Lecture 3: Absorption

Question 1

How does absorption work?

Answer 1

• Absorption involves energy level transitions.
• The Boltzmann distribution is recovered by relaxation.
• EM radiation stimulates absorption and emission equally, that is why we need a population difference to see an effect.
• The sensitivity depends on the population difference..

Question 2

What data output do we get from absorption?

Answer 2

• Data output is a spectrum of energy absorbed vs frequency/wavelength. We get resonances of broad peaks or unresolved lines.
• The width of peaks depends on the lifetime of the excited state.
• If a decay is fast, position is uncertain and the line is broad. If decay is slow, lines are sharp.
• The linewidth of peaks directly affects the spectral resolution and hence the information content of the spectrum.

Question 3

How does UV/Vis spectroscopy work?

Answer 3

UV/vis spec can detect electronic transitions. It has a range of 200-750 nm.
• It can detect molecules called chromophores which absorb in this range. Examples are: aromatic amino acids, nucleotide base and NADH.
• Its advantages are it is inexpensive, simple, very fast and very sensitive (micromolar range). It can find a sample concentration with the Beer-Lambert law. It also requires very little training.
• The equilibrium position for excited electrons tends to be greater because excited orbitals are further from the nuclei.
There are two main set ups.
1. Standard spectrophotometer: can only measure one wavelength at a time using a mono-chromator.
2. Diode array spectro-photometer: can measure many wavelengths at the same time, much faster. It uses a prism.

Question 4

How can use UV/Vis spectroscopy?

Answer 4

The Beer-Lambert law can be used to find concentration. We find the absorption spectrum of a molecule of an interest, so we can find the maximum.

A=log⁡( I0/It)

A= εcl

Absorption spectra can change based on chemistry or based on the environment. Interactions between chromophores in native double-stranded DNA reduces the absorption intensity. 
Transition metals can be viewed using this technology. The spectra can be changed by geometry and ligands. This is very useful for metals in proteins such as zinc in carbonic anhydrase. 
UV/vis can also be used to measure rapid reactions using a stopped flow device. 
Ultra-fast lasers (10-9 - 10-15 s) can measure very fast reactions (<10-13). This has been used for studying rhodopsin, LHC and photosystems. 
Peptide bond absorption occurs at 200nm. However, we can calculate the BL coefficient for 230 nm where trp, thr, phe and cysteine (S-S) absorbs. εprot= ntrp x εtrp + ntyr x εtyr + ncystine x εcystine
Nucleic acids absorbs from 230-290 nm, which comes mainly from the bases.

Question 5

What is the isosbestic point?

Answer 5

Isosbestic points are points at which the observed spectrum where the BL coefficient is the same for two molecules in equilibrium. Using two of these points, we can find the absolute concentration of the two molecules.

Question 6

What is IR spec?

Answer 6

IR spec detects molecular vibrations.
• It is used for H-bonding, secondary structure, H/D exchange and ionization states.
• However, it does have a number of limits. It has a strong absorption from solvents and cells. It also has a large number of spectral peaks, which gives problems with resolution and peak assignment.
Measurement is very simple.
1. IR is emitted using a heated metal carbide, giving multiple wavelengths.
2. The radiation is monochromated using a prism or grating.
3. The radiation hits the sample in cells which don’t absorb e.g. sodium chloride.
4. The heat is detected, and a spectrum is found.
• The kinetic isotopic affect is often exploited as a way to assign observed bands to specific groups. The KIE occurs because there is a change in vibrational frequency caused by the change in mass of the isotope (refer to above equation).