SpectroscopyEnergy of a molecule can be written (to a

reasonable approximation)E = Ekinetic+Erotational+Evirbational+Eelectronic+Enuclear

Molecules, M, tend to sit in their lowest el and vib state. When electromagnetic radiation is incident on M, if emr is resonant with an energy gap to another state of M, then M can be ‘pushed’ into new level: spectroscopy is the measure of that energy and how easily it happens.

Microwave: rotations; Infrared: vibrations; UV-visible: electronic (+vibns).

AbsorbanceBonds Molecules SpectroscopyMolecules are 'glued together' by electrons between the atoms. ~ Two electrons per bond.Approximation: electrons can be put into independent orbitals (with defined spatial characteristics). Two of Opposite spin.Two types of bonds are important for bio molecules:

(sigma) bonds look like s-orbitals when viewed along the bond axis.

(pi) bonds look like p-orbitals (dumbbells) when viewed down the bond axis

Also non-bonding pairs, e.g. lone pairs on N & OUltra violet/visible (UV/vis): how much energy is

required to push electrons to new orbitals.Infra red (IR): strong bonds high cm1, bends low cm1

electron systems

electrons are held less tightly than electrons so require less energy to excite to unoccupied orbitalsMany biological systems have alternating = and bonds

making conjugated systems: electrons held even less tightly coloured compounds.

E.g. -carotene

Most biomolecule spectroscopy used is or n

Electrons bonds structure

.

UV/visible light: ~ 180 nm – 800 nm, energy hhc/causes electrons to go to higher energy levels.

required depends on electron rearrangement needed.

In solution: broad bands due to diff. vibrational levels in excited state &molecules having slightly different energy levels.

h

UV: –350 nmVis: 400 nm –

Excited electronic state

Ground electronic state

Ground vibn’l levelr

Absorption spectra of DNA & proteins

With proteins and DNA the transitions we usually study are n * and * so can access them with normal spectrometers.

Below 200 nm need nitrogen purging because O2 absorbs

Polarization of transitionsDirection electrons move during a transitionOften defined by the symmetry of the molecule * transitions are in the plane of the chromophore

AbsorbanceAbsorbance: A = log(Io/I) = log(intensities in/out) Beer Lambert A = cl, extinction coefficient (units?),

c concentration, l length (cm) Oriented samples and polarised light: only light whose electric field pushes the electrons along the polarisation direction causes a transition

OH

µ

µ

µ

µ

ProteinsFar UV (250 – 180 nm) dominated by peptide group

Many buffers absorb here — so beware!First n* 210 – 220 nm, weak (~100 cm1dm3mol1)First * 180 nm, stronger ( ~ 7000 cm1dm3mol1)

In -helix only has a component at 208 nm?? n* transition 175 nm, charge transfer between amidesSide chains absorb in this region: aromatics, Asp, Glu, Asn, Gln, Arg & His. Usually small.

O

N C

*n*

ProteinsNear UV — 'aromatic' side chains. Tryptophan: Indole side chain, absorbs 240 – 290 nm,

3 or more transitions, ~5,000 cm1dm3mol1

Tyrosine: 274 nm, ~1400 cm1dm3mol1

Phenyl alanine: 250 nm, ~200 cm1dm3mol1

Cystine (disulfides): 250–270 nm, ~300 cm1dm3mol1

pH: tyrosine & tryptophan have protonation sites thatdirectly affect conjugation of chromophore

tyrosine -OH, pKa ~ 10.9 shifts from 275 nm to 295 nm

hemes, flavins, pyridoxal phosphate, metalloprotiens have intense UV or visible absorption bands.

Protein concentration from UV absorbance(280 nm, tryptophan) = 5,700 mol1 dm3 cm1

(280 nm, tyrosine) = 1,300 mol1 dm3 cm1

So can determine protein concentration using: 280 = (nW (5690) + nY(1280) + nC(120))

Equation is valid provided:- · no contribution from light scattering· no other chromophore (e.g. cofactor) in the protein· no other absorbing contaminant, e.g. nucleic acidsAlt. 1 mg/mL av. abs = 1.1±0.5Or 1.55 A280 - 0.76 A260 = mg protein/mL (accounts for nucleic acid contamination) up to 100% errorOr use chemistry to create visible molecules from reaction with peptide backbone need standards with similar

response

Absorbance spectra

Nucleic acids

Near UV absorbance of nucleic acids due (almost) exclusively to planar purine and pyrimidine bases

Backbone from 190 nmEach 'simple' band observed is a number of transitionsLigands bind change DNA

A and ligand A.Intercalators upon DNAbinding: red shift (4-20nm) & hypochromoism ( up to 50% A)and less structured spectrum

P OO

O

H2C O N

O

P OO

O

H2C O

O

H

N

O

Me

H

O H

N

N

N

NN

H

PO O

O

CH2O

O

PO O

O

CH2O

O

HN

N

ON

H N

NN

N

N

H

O

H

3'

5'

5'

3'

Cytosine

Guanine

AdenineThymine

DNA melting curves

A

40 80/CdA/dt

DNA absorbance as a functionof Temperature: helix coiltransition.

DSSS + ~ 10% Abs.Tm is 50:50 DS:SS.

Maximum of derivative is ~ Tm

Determine thermodynamic data from shape of curve.AT rich Tm lower than GC rich

A increases due to loss of- stacking interaction (cf. intercalators)

Protein infra red absorbance

Use cmUse cm11 as energy unit as energy unitAmide I C=O stretch: solution ~ 1690, solid ~ 1650 cmAmide I C=O stretch: solution ~ 1690, solid ~ 1650 cm11

Amide II N-H bend: solution ~ 1600, solid ~ 1640 cmAmide II N-H bend: solution ~ 1600, solid ~ 1640 cm11

-helix + unordered: 1650 cm-helix + unordered: 1650 cm11

-sheet:1618, 1632, -sheet:1618, 1632, 1661 cm1661 cm11

-turns: 1660-turns: 16601679 cm1679 cm11

non H-bonded C=O: non H-bonded C=O: 1700 cm1700 cm11

Use DUse D22O in most cases.O in most cases.

CaF2 cellsBSA 20 mg/mL0.1 mm pathlengthD2O

Extinction coefficients

varies with species, , and concentration.To determine (ideally) 3 independent weighings +

2 serial dilutions Beer Lambert Law.Often use single point, but beware!

Watch effects of pH — changes species & hence .

Binding constants from UV

))((

)1((

)(

2121

1211

2211

xc

xxc

xxcA

tot

tot

tot

usually changes when two molecules bind. Simple e.g. change pH to add or take proton.

1: extinction coefficient of species 1 x1:mole fraction of species 1

A: total absorbance ctot: total analyte concentration

etcIsosbestic points (constant absorbance during a

titration where the total concentration of analyte stays the same, but species is in more than one form)

means that it is in two states.

)/()( 2121 totc

Ax

Determine the concentration of p-nitrophenol (PH) and its conjugate base (P-) at pH = 7.2 from the data at 315 nm.

A315(pH=4.5)=0.55; A315(pH=10.3)=0.08; A315(pH=7.2)=0.30A(pH=7.2) = {[PH]7.2(PH@315) + [P-]7.2(P@315)}l

(PH@315)= 0.55/(5105) = 11,000 moles1 dm+3 cm1

(P@315)= 0.08/(5105) = 1,600 moles1 dm+3 cm1

[PH]7.2=(5105)x1 ; [P]7.2=(5105)x2=(5105)(1x1)

A(pH=7.2) = 0.30 = 5105 [11,000x1+1,600(1-x1)]X1={0.30/(5105)1,600}/{11,0001,600} = 0.47X2=10.47=0.53

[PH]7.2=(2.3105); [P]7.2=(2.7105)

NB if pH = 7.2, then [H+] = 107.2

So equilibrium association constant for H+ and P is:

Kassoc=[PH]/{[P][H+]}=(2.3105)/{(2.7105) 107.2}=1.4107

Roles in spectroscopy of:1. Electromagnetic spectrum, esp. frequencies and

wavelengths of UV, visible and IR2. Lenses3. Mirrors4. Gratings and monochromaters5. Prisms and quarter/half wave plates and

monochromaters6. Photoelastic modulators7. Diode detectors8. Photomultiplier tubes9. Why does Beer-Lambert law work (relate to

definition of absorbance)10. IR light source11. Data processing of photon count to plot on

screen.12. Compensation of lamp energy variations