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 +