Dong-Sun Lee / cat-lab / SWU
2010-Fall Version
Chapter 26 A
Molecular Absorption
Spectrometry
Application of UV-Visible spectrometry
Absorbing species
Organic compounds :
Any compound containing one or more of the chromophore groups is
potentially feasible.
Chromophores are unsaturated organic functional groups that absorb in the
ultraviolet or visible region.
Many nonabsorbing organic analytes can be determined by using them to
react with a chromophoric reagent to yield a product that absorbs in the UVvisible region.
Absorption spectra for typical organic compounds.
UV-visible spectra of benzene,
naphthalene, and anthracene in
ethyl alcohol.
Inorganic compounds :
Few inorganic substances absorb strongly.
Converting inorganic ion to a highly absorbing substance by addition of complexing
agent. Colors arise from
d d transition within metal ion
n  * and   * transition within the ligand
Typical chelating reagents for absorption (a) diethyldithiocarbamate, (b) diphenylthiocarbazone.
Absorption spectra of aqueous solutions
of transition metal ions.
Absorption spectra of aqueous solutions
of rare earth ions.
Charge transfer absorption
A charge transfer complex is a strongly
absorbing species that is made up of an
electron-donating species bonded to an
electron-accepting species.
Absorption spectra of aqueous charge
transfer complexes.
Serum iron determination
Step 1. Reduce Fe3+ in transferrin to Fe2+ by hydroxylamine hydrochloride, thioglycolic
acid, or ascorbic acid.
Step 2. Add trichloroacetic acid to precipitate proteins.
Step 3. Transfer a measured volume of supernatant liquid to a fresh vessel and add
buffer plus excess ferrozine to form a purple complex.
Visible absorption spectrum of the complex (ferrozine)3Fe(II) used in the colorimetric analysis of iron.
Spectra for reduced cytochrome C obtained with four spectral bandwidth: 1) 20 nm,
2) 10 nm, 3) 5 nm, 4) 1 nm. At bandwidths < 1 nm, peak noise became pronounced.
Spectra of cerium(IV) obtained with a spectrophotometer having (A) glass optics and
(B) quartz optics. The false peak in A occurs when stray radiation is transmitted at long
wavelengths.
Error curves for various categories of
instrumental uncertainties.
Errors in spectrophotometric measurements
due to dark current noise and cell positioning
imprecision in a research quality instrument.
Experimental curves relating relative concentration uncertainties to absorbance for
two spectrometer.
Procedural details
1) Forming an absorbing species
2) Selection of measurement wavelength : Obtaining absorption spectra
Maximum sensitivity is realized when max for the analyte is selected, because the
change in absorption per unit concentration is the greatest.
3) Determination of the relationship between absorbance and concentration.
A. Calibration curve
B. Standard addition method
Calibration curve
Standard addition method
Simultaneous analysis of mixture
The total absorbance of a solution at any given wavelength is equal to the sum
of the absorbances of the individual components in the solution.
A1 = Ax,1 + Ay,1 + Az,1 …. = x1bCx + y1bCy + z1bCz ….
A2 = Ax,2 + Ay,2 + Az,2 …. = x2bCx + y2bCy + z2bCz ….
Cx =
Cy =
A1
y1b
A2
y2b
x1b
y1b
x2b
y2b
x1b
A1
x2b
A2
x1b
y1b
x2b
y2b
Two cases for analysis of a mixture.
(a)
Spectra of the pure components have
substantial overlap.
(b)
Regions exist in which each component
makes the major contribution.
Visible spectrum of MnO4– , Cr2O72– , and an unknown mixture
containing both ions.
Isosbestic point
A wavelength at which the absorbance spectra of two species cross each other. The
appearance of isosbestic points in a solution in which a chemical reaction is occurring is
evidence that there are only two components present, with a constant total concentration.
A465 = HIn b[HIn] + In– b [In–]
Absorption spectra of 0.37 mM methyl red as a function of pH between pH 4.5 and 7.1.
Spectrophotometric titrations
A photometric titration curve is a
plot of absorbance ( corrected for
volume change ) as a function of
titrant volume. The curve consists
of two straight-line portion of
differing slopes, one before and
one after the equivalence point.
The end point is taken as the
extrapolated intersection of the two
straight lines.
Spectrometric end-point detection
has been applied to all types of
reactions.
Although
stepwise
titration of mixtures is possible.
Curve for the spectrometric titration at 745 nm for
100 ml of a solution 0.002 M in both Bi3+ and Cu2+
with 0.100M EDTA.
Typical photometric titration curves. Molar absorptivities of analyte titrated, product,
and the titrant are A, P, T, respectively.
(a) Spectrophotometric titration of 30.0 ml of EDTA in acetate buffer with CuSO4
in the same buffer.
Upper curve: [EDTA] = [Cu2+] = 5.00 mM. Lower curve: [EDTA] = [Cu2+] =
2.50 mM.
(b) Trans formation of data to mole fraction format.
Methods for obtaining the stoichiometry of complex
(determination of the composition of complexes)
M + nL = MLn
1) Continuous-variation method
(Job’s method)
This method is based on the measure-ment
of a series of solutions in which molar
concentrations of two reactants vary but
their sum remains constant. The absorbance
of each solution is measured at a suitable
wavelength, corrected for any absorbance
the solution would have if no reaction
occurred, and plotted versus the mole
fraction of one reactant. A maximum in
absorbance occurs at the mole ratio
corresponding to the combining ratio of the
reactants.
Corrected A = measured A – AM – AL
Continuous variations plots for 1:3, 1:2 and 1:1
metal to ligand complexes.
2) Mole-ratio method
A series of solution is prepared in
which the analytical concentration
of one reactant is held constant
while that of other is varied. A plot
of absorbance versus mole ratio of
the reactants is then prepared. If the
reaction is sufficiently complete,
two straight lines of different slopes
are obtained. The intersection of the
extrapolated lines corresponds to
the combining ratio in the complex.
Unlike the method of continuous
variations, the measured absorbance
does not have to be corrected by
subtracting the absorbance.
Mole-ratio plots for 1:1 and 1:2 metal-to-ligand
complexes.
3) Slope-ratio method
This method, used mainly in studying weak complexes, requires that the formation reaction can be
forced to completion with a large excess of either metal or ligand. Two sets of solutions are
prepared : The first contains various amounts of metal ion each with the same large excess of
ligand, while the second consists of various amounts of ligand each with the same large excess of
metal. For the reaction
xM + yL = MxLy
when L is present in large excess, the concentration of product formed is limited by the
concentration of the metal, or
[MxLy] = CM / x
If Beer’s law obtains,
A = b[MxLy] = bCM / x
and a plot of A versus CM will yield a straight line with a slope of b/x.
Similarly, for the solutions containing M in large excess,
[MxLy] = CL / y
A = b[MxLy] = bCL / y
The ratio of the two slopes is the combining ratio for the reaction
(bCM / x )(bCL / y) = x / y
Measuring an equilibrium constant : the Scatchard plot
P + X = PX
K = [PX] / [P][X]
Po = [P] + [PX]

[P] = Po – [PX]
 [PX] / [X] = K [P]
= K (Po – [PX])
A Scatchard plot is a graph of [PX]/[X] versus [PX]. The slope is –K.
The absorbaance of the solution at some wavelength is the sum of absorbances
of PX and P :
A = PX [PX] + P [P]
A = PX [PX] + Po [Po] – P [PX] = PX [PX] + Ao – P [PX]
A = [PX](PX – P ) + P Po = [PX]  + Ao
[PX] = ( A – Ao ) /  = A / 
 A /  [X] = K (Po – A /  )
 A / [X] = K  Po – KA
Scatchard plot of iron(III) complexes of fluoro-oxo-quinoline. (A) Ciprofloxacin, (B)
enoxacin, C) ofloxacin, (D) norfloxacin.
Dong-Sun Lee et al. J. Pharm. Biomed. Anal., 12(2), 157-164, 1994.
Derivative spectroscopy
If a spectrum is expressed as absorbance A as a function of wavelength , the derivative
spectra are
Zero order
A = f()
A =  bC
First order
dA / d = f’()
dA / d = (d/d )bC
Second order
d2A / d2 = f’’()
d2A / d2 = (d2/d2 )bC
The first derivative spectrum start and finish at zero, passes through zero at max of the
absorption band with first a positive and then a negative band, with the maximum and
minimum at the same wavelengths as the inflection points in the absorption band. This
bipolar function is characteristic of all the odd-order derivatives.
The most characteristic feature of the second order derivative is a negative band with the
minimum at the same wavelength as the maximum on the zero-order band. It also shows
two additional positive “satellite” bands on either side of the main band. The presence of
a strong negative or positive band, with the minimum or maximum at the same
wavelength as max of the absorbance band, is characteristic of the even-order derivatives.
Note that the number of bands observed is equal to the derivative order plus one.
First to fourth derivatives of an 500 nm Gaussian band.
Resolution of two overlapping bands in the fourth derivative.
Baseline shift elimination and suppression of scattering using the first derivative.
max = 260 nm
DNA
ADNA = Aadenine + Aguanine + Acytocine + A thymine
Structure of part of a DNA chain and its UV-vis spectrum.
The Edman method of NH2-terminal
amino acid analysis
 max = 266 nm
PTH-Glu
A
 max = 265 nm
PTH-Asp
dA/d
 (nm)
Determination of serum GOT and GPT
Principle :
GOT
-ketoglutarate + aspartate  oxalacetate + glutamate
COOH
COOH
COOH
COOH
CH2
CH2
CH2
CH2
CH2
CHNH2
C=O
CH2
C=O
COOH
COOH
CHNH2
COOH
GPT
-ketoglutarate + alanine
COOH
 pyruvate + glutamate
COOH
COOH
COOH
COOH
CH2
CHNH2
C=O
CH2
CH2
COOH
CH3
CH2
C=O
CHNH 2
COOH
COOH
oxalacetate ( or pyruvate) + dinitrophenylhydrazine  hydrazone (colored)
spectrometry
Diagnostic interpretation :
SGOT
SGPT
Normal value ( units/ml)
8 ~ 40 (mean 22)
5 ~ 30 (mean 16 )
Myocardial infarction
50 ~ 400
35 ~ 100
Necrosis of hepatic cell
50 ~ 1000
100 ~ 2000
Apparatus :
UV-vis spectrometer
cuvet
water bath ( incubator)
pipet ( 0.2ml, 1ml, 10 ml)
test tube ( 15~ 20 ml), beaker
Reagents :
1) phosphate buffer( 0.1M, pH 7.4) : mix 420 ml of 0.1M disodium phosphate and 80
ml of 0.1M potassium dihydrogen phosphate
2) pyruvate (2mM/L, for standard curve) : dissolve 22.0 mg of sodium pyruvate in 100 ml of
phosphate buffer.
3) -ketoglutarate( 2mM/L), dl-aspartate( 200mM/L) for GOT substrate and standard curve :
place 29.2 mg of ketoglutaric acid and 2.66g of dl aspartic acid in a small beaker. Add 1N sodium
hydroxide until the solution is complete. Adjust to a pH of 7.40 with sodium hydroxide, transfer
quantitatively with buffer to a 100 ml volumetric flask, and then dilute to the mark with buffer
solution.
4) -ketoglutarate( 2mM/L), dl-alanine( 200mM/L) for GPT substrate and standard curve :
place 29.2 mg of ketoglutaric acid and 1.78g of dl-alanine in a small beaker. Add 1N sodium
hydroxide until the solution is complete. Adjust to a pH of 7.40 with sodium hydroxide, transfer
quantitatively with buffer to a 100 ml volumetric flask, and then dilute to the mark with buffer
solution.
5) 2,4 dinitrophenylhydrazine ( 1mM/L) : dissolve 19.8 mg of 2,4 dinitrophenylhydrazine in 100
ml of 1N HCl
6) 0.4N NaOH solution.
Procedure
1) One ml of the desired substrate is pipetted into a test tube.
* GOT substrate : -ketoglutarate, dl-aspartate
GPT substrate : -ketoglutarate, dl-alanine
2) and placed in a water-bath at constant temperature(37oC) for 10 minutes.
3) Upon the addition of 0.2 ml of serum, the contents are mixed.
4) and after an incubation period of exactly 60 min for GOT, or 30 min for GPT, the tube is removed
from the water bath.
5) One ml of 2,4-dinitrophenylhydrazine reagent is added immediately, thereby stopping the reaction.
6) After the tube is permitted to stand at room temperature for a minimum of 20 min,
7) 10 ml of 0.4N NaOH are added, a rubber stopper is inserted, and the contents are mixed by
inversion.
8) At the end of 10 min, the absorbance of the solution is measured at 505 nm, using water as the
blank.
Reference : Stanley Reitman, Sam Frankel ; A colorimetric method for the determination of serum
glutamic oxaloacetic and glutamic pyruvic transaminases, Am. J. Clin. Path., 28, 56-63, 1957.
Absorption spectrum of equimolar alkaline solution of the 2,4-dinitrophenylhydrazines of
–ketoglutarate, oxalacetate, and pyruvate.
UV-visible spectrum of Fe(III)
complex of salisylic acid.
Visible spectrum of Fe(III)
complex of salisylic acid.
Flow Injection Analysis (FIA)
In FIA, a sample is injected into a moving liquid stream to which various reagents can be
added. After suitable time, the reacted sample reaches a spectrophotometric cell detector.
(Left) Schematic diagram of FIA, showing two different reagent addition schemes.
(Right) FIA system with enlarged view of chemistry section.
A dialysis flow module.
The membrane is supported
between two grooved Teflon
blocks.
FIA apparatus for the determination of caffeine
in acetylsalicylic acid preparation.
FIA of ppb levels of H2O2 in air.