Dong-Sun Lee / cat-lab / SWU

Chapter 26 AMolecular Absorption

Spectrometry

Application of UV-Visible spectrometry

2010-Fall Version

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 UV-visible 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.

Absorption spectra of aqueous charge transfer complexes.

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.

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.

A1 = Ax,1 + Ay,1 + Az,1 …. = x1bCx + y1bCy + z1bCz ….

A2 = Ax,2 + Ay,2 + Az,2 …. = x2bCx + y2bCy + z2bCz ….

A1 y1b

Cx = A2 y2b

x1b y1b

x2b y2b

x1b A1

Cy = x2b A2

x1b y1b

x2b y2b

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.

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 inters

ection of the two straight lines.

Spectrometric end-point detection has been applied to all types of reactions. Although stepwise titration of mixtures is possible.

Spectrophotometric titrations

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 – KA

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 / d2 = f’’() d2A / d2 = (d2/d2 )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.

Structure of part of a DNA chain and its UV-vis spectrum.

max = 260 nm

DNA

ADNA = Aadenine + Aguanine + Acytocine + A thymine

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 COOH

-ketoglutarate + alanine pyruvate + glutamate

COOH COOH COOH COOH

CH2 CHNH2 C=O CH2

CH2 COOH CH3 CH2

C=O CHNH2

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.