Quiz 2 Material Flashcards
1
Q
Define spectroscopy.
A
The study of the interaction (absorption &/or emission) of electromagnetic radiation with matter
2
Q
How do spectroscopy methods differ?
A
-
Region of spectrum
- UV
- visible
- infrared
- microwave
- radio
-
Type of radiation-matter interactions
- Absorption
- Emission
-
What is analyzed
- Molecule
- Atom
3
Q
Describe light as a wave. [5]
A
- Wavelength, λ → the distance between successive maxima
- Amplitude → magnitude of the electric vector at the wave maxima
- Frequency, v → # of occurrences per unit time e.g., cycles per second
- Wavenumber, ṽ → wavelengths per unit length
- Wavelength and frequency are related by the speed of light, which we treat as a constant, c = 3x108m/s
4
Q
Describe waves of light & interference.
A
- Maximum constructive interference (a) waves are in phase → amplitude = 2A
- Interference (b) 90° out of phase → amplitude = 1.4A
- Minimum destructive interference (c ) → waves 180° out of phase → amplitude = 0
5
Q
Describe light as wavelike particles.
A
6
Q
What is photon flux?
A
7
Q
Describe the properties of light. [8]
A
7
Q
Describe the properties of light. [8]
A
8
Q
Energy is proportional to frequency (and wavenumber)
True or False?
A
True.
9
Q
Energy is inversely related to wavelength.
True or False?
A
True.
10
Q
Energy is inversely related to frequency (and wavenumber).
True or False?
A
False.
Energy is proportional to frequency (and wavenumber).
11
Q
Energy is proportional to wavelength.
True or False?
A
False.
Energy is inversely related to wavelength.
12
Q
Describe the relationship between energy, frequency, and wavelength.
A
- Energy is proportional to frequency (and wavenumber).
- Energy is inversely related to wavelength.
13
Q
Discuss the energy of visible light.
A
- Mostly food analysis focuses on ultraviolet, visible, and infrared.
14
Q
Describe transitions used for quantization of energy. [4]
A
- A given transition corresponds to a certain energy.
- Transitions:
- Translational
- Rotational
- Vibrational
- Electronic
- A photon of a particular energy causes a given transition.
15
Q
Describe how the internal energy of a molecule or atom varies.
A
- atoms and molecules exist predominantly in their ground state
- a species struck by a photon may absorb the photon
- the species energy is increased by an amount equal to the photon energy, hv
- the internal energy of a molecule or atom varies in a series of discrete steps
- “The set of available energy levels for any given atom or molecule will be distinct for that species.”
16
Q
When increasing energy, what are the transitions that occur?
A
- Only electronic transitions occur when considering the transitions of atoms.
- When considering molecules, all three types of transitions are important.
17
Q
What wavelength of light is associated with electronic transitions?
A
UV-VIS light (180 - 750nm)
18
Q
What wavelength of light is associated with vibrational energy?
A
IR light (0.78 - 300μm)
19
Q
What wavelength of light is associated with rotational energy?
A
Microwave light (0.75 - 3.75mm)
20
Q
Describe the wavelength regions, spectroscopic methods, and associated transitions. [7]
A
21
Q
Describe absorption of energy in spectroscopy.
A
- Energy from a photon of electromagnetic radiation is transferred to the molecule or atom.
- Molecule/atom goes from the ground state to an excited state.
- An absorption spectrum characteristic for a particular molecule/atom
22
Q
Why do we see continuous spectra, and not discrete bands in spectroscopy?
A
Smearing
23
Q
What is the source of nonbonding valence electrons?
A
N, O, S, P
24
What is the source of pi(bonding)-electrons?
Double, triple bonds
25
What is a pigment?
Molecules that absorb VIS light
26
UV-VIS light has enough energy to cause […].
Outer shell electronic transitions
27
Conjugation lowers excitation energy (longer wavelength).
True or False?
True.
* electron delocalization lowers the required excitation energy
* absorption shifts +30nm for each additional conjugated double bonds
* Generally a system of greater than or equal to 7 conjugated double bonds will absorb VIS (e.g., flavonoids and carotenoids)
28
Conjugation increases excitation energy (shorter wavelength).
True or False?
False.
* electron delocalization lowers the required excitation energy
* absorption shifts +30nm for each additional conjugated double bonds
* Generally a system of greater than or equal to 7 conjugated double bonds will absorb VIS (e.g., flavonoids and carotenoids)
29
Describe the UV absorption of proteins.
* Below 200nm, the backbone of the protein is being excited
* Higher wavelengths are due to the side chains of the protein being excited.
30
Describe the absorption of chlorophyll (a pigment; highly conjugated system).
* Pigments absorb in the visible region.
31
What is fluorescence?
Absorption + Emission = fluorescence
Fluorescence = radiative decay (emitted photon = lower E than absorbed photon)
32
Describe the population of states.
Boltzmann Distribution → the population of a state decreases exponentially with the energy of the state.
33
Which phenomena associated with light are most readily explained by considering the wave nature of light? [3]
* Interference, diffraction, and refraction.
* The phenomena is associated with _light propagation_ → includes refraction, diffraction, and interferences, all of which are important in spectroscopy.
34
Which phenomena associated with light are most readily explained by the particle nature of light? Explain these phenomena based on your understanding of the quantum nature of electromagnetic radiation.
* Interaction of light with matter, which is the basis of absorption and emission spectroscopy
* Potential energy spacing between allowed internal energy levels are characteristic of a species (= qualitative ‘fingerprint’)
* The phenomena associated with the interaction of light with matter, absorption being the most important for spectroscopy.
* Experiments related to the photoelectric effect illustrate that the light is quantized. Absorption is a simple capture of this bundle of energy, which must match the energy differences in the molecule/atom.
35
What does it mean to say that the energy content of matter is quantized?
* Potential or internal energy of an atom or molecule does not vary in a continuous manner but rather in a series of discrete steps.
* The quantum nature of atoms and molecules puts limitations on the energy levels that are available to them.
* The energy levels of matter are not continuous. For any given matter there are specific energy levels in which it can reside. If plotted, the energy levels would have a digital type readout rather than an analogue type readout.
36
Molecular absorption of radiation in the **UV-VIS range** results in transitions between what types of energy levels?
Electronic energy levels
Higher energy levels compared to IR range.
37
Molecular absorption of radiation in the **IR range** results in transitions between what types of energy levels?
Vibrational energy levels
Lower energy level compared to UV-VIS.
38
How do the allowed energy levels of molecules differ from those of atoms? Answer with respect to the diagram.
* Atoms → only electronic transitions
* Molecules → all three transitions are relevant
* Molecules will have many more energy levels due to the vibrational and rotational energy levels that occur in multi-atom structures.
* Atoms will have only electronic levels. Within each electronic energy level, molecules will have their vibrational and rotational energy levels.
39
In fluorescence spectroscopy, why is the length of the emitted radiation longer than the wavelength of radiation used for excitation of the analyte?
* In most cases, only a fraction of the energy difference between the excited and ground states is lost in the emission process.
* The other fraction of excess energy is dissipated as heat during vibrational relaxation.
* The excited species undergoes vibrational energy relaxation down to the lowest vibrational energy level within the excited electronic state, and then undergoes a transition to the ground electronic state through the emission of a photon.
* The photon emitted will have an energy that equals the energy difference between the lowest vibrational level of the excited electronic state and the ground electronic state level it descends to.
* The fluorescing molecule may descend to any of the vibrational levels within the ground electronic state.
* The wavelength of the emitted light is longer because typically some of the energy associated with the absorbed light is dissipated as heat (vibrational relaxation) and thus, the emitted light is of lower energy. The lower energy associated with the photons of the emitted light means that the corresponding light waves are of longer wavelength
* E = hv = hc/wavelength
40
What are the wavelength limits of Ultraviolet?
10 - 380 nm
41
What are the wavelength limits of visible light?
380 - 750 nm
42
What types of spectroscopy can be used with ultraviolet?
Absorption
Emission
Fluorescence
43
What types of spectroscopy can be used with visible light?
Absorption
Emission
Fluorescence
44
What types of transitions in chemical systems with similar energies are there in UV-VIS?
Outer-shell electrons in atoms, bonding electrons in molecules.
45
What is the principle of UV-VIS spectroscopy?
What is an inconvenience?
What is an advantage?
1. pass a beam of photons through a sample
2. measure the amount of light that is transmitted (or absorbed)
3. relate the %T (or %Abs) to [analyte]
* Inconvenience → T is NOT linear with concentration
* Advantage → A is linear with concentration (*within limits*)
46
How is transmittance, T calculated?
47
How is absorbance, A calculated?
48
What are assumptions associated with UV-VIS spectroscopy?
What is a solution to this problem?
**A = log(P0/P) = alc**
* **Assumption** → Attenuation of beam solely due to absorption by sample = not completely valid! (i.e., light scattering, light reflection)
* **Solution** → measure the light exiting a **_reference sample_** (e.g., cuvette + solution, no analyte); use this as P0; ‘zeroing’ the instrument
49
In UV-VIS spectroscopy, discuss deviations from Beer-Lambert Law [3].
* **Analyte concentration \> 10mM**
* Intermolecular distances decrease
* Crowding
* Electrostatic interactions
* **Chemical processes**
* Reversible association/dissociation of molecules
* Ionization (unbuffered system)
* **Polychromatic light**
* Absorptivity is defined at a specific wavelength (monochromatic light)
* Multiple wavelengths = different absorption
50
Describe sample preparation for UV-VIS spectroscopy.
* Homogenization and clarification (e.g., centrifuge, filter)
* Chemical modification → absorb at desirable wavelength range
* Reference/blank sample = similar preparation/modifications w/o analyte
* Sample cell:
* Quartz cuvette (UV, VIS)
* Plastic (UV-VIS), 23-900nm
* Fused silica (UV)
* Silicate glass (VIS)
* Plastic (VIS)
* Match the application (wavelength range) with the correct cuvette material)
51
Describe why wavelength selection is important in UV-VIS spectroscopy. [2]
Maximize sensitivity
Greater adherence to Beer's Law (less variation in absorptivity)
52
Describe calibration of UV-VIS spectroscopy instruments.
* 0% transmittance → block the detector
* 100% transmittance → measure the reference cell (set as P0)
53
Describe calibration curves in UV-VIS spectroscopy.
* **Linear** → obey Beer's Law
* **Non-linear** → due to concentration-dependent changes in chemistry of system (change in absorbance per unit change in concentration is _not_ constant → due to limitations in instrument
54
Discuss relative error in UV-VIS spectroscopy.
* Get lower relative errors with measurements at intermediate transmittance (%T = 15-65%; A = 0.2-0.8)
* Relative errors will be larger outside this range.
55
Describe the light source & features in UV-VIS spectroscopy instrumentation.
56
Describe the wavelength isolator/dispersion element of a UV-VIS spectroscopy instrument.
57
Describe the typical phototube design of a UV-VIS detector.
58
Describe double-beam spectrometer in UV-VIS spectroscopy.
* Measure reference and sample simultaneously
* Minimize errors due to drift/light fluctuations
* But, also lowers the intensity (Beam sharing)
59
Which compounds absorb 200-800nm?
60
What compounds give fluorescence?
Describe properties of fluorescent molecules. [5]
* Generally molecules that fluoresce are **highly conjugated** systems.
* Properties include:
* Maximum excitation and maximum emission wavelengths
* Stokes shift (λEMISSION − λEXCITATION)
* Extinction coefficient
* Quantum yield (conversion of absorption → emission)
* Lifetime (duration of excited state)
61
Describe fluorescence spectroscopy instrumentation and its differences from UV-VIS absorption spectrophotometer.
* Two wavelength isolators (for excitation and emitted light)
* Detector is 90° with regard to light source
62
Which is more sensitive?
Fluorescence or UV-VIS absorption spectroscopy?
Fluorescence is 10-1000x more sensitive.
63
Compare cuvettes - absorption vs. fluorescence.
64
Describe fluorescence spectroscopy applied to concentration.
65
Describe fluorescence applied to protein unfolding.
66
Describe fluorescent dye-binding experiments.
* Exposed non-polar surface area → increase in fluorescence means that there is binding to the protein because there are some hydrophobic surfaces exposed
* Amyloid fibrils → increase in fluorescence due to dye binding to beta-sheet structures of the amyloid fibrils
67
Describe how fluorescence can be used to determine egg freshness.
68
Describe how UV-VIS may be used for quantitative & qualitative measurements.
* **Qualitative**
* Determine the presence of particular analytes
* Comparison of samples based on relative changes in amplitude
* **Quantitative**
* Determine the amount of analyte in the sample
* Relate intensity to [analyte] using a known factor
69
Why is it common to use absorbance values rather than transmittance values when doing quantitative UV-VIS spectroscopy?
* T and %T are not directly proportional to the concentration of the absorbing analyte in the sample solution (i.e., nonlinear relationship). Under appropriate conditions, absorbance is directly proportional (i.e., linear relationship) to the concentration.
70
For a particular assay, your plot of absorbance vs. concentration is not linear. Explain possible reasons for this.
* Analyte concentration may be too great, so its absorptivity is altered → Beer's Law generally is only valid for relatively dilute samples.
* There may be reversible association-dissociation of analyte molecules or the ionization of a weak acid in an unbuffered solvent. Since different forms of the analyte have different absorptivities , the linear relationship may not hold.
* Radiation passing through the sample may be polychromatic (vs. monochromatic)
71
What criteria should be used to choose an appropriate wavelength at which to make absorbance measurements, and why is that choice so important?
* Choose the wavelength at which the analyte demonstrates maximum absorbance, and where absorbance does not change rapidly with changes in wavelength, if possible. This provides maximum sensitivity (i.e., absorbance change per unit change in analyte concentration), and greater adherence to Beer's law.
72
In a particular assay, the absorbance reading on the spectrophotometer is 2.033 and for another sample is 0.032. Would you trust these values? Why or why not?
* The 2.033 value is too high and the 0.032 value is too low to be ideal for good precision. Ideally use range of ~0.2 to 0.8 absorbance units (maybe up to 1.0 to 1.5 absorbance units). With too high or too low of values, relative concentration uncertainty is too high.
73
Explain the differences between electromagnetic radiation in the UV and VIS ranges. How does quantitative spectroscopy using the UV range differ from that using the VIS range?
* UV range ~160nm through 375nm → use quartz sample holders (because glass absorbs radiation below 350nm)
* Vis range ~380 nm through 720 nm
* Other light sources, such as Xenon lamps, which can cover UV-VIS (and even IR). Also, quartz cuvettes can be used in UV and VIS. So, if you have the correct lamp and a quartz cuvette, there really is no difference between UV and VIS, other than **wavelength**.
74
What is actually happening inside the spectrophotometer when the analyst ‘sets’ the wavelength for a particular assay?
* You are adjusting the monochromator to set the wavelength
* A monochromator disperses radiation, so radiation of a specific wavelength can be made to exit the monochromator and be directed to the sample.
* A monochromator consists of entrance and exit slits, concave mirrors, and a dispersing element.
* It is necessary to obtain monochromatic radiation that passes through the sample.
* Beer's law, which relates absorbance to concentration, holds only for monochromatic radiation
75
Considering a typical spectrophotometer, what is the effect of decreasing the exit slit width of the monochromator on the light incident to the sample?
* Light incident to the sample will be more monochromatic (narrower band width), but of lower radiant power (less light passes through monochromator).
76
Describe the similarities and differences between a phototube and a photomultiplier tube. What is the advantage of one over the other?
* Similarity → both detectors function by converting the energy associated with incoming photons into electrical current
* Advantage of photomultiplier tube is that signal is continuously amplified, so radiation can be less intense and still be detected (= more sensitive than phototube)
77
Your lab has been using an old single-beam spectrophotometer that must now be replaced. You obtain sales literature that describes single-beam and double-beam instruments.
What are the basic differences between a single-beam and double-beam spectrophotometer, and what are the advantages and disadvantages of each?
Single-beam → radiant beam follows only one path (i.e., from source through the sample to the detector)
Double beam → radiant beam is split so half the beam goes through one cell-holding compartment and the other half of the beam through the second, or beam is alternatively passed through the sample and reference cells by means of a rotating sector mirror
Advantages of double beam → can simultaneously measure and compare relative absorbance of a sample and a reference cell; can compensate for deviations or drifts in the radiant output of the source.
Disadvantage of double beam → radiant power of the incident beam is decreased because beam is split, causing inferior signal-to-noise ratios
78
Explain the similarities and differences between UV-VIS spectroscopy and fluorescent spectroscopy with regard to instrumentation and principles involved.
What is the advantage of using fluorescence spectroscopy?
**Similarities** → essentially the same instrumental components; both are based on absorption of radiant energy by the analyte, and the detection of radiant energy at the detector.
**Differences** → Fluorescence spectroscopy depends on measuring radiation emitted by the analyte as it relaxes from an excited electronic energy level to its corresponding ground state. The analyte is originally activated to the higher energy level by the absorption of radiation in UV or VIS range. Fluorometers have two wavelength selectors (i.e., excitation beam and emission beam versus 1 for spectrometer). Fluorometers have detector arranged so that emitted radiation is travelling at an angle of 90° relative to the axis of the excitation beam (to minimize signal interference due to transmitted source radiation and radiation scattered from the sample).
Advantage of fluorescence spectroscopy → 10-1000x more sensitive than UV-VIS absorption spectroscopy; has helpful applications regarding protein unfolding; amyloid fibrils; and egg freshness
79
What are the wavelength limits and types of transitions in Infrared Spectroscopy
0.075 - 1000um
Vibrational positions of atoms in molecular bonds
80
IR covers a broad region of light compared to UV and VIS.
Describe the three sections and identify the most useful.
81
When can a molecule absorb IR radiation?
If it vibrates such that its charge distribution (therefore its electric dipole moment) changes during vibration
82
What is harmonic approximation?
A model to approximate the energy of a vibrational transition (IR spectroscopy)
IR absorption is unique to each group of atoms.
Different bond strengths and different masses give rise to discrete energy levels
83
What is fundamental absorption?
Frequency of radiation that will make vibrating molecular functional group move from _lowest vibrational state_ to _first excited state_.
84
What are overtones?
Absorption of radiation to move vibrating molecular functional group to _higher excited states_ → 2-3x higher energy = less likely (less absorption at these frequencies)
85
Mid-IR generally involves overtones.
True or False?
False.
Mid-IR generally involves fundamental absorption.
86
Mid-IR generally involves fundamental absorption.
True or False?
True.
87
Near-IR generally involves overtones.
True or False?
True.
88
Near-IR generally involves fundamental absorption.
True or False?
False.
Near-IR generally involves overtones.
89
What are the two main Mid-IR instrument designs?
Dispersive IR
Fourier Transform IR
90
Describe dispersive IR.
91
Describe Fourier Transform IR.
* No means to select a single wavelength to shine on the sample
* The sample is exposed to all wavelengths at once
* Radiation is _not dispersed_; instead, uses an **interferometer**
* All wavelengths pass through the sample and arrive at the detector simultaneously.
* Mathematical treatment (FT) converts results into IR absorption spectrum
92
What are the advantages of Fourier Transform IR over Dispersive IR? [4]
* Faster
* More sensitive
* Better wavelength resolution
* Better wavelength accuracy
93
Describe FTIR instrument design.
* IR beam is split then recombined using mirrors
* Intensity of radiation reaching detector varies as function of optical path difference as one mirror is moved → causes interference
* Interferogram → intensity as a function of optical path difference
* Converted to intensity vs wavenumber using FFT
94
What are the light sources used in FTIR instrument design?
* Inert solids heated electrically to 1000-1800°C
* Three sources → Nernst glower, Globar, Nichrome coil
95
What are detectors in FTIR instrument design?
* **Variations of thermocouples** → output voltage varies with temperature changes caused by radiation striking detector
* **Several types** → Golay, pyroelectric, semiconductor
96
Describe MID-IR sample handling for measuring liquids.
Transmission IR
* Used to measure liquids
* High absorptivity, thus use very small pathlengths (0.01 - 1.0 mm)
* Can be used for solids if sample is ground and pelleted with potassium bromide, or dispersed in Nujol mineral oil
* Cell windows are made of non-absorbing materials (e.g., halide, sulfide salts, zinc selenide).
97
Describe MID-IR sample handling for measuring solids, pastes, and viscous liquids (e.g., peanut butter).
* Attenuated Total Reflectance IR (ATR IR)
* Measures total energy reflected from surface of sample in contact with IR transmitted crystal
98
Functional group spectroscopy = Near-IR spectra.
True or False?
False.
Functional group spectroscopy = Mid-IR spectra.
99
Functional group spectroscopy = Mid-IR spectra
True or False?
True
100
Describe Near-IR spectra.
* higher energy
* spectra consist mainly of overtones
* Lower intensity
* Broader absorption
* Arise primarily from functional groups that have an H atom attached to a C, N, or O
* e.g., common groups in water, organic compounds
101
Describe Near-IR instrumentation.
* Design
* Dispersive (common)
* FT-IR
* Mode
* Transmission (Liquids; quartz cuvette)
* Reflectance:
* **Specular** (light reflects back towards light source) → no information; not measured
* **Diffusive** (light reflects in random angles) → penetrates sample & partially absorbed; provides information (is measured)
* Used for solid foods
102
What is the equation for reflectance?
103
**Quantitative** analysis using Mid-IR & Near-IR relies on […].
* Instrument calibration with known standards, or identical food products
104
Quantitative analysis using Mid-IR or Near-IR requires measurements […].
At 2 or more wavelengths due to overlapping NIR absorption bands
105
Quantitative analysis in Mid-IR & Near-IR uses […] to relate data to concentration, and […] to calibrate IR instrument
Quantitative analysis in Mid-IR & Near-IR uses **multivariate statistical techniques** to relate data to concentration, and **multivariate regression approaches** to calibrate IR instrument by comparing IR data to data from conventional methods of analysis.
e. g., partial least squares (PLS), principal component regression (PCR)
e. g., for protein, use Kjeldahl or Dumas methods
106
Describe **qualitative** analysis in Mid-IR & Near-IR applications in food.
* Compare spectra of _sample_ and _reference sample_, for example:
* Distinguish between wheat types (hard red spring vs. hard red winter)
* Distinguish between orange juice and other juices
* Authentication of olive oil
107
Discuss specific applications of automated FTIR instruments.
108
Lighter atoms give lower frequencies.
True or False?
False.
Lighter atoms give higher frequencies
109
Lighter atoms give higher frequencies.
True or False?
True.
110
Heavier atoms give lower frequencies.
True or False?
True.
111
Heavier atoms give higher frequencies.
True or False?
False.
Heavier atoms give lower frequencies.
112
Stronger bonds give higher frequencies, and weaker bonds give lower frequencies.
True or False?
True.
113
Stronger bonds give lower frequencies, and weaker bonds give higher frequencies.
True or False?
False.
Stronger bonds give higher frequencies, and weaker bonds give lower frequencies.
114
Higher energy → greater amplitude.
True or False?
True.
115
Higher energy → lesser amplitude.
True or False?
False.
Higher energy → greater amplitude.
116
Describe FTIR to study secondary protein structure.
117
What is synchrotron radiation?
* Electron accelerator and storage ring
* Accelerating electrons gives off EM radiation
* Very intense light source
118
IR instrumentation is never portable.
True or False?
False.
119
IR instrumentation can be portable.
True or False?
True.
120
What is the primary method for measuring the mineral content (& toxicants like heavy metals) of food?
Atomic absorption & atomic emission spectroscopy
Note → minerals are present as minor constituents → need to quantify against a background of other components
121
When is atomic absorption spectroscopy used in mineral analysis rather than titration, colorimetric assays, or ion selective electrodes? [3]
* several minerals determined
* large sample number
* typically for nutrition labelling
122
How are samples prepared for atomic absorption analysis?
Most samples are wet ashed
123
What does AAS measure?
124
What does AES measure?
125
Compare atomic versus molecular absorption/emission.
Atomic only has electronic transitions!
126
Minerals do not have complex line spectra.
True or False?
False.
Each element has a unique set of energy levels = unique line spectra
127
Minerals have complex line spectra.
True or False?
True.
128
In line spectra there tend to be more emission spectra than absorption spectra.
True or False?
True.
129
In line spectra there tend to be less emission spectra than absorption spectra.
True or False?
False.
130
Emission and absorption spectra are often not unique for each element.
True or False?
False.
They are unique.
131
Describe the basic components [6] of atomic absorption spectroscopy instrumentation.
1. Light source
2. Beam chopper
3. Atomizer (+ nebulizer)
4. Monochromator
5. Detector
6. Readout device (CPU)
132
Describe the light source for AAS.
Hollow Cathode Lamp (HCL)
Cathode is made of the element to be measured.
133
What is the principle of atomization in AAS?
* Introduce sample as small droplets/fine mist, at high temperatures
* Solvent evaporates
* Sample vaporizes
* Molecules decompose to atoms
* **Flame atomizer**
* Nebulizer: sample → fine mist
* mix with oxidant-fuel (air-acetylene)
* introduce to flame, 5-10cm long
* **Graphite furnace**
* sample (~ul) carried through graphite tube
* electrically heated
134
What is considered ideal in atomization? [2]
Maximize atomization while minimizing ionization
Neutral atoms and ions have different line spectra
135
Describe the factors that affect the frequency of vibration of a molecular functional group and thus the frequencies of radiation that it absorbs.
Also, explain how the fundamental absorption and overtone absorptions of a molecule are related.
* Factors include strength of the bond and the mass of the molecular system.
* Fundamental absorption is the initial frequency of vibration of the bond.
* Overtone absorption are of a frequency 2-3 times that of the fundamental frequency; intensity of these absorptions is much lower than the fundamental absorption, since these transitions are less favoured.
136
Describe the essential components of an FT mid-IR spectrometer and their function, and compare the operation of the FT instrument to a dispersive instrument.
What advantages do FT instruments have over dispersive IR spectrophotometers?
FT Instrument: IR source: interferometer (splits then recombines a light beam); mirrors and mirror drive; sample cell ;detector; amplifier; computer
FT operation: radiation is not dispersed, but rather all wavelengths arrive at the detector simultaneously, a mathematical treatment (a fourier transfrom) is used to convert the results into a typical IR spectrum → uses an interferometer to split then recombine a light beam
Dispersive instrument → use a monochromator to disperse the individual frequencies of radiation and sequentially pass them through the sample so that the absorption of each frequency can be measured
Advantages of FT instruments (vs. dispersive) → can acquire spectra more rapidly, with greatly approved signal-to-noise ratio
137
Of the three antioxidants butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate, which would you expect to have a strong IR absorption band in the 1700-1750cm-1 spectral region?
A strong absorption band in the 1700-1750cm-1 region is indicative of the presence of a carboxyl group. Single propyl gallate is the only one of the three compounds to have a carboxyl group in its structure, only propyl gallate sould exhibit a strong band in this region.
138
Describe the two ways in which radiation is reflected from a solid or granular material.
Which type of reflected radiation is useful for making quantitative measurements on solid samples by near-IR spectroscopy?
How are near-IR reflectance instruments designed to select for the desired component of reflected radiation?
Radiation is reflected form a solid or granular material by:
**Specular reflection** → the mirror-like reflectance when radiation is reflected from a sample surface.
**Diffuse reflectance** → when radiation penetrates through the surface of the sample, and is reflected off several sample particles before it exits the sample; diffuse reflectance is useful for NIR reflectance spectroscopy
NIR reflectance instruments are designed with filters selected to transmit wavelengths that are known to be absorbed by the sample constituents.
139
Describe the steps involved in calibrating a near-IR reflectance instrument to measure the protein content of wheat flour.
Why is it usually necessary to make measurements at more than one wavelength?
* Select a set of calibration (training samples) → representative, contain constituents of interest, with uniform distribution of concentrations
* Analyze by classical analytical method for the constituent and obtain spectral data on each sample with the NIR instrument at all available wavelengths
* Use multiple linear regression to select optimum wavelengths for measurement (select 2-6 wavelengths, and check selection to see if reasonable from a spectroscopic standpoint)
* Test regressions using all possible combinations of wavelengths being tested, to get combination that provides the best results (i.e., maximize correlation coefficient and minimize standard error).
* Test the calibration obtained using the instrument to predict the composition of a set of test samples that are independent of the calibration set
140
AAS and AES instruments rely on energy transitions in atoms of elements being measured.
What is an ‘energy transition’ in this context and why can it be used to detect and quantify a given element in a sample containing multiple elements?
What is the source of energy that produces this energy transition in an AAS instrument? In an AES instrument?
Energy transition → is the energy change associated with a transition between two energy levels for an atom (e.g., ground and excited states). Atoms absorb or emit radiance of discrete wavelengths because the allowed energy levels of electrons in atoms are fixed and distinct. In other words, each element has a unique set of allowed electronic transitions and therefore a unique spectrum, enabling accurate identification and quantification even in the presence of other elements.
In AAS → the source of energy is radiation from a hollow cathode lamp (or an electrode-less discharge lamp)
In AES → the source of energy is flame (heat) or inductively coupled plasma torch (radiofrequency power).
141
Describe the process of ‘atomization’ as it pertains to AAS and AES analyses.
Atomization (i.e., separating particles into individual molecules and breaking molecules into atoms) is required before atomic absorption or emission measurements can be made. The sample solution goes through the process of desolvation, vaporization, atomization, and ionization.
142
Your boss wants to purchase an AAS instrument for your analytical lab because it is cheaper but you want an ICP-OES instrument because it is more versatile and will greatly increase your sample throughput. To convince your boss, you need to educate him.
## Footnote
**Explain the underlying principles of operation for an ICP-OES instrument in language your boss can understand. Describe the instrument you want to purchase.**
**Principles** → measures emission of energy (at specific wavelength) as atom excited by plasma returns to ground state → amount of radiant energy emitted is proportional to concentration of specific element.
**Instrumentation** → atomization-excitation source → ICP torch (plasma); Echelle optics (or monochromaters for older PMT-based instruments) → solid-state array detectors → computer/readout device
143
Your boss wants to purchase an AAS instrument for your analytical lab because it is cheaper but you want an ICP-OES instrument because it is more versatile and will greatly increase your sample throughput. To convince your boss, you need to educate him.
## Footnote
**Explain how AAS differs in instrumentation and principle of operation from ICP-OES.**
FOR AAS:
* Use hollow cathode lamp as a radiation source to excite atoms (vs. ICP torch in ICP-OES)
* Use flame (or graphite furnace) only to convert molecules to atoms (vs. plasma torch in ICP-OES)
* Measure amount of energy absorbed as atoms go from ground state to excited state (vs. measuring emitted light from atoms as they relax from excited state to ground state)
144
Your boss wants to purchase an AAS instrument for your analytical lab because it is cheaper but you want an ICP-OES instrument because it is more versatile and will greatly increase your sample throughput. To convince your boss, you need to educate him.
## Footnote
**Can you make the case that costs for an ICP-OES would be lower over the long term?**
ICP-OES offers a number of advantages:
* Multiple element capabilities in a single sample with a single aspiration
* Higher throughput
* Can detect more elements
* Detection limits generally better (compared to flame AAS)
* Better for refractory compounds (i.e., those stable at high temperatures)
* Fewer non-spectral interferences
* Wider analytical working range
* Use of explosive fuel gas not required; less likely to have costly accidents.
145
Your boss wants to purchase an AAS instrument for your analytical lab because it is cheaper but you want an ICP-OES instrument because it is more versatile and will greatly increase your sample throughput. To convince your boss, you need to educate him.
## Footnote
**For most types of food samples other than clear liquids, what type of sample preparation and treatment is generally required before using ICP-OES or AAS for analysis?**
* Ash sample, solubilize ash in water or dilute acid, dilute to volume, and analyze.
146
You are training a new technician in your laboratory on mineral analysis by AAS and ICP-OES.
Briefly describe HCL in AAS.
* Generates light of specific wavelength to be absorbed by the element of interest.
147
You are training a new technician in your laboratory on mineral analysis by AAS and ICP-OES.
Briefly describe Plasma in ICP-OES.
* Atomization-excitation source
148
You are training a new technician in your laboratory on mineral analysis by AAS and ICP-OES.
Briefly describe Echelle optical system in ICP-OES
* Utilzies a prism and a grating to create a two-dimensional spectrum with a wide-wavelength range.
149
You are training a new technician in your laboratory on mineral analysis by AAS and ICP-OES.
Briefly describe nebulizer in AAS and ICP-OES.
* Converts sample solution to fine mist or aerosol.
150
In the quantitation of Na by atomic absorption, KCl, or LiCl was not added to the sample.
Would you likely over- or under-estimate the true Na content? Explain why either KCl or LiCl is necessary to obtain accurate results.
* Underestimate, due to ionization interference, since Na is easily ionized
* Add ionization suppressants to increase the concentration of electrons in the flame, to shift equilibrium to the left
151
Give five potential sources of error in sample preparation prior to atomic absorption analysis.
* Not using distilled and/or deionized H2O
* Not using reagent grade chemicals
* Not using sufficiently cleaned lab-ware
* Using glass rather than plastic lab-ware
* Not diluting sample properly
152
You are performing iron analysis on a milk sample using AAS. Your results for the blank are high. What could be causing this problem, and what is a possible remedy?
The problem is most likely caused by contamination. Check to make sure the reagents are pure and that the lab-ware was all acid washed.
Consider checking the accuracy of your assay by analyzing a standard reference material purchased from NIST.
153
The detection limit for calcium is lower for ICP-OES than it is for flame AAS. How is the detection limit determined, and what does it mean?
* Detection limit (or LOD) is usually defined as the lowest concentration of an analyte that can be distinguished from the appropriate blank with a given level of confidence.
* To determine the LOD for a particular element using a particular instrument, one would simply prepare the appropriate blank, tune the instrument to measure the element, and analyze the blank several times. Then, calculate the mean and standard deviation of the reading and plug the values into the above equation. Variability in the blank signal is often referred to as ‘noise’. Therefore, the noise in the AAS signal is generally higher than the noise in an ICP-OES signal.
154
Discuss AAS calibration and use
155
Discuss AAS calibration and use
* Absorbance vs. concentration will deviate from linearity predicted by Beer's law when concentration exceeds a certain level.
* Properly constructed calibration curves using pure standards are essential for accurate quantitative measurements.
* If values for linear ranges are not provided by manufacturer, the linear range of an element should be established by running a series of standards of increasing concentration and plotting absorbance versus concentration.
* The concentration of the unknown sample solution should be adjusted so that the measured absorbance always falls within the linear range of the calibration curve.
* If values are provided by the manufacturer, measured absorbances deviating from these values indicate appropriate adjustments are required (e.g., flame characteristics modified, lamp alignment changed, etc.)
156
Describe spectral interferences in AAS.
* **Spectral overlap** = absorption of source radiation by other elements
* Use different wavelength, or narrow monochromator slit width
157
Describe non-spectral interferences in AAS. [4]
* **Variations in transport of sample → flame atomizer**
* Results in variations in sample amount
* Prepare standards identically as samples (e.g., viscosity, vapour pressure)
* **Suppression of solute volatilization** (e.g., Ca suppressed by P)
* **Formation of thermostable oxides & complexes that don't decompose**
* **Ionization of element = lower absorption**
* Use ionization suppressors - easily ionized elements (K, Cs, Li)
158
Describe the principle of AES.
* Sample solution is volatilized, then atomized, and excited by heat (flame), light (laser), electricity (ars or sparks), or radio waves (inductively coupled plasma, ICP)
* Atoms in an excited state emit energy (specific wavelengths for each element) when they return to a lower energy state/ground state
* Amount of radiation emitted is proportional to concentration.
159
Describe AES using inductively coupled plasma (ICP).
* **Flame AES**
* Flame = atomization & excitation source
* Useful only for elements of relatively low excitation energy (Na, K)
* **Inductively coupled plasma-optical emission spectroscopy**
* Plasma = atomization & excitation source
* Argon gas in a quartz tube is heated using rf power applied to copper coil
* Induces a magnetic field
* Ar, ions, and electrons are accelerated in a circular path → coupled to magnetic field
* Produces high energy electrons and argon ions (6000-7000K)
* Excites elements in the sample
160
Describe Echelle Optical System.
* Uses two dispersing components in series:
* Prism (x-axis) and diffraction grating (y-axis)
* Results in 2-D dispersion of light
* Allows high bandpass and resolution
* Detection using a solid-state array detector:
* Complementary metal oxide semiconductor
* Charge coupled device
* Charge injection device.
161
Describe spectral interferences in ICP-OES.
* Background shift interference
* Certain ions may increase background
* Perform background subtraction
* Spectral overlap interference
* Overlap of emission lines of different elements
* Choose a different emission line, or use a correction factor
162
Describe nonspectral interferences in ICP-OES.
Not an issue
163
Describe sample preparation for atomic spectroscopy.
* **Ashing**
* Remove organic material; solubilize minerals
* Less volatilization & better solubility with wet ashing
* **Reagents**
* Need highly pure chemical reagents (for wet ashing) and water (to prepare sample and standard solutions)
* Need to use reagent blanks
* **Standards**
* Must be prepared carefully (similar to sample)
* Run standards frequently
* Check with standard reference materials
* **Labware**
* Plastic containers preferable to glass (can absorb metals)
* Must be washed and rinsed carefully, and soaked in acid
164
Describe the order of components in UV-VIS spectroscopy.
UV or visible lamp → monochromator → sample → detector → readout
165
Describe the order of components in fluorescence spectrometry.
UV or visible lamp → monochromator → sample → monochromator → detector → readout
166
Describe the order of components in Mid-IR spectrometry.
For dispersive systems: mid-IR source → grating → slit → sample → detector → readout
For FT systems: mid-IR source → interferometer → (He:Ne alignment laser, beam splitter, movable mirror, fixed mirror) → sample → detector → readout
167
Describe the order of components in Near-IR spectrometry.
For dispersive systems: near-IR source → grating → slit → sample → detector → readout
For FT systems: near-IR source → interferometer → (He:Ne alignment laser, beam splitter, movable mirror, fixed mirror) → sample → detector → readout
168
Describe the order of components in atomic absorption.
HCl → sample inserted in flame → monochromator → detector → readout
169
Describe the order of components in atomic emission.
Sample inserted in plasma → monochromator (or echelle optics) → detector → readout
170
Why do fluorometers have detector arranged so that emitted radiation is travelling at an angle of 90° relative to the axis of the excitation beam?
To **minimize signal interference** due to transmitted source radiation and radiation scattered from the sample.
