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  1. Dong - Sun Lee / cat - lab / SWU 2010-Fall Version Chapter 26 A Molecular Absorption Spectrometry Application of UV-Visible spectrometry

  2. 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.

  3. UV-visible spectra of benzene, naphthalene, and anthracene in ethyl alcohol. Absorption spectra for typical organic compounds.

  4. 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.

  5. Absorption spectra of aqueous solutions of transition metal ions. Absorption spectra of aqueous solutions of rare earth ions.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Experimental curves relating relative concentration uncertainties to absorbance for two spectrometer.

  12. 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

  13. 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 …. A1 y1b Cx = A2y2b x1b y1b x2b y2b x1b A1 Cy = x2b A2 x1b y1b x2b y2b

  14. Two cases for analysis of a mixture. • Spectra of the pure components have substantial overlap. • Regions exist in which each component makes the major contribution.

  15. Visible spectrum of MnO4–, Cr2O72– , and an unknown mixture containing both ions.

  16. 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 = HInb[HIn] + In– b [In–] Absorption spectra of 0.37 mM methyl red as a function of pH between pH 4.5 and 7.1.

  17. 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.

  18. Typical photometric titration curves. Molar absorptivities of analyte titrated, product, and the titrant are A, P, T, respectively.

  19. 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.

  20. 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.

  21. 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.

  22. 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 containingM 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

  23. 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

  24. 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.

  25. 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 negativeor positive band, with the minimum or maximum at the same wavelength as maxof 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.

  26. First to fourth derivatives of an 500 nm Gaussian band.

  27. Resolution of two overlapping bands in the fourth derivative.

  28. Baseline shift elimination and suppression of scattering using the first derivative.

  29. max = 260 nm DNA ADNA = Aadenine + Aguanine + Acytocine + Athymine Structure of part of a DNA chain and its UV-vis spectrum.

  30.  max = 266 nm PTH-Glu The Edman method of NH2-terminal amino acid analysis A  max = 265 nm PTH-Asp dA/d  (nm)

  31. 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

  32. 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

  33. 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.

  34. 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.

  35. Absorption spectrum of equimolar alkaline solution of the 2,4-dinitrophenylhydrazines of –ketoglutarate, oxalacetate, and pyruvate.

  36. UV-visible spectrum of Fe(III) complex of salisylic acid. Visible spectrum of Fe(III) complex of salisylic acid.

  37. 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.

  38. (Left) Schematic diagram of FIA, showing two different reagent addition schemes. (Right) FIA system with enlarged view of chemistry section.

  39. A dialysis flow module. The membrane is supported between two grooved Teflon blocks. FIA apparatus for the determination of caffeine in acetylsalicylic acid preparation.

  40. FIA of ppb levels of H2O2 in air.