lecture 6 Flashcards
How can the EM spectrum be divided up?
Near IR , mid IR and far IR
What can near IR detect?
quantitative determination of species such as proteins, fats, low low-molecular-weight hydrocarbons and water. Further use in the agricultural products, food, petroleum and chemical industries
What can Mid IR detect?
Most popular of the IR fields, used in determining structure of organic and biochemical compounds
What can far IR detect?
less popular though it has found uses in inorganic studies
What causes vibrational motions?
due to the attraction and repulsion of charges in the atoms of a molecule
What is a vibrational motion?
- where the bonds between atoms in a molecule move
What is the vibrational motion for simple atomic molecules?
have one bond and one vibrational bond
What are the 6 different types of vibrational motion?
- symmetric stretch
- asymmetric stretch
bend - vibration in the x
- vibration in the y
- vibration in the z
What are the type of stretching vibrational modes?
- symmetrical
- asymmetrical
What are the types of being vibrational modes?
- scissoring
- rocking
- wagging
- twisting
What happens f the radiation matches the vibrational frequency of the molecule?
then the radiation will be absorbed
How can a sample be “IR active”
it must be associated with changes in the dipole moment
What is permanent dipole?
when 2 atoms have substantially different electronegativity, so one atom attracts more electrons than the other, for example in the image here, which shows electron density
What is an instantaneous dipole?
occur due to chance when electrons happen to be more concentrated in one place than another in a molecule
What is an induced dipole?
occurs when a molecule with a permanent dipole repels another molecule’s electrons, inducing a dipole in that moment – called polarised.
What is the Four transform infrared spectroscopy?
A mathematical process, which decomposed a function of time into frequencies that make it up
What does the Fourier transform actually do?
Converts the raw data into an actual spectrum
What is the Fourier transformer based off?
The Michelson interferometer
What does the Fourier transform need?
Beam splitter; 2. Fixed mirror; 3. Movable mirror
How does the Fourier transform Infrared Spectroscopy (FTIR) work ?
- IR hits the beam splitter , transmits and reflects the other half
- 2 beams reflected by fixed and moving mirror recombine to form a resultant beam . Creates and interference pattern
- combined beam reaches detector by passing through the sample
- spectrum is called an interferogram
What are the 3 main uses of IR spectroscopy?
- identification of functional groups
- Secondary structure information
- difference spectra
How can you identify functional groups from the IR spectrum?
- graph with group frequency region and a fingerprint region
How do breathalysers work?
collect evidence to determine levels of alcohol in breath with IR spectrometers
How can secondary structure be determined by IR spec?
- proteins held together by H bonds
- these bonds absorb radiation n
What is the main bond that absorbs IR radiation?
- The amide bond
What happens when there is decomposition of the amide 1 bond?
β-sheet: 2 peaks at 1630 and 1690 cm−1
α-helix: peak at 1652 cm−1
Random coil: 1660 and 1700 cm−1
What is difference spectra?
- looks at the difference between two spectra acquired in two different states of a protein
- i.e. the same protein but in different environments
How is difference spectra measured?
- an initial spectrum is measured without light
- then light or chemicals is added
- the difference between these spectra is measured and compared
What is advantage of the difference spectra method?
- Not only allows the meaurement of the difference between the final and initial states, but also the following of the the reaction time
What is another advantage of the difference spectra method?
- comparison between resting and active state when light is added
what differences might we observe In difference spectra between 2 samples of the protein in different conditions?
- Dark/light
- Different buffer pH
- Bound to different small molecules
What differences might we observed in difference spectra in the same sample before/after activation?
- Intrinsic light reaction
- Electrochemical trigger
- After release of a photo-activated substrate
What can the difference in bond peaks determine
the bond type - but not which exact bond in the protein it is
How do e determine which exact bond in the protein it is?
- Site directed mutagenesis
- Isotopic labelling
