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    Advanced Analytical Chemistry Advanced Analytical Chemistry: Dr. Carlos D Garcia

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    Tanushri Banerjee
    This document provides an overview of advanced analytical chemistry techniques, including UV-Vis spectrometry and molecular luminescence spectrometry. It discusses topics like Beer's law, applications of UV-Vis such as quantitative analysis and kinetic methods, and factors that influence fluorescence. Instrumentation for techniques like fluorescence, phosphorescence, and chemiluminescence are also covered. The document aims to introduce students to spectroscopic principles and their uses in analytical chemistry.

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    Advanced Analytical Chemistry Advanced Analytical Chemistry: Dr. Carlos D Garcia

    Uploaded by

    Tanushri Banerjee
    35 views16 pages
    This document provides an overview of advanced analytical chemistry techniques, including UV-Vis spectrometry and molecular luminescence spectrometry. It discusses topics like Beer's law, applications of UV-Vis such as quantitative analysis and kinetic methods, and factors that influence fluorescence. Instrumentation for techniques like fluorescence, phosphorescence, and chemiluminescence are also covered. The document aims to introduce students to spectroscopic principles and their uses in analytical chemistry.

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    This document provides an overview of advanced analytical chemistry techniques, including UV-Vis spectrometry and molecular luminescence spectrometry. It discusses topics like Beer's law, applications of UV-Vis such as quantitative analysis and kinetic methods, and factors that influence fluorescence. Instrumentation for techniques like fluorescence, phosphorescence, and chemiluminescence are also covered. The document aims to introduce students to spectroscopic principles and their uses in analytical chemistry.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    35 views16 pages

    Advanced Analytical Chemistry Advanced Analytical Chemistry: Dr. Carlos D Garcia

    Uploaded by

    Tanushri Banerjee
    This document provides an overview of advanced analytical chemistry techniques, including UV-Vis spectrometry and molecular luminescence spectrometry. It discusses topics like Beer's law, applications of UV-Vis such as quantitative analysis and kinetic methods, and factors that influence fluorescence. Instrumentation for techniques like fluorescence, phosphorescence, and chemiluminescence are also covered. The document aims to introduce students to spectroscopic principles and their uses in analytical chemistry.

    Copyright:

    Attribution Non-Commercial (BY-NC)
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    of 16

    Advanced Analytical Chemistry

    CHE 5263

    Dr. Carlos D Garcia


    Department of Chemistry The University of Texas at San Antonio One UTSA Circle, San Antonio, TX 78249

    http://www.utsa.edu/chem/faculty/carlosgarcia/garcia.html

    Outline
    Intro to UV-Vis Spectrometry Applications of UV-Vis

    Sample Problems

    Molecular Luminescence Spectrometry

    Introduction to UV-Vis Spectrometry


    Most relevant magnitudes

    Equations leading to Beers Law

    T =P

    PO

    A = log P

    PO

    A = .b.C

    Introduction to UV-Vis Spectrometry


    To solve mixtures you MUST remember that:

    AT = A1 + A2 + ...+ An = 1.b.C1 + 2 .b.C2 + ...+ n .b.Cn

    Apparent chemical deviations Arise when analyte dissociates, associates, or somehow reacts Leads to a product with a different and/or MAX

    Color #1

    HIn In + H +
    Color # 2

    Introduction to UV-Vis Spectrometry


    Quick problem An student is determining the [phenolphthalein] by spectrophotometry. If he dissolved the sample in a borate buffer of pH=9.3... What fraction of the sample is not measured?

    HPhen Phen + H +
    transp pink

    Ka = 2 x 10-10

    Can you demonstrate that mathematically? What pH should be used? What buffer would give you such pH?

    Introduction to UV-Vis Spectrometry


    Deviations due to polychromatic radiation Typically this is not a problem Assuming that L-Bs law is valid at both

    Am = log

    (P0' + P0" ) (P0' .10 'bC + P0" .10 "bC )

    Deviations due to stray radiation Just close the cover! Deviations due to mismatched cells CellSAMPLE CellBLANK Corrected with a simple blank

    Introduction to UV-Vis Spectrometry


    Noise as a function of Transmittance Concentration uncertainty RSD of c (Sc/c) vs ST (determined experimentally)

    c=

    0.434 lnT b

    Sc 0.434ST = c T .logT
    Classification and Sources of noise

    Introduction to UV-Vis Spectrometry


    Typical arrangement

    Applications of UV-Vis Spectrometry


    Signal = 8.7 x 10-19 P A Exitation / relaxation

    M + h M * M* M + heat

    Environment has a HUGE influence Electronic, vibrational, and rotational Electronic Electronic (smoothed) Most organic compounds will absorb light

    Applications of UV-Vis Spectrometry

    Applications of UV-Vis Spectrometry


    Typical absorption spectra Organic molecules n * (low) * (1-15K L mol-1 cm-1) Inorganic ions Transitions in the d electrons (low) Charge-Transfer Complexes Typically large

    Applications of UV-Vis Spectrometry


    Quantitative Applications The solvent selection is critical Consider solubility and compatibility with cuvettes

    Applications of UV-Vis Spectrometry


    Applications Absorbing species (this is the easy part) Non-absorbing species (derivatization) Standard addition method Mixtures Titration curves Kinetic methods Continuous variations method Mole-Ratio method Slope-Ratio method

    Applications of UV-Vis Spectrometry


    Standard Addition Method Very useful when sample is in a complex matrix Measurements done to the sample + spiked sample You want to reach 5X the initial signal
    1.000 0.900 0.800 0.700 Absorbance, A 0.600 0.500 0.400 0.300 0.200 0.100 0.000 -2.00 0.00 2.00 4.00 6.00 8.00 10.00 Volume of standard solution, mL

    (V ) C CS = S 0 S VX

    C SC = SV S V X

    Applications of UV-Vis Spectrometry


    Solving for a mixture Both species (N and M) absorb differently Should select two wavelengths (1 and 2) Perform 4 calibration curves Calculate 4 absorptivity coefficients M1 / M2 / N1 / N2 Measure the absorbance at both wavelengths (A1 and A2) A1 = AM1 + AN1 A1 = M1.b.CM + N1.b.CN A2 = AM2 + AN2 A2 = M2.b.CM + N2.b.CN

    Applications of UV-Vis Spectrometry


    Titration curves Spectrophotometry is used to determine the final point The R P

    Applications of UV-Vis Spectrometry


    Kinetic model Dynamic conditions Enzymes used to catalyze the reactions (clinical chemistry) Determination of [E]: [S] >> Km Determination of [S]: [S] << Km

    k1 k1 k 2 [E]0 [S] d[P] = k1 + k 2 dt + [S] k1

    k2

    E + S ES P + E

    k1 + k 2 = Km k1

    Applications of UV-Vis Spectrometry


    The method of Continuous Variations Used to calculate stoichiometry ratios [M+] = [L] are mixed VT and NT are constant

    Molecular Luminescence Spectrometry


    Variety of methods where molecules are exited and emit light Fluorescence Phosphorescence Chemiluminescence Electroluminescence Advantages / Disadvantages Sensitivity (LOD 103 better than absorption spectrometry) Wide linear concentration range Selectivity Limited applicability

    http://onlinelibrary.wiley.com/doi/10.1002/anie.201002974/pdf

    Molecular Luminescence Spectrometry


    Why molecules emit light? Electrons are exited by absorption of UV and then relax Quick absorption (10-14 / 10-15 s) Fluorescence (10-5 / 10-10 s) Phosphorescence (10-4 / 10 s)

    10

    Molecular Luminescence Spectrometry


    What are the most significant variables? Quantum yield (depends on the rate constants)
    kf ki kec kic kpd kd fluorescence intersystem crossing external conversion internal conversion predissociation dissociation

    kf k f + k i + k ec + k ic + k pd + k d

    Transition form the lowest vibrational level of S1 to one of the levels in S0


    Occurs at > 250 nm (as most organic molecules get dissociated at those energy levels)

    Chemical structure
    Look for aromatic electrons with low-energy * transitions Structural rigidity (the more the better)

    Temperature, solvent, and pH

    Molecular Luminescence Spectrometry

    11

    Molecular Luminescence Spectrometry


    Effect of concentration Fluorescence (F) depends on
    f
    K P0 P quantum efficiency of the process geometry and other factors power of the incident beam power of the beam after the cell (b)
    F = f K " ( P0 P )

    Constant (K)

    If we also consider the Beers law

    P P0

    = 10 bC

    F = 2.303 f K " bC P0

    Quenching
    F0 F = 1+ K d [ Q ]

    Molecular Luminescence Spectrometry


    Typical Spectra (3D representation)

    http://www.spectra-magic.de/Anwendungen/L1_Luminmescence_static_E.htm

    12

    Molecular Luminescence Spectrometry


    Instrumentation Sources (Hg, Xe, laser, LED) Monochromator Transducer Cells and components

    Molecular Luminescence Spectrometry


    Instrumental design

    13

    Molecular Luminescence Spectrometry


    Applications Inorganic species (upon the addition of a chelating agent) Not very used

    Molecular Luminescence Spectrometry


    Applications Organic species (wide number of applications) Adenine, Gianine Anthralinic acid Aromatic polycyclic hydrocarbons Nerve agents Proteins (GFP) Tryptophan (native) Can help to identify structural changes in the structure

    14

    Molecular Luminescence Spectrometry


    Chemiluminescence Simple species that release light upon reaction Lots of applications! Gases (NO, h 600-2800 nm) Sulfur compounds (SO2) Inorganic species (strong oxidants) Coupled to anything that gives H2O2

    A + B C *+ D C * C + h

    Molecular Luminescence Spectrometry


    Chemiluminescence Light produced by a biochemical reaction Requires "luciferin" and "luciferase Good detection limits (fmol)

    15

    Molecular Luminescence Spectrometry


    Fluorescence resonance energy transfer (FRET) Non-radiative energy transfer between two different molecules Exited molecule (donor) interacts with an acceptor (that emits at a ) They should be < 10 nm apart Protein-protein interactions Conformational changes Coupled to biochemical reactions (BRET)

    16

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