Spectroscopy and Spectrofluorimetry

Introduction
Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic
radiation by atoms or molecules (or atomic or molecular ions) to qualitatively or quantitatively
study the atoms or molecules, or to study physical processes. The interaction of radiation with
matter can cause redirection of the radiation and/or transitions between the energy levels of the
atoms or molecules. A transition from a lower level to a higher level with transfer of energy from
the radiation field to the atom or molecule is called absorption. A transition from a higher level
to a lower level is called emission if energy is transferred to the radiation field, or nonradiative
decay if no radiation is emitted. Redirection of light due to its interaction with matter is called
scattering, and may or may not occur with transfer of energy, i.e., the scattered radiation has a
slightly different or the same wavelength.

Interaction of Light with Matter

The diagram on the right illustrates the
energy level structures of atoms and molecules.
We need to pay our attention to how they interact
with electromagnetic waves. Such interactions are at
the very heart of spectroscopy.
There are lots of spectroscopic processes. The
important ones are:
 Absorption
 Fluorescence
 Rayleigh scattering
 Raman scattering
 Refraction
They appear to be different but in fact they are all closely related. We would like to examine
these in much greater depth to understand them and their relationships.

Absorption
When atoms or molecules absorb light, the incoming energy excites a quantized structure
to a higher energy level. The type of excitation depends on the wavelength of the light. Electrons
are promoted to higher orbitals by ultraviolet or visible light, vibrations are excited by infrared
light, and rotations are excited by microwaves.
An absorption spectrum is the absorption of light as a function of wavelength. The
spectrum of an atom or molecule depends on its energy level structure, and absorption spectra
are useful for identifying of compounds.
Measuring the concentration of an absorbing species in a sample is accomplished by
applying the Beer-Lambert Law.
The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and
concentration of an absorbing species. The general Beer-Lambert law is usually written as:
A=λbc
where A is the measured absorbance, λ is a wavelength-dependent absorptivity
coefficient, b is the path length, and c is the analyte concentration. When working in
concentration units of molarity, the Beer-Lambert law is written as: A = ε b c
where ε is the wavelength-dependent molar absorptivity coefficient with a unit of M-1 cm-1.

Experimental measurements are usually made in terms of transmittance (T), which is defined as:
T = I / Io
where I is the light intensity after it passes through the
sample and Io is the initial light intensity. The relation
between A and T is: A = -log T = - log (I / I o).
Modern absorption instruments can usually display
the data as transmittance, % transmittance, or absorbance.
An unknown concentration of an analyte can be determined Absorption of light by a sample
by measuring the amount of light that a sample absorbs and
applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can
be determined using a working curve of absorbance versus concentrations derived from
standards.

Limitations of the Beer-Lambert law

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors.
Causes of nonlinearity include:
 deviations in absorptivity coefficients at high concentrations (>0.01 M) due to
electrostatic interactions between molecules in close proximity
 scattering of light due to particulates in the sample
 fluoresecence or phosphorescence of the sample
 changes in refractive index at high analyte concentrations
 shifts in chemical equilibrium as a function of concentrations
 non-monochromatic radiation, deviations can be minimized by using a relatively flat part
of the absorption spectrum such as the maximum of an absorption band

Electromagnetic Spectrum
Type of Radiation Frequency Range (Hz) Wavelength Range Type of Transition
γ-rays 1020-1024 <10-12 m nuclear
x-rays 1017-1020 1 nm-1 pm inner electron
ultraviolet 1015-1017 400 nm-1 nm outer electron
visible 4-7.5x1014 750 nm-400 nm outer electron
near-infrared 1012-4x1014 2.5 um-750 nm outer electron, molecular
vibrations
infrared 1011-1012 25 um-2.5 um molecular vibrations
microwaves 108-1012 1 mm-25 um molecular rotations,
electron spin flips*
radio waves 100-108 >1 mm nuclear spin flips*

Emission
Atoms or molecules that are excited to high energy levels can decay to lower levels by
emitting radiation (emission or luminescence). For atoms excited by a high-temperature energy
source this light emission is commonly called atomic or optical emission, and for atoms excited
with light it is called atomic fluorescence. For molecules it is called fluorescence if the transition
is between states of the same spin and called phosphorescence if the transition occurs between
states of different spin. In simple terms, phosphorescence is a process in which energy absorbed
by a substance is released relatively slowly in the form of light.
The emission intensity of an emitting substance is linearly proportional to analyte
concentrations (at low concentration range), and is useful for quantitating emitting species.

Scattering
When electromagnetic radiation passes through matter, most of the radiation continues in
its original direction, but a small fraction is scattered in other directions. Light that is scattered at
the same wavelength as the incoming light is called Rayleigh scattering. Light that is scattered in
transparent solids due to vibrations (phonons) is called Brillouin scattering. Brillouin scattering
is typically shifted by 0.1 to 1 cm-1 from the incident light. Light that is scattered due to
vibrations in molecules or optical phonons in solids is called Raman scattering. Raman scattered
light is shifted by as much as 4000 cm-1 from the incident light.

Raman Effect
The Raman effect occurs
when light impinges upon a
molecule and interacts with the
electron cloud of the bonds of that
molecule. The incident photon
excites one of the electrons into a
virtual state. For the spontaneous
Raman effect, the molecule will be
excited from the ground state to a
virtual energy state, and relax into
a vibrational excited state, which
generates Stokes Raman scattering.
If the molecule was already in an
elevated vibrational energy state,
the Raman scattering is then called anti-Stokes Raman scattering.
A molecular polarizability change, or amount of deformation of the electron cloud, with
respect to the vibrational coordinate is required for the molecule to exhibit the Raman effect. The
amount of the polarizability change will determine the Raman scattering intensity, whereas the
Raman shift is equal to the vibrational level that is involved

Spectrofluorometer
Two general types of instruments exist:
 Filter fluorometers use filters to isolate the incident light and fluorescent light.
 Spectrofluorometers use diffraction grating monochromators to isolate the incident light
and fluorescent light.
Both types utilize the following scheme: The light from an excitation source passes through a
filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by
the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in
all directions. Some of this fluorescent light passes through a second filter or monochromator
and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the
risk of transmitted or reflected incident light reaching the detector.
 Various light sources may be used as excitation sources, including lasers, photodiodes,
and lamps; xenon arcs and mercury vapor lamps in particular. A laser only emits light of high
irradiance at a very narrow wavelength interval, typically under 0.01 nm, which makes an
excitation monochromator or filter unnecessary. The disadvantage of this method is that the
wavelength of a laser cannot be changed by much. A mercury vapor lamp is a line lamp,
meaning it emits light near peak wavelengths. By contrast, a xenon arc has a continuous
emission spectrum with nearly constant intensity in the range from 300-800 nm and a sufficient
irradiance for measurements down to just above 200 nm.
Filters and/or monochromators may be used in fluorimeters. A monochromator transmits
light of an adjustable wavelength with an adjustable tolerance. The most common type of
monochromator utilizes diffraction grating, that is, collimated light enters a grating and exits
with a different angle depending on the wavelength. The monochromator can then select which
wavelengths to transmit. For allowing anisotropy measurements the addition of two polarization
filters are necessary: One after the excitation monochromator or filter, and one before the
emission monochromator or filter.
As mentioned before, the
fluorescence is most often measured at a
90° angle relative to the excitation light.
This geometry is used instead of placing the
sensor at the line of the excitation light at a
180° angle in order to avoid interference of
the transmitted excitation light. No
monochromator is perfect and it will
transmit some stray light, that is, light with
other wavelengths than the targeted. An
ideal monochromator would only transmit
light in the specified range and have a high wavelength-independent transmission. When
measuring at a 90° angle, only the light scattered by the sample causes stray light. This results in
a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000
when compared to the 180° geometry. Furthermore, the fluorescence can also be measured from
the front, which is often done for turbid samples.
The detector can either be single-channeled or multichanneled. The single-channeled
detector can only detect the intensity of one wavelength at a time, while the multichanneled
detects the intensity at all wavelengths simultaneously, making the emission monochromator or
filter unnecessary. The different types of detectors have both advantages and disadvantages.
The most versatile fluorimeters with dual monochromators and a continuous excitation
light source can record both an excitation spectrum and a fluorescence spectrum. When
measuring fluorescence spectra, the wavelength of the excitation light is kept constant,
preferably at a wavelength of high absorption (i.e., maximum absorbance, ex), and the emission
monochromator scans the spectrum. For measuring excitation spectra, the wavelength passing
through the emission filter or monochromator is kept constant and the excitation monochromator
is scanning. The excitation spectrum generally is identical to the absorption spectrum as the
fluorescence intensity is proportional to the absorption.

Excitation sources
A xenon arc lamp is an artificial light source. Powered by electricity, it uses ionized
xenon gas to produce a bright white light that closely mimics natural daylight. Xenon arc lamps
can be roughly divided into three categories:
 Continuous-output xenon short-arc lamps
 Continuous-output xenon long-arc lamps
 Xenon flash lamps (which are usually considered
separately)
Each consists of a glass or fused quartz arc tube with tungsten
metal electrodes at each end. The glass tube is first evacuated
and then re-filled with xenon gas. For xenon flashtubes, a third
"trigger" electrode usually surrounds the exterior of the arc
tube.
Our spectrofluorometer is equipped with Continuous- 15 kW xenon short-arc lamp
output xenon short-arc lamps where the spectrum is between
200 nm and 650 nm.

Monochromator designs
A typical monochromator design is
shown on the right. It consists of a
diffraction grating (dispersing element),
slits, and spherical mirrors. The light source
emits a broad spectrum of radiation as
represented by the multi-colored line from
the lamp to the grating. (The yellow color of
the light source represents all colors.) The
diffraction grating disperses light by
diffracting different wavelengths at different
Schematic of a Czerny-Turner monochromator
angles. The grating is positioned so that
green light passes through the exit slit and
all other colors are blocked. The particular wavelength that passes through the monochromator is
selected by rotating the angle of the grating. The mirror and slit positions remain fixed. If this
grating was rotated clockwise slightly, what color light would pass through the exit slit?
Scanning a spectrum is accomplished by rotating the grating with a motor. The detector
measures the power of the light that strikes it, converting the light power to an electrical signal.

Monochromator parameters
 Bandpass: The wavelength range that the monochromator transmits.
 Dispersion: The wavelength dispersing power, usually given as spectral range / slit width
(nm/mm). Dispersion depends on the focal length, grating resolving power, and the grating
order.
 Resolution: The minimum bandpass of the spectrometer, usually determined by the
aberrations of the optical system.
 Acceptance angle (f/#): A measure of light collecting ability, focal length / mirror diameter
 Blaze wavelength: The wavelength of maximum intensity in first order.

Photomultiplier Tube (PMT)

 Photomultiplier tubes (PMTs) convert photons to an electrical signal. They have a high
internal gain and are sensitive detectors for low-intensity applications such as fluorescence
spectroscopy.
Design. A PMT consists of a
photocathode and a series of dynodes in an
evacuated glass enclosure. When a photon
of sufficient energy strikes the
photocathode, it ejects a photoelectron due
to the photoelectric effect. The
photocathode material is usually a mixture
of alkali metals, which make the PMT
sensitive to photons throughout the visible
region of the electromagnetic spectrum. Schematic of a PMT
The photocathode is at a high negative
voltage, typically -500 to -1500 volts. The Typical specifications of PMT
photoelectron is accelerated towards a series Wavelength range:1 110-1100 nm
of additional electrodes called dynodes. These 2
Quantum efficiency (Q.E.): 1-10%
electrodes are each maintained at successively Response time: 1-20 ns
less negative potentials. Additional electrons 1
Wavelength sensitivity depends on
are generated at each dynode. This cascading wavelength. UV-sensitive PMTs must have
effect creates 105 to 107 electrons for each UV-transmitting windows.
photoelectron that is ejected from the 2
The Q.E. is the (number of electrons ejected
photocathode. The amplification depends on by the photocathode / number of incident
the number of dynodes and the accelerating photons).
voltage. This amplified electrical signal is
collected at an anode at ground potential, Schematic state energy level diagram
which can be measured. S is singlet and T is triplet. The S0 state is the ground
Phototubes are similar to PMTs, state and the subscript numbers identify individual
but consist of only a photocathode and states.
anode. Since phototubes do not have a
dynode chain to provide internal
amplification, they are used in less
sensitive applications such as absorption
spectrometers.

Emission spectra
Timescale
1. Fluorescence
S0 → Sn absorption
Sn → S1 internal conversion (10-11 - 10-14 sec)
Characteristics of Excited States
S1 → S0 + h fluorescence (10-7 - 10-9 sec)
a. Energy
S1 → Tn intersystem crossing (10-8 sec)
b. Lifetime
S1 → S0 internal conversion (10-5 - 10-7 sec)
c. Quantum Yield
d. Polarization T1 → S0 + h phosphorescence (10 - 10-3 sec)
T1 → S0 internal conversion (10 - 10-3 sec)
2. Phosphorescence occurs at longer wavelength than does fluorescence.
Often, the emission band is red-shifted
relative to the absorption band: "Stokes shift"

3. Quantum Yield (QY) = F

FF = number of fluorescence quanta emitted
divided by number of quanta absorbed to a
singlet excited state;
FF = ratio of photons emitted to photons
absorbed

Excited states decay exponentially with time.

I = I0e-t/τ
I0 is the initial intensity at time zero,
I is the intensity at some later time t,
and τ is the lifetime of the excited state (i.e., the
fluorescence lifetime).
Also, kF = 1/ τ, where kF is the rate constant for
fluorescence.

Quantum yield is the ratio of photons emitted to photons absorbed by the system: FF = kF / kF +
kISC + knr + kq + kr

4. Polarization The degree of polarization is defined as

Molecule of interest is randomly oriented follows:
in a rigid matrix (organic solvent at low
temperature or room temperature polymer). Plane
polarized light is used as the excitation source.
where I|| and I┴ are the intensities of the
Polarization of fluorescence of phenol in
observed parallel and perpendicular
propylene glycol at -70°C shows that the
components, respectively. α is the angle
transition moments of the corresponding
between the emission and absorption
absorption bands are mutually perpendicular.
transition moments. If α is 0°, then P =
+1/2, and if α is 90°, then P = -1/3.
 Phosphorescence is usually slow (seconds),
therefore, quenching by impurities including oxygen usually makes phosphorescence
difficult to observe. Low temperature glasses and rigorous exclusion of oxygen are usually
necessary to observe phosphorescence. Since this condition is not biological, fluorescence
is the primary emission process of biological relevance.
 Relative quantum yields are determined by using a standard such as quinine sulfate in 1 N
H2SO4 (fF = 0.70), or fluorescein in 0.1 N NaOH (fF = 0.93). The areas under the emission
band of the standard relative to the sample are compared. It is, of course, important that the
 absorption at lex are matched.
 Excited-state decay rates can be measured by exciting the sample with a short pulse of light
and monitoring the emission as a function of time.

Fluorescence decay of a pure sample follows a single exponential decay. The dark line
shows the excitation pulse. Time correlated single photon counting was used to obtain this data.
This technique counts the number of emitted photons hitting a detector at times, t, following
excitation.
One critical difference between steady-state and kinetic measurements of fluorescence is
that the value of tF is not a function of concentration of the sample while the value of FF is
concentration dependent. Only at low concentration is the value of FF linearly dependent on
concentration. The reason is the so-called inner filter effect.

5. The inner filter effect

At low concentration, the emission of light is uniform from the front to the back of
sample cuvette. At high concentration, more light is emitted from the front than the back. Since
emitted light only from the middle of the cuvette is detected the concentration must be low to
assure accurate FF measurements.

Fluorescence characteristics of chromophores found in proteins and nucleic acids. Generally,

quantum yields are low and lifetimes are short.
Absorption Fluorescence  
Substance Condition lmax (nm) lmax (nm) fF tF (nsec)
Tryptophan H2O, pH 7 280 348 0.20 2.6
Tyrosine H2O, pH 7 274 303 0.14 3.6
Phenylalanine H2O, pH 7 257 282 0.04 6.4
-4
Adenine H2O, pH 7 260 321 2.6 • 10 <0.02
Guanine H2O, pH 7 275 329 2.6 • 10-4 <0.02
-4
Cytosine H2O, pH 7 267 313 0.8 • 10 <0.02
-4
Uracil H2O, pH 7 260 308 0.4 • 10 <0.02
NADH H2O, pH 7 340 470 0.019 0.40
Define: lmax (nm): maximum wavelength of the absorption; lmax (nm): maximum wavelength of
the emission; fF: Quantum yield; tF (nsec): lifetime of the excited state.
 Fluorescence emission spectra of human serum albumin (solid line), tryptophan alone
(dashed line), and an 18:1 molar ratio of tyrosine to tryptophan (gray line): Excitation at 245 nm.
The 18:1 sample approximates the relative occurrence of these amino acids in the protein. Note
that the spectrum of the protein closely resembles that of pure tryptophan because tyrosine
sensitivity is low and its emission is most likely quenched by tryptophan (via energy-transfer
mechanism). (Dr. Rima: where is the figure for this paragraph?)

Commonly, fluorescent probe

molecules are used to characterize
protein and nucleic acids.
Sensitivity is higher.
lmax is also different from biomolecule
so selective excitation is possible.

Fluorescence generally is much

more sensitive to the environment of
the chromophore than is light
absorption. Therefore, fluorescence is
an effective technique for following the
binding of ligands or conformational changes.
The sensitivity of fluorescence is a consequence of the relatively long time a molecule
stays in an excited singlet state before deexcitation. Absorption, or CD, is a process that is over
in 10-15 sec. On this time scale, the molecule and its environment are effectively static. In
contrast, during the 10-9 to 10-8 sec that a singlet remains excited, all kinds of processes can
occur, including protonation or deprotonation reactions, solvent-cage relaxation, local
conformational changes, and any processes coupled to translational or rotational motion.
A number of fluorescent molecules have a very convenient property in aqueous solution
their fluorescence is very strongly quenched, but in a nonpolar or a rigid environment (like in a
protein or nucleic acid) a striking enhancement is observed. In addition protein protects the probe
from quenchers such as oxygen.
F0/F = fo/f = [kF + kIC + kISC + kq(Q)]/(kF + kIC + kISC) = 1 + kqt0(Q),
where F = fluorescence in the presence of quencher, F0 = fluorescence in the absence of
quencher.
Therefore, a plot of F0/F versus concentration of Q will yield a value for kq.
Quenching of tryptophan fluorescence by collision with oxygen:
Tryptophan: kq = 12 • 109 M-1 sec-1 (diffusion controlled)
Carbonic anhydrase: kq = 2.6 • 109 M-1 sec-1
What are the purposes of these 5 Figures?
Experiment

Excitation and emission of anthracene

Emission spectrum
a. Select the convenience excitation wavelength (λex) for the anthracene solution

b. Keep the excitation wavelength ((λex) constant and scan the emission wavelength
between λ > than λex and < 2 λex. This operation is to avoid the incident light going
directly into the photomultiplier.
Excitation spectrum
a. Select the wavelength at the maximum emission and keep constant at λem.

b. Scan λex from 200 nm to λex < λem maximum.

Conclusion
a. Compare the maxima in the emission spectrum and in the excitation spectrum
b. Calculate the values of those maxima in nm-1 (Dr. Rima: please clarify this questions or
show it how you do it?)
c. Mention the Rayleigh and Raman diffusion
d. Plot the fluorescence intensities in function of the concentrations
e. Determine limit of detection: Follow the intensity of the fluorescence with the dilution
until the signal cannot be detected. It is often defined by 3-fold of background noises.

References

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for Biology and Medicine”. Dekker, New York, 1971.
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Bacon, Boston, 1978, chap. 9.
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York, 1975, chap. 4.
5. J.E. O’Reilly, J. Chem. Ed., 53, 191 (1976).
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10. G.G. Guilbault, “Practical Fluorescence: Theory, Methods, Techniques”. Dekker, New
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2002