FORS-2107EL Study Guide - Quiz Guide: Statistical Hypothesis Testing, Morphine, Cardiac Arrhythmia
27 Jun 2018
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FORS-2107 - Quiz 2 Review
Spectroscopy
Definitions and Terminology
-Sensitivity: how much detection the method is capable of when there is a change in
concentration in a specific analyte
-Limit of detection (LOD): the lowest concentration of a compound that can be
detected and differentiated from all the other components present in the compound
(matrix), this is an actual measurable amount
-Selectivity: the ability of the method to discriminate between two different atoms/ions/
compounds
-Sensitivity and selectivity may be interdependent, therefore increasing the selectivity
of a method may lead to reduced background signal which increases sensitivity
-Spectroscopy typically involves the measurement of the interaction between matter
and radiation (ex. light)
Types of Optical Spectroscopy
-UV-Vis absorbance spectroscopy
-IR absorbance spectroscopy (IR)
-Atomic absorption spectroscopy (AAS)
-Fluorescence spectroscopy
UV-Vis absorbance spectroscopy
Absorbance
-Absorbance: absorption of photons released, that are of a wavelength within the UV
and visible regions of spectrum, which will involve promotion of electrons to higher
energy levels
-The electron is promoted, then it goes back down to a lower level, it will release
energy which can be measured, it releases energy by having collisions with near by
particles and heat
-Sensitivity and selectivity are governed by choice of wavelength
-Absorption may be quantitatively characterized by Beer’s Law:
-Absorbance: A(λ) = ƐbC
-Where
-A = absorbance
-Ɛ = molar absorptivity (L / mol*cm) at a given wavelength
-b = length of path that light (cm)
-C = concentration of sol’n being analyzed (mol/L)
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Absorbance Spectra
-The graph on the left is the descriptor, shown in
wavelengths vs absorbance
-From the left graph we can calculate Ɛ (molar
absorptivity), since C (concentration) and b (length of
path) will be constant
-On the graph on the right, A(λ1) is more sensitive than
that at λ2 since sensitivity is the slope of the standard
curve, therefore λ1 will have a larger sensitivity due to larger slope
Absorption Spectroscopy Instrumentation
-Overall, we need to ensure that light of a single
wavelength only, passes through the sample, with
reasonable intensity, and reaches the detector for
intensity measurement, so we can use a diffraction
grading to choose what wavelength hits the source
-This requires
-Light source (laser, continuum source)
-Wavelength isolation (grating, prism)
-Focusing Optics (mirrors, lenses)
-Something that will stop the dispersion of light
so it remains in the box we are doing the experiment on
-Detector
-Advantages
-We can find the absorption spectrum, which are (unique) characteristics of the
compound we are analyzing
-Technology widely available
-May be used as detection scheme in chromatography which will improve selectivity
- Disadvantages
-Multiple species may absorb at a given wavelength - so selectivity can be low
- Ɛ (molar absorptivity) is highly environmentally sensitive
-Matrix effects, therefore other components in the solution, may be problematic
Infrared Absorbance Spectroscopy (IR)
-Infrared (IR) spectroscopy is a form of absorbance spectroscopy which makes use of
infrared radiation as the incident light used
-Unlike UV-visible spectrophotometry, absorption of IR radiation excites vibrational
energy levels of absorbing molecules, so when electrons come down from excited
state, they will release less energy from collisions (Absorption) and more in
vibrational energy
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-The different functional groups and bonds in a molecule will have different vibrational
energies, so each chemical structure has a characteristic IR spectrum, therefore this
is a descriptor for the compound
-IR spectrum is typically plotted as transmittance (absorbance) vs wavelength where
wavelength is plotted as 1/ wavelength to give numerical values instead of a
frequency to make the graph more convenient to analyze, whereas UV-visible
absorbance spectrum is plotted absorbance vs wavelength
There are two important regions to IR spectroscopy graphs:
-A group frequency region gives you bands that
are characteristic to particular functional groups
-The fingerprint regions contains features that will
tell you about characteristics of the connection
(bonds) of the molecules between the functional
groups
-Advantages
-Compounds have characteristic (identifiers) IR
spectra, these spectra may be useful in identification of substances
-Since IR is a form of absorption spectroscopy, so through beer’s law is possible (A
= epsilon*b*C)
-Disadvantages
-Accuracy and precision may be lower then that encountered with UV-vis
techniques
Atomic Absorption Spectroscopy (AAS)
-In some cases, knowledge of the elemental composition
(relative) of a sample is required, here we can use atomic
spectroscopy
-Incident light made up of the emission spectrum of the element
is used as the source of light
-Absorbance of the incident light by the atoms in the flame
proceeds in a similar fashion to molecular absorbance, governed by beer’s law
Fluorescence
-Fluorescence is a form of luminescence which occurs when an electron is excited
through photon absorption, and when it comes down from the excited state, the
energy is release through
-emission of a photon, and collision, and heat
-Here λemitted > λabsorbed (stokes shift) so light emitted is greater then light absorbed,
due to photon released
-Things that are not fluorescent release all their excitation energy through heat, so if
heat is added then we can reduce the fluorescence released
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