CE70009 - Product Characterisation Flashcards
How do monochromatic and polychromatic and polarized light differ?
Mono - one wavelength
Poly - multiple wavelengths
Polarised - waves all in one direction
What’s collimated light?
When light rays are parallel to each other
What’s coherent light?
When there is no interference between waves
List key parts of transmission microscope:
Ocular / eyepiece
Objective lens
Stage
Condenser lens
Condenser diaphragm
Focusing knobs
How is numerical aperture, NA, calculated?
NA = nsinØ
n = refractive index of medium in which lens is working, = 1.0 for air, 1.33 for water, and up to 1.56 for oils
How does resolution, light transmission, working distance, and depth of field vary with numerical aperture, NA?
Resolution increases with NA
Light transmission decreases with NA
Working distance decreases with NA
Depth of field decreases with NA
What is bright and dark field illumination?
Bright field microscopes usually have halogen lamp or LED light sources. This type of microscope tends to have low contrast owning to the biological samples transmitting most of the light. Staining if often required to combat this problem, which comes with the disadvantage that live imaging is difficult due to staining killing the cells.
Dark field microscopy is generally preferred therefore over light field. With a dark field microscope a special aperture is used to focus incident light meaning the background stays dark. The light does not pass directly through the sample being studied. Instead light is reflected off the specimen, making it appear to be emitting light. Brightfield microscopy shows clear magnification while the dark field image shows minute details.
Bright field – sample is dark while background is bright
Dark field: sample is bright while background is dark. Light is shone from top and we look at the light that is reflected back. Hollow cone of light with great obliquity. no transmission: reflection or scattering
What’s confocal microscopy?
An optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation.
Confocal microscopy offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labelled with one or more fluorescent probes.
When fluorescent specimens are imaged using a conventional widefield optical microscope, secondary fluorescence emitted by the specimen that appears away from the region of interest often interferes with the resolution of those features that are in focus. This situation is especially problematic for specimens having a thickness greater than about 2 micrometers. The confocal imaging approach provides a marginal improvement in both axial and lateral resolution, but it is the ability of the instrument to exclude from the image the “out-of focus” flare that occurs in thick fluorescently labelled specimens, which has caused the recent explosion in popularity of the technique.
In confocal microscopy a pinhole is used in the focal plane both at illumination and at detection.
In this way out of focus emitted light is effectively rejected by the detection pinhole and an increased resolution is obtained .
How does a TEM (transmission electron microscope) work?
• Gun emits electrons
• Electric field accelerates
• Magnetic (and electric) field control path of electrons
• Electron wavelength @ 200KeV = 2x10-12 m
• Resolution normally achievable @ 200KeV = 2 x 10-10 m = 2Å
• High Vacuum
The sample will appear dark. Shadowing technique.
Very thin sample required
How is the wavelength of electrons determined?
By accelerating voltage (V) on the filament from which they were emitted.
Wavelength = 0.1*(150/V)^0.5
Very high voltages (~100kV) required.
Why is high vacuum needed in TEM?
For transmission electron microscopy, vacuum needed as the mean free path of electrons is very short in air.
High vacuum of 10^-5 mbar aimed for.
Also
- tungsten filaments burn out in air
- columns must be kept dust free
The vacuum is achieved via pumps
What is SEM (scanning electron microscopy) used for?
• Topography and morphology
• Chemistry
• Crystallography
• Orientation of grains
• In-situ experiments:
– Reactions with atmosphere
– Effects of temperature
In brief: we shoot high-energy electrons and analyze the outcoming electrons/x-rays
• A SEM typically has orders of magnitude better depth of focus than a optical microscope making SEM suitable for studying rough surfaces
• The higher magnification, the lower depth of focus
What are issues with SEM?
Needs to be conducted in vacuum.
Samples need to be dry.
If electrons get absorbed by the sample, the surface becomes negative and repels further electrons. So, the sample should be coated with a conductor e.g. gold.
What is STM?
How does it work?
Scanning tunnelling microscopy
A scanning tunnelling microscope (STM) is an instrument for imaging surfaces at the atomic level.
The STM is based on the concept of quantum tunnelling. When a conducting tip is brought very near to the surface to be examined,
a voltage difference applied between the two can allow electrons to tunnel through the vacuum between them. The resulting tunnelling current is a function of tip position, applied voltage, Information is acquired by monitoring the current as the tip’s position scans across the surface, and is usually displayed in image form.
STM can be a challenging technique, as it requires extremely clean and stable surfaces, sharp tips, excellent vibration control, and sophisticated electronics.
STM requires a conducting material to tunnel.
What is AFM?
How does it work?
Atomic force microscopy
AFM consists of scanning a sample with a probe mounted on a cantilever and determining the surface topography from the deflection of laser light on the probe with a position sensitive detector.
The tip (or the sample) is positioned using an extremely precise piezo-electric unit, reaching Å resolution (in x,y,z).
The probe-sample interactions, which flex the cantilever, are repulsive at short distances. The tip can either be in permanent contact with the sample – contact mode –, or oscillating at its resonance frequency (~100 kHz), tapping briefly the sample – tapping mode.
Further, a feedback mechanism keeps the oscillating amplitude constant.
What are issues with AFM (atomic force microscopy)?
As the sharp point moves along the surface of the sample, if the sample is soft, the point may cause the sample surface to deform.
AFM may then detect what is under the surface instead of the surface itself.
What are 3 common types of scattering techniques to identify particle size?
Light scattering
X-ray scattering
Neutron scattering
What are the issues with scattering?
You can’t identify the structure inherently from the scatter / crystal pattern. Estimates and guesses must be made.
What are the 4 outcomes of shining a light on a substance?
Reflection
Transmission (passing through)
Absorption
Scattering
What is the key law of reflection?
Angle of incidence = angle of reflectiom
What is snells law?
Snells law calculates n, the refractive index
Sin (i) / Sin (r) = n
Where I and r are angle if incidence and refraction
What does the Beer-Lambert law for absorption show?
The Beer-Lambert Law (also called Beer’s Law) is a relationship between the attenuation of light through a substance and the properties of that substance.
A = ƐlC
A - absorbance
Ɛ - molar absorptivity
l - length of light path
c - concentration
What is constructive and destructive interference?
Constructive - waves in phase amplify each each other
Destructive - waves out of phase cancel each other out
What is the condition for constructive interference?
n * λ= 2 d sin (Ø /2)
Bragg equation
Where:
λ - wavelength
d - spacing between crystal lattice planes
What is reciprocal space?
Reciprocal space (also called k-space) provides a way to visualize the results of the Fourier transform of a spatial function.
It’s a mathematical space that represents spatial frequencies rather than physical positions.
In scattering, what is q?
q is the distance of the scattered light,
q = 4piSinø / lambda
Where ø is the angle made by scattering, lambda is the light wavelength
Also,
q = |ks| - |ki|
Where:
ks - scattered beam wave vector
ki - incident beam wave vector
What is the intensity of coherently, elastically scattered radiation dependant on?
I(q) = N(delta*density * V)^2 * P(q)S(q)
N = molecules/unit volume
V = molecular volume
P(q) = form factor / particle shape
S(q) = structure factor / inter-particle correlation distances
Delta mean density = density (r)- density s = the scattering density difference between the scattering particle and solvent
What do form factor, P, and structure factor, S, represent?
P - particle shape
S - inter-particle correlation distances
Scattering measures both S+P in ‘reciprocal’ space
What is the relationship between q and d?
small q ~ large d [large q ~ small d]
small lambda ~ large q ~ small d
Where:
q - the magnitude of the scattering vector
d - particle diameter
lambda - wavelength
To summarize:
A larger q often corresponds to smaller structural features or finer details within the sample.
Smaller wavelengths (λ) typically lead to larger q values in scattering experiments, enabling the investigation of smaller structural elements.
What is Rayleigh scattering?
Rayleigh scattering is the elastic scattering of light by molecules and particles much smaller than the wavelength of the incident light.
Rayleigh scattering intensity has a very strong dependence on the size of the particles (it is proportional the sixth power of their diameter). It is inversely proportional to the fourth power of the wavelength of light, which means that the shorter wavelength in visible white light (violet and blue) are scattered stronger than the longer wavelengths toward the red end of the visible spectrum.
This type of scattering is therefore responsible for the blue colour of the sky during the day and the orange colours during
sunrise and sunset.
What are the limits to Rayleigh scattering?
Rayleigh scattering only works for particles less than lambda/10, so microemulsions, nanoparticles and polymers can be sized in this way, note though the very strong dependence on d, so experiments are seriously effected by very small amounts of dust.
What is low angle scattering?
Small-angle scattering (SAS) is a technique based on deflection of collimated radiation away from the straight trajectory after it interacts with structures much larger than the wavelength of the radiation.
The deflection is small (0.1-10°) hence the name small-angle.
For particles larger than the wavelength of light, the light scatters from the edge of the particle at an angle which is dependent on the size of the particle.
Larger particles scatter light at relatively smaller angles than light scattered from smaller particles. From observing the intensity of light scattered at different angles, we can determine the relative amounts of different size particles.
As the particles get close to or smaller than the wavelength of light, more of the light intensity is scattered to higher angles and back-scattered.
What is the most suitable method to determine particle size from light scattering measurements?
Fraunhofer diffraction is the simplest method of determining particle size from light scattering measurements. It applies to particles larger than approximately one micron.
In optics, the Fraunhofer diffraction equation is used to model the diffraction of waves when plane waves are incident on a diffracting object, and the diffraction pattern is viewed at a sufficiently long distance (a distance satisfying Fraunhofer condition) from the object (in the far-field region), and also when it is viewed at the focal plane of an imaging lens.
What scattering technique do we use for particles in solution between 1 micron and 50 nm?
Dynamic light scattering
What is dynamic light scattering?
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. (Useful for particles between 50 nm and 1 um)
When in solution, macromolecules are buffeted by the solvent molecules. This leads to a random motion of the molecules called Brownian motion.
As light scatters from the moving macromolecules, this motion imparts a randomness to the phase of the scattered light, such that when the scattered light from two or more particles is added together, there will be a changing destructive or constructive interference. This leads to time-dependent fluctuations in the intensity of the scattered light.
In a QELS (Dynamic Light Scattering) measurement, the time-dependent fluctuations in the scattered light are measured by a fast photon counter. The fluctuations are directly related to the rate of diffusion of the molecule through the solvent. Therefore, the fluctuations can be analyzed to determine a hydrodynamic radius for the sample
The smaller the particles, the faster the fluctuations.
(QELS = Quasi Elastic Light Scattering aka photon correlation spectroscopy)
What is PCS?
Photon correlation spectroscopy
How is gamma related to the magnitude of scattering vector, q?
Γ can be converted to the diffusion constant D for the particle via the relation:
D=Γ/q^2
Where:
Γ - decay rate
q - magnitude of scattering vector
D - diffusion constant
What is q in scattering measurement techniques?
The magnitude of the scattering vector
What are problems with light scattering?
• Only works for single particle scattering, multiple scattering much harder to analyse, thus only works for very dilute suspensions.
• Apart from DLS only relatively large particles can be studied, no information about the nature of particles or their structure can be obtained.
• To go smaller or to investigate smaller features we need to use light of shorter wavelength, such as X-rays or neutrons
How does refractive index effect how we see objects?
Seeing objects requires a difference in refractive index between the object and its surroundings
n (air) ~ 1
n (water) ~ 1.33
n (glass) ~ 1.50
n (toluene) ~ 1.49
Glass beads:
• nearly disappear in toluene
• best visible in air.
Any radiation can be used in the same way as light to “see”. The requirement is a difference between the way the radiation interacts with the atoms and molecules in the object and its surroundings, i.e. CONTRAST.
Explain the fundamentals of neutrons:
• Neutrons have zero charge and negligible electric dipole and therefore interact with matter via nuclear forces (to do with nucleus of atom)
• Nuclear forces are very short range (a few fermis, where 1 fermi = 10-15 m) and the sizes of nuclei are typically 100,000 smaller than the distances between them.
• Neutrons can therefore travel long distances in material without being scattered or absorbed, i.e. they are and highly penetrating (to depths of 0.1-0.01 m).
• Example: attenuation of low energy neutrons by Al is ~1%/mm compared to >99%/mm for x-rays
• They have amplitude and phase
• They can be scattered elastically or inelastically
What happens in elastic scattering?
Elastic scattering changes direction but not the magnitude of the wave vector.
Inelastic scattering changes both direction and magnitude of the neutron wave vector.
It is the elastic, coherent scattering of neutrons that gives rise to small-angle scattering
How is the scattering cross section of an atom found?
The scattering cross section of an atom, σ (sigma)
σ = 4πb^2
Where b is the scattering length of the nucleus and measures the strength of the neutron-nucleus interaction.
..as if b were the radius of the nucleus as seen by the neutron.
How is scattering length of a nucleus, b, found?
• For some nuclei, b depends upon the energy of the incident neutrons because compound nuclei with energies close to those of excited nuclear states are formed during the scattering process.
• This resonance phenomenon gives rise to imaginary components of b. The real part of b gives rise to scattering, the imaginary part to absorption.
• b has to be determined experimentally for each nucleus and cannot be calculated reliably from fundamental constants.
How do isotopes affect neutron scattering lengths?
Neutron scattering lengths for isotopes of the same element can have very different neutron scattering properties.
This is NOT the case for light scattering.
How is scattering length density found?
The average scattering length density rho for a particle is simply the sum of the scattering lengths (b)/unit volume
What is contrast (or solvent) matching?
Matching the scattering density of a molecule with the solvent.
This facilitates the study of one component by rendering the other as “invisible”
What’s DLS?
Dynamic light scattering
What particle sizes is light microscopy suitable for?
Around 100 nm. Visible light range.
What particle size is electron microscopy suitable for?
Around 0.1 nm. Atomic level.
Why is Fourier transformation required?
The Fourier transform is central in scattering. In scattering, an incident wave travels through a sample. All of the entities in the sample act as scattering sources, giving rise to secondary waves that interfere with one another.
The transform inverts the units of the input variable. For instance, when the input stream represents time, the Fourier space will represent frequency (1/time).
What is numerical aperture?
In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light.
Numerical aperture is commonly used in microscopy to describe the acceptance cone of an objective.
What is resolution?
In microscopy, the term “resolution” is used to describe the ability of a microscope to distinguish details of a specimen or sample.
In other words, the minimum distance between 2 distinct points of a specimen where they can still be seen by the observer or microscope camera as separate entities.
Compare AFM and STM (atomic force and scanning tunnelling microscopy)
AFM:
- Works in liquid and gas conditions
- The AFM works on the interaction forces between the tip and the sample surface.
- AFM can image both conductive and non- conductive samples.
- Sub-nanometer resolution in all modes
STM:
- Only works in high vacuum
- STM is a sort of microscopy that scans the surface of a sample using a conductive tip.
- Only images conductive samples.
- Atomic resolution
AFM has a higher topographic contrast, direct height measurement, and superior surface characteristics than STM.
AFM can offer data on a sample’s physical and mechanical properties, such as height, roughness, and elasticity. STM can provide data on a sample’s electronic properties, such as conductivity and electronic structure.
How do displays in real space and reciprocal space vary?
Real space is where the substance’s atoms actually are. Reciprocal space shows the diffraction pattern from the atoms, which may not change in accordance with changes in real space.
E.g. if atoms get closer together, the constructive pattern of the diffracted waves get further apart.
How is hydrodynamic radius (effective radius of an ion in a solution measured by assuming that it is a body moving through the solution and resisted by the solution’s viscosity) calculated?
rh = kT/6πηD
Where:
k is Boltzmann’s constant
T is the temperature in K
η is the solvent viscosity.
rh is hydro’ radius
D is diffusion constant
What does contrast refer to in microscopy?
The difference in scattering length density between particles and solvent
What is spectroscopy?
The study of matter using electromagnetic radiation.
What is Planck’s equation?
What does it show?
E = hv = hc/λ = hcv*
Where E is the energy of a particle of light (photon), v is its frequency, and h is Planck’s constant.
c is speed of light and λ is wavelength.
The wave number is represented by the Greek letter nu (ν) with a tilde over it. Here, it is written as v*
What’s vibrational spectroscopy?
A method of measuring molecular vibrations
What conditions are necessary for exchanges (emission and absorption) to occur between light and matter?
The energy of absorbed radiation must exactly match that of a transition in an atom or molecule.
If the frequency of absorbed radiation exactly matches the frequency of vibration of a functional group in a molecule, the emitted wave is reduced in amplitude. This is due to IR absorption.
What happens when IR is absorbed by a chemical bond?
The bond will experience a jump / increase in energy (in eV).
The amplitude of the vibrations of the chemical bonds will increase. Amplitude of vibration changes, not frequency.
Note - if the bond was not vibrating in the first place, there would be no interaction between the bond and the IR waves.
Define IR spectrum:
A representation of what electromagnetic radiation is absorbed or emitted by a sample
What are the regions of the IR spectrum?
Near IR (near to visible): 14000 - 4000 /cm Overtones and harmonics
Mid IR: 4000 - 400 /cm Molecular vibrations
Far IR: 400 - 10 /cm Rotations / low energy vibrations
How is the energy of a harmonic oscillator calculated? (Simple solution to Schrodinger wave equation)
E = (v+0.5)hv’
v’ = (1/2π)*(k/m)^0.5
Where:
E - energy of the state corresponding to integer v
v - quantum number (integer)
ν’ - frequency
h - planck constant
k - spring/force constant
m - mass
In equation 2, v may be replaced by v* (wave number), becoming (1/2πc)*(k/m)^0.5.
Mass may be replaced by mu. ((mu = (m1m2)/(m1+m2))
What is the vibrational selection rule / rule for quantum number jumps?
For harmonic oscillators, Δv = +/- 1
Jump in only 1 neighbouring energy level can be made at once.
What is μ in spectroscopy?
μ = m1*m2/(m1+m2)
Where μ is the reduced mass if both ends of a diatomic molecule are moving. m are the molecule masses.
How do energy levels for the anharmonic vibrations of real molecules differ to those of harmonic vibrations?
For anharmonic vibrations, as energy increases, the ‘jump’ between energy levels decreases.
Whereas, the jump for harmonic vibrations remains the same. The harmonic model is suitable for real molecules, especially at lower energy levels.
The bonds in actual molecules are not obeying Hook’s law exactly.
The force needed to compress a bond by a definite distance is larger
than the force required to stretch this bond, thus potential is anharmonic.
Define IR active:
Able to absorb IR radiation
Define fundamental transition:
Transition from ground state to first excited state (0 -> 1)
Define anharmonic motion:
Oscillatory motion in which the restoring force is not proportional to the displacement
Define the selection rule for IR activity:
The motion corresponding to a normal mode should be accompanied by a change of dipole moment
∂μ / ∂r ≠ 0
The selection rule for infrared (IR) activity in molecular spectroscopy is a set of guidelines that determine whether a vibrational mode of a molecule will interact with infrared radiation and produce an IR absorption or not.
Here are the key points of the selection rule for IR activity:
- A change in the molecular dipole moment is required. In other words, the molecule must oscillate or vibrate in such a way that the distribution of charge within the molecule changes.
- Symmetry consideration: the vibrational mode must also have the appropriate symmetry properties to interact with IR radiation. Symmetry is a fundamental concept in molecular spectroscopy, and only certain vibrational modes with specific symmetries will be IR active.
- The mode must be associated with a net change in the molecular dipole moment: In a molecule, there may be multiple vibrational modes, but not all of them will be IR active. Only those modes that result in a net change in the molecule’s dipole moment will be IR active.
- The intensity of the IR absorption is directly proportional to the magnitude of the change in the molecular dipole moment during the vibrational transition. Larger changes in dipole moment result in more intense IR absorption bands.
Define normal mode:
A characteristic vibrational pattern of a molecule or group of atoms.
Each normal mode has a discrete vibrational frequency
In general, a non-linear molecule with N atoms has 3N – 6 normal modes of vibration, but a linear molecule has 3N – 5 modes, because rotation about the molecular axis cannot be observed.
A diatomic molecule has one normal mode of vibration, since it can only stretch or compress the single bond.
How can diatomic molecules move?
By rotation, vibration, and translation
A molecule with N atoms has 3N ‘degrees of freedom’
What is the dipole moment?
How do you calculate dipole moment, μ?
It is a measure of the asymmetry of charge distribution on the two atoms (for diatomic molecules)
μ = q * r
Where q is charge and r is distance (bond length)
∂μ / ∂r ≠ 0 during molecular vibration
Why is CO2 a greenhouse gas? (Consider IR)
CO2 molecules can vibrate in ways that simpler nitrogen and oxygen molecules cannot, which allows CO2 molecules to capture the IR photons.
CO2 has 3N-5 (3*3-5 = 4) vibrations as it is a linear molecule - symmetric, antisymmetric, and 2 bending modes.
How many vibration modes do linear and non-linear molecules have?
Linear: 3N - 5
Non-linear: 3N-6
Where N is the number of atoms
What’s a degenerate vibration?
A degenerate vibration refers to a situation where two or more vibrational modes of a molecule have the same vibrational frequency.
These degenerate vibrational modes have identical vibrational frequencies and are often associated with the same symmetry properties.
Degeneracy in molecular vibrations can occur when the symmetry of a molecule allows for multiple vibrational modes with the same energy.
For example, consider a diatomic molecule like oxygen (O2). It has two atoms, and when it vibrates, it can do so in different ways. In the case of O2, there are two vibrational modes associated with stretching the O-O bond. These two modes have the same vibrational frequency, and they are said to be degenerate. This degeneracy is a result of the symmetry of the molecule.
How is absorbance calculated using the Beer-Lambert law?
A = εcl
Where:
ε - molar absorptivity (or absorption or extinction coefficient)
c - concentration
l - length (or thickness or pathlength)
The absorption of a species is proportional to its concentration
A = ln (I0/I) = (ac)c(ab)*b
Where:
I0 - incident intensity
I - emitted intensity
c - concentration
b - path length (l)
ab and ac are constants of proportion ?*
What are the the IR sampling methods? (3)
Transmission (liquid films between transparent salt plates)
Reflection (specular or diffuse. Used for surface coatings, powdered, and textured samples. Surface sensitive)
Attenuated total reflection (ATR) (small penetration depth. Ideal for strongly absorbing samples. Surface sensitive) [Regarding ATR, dp refers to penetration depth of IR beam in the sample - typically 0.2 - 3 um].
What is dp, when looking at IR spectroscopy?
Penetration depth (of IR into the material)
[Equation to calculate is within notes]
How does FT-IR work?
Source of Infrared Radiation:
FT-IR spectroscopy begins with an IR radiation source, typically a thermal source like a heated filament or a globar. This source emits a broad spectrum of IR radiation that covers a range of frequencies.
Sample Interaction:
The IR radiation is directed onto the sample being analyzed. The sample may be in various forms, such as a solid, liquid, or gas. Molecules in the sample interact with the incident IR radiation.
Absorption of IR Radiation:
When the IR radiation interacts with the sample, molecules absorb energy at specific frequencies corresponding to their vibrational and rotational modes. Different chemical bonds (e.g., C-H, O-H, C=O) have characteristic vibrational frequencies, and these are what FT-IR spectroscopy detects.
Detector:
The transmitted light, which has not been absorbed by the sample, reaches a detector. The detector records the intensity of the transmitted IR radiation as a function of frequency.
Interferometer:
The key innovation in FT-IR spectroscopy is the interferometer. Instead of measuring the intensity of IR radiation at each frequency one by one, FT-IR uses an interferometer to obtain an interferogram, which contains information about all frequencies simultaneously.
Fourier Transformation:
The interferogram is then subjected to a mathematical technique called Fourier transformation. This transformation converts the data from the time domain (as a function of time) into the frequency domain (as a function of frequency).
Spectrum Generation:
The Fourier-transformed data results in an IR spectrum, which is a graph of intensity (absorbance) versus frequency (in wavenumbers, typically measured in cm⁻¹). Peaks in the spectrum represent the frequencies at which the sample absorbed IR radiation.
Data Analysis:
The obtained IR spectrum is analyzed to identify the functional groups and chemical bonds present in the sample. Each peak corresponds to a specific vibrational or rotational mode of the molecules in the sample.
Guided inquiry Q1: Do you know any spectroscopic technique?
Visible light/UV
IR
Near-IR
X-ray (crystallography or absorption spec)
Neutron
Raman scattering
Mass spec
Fluorescence
Mössbauer (gamma-ray spec)
EPR spectroscopy, also known as electron spin resonance
(They can be categorised into types: absorption, emission, fluorescence, nuclear magnetic resonance, scattering)
Guided inquiry Q2: What is on the x-axis of an IR spectrum?
Typically, wavenumber ( /cm) is along the x-axis of a spectrum.
The y-axis is typically a measure of transmittance.
For a UV/visible light spectrum, x and y axis may be wavelength and absorbance respectively.
Guided inquiry Q3: Describe the IR spectrum of CO2
x-axis: wavenumber (/cm)
y-axis: transmittance (%)
CO2 is a linear molecule therefore has 4 vibrational modes (3*number of molecules - 5).
However, not all of these vibrations absorb IR. The symmetrical stretch of CO2 is inactive in the IR because this
vibration produces no change in the dipole moment of the molecule. In order to be IR active, a vibration must cause a change in the dipole moment of the molecule.
Only two IR bands (2350 and 666 cm–1) are seen for carbon dioxide, instead of four corresponding to the four fundamental vibrations.
Carbon dioxide is an example of why one does not always see as many bands as implied by our simple calculation.
In the case of CO2, two bands are degenerate, and one vibration does not cause a change in dipole moment.
Other reasons why fewer than the theoretical number of IR bands are seen include:
1) An absorption is not in the 4000–400 cm–1 range.
2) An absorption is too weak to be observed.
3) Absorptions are too close to each other to be resolved on the instrument.
4) Weak bands which are overtones or combinations of fundamental vibrations are observed.
Which of the following molecules display an IR spectrum?
HCl, CO2, C6H6, SF6, N2, O3
HCl (the stretch of bond introduces a difference in the dipole moment of the molecule)
CO2 (bands made by the doubly degenerate bend and asymmetric stretch)
C6H6 (Arenes have absorption bands in the 650-900 cm−1 region due to bending of the C–H bond out of the plane of the ring. Arenes also possess a characteristic absorption at about 3030-3100 cm−1 as a result of the aromatic C–H stretch. Two bands (1500 and 1660 cm−1) caused by C=C in plane vibrations are the most useful for characterization as they are intense)
SF6 (has 2 stretching modes and 4 bending bonds)
O3 (Symmetric Stretch (ν1) - stretching of both O-O bonds while the central oxygen remains stationary. It is the strongest IR absorption band in the ozone spectrum, typically occurring around 1,100 cm⁻¹. Asymmetric Stretch (ν3) - the two O-O bonds stretch in opposite directions while the central oxygen atom remains stationary. This is another strong IR absorption band, typically occurring around 1,250 cm⁻¹. Bend (ν2) - involves the bending of the O-O-O angle. The bend mode is typically observed at around 700 cm⁻¹.
(N2 is a symmetrical molecule so there is no change in dipole moment as it vibrates.
Which of the following molecules display a Raman spectrum?
HCl, CO2, C6H6, SF6, N2, O3
HCl (contains polarizable bonds)
C6H6
CO2 (it has polar covalent bonds that have significant polarizability)
SF6 (polar molecule with polar S-F bonds)
N2
O3 (significant polarizability)
N2 and C6H6 can also produce Raman spectra, but they are generally weaker due to their nonpolar characteristics.
How would you proceed to determine the amount of oleic acid in water?
Sketch curves if needed and indicate the procedure step by step.
Transmission or ATR infrared spectroscopy could be used for this.
First a calibration curve could be made that would take advantage of the Beer Lambert law. The calibration involves the preparation of standards of known concentration. If properly done, a linear trend is found.
Second, this curve could then be used to determine the amount of oleic acid in the aqueous sample. It should be noted that the absorbance measured for the sample should be within the range of absorbance measured for the standards.
Determining the amount of oleic acid in water using spectroscopy typically involves measuring the absorbance of light at specific wavelengths, as oleic acid has characteristic absorption peaks in the UV-Visible spectrum. Here’s a step-by-step procedure for this analysis:
Materials and Equipment:
Oleic acid sample
Deionized or distilled water
Spectrophotometer
Quartz cuvettes or sample cells
UV-Visible spectrophotometer
Appropriate solvent (e.g., ethanol) for preparing oleic acid solutions
Chemical balance
Pipettes and pipette tips
Clean glassware and lab supplies
Procedure:
Prepare Oleic Acid Solutions:
a. Weigh an appropriate amount of oleic acid using a chemical balance to make a stock solution (typically in milligrams or grams).
b. Dissolve the oleic acid in a suitable solvent (e.g., ethanol) to make a concentrated stock solution.
Dilute the Stock Solution:
a. Prepare a series of dilute oleic acid solutions by taking specific volumes of the stock solution and diluting them with deionized or distilled water. The concentrations of these solutions should cover the expected range of oleic acid concentrations.
Blank Solution:
a. Prepare a blank solution by using the same solvent (e.g., ethanol) and deionized or distilled water. This blank solution will be used as a reference to account for the solvent’s absorption.
Instrument Setup:
a. Turn on the UV-Visible spectrophotometer and allow it to warm up according to the manufacturer’s instructions.
b. Set the spectrophotometer to the appropriate wavelength range for oleic acid analysis, typically in the UV-Visible range.
c. Use a quartz cuvette or sample cell that is compatible with the spectrophotometer.
Calibration:
a. Measure the absorbance of the blank solution at the selected wavelength(s). This establishes a baseline for the solvent.
b. Measure the absorbance of each of the dilute oleic acid solutions at the same wavelength(s).
Create a Calibration Curve:
a. Plot the concentration of oleic acid (x-axis) against the absorbance values (y-axis) for the standards.
b. Use this calibration curve to determine the concentration of oleic acid in the samples.
Sample Measurement:
a. Prepare a sample solution by diluting the unknown oleic acid in water.
b. Measure the absorbance of the sample solution at the same wavelength(s) used for the standards.
Calculate Oleic Acid Concentration:
a. Use the calibration curve to determine the concentration of oleic acid in the sample based on its absorbance.
Data Analysis:
a. Record the results, and if necessary, repeat the measurements for the sample to ensure accuracy and precision.
b. Calculate the concentration of oleic acid in the sample based on the calibration curve.
Reporting:
a. Report the concentration of oleic acid in the sample, including the units used (e.g., mg/L or g/L).
https://www.sciencedirect.com/science/article/pii/S0022286019302509
Cocoa butter exhibits several polymorphs. Which technique(s)
could you use to identify the phase(s) in a sample?
Raman spectroscopy - Raman spectroscopy is a non-destructive technique that can provide information about the molecular vibrations and crystal structures of materials. Different polymorphs exhibit distinct Raman spectra due to variations in their crystal lattice structures. By comparing the Raman spectra of the sample with reference spectra of known cocoa butter polymorphs, you can identify the phases present.
X-ray Diffraction (XRD) - XRD can provide detailed information about the arrangement of atoms or molecules in a sample. By analyzing the XRD pattern of cocoa butter, you can determine which polymorph(s) are present in the sample.
The above two are the most important techniques. The below could also be used.
Differential Scanning Calorimetry (DSC) - DSC is commonly used to analyze the thermal behavior of materials, including phase transitions. Different polymorphs of cocoa butter have distinct melting points and enthalpies of fusion. By performing a DSC analysis, you can identify the phase transitions and infer the presence of specific polymorphs.
Polarized Light Microscopy (PLM) - PLM is a microscopy technique that involves the use of polarized light to observe the birefringence patterns of crystalline materials. Different cocoa butter polymorphs exhibit characteristic birefringence patterns under polarized light. By comparing the observed patterns with reference images of known polymorphs, you can identify the phases.
Infrared Spectroscopy (IR) - Infrared spectroscopy can provide information about the functional groups and molecular vibrations in a sample. Different cocoa butter polymorphs may exhibit variations in their IR spectra. By comparing the IR spectra of the sample with reference spectra of known polymorphs, you can gain insights into the phases present.
Microscopy Techniques (Optical and Electron Microscopy) - Microscopy techniques, including optical and electron microscopy, can be used to visually examine the crystalline structure of cocoa butter. Differences in crystal morphology, size, and arrangement can help identify specific polymorphs.
Solid-State NMR Spectroscopy - Solid-state nuclear magnetic resonance (NMR) spectroscopy is a technique that can provide information about the local structure and interactions within a solid sample. It can be used to investigate the differences in the molecular environment of cocoa butter polymorphs.
What is polymorphism?
Polymorphism is the ability of a specific chemical composition to crystallize in more than one form. This generally occurs as a response to changes in temperature or pressure or both. The different structures of such a chemical substance are called polymorphic forms, or polymorphs.
A solid material with at least 2 different molecular arrangements that gives distinct crystal species.
What is ATR?
Attenuated total reflection.
ATR uses a property of total internal reflection resulting in an evanescent wave. A beam of infrared light is passed through the ATR crystal in such a way that it reflects at least once off the internal surface in contact with the sample. This reflection forms the evanescent wave which extends into the sample. The penetration depth into the sample is typically between 0.5 and 2 micrometres.
Attenuated total reflection (ATR) is a sampling technique used in conjunction with infrared spectroscopy which enables samples to be examined directly in the solid or liquid state without further preparation.
How is dipole moment induced by electric field calculated?
μ = αE
The polarizability, a, represents the ability of an applied electric field
E to induce a dipole moment μ in atom or molecule. Polarizabilities
of atoms are isotropic, whereas polarizabilities of molecules may
vary with positions of atoms in the molecule, depending on
molecule’s symmetry.
What is Stokes shift?
The term Stokes shift is used in Raman spectroscopy where it describes whether the Raman scattered radiation is at lower energy (Stokes shifted) or higher energy (anti-Stokes shifted) than the Rayleigh scattered radiation.
When radiation is scattered from a molecule the majority of photons scatter elastically with no change in the vibrational energy of molecule during the scattering process (Rayleigh scattering).
In Stokes Raman scattering the molecule gains a quantum of vibrational energy from the photon during the scattering process and the Stokes radiation, therefore, has a longer wavelength than the incident radiation (less energy)
In anti-Stokes Raman scattering the reverse occurs, with the molecule losing a quantum of vibrational energy during the scattering process and the anti-Stokes radiation, therefore, has a shorter wavelength than the incident (more energy)
Raman peaks are characterised by their wavenumber shift away from the incident radiation, with Stokes peaks having a positive wavenumber shift and anti-Stokes shifts being negative.
If we excite with a laser at 532 nm, where should the Stokes vibrational oxygen Raman peak appear?
Stoke shift / line: v0 - vs
Calculate the oxygen Raman wavelength: according to the Raman shift is 1556 cm-1
With an excitation wavelength at 532 nm the oxygen Stokes line will appear at 1/532 nm – 1556 cm-1
= 18797 cm-1– 1556 cm-1= 17241 cm-1 = 580 nm
How does Raman microscopy work?
Laser Excitation: A monochromatic laser source, typically in the visible or near-infrared range, is directed onto the sample. The laser light is focused on the sample, creating a small, intense beam at the point of interest. This focused laser beam serves as the excitation source.
Raman Scattering: When the laser light interacts with the sample, a small fraction of the incident photons undergoes inelastic scattering, known as Raman scattering. In this process, the energy of the photons is either increased (Stokes scattering) or decreased (anti-Stokes scattering) by an amount corresponding to the energy levels of molecular vibrations and rotations in the sample.
Raman Shift: The energy difference between the incident laser light and the scattered light is called the Raman shift. It is measured in wavenumbers (cm⁻¹) or nanometers (nm) and is characteristic of the vibrational and rotational modes of specific chemical bonds within the molecules in the sample. By analyzing the Raman shifts, you can obtain information about the chemical composition and molecular structure of the sample.
Spectrograph: The Raman-scattered light is collected and directed into a spectrograph. The spectrograph disperses the collected light into its constituent wavelengths, creating a Raman spectrum. This spectrum represents the intensity of the scattered light at different Raman shifts.
Detection: The Raman spectrum is detected by a sensitive detector, such as a charged-coupled device (CCD) or a photomultiplier tube (PMT). The detector records the intensity of the Raman signals across the entire spectrum.
Data Analysis: The Raman spectrum provides valuable information about the molecular vibrations and chemical bonds present in the sample. By analyzing the peaks and patterns in the spectrum, researchers can identify the chemical compounds present, understand their molecular structures, and even quantify their concentrations.
Raman microscopy offers several advantages, including the ability to perform non-destructive and label-free chemical analysis of a wide range of materials, including solids, liquids, and gases. It can be used in various fields, such as materials science, biology, chemistry, and pharmaceuticals, for characterizing and studying a wide range of samples at the microscopic level.
What’s a microtome?
An instrument for cutting extremely thin sections of material for examination under a microscope.
What is Raman band normalisation for?
For quantitative analysis Raman band normalisation is required in order to correct spectra for changes in focus, alignment, laser intensity, and signal attenuation, etc.
Therefore, to compare the two different bands from depth profiles it is necessary to account for possible laser power fluctuations, which was accomplished using band area normalisation.
Example for PET:
In order for the 1096 cm-1 band (crystalline PET) to be assessed it is
necessary to normalise its intensity with respect to a band that is unaffected by crystallinity.
The band at 795 cm-1 was selected for normalization as it is known to be insensitive to conformation or crystallinity.
Band normalisation is performed by dividing the area of the the 1096 cm-1 band by the area of the band at 795 cm-1
What is SERS?
Surface-enhanced Raman spectroscopy or surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures.
What’s a surface plasmon?
A collective oscillation of surface conduction electrons in materials
with a negative real and small positive imaginary dielectric constant
What is TERS?
Tip-enhanced Raman spectroscopy (TERS) is a variant of surface-enhanced Raman spectroscopy (SERS).
TERS uses a metallic-coated tip—typically on a scanning probe / atomic force microscope—to enhance the Raman signal from molecules within a few nanometres of the tip. You can determine the spectrum originating from molecules solely in the small volume close to the tip.
This is done by comparing the spectrum from the surface with and without the tip present. This gives a much higher spatial resolution than normal Raman scattering (nanometre-scale, rather than about 0.2 µm).
