SPECTROSCOPY -- UV/Vis

I. Interaction of electromagnetic radiation (light) with molecules

A. Molecules have different states (configurations) characterized by differences in energy

B. Light has energy determined by its wavelength, l, or frequency, n

c / l = n and E = h n (c = 3 x 10¹⁰ cm/s, the speed of light; h = Planck's constant)

Thus, there is an inverse relationship between wavelength and Energy. In the diagram below, gamma rays have the largest energy and radiowaves the smallest.

<-----Energy

C. If light striking a molecule in State 1 has an energy equal to the difference in energy between molecular State 1 and State 2, there is a probability that a molecule will absorb a photon and jump to the higher energy state, State 2.

The molecule can jump back to state 1 by:

1) reemitting a photon of the same energy (or lower energy in the case of fluorescence) or

2) Transferring energy to another molecule

D. Light waves are described by sinusoidally oscillating electric vectors ,E, and perpendicular magnetic vectors, H. Hence,.they are called Electromagnetic radiation

The probability of absorption is related to the orientations of E (and also H) vectors with respect to the molecule. For now, assume that light waves and molecules are randomly oriented.

E. What magnitudes of energies are involved?

1. UV (ultraviolet) radiation: l = 190 - 400 nm Visible: l = 400 - 800 nm
2. 
   This corresponds to approximately 95 kcal/mole of photons, the magnitude of energy in a covalent bond
3. Energies of these magnitudes correspond to electronic transitions ==> promotion of an electron from one molecular orbital to an unoccupied molecular orbital of higher energy. Absorption of a photon of light has a high probability of occuring when the orientation of the electric vector of the light wave, E, is oriented in a direction which will induce a movement of an electron from one molecular orbital to another oribital of higher energy. Another way of stating this is to say that E is oriented along a Transition Dipole of the molecule.

F. For a single type of transition between two well-defined states, the Absorbance spectrum would be a very sharp peak.

In practice, there are a range of possible transitions from various vibrational energy levels of the ground state to various vibrational energy levels in the first excited state ==> a series of narrow absorption peaks of similar energies; these cannot be resolved resulting in a single broad and sometimes complex peak for each type of electonic transition.

II. Measurements

A. Instrumentation

1. Single Beam Spectrophotometer
   

2. Split Beam Spectrophotometer
   

Can have 2 detectors or use 1 detector and read I and Io successively

B. Quantitatively:

For light passing through a solution, the rate at which photons are absorbed as a function of distance, l, through the sample cuvette can be thought of as a 1st order reaction and is proportional to the concentration, c, of absorbing molecules with a rate constant we'll call e'.

Thus:

change to log10

==> A_l = e_l c l where A and e are for a specific wavelength, l

This is the Beer-Lambert Law or (more commonly) Beer's Law

III. What Electronic properties lead to absorption of Light?

A. Look at a simple molecule -- Formaldehyde ==> H₂C=O

1. Electronic Configuration:
   C -- 1s² 2s² 2p_y¹ 2p_x¹ 2p_z⁰ ----->1s² (3sp²)³ 2p_z¹
   O -- 1s² 2s² 2p_y² 2p_x¹ 2p_z¹
   C uses 3 sp² hybrid orbitals to form 3 s-bonds with O and the 2 H's; the remaining 2p_z orbital forms a p bond with O. O has the 2p_y atomic orbital which is not involved in bonding, and it contains a non-bonding pair of electrons.
   

2. Molecular Orbital Diagram -- only higher energy orbitals are shown
   
   • n = non-bonding orbital (O 2p_y)
   • p = bonding orbital (C 2p_z + O 2p_z)
   • p* = anti-bonding orbital (C 2p_z - O 2p_z ) -- has a node between the two atoms
3. Quantum Mechanics:
   • p ---> p* transition is "allowed" and will occur when E is parallel to the x axis
   • n ---> p* transition is "symmetry forbidden" ==> transition does not induce a dipole change in the molecule. This transition does occur because of limitations in theory used to predict transitions but with very low probability, <1% of p ---> p*.

B. What about Proteins? Protein absorbances come from 3 sources:

1. Peptide Bond
   

   Asp, Glu, Asn, Gln, Arg, and His sidechains have absorption in this region also (190 -- 230 nm) but is very weak compared to peptide bond p --> p*. Absorption in the 190-230 nm region can be and is used to quantitate protein/peptide concentrations, but this is complicated by many compounds used in buffers which also absorb at these wavelengths.

2. Aromatic Amino Acids -- most useful
   Phe e₂₅₀ = 400 symmetry forbidden p --> p*
   Tyr e₂₇₄ = 1400 p --> p*
   Trp e₂₈₀ = 4500 at least 3 different transitions. A₂₈₀ is one of the most commonly used methods to measure protein concentration (aside from colorimetric methods such as the Lowry or Bradford dye binding assays), but this method is obviously very sensitive to differences in amino acid composition.
3. These transitions can change with pH; especially Tyr
   

4. Prosthetic Groups
   Nucleotides --> e.g. FMN, NAD
   Heme
   Cu
   Retinal etc..

C. Estimating Protein Concentration

1. Generally measure A₂₈₀ and assume an average composition of Tyr and Trp:
   Approximately 1 mg/ml ---> 1.0 A
2. Measure absorbance of peptide groups at l = 230 nm: 1 mg/ml ---> 3.0 A
   This is not commonly used because many other groups absorb in this wavelength region.

D. Nucleic Acids:

Nucleotide spectra are complicated to analyze quantitatively because there are many non-bonded electrons. Expect several different p --> p* and n --> p* transitions at each region between 200 nm and 300 nm

1. All nucleotides have l_max near 260 nm which is not affected by sugar phos. configuration ==> can measure nucleic acids at 260 nm to estimate concentration. e₂₆₀ =~ 1 x 104 M-1 ===> very sensitive and can measure concentrations down to approx. 3 µg/ml
   

2. Hyperchromism -- A₂₆₀ is lower for dsDNA than for ssDNA or for individual nucleotides. Results from stacking of bases in the double helical conformation; quantitative explanation is very complicated. (see Figure above)

IV. Optical Activity: Circular Dichroism (CD) and Optical Rotary Dispersion (ORD)

A. Both types of spectroscopy study the effects of chiral molecules on plane polarized light.

Chiral Molecules can exist in two forms which are non-superimposable mirror images of one another.
Plane polarized light can be described by 2 components of circularly polarized light whose electric vectors rotate in opposite directions during propagation.

E_L -- rotates counter-clockwise during propagation

E_R -- rotates clockwise during propagation

The sum of these two is a wave whose E oscillates in the same plane during propagation

Non-chiral molecules interact with Left Circularly Polarized Light (LCPL) and Right Circularly Polarized Light (RCPL) in exactly the same way.

1. Chiral molecules, however, transmit RCPL: and LCPL differently; n_R (index of refraction for RCPL) is not the same as n_L and one is slowed down with respect to the other; this is a result of the fact that chiral molecules have their electrons distributed in an asymmetric way so that they interact differently with RCPL than with LCPL. Furthermore, the two forms of chiral molecules (which are mirror images of one another) interact with RCPL and LCPL in opposite ways. For example, if n_R > n_L ==> RCPL is retarded with respect to LCPL and the plane of polarization (the orientation of E for the sum of RCPL and LCPL) will be rotated to the left. The chiral molecule with opposite hand of the one described in this example will have n_R < n_L and will rotate plane polarized light to the right.
2. Amount of retardation -- hence the amount of rotation -- is directly proportional to the concentration in gm/cm3, c, and the path length in decimeters, d.

where [a] is the specific rotation in gm/cm³;
[M] is the specific rotation per mole of protein; and
[m] is the specific rotation per amino acid residue (Mo is the mean MW per aa-residue)

[a] measured as a function of l is called the Optical Rotary Dispersion Spectrum or ORD

B. Circular Dichroism (CD)

If l is at an absorption band of the molecule being studied, then e_L does not equal e_R ====> e_L - e_R = De, the Circular Dichroism

1. CD is measured first by passing LCPL and then RCPL through the sample and subtracting, A_L - A_R which is directly proportional to e_L - e_R = De
2. Plane polarized light becomes Elliptically Polarized Light
   

3. One measurement of CD is the Ellipticity = Q_l = Tan^-1(b/a)
4. 
5. ORD and CD contain the same information and are related by Kronig-Kramers Transforms. CD is the most commonly used technique.
6. CD is a very sensitive probe of macromolecule conformation. All amino acids are chiral except for glycine; when a protein folds up into regular secondary structure, the result is a very chiral molecule. This is especially true for a-helices which normally form with a right-handed helix ===> not only the components are chiral, but the conformation is chiral as well. Different conformations have characteristic CD spectra (see figure below). One can measure the CD spectum of a protein and determine how much of each type of secondary structure (a-helix, b-sheet, random coil) by finding which combination of the basis CD spectra for each type adds up to give a spectrum which is the same as the spectrum of the protein. Similarly, one can measure conformation and conformational changes in nucleic acids by CD.