26 Molecular Absorption Spectrometry

26A Ultraviolet and Visible Molecular Absorption Spectroscopy

26A-1 Absorbing species
Molecules Absorbing UV/Visible radiation
Qualitative
Quantitative
Absorption by organic Compounds
(1) shared electrons that participate directly in bond formation and are thus
associated with more than one atom
(2) unshared outer electrons that are largely localized about such atoms as oxygen,
the halogen, sulfur and nitrogen.
Species with unsaturated bonds generally absorb in the UV
Chromophore: unsaturated organic functional groups that absorb in the
UV/visible region.
Table 26-1 Absorption Characteristics of Some Common Organic Chromophores
Chromophore
Alkene
Conjugated alkene
Alkyne

Example
C6H13CH=CH2
CH2=CHCH=CH2
C5H11CCCH3

Solvent
n-Heptane
n-Heptane
n-Heptane

Carbonyl

CH3(C=O)CH3

n-Hexane

CH3(C=O)H

n-Hexane

Carboxyl
Azo
Nitro
Nitrate
Nitroso

CH3(C=O)NH2
CH3COOCH
CH3N=NCH3
CH3NO2
C2H5ONO2
C4H9NO

Water
Ethanol
Ethanol
Isooctane
Dioxane
Ethyl ether

Aromatic

Benzene

n-Hexane

max, nm
177
217
178
196
225
186
280
180
293
214
204
339
280
270
300
665
204
256

max
13,000
21,000
10,000
2,000
160
1,000
16
Large
12
60
41
5
22
12
100
20
7,900
200

Table 26-2 Absorption by Organic Compounds Containing Unsaturated Heteroatoms

Compound
CH3OH
(CH3)2O
CH3Cl
CH3I

max, nm
167
184
173
258

max
1480
2520
200
365

Absorption by Inorganic Species

Charge-Transfer Absorption

Compound
(CH3)2S
(CH3)2NH2
(CH3)3N

Fig. 26-2, 3
Fig. 26-4
168

max, nm
229
215
227

max
140
600
900

Fig. 26-2 Absorption spectra of Fig. 26-3 Absorption spectra

aqueous solutions of several
of aqueous solutions of
Fig. 26-1 Absorption spectra
transition metal ions.
rare earth ions.
for typical organic compounds.

Fig. 26-4 Absorption spectra of aqueous charge-transfer complexes.

Quantitative: UV/visible spectrophotometry

Qualitative: infrared spectrophotometry

26A-2 Qualitative Applications of UV/Visible Spectroscopy

Table 26-1
Solvents (Fig. 24-14, Table 26-3)
The Effect of Slit Width (Fig. 26-5)
The Effect of Scattered Radiation at the Wavelength Extremes of a
Spectrophotometer (Fig. 26-6)
Table 26-3 Solvents for the ultraviolet and visible regions
Solvent

Lower wavelength
Solvent
limit, nm

Lower wavelength
Solvent
limit, nm

Lower wavelength
limit, nm

Water
180
Cyclohexane
200
Acetone
330
Ethanol
220
Cellosolve
320
Dioxane
320
Diethyl ether
Hexane
200
210
CCl4
260
Fig. 26-5 Spectra for reduced cytochrome c obtained with four spectral bandwidths: (1) 20 nm,
(2) 10 nm, (3) 5 nm, and (4) 1 nm. At bandwidths < 1 nm, peak noise became pronounced.
169

Fig. 26-6 Spectra of cerium(IV) obtained with a spectrophotometer having glass optics (A) and
quartz optics (B). The false peak in A occurs when stray radiation is transmitted at long
wavelengths.

26A-3 Quantitative
Applications
1.
2.
3.
4.
5.

Wide applicability.
High sensitivity
Moderate to high selectivity
Good accuracy
Easy and convenience
Fig. 26-5

Fig. 26-6

Direct measurement
Nitrate in nature water: NO3-: 220 nm
1. Absorbance at 220 nm
2. Absorbance at 270 nm (correct for the interference)
Indirect measurement
12 MoO42- + H2PO4- + 24 H+ [H2PMo12O40]- + 12 H2O
12-molybdophosphoric acid, 12-MPA (yellow)
ascorbic acid or stannous chloride
molybdenum blue (650~890 nm strong absorbance)
Application to Absorbing Species
common organic chromophore (Tab. 26-1)
inorganic species: ions of transition metals and their complexes (color), nitrite, nitrate,
chromate ion, oxides of nitrogen, element halogens and ozone.
Applications to Nonabsorbing Species
react with chromophoric reagents products that absorb strongly in the UV and
visible regions
Inorganic reagent
Analyte
Organic chelating agent Analyte
thiocyanate
Fe, Co, Mo
diethyldithiocarbamate
Cu
anion of H2O2e
Ti, V, Cr
diphenylthiocarbazone
Pb
iodide
Bi, Pd, Te
1,10-phenanthroline
Fe
Ce(IV)
low-molecular-weight aliphatic alcohol
dimethylglyoxime
Ni

Fig. 26-7 Typical chelating reagents for absorption. (a) Diethyldithiocarbamate. (b)
Diphenylthiocarbazone.
170

Procedural Details
development of conditions that yield a reproducible relationship (preferably linear)
between absorbance and analyte concentration.
1. Wavelength Selection
made a wavelength corresponding to an absorption peak maximum sensitivity
2. Variables That Influence Absorbance
solvent, pH of the solution, temperature, high electrolyte concentrations and
presence of interfering substances.
3. Determination of the Relationship Between Absorbance and Concentration
Calibration standards
The measured absorbance of a given solution will usually vary somewhat from
instrument to instrument. Thus, determination should never be based on molar
absorptivities found in the literature.
4. The Standard Addition Method
standard addition:
a known amount of analyte + a second aliquot sample
the difference in Abs. calculate the analyte conc. of the sample
Plot of As as a function of Vs
As = mVs + b ,

m = kcs ,

b = k Vxcx

2
2
2
2
m
kcs
S c S m Sb
S m Sb
bcs

=
cx =
, = + sc = c x +
b kVx c x
mVx
m b
cx m b

Ex. 26-1 10-mL aliquots of a natural water sample were pipetted into 50.00-mL volumetric
flasks. Exactly 0.00, 5.00, 10.00, 15.00 and 20.00 mL of a standard solution containing
11.1 ppm of Fe3+ were added to each, followed by an excess of thiocyanate ion to give the
red complex Fe(SCN)2+. After dilution to volume, absorbances for the five solutions,
measured with a photometer equipped with a green filter, were found to be 0.240, 0.437,
0.621, 0.809 and 1.009, respectively (.0982-cm cells). (a) What was the [Fe3+] in the water
sample? (b) Calculate the standard deviation of the slope, the intercept and the [Fe].
(a) cs =11.1 ppm, Vs = 10.00 mL, Vt = 50.00 mL
m = 0.03820, b = 0.2412
As = 0.03820 Vs + 0.2412

bcs
(0.2412)(11.1 ppmFe3+ )
cx =
=
= 7.01 ppmFe3+
mVx (0.03820/mL)(100mL)
(b) sm = 3.07 10-4 and sb = 3.76 10-3
2

sc = c x

Sm Sb
+
m b

3.07 10 4 3.76 10 3
+
= 0.12 ppmFe3+
= 7.01
0.03820 0.2412
171

Fig. 26-8 Determination of Fe3+

as the Fe(SCN)2+ complex.

Ex. 26-2 The single-point standard addition method was used in the determination of
phosphate by the molybdenum blue method. A 2.00-mL urine sample was treated with
molybdenum blue reagents to produce a species absorbing at 820 nm, after which the
sample was diluted to 100.00 mL. A 25.00-mL aliquot gave an absorbance of 0.428
(solution 1). Addition of 1.00 mL of a solution containing 0.0500 mg of phosphate to a
second 25.0-mL aliquot gave an absorbance of 0.517. Use these data to calculate the
number of milligrams of phosphate per milliliter of the sample.
A1 = bcx

A2 =

b Vx c x
Vt

b Vs c s
Vt

b(Vx c x + Vs c s )
Vt

A1c s Vs
0.428 0.0500mgmL1 1.00mL
cx =
=
= 0.00780mgmL1
A 2 Vt A1Vx 0.517 26.00mL 0.428 25.00mL
conc. of phosphate = 0.00780 mgmL-1 100/2.00 = 0.390 mg/mL
6. Analysis of Mixture (Fig. 26-9)
Atotal = A1 + A2 + + An = 1bc1 + 2bc2 + +nbcn
A1 = M1bcM + N1bcN
A2 = M2bcM + N2bcN
Fig. 26-9 Absorption spectrum of a two-component mixture
(M+N), with spectra of the individual components.
Wavelengths 1 and 2 are chosen for the analysis because the
individual component spectra are significantly different at
these two wavelength.
Ex. 26-3 Palladium (II) and gold (III) can be analyzed simultaneously through reaction with
methiomeprazine (C19H24N2S2). The absorption maximum for the Pd complex occurs at
480 nm, while that for the Au complex is at 635 nm. Molar absorptivity data at these
wavelengths are
Molar Absorptivity,
480 nm
635 nm
3
Pd complex
3.55 10
5.64 102
Au complex
2.96 103
1.45 104
A 25.0-mL sample was treated with an excess of methiomeprazine and subsequently diluted to
50.0 mL. Calculate the molar concentrations of Pd(II), CPd, and Au(III), CAu, in the sample if
the diluted solution had an absorbance of 0.533 at 480 nm and 0.590 at 635 nm when measured
in a 1.00-cm cell.

At 480 nm: 0.533 = (3.55 103)(1.00) CPd + (2.96 103)(1.00) CAu

C Pd =

0.533 2.96 103 C Au

3.55 103

At 635 nm: 0.590 = (5.64 102)(1.00) CPd + (1.45 104)(1.00) CAu

172

0.590 = 5.64 10

(0.533 2.96 103 ) C Au

3.55 103

+ 1.45 10 4 C Au

= 0.0847 4.70 10 2 C Au + 1.45 10 4 C Au

C Au =

(0.590 0.0847)
1.403 10

C Pd = 0.53

= 3.60 10 5 M

2.96 103 3.60 10 5

3.55 10

= 1.20 10 4 M

Au(III) = 7.20 105 and Pd(II) = 2.40 10-4 M

26A-4 Photometric and Spectrophotometric Titrations

one or more of the reactants or products absorb radiation or that an absorbing
indicator be present.
Titration Curves
plot of absorbance (correct for volume
change) as a function of titrant volume:
consists of two straight-line regions with
different slopes
: intersection of extrapolated linear
portions of the two lines
Fig. 26-12 Typical photometric titration
curves. Molar absorptivities of the analyte
titrated, the product and the titrant are A, P
and T, respectively.
(Fig. 26-12)
Analyte
Titrant
(a)
nonabsorbing ( = 0)
absorbing ( > 0)
thiosulfate
triiodide
(b)
colorless ( = 0)
( = 0)
iodide
iodate ion

Product
( = 0)
Absorbing ( > 0)
triiodide

Application of Photometric Titrations

more accurate than a direct photometric determination
presence of other absorbing species may not interfere
more dilute solution may be titrated
photometric end point applied to all types of reaction
ex: successive titration of Bi(III) and Cu(II). At 745
nm, cations, reagent and the Bi complex no absorb,
but the Cu complex absorbance additional
reagent cause no further abs. change
two well-defined end points.
Fig. 26-13 Photometric titration curve at 745 nm for 100 mL of a solution that was 2.0 10-3
M in Bi3+ and Cu2+
173

26B Automated Photometric and Spectrophotometric Methods

26C Infrared Absorption Spectroscopy
Qualitative > Quantitative

26C-1 Infrared Absorption Spectra

exception of a homonuclear species, such as molecular H2, O2 and N2, all moleculars,
organic and inorganic, absorb IR radiation.
Fig. 26-20
Infrared spectrum for
n-butanal (nbutyraldehyde).

174