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UV-visible spectroscopy How They Work. What is Spectroscopy? The study of molecular structure and...
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Transcript of UV-visible spectroscopy How They Work. What is Spectroscopy? The study of molecular structure and...
![Page 1: UV-visible spectroscopy How They Work. What is Spectroscopy? The study of molecular structure and dynamics through the absorption, emission and scattering.](https://reader036.fdocuments.us/reader036/viewer/2022062421/56649d045503460f949d780c/html5/thumbnails/1.jpg)
UV-visible spectroscopy
How They Work
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What is Spectroscopy?
• The study of molecular structure and dynamics through the absorption, emission and scattering of light.
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What is Light?
• According to Maxwell, light is an electromagnetic field characterized by a frequency f, velocity v, and wavelength λ. Light obeys the relationship
f = v / λ.
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The Electromagnetic Spectrum
= c / E = h
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Spectroscopy
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)
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Transmission and Color
The human eye sees the complementary color to that which is absorbed
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Absorbance and Complementary Colors
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Two-Component Mixture
Example of a two-component mixture with little spectral overlap
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Two-Component Mixture
Example of a two-component mixture with significant spectral overlap
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Influence of 10% Random Error
Influence on the calculated concentrations• Little spectral overlap: 10% Error• Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%)
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Absorption Spectra of Hemoglobin Derivatives
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Light Sources
UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
InfraRed (IR) Spectrophotometer
1. Carborundum (SIC)
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Dispersion Devices
• Non-linear dispersion• Temperature sensitive
• Linear Dispersion• Different orders
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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.
Polychromatic Ray
Infrared
RedOrange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic Ray
SLIT
PRISM
Polychromatic Ray
Monochromatic Ray
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Photomultiplier Tube Detector
Anode
• High sensitivity at low light levels• Cathode material determines spectral sensitivity• Good signal/noise• Shock sensitive
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The Photodiode Detector
• Wide dynamic range• Very good
signal/noise at high
light levels• Solid-state device
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Schematic Diagram of a Photodiode Array
• Same characteristics as photodiodes• Solid-state device• Fast read-out cycles
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Conventional Spectrophotometer
Schematic of a conventional single-beam spectrophotometer
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Conventional Spectrophotometer
Optical system of a double-beam spectrophotometer
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Conventional Spectrophotometer
Optical system of a split-beam spectrophotometer
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Definition of Resolution
Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths
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Instrumental Spectral Bandwidth
The SBW is defined as the width, at half the maximum intensity, of the band of light leaving the monochromator
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Natural Spectral Bandwidth
The NBW is the width of the sample absorption band at half the absorption maximum
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Transmission Characteristics of Cell Materials
Note that all materials exhibit at least approximately 10% loss in transmittance at all wavelengths
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Cells
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer
NaCl
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Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume
Cell Types I
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Cell Types II
Micro cell (a) for very small volumes and flow-through cell (b) for automated applications
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Transmittance and Concentration The Bouguer-Lambert Law
PathlengthConsteIIT 0/
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Transmittance and Path Length: Beer’s Law
ionConcentratConsteIIT 0/
Concentration
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The Beer-Bouguer-Lambert Law
cbIIIITA /log/loglog 00
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BEER LAMBERT LAW
Glass cell filled with concentration of solution (C)
IILight
0
As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.
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R- Transmittance
R = 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).
I
I0
I
I0
I0
I
1
T
I
I0
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A CL = KCL by definition and it is called the Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of solute per liter of solution
A = ECL
E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution
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E =Absorbance x Liter
Moles x cm
E differs from K (Specific extinction Coefficient) by a factor of molecular weight.
UNITS
A = ECL
A = No unit (numerical number only)
E =
Liter
Cm x Mole
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L = Cm
C = Moles/Liter
A = KCL
A = No unit C = Gram/Liter L = Cm
A = ECL = (Liter
Cm x Mole) x
Mole
Literx Cm
K=Liter
Cm Gram
A = KLC = (Liter
Cm x Gram
Gram
Literx Cm) x
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STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHOD
1. Run the sample for spectrum
2. Obtain a monochromatic wavelength for the maximum absorption wavelength.
3. Calculate the concentration of your sample using Beer Lambert Equation: A = KCL
Wavelength (nm)
Absorbance
0.0
2.0
200 250 300 350 400 450
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Slope of Standard Curve = AC
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
Absorbance at 280 nm
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where becomes non-linear.
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SPECTROMETRIC ANALYSIS USING STANDARD CURVE
1 2 3 4
0.4
0.8
1.2Absorbance at 540 nm
Concentration (g/l) glucose
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
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• 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.
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Make a dilution series of a known quantity of analyte and measure the Absorbance. Plot concentrations v. Absorbance.
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What concentration do you think the unknown sample is?
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In this graph, values above A=1.0 are not linear. If we use readings above A=1.0, graph isn’t accurate.
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The best range of this spectrophotometer is A=0.1 to A=1.0, because of lower errors. A=0.4 is best.
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Relating Absorbance and Transmittance• Absorbance 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)
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Precision and Accuracy
Precision – Precision + Precision – Precision +
Accuracy – Accuracy – Accuracy + Accuracy +