1.1 Range of molar absorptivity
M  h excitation

 M *
Electronic excitation of outer valence (i.e. bonding) electron
How probable for this electronic excitation? (allowed transition, or forbidden)
judged by the range of 
 = 104 -105 L mol-1 cm-1, strong absorption
 < 103 L mol-1 cm-1, low intensity
1.2 Which electron get excited?
1.2.1 Organic molecules
,  (bonding) and n (non-bonding) orbitals
*, * (anti-bonding) orbitals
  * E large ( < 150 nm, out of range)
 = 10 -10,000 Lmol-1cm-1
n  * E smaller ( = 150 - 250 nm)
 = 200-2000 Lmol-1cm-1
  *
n  * E smallest ( = 200 - 700 nm)
 = 10-10,000 Lmol-1cm-1
Ideal for UV-Vis spectrometry of organic
chromophore
1.2.2 Inorganic molecules
Most transition metal ions are colored (absorption in Vis) due to d  d electronic
transition
Fig. 14-3 (p.370)
Fig. 14-4 (p.371)
1.2.3 Charge-transfer absorption
AD  h excitation

 A D 
A: electron donor, metal ions
D: electron acceptor, ligand
 > 10,000
Fig. 14-5 (p.371)
2.1 Approximation of T and A
A = -log T = log (P0/P) = ··b·c
: molar absorptivity at one particular
wavelength (L·mol-1cm-1)
b: path length of absorption (cm)
c: molar concentration (mol·L-1)
Fig. 6-25 (p.158)
Light loss due to reflection (17.3%),
scattering, …
Fig. 13-1 (p.337)
Psolution P
T

Psolvent P0
Psolvent
P0
A  log
 log
Psolution
P
2.2 Application of Beer’s law to mixtures
Absorbance is additive
Atotal
= A1 + A2 + …
= 1bc1 + 2bc2
For a 2-component mixture, we measure the absorption at two different wavelength,
respectively
A1 = 1,1·b·c1 + 2,1·b·c2
A2 = 1,2·b·c1 + 2,2·b·c2
2.3 Limitations of Beer’s law
2.3.1 Real deviations
At low concentration
A = -log T = log (P0/P) = ··b·c
At c > 0.01 M
solute-solute interaction, hydrogen-bond, …
can alter the electronic absorption at a given wavelength
 dilute the solution
2.3.2 Chemical effects
analyte associates, dissociates or reacts with a solvent to
give molecule with different 
Example: acid-base equilibrium of an indicator
HIn  H   In
K a  1.2  105
430
HIn
In-
570
6.30 x102
7.12x103 (measured in HCl solution)
2.06 x 1049.61 x102
(measured in NaOH solution)
What’s the absorbance of unbuffered solution at c = 2 x 10-5M?
[ H  ][ In  ]
Ka 
 1.42 10 5
[ HIn ]
[ H  ]  [ In  ]

[ HIn ]  c  [ In ]

[ In  ]  1.12 10 5 M
[ HIn ]  0.88 10 5 M
A430   In  , 430b[ In  ]   HIn , 430b[ HIn ]  0.236
A570   In  ,570b[ In  ]   HIn ,570b[ HIn ]  0.073

HIn  H  In

Fig. 13-3 (p.340)
=[HIn] + [In-]
2.3.3
Instrumental deviations due to polychromatic radiation
Beer’s law applies for monochromatic absorption only.
If a band of radiation consisting of two wavelength ’, and ”
Assuming Beer’s law applies to each wavelength
For first wavelength 
P'
A'  log 0'   ' bc
P
'
P'  P010 'bc
For the second wavelengt h "
P"  P0" 10 "bc
M easured absorbance
P0'  P0"
P0'  P0"
Am  log
 log '  'bc
P' P"
P010
 P0" 10 "bc
Fig. 7-11 (p.176)
2.3.3
Instrumental deviations due to polychromatic radiation
Measured absorbance
P0'  P0"
P0'  P0"
Am  log
 log ' bc
P' P"
P010  P0" 10 "bc
′
″
′
″
′
″
Non-linear calibration curve
Fig. 13-4 (p.341)
How to avoid :
Select a wavelength band near its maximum absorption where
the absorptivity changes little with wavelength
Fig. 13-5 (p.341)
2.3.4
Other physical effects
stray light – the scattering, reflection radiation from the instrument, outside the
nominal wavelength band chosen for measurement
mismatched cell for the sample and the blank
3.1 Standard deviation of c
1
c   log T
b
c
0.434

T
bT
c 2 2
2
 c  ( ) T
T
sc   c2
sT   T2
sc 0.434 sT

c
T log T
3.2 Sources of instrumental noise
Case I
Limited readout resolution (31/2-digit displays  0.1% uncertainty from 0%T -100% T)
Thermal noise in thermal detector, etc (particularly for IR and neat IR spectrophotometer)
sT  k1
Case II
Shot noise in photon detector (random emission of photon from the light source or
random emission of electrons from the cathode in a detector)
Case III
Flicker noise,
sT  k2 T 2  T
sT  k3T
Fail to position sample and blank cells reproducibly in replicate
measurements (as a result, different sections of cell window are exposed to radiation,
and reflection and scattering losses change)
3.2 Sources of instrumental noise
Fig. 13-3 (p.344)
4.1 Designs
a. Single beam
b. Double-beam-in-space
c. Double-beam-in-time
Advantage of double beam
configuration
•
Compensate for fluctuation in the
radiant output, drift in transducer,
etc.
•
Continuous recording of spectra
Fig. 13-13 (p.352)
Shimadzu UV-2450
Spectrophotometer
Wavelength Range
190 to 900nm (performance guaranteed range). Extendable to 1,100nm through the use of the optional photomultiplier. (The measurable range
maybe restricted in the shorter wavelength side depending on the type of photomultipler used.)
Monochromator
System
UV-2450: Single monochromator with a high-performance blazed holographic grating in the aberration corrected Czerny-Turner mounting.
Resolution
0.1nm
Spectral Bandwidth
0.1, 0.2, 0.5, 1, 2 and 5nm
Wavelength
Accuracy
±0.3nm
Wavelength
Repeatability
±0.01nm
Wavelength
Scanning Speed
FAST, MEDIUM, SLOW, and SUPER SLOW
Light Source
50W halogen lamp (2,000 hours of life) and D2lamp (500 hours of life)
Light Source lamp
switching
Selectable between 282nm and 393nm
Stray Light
UV-2450: Less than 0.015% at 220nm and 340nm
Detector
Photomultiplier R-928
Photometric System
Double beam, direct ratio system with dynode feedback
Photometric Mode
Absorbance (Abs.), transmittance (%), reflectance (%) and energy (E).
Photometric Range
Abosrbance: -4~5 Abs. (0.001 Abs. increments)
Transmittance: 0~999.9% (0.01 increments)
Reflectance: 0~999.9% (0.01% increments)
Photometric
Accuracy
±0.002Abs(0~0.5Abs), ±0.004Abs(0.5~1Abs),±0.3T (0~100%T)
(all determined with NIST 930D standard filter)
Photometric
Repeatability
0.001Abs (0~0.5Abs), ±0.1%T
Baseline Correction
Selectable with storage in firmware
Baseline Flatness
Within ±0.001Abs.
(excluding noise, 2nm slit width and SLOW wavelength scanning speed)
Drift
Less than 0.0004 Abs. per hour (after 2 hours warm-up)
Dimensions
570 (W) x 660 (D) x 275 (H) (mm)
Weight
36kg
Power Supply
AC 100V/120V/220V/240V, 50/60Hz 250VA (swithc-selectable)
Shimadzu UV-2450
Spectrophotometer
d. Effects of monchromator exit slit width on spectra
Narrow exit slit width improves the spectrum resolution
but it also significantly reduce the radiant power
Trade-off between resolution and S/N ratio
e. Multichannel spectrometer
No monochromator,
but disperses transmitted light and
measures “all wavelength at once”
Fig. 13-15 (p.353)
No Scanning-simple and fast
More expensive
Limited resolution
5.1 Quanlitative spectra
Solvent effects on the UV-Vis spectra
Polar solvents “blur” vibrational features
Polar solvents shift absorption maxima
n  *
blue shift
  *
red shift
UV-Vis not reliable for qualitative
but excellent for quantitative
analysis

-
5.2 Quantitative analysis
- Determining the relationship between A and c
External Standards
Standard-Addition
5.3 Spectrophotometric kinetics
A+ R Û P
Fig. 14-14 (p.382)
Fig. 14-16 (p.384)