ChE ELNT: Nanotechnology

Lecture 3
Characterisation of Nanomaterials
Molecular Spectroscopy
(Infrared, UV-Vis, Fluorescence)
Dr. Lorico DS Lapitan Jr.
g-rays X-rays UV IR Microwave Radio

Visible

Department Of Chemical Engineering, CHE ELNT

University Of Santo Tomas, España, Manila 5CHE A-B-C
INFRARED (IR) SPECTROSCOPY
Theory and Interpretation of
IR Spectra
 SPECTROSCOPY - Study of spectral
information

Physical response Detecting

Molecule
stimulus instrument

Visual (most common)

representation, or
Spectrum

Upon irradiation with infrared light, certain bonds respond

by vibrating faster. This response can be detected and
translated into a visual representation called a spectrum.
 ELECTROMAGNETIC SPECTRUM
Most organic spectroscopy uses electromagnetic energy, or radiation,
as the physical stimulus.

Electromagnetic energy (such as visible light) has no detectable mass

component. In other words, it can be referred to as “pure energy.”

Other types of radiation such as alpha rays, which consist of helium

nuclei, have a detectable mass component and therefore cannot be
categorized as electromagnetic energy.

The important parameters associated with electromagnetic radiation are:

• Energy (E): Energy is directly proportional to frequency, and inversely

proportional to wavelength, as indicated by the equation below.
• Frequency (m)
• Wavelength (l)
E = hm
EFFECT OF ELECTROMAGNETIC RADIATION
ON MOLECULES

Graphics source: Wade, Jr., L.G. Organic Chemistry, 5th ed. Pearson Education Inc., 2003
 Infrared radiation is largely thermal energy.
It induces stronger molecular vibrations in covalent bonds, which can
be viewed as springs holding together two masses, or atoms.

Specific bonds respond to (absorb) specific frequencies

Graphics source: Wade, Jr., L.G. Organic Chemistry, 5th ed. Pearson Education Inc., 2003
VIBRATIONAL MODES
• Covalent bonds can vibrate in several modes, including stretching,
rocking, and scissoring.
• The most useful bands in an infrared spectrum correspond to
stretching frequencies, and those will be the ones we’ll focus on.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 5th ed. Pearson Education Inc., 2003
TRANSMISSION vs. ABSORPTION
When a chemical sample is exposed to the action of IR LIGHT, it can
absorb some frequencies and transmit the rest. Some of the light can
also be reflected back to the source.

Transmitted light
IR Chemical
Detector
source sample

From all the frequencies it receives, the chemical sample

can absorb (retain) specific frequencies and allow the
rest to pass through it (transmitted light).

The detector detects the transmitted frequencies, and by doing so also reveals the
values of the absorbed frequencies.
AN IR SPECTRUM IN ABSORPTION MODE
The IR spectrum is basically a plot of transmitted (or absorbed) frequencies
vs. intensity of the transmission (or absorption). Frequencies appear in the
x-axis in units of inverse centimeters (wavenumbers), and intensities are
plotted on the y-axis in percentage units.

The graph above shows a spectrum in absorption mode.

AN IR SPECTRUM IN TRANSMISSION MODE

The graph above shows a spectrum in transmission mode.

This is the most commonly used representation.
CLASSIFICATION OF IR BANDS
IR bands can be classified as strong (s), medium (m), or weak (w),
depending on their relative intensities in the infrared spectrum. A strong
band covers most of the y-axis. A medium band falls to about half of the
y-axis, and a weak band falls to about one third or less of the y-axis.
INFRARED ACTIVE BONDS
Not all covalent bonds display bands in the IR spectrum. Only polar
bonds do so. These are referred to as IR active.
The intensity of the bands depends on the magnitude of the dipole
moment associated with the bond in question:
• Strongly polar bonds such as carbonyl groups (C=O) produce strong
bands.
• Medium polarity bonds and asymmetric bonds produce medium
bands.
• Weakly polar bond and symmetric bonds produce weak or non
observable bands.
INFRARED BAND SHAPES
Infrared band shapes come in various forms. Two of the most common
are narrow and broad. Narrow bands are thin and pointed, like a
dagger. Broad bands are wide and smoother.

A typical example of a broad band is that displayed by O-H bonds, such

as those found in alcohols and carboxylic acids, as shown below.
INFORMATION OBTAINED FROM IR SPECTRA

• IR is most useful in providing information about the presence or

absence of specific functional groups.

• IR can provide a molecular fingerprint that can be used when

comparing samples. If two pure samples display the same IR
spectrum it can be argued that they are the same compound.

• IR does not provide detailed information or proof of molecular

formula or structure. It provides information on molecular fragments,
specifically functional groups.

• Therefore it is very limited in scope, and must be used in conjunction

with other techniques to provide a more complete picture of the
molecular structure.
Functional Groups
IR ABSORPTION RANGE
The typical IR absorption range for covalent bonds is 600 - 4000 cm-1. The graph
shows the regions of the spectrum where the following types of bonds normally
absorb. For example a sharp band around 2200-2400 cm-1 would indicate the
possible presence of a C-N or a C-C triple bond.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 5th ed. Pearson Education Inc., 2003
 THE FINGERPRINT REGION
Although the entire IR spectrum can be used as a fingerprint for the purposes of
comparing molecules, the 600 - 1400 cm-1 range is called the fingerprint region.
This is normally a complex area showing many bands, frequently overlapping each
other. This complexity limits its use to that of a fingerprint, and should be ignored by
beginners when analyzing the spectrum. As a student, you should focus your
analysis on the rest of the spectrum, that is the region to the left of 1400 cm-1.

Focus your analysis on this region. This is where most stretching Fingerprint region: complex and difficult to
frequencies appear. interpret reliably.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
IR SPECTRUM OF ALKANES
Alkanes have no functional groups. Their IR spectrum displays only C-C and C-H
bond vibrations. Of these the most useful are the C-H bands, which appear
around 3000 cm-1. Since most organic molecules have such bonds, most organic
molecules will display those bands in their spectrum.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 5th ed. Pearson Education Inc., 2003
IR SPECTRUM OF ALKENES
Besides the presence of C-H bonds, alkenes also show sharp, medium bands
corresponding to the C=C bond stretching vibration at about 1600-1700 cm-1.
Some alkenes might also show a band for the =C-H bond stretch, appearing
around 3080 cm-1 as shown below. However, this band could be obscured by the
broader bands appearing around 3000 cm-1 (see next slide)

Graphics source: Wade, Jr., L.G. Organic Chemistry, 5th ed. Pearson Education Inc., 2003
IR SPECTRUM OF ALKENES
This spectrum shows that the band appearing around 3080 cm-1 can be obscured
by the broader bands appearing around 3000 cm-1.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
 IR SPECTRUM OF ALKYNES
The most prominent band in alkynes corresponds to the carbon-carbon
triple bond. It shows as a sharp, weak band at about 2100 cm-1. The
reason it’s weak is because the triple bond is not very polar. In some
cases, such as in highly symmetrical alkynes, it may not show at all due to
the low polarity of the triple bond associated with those alkynes.

Terminal alkynes, that is to say those where the triple bond is at the end of
a carbon chain, have C-H bonds involving the sp carbon (the carbon that
forms part of the triple bond). Therefore they may also show a sharp, weak
band at about 3300 cm-1 corresponding to the C-H stretch.

Internal alkynes, that is those where the triple bond is in the middle of a
carbon chain, do not have C-H bonds to the sp carbon and therefore lack
the aforementioned band.

The following slide shows a comparison between an unsymmetrical

terminal alkyne (1-octyne) and a symmetrical internal alkyne (4-octyne).
IR SPECTRUM OF ALKYNES

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
IR SPECTRUM OF A NITRILE
In a manner very similar to alkynes, nitriles show a prominent band around 2250
cm-1 caused by the CN triple bond. This band has a sharp, pointed shape just
like the alkyne C-C triple bond, but because the CN triple bond is more polar, this
band is stronger than in alkynes.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
IR SPECTRUM OF AN ALCOHOL
The most prominent band in alcohols is due to the O-H bond, and it appears as a
strong, broad band covering the range of about 3000 - 3700 cm-1. The sheer size
and broad shape of the band dominate the IR spectrum and make it hard to miss.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
IR SPECTRUM OF ALDEHYDES AND KETONES
• Carbonyl compounds are those that contain the C=O functional
group. In aldehydes, this group is at the end of a carbon chain,
whereas in ketones it’s in the middle of the chain. As a result, the
carbon in the C=O bond of aldehydes is also bonded to another carbon
and a hydrogen, whereas the same carbon in a ketone is bonded to
two other carbons.

• Aldehydes and ketones show a strong, prominent, stake-shaped band

around 1710 - 1720 cm-1 (right in the middle of the spectrum). This
band is due to the highly polar C=O bond. Because of its position,
shape, and size, it is hard to miss.

• Because aldehydes also contain a C-H bond to the sp2 carbon of the
C=O bond, they also show a pair of medium strength bands positioned
about 2700 and 2800 cm-1. These bands are missing in the spectrum
of a ketone because the sp2 carbon of the ketone lacks the C-H bond.
IR SPECTRUM OF ALDEHYDES AND KETONES

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
IR SPECTRUM OF A CARBOXYLIC ACID
A carboxylic acid functional group combines the features of alcohols and ketones
because it has both the O-H bond and the C=O bond. Therefore carboxylic acids
show a very strong and broad band covering a wide range between 2800 and
3500 cm-1 for the O-H stretch. At the same time they also show the stake-shaped
band in the middle of the spectrum around 1710 cm-1 corresponding to the C=O
stretch.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
 IR SPECTRA OF AMINES
The most characteristic band in amines is due to the N-H bond stretch, and it appears as a
weak to medium, somewhat broad band (but not as broad as the O-H band of alcohols). This
band is positioned at the left end of the spectrum, in the range of about 3200 - 3600 cm-1.

Primary amines have two N-H bonds, therefore they typically show two spikes that make this
band resemble a molar tooth. Secondary amines have only one N-H bond, which makes
them show only one spike, resembling a canine tooth. Finally, tertiary amines have no N-H
bonds, and therefore this band is absent from the IR spectrum altogether. The spectrum
below shows a secondary amine.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
 IR SPECTRUM OF AMIDES
The amide functional group combines the features of amines and ketones because
it has both the N-H bond and the C=O bond. Therefore amides show a very
strong, somewhat broad band at the left end of the spectrum, in the range between
3100 and 3500 cm-1 for the N-H stretch. At the same time they also show the
stake-shaped band in the middle of the spectrum around 1710 cm-1 for the C=O
stretch. As with amines, primary amides show two spikes, whereas secondary
amides show only one spike.

Graphics source: Wade, Jr., L.G. Organic Chemistry, 6th ed. Pearson Prentice Hall Inc., 2006
ULTRAVIOLET AND VISIBLE
SPECTROSCOPY
Theory and Application of UV-Vis
to Characterisation of Au and Ag
Plasmonic Nanomaterials
Visible Spectroscopy
WHAT IS COLOUR?
Colour is a sensation which occurs when light enters the eye and
focuses on the retina at the back of the eye. The light actually initiates a
photochemical reaction in the nerve cells attached to the retina. These
transmit impulses to the brain and stimulate our sense of colour.

CONES - Give colour and RETINA

three types which pick up

red, blue and green BRAIN

RODS - Give grey/black and

= CONES
also used for night vision. = RODS

All the colours we actually sense are made up of these three colours
together with white and grey and black.
Visible Spectroscopy
COMPOSITION OF WHITE LIGHT
• Sunlight is white light and covers a wavelength range of 380-750nm. A simple
physics experiment shows that white light is actually a composition of a range of
colours i.e., light of different energies and hence wavelengths.

When white light falls on an

object the colour detected by
the eye will depend upon the Red
ABSORPTION/REFLECTION Orange
properties of the material WHITE
Yellow
in the object; LIGHT
Green
Blue
Indigo
Violet

• If the material completely REFLECTS all light it appears WHITE

• If the material absorbs a constant fraction of the light across the spectrum it
appears GREY.
• If the material completely ABSORBS all the light it appears BLACK
Visible Spectroscopy
When a sample only absorbs light of a single wavelength
the eye sees COMPLEMENTARY colours.
Wavelength Range Colour Colour Seen By
Absorbed Absorbed Eye
380 - 430 Violet Yellow - Green
430 - 480 Blue Yellow
480 - 490 Green - Blue Orange
490 - 500 Blue - Green Red
500 - 560 Green Purple
560 - 580 Yellow - Green Violet
580 - 590 Yellow Blue
590 - 610 Orange Green - Blue
610 - 750 Red Blue - Green

LOW HIGH
UV-Visible Spectroscopy
UV Radiation – Wavelength range 220 - 380nm

VISIBLE Radiation – Wavelength range 380 - 780nm

Substances can absorb varying amounts of UV and/or

Visible radiation at particular wavelengths – Coloured
compounds absorb energy in both UV and visible region of
the electromagnetic spectrum.

Substances can be liquids or solids and measurements are

made with instruments called SPECTROPHOTOMETERS
or SPECTROMETERS.
UV-Visible Spectroscopy: Instrumentation
1. The construction of a traditional UV-VIS spectrometer is very
similar to an IR, as similar functions – sample handling,
irradiation, detection and output are required
2. Here is a simple schematic that covers most modern UV
spectrometers:

log(I0/I) = A
I0 I
sample
UV-VIS sources

200 700

detector
l, nm

monochromator/
reference

beam splitter optics I0 I0

UV-Visible Spectroscopy: Instrumentation

3. Two sources are required to scan the entire UV-VIS band:

• Deuterium lamp – covers the UV – 200-330
• Tungsten lamp – covers 330-700

4. As with the dispersive IR, the lamps illuminate the entire

band of UV or visible light; the monochromator (grating or
prism) gradually changes the small bands of radiation sent
to the beam splitter

5. The beam splitter sends a separate band to a cell containing

the sample solution and a reference solution

6. The detector measures the difference between the

transmitted light through the sample (I) vs. the incident light
(I0) and sends this information to the recorder
UV-Visible Spectroscopy: Instrumentation
Sample Handling

1. Virtually all UV spectra are recorded solution-phase

2. Cells can be made of plastic, glass or quartz

3. Only quartz is transparent in the full 200-700 nm range;

plastic and glass are only suitable for visible spectra

4. Concentration (we will cover shortly) is empirically

determined

A typical sample cell (commonly called a cuvette):

UV-Visible Spectroscopy – Theory
INCIDENT LIGHT TRANSMITTED LIGHT
254nm 254nm
SAMPLE
Intensity (I o ) Intensity (I t )

• If a particular wavelength of UV or Visible radiation can be isolated from the

source and passed through a sample which can ABSORB some of the radiation
then the TRANSMITTED light intensity (It ) will less than the INCIDENT light
intensity (Io).

• The amount of light transmitted with respect to the incident light is called
TRANSMITTANCE (T) ie.,
T=
It

Io

• Sample can absorb all or none of the incident light and therefore
• transmittance often quoted as a percentage eg.,

It
% T= X 100
Io
UV-Visible Spectroscopy – Theory
ABSORBANCE A = - log10 T
It B
A = - log10
I A
o
Io 0
A = log10 220 380
Wavelength(nm)
It

By plotting Absorbance vs wavelength an ABSORBANCE SPECTRUM is

generated. The absorbance spectra for the same compounds A and B are
shown.

With the advantage that absorbance measurements are usually linear with
Concentration, absorbance spectra are now used
The Laws of Spectrophotometry
There are two very important basic laws and a third one which is a
combination of the two.

LAMBERTS LAW – ABSORBANCE (A) proportional to the PATHLENGTH (l)

of the absorbing medium.

BEERS LAW - ABSORBANCE (A) proportional to the CONCENTRATION (c)

of the sample.

BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l

A  cl

A = Ecl (A is a ratio and therefore has no units)

The constant E is called the MOLAR EXTINCTION COEFFICIENT
UV-Visible Spectroscopy – Theory
UNITS OF THE MOLAR EXTINCTION COEFFICIENT
• CONCENTRATION (c) - Moles litre-1
• PATHLENGTH (l) - cm

A = Ecl Hence E =A
cl
E = 1 ˛

mole litre-1 x cm

E = mole-1 litre x cm -1
But 1 litre = 1000 cm3
E = 1000 mole-1 cm3 x cm -1
Hence Units of E = 1000 cm2 mole -1
 UV-Visible Spectroscopy – Theory
IMPORTANCE OF THE BEER LAMBERT LAW
A = Ecl but if E and l are constant

ABSORBANCE  CONCENTRATION and should be linear relationship

Prepare standards of the analyte to be quantified at known concentrations
and measure absorbance at a specified wavelength.

Prepare calibration curve.

ABSORBANCE AT 300nm
From measuring absorbance of sample x
x
Concentration of analyte in sample
can be obtained from the calibration curve x
x
E can be obtained from the slope of the
calibration curve for a given wavelength (l) x
CONCENTRATION (moles litre-1 )
UV-Visible Spectroscopy – Theory
RULES FOR QUANTITATIVE ANALYSES
x

ABSORBANCE AT 300nm
At high concentrations the calibration curve may x
deviate from linearity – Always ensure your
concentration of the sample falls within the linear x
range – if necessary dilute sample
x
Absorbance not to exceed 1 to reduce error* x
CONCENTRATION (moles litre-1 )

CHOOSE CORRECT WAVELENGTH A

An analyte may give more than one absorbance C lmax
0.6
B
maxima (lmax) value.

Many compounds absorb at 220-230nm hence do

not use A

Need to choose wavelength more specific 0

to compound (SELECTIVITY) and if more 220 380
than one select one with highest absorbance Wavelength (nm)
as this gives less error – hence use C
Optical Properties of Nanomaterials
Optical Properties of Nanomaterials
• Colour of a nanoparticle solution is dependent on
nanoparticle size.
Surface Plasmon Resonance
When a nanoparticle is much smaller than the
wavelength of light, coherent oscillation of the
conduction band electrons induces by the interaction
with an electromagnetic field. This is called Surface
Plasmon Resonance (SPR)
Size Dependence

P. N. Njoki, I.-I. S. Lim, D. Mott, H.-Y. Park, B. Khan, S.

Mishra, R. Sujakumar, J. Luo and C.-J. Zhong, The Journal
of Physical Chemistry C, 2007, 111, 14664-14669
Surrounding Medium
The surface plasmon
resonance peak changes
with it own dielectric
properties and those of its
local environment including
the substrate, solvent, and
adsorbates.

Spectral shift for individual blue (roughly spherical) silver nanoparticles.

Typical blue particles spectrum as it is shifted from (a) air to (b) 1.44 index
oil, and successive oil treatments in 0.04 index incremental increases.
Particle Density
Beginning from the left
“glass doped with gold
nanoparticles” and spacing
between them is large.
In the right side figure “bulk
gold is doped with glass”. As
the spacing is reduced,
dipole interactions become
increasingly important.
 Silver Nanoparticles Optical Properties:
The Effect of Size on Optical Properties
• Smaller nanospheres
primarily absorb light
and have peaks near
400 nm, while larger
spheres exhibit
increased scattering
and have peaks that
broaden and shift
towards longer
wavelengths (known as
red-shifting)

Paramelle, D., Sadovoy, A., Gorelik, S., Free, P., Hobley, J., & Fernig, D. G. (2014). A rapid method to estimate the concentration
of citrate capped silver nanoparticles from UV-visible light spectra. Analyst, 139(19), 4855-4861.
 Silver Nanoparticles Optical Properties:
The Effect of Local Refractive Index on Optical
Properties
As the refractive index near the nanoparticle surface
increases, the nanoparticle extinction spectrum shifts to
longer wavelengths (known as red-shifting).

The figure displays the extinction

spectrum of a 50 nm silver
nanosphere as the local refractive
index is increased. Increasing the
refractive index from 1.00 to 1.60
results in an extinction peak shift of
over 90 nm, moving the peak from
the ultraviolet to the visible region of
the spectrum.
Silver Nanoparticles Optical Properties:
The Effect of Aggregation on Optical Properties
As the particles destabilize, the original extinction peak will
decrease in intensity (due to the depletion of stable
nanoparticles), and often the peak will broaden or a
secondary peak will form at longer wavelengths (due to the
formation of aggregates).
In the figure, the extinction spectrum of 50 nm
is monitored as sodium carbonate is added to
the solution (20 mM salt concentration). The
rapid and irreversible change in the extinction
spectrum clearly demonstrates that the
nanoparticles are agglomerating.
Gold Nanoparticles Optical Properties:
The Effect of Size on Optical Properties

Smaller Au
nanospheres primarily
absorb light and have
peaks near 520 nm,
while larger spheres
exhibit increased
scattering and have
peaks that broaden
significantly and shift
towards longer
wavelengths (known
as red-shifting).
 Gold Nanoparticles Optical Properties:
The Effect of Local Refractive Index on Optical
Properties
As the refractive index near the nanoparticle surface increases,
the nanoparticle extinction spectrum shifts to longer wavelengths
(known as red-shifting).
The figure displays the
calculated extinction
spectrum of a 50 nm gold
nanosphere as the local
refractive index is increased.
Increasing the refractive index
from 1.00 to 1.60 results in an
extinction peak shift of over
40 nm, moving the peak from
the green to the red region of
the spectrum.
 Gold Nanoparticles Optical Properties:
The Effect of Aggregation on Optical Properties
As the particles destabilize, the original extinction peak will
decrease in intensity (due to the depletion of stable nanoparticles),
and often the peak will broaden or a secondary peak will form at
longer wavelengths (due to the formation of aggregates).
In the figure, the extinction spectrum of
12 nm functionalized gold that has
carboxy (-COOH) groups on the surface
is monitored as the solution pH is
changed from 6.5 to 3. As the solution
becomes more acidic, the carboxy group
is protonated and the zeta potential is
reduced resulting in destabilized
nanoparticles that aggregate. The rapid
change in the extinction spectrum as the
pH is decreased clearly demonstrates
that the nanoparticles are
agglomerating.
Gold nanoparticles for applications in cancer
radiotherapy: Mechanisms and recent advancements

Her, S., Jaffray, D. A., & Allen, C. (2017). Gold nanoparticles for applications in cancer radiotherapy: Mechanisms and recent
advancements. Advanced drug delivery reviews, 109, 84-101.
Gold nanoparticles in radiation research:
potential applications for imaging and
radiosensitization

Dorsey, J. F., Sun, L., Joh, D. Y., Witztum, A., Kao, G. D., Alonso-Basanta, M., ... & Tsourkas, A. (2013). Gold
nanoparticles in radiation research: potential applications for imaging and radiosensitization. Translational cancer
research, 2(4), 280.
 Tumors Grow Blood Vessels
Tumors need blood to grow larger than ~2mm in size

Peer, D, et al. Nature Nanotechnology 2007, 2, 751-760

Enhanced Permeation and Retention Effect
Tumors have “leaky” blood
vessels, which allow relatively
large nano-sized “pills” to
enter. This is called
Enhanced Permeability and
Retention (EPR) Effect .
Normal blood vessels are not
“leaky” and nano-particles are
prevented from entering.
This allows one to selectively
target tumors.

Duncan, R. Nature Reviews Cancer 2006, 6, 688-701

FLUORESCENCE SPECTROSCOPY

Theory and Application of

Fluorescence Spectroscopy for
Characterisation of Quantum Dots
Emission Spectroscopy
• A characterization technique that measures the
emission of radiation by a material that has
been excited.

• Fluorescence spectroscopy is one type of

emission spectroscopy which records the
intensity of light radiated from the material as a
function of wavelength. It is a nondestructive
characterization technique.
Fluorescence Spectroscopy: Theory
• non-radiative decay (loss
Excited Electron from of energy through heat) Electron in the Ground
Ground State State
• radiative decay (loss of
energy through light)

Emission of luminescence photon for Group 12-16 semiconductor quantum dot.

Fluorescence Spectroscopy: Instrumentation
• A fluorimeter is a device that records the fluorescence intensity
as a function of wavelength.
Fluorescence Spectroscopy: Instrumentation
1) The light source
The excitation energy is provided by a light source that can
emit wavelengths of light over the ultraviolet and the visible
range.
• lasers (high irradiance at a very narrow wavelength
interval)
• xenon arcs (continuous emission spectrum between the
ranges of 300 - 800 nm)
• mercury-vapor lamps (discrete line source).
Fluorescence Spectroscopy: Instrumentation
2) The diffraction grating and primary filter

• The diffraction grating splits the incoming light source into

its component wavelengths.

• The monochromator can then be adjusted to choose with

wavelengths to pass through. Following the primary filter,
specific wavelengths of light are irradiated onto the sample
Fluorescence Spectroscopy: Instrumentation
3) Sample cell and sample preparation
• A proportion of the light from the primary filter is absorbed
by the sample.

• After the sample gets excited, the fluorescent substance

returns to the ground state, by emitting a longer
wavelength of light in all directions. Some of this light
passes through a secondary filter.
Fluorescence Spectroscopy: Instrumentation
3) Sample cell and sample preparation
The choice of cuvette depends on three factors:

• Type of solvent - For aqueous samples, specially

designed rectangular quartz, glass or plastic cuvettes are
used. For organic samples glass and quartz cuvettes are
used.
• Excitation wavelength – Depending on the size and
thus, bandgap of the Group 12-16 semiconductor
nanoparticles, different excitation wavelengths of light are
used. Depending on the excitation wavelength, different
materials are used
Fluorescence Spectroscopy: Instrumentation

3. Cost – Plastic cuvettes are the least expensive and can

be discarded after use. Though quartz cuvettes have the
maximum utility, they are the most expensive, and need to
reused. Generally, disposable plastic cuvettes are used
when speed is more important than high accuracy.
Fluorescence Spectroscopy: Instrumentation
4) Secondary filter
• The secondary filter is placed at a 90° angle to the original
light path to minimize the risk of transmitted or reflected
incident light reaching the detector. From the secondary filter,
wavelengths specific to the sample are passed onto the
detector.

5) Detector
• The detector can either be single-channeled or multichannel.
• The single-channeled detector can only detect the intensity
of one wavelength at a time, while the multichannel detects
the intensity at all wavelengths simultaneously, making the
emission monochromator or filter unnecessary.
Fluorescence Spectroscopy: Instrumentation
6) Output
The output is the form of a plot of intensity of emitted light
as a function.
Quantum Yields (ΦF)
• The fluorescence quantum yield can be calculated by
the ratio of photons absorbed to photons emitted by
the system.

• The quantum yield gives the probability of the excited

state getting relaxed via fluorescence rather than by
any other non-radiative decay.

• The most well-known method for recording quantum

yield is the comparative method which involves the use
of well characterized standard solutions.
Quantum Yields (ΦF)
• If a test sample and a standard sample have similar
absorbance values at the same excitation wavelength,
it can be assumed that the number of photons being
absorbed by both the samples is the same.

• This means that a ratio of the integrated fluorescence

intensities of the test and standard sample measured
at the same excitation wavelength will give a ratio of
quantum yields. Since the quantum yield of the
standard solution is known, the quantum yield for the
unknown sample can be calculated.
Quantum Yields (ΦF)

Absorbance
A plot of integrated fluorescence intensity versus absorbance at the excitation
wavelength. The slope of the graphs are proportional to the quantum yield of the
different samples. Quantum yield is then calculated using an equation, where
subscripts ST denotes standard sample and X denotes the test sample; QY is the
quantum yield; RI is the refractive index of the solvent.
 Quantum Yields (ΦF)
The assumption used in the comparative method is valid only in the Beer-Lambert law linear
regime. Beer-Lambert law states that absorbance is directly proportional to the path length
of light travelled within the sample, and concentration of the sample. The factors that affect
the quantum yield measurements are the following:
• Concentration – Low concentrations should be used (absorbance <
0.2 a.u.) to avoid effects such as self quenching.
• Solvent – It is important to take into account the solvents used for the
test and standard solutions. If the solvents used for both are the same
then the comparison is trivial. However, if the solvents in the test and
standard solutions are different, this difference needs to be accounted
for. This is done by incorporating the solvent refractive indices in the
ratio calculation.
• Standard samples – The standard samples should be characterized
thoroughly. In addition, the standard sample used should absorb at
the excitation wavelength of the test sample.
• Sample preparation – It is important that the cuvettes used are
clean, scratch free and clear on all four sides. The solvents used
must be of spectroscopic grade and should not absorb in the
wavelength range.
• Slit width – The slit widths for all measurements must be kept
constant.
Qualitative Information
• Useful qualitative information such as size distribution,
shape of the particle and presence of surface defects can
be obtained.

• The full width at half maximum (FWHM) is given by the

difference between the two extreme values of the
wavelength at which the photoluminescence intensity is
equal to half its maximum value. From the full width half
max (FWHM) of the fluorescence intensity Gaussian
distribution, it is possible to determine qualitatively the
size distribution of the sample.
Qualitative Information
• For a Group 12-16 quantum dot sample if the FWHM is
greater than 30, the system is very polydisperse and has
a large size distribution. It is desirable for all practical
applications for the FWHM to be lesser than 30.

Emission spectra of CdSe QDs showing the full width half maximum (FWHM).
Quantum Dots
• Quantum dots are usually regarded as
semiconductors by definition.
• Similar behavior is observed in some metals.
Therefore, in some cases it may be acceptable to
speak about metal quantum dots.
• Typically, quantum dots are composed of groups
II-VI, III-V, and IV-VI materials.
• QDs are bandgap tunable by size which means
their optical and electrical properties can be
engineered to meet specific applications.
Quantum Dots (QD)
• Nanocrystals (2-10 nm) of
semiconductor compounds
• Small size leads to
confinement of excitons
(electron-hole pairs)
Eg
• Quantized energy levels

• Examples: CdSe, PbSe, PbTe,

InP
Quantum Dots (QD)
• When the quantum dots are illuminated by UV light, some of the
electrons receive enough energy to break free from the atoms.

• When these electrons drop back into the outer orbit around the atom
(the valence band), they emit light.

• The colour of that light depends on the energy difference between the
conductance band and the valence band.
Quantum Dots

Colloidal quantum dots irradiated

with a UV light. Different sized
quantum dots emit different colour
light due to quantum confinement.
Quantum Dots
Absorption and emission occur at specific wavelengths,
which are related to QD size

Eg
 QD Synthesis: Colloidal Methods
• Example: CdSe quantum
dots
• 30mg of Elemental Se and 5mL of
octadecene are used to create a stock
precursor Se solution.
• 0.4mL of Trioctylphosphine oxide (TOPO) is
added to the Se precursor solution to
disassociate and cap the Se.
• Separately, 13mg of CdO, 0.6mL of oleic
acid and 10mL of octadecene were
combined and heated to 225oC
• Once the CdO solution reaches 225oC,
room-temperature Se precursor solution
was added. Varying the amount of Se
solution added to the CdO solution will
result in different sized QDs.

Journal of Chemcial Education. Vol. 82 No.11 Nov 2005

 QD Synthesis: Colloidal Methods

J. Am. Chem. Soc., 2003, 125, 12567

QD Synthesis: Colloidal Methods

Adv. Mater, 2007, 19, 376

QD Synthesis: Colloidal Methods
 Applications of QDs: Biological
• A broad absorption and narrow emission spectrum means a single
excitation source can be used to excite QDs of different colors making
them ideal for imaging multiple targets simultaneously.

CdSe/ZnS QDs used to image cancer cells in a live mouse.

Gao, Xiaohu. "In vivo cancer targeting and imaging
with." Nature Biotechnology 22(2004): 8.
Bibliography
• A. T. R. Williams, S. A. Winfield, and J. N. Miller, Analyst,
1983, 108, 1067.
• G. Schmid, Nanoparticles: From Theory to Application,
Wiley-VCH, Weinham, (2004).
• J. Y. Hariba, A Guide to Recording Fluorescence Quantum
Yield, Jobin Yvon Hariba Limited, Stanmore (2003).
• C. Qing Zhu, P. Wang, X. Wang, and Y. Li, Nanoscale Res.
Lett.., 2008, 3, 213