UV-visible spectroscopy

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UV-visible spectroscopy. How They Work. What is Spectroscopy?. The study of molecular structure and dynamics through the absorption, emission and scattering of light. What is Light?. - PowerPoint PPT Presentation


  • UV-visible spectroscopyHow They Work

  • What is Spectroscopy?The study of molecular structure and dynamics through the absorption, emission and scattering of light.

  • What is Light?According to Maxwell, light is an electromagnetic field characterized by a frequency f, velocity v, and wavelength . Light obeys the relationship

    f = v / .

  • The Electromagnetic Spectrumn = c / lE = hn

  • SpectroscopySpectral Distribution of Radiant EnergyWave Number (cycles/cm)

  • Transmission and ColorThe human eye sees the complementary color to that which is absorbed

  • Absorbance and Complementary Colors

  • Two-Component MixtureExample of a two-component mixture with little spectral overlap

  • Two-Component MixtureExample of a two-component mixture with significant spectral overlap

  • Influence of 10% Random ErrorInfluence on the calculated concentrations Little spectral overlap: 10% Error Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%)

  • Absorption Spectra of Hemoglobin Derivatives

  • Light Sources UV Spectrophotometer1.Hydrogen Gas Lamp2.Mercury LampVisible Spectrophotometer1.Tungsten LampInfraRed (IR) Spectrophotometer1.Carborundum (SIC)

  • Dispersion Devices Non-linear dispersion Temperature sensitive Linear Dispersion Different orders

  • Dispersion of polychromatic light with a prism Prism - spray out the spectrum and choose the certain wavelength (l) that you want by moving the slit.

  • Photomultiplier Tube DetectorAnode High sensitivity at low light levels Cathode material determines spectral sensitivity Good signal/noise Shock sensitive

  • The Photodiode Detector Wide dynamic range Very good signal/noise at high light levels Solid-state device

  • Schematic Diagram of a Photodiode Array Same characteristics as photodiodes Solid-state device Fast read-out cycles

  • Conventional SpectrophotometerSchematic of a conventional single-beam spectrophotometer

  • Conventional SpectrophotometerOptical system of a double-beam spectrophotometer

  • Conventional SpectrophotometerOptical system of a split-beam spectrophotometer

  • Definition of ResolutionSpectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths

  • Instrumental Spectral BandwidthThe SBW is defined as the width, at half the maximum intensity, of the band of light leaving the monochromator

  • Natural Spectral BandwidthThe NBW is the width of the sample absorption band at half the absorption maximum

  • Transmission Characteristics of Cell MaterialsNote that all materials exhibit at least approximately 10% loss in transmittance at all wavelengths

  • Cells UV SpectrophotometerQuartz (crystalline silica)Visible SpectrophotometerGlassIR SpectrophotometerNaCl

  • Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volumeCell Types I

  • Cell Types IIMicro cell (a) for very small volumes and flow-through cell (b) for automated applications

  • Transmittance and Concentration The Bouguer-Lambert Law

  • Transmittance and Path Length: Beers LawConcentration

  • The Beer-Bouguer-Lambert Law

  • BEER LAMBERT LAW As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.

  • R- TransmittanceR = I0 - original light intensity I- transmitted light intensity% Transmittance = 100 x Absorbance (A) or optical density (OD) = Log = Log = 2 - Log%T Log is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution).

  • A CL = KCL by definition and it is called the Beer Lambert Law.A = KCLK = Specific Extinction Coefficient ---- 1 g of solute per liter of solutionA = ECLE =Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution

  • E differs from K (Specific extinction Coefficient) by a factor of molecular weight.

    UNITS A = ECL A = No unit (numerical number only)

  • L = Cm C = Moles/Liter

    A = KCLA = No unit C = Gram/LiterL = Cm

  • STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHODRun the sample for spectrum2.Obtain a monochromatic wavelength for the maximum absorption wavelength.3.Calculate the concentration of your sample using Beer Lambert Equation: A = KCL

  • There is some A vs. C where graph is linear.NEVER extrapolate beyond point known where becomes non-linear.

  • SPECTROMETRIC ANALYSIS USING STANDARD CURVE Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

  • Every instrument has a useful range for a particular analyte. Often, you must determine that range experimentally. This is done by making a dilution series of the known solution. These dilutions are used to make a working curve.

  • Make a dilution series of a known quantity of analyte and measure the Absorbance. Plot concentrations v. Absorbance.

  • What concentration do you think the unknown sample is?

  • In this graph, values above A=1.0 are not linear. If we use readings above A=1.0, graph isnt accurate.

  • The best range of this spectrophotometer is A=0.1 to A=1.0, because of lower errors. A=0.4 is best.

  • Relating Absorbance and TransmittanceAbsorbance rises linearly with concentration. Absorbance is measured in units.Transmittance decreases in a non-linear fashion. Transmittance is measured as a %.Absorbance = log10 (100/% transmittance)

  • Precision and AccuracyPrecision Precision +Precision Precision +Accuracy Accuracy Accuracy +Accuracy +