FT-IR Spectroscopy

FT-IR

• Fourier transform infrared spectroscopy is a technique used to collect infrared spectra.

Old vs. New Spectrum

• Old spectrum collection involved a grating, resulting in less clean and sharp data.
• New spectrum collection uses FT-IR, producing sharper, more detailed features.

Interferogram

• An interferogram is created using an IR radiation source, a half-silvered mirror (beam splitter), a fixed mirror, a translating mirror, and a sample.
• Time domain data from the interferogram is converted into a spectrum, with the x-axis representing cm-1.

Transmission Mode

• In transmission mode, IR radiation is transmitted through the sample.

FT-IR as an Instrument

• FT-IR functions as a single-beam instrument.
• It involves taking a background spectrum to account for noisy backgrounds and then a sample measurement.
• Software subtracts the background from the sample measurement.
• Multiple scans are taken and ensemble averaging is used to increase the signal-to-noise ratio (S/N).

Sample Holders

• For gases, sample holders use windows made of KBr, NaCl, or CaF2 (salt plates).
• For liquids, a neat liquid is placed on a NaCl or KBr plate, then pressed with a second plate on top creating a sandwich.
• For solids, the sample is mixed with KBr powder in a mortar and pestle, then pressed into a pellet using a pellet press.

ATR

• ATR stands for Attenuated Total Reflection.
• The difference between diamond tip and ATR crystal results in Total Internal Reflection (TIR).
• The ATR crystal allows IR radiation to travel into the sample and back out many times, with part of the IR light being absorbed.

IR Light Sources

• Black body radiation involves a heated material that emits light.
• IR sources include the Nernst glower (a ceramic heated to incandescent), silicon carbide (acting as a black body), coiled wire sources, Globar sources and solid state sources.

Pyroelectric Detectors

• Pyroelectric detectors generate an electrical signal when heated or cooled.
• Pyroelectric detectors can be noisy.
• Examples include LiTaO3 and DLATGS (Deuterated lanthanum alpha-alanine doped triglycine sulfate).
• A black coating absorbs IR radiation.

Comparing IR Detectors

• A pyroelectric detector is noisy.
• LiTaO3 is noisier than DLaTGS but more rugged and durable.
• DLaTGS can provide 4x higher S/N than LiTaO3.
• Ensemble averaging can increase S/N if we cannot change detectors.

Beer's Law

• UV/Vis has high sensitivity.
• FT-IR has poor sensitivity.

Qualitative Measurements

• FT-IR can be used for qualitative measurements, but is not as sensitive as UV/Vis.
• FT-IR is best for qualitative measurements.

Raman

• Raman is a complementary technique to FT-IR spectroscopy for qualitative organic structure identification.
• Water is not an interference.
• Aqueous samples can be analyzed.
• Glass or quartz sample holders can be used.
• It allows for rapid and non-destructive analysis, including measuring contents of a bottle without opening it.

Raman History

• Historically, the sun was used as a source, with Raman filtering green light through a sample of CHCl3 (chloroform).
• Most of the light was green, but a small portion was yellow, representing inelastic Raman scattering with some energy loss.

Raman Signal

• Raman signal is light that is inelastically scattered.
• Both elastic.
• Laser wavelength will also be scattered (rayleigh scatter).
• 999,999 photons for every 1 raman scattered photon.

Raman Effect

• A small amount of scattered light has a different wavelength than the incident light.
• The change is due to the chemical structure of the molecule.
• The signal that is detected is the inelastically scattered light (Raman).

Raman Shift

• The shift in lambda is caused by absorption of a small amount of the photons energy.
• That energy corresponds to a vibrational level.

Comparing FT-IR to Raman

• For FT-IR, a molecule must experience a change in the dipole moment to be IR active.
• For Raman, no change in dipole moment is necessary.
• Examples are 12 and O2, which are Raman active.

Raman Spectra

• Raman spectra look similar to IR spectra.
• Both have peaks due to exciting vibrational levels.
• For some functional groups that give weak (or zero) IR signals, a strong Raman signal can be detected.
• Excitation line= rayleigh scattering

FT (fourier transform)

• A math algorithm that converts a signal in the time domain to the frequency domain.
• It greatly improves S/N and can isolate a weak signal from environmental noise.
• It is useful when the instrument is detector noise limited, such as with IR, microwave, and NMR.

Frequency Dispersion

• Frequencies are dispersed mathematically rather than physically.
• It is a Non-dispersive instrument.
• No grating.
• No need for a monochromator.
• Uses an interferometer instead of a grating.
• Requires a powerful computer.
• Fourier transform spectroscopy is done by including an interferometer in the instrument.

Michelson Interferometer

• Incoming light is split with a beamsplitter and then recombines to cause interference which results in an interferogram.
• Fourier transform converts the interferogram (time domain) into a spectrum (frequency domain).
• f= 2 v(mirror)/ lambda.

Resolution

• What kind of resolution can we get with the fourier transform dispersing our radiation.
• It depends on the distance the translating mirror travels.

FT Advantages

• FT provides an advantage over physical dispersion with monochromator.
• When instrument is detector noise limited- same amount of noise in signal whether you are measuring a small signal intensity or a large one.
• Jacquinot advantage is because scattering light mathematically you do not use as many optics that can cause light to be lost (reflection losses and narrow slits).
• Higher signal, same noise= higher S/N and better resolution.
• Fellgett advantage is because you are measuring all the wavelengths simultaneously, you can save time. Or can use time wisely by taking numerous scans and using ensemble averaging to improve S/N.
• Interferometers are very precise- great wavelength reproducibility and resolution.
• Makes it easier to see fine spectral features.
• The bottom line on detectors is Diffraction grating monochromatic with CCDs are best for UV and visible light.
• They can provide good S/N in real times.
• They are cheap, small, and can be portable.

FT is best for IR

• FT is best for IR because it improves S/N.
• IR detectors are noisy.
• FT is best for techniques that are detector noise limited including NMR and microwave spectra.

NMR

• 1952 nobel prize.
• 1953 first instrument was sold.
• It is the most powerful tool available for structural elucidation.
• Provides structural info about how the molecule is put together.
• Either the atomic number, the mass number, or both must be odd.
• PURE SAMPLE.
• Structural qualitative analysis.
• NMR: all about the nucleus.
• A spinning nucleus creates a magnetic field. In the absence of an external magnetic field (Bo) all energies are identical.
• In an NMR, an external magnetic field is applied.
• The application of magnetic (external) field causes the energy level to split.
• The magnetic moment of the nucleus becomes oriented in one of two directions.
• The nuclear magnetic energy levels of an atom located in a magnetic field will be split by the field
• The difference will correspond to radio frequency.
• Between 3KHz and 300GHz
• Magnetic moment.
• Created by the spinning nucleus
• Magnetogyric ratio.
• Constant for a particular isotope.
• In the RF range.
• This is the absorption frequency, also called the resonance frequency, also called the Larmor frequency.
• It is dependent on both the magnet in the NMR instrument and what kind of isotope you are investigating.
• Comparison of data for different RF.
• Higher magnetic field= higher resolution.

Nuclei and Signal

• Nj is the number of higher energy protons (M-12).
• No is the number of lower energy protons (M+1/2).
• We need a difference between the two to get a net signal.
• An excess of lower energy nuclei is needed to create an NMR signal (Nj>No). Very little difference between the two.
• We do what we can to increase S/N. Decrease temp. Increase magnetic field.
• A nucleus absorbs RF energy.
• The frequency of the precession (staggering spin) is called the Larmor frequency.
• This is the frequency difference between split energy levels.
• It is the frequency absorbed then emitted by the nuclei.
• It is in the RF range.
• After the energy is absorbed.
• The nucleus wants to get rid of it.

Relaxation

• We call this relaxation.
• In NMR this is the emission of the RF energy.
• The emitted RF energy is measured as the NMR signal.
• It is slightly different for different functional groups.
• Fourier transform NMR.

Fourier Transform

• The RF energy is pulsed, allowing the nuclei to relax before being excited again.
• The signal can be observed as a decay from the excited state, which lasts as long as the relaxation processes are going on.
• This is called a free induction decay.
• FID = It is an interferogram. Superconducting magnet
  • Niobium/tin or titanium bathed in liquid He
  • High field strength, stable, relatively small size
• Sample cell: glass tube, spinning in a magnetic field
• For H NMR use solvent with no H
  • CDC13
    • 2-15% solution RF transmitter/receiver
• RF receiver
• Coiled around the sample tube
• Signal received is very small, it must be amplified
• NMR signal is detector noise limited
• Ensemble averaging can help increase S/N

Fluorescence

• Types (light is emitted)
  • Fluorescence
  • Phosphospherence
  • Chemiluminescence
  • Bioluminescence
• The intensity of the light is proportional to the concentration of the analyte
• Fluorescence
  • The molecule emits photons, some amount of time after absorbing photons
  • The emitted light is the luminescence
• It is different than UV Vis spectroscopy because the wavelength of the emitted light is longer than the wavelength of absorbed light
• Some energy has been lost
• Not all molecules fluoresce

Fluorescence sensitivity

• Fluorescence is a more sensitive technique than UV/vis absorption- one of the most sensitive techniques we have
• The intensity of the fluorescence can be measured independently of the source intensity (beers law)
• Very intense light sources can be used to increase the fluorescence signal: lasers, pulsed Xe lamp
• Quantum yield
  • Photons emitted/absorbed
  • Strongly fluorescent compounds have Q- 1
  • Weakly fluorescent compounds have Q near 0
• Electronic transitions
• One feature fluorescent molecules have is delocalized electrons
• For this reason transitions are typically
  • Pi to pi star
  • N to pi star
• Quinine
  • Exc 350nm
  • Em 450nm
  • Q= 0.5-0.6
• Another fluorescent molecule
  • Green fluorescent protein
  • Intramolecular bonding leads to rigidity

External Fluoresence Influences

• External influences on a molecules fluorescence
  • Decrease temp will increase fluorescence
  • If solvent contains heavy atoms this will decrease fluorescence
• Increase dissolved O2 will quench fluorescence
  • Quench means decrease pH
  • Both em and fluorescence intensity can change depending on whether the molecule is protonated or not

Other Factors

• Excitation and emission spectra
  • The absorbed photons have shorter wavelengths than fluorescence (emitted) photons
• Instrumentation
  • Fluorimeter
  • Exc UV
  • Em vis
  • Light source, slit, mirrors, diffraction grating, sample cuvette (quartz), mirrors, diffraction grating, detector (PMT)
  • Simpler filter-based instrumentation
  • Biochemists
  • Pulsed Xe lamp
• Fluorescent tags
  • Proteins can be tagged
  • Fluorescein
    • Exc 494nm
    • Em 521nm
  • Reacts with protein amine groups

Bioluminescence

• A biochemical reaction provides energy to excite fluorescence.