drifts and is off by a little in wavelength

14. The Single-Beam Spectrometer

15. How Do UV spectrometers work?
Rotates, to achieve scan
Matched quartz cuvettes
Sample in solution at ca M.
System protects PM tube from stray light
D2 lamp-UV
Tungsten lamp-Vis
Double Beam makes it a difference technique
Two photomultiplier inputs, differential voltage drives amplifier.

16. Optics of the Grating Monochromator
The polychromatic light is separated into monochromatic wavelengths by diffraction. n = d(sin  + sin )

17. Optics of the Grating Monochromator
In the equation n = d(sin  + sin ) n is the order of the diffraction n = 1, 2, 3 etc, d is the number of lines etched on the grating,  is the angle of the incident beam and  is the angle of the emerging beam.

18. The photodiode array detector

19. The photodiode array detector

20. UV Instrumentation Key components: Light Source Monochromator
Sample/reference holder
Radiation detection
Readout device

21. Spectroscopic Procedure
We should always try to keep the absorbance reading of our sample below 1.
Because % transmittance is related logarithmically with concentration, it means that from 1-99% transmittance we can detect ~ 2 orders of magnitude in analyte concentration.
Any orders of magnitude greater than that will be detected in the range of 0-1% T.
In order to maximize accuracy, you should dilute the solution if you have to so that the transmittance reading is not maxed out in that region.

22. Spectroscopic Procedure
You should always try to keep the absorbance reading of your sample below 1.
Because % transmittance is related logarithmically with concentration, it means that from 1-99% transmittance you can detect ~ 2 orders of magnitude in analyte concentration.
Any orders of magnitude greater than that will be detected in the range of 0-1% T.
In order to maximize accuracy, you should dilute the solution if you have to so that the transmittance reading is not maxed out in that region.

23. An Electronic Spectrum
Make solution of concentration low enough that A≤ 1
(Ensures Linear Beer’s law behavior)
Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent.
Can extract the  value if conc. (M) and b (cm) are known
UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV.
Absorbance
Wavelength, , generally in nanometers (nm)
0.0
400
800
1.0
200
maxwith certain extinction  UV
Visible

24. Ultraviolet Spectroscopy
nm photons excite electrons from a  bonding orbital to a * antibonding orbital.
Conjugated dienes have MO’s that are closer in energy.
A compound that has a longer chain of conjugated double bonds absorbs light at a longer wavelength =>

25. Absorption Characteristics of Some Common Chromophores
Example
Solvent
lmax (nm)
emax
Type of transition
Alkene
n-Heptane
177
13,000
pp*
Alkyne
178
196
225
10,000
2,000
160 _
Carbonyl
n-Hexane
186
280
180
293
1,000 16
Large 12
ns*
np*
Carboxyl
Ethanol
204 41
Amido
Water
214 60
Azo
339 5
Nitro
CH3NO2
Isooctane 22
Nitroso
C4H9NO
Ethyl ether
300
665
100 20
Nitrate
C2H5ONO2
Dioxane
270

26. Solvents for UV (showing high energy cut-offs)
Water 205
CH3CN 210
C6H
Ether 210
EtOH 210
Hexane 210
MeOH 210
Dioxane 220
THF 220
CH2Cl2 235
CHCl3 245
CCl4 265
benzene 280
Acetone 300
Various buffers for HPLC, check before using.

27. Deviation from Beer’s Law
Beer’s law is only valid for low concentration, up to 10 mM;
The intermolecular distances in a given solution will decrease, eventually reach a point at which neighboring molecules mutually affect the charge distribution of the other affect 
Chemical processes such as the reversible association-dissociation of analyte molecules, or the ionization of a weak acid in an unbuffered solvent.
Instrumentation limitation-incident beam may be polychromatic .

28. Background Correction
Processes other than analyte absorption
result in significant decrease in the power
of the incident beam;
Reference cell is used to correct
these processes;
Reference cell is often prepared by
adding distilled water to an absorption
cell;
The reference cell is then placed in the
path of the light beam, and the power
of the radiation exiting the reference cell
is measured and taken as P0 for the sample cell.

29. Calibration Curves
Linear calibration curve;
Nonlinear calibration

30. An Example--Pulegone Frequently plotted as log of molar extinction 
So at 240 nm, pulegone has a molar extinction of 7.24 x 103
Antilog of 3.86

31. Example
A solution containing a compound of formula weight 280 g/mol absorbed 65.0% of the light with 450 nm wavelength in a 2.00 cm cell at a concentration of 15.0 × 10-3 g/L. Calculate its molar absorptivity at 450 nm.