Final Flashcards
1
Q
What is spectroscopy??
A
deals with the production, measurement, and interpretation of spectra arising from interaction of electromagnetic radiation with matter
2
Q
Interference
A
Two waves cross one another, they result in an instantaneous wave, at the point of intersection, whose amplitude is the algebraic sum of the amplitudes of the individual waves at the point of intersection.
3
Q
Types of interference
A
Maximum Constructive Interference (Wavelength doubled)
Maximum Destructive Interference (Wavelength is 0)
Interference
4
Q
Energy and Wavelength of functional groups
A
Compounds with resonance have less energy and a higher wavelength (phenyl)
Compounds with no resonance have more energy and a lower wavelength (methyl)
5
Q
Energy Level Transitions
A
Gaining Energy (E0 -> E1): Adsorption
Releasing Energy (E1 -> E0): Emission
6
Q
Relative Transmittance
A
T = P/P0
Index of analyte concentration (fragment of incident light absorbed by solution)
7
Q
Deviation from Beer’s Law
A
- Only valid for diluted solution (low concentration) up to 10 mM
- Chemical processes such as reversible association-dissociation of analyte molecules
- Instrumentation limitation
Only applied to monochromatic radiation
8
Q
What are the different energy levels
A
Largest: Electronic Energy Levels
Middle: Vibrational Energy Levels
Smallest: Rotational Energy Levels
9
Q
UV/Vis components
A
Light Source, Monochromator, Sample/reference holder, radiation detector, readout device
10
Q
Sample-holder or cuvette for UV/Vis
A
Material: not absorb any radiation in the spectral region being used
Quartz, glass, plastics
Dimension: path lengths ranging from 1 to 100 mm are commercially available
Narrow cells (4mm width) for limiting amount of sample solution
11
Q
Choosing appropriate wavelength for UV/Vis
A
Expect to choose wavelength at which the analyte has maximum absorbance and where the absorbance does not change rapidly with changes in wavelength.
One where is there maximum sensitivity and better adherence to Beer’s Law
12
Q
What range is UV/Vis in
A
200-700 nm
13
Q
UV/Vis Light Source
A
Stable, sufficient energy for detection, cover the entire designated wavelength.
Visible: tungsten filament lamp
UV: deuterium electrical discharge lamps, used with quartz sample holders, glass absorb radiation below 350 nm
14
Q
Monochromator
A
Isolates the specific, narrow, continuous group of wavelengths to be used in the assay.
Monochromatic: single frequency and wavelength of radiation
Has entrance and exit slits, concave mirrors, and a dispersing element
15
Q
Radiation detector
A
Produce electrical signal when struck by photons to turn energy into an electrical current.
This signal is proportional to radiant power.
Examples: Phototube, photomultiplier tube, photodiode detector
16
Q
Fluorescence spectroscopy
A
More sensitive than UV/Vis Spectroscopy (1-3 orders of magnitude)
Absorbs energy from radiation in the UV/Vis range, radiation is simultaneously emitted when analyte relaxes.
17
Q
Fluorescence Spectroscopy Components
A
Same as UV/Vis but with 2 monochromators
Emission and Excitation Monochromator
18
Q
Fluorescence
A
Moving from En -> E0
Emitting visible light after absorbing UV (non-visible) light
19
Q
Phosphorescence
A
Intersystem crossing
Moving from the triplet exited state to the ground state
Longer Fluorescence Lifetime
20
Q
Infared Spectroscopy
A
Measurement of the absorption of different frequencies of IR radiation by the matter
21
Q
IR energy level
A
Stays in the ground state and only moves between rotational and vibrational levels
22
Q
IR Molecular Vibrations
A
Bending and stretching of bonds
This molecular asymmetry is a requirement for excitation by IR because symmetric molecules do not display absorbance in IR regions.
Think of molecules like a spring (Hooke’s Law)
23
Q
IR Regions
A
Near-IR (12,500-400 1/cm)
Mid Ir (400-650 1/cm)
far-IR (650-100 1/cm)
24
Q
IR Functional groups
A
Identify functional groups by their frequency on the spectrum
25
Mid-IR Instrumentation
Fourier transform instrument (FTIR): all wavelengths arrive at detector simultaneously.
Nichrome wire light source
Michelson Interferometer: beam split by a splitter and recombined by reflecting back split beams with mirrors.
Thermocouple Detector: output voltage varies with changes caused by varying levels of radiation striking the detector.
26
Quantitative IR and deviations from Beer's Law
Difficult to obtain reliable quantitative data based on IR
Deviations from Beer's law: low intensity of IR source, narrow bands and wide slit required, require calibration sources
27
Attenuated Total Reflectance FTIR
Measuring thick solid, viscous liquid
Surface sensitive technique
28
Photoacoustic IR
Measure effect of absorbed energy (sound at different wavelength)
Used for Gas, liquid, solid, suitable for highly absorbing samples.
29
Near-IR
Quantitative analysis of sample that minimizes impact of size and shape on sample particles.
Can not use quartz (does not absorb radiation)
Range of 700nm to 2500nm
30
Near-IR Absorption Bands
Bands are broad and overlap which give a complex spectrum.
C-H, N-H, and O-H have sufficient intensity.
Determine constituents by where these bonds peak.
31
Near-IR Sample preparation
The food tightly packed into a cell against a quartz window, thereby providing a smooth, uniform surface from which reflection can occur.
32
Rheology
A science of deformation and flow of all materials
Measuring force/deformation as a function of time
33
Viscosity and Stress
Viscosity: define as the internal resistance to flow
Stress: the measurement of force divided by area
34
Normal Stress
Force directly perpendicular to a surface, tension or compression (chewing gum and kneading)
35
Shear Stress
Force parallel to the sample surface (spreading butter on a slice of toast)
36
Newtonian Fluid
Viscosity does not change as shear rate and time changes
Ex: water, air, oil, honey
37
Non-Newtonian Fluid
Viscosity changes as a function of shear rate
Stress = Newtonian Viscosity x Shear Rate
Most foods are Non-Newtonian
38
Apparent Viscosity
Shear-dependent viscosity.
Apparent Viscosity = Stress/Shear Rate
39
Shear thinning
As shear rates increases, viscosity decreases.
Pseudoplastic if time independent
Thixotropic if time dependent
40
Shear Thickening
As shear rate increases, viscosity increases
Dilatant if time independent
Anti-thixotropic is time dependent.
41
Herschel-Buckley Model
n: flow behavior index
σ0: yield stress (PA)
K: consistency index (Pa s)
σ = Kγ^n + σ0
42
Newtonian Model
n = 1
K = μ
σ0 = 0
σ = μγ^1
43
Power Law Model
σ0 = 0
σ = Kγ^n
Show no yield stress and a non-linear relationship between shear stress and shear rate
44
Bingham Plastic Model
n = 1
K = μpl
σ = σ0 + μplγ^1
Relationship between shear stress and shear rate is linear once flow is established.
45
Rotational Viscometry
A known test fixture in contact with a sample, and through some mechanical, rotational means, the fluid is sheared by the fixture.
46
Rheometer
Measure the viscosity, relationship between shear rate and shear stress
47
Rheometry primary assumptions
Laminar flow: synonymous with streamline flow
Steady state: no net changes in system over time
no-slip boundary condition: when test fixture is immersed in fluid sample, walls of fixture and sample serve as boundaries for the fluid.
48
Steady shear mode viscometer
Steady shear: the sheared fluid viscosity, contained between the boundaries, remains constant at any single position, the velocity gradient across the fluid is a constant
49
Concentric Cylinder
Uses torque to generate rotation about an axis
Advantages: Good for low-viscosity fluids, suspensions, and dilute samples and large SA increase sensitivity at low shear rates
Disadvantage: Large sample is required
50
Brookfield Viscometer
An example of a concentric cylinder rheometer
Records shear stress at different rpms
51
Cone and Plate
Permits shear rate and shear stress to remain constant for any location of sample in fluid gap.
Advantages: Good for high shear rates, medium and high viscosity samples, small sample required, and cleanup is easy
Disadvantages: Large particles interfere with sensitivity, must maintain constant gap height
52
Steady Shear Rotational Viscometry Procedure
1. Test fixture selection
2. Speed (Shear rate) selection.
3. Data collection
(Torque and speed)
4. Shear calculations.
(Shear stress and shear rate)
5. Model parameter determination
(Applicable rheological model and parameters: viscosity, yield stress, flow behavior index)
53
Small strain testing
Goal is to apply minimal amount of strain or stress required to measure rheological behavior while preventing damage to sample.
Used to understand properties of a food network.
54
Large strain testing
Used to determine the large strain and fracture food properties.
Used to give indication of sensory texture or product durability.
55
Texture Profile Analysis
Data related to hardness, cohesiveness and other sensory parameters.
two-cycle compression test
56
Texture Analyzer
Used to create correlation between sensory perception and physical properties.
Texture parameters: crispy, crunchy, chewy, sticky, elasticity, yield point, ripeness, gel strength,
57
Gas Chromatography
Used for analysis of: FAs, triglycerides, sterols, gasses, solvent analysis, water etc.
Good for thermally stable volatile compounds
58
GC Factors to consider
Temperature at injection point
Concentration of compounds
Enzymes
Microbial growth
Chemical reactions
59
Direct headspace sampling
Using gas-tight syringe, injected directly GC
Good for rapid analysis of very low boiling point compounds
Has a low sensitivity
60
Purge and trap headspace isolation
Has a higher sensitivity for trace analysis.
Purge a gas into samples -> volatile compounds move into headspace vapors -> pass a cryogenic or adsorption trap -> trap is heated, carrier gas introduced the volatile compounds into the column.
61
Headspace trap materials
synthetic porous polymers
62
Distillation methods of isolation
Effective at isolating volatile compounds from foods for GC analysis. Product moisture or outside steam is used to head and codistill volatiles from a food product.
63
Solvent extraction for GC isolation
Preferred for recovery of volatiles from food.
Recovery depends on solubility of solute in different solvents.
Involves use of organic solvent and water (2 immiscible phases)
64
Solid-phase microextraction (SPME)
Microextraction technique which employs a thin film on sorption polymers on a fine fused silica fiber.
Advantages: Less solvent required and minimal solvent evaporation, faster, more precise and more accurate, less cost and contamination, automated
65
Solid-phase microextraction (SPME) method
1. Phase is bound onto a fine fused silica filament.
2. The fiber is immersed in a sample/headspace of sample.
3. Fiber is pulled into a protective metal sheath, removed from sample, and forced through septum of GC after desired extraction.
4. Adsorbed volatiles are thermally desorbed from the fiber
66
SPME fiber
Having varying film thickness and coating of polymer
Thicker (100 micrometer) - volatiles, thinner - large molecules
Ex: polar phase (polyacrylate and carbowax coating)- polar compounds
67
Components of GC
Gas supply and regulators
Injection port
Oven
Column
Detector
Electrometer
Recorder/data handling system
68
Gas Supply and Regulators
Common carrier gas: nitrogen, helium, hydrogen
Detector Gas: air and hydrogen
Must use high purity gases
Gas lines, regulators, and fittings must be of good quality
69
Injection port
A place for sample introduction, vaporization, some dilution/splitting.
Injection by syringe manually: might cause poor precision, or automatically (autosampler).
Variance minimized by internal/external sample
70
Split injection
Splitting injection allows a portion of sample to go through column.
20 degrees Celsius above maximum column oven temperature.
71
Splitless injection
Whole sample enters column.
Used to increase sensitivity.
72
GC Direct Injection Factors to Consider
Thermal degradation
Damage to GC column
Effect of water vapor
Contamination
Vaporization speed
73
When should sample derivatization occur?
Low in volatility
Poor separation due to polarity
Unstable
74
GC Oven
Control temperature of column.
Determined by interaction of analyte with the stationary phase and boiling point for separation of compounds.
Higher Temp: may cause faster elution but poorer resolution.
75
Packed GC Column
Solid support: silane-treated diatomaceous earth/
Liquid coating (loading) depends on analysis, impact retention time, resolution, baseline.
Liquid coating may be lost at high temperature which causes increase of baseline.
76
Stationary phase of GC Column
Polysiloxane-basd
Choose polar phase to separate polar compounds and phenyl-based column to separate aromatic compounds.
77
GC Capillary Column
Packing: hollow fused silica glass.
Types: Megabore column (0.53 mm i.d); normal (0.2- 0.32 mm i.d); microbore (0.1 mm i.d)
Liquid coating: chemically bonded to the glass wall.
Liquid phase (film) thickness and separation
Thicker film -> longer interaction with stationary phase and better separation
Thinner film -> column bleeding and a poor baseline
78
Separation and resolution of GC Column
Capillary column has better resolution than packed.
Longer column gives better resolution but is not obvious for a capillary column.
Separation is achieved due to the high efficiency of capillary columns, even though the stationary phase is not optimal.
79
Thermal Conductivity Detector
Temperature difference between carrier gas and mixed gas eluted from column directly related to temperature change of filament in the detector.
Carrier gas choice: hydrogen or helium, sometimes nitrogen.
Universal and non-destructive detector; low sensitivity.
Good for further analysis and separation of water, CO.
80
Flame Ionization Detector
Analytes from column are burnt into hydrogen flame, forming ions which are detected by electrodes -> currents.
Good sensitivity, for quantitative study but destructs compounds.
Good for organic compounds containing C-H or C-C bonds, popular in food analysis.
No response to H20, NO2, H2S
81
Electron Capture Detector
Detecting decrease of a standing current generated from the flowing of carrier gas only.
Good for Halocarbons, compounds with double bonds.
High sensitivity and specificity, but narrow linear range
82
Flame Photometric Detector
Burn analyte, measure wavelength of illuminated light.
When S or P is present, characteristic wavelength of light is detected.
Photo multiplies tube (PMT) amplifies the current and transfers to recorder.
High sensitivity and specificity.
Used for heavy metals and compounds containing S and/ or P (pesticides and aroma)
83
Photoionization Detector
UV light ionizes the analyte to form ions, ions are erected by the electrodes to form a current.
Small current is magnified by electrometer of the GC/
Sensitive, non-destructive
Common in flavor analysis
84
Separation of GC Column
N2 is the most efficient carrier gas, but its minimum occurs in low u.
Hydrogen is the best choice.
85
Degradation of 2,4-Decadienal
2,4-Decadienal is a key component in chicken, lamb, beef, and French fries
86
Mass Spectrometry
An analytical tool used for measuring the molecular mass of a molecule.
Ionization of molecules
Separation of ions based on mass-to charge ratio.
Detected under electrostatic field.
87
MS Sample Introduction for Pure Compounds
Direct Injection: for gasses or volatile liquids
Same as injection in GC
Direct Injertion Probe: for somewhat volatile solid
The sample is placed in a small cup on the tip of the prove.
88
MS Sample Introduction for Mixtures
GC-MS or LC-MS through an interface which removes excess GC carrier gas or HPLC solvent.
89
Electron impact (EI) ionization
A beam of electrons emitted from a heated filament composed of rhenium or tungsten metal.
When a direct current is applied to the filament (~70 eV), it heats and emits electrons that move across the ion chamber toward a positive electrode.
Emitted electrons extract an electron from sample compound molecules, forming ionized molecules.
The ionized molecules can further fragment into smaller molecular fragments due to high energy
90
MS Ionization Process
M + e -> M+ + 2e
91
MS Ionization
Both negatively and positively charged molecules of various sizes unique to each compound are formed.
92
MS Repeller plate
Repeller plate is positively charged: positive fragments are repelled and move toward the quadrupole mass analyzer and these fragments are analyzed
93
MS Ionization Acceleration
Accelerating and focusing plates: to increase the energy of charged molecules and to focus the beam of ions.
A maximum number of ions with same kinetic energy reaches the mass analyzer.
94
Chemical Ionization (CI)
Virtually the same ion source device as EI, except, CI uses tight ion source, and reagent gas.
Reagent gas (methane, ammonia, isobutane) is first subjected to electron impact. Then sample ions are formed by interaction of reagent gas ions and sample molecules. Process involved ion-molecule interaction.
95
CI Advantages
Generate fewer fragments -> simpler spectrum.
Characteristic [MH]+ peak -> determine molecular weight.
Useful technique for Mw determination of unknown compounds, or when no molecule ion is observed in EI mass spectrum.
96
CI Process
Depending on mode, MH+ (+ CI) or [M-H]- (- CI) are often observed. Unlike molecular ions obtained in EI method, MH+ and [M-H]- detection occurs in high yield and less fragment ions are observed.
97
Mass Analyzer
Heart of MS
Separating charged fragments based on m/z
Dictating the mass range, accuracy, and sensitivity
98
Types of Mass analyzers
Magnetic sectors
Quadrupoles
Ion traps
Time-of-flight (TOF)
Fourier-transform ion cyclotrons (FT-ICR)
99
Magnetic Sensor
Use magnetic field to separate ions based on m/z.
Deflection depends on ion mass and charge, magnetic field strength and velocity of ions.
To detect multiple ions and produce the typical mass spectra, the magnet field strength is changed over the analysis time -> all possible m/z rations are seen.
Popular high-resolution instrument
100
Quadrupole Mass Analyzer
Quadrupole: “four rods” are used to generate two equals but out-of-phase electric potentials.
One is alternating current (AC) frequency of applied voltage that falls in radiofrequency (RF) range, the other is direct current (DC)
The potential difference -> an oscillating electric field between two of the opposite rods, equal but opposite charges.
By adjusting the potentials on the rod, only selected ions can be detected.
Routinely used in quantitative analysis.
Low resolution but cheap
101
GC-MS
To identify unknown or determine the purity.
GC column is connected directly to the MS via a heated capillary transfer line which is kept hot enough to avoid condensation of the volatile component eluting from GC column.
Ex: the separation of methyl esters from six long-chain FAs mixture
102
LC-MS
LC-MS interface facilitates removal of solvent.
Applied energy is only for evaporation and compound integrity is maintained.
Heat energy applied in evaporation does not contribute to degradation of any thermally labeled species present in LC eluant.
103
Electrospray Interface
Compressed nitrogen gas at high velocity is introduced to facilitate nebulization of the LC effluent, generating a high spray of highly charged droplets. Droplets go through desolvation to form analyte ions when they reach the sample cone.
Highly sensitive with limits of detection at femtogram (10^-15) level and a linear dynamic range between 3 and 4 orders of magnitude.
104
Molecular absorption of radiation in the UV-Vis range results in transitions between what types of energy levels?
Electrical
105
Molecular absorption of radiation in the IR range results in transitions between what types of energy levels?
Vibrational and Rotational
106
Similarities and differences between UV-Vis and Fluorescence spectroscopy
Similarities: components are essentially the same and both are based on absorption of radiant energy by the analyte, and the detection of radiant energy at the detector.
Differences: Has 2 monochromators, fluorescence spectroscopy depends on measuring radiation emitted by the analyte as it relaxes from an excited electronic energy level to its corresponding ground state.
107
Major ions expected in EI mass spectrum of ethanol?
CH3-CH2-OH+, CH2-0H+, CHO+, CH3 +
108
Convert rpm to radians
(x revolution/min) (1 min/60 seconds) (2pi radians/revolution)
109
Fluorometer
Instrument used for Fluorescence Spectroscopy
Light Source: Tungsten filament lamp
Has 2 monochromators (Emission and Excitation)
Sample/Reference Holder
Radiation detector
110
Can IR Spectroscopy be used to find both qualitative and quantitative data?
Yes
Mid-IR is the most common.
Near-IR is possible.
Quantitative is more difficult to obtain than qualitative.
111
Convert Pascals seconds to cp
Answer]: 1N = 1kg m/s2
1 poise=0.1 kg m-1 s^-1 = 100 cp
1 Pa s=1 N m^-2 s = 1 kg m^-1 s^-1 = 10 poise =1000 cp
112
Components of Mass Spectrometer
Sample Introduction, Ion Source, Mass Analyzer, Detector, Data System
113
Beer’s Law
A = abc
A: absorptivity (1/cm)
b: pathlength through solution(cm)
c: concentration of absorbing species (M, mM)
114
Strain
ε=∆L/L
