Spectrophotometry

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Spectrophotometry
Determining the concentration or absorbance
characteristics of solutions by using an
instrument that produces monochromatic light.
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Uses of Spectrophotometry
Concentration of Both Inorganic and Organic
Molecules
Absorption Spectra
Kinetic Assays
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Absorbance and Color
Absorbance
Concentration
http//www.scieng.flinders.edu.au/cml/cml1b.htm
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Why Does a Solution Appear Colored ?
It absorbs visible light from certain wavelengths
and reflects light from other wavelengths.
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Principles of Absorbance
Beer - Lambert Law log10
(I0 / I) A ? l C I0 incident radiation, I
radiation transmitted A absorbance ?
extinction coefficient (units of the molar
absorptivity are M-1 cm-1) at a given ?. l
pathlength in cm. C concentration, M
http//www.wellesley.edu/Biology/Concepts/absorpti
onspectrophotomet.html and Wharton and McCarty,
1972
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Absorbance of an NADH Solution
Extinction Coefficient at 340 nm 6.220 mM-1
cm-1 At 0.1 mM NADH Absorbance 6.220 mM-1 cm-1
0.1 1 cm 0.622.
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Standard Curve
Terms to know Standard A solution of known
concentration prepared from a chemical
preparation. Sample A solution prepared from the
tissue we are studying.
We need to generate a mathematical relationship
between the absorbance of the solution and the
concentration of the substance we are trying to
measure. The relationship is called the standard
curve.
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Determining the Zero Point
Both the solvents used to dissolve the substance
and the cuvette used to hold the solution absorb
and reflect light. Therefore, to determine the
absorbance of a solution we must compare its
absorbance relative to a blank solution. The
transmittance / absorbance of this blank is
called the zero.
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Measurement Goals
Our standards should mimic our samples as close
as possible. We try to achieve a linear
relationship between absorbance and
concentration. We also try to have the samples
fall (i.e., have absorbances) between our lowest
standard and a higher standard. Samples falling
between our zero and the first standard are not
as reliable.
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A Standard Curve
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Interference
Many substances in the solution (both the
standards and samples) are likely to absorb at
the wavelength(s) being used.
If the interfering substances are equally present
in the standards and samples, then the problem is
minimized. If only the samples have the
interfering compound or a few samples have the
interfering compound, then a problem exists.
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How to Deal with Interference
Sample purification. Centrifugation or
filtration is often used to deal with particulate
interference.
To handle chemical interference, selected cleanup
procedures are used.
Dual wavelengths are often used to subtract out
interfering substances. For example, measurement
at 280 nm and 260 nm are used to account for
protein interference of DNA measurements.
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Instrumentation
Spectrophotometer Contains 1. Radiation Source -
H, De, W 2. Monochromator - often a quartz prism
transparent to ? gt 180 3. Radiation Detector -
photometer
Colorimeter - uses colored filters
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Sources of Light
Hydrogen or Deuterium - UV Lamp, 180 nm to 340 nm
Tungsten - Visible Lamp 340 nm to 1000 nm
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Potential Problems
Slit Width - If too wide, spectral purity is
compromised.
Calibration - If wavelength listed is not actual
wavelength, errors result.
Stray Light and Turbidity
Deviations from Beers Law. Best to stay
between 0.1 and 1.0 absorbance.
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Cuvettes
Below 330 nm, silica or quartz cuvettes are
required.
If cuvettes are mismatched, measurement errors
may result.
Standard volume is 3 ml but some are designed for
0.1 to 1.0 ml.
Microplates are used when volumes are below 200
?L.
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Absorption Spectra
Absorbance as a Function of Wavelength, ?
The energy or wavelength required for electronic
excitation is unique for each chromophore.
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Light Absorption By Pigments
400
500
600
700
Wavelength (nm)
400
500
600
700
Wavelength (nm)