Pharmaceutical Analysis (WBFA035-05)

Lecture 8, Part B
Basics of Spectrophotometry:
Ultraviolet (UV) - Visible (Vis) Absorbance

• Harris (10th Ed.): Chapter 18 (Section 18-1 to 18-5)

 Measuring absorption of light: Absorbance

Light with many Light with just ONE 

different  selected for sample
P
Transmittance T
(fraction transmitted light): ......... P0
 P0 
Absorbance: ……………….......... A  log   - log(T)
P
Lambert-Beer Law: ……...……… A  ελ  b  c
 = molar absorptivity (M-1 cm-1) – wavelength-specific
c = concentration of the absorbing analyte (M)
b = optical pathlength through sample (cm)
Absorption of light by chemical species (e.g. molecules)
V4
V3 Electronic excited
V2 state (S1)
S1
V1
V0
Energy

Notation for this transition:

S0(V1, g1)  S1(V2, g2)

Electronic ground
V4 state (S0)
V3
Vibration levels
S0 V2
g3 g4 V1
g2 Rotation levels
g1
V0
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What does an absorbance spectrum look like?

CH2=CH-CH=CH2
butadiene

http://www.chemguide.co.uk/analysis/uvvisible/specbutadiene.gif
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What happens when a molecule absorbs light
in the UV-Vis?

 Electrons absorb energy from radiation in the UV-Vis

regions of the electromagnetic spectrum
 Electronic transitions differ, depending on electron type:
• we are interested in ,  and n electrons
• ,  electrons: are bonding electrons
• n electrons: lone-pair, non-bonding electrons (for
instance, in O or N)

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Atomic orbitals overlap to form covalent bonds

s
“bonding” orbitals “antibonding” orbitals

• Continuous electron motion minimizes repelling force between two

positively charged nuclei
• Single bond: molecular  orbital; double bond: 1 x , 1 x 
molecular orbitals

Source: Skoog, Holler, Nieman “Principles of 6

Instrumental Analysis: 5th Edition”, p. 331
Physicochemical fundamentals of spectroscopy
(hydrogen)

excited state
s  *

p 1sA 1sB

d  ground
state

Orbitals Hydrogen: 2H H2
 Electronic excited states
(values for lmax and el(max))
Lambert-Beer Law: A absorbance (dimensionless)
c concentration (mol·L-1)
 P0  b pathlength (cm)
A = ε   b  c = log    molar absorptivity
P (cm-1·mol-1·L)
Excited state with lowest energy is p*
Chromophore transition type lmax (nm) el(max)
−C=C− …………….. p p* ~170 ~15.000

transition is not very likely

−C≡C− …………….. p p* ~180 ~10.000
−C=O …………….. n p* ~280 ~15

“forbidden”
O
…………….. n p* ~200 ~40
OH
O
…………….. n p* ~215 ~60
NH2

− NO2 …………….. n p* ~280 ~20

n s* <170 <1000
 Electronic excited states
(conjugation and the  →  * transition)

P*
Energy

ethene butadiene hexatriene benzene

lmax(nm)
171 217 268 254
 Electronic excited states
(solvent polarity and absorbance wavelength)
All these energy levels
p*
decrease when moving from
non-polar to polar solvent:
n
• n decreases more than p*
p • n decreases more than p
non-polar polar • p* decreases more than p
solvent solvent • p decreases the least

Consequences:
• DE(n-p*) increases  lmax(n-p*) decreases
(hypsochrome effect) blue shift
• DE(p-p*) decreases  lmax(p-p*) increases
(bathochrome effect) red shift
 Electronic excited states
(auxochrome; absorbance wavelength and molar absorptivity)

X
lmax(E2) e(E2) lmax(B) e(B)
X= H 204 7900 254 200
CH3 207 7000 261 300
These are the
auxochromes OH 211 6200 270 1450
– have lone
pair electrons O- 235 9400 287 2600
NH2 230 8600 280 1430

NH3+ 203 7500 254 160

• Auxochromes do not themselves absorb light, but modify the ability of
chromophores to absorb light: 1) they often cause shifts to longer wavelengths of
chromophore absorbance (bathochrome effect) 2) chromophores often exhibit
increased absorbance (hyperchrome effect)
 Electronic excited states
(auxochrome; absorbance wavelength and molar absorptivity)
X = auxochrome
350
Chromophore
300 hyperchromic
250
e (L/mol/cm)

200 hypsochr. bathochr.

(blue shift) (red shift)
150
hypochr.
100
50
0
220 230 240 250 260 270 280
wavelength (nm)
Application: Analysis of iron as (ferrozine) 3Fe(II)
complex
• First:
determine λ of
maximum
absorbance
• Strong (!)
absorbance
(large ) at
562 nm

A  ελ  b  c
Harris 8th Ed., figure 17-8

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Application: Analysis of iron as (ferrozine) 3Fe(II)
complex •
Set spectrophotometer to
562 nm
• Measure absorbance of
solutions having known
concentrations at 562 nm
• Fe(II) concentrations in
M range
• Concentrations in the M
range are generally the
smallest limit of detection
possible for absorbance
measurements

A  ελ  b  c
Harris 8 Ed., figure 17-9
th

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Lambert-Beer Law

A  ε  b  c = -log(T)
λ

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Absorbance measurements: Limits
• Solution concentrations should not exceed 0.01 M
• At higher concentrations, analyte molecules are too
close to one another
• This can result in significant changes in the electron
distribution of molecules that affect molecular
absorbance properties
• If the analyte undergoes reactions or interactions with
solvent molecules, products may be formed that have
changed absorbance properties

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Lambert-Beer Law: Calculations
Example:
A solution with a [KMnO4] of 7.25  10-5 M has a
transmittance of 44.1 % at a  of 525 nm in a cuvette of
pathlength 2.10 cm.

Calculate the absorbance of this solution:

A = -log T = log (P0/P)

A = -log T = -log 0.441 = -(-0.3554) = 0.355

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Lambert-Beer Law: Calculations
Example:
A solution with a [KMnO4] of 7.25  10-5 M has a
transmittance of 44.1 % at a  of 525 nm in a cuvette of
pathlength 2.10 cm.

Calculate the molar absorptivity, :

e = A / (bc) = 0.3554 / (2.10 cm  7.25  10-5 mol L-1)

 = 2.33  103 L mol-1 cm-1 Always write

the units!!
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