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Transcript of 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?
• 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.
The Electromagnetic Spectrum
= c / E = h
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
Transmission and Color
The human eye sees the complementary color to that which is absorbed
Absorbance and Complementary Colors
Example of a two-component mixture with little spectral overlap
Example of a two-component mixture with significant spectral overlap
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%)
Absorption Spectra of Hemoglobin Derivatives
1. Hydrogen Gas Lamp
2. Mercury Lamp
1. Tungsten Lamp
InfraRed (IR) Spectrophotometer
1. Carborundum (SIC)
• 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 Detector
• 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
Schematic of a conventional single-beam spectrophotometer
Optical system of a double-beam spectrophotometer
Optical system of a split-beam spectrophotometer
Definition of Resolution
Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths
Instrumental Spectral Bandwidth
The SBW is defined as the width, at half the maximum intensity, of the band of light leaving the monochromator
Natural Spectral Bandwidth
The NBW is the width of the sample absorption band at half the absorption maximum
Transmission Characteristics of Cell Materials
Note that all materials exhibit at least approximately 10% loss in transmittance at all wavelengths
Quartz (crystalline silica)
Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume
Cell Types I
Cell Types II
Micro 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: Beer’s Law
The Beer-Bouguer-Lambert Law
cbIIIITA /log/loglog 00
BEER LAMBERT LAW
Glass cell filled with concentration of solution (C)
As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.
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).
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
E =Absorbance x Liter
Moles x cm
E differs from K (Specific extinction Coefficient) by a factor of molecular weight.
A = ECL
A = No unit (numerical number only)
Cm x Mole
L = Cm
C = Moles/Liter
A = KCL
A = No unit C = Gram/Liter L = Cm
A = ECL = (Liter
Cm x Mole) x
A = KLC = (Liter
Cm x Gram
Literx Cm) x
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
200 250 300 350 400 450
Slope of Standard Curve = AC
1 2 3 4 5
Absorbance at 280 nm
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where becomes non-linear.
SPECTROMETRIC ANALYSIS USING STANDARD CURVE
1 2 3 4
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
• 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 isn’t 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 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)
Precision and Accuracy
Precision – Precision + Precision – Precision +
Accuracy – Accuracy – Accuracy + Accuracy +