Spectroscopic and

biophysical techniques
UE Micro and Nanotechnologies for Health

Marco Maccarini

marco.maccarini@univ-grenoble-alpes.fr
 Content

• Introduction

• UV-Vis absorption spectroscopy

• IR absorption spectroscopy
 Spectroscopy

• The branch of science concerned with the

investigation and measurement of spectra produced
when matter interacts with or emits electromagnetic
or other types of radiation.

• Absorption/Emission

• Scattering/Diffusion
Spectroscopy

?
 Theorem Fluctuation Dissipation

• The fluctuation-dissipation theorem relies on the

assumption that the response of a system in
thermodynamic equilibrium to a small applied
force is the same as its response to a
spontaneous fluctuation

What is a spontaneous fluctuation?

Example: protein
 Electromagnetic radiation

E = E0 cos 2⇡⌫t c=⌫

The interaction between the electromagnetic waves and the matter occurs as
an interaction between the electric or magnetic field of the wave with the electrical
or magnetic field inside the matter

By SuperManu - Self, based on Image:Onde electromagnetique.png, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=2107870

 Type of interactions

Sample
Incident Radiation I0 Transmitted Radiation

Absorbed
Radiation

Rayleigh scattering

Raman scattering

Scattered in different directions

Re-emitted
(fluorescence/phosphorescence)
 Spectral regions

Υ rays UV IR Radiowaves
X rays Vis μwave
λ [cm]
10-11 10-9 10-7 4∙10-5 8∙10-5 10-1 10 105

3∙1021 3∙1019 3∙1017 7.5∙1014 4∙1014 3∙1011 3∙109 3∙105

Freq[Hz]
c
⌫=
 Spectral regions

• Υ rays: probe energy transition between different nuclear energy

states - very high energy, little biological interest
• x rays: probe energy transition of electrons in the inner states -
high energies
• UV-Vis spectroscopy: probe energy transitions between
electrons in the valence shell. Many biological molecules
(proteins, amino-acids, nucleotides) absorb in this energy range
• IR spectroscopy: probe energies corresponding to molecular
vibrational states (oscillation of atoms around equilibrium
positions)
• NMR: probes energy transitions between different states of
nuclear spin.
 Type of interactions

Sample
Incident Radiation I0

Absorbed
Radiation

Re-emitted
(fluorescence/phosphorescence)
 Energy levels in atoms

E3

X -
-
- E2

E0
E1 E
2
E1 -
E3

E0
-

Only if the energy of the radiation equals the difference in energy

between two levels the radiation can be absorbed
 Energy levels in molecules
Equation (E1.3) is
quantity, A(λ), is calle
ple.
• The OD
electrons can
of be
small measure
molecules
energy

1
have different energetic states
• S of about
ground
0 state0.1 to ≤ 3. No
• S1intensity
first excited is transmitted.
Conformational

state
nery

0 • Vibrational" states
ε = ε / e, where e = 2.
• The interaction with EM wave can
induce transition between different
energy states

r e E1.3 The UV--visib

• This happens when the energy of
the EM wave matches the energy
difference between the two states
Conormaional coordinae
The UV--visible spectra
Conformational state −1
100--1000 kJ mole b
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Absorption

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Absorption

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Absorption

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Absorption

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Emission

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Emission

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Relaxation

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Relaxation

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Relaxation

Em

En
 Absorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)

Relaxation

Em

En
Probability of transitions - selection rules
• Every system is characterised by a number of energy levels
• Biological systems contains 1017-1020 molecules
• It is important to know the distribution of the energy of these
molecules (i.e. how many molecules are in each energy level)
• At equilibrium (in the absence of radiation) the distribution of the
energy populations follows the Boltzmann statistics.
• For two states A and B of energy Ea and Eb (Ea>Eb)
✓ ◆
Na ga E a Eb
= exp
Nb gb RT

• Electronic and vibrational not populated at room temperature

• NMR and rotational energy level populated at room temperature
Probability of transitions - selection rules
• Not all the transition between different energy levels in the system
are allowed. There are selection rules (complex theory).

• The selection rules are probability of particular spectroscopic

transition to take place

1.Bohr condition: the energy of the interacting radiation should equals

the energy difference between the initial and final energy level

h⌫ = Ef Ei

2.The probability of a transition between to states m and n is equal in

the two directions.

Pm!n = Pn!m
 Probability of transitions - selection rules
Adsorption and emission depend from the populations of the two
energy levels.
Question:
what happens if the two energy levels are equally populated?

Em

En
 Probability of transitions - selection rules
Adsorption and emission depend from the populations of the two
energy levels.
Question:
what happens if the two energy levels are equally populated?

Em

En
Answer:
There must be a different in the population to observe emission/
absorption.
If not the system will be perfectly transparent to the radiation
 Transition probability - selection rules

3. The probability of transition between two energy levels is

proportional to the transition moment

The transition moment determine the selection rules, which define the
necessary condition for a transition to be allowed.

They are linked to the symmetry of the energetic states, depend on

materials and on the type of radiation and interaction
 Adsorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)
• T, number of molecules that undergo the energy transition

T (n ! m) = Bmn Nn ⇢ Bmn = Bnm probability of absorption

T (m ! n) = Bnm Nm ⇢ Nn,m population of the energy level

ρ energy density of the radiation

Em

En
 Adsorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)
• T, number of molecules that undergo the energy transition

T (n ! m) = Bmn Nn ⇢ Bmn = Bnm probability of absorption

T (m ! n) = Bnm Nm ⇢ Nn,m population of the energy level

ρ energy density of the radiation

Em

En
 Adsorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)
• T, number of molecules that undergo the energy transition

T (n ! m) = Bmn Nn ⇢ Bmn = Bnm probability of absorption

T (m ! n) = Bnm Nm ⇢ Nn,m population of the energy level

ρ energy density of the radiation

Em

En
 Adsorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)
• T, number of molecules that undergo the energy transition

T (n ! m) = Bmn Nn ⇢ Bmn = Bnm probability of absorption

T (m ! n) = Bnm Nm ⇢ Nn,m population of the energy level

ρ energy density of the radiation

Em

En
 Adsorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)
• T, number of molecules that undergo the energy transition

T (n ! m) = Bmn Nn ⇢ Bmn = Bnm probability of absorption

T (m ! n) = Bnm Nm ⇢ Nn,m population of the energy level

ρ energy density of the radiation

Em

En
• Equilibrium: system become transparent to radiation
 Adsorption, saturation and relaxation
• Let’s consider a system that has two energy levels n and m
• Em-En is the energy difference (Em>En)
• T, number of molecules that undergo the energy transition

T (n ! m) = Bmn Nn ⇢ • Spontaneous transitions (very

T (m ! n) = Bnm Nm ⇢ high for UV-vis and IR)
• Low for NMR

Em

En
 Lambert-Beer Law
• The absorbance depends on thickness and concentration

I0 I

I0 I

I0 I

I0 I
 Lambert-Beer Law
• How the absorbance depends on thickness and concentration
Z Z
dI = KIdx dI = KIdx dx

ln I = Kx + C f or x = 0 I = I0

C= ln I0

I0
ln = Kx ! I = I0 exp ( Kx)
I

K = ✏C ✏ molar extinction coefficient

C concentration
 Lambert-Beer Law
• How the absorbance depends on thickness and concentration

✓ ◆
I0
A = log = ✏Cl
I

A Absorbance or Optical Density

I0 intensity of the incident radiation
I intensity of the transmitted radiation
✏ molar extinction coefficient M-1cm-1

C concentration mole/l

l thickness of the sample

 Lambert-Beer Law
• In the presence of different molecular species i

X
A=l (✏i Ci )

• The measure of the absorbance must be corrected for that of

the solvent

C = (A Asolv )/✏l

• The law of Lambert-Beer can help us to determine the

concentration of a given substance in solution
 Spectroscopy UV-Vis
• UV-Vis spectroscopy consists in measuring of the intensity
of absorbed/emitted radiation as a function of the
frequency or wavelength
• The wavelengths of the used radiation are: UV (184-400nm),
Visible (400-800nm)
• The UV-Vis spectroscopy probes energy level
characteristic of valence electron
• Used for quantitative determination of different molecules like
organic compounds or biological macromolecules
• Concentration of a given substance (Lambert-Beer Law)
 Franck-Condon Principle

vibrational energy level

• Electronic transitions ~ 10-15 sec

• Vibrational transitions ~ 10-11 sec 1000 times slower!
 exit slit
UV-Vis spectrometer

detector
2
1
ion grating

• It shines light through a sample light at wavelength between

200 and 800 nm
• It measures the absorption as a function of the wavelength
• It obtains an absorption spectrum
3
 Spectroscopy UV-Vis: Example
Electrons
Energy
1.0
λmax=217nm

0.8

Butadine
Absorbance

0.6

0.4
} ΔE

0.2

Absorption spectrum
0.0
160 180 200 220 240 260 280 300 Ground state Excited state
wavelength (nm)

c
E = h⌫ c= ⌫ E=h
 = ε / e, transitions,
εelectronic where e = 2.303.mol
Pub
respectively. (After Cantor
Spectroscopy UV-Vis: Example and Schimmel, 1980.)
of 4
tisti
r e E1.3 The UV--visible spe
as o
(a)
Conormaional
Energy
coordinae
The UV--visible spectral range E
λmax=217nm
100--1000 kJ mole−1 between 5 kJs
1.0

Absorbance
Fig. E1.2 Potential energy
0.8 ground (lowest-energy) and first tem
diagram of the lowest (S0 )
first excited (S1 )
andButadine
in small molecules. These eneE
Absorbance

0.6

ambient temperature (2.5 kJwhemo

0.4
electronic states in a
molecule. r, v, and e are
}
aelenh ΔE
Each electronic state is itsel
(b)
rota
rotational, vibrational and

Absorbance
tional and vibrational energies a sh
electronic transitions,
0.2
524 E Optical spectroscopy
molecules, the energy differen are
respectively. (After Cantor
0.0
of 40 kJ mole−1 , also larger rota than
160 180 200 220 240
and Schimmel, 1980.)
wavelength (nm)
260 280 300

tistical
Equation (E1.3) is the Beer--Lambert law.Ground
The quantum
dimensionless
state
aelenh
mechanics
(measured)
Excited state spe
tha
quantity, A(λ), is called the absorbance or optical density (OD) of the sam- of th
as occupying the lowest vibratio
ple. OD can be measured accurately
(a) by modern spectrophotometers in the range
1
Energy (c) corresponding
states
of about 0.1 to ≤ 3. Note that for a value of 3, a fraction of only 1/1000 of the is b

Absorbance
env
Absorbance

−1ε, are related by

5 kJ mole
intensity is transmitted. The molar extinction coefficients, ε " and , so that many rotat
nery

0
ε = ε" / e, where e = 2.303. Published values are usually given for ε. a sm
temperature.
Electronic transitions, such aF
r e E1.3 The UV--visible spectral range aelenhof the appropriateof s
aelenh
wavelength when light
wavelength wa
The UV--visible spectral range corresponds to transition energies of the order of broth
Conormaional coordinae
−1
100--1000 kJ mole between (b)
rotational or vibrational
Fig. E1.3 Absorption
states, which matches the difference between the
states,
e
 Spectroscopy UV-Vis: Example

1.0
λmax=217nm

0.8
Absorbance

0.6 Butadine

0.4

0.2

0.0
160 180 200 220 240 260 280 300

wavelength (nm)

A substance that absorbs in the UV range is colourless!

Spectroscopy UV-Vis: β-carotene
λmax=450-500 nm
orange
 Spectroscopy UV-Vis: β-carotene

Color wheel Complementary

colours
 Spectroscopy UV-Vis: β-carotene

Orange component
Blue component
reflected
absorbed

Color wheel Complementary

colours
Spectroscopy UV-Vis: β-carotene
λmax=450-500 nm
orange
 Chromophores
Chromophores: the part of a molecule responsible for
absorbing radiation
Examples

c
=
⌫
528
Other Examples
E Optical spectroscopy

Fig. E1.7 UV absorption 8000

spectra of poly-L-lysine
hydrochloride in aqueous
solution: random coil at
6000
pH 6.0, 25 ◦ C; helix at pH β
10.8, 25 ◦ C; β-form at pH e Random coil
10.8, 52 ◦ C. The x-axis is
the wavelength in nano- 4000
elix
metres (the ‘milli-micron’
is an old fashioned unit,
now replaced by ‘nano- 2000
metre’). (From Rosenheck
and Doty, 1961, reviewed
by Wetlaufer, 1962.) 0
180 190 200 210 220 230 240 250
l (µm)

The molar extinction coefficient of a lysine polypeptide, express

acid residue, is shown Fig. E1.7 as a function of wavelength in
peptide group absorption.
Other Examples (amino acids) E1 Visible and IR absorption spectroscopy 529

5000 5000 Fig. E1.8 The far-UV

absorption spectra of
amino acids in aqueous
4000 4000 solution at pH 5, except
for cystine (pH 3.0). The
Molar exincion coeicien chloride ion contribution
3000 3000 C ()
was subtracted from the
. C1 basic amino acids
measured as hydrochlo-
2000 2000 rides. TRY is an old-
TR fashioned abbreviation
MT for tryptophan. CYS (-SH)
1000 1000 R is cysteine. CYS is cystine.
A Other abbreviations are
the usual ones. (From
AA R
0 0 Sussman and Gratzer,
1850 1950 2050 2150 2250 1850 1950 2050 2150 2250 1962, reviewed by
12 500
60 000 Wetlaufer, 1962.) (Figure
reproduced with
10 000 50 000 permission from Elsevier.)
Molar exincion coeicien

40 000 TR
7500
. C1 TR
30 000
AR
5000 C1
20 000
C
2500
10 000
AR .
C1 4
0 0
1850 1950 2050 2150 2250 1850 1950 2050 2150 2250

l () l ()
 IR Spectroscopy
• It studies the vibrational motions of the atoms in a molecule
• The wavelength used is in the range 2500-16000 nm
• Molecules absorb specific frequencies, characteristic of their
structure (resonant frequencies)

Symmetric stretching Asymmetric stretching Scissoring

 IR Spectroscopy

100
Transmittance (%)

50

H-C≡C(CH2)5CH3 1-Octyne
0
4000 3000 2000 1500 1000 500
wavenumber (1/cm)

1
Transmittance vs wavenumber ⌫˜ =
[cm]
 Wavenumber

1
⌫˜ = high wavenumber = low frequency
[cm]

c= ⌫ speed of light = wavelength * frequency

c 1
⌫= = c = ⌫˜c
 IR Spectroscopy
1-Octyne
100
Transmittance (%)

50

H C

0
H-C≡C(CH2)5CH3
4000 3000 2000 1500 1000 500
wavenumber (1/cm)

1
Transmittance vs wavenumber ⌫˜ =
[cm]
 IR Spectroscopy
1-Octyne
100
Transmittance (%)

50

H C

0
H-C≡C(CH2)5CH3
4000 3000 2000 1500 1000 500
wavenumber (1/cm)

1
Transmittance vs wavenumber ⌫˜ =
[cm]
 IR Spectroscopy
1-Octyne
100
Transmittance (%)

C≡C
50

Diagnostic region Fingerprint region

H-C H-C≡C(CH2)5CH3
0
4000 3000 2000 1500 1000 500
wavenumber (1/cm)

1
Transmittance vs wavenumber ⌫˜ =
[cm]
Molecules as masses connected by springs

H C

Wall

x0
=equilibrium position
Molecules as masses connected by springs

H C Fspring=-KΔx

K = strength of the spring (bond)

Δx= x-x0

Wall

m m

x0
Molecules as masses connected by springs

H C Fspring=-KΔx

r
1 K
⌫= =frequency of oscillation
2⇡ m
Wall

m m

x0
Molecules as masses connected by springs
r
1 K
⌫= =frequency of oscillation
2⇡ m

K higher higher frequency of oscillation

K lower lower frequency of oscillation

m higher lower frequency of oscillation

m lower higher frequency of oscillation
Molecules as masses connected by springs

Wall
(infinite
mass) r
1 K
m ⌫=
2⇡ m

m1 m2
m M=
H C
m1 + m2
m1 m2
M= reduced mass
 Molecules as masses connected by springs
r r
H C 1 K 1 K
⌫= ⌫=
m2 m1 2⇡ m 2⇡ M

m1 m2
Reduced mass: M=
m1 + m2
m1 m2 12 · 1
MC-H = = = 0.923
m1 + m2 12 + 1

m1 m2
MC-C = =?
m1 + m2
 Molecules as masses connected by springs
r r
H C 1 K 1 K
⌫= ⌫=
m2 m1 2⇡ m 2⇡ M

m1 m2
Reduced mass: M=
m1 + m2
m1 m2 12 · 1
MC-H = = = 0.923
m1 + m2 12 + 1
r
m1 m2 12 · 12 1 K
MC-C =
m1 + m2
=
12 + 12
=6 M higher ⌫= lower
2⇡ M
r
MC=C =
m1 m2
=
12 · 12
=6 K higher 1 K higher
m1 + m2 12 + 12 ⌫=
(~2*K) 2⇡ M
• Stronger bond correspond to higher frequencies of vibration
• Lighter atoms correspond to higher frequency of vibration
 Typical wavenumber values
r
H C 1 K
⌫=
m2 m1 2⇡ M

⌫ [cm-1]

C-H 3032

C-O 1109

C=C 1682

C≡C 2026

C=O ~1700-1800
 Typical wavenumber values
double bond
bond with H triple bond
100 single bond
C-H C≡C C=C C-O
80
Transmittance [%]

60
X-H

40

20

0
4000 3500 3000 2500 2000 1500 1000 500
wavenumber [1/cm]
 Typical wavenumber values

≡C-H =C-H -C-H

3300cm-1 3100cm-1 2900cm-1

• the IR absorption frequency of a bond depends also on the

surrounding environment.
• In this case the hybridisation of the C atom
 Typical infrared absorpiton values for various types of bonds

13/11/2018 Infrared Spectroscopy: A Quick Primer On Interpreting Spectra — Master Organic Chemistry

An even more compressed overview looks like this: (source)

3600 – 2700 cm-1 X-H (single bonds to hydrogen)

2700 – 1900 cm-1 X≡X (triple bonds)
-1
 O-H bond
• OH bonds between hydroxyl groups lead so tome variations in
O-H bond strength
• wider range of vibrational energies around 3400-3200 cm-1
13/11/2018 I a S :AQ P O I S a Ma O a C

[S a no .O a a a a
 .

A a C=O stretch
a a , N-H a a
a aa ( ). T a a a a aa a a
a a a a N-H .

Ne , peaksa are strong

a C=O a , a 1630-1800 -1.. T a a
• C=O and sharp
a a a a a a a a , a a
• they appear
- a aina a .region around 1630-1800 cm
-1

T a 80/20 a a : a .
Examples: decane and 1-octyne

-C-H

C≡C

≡C-H
Examples: and alkene (1-Hexene)

C=C
=C-H -C-H

CH vs =CH vs ≡C-H