IR Spectroscopy Flashcards
Why is FRET extremely sensitive to small changes in distance?
The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
What is IR spectroscopy?
- Infrared spectroscopy is the analysis of infrared light interacting with a molecule.
- IR Spectroscopy measures the vibrations of atoms, and based on this it is possible to determine the functional groups.
What wavelengths are used in IR spectroscopy?
Electromagnetic energy with wavelengths longer than visible light but shorter than microwaves (0.8 – 100 um)
How is wavenumber related to frequency and wavelength?
- Higher wavenumber corresponds to shorter wavelengths (higher energy), while lower wavenumber corresponds to longer wavelengths (lower energy).
IR radiation can be measured in terms of its […]
Frequency
Compare the absorption peaks within the IR region compared to UV-Vis regions.
The absorption peaks within this IR region are
usually sharper when compared with absorption
peaks from the UV-Vis regions
How can we determine functional groups with IR spectroscopy?
- To determination of functional groups within a sample since different functional group absorbs different particular frequency of IR radiation.
- Each molecule has a characteristic spectrum often referred to as the fingerprint.
Describe the IR spectrum.
- A plot of % transmittance or absorption versus wavenumber
- The higher the wavenumber, the shorter the wavelength
- Can convert between %T and A using same equations as in UV/Vis spectroscopy
Describe the IR transmittance and absorbance spectra.
They are mirror images about the x-axis.
Can convert results between absorbance and transmittance using same equations as in UV/Vis spectroscopy.
Practice.
Convert 56% transmittance to absorbance.
Convert 0.252 absorbance to transmittance.
What is the theory behind IR spectroscopy?
- IR spectra are results of low energy absorption derived from vibrations and to some extent rotations of molecules
- Absorption is possible when a net change in the dipole moment
- Caused by vibrations (or rotations) of the molecule
Define: fundamental absorption and overtones.
- Fundamental absorption: frequency of radiation that will make vibrating molecular functional group move from lowest vibrational state to first excited state (related to mid-IR absorption)
- Overtones: absorptions of radiation to move vibrating molecular functional group to higher excited states (related to near-IR absorption)
A molecule can absorb IR radiation if…
It vibrates such that its charge distribution (therefore, its electric dipole moment) changes during vibration.
Overtones are measured with […]
near-IR absorbance
Fundamental absorption is related to […]
mid-IR absorbance to first excited state
What type of stretches are more likely to result in a change in dipole moment?
Asymmetric stretches
Stretching motions vibrate at higher frequencies than bending motions.
True or False?
True.
Bending motions vibrate at higher frequencies than stretching motions.
True or False?
False.
Stretching motions vibrate at higher frequencies than bending motions.
Asymmetric stretches are more likely than symmetric stretches to result in a change in dipole moment.
True or False?
True.
Symmetric stretches are more likely than asymmetric stretches to result in a change in dipole moment.
True or False?
False.
Asymmetric stretches are more likely than symmetric stretches to result in a change in dipole moment.
Describe characteristic IR wavenumbers for these functional groups.
Which are sp^3, sp^2, and sp?
Describe the characteristic IR wavenumbers.
What are the characteristic mid-IR and near-IR absorption frequencies of fats?
What are the characteristic mid-IR and near-IR absorption frequencies of water?
- Raman-spectroscopy is considered more powerful than near-IR because it will not detect water like near-IR does.
What are the characteristic mid-IR and near-IR absorption frequencies of proteins?
What are the characteristic mid-IR and near-IR absorption frequencies of carbohydrates?
What are the characteristic mid-IR and near-IR absorption frequencies of aromatics?
What are the characteristic mid-IR and near-IR absorption frequencies of alcohols?
What are the characteristic mid-IR and near-IR absorption frequencies of ethers?
What are the types of IR instrumentation?
- Two types: dispersive and Fourier transform
What is dispersive IR instrumentation?
- Has components similar to UV-Vis spectrometers
- i.e., radiation source
- monochromator or filter
- sample holder
- detector connected to amplifier system
Largely now replaced by Fourier-Transform IR
What is Fourier Transform IR Spectrophotometery?
- Uses an interferometer rather than a monochromator, and polychromatic IR light to generate an interferogram
Define: polychromatic IR light
IR light of many wavelengths
Used in FT-IR
Define: interferometer
Set of mirrors used to manipulate and cause interference between two light beams from one original source.
Used in FT-IR
What is an interferogram?
Plot of signal intensity versus time/distance
Generated by FT-IR
Describe FT-IR instrumentation.
How is an interferogram generated?
- Mathematically decoded by a Fourier transformation
- Before the FT, the interferogram is intensity of signal vs. time (or mirror position)
- Mirror position changes at a fixed velocity
- Before the FT, the interferogram is intensity of signal vs. time (or mirror position)
- After the FT, intensity (absorbance/transmittance) vs. frequency
- An IR spectrum
- The FT takes the signal, and determines the amount of each wavelenght/wavenumber that went into the signal
What does Fourier transformation acheive?
The FT takes the signal and determines the amount of each wavelength/wavenumber that went into the signal.
What are the advantages of FT-IR compared to mid-IR dispersive instruments? [4]
- Faster
- More sensitive
- Better wavelength resolution and wavelength accuracy
- Radiation is not dispersed; instead - all wavelengths arrive at detector simultaneously (mathematical treatment (called FT) converts results into IR spectrum
Describe the sample handling techniques for transmission IR spectroscopy.
- Commonly used to measure liquids
- 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 nonabsorbing materials (e.g., halide; sulfide salts; zinc selenide)
Describe the sample handling techniques for attenuated total reflectance.
- Little or no sample preparation, widely applied sample techniques.
- Commonly used to measure solids, pastes, and viscous liquids.
- Measures total energy reflected from surface of sample in contact with IR transmitting crystal
What is attenuated total reflectance?
- IR sampling technique that allows for solids and liquids to be analyzed directly.
- Relies on [attenuated] total internal reflection.
- Evanescent wave internall reflects into sample
Evanescent = exponential wave decay as function of penetration depth.
What is an evanescent wave?
Evanescent = exponential wave decay as function of penetration depth.
Relates to attenuated total reflectance (ATR)
What are the advantages of attenuated total reflectance?
- No need for sample to be thin.
- Radiation is not transmitted to the sample
- No need to dilute the sample
- Penetration depth of the radiation is limited to a few micrometers.
- The physical state of the sample is an important factor: intimate contact with ATR crystal
Describe the ATR sampling accessory.
- Sample is pressed at moderate tightness between plate and pin.
- Handle will click once adequate torque has been applied.
- Prevents damage to crystal/accessory
What materials are used for ATR crystals?
- Germanium
- ZnSe (zinc selenide)
- Silicon (far-IR region)
- Diamond
- Diamond coated ZnSe
Must have higher refractive index than sample investigated.
What do we need to take account of when selecting a crystal material for ATR?
- pH
- Hardness
- IR absorption
- etc.
Can IR be portable?
Yes, may be on-line and in-field.
Describe mid-IR spectroscopy applications in food science.
- Obey Beer’s law (though greater deviations than in UV-Vis spectroscopy due to low intensities of IR sources).
- (1) Identify specific components: “fingerprint”
- (2) Detect changes in components: chemical and physical
- (3) Quantification/qualification of components: proteins; lipids; CHO; moisture
Describe how these peaks may be identified as potato, whey, and vegetable oil.
What is a MIRA IR milk analyzer?
- Analyze hundreds of samples/hour
- Measures fat, protein, and lactose content simultaneously (AOAC International Official Method)
- Automated instrument
- Homogenize milk fat globules to minimize light scattering.
- Pump milk into flow-through cell IR beam passed through sample
Describe quantitative analysis using NIR instruments.
- Necessary to take measurements at 2 or more wavelengths because of overlapping nature of near-IR absorption bands
- Use multivariate statistical techniques (chemometrics) to relate data to concentration (partial least squares and principal components regression)
Describe qualitative analysis using NIR spectroscopy.
- Classify a sample into one of two or more groups (rather than quantifying a constituent)
- Classification techniques used to group similar spectrum (principal component analysis; discriminant analysis)
- Food applications:
- Wheat type: hard red spring vs. hard red winter
- Orange juice vs. other juice
- Authentication of sources of olive oil
- Discrimination of beef
What are the advantages of IR spectroscopic assay? [5]
- Sensitive
- Fast response time
- Nondestructive measurement (short and simple sample preparation)
- Inexpensive instrumentation
- Broad applications
However, it is difficult to analyze spectra and quantification requires a complicated calibration.
What are the disadvantages of IR spectroscopic assays? [2]
- Difficult to analyze spectra (overlap of bands [overtones]; poor signal-to-noise ratio; baseline fluctuations)
- Quantification requires a complicated calibration
However, it is sensitive, fast, non-destructive, inexpensive, and has broad applications.
What are the key concepts of FRET?
FRET stands for Förster Resonance Energy Transfer (sometimes called Fluorescence Resonance Energy Transfer). It is a technique used to study molecular interactions at a very close range (1–10 nm).
Donor & Acceptor: The donor is a fluorescent molecule that, when excited, can transfer energy to an acceptor molecule if they are close enough.
Distance Dependence: The efficiency of energy transfer is highly dependent on the distance between the donor and acceptor (∝ 1/d⁶), making it useful for studying molecular interactions.
Spectral Overlap: The emission spectrum of the donor must overlap with the absorption spectrum of the acceptor for efficient transfer.
Quenching & Signal Change: When FRET occurs, the donor’s fluorescence decreases, and the acceptor’s fluorescence increases, which can be measured to infer molecular proximity and interactions.
FRET occurs when an excited donor fluorophore transfers its energy to an acceptor fluorophore through non-radiative dipole-dipole coupling.
