What is Spectroscopy? The study of the interaction of electro- magnetic radiation with matter. We use spectroscopy as a tool to look indirectly at molecules.

What is Spectroscopy?

• The study of the interaction of electro-magnetic radiation with matter.

• We use spectroscopy as a tool to look indirectly at molecules.

Spectroscopy

Electromagnetic Radiation

E = Electric field

H

E

H = Magnetic field

y

x

z

Direction ofpropagation

Wavelength

• Transports energy.

• Electric and magnetic fields oscillate: that’s the “wave”.

• Moves at speed of light.

• Wavelength, frequency, energy all related.

WavelengthDistance between crests

AmplitudeHalf the height of trough to

crest

FrequencyNumber of crests that pass a point in space every second

E = h c / = c / = c /

c = speed of light (3 x 108 m/s)E = energy = wavelength = frequency h = Planck’s constant (4 x 10-15 eV sec)

Spectroscopy

Wavele

ng

th

(nm

)

En

erg

y a

s w

aven

um

bers

(c

m-1)

Microwave

Infrared

Visible

Ultraviolet

X-rays

Vibrational

transitions

104

105

103

102

10

1

103

102

104

105

106

107

Rotationaltransition

s

Electronictransition

sInner shell

Electronictransition

sOuter shell

Each region

Differentspectroscopic technique

The spectroscopic technique used to study a particular transition will depend on the energy difference between the ground and excited states (E).

Different frequencies of light (electromagnetic radiation) interact with different aspects molecular motion (potential

and kinetic energy).

IR: molecular vibrations

UV-Vis: valance electroni

c excitatio

n

X-ray: core

electron excitation

Radio waves:Nuclear spin states(in a magnetic field)

Transitions between energy levels which involve electrons, electronic transitions, between

molecular orbitals.

Ultraviolet-Visible Spectroscopy

In UV/Vis spectroscopy we talk in terms of the

wavelength () of the transition.

Nuc

Level 1

Level 2

Level 3

Gain energyMove to level 2 or

3

Lose energyDrop to level 2 or

1

Energy levels of electrons

Transitions occur between200-400 nm in the UV

region400-750 nm in the visible

regionFor a transition to occur the incident radiation must have the correct energy to excite the molecule from the ground state to a higher, ‘excited’ state

Molecular Geometry

En

erg

y

S0 (Ground Estate)

S1 (Excited Estate)

rotational levels

vibrational levels

UV-Vis Absorption Spectroscopy

h

Important Terms and Symbols in UV-Vis Absorption

SpectroscopyTerm and Symbol

Radiant Power (P, P0)

Absorbance (A)

Transmittance (T)

Path length (b)

Absorptivity (a)

Molar absorptivity ()

Definition

Energy of radiation

Log (P0/P)

P/P0

-

A/lc

A/lc

Alternative Name and Symbol

Radiation Intensity (I, I0)

Optical density (OD); Extinction (E)

Transmission (T)

l, d

Extintion coefficient (k)

Molar Extintion coefficient

Beer-Lambert Law

A c l=

= absorbance (no units).

= Molar extintion coefficient (M-1 cm-1).

= Concentration (M).

= pathlength (cm).

A

c

l

Beer-Lambert Law

I0

I

S

l

dx

Block of absorbing matter

A beam of incident intensity I0 passes through length l of an absorbing medium, which decreases the transmitted intensity to I.

Considering a cross-section of area S and infinitesimal thickness dx containing dn molecules, each of which has a cross-sectional surface of photon capture of a.

Based on the probability of photon capture, we can imagine the total area of the molecular photon capturing surface to be dS, and thus the ratio of photon capture surface area to total surface area is dS/S.

This ratio represents the probability of photon capture.

Beer-Lambert Law

I0

I

S

ldx

The intensity entering the section is Ix, proportional to number of photons per square centimeter. Loss of intensity through absorption is given by dIx, which is an absolute quantity; thus the relative loss of power is:

= probability of photon capture.

dS

S

Now consider the light intensity passing through distance dx.

dIx

Ix(minus sign indicates loss)

dIx

Ix

dS

S=

The relative loss of power must be equal to the relative probability for capture.

Beer-Lambert Law

I0

I

S

ldx

Note that dS is the sum of all capture areas for all molecules, and must therefore be proportional to the number of molecules

dS = adn (concept of absorptivity)

where a is the photon capture cross section (molecular absorptivity co-efficient).Integrating over all of the molecules within the entire block (to obtain the total absorbance over an infinite number of slices of thickness dx), we get:

dIxIx

=Io

I adnS0

n

which gives:II0

=anS

-ln

Now convert to base 10 and invert the fraction to change the sign:

I0

I=

an2.303S

log

Beer-Lambert Law

I0

I

S

ldx

The cross-sectional area S can be expressed as a volume (cm3 or mL) divided by a length (pathlength b in cm), thus:

Finally, since n/V has units of concentration (molecules per cm3), we can convert n/V to moles per liter according to:

Which leads to:I0

I=

anl2.303V

log

=Vl

S in cm2

Combining everything together, we get:

1000 cm3/L

V(cm3)=c

n6.02 x 1023

mol x

6.02 x 1023 alc2.303 x 1000

I0

I=log

Beer-Lambert Law

I0

I

S

ldx

6.02 x 1023 acl2.303 x 1000

I0

I=log

Finally, collecting all of the

constants into a single term, , we get:

A c lI0

I=log =

A c l=

= absorbance (no units).

= Molar extintion coefficient (M-1 cm-1). (wavelength dependent)

= Concentration (M).

= pathlength (cm).

A

c

l

1) Real Limitations• Beer-Lambert law is derived on the basis of having a

dilute solution. At high concentrations, (>0.01 M) intermolecular interactions can occur between molecules (dipole-dipole interactions), directly altering

• Intermolecular interactions can also occur when secondary species (i.e., salts) interact with charged chromophores, thus altering

• The value of is also dependent on refractive index of the medium, thus solvent effects on absorbance can be observed (correct using n/(n2 + 2)2 instead of ).

Limitations / Beer-Lambert Law

2) Apparent Chemical Deviations• Arise as a result of chemical reactions involving the

absorbing sample (association, dissociation, oxidation, reduction, addition, elimination, etc…).

Limitations / Beer-Lambert Law

Classic example is pH sensitive chromophores (indicator dyes). Figure shows the absorbance at 430 nm and 570 nm for a dye that undergoes a protic equilibrium that depends on total dye concentration.

0.00 4.00 8.00 12.00 16.000.000

0.200

0.400

0.600

0.800

1.000

Ab

sorb

ance

Indicator (M x 105)

= 430 nm

= 570 nm

Limitations / Beer-Lambert Law3) Instrumental Deviations due to

Polychromatic Radiation• Strict adherence to Beer-Lambert Law requires

perfectly monochromatic light.

• In cases where the spectral band width of the incoming light is many nanometers, then the sample can have a range of values.

In practice:• Narrower spectral

bandwidth will lead to better linearity.

• Taking measurements at the maximum wavelength

(at max) will lead to better linearity.

Ab

so

rba

nc

e

Ab

so

rba

nc

e

Wavelength Concentration

Band B

Band A

Ba

nd

B

Ba

nd

A

Limitations / Beer-Lambert Law4) Instrumental Deviations due to Stray

Light• Imperfections in monochromator gratings often lead to

0.005 – 0.2% stray light.

• Differs greatly in wavelength from the value of the principal radiation, and may not even pass through sample (i.e., depends on how light-tight the instrument is).

• Observed absorbance in presence of stray light is given by:

A’I0 + Is

I + Is

log=

Where Is is the intensity of non-absorbed stray radiation. This leads to a negative deviation in A vs C, as shown in the Figure0 2.5 5.0 7.5 10.0

1.0

2.0

Ab

sorb

ance

Concentration (M x 103)

5 %

1 %

0.2 %0.0 %

Is

I0

x 100 %

• Measurement of absorbance by molecular samples requires that the sample be placed in some kind of recipient.

• Reflection of light occurs at four different interfaces, leading to ~8.5% loss in transmitted light.

• Attenuation of light can also occur via scattering or absorbance by species other than the solution.

Reflection losses at interfaces

Incident beam, I0

Emergent beam, I

Scattering losses in solution

Reflection losses at interfaces

Instrument components: UV-VIS

Instruments for absorption measurements

signal processo

r

optical

source

h sample

h

detect

or

selector

SourcesA source must:

• Generate a beam of radiation with sufficient power for easy detection and measurement.

• Provide output power that is both stable and intense.

Instrument components: UV-VIS

Types of spectroscopic sources

1. continuous sources

Wavelength

Rela

tive e

nerg

y

H2 and D2 lamp / Xe arc lamp

Tungsten filament lamps

2. lines sources

Wavelength

Inte

nsit

y

Hollow cathode lampHg vapor lamp / Laser

Instrument components: UV-VIS

Sources

6000 K

4000 K3000 K

2000 K

Xenon arcCarbon

arcTungsten lampNerst

glower

Rela

tive

en

erg

y

Wavelength (nm)

500 1000

1500

2000

2500

3000

1

10

102

103

104

Sample containers

Instrument components: UV-VIS

Glass - 340 - 2500 nmQuartz - 200 - 2500 nm

Polystyrene - VisibleMetacrylate - UV

1. Filters

1.1 Interference filters

1.2 Absorption filters

Wavelength selectors

Instrument components: UV-VIS

The simplest method for isolating a narrow band of radiation.

Work by selectively absorbing radiation from a narrow region.

Use constructive and destructive interference to isolate a narrow range of wavelengths.

2. Monochromators

2.1 Prism

Wavelength selectors

Instrument components: UV-VIS

2.2 GratingsFinely grooved highly reflective surface (Diffraction).

Quartz or glass cut at an angle (Refraction).

Collimating mirrors

Entrance slit Exit slit

Grating surface

>

Wavelength selectors

Instrument components: UV-VIS

0.5

1.0

Ab

sorb

an

ce

Wavelength

Nominal wavelength

Effective bandwidt

h

1/2 Peak heigh

t

- +h--

e-

DetectorsInstrument components: UV-VIS

The detector changes the radiation transmitted from the UV-Vis Spectrophotometer into a current or voltage.

1. Phototube

3. Photodiode array

2. Photomultiplier

Light strikes photocathode (-) that emits electrons.Electrons are accelerated towards the anode (+).Current proportional to photons.

Electrons that are accelerated towards a series of increasingly positive anodes (+).Electrons are directed towards the collecting anode.

Multi-element detector composed of an array of solid-state detectors.

Signal processors

Instrument components: UV-VIS

Amplifies the voltage produced by the detector.

Converts the voltage into absorbance units.

Instruments: UV-VISSingle beam UV-Vis spectrophotometer

Instruments: UV-VISDouble beam UV-Vis spectrophotometer

Instruments: UV-VIS

Ultraviolet-Visible SpectroscopyBonding electrons appear in and molecular

orbitalsnonbonding in n

En

erg

y

n

n

n

Antibonding

Antibonding

Nonbonding

Bonding

Bonding

Electronic transitions can occur between various states.

The energy of the transitions increases in the following order:

(n *) < ( *) < (n *) < ( *)

Electronic Transitions

• Most absorption spectroscopy of organic compounds is based on these transitions.

• The absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm).

• These transitions need an unsaturated group in the molecule to provide the p electrons.

• Excitation from the ground state to the excited state requires EM radiation with a wavelength of 150 nm.

• Not useful for routine spectroscopy

• The number of organic functional groups with n * peaks in the UV region is small.

• Excitation requires EM radiation with a wavelength in the range 150 - 250 nm.

* bandn * band

n * band

* band

ChromophoreGroup on molecule responsible for electronic transition

• These molecules generally contain conjugated systems.

• Adjacent double bonds are conjugated.

• The net effect is to bring the ground state and excited states closer in energy.

• The longer the chain of conjugation the longer the wavelength of the absorption band.

Bathochromic shift

Hypsochromic shift

Hyperchromic effect

Hypochromic effect

Shift in absorption to a longer wavelength.

Shift in absorption to a shorter wavelength.

An increase in the intensity of absorption.

An decrease in the intensity of absorption.

Some Spectroscopic Terminology

• When one absorbing species reacts to form another absorbing species, there usually is a point(s) at which the spectra cross. This is the isobestic point.

• Example, acid/base indicators.

Isosbestic Point

There are 3 classes of protein chromophores peptide bonds, amino acid side chains and prosthetic

groups.

Most proteins and nucleic acids are colorless in the visible region, however they absorb in the near-UV region. Chromophores in proteins and nucleic acid absorb light only at wavelengths lower than 300 nm. On the other hand, measurements have to be done in wavelengths longer than 170 nm, below which the absorbance of water becomes too high.

Absorption Spectra of Biopolymers

UV absorption spectra of proteins

For a typical protein there is a distinctive absorption peak at 280 nm (due to the * transitions in aromatic amino acids), a stronger one at 190 (due to the * transition in the amide group), and a shoulder at 210-220 nm (due to the weaker n π* transition in the amide group).

Ab

sorb

an

ce

Wavelength (nm)

2.0

1.5

1.0

0.5

0

0.4

0.3

0.2

0.1

250 300200

4.5 x 10-7 M

2.25 x 10-6 M

Chromophores in proteins

The peptide bond

Amino acid side chains

• A number of amino acids – Asp, Glu, Asn, Gln, Arg & His have electronic transitions around 210 nm. Usually these cannot be observed in proteins because they are masked by Intense peptide bond absorption.

• The most useful range for proteins is above 230nm where there are absorptions from the aromatic side chains of Phe, Tyr and Trp.

• The absorption of Phe is low and if Tyr & Trp are present in a protein it contributes little to the absorption above 230nm. The disulphide group of Cys also has a weak absorption ~250 nm which can be important in protein optical activity.

• The absorption spectra of Tyr & Trp contain contributions from at least three electronic transitions. Assignment of individual transitions in proteins containing many Tyr and Trp in a variety of environments is not possible.

Amino acid side chainsThe aromatic Trp, Tyr, and Phe are the only residues absorbing significantly at wavelengths higher than 230 nm. The maximum at 280 nm is due to Trp and Tyr.

-

Mola

r ab

sorp

tivit

y (

M-1 c

m-1)

Tyrosine

Phenylalanine

Tryptophan

200 220 240 260 280 300 320

100005000

2000

1000

10

20

50

100

200

500

4000020000

4000

3000

2000

1000

0190 200 210 220 230

Alanine

Lysine HCl

Methionine

Wavelength (nm)

Absorption Spectra of Biopolymers

The local environment of the amino acids in a native protein depends on the electric properties of the peptide chain near by and the associated solvent. The resulting change in spectra is sufficient to diagnose whether a protein is folded or not, but CD is more informative.

SECONDARY STRUCTURE

UV spectra of poly-L-lysine hydrochloride (random coil) changes upon formation of secondary structure (raising pH induces helix formation - reducing the net positive charge of the lysine side chain -; raising the temperature induces beta sheet).

Many proteins include prosthetic groups such as iron porphyrins in hemeproteins, flavin in flavoproteins, or phosphate groups, that have strong absorption in the visible and near UV. Because these bands are sensitive to the environment, they can be used in the study of enzymatic reactions.

Absorption Spectra of Biopolymers

x 1

0-4

Chromophores in genetic material

The strong near-UV absorption of nucleic acids is due to the purine and pyrimidine bases. The electronic states of these bases are much more complex than those of protein chromophores, and apparent gaussians in the spectra are really composites of several electronic transitions.

The nucleotides all have similar absorption spectra, dominated by an absorption peak at 260 nm and a shoulder at 200 nm.The free base, the nucleoside (attached to sugar), the nucleotide (attached to a sugar phosphate), and the denatured polynucleotide all have similar spectrum.

In general polynucleotides and nucleic acids absorb less per nucleotide that their constituent nucleotides. Also, native double-stranded DNA absorbs less per nucleotide than denatured (melted strands). This results from the interactions between adjacent bases. Stacked base pairs in a double helix absorb less than partially stacked bases in a single strand, which absorb less than the mononucleotides.

Absorption Spectra of BiopolymersUV absorption spectra of nucleic acids

180

200

220

240

260

280

300

0

0.2

0.4

0.6

0.8

1.0E. coli DNA 82°CEnzymatic digest 25°CNative 25°C

(nm)

Ab

sorb

an

ce

What determines absorbance?• The absorbance spectrum for a chromophore under standard

conditions is only partly determined by its chemical structure.

• The environment of the chromophore also affects the precise spectrum obtained.

• pH• Solvent polarity• Orientation effects

• Chromophores can act as reporter molecules which can give information about their immediate environment.

• Defined conditions (pH and solvent).

• Protonation/deprotonation effects resulting from pH changes or oxidation/reduction effect electron distribution in chromophores.

• Solvent polarity –Altered spectra in different solvents (DMSO, dioxane, ethylene glycol, glycerol and sucrose) compared to water = Solvent perturbation.

• Orientation effects result from the relative geometry of neighboring chromophore molecules. E.g. hyperchromicity of nucleic acids.

Applications of absorption spectra

Quantitative

Reaction rate calculation

Standard curve

Structural studies

Enzymatic

Identification

Beer-Lambert law to determine concentration.

Continuous or stopped.

measure concentration.

folding, assembly, denaturation, ligand binding

pathways, reaction intermediates

of compounds (vitamins, hormones) not much use for proteins/nucleic acids, unless they contain groups with absorption in the visible region of spectrum.

Immunodetection commercial detection kits.