SPEKTROFOTOMETER INFRAMERAH
Spektrofotometer inframerah pada umumnya
digunakan untuk :
Menentukan gugus fungsi suatu senyawa
organik
Mengetahui informasi struktur suatu senyawa
organik dengan membandingkan daerah
sidik jarinya
SPEKTROFOTOMETER
INFRAMERAH
Spektroskopi
Pengukuran pada spektrum inframerah dilakukan pada
daerah cahaya inframerah tengah (mid-infrared) yaitu pada
panjang gelombang 2.5 - 50 µm atau bilangan gelombang
4000 - 200 cm-1. Energi yang dihasilkan oleh radiasi ini
akan menyebabkan vibrasi atau getaran pada molekul. Pita
absorbsi inframerah sangat khas dan spesifik untuk setiap
tipe ikatan kimia atau gugus fungsi. Metoda ini sangat
berguna untuk mengidentifikasi senyawa organik dan
organometalik.
Spektroskopi
Spektrum yang dihasilkan berupa grafik yang
menunjukkan persentase transmitan yang bervariasi pada
setiap frekuensi radiasi inframerah.
Interaksi dengan cahaya
A Simple Analogy
If you have a weight on a spring, you can set it into
motion by tapping it at its natural resonance
frequency. The best way to input energy to it is to
tap it 90 degrees out of phase. We can diagram the
experiment as follows:
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.
Absorption
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.
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 (see atomicemission spectroscopy),
For atoms excited with light it is called atomic
fluorescence (see atomic-fluorescence
spectroscopy).
Emission
For molecules it is called fluorescence if the
transition is between states of the same spin
and phosphorescence if the transition occurs
between states of different spin.
The emission intensity of an emitting substance is
linearly proportional to analyte concentration
at low concentrations, 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.
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.
Atomic-Absorption Spectroscopy (AA)
Introduction
Atomic-absorption (AA) spectroscopy uses the
absorption of light to measure the concentration of
gas-phase atoms.
Since samples are usually liquids or solids, the
analyte atoms or ions must be vaporized in a flame
or graphite furnace.
Atomic-Absorption Spectroscopy (AA)
The atoms absorb ultraviolet or visible light and
make transitions to higher electronic energy
levels. The analyte concentration is
determined from the amount of absorption.
Applying the Beer-Lambert Law directly in AA
spectroscopy is difficult due to variations in
the atomization efficiency from the sample
matrix, and nonuniformity of concentration
and path length of analyte atoms (in graphite
furnace AA).
Atomic-Absorption Spectroscopy (AA)
Concentration measurements are
usually determined from a working
curve after calibrating the instrument
with standards of known
concentration.
Atomic-Absorption Spectroscopy (AA)
Atomic-absorption spectroscopy (AAS) and
atomic-emission spectroscopy (AES) both rely
on the analyte existing as free atoms in the gas
phase. There are two common types of
interferences that reduce the concentration of
free gas-phase atoms: ionization and the
formation of molecular species.
Atomic-Absorption Spectroscopy (AA)
Note that the distribution of gas-phase atoms between the
ground and excited states is a physical property that depends
on the temperature of the environment. This distribution will
affect the analytical signal, but it is not a chemical
interference.
Concentration measurements are usually determined from a
working curve after calibrating the instrument with standards
of known concentration. To prevent any bias due to
differences between the standards and the samples, any
reagents that are added to reduce chemical interferences
should be added to the standards as well as the sample
solution.
Atomic-Absorption Spectroscopy (AA)
Preventing Ionization
Since samples are usually liquids or solids, the
sample must be vaporized and atomized in a hightemperature source such as a flame, graphite
furnace, or plasma. This high-temperature
environment can also lead to ionization of the
analyte atoms.
Atomic-Absorption Spectroscopy (AA)
Analyte ionization can be suppressed by adding a
source of electrons, which shifts the equilibrium of
the analyte from the ionic to the atomic form:
Analyte <--> Analyte+ + eCesium and potassium are common ionization
suppressors that are added to analyte solutions.
These atoms are easily ionized and produce a high
concentration of free electrons in the flame or
plasma.
Atomic-Absorption Spectroscopy (AA)
Preventing Refractory Formation
Some elements can form refractory compounds that
are not atomized in flames or plasmas. An example
is the presence of phosphates, which interferes
with calcium measurements due to formation of
refractory calcium phosphate:
3 CaCl2 (aq) + 2 PO43- (aq) --> Ca3(PO4)2 (s) + 6 Cl- (aq)
Atomic-Absorption Spectroscopy (AA)
Formation of refractory compounds can be
prevented or reduced by adding a releasing
agent. For calcium measurements, adding
lanthanium to the sample (and standard)
solutions binds the phosphate as LaPO4. LaPO4
has a very high formation constant Kf and
effectively ties up the phosphate interferent.
Instrumentation of AAS
Light source
The light source is usually a hollow-cathode
lamp of the element that is being measured.
Lasers are also used in research instruments.
Since lasers are intense enough to excite
atoms to higher energy levels, they allow AA
and atomic fluorescence measurements in a
single instrument.
The disadvantage of these narrow-band light
sources is that only one element is
measurable at a time.
Instrumentation of AAS
Light separation and detection –
AAS use monochromators and detectors for uv
and visible light. The main purpose of the
monochromator is to isolate the absorption
line from background light due to
interferences.
Simple dedicated AAS instruments often replace
the monochromator with a bandpass
interference filter. Photomultiplier tubes are
the most common detectors for AAS.
Instrumentation of AAS
Atomizer
AA spectroscopy requires that the analyte atoms be in
the gas phase. Ions or atoms in a sample must
undergo desolvation and vaporization in a hightemperature source such as a flame or graphite
furnace.
Flame AA can only analyze solutions, while graphite
furnace AA can accept solutions, slurries, or solid
samples.
Sample solutions are usually aspirated with the gas
flow into a nebulizing/mixing chamber to form small
droplets before entering the flame.
Instrumentation of AAS
Excitation
-- A flame provides a high-temperature source
for desolvating and vaporizing a sample to
obtain free atoms for spectroscopic analysis.
In atomic absorption spectroscopy ground state
atoms are desired.
For atomic emission spectroscopy the flame
must also excite the atoms to higher energy
levels.
Instrumentation of AAS
The graphite furnace has several advantages over a
flame.
1. It is a much more efficient atomizer than a flame and
it can directly accept very small absolute quantities
of sample.
2. It also provides a reducing environment for easily
oxidized elements. Samples are placed directly in
the graphite furnace and the furnace is electrically
heated in several steps to dry the sample, ash
organic matter, and vaporize the analyte atoms.
Instrumentation of AAS
Temperatures of Some Common Flames
Fuel
Oxidant
Temperature (K)
Hydrogen
Air
2000-2100
Acetylene
Hydrogen
Air
Oxygen
2100-2400
2600-2700
Acetlylene
Nitrous Oxide
2600-2800
Instrumentation of AAS
A flame atomic-absorption spectrometer
a graphite-furnace atomicabsorption spectrometer
Atomic-Fluorescence Spectroscopy (AFS)
Introduction
Atomic fluorescence is the optical emission from gasphase atoms that have been excited to higher
energy levels by absorption of electromagnetic
radiation.
The main advantage of fluorescence detection
compared to absorption measurements is the
greater sensitivity achievable because the
fluorescence signal has a very low background. The
resonant excitation provides selective excitation of
the analyte to avoid interferences.
Atomic-Fluorescence Spectroscopy (AFS)
AFS is useful to study the electronic structure of
atoms and to make quantitative measurements.
Analytical applications include flames and
plasmas diagnostics, and enhanced sensitivity in
atomic analysis.
Instrumentation of AFS
Analysis of solutions or solids requires that the
analyte atoms be desolvated, vaporized, and
atomized at a relatively low temperature in a heat
pipe, flame, or graphite furnace.
A hollow-cathode lamp or laser provides the
resonant excitation to promote the atoms to
higher energy levels. The atomic fluorescence is
dispersed and detected by monochromators and
photomultiplier tubes, similar to atomic-emission
spectroscopy instrumentation.
Quantitative Fluorimetry
Introduction
Light emission from atoms or molecules can be used to
quantitate the amount of the emitting substance in a
sample. The relationship between fluorescence intensity
and analyte concentration is:
F = k * QE * Po * (1-10[-epsilon*b*c])
where F is the measured fluorescence intensity, k is a
geometric instrumental factor, QE is the quantum efficiency
(photons emitted/photons absorbed), Po is the radiant
power of the excitation source, epsilon is the wavelengthdependent molar absorptivity coefficient, b is the path
length, and c is the analyte concentration (epsilon, b, and c
are the same as used in the Beer-Lambert law).
Quantitative Fluorimetry
Expanding the above equation in a series and
dropping higher terms gives:
F = k * QE * Po * (2.303 * epsilon * b * c)
This relationship is valid at low concentrations
(<10-5 M) and shows that fluorescence intensity
is linearly proportional to analyte concentration.
Determining unknown concentrations from the
amount of fluorescence that a sample emits
requires calibration of a fluorimeter with a
standard (to determine K and QE) or by using a
working curve.
Quantitative Fluorimetry
Limitations
Many of the limitations of the Beer-Lambert law also
affect quantitative fluorimetry. Fluorescence
measurements are also susceptible to inner-filter
effects. These effects include excessive absorption of
the excitation radiation (pre-filter effect) and selfabsorption of atomic resonance fluorescence (postfilter effect).
Specific fluorescence techniques
Atomic fluorescence spectroscopy (AFS)
Molecular laser-induced fluorescence (LIF)
Atomic Emission Spectroscopy (AES, OES)
Introduction
Atomic emission spectroscopy (AES or OES) uses quantitative
measurement of the optical emission from excited atoms to determine
analyte concentration. Analyte atoms in solution are aspirated into the
excitation region where they are desolvated, vaporized, and atomized
by a flame, discharge, or plasma. These high-temperature atomization
sources provide sufficient energy to promote the atoms into high
energy levels. The atoms decay back to lower levels by emitting light.
Since the transitions are between distinct atomic energy levels, the
emission lines in the spectra are narrow. The spectra of multielemental samples can be very congested, and spectral separation of
nearby atomic transitions requires a high-resolution spectrometer.
Since all atoms in a sample are excited simultaneously, they can be
detected simultaneously, and is the major advantage of AES
compared to atomic-absorption (AA) spectroscopy.
Instrumentation of Atomic Emission Spectroscopy (AES, OES)
Instrumentation
As in AA spectroscopy, the sample must be converted to free atoms,
usually in a high-temperature excitation source. Liquid samples are
nebulized and carried into the excitation source by a flowing gas. Solid
samples can be introduced into the source by a slurry or by laser
ablation of the solid sample in a gas stream. Solids can also be
directly vaporized and excited by a spark between electrodes or by a
laser pulse. The excitation source must desolvate, atomize, and excite
the analyte atoms. Since the atomic emission lines are very narrow, a
high-resolution polychromatic is needed to selectively monitor each
emission line. :
UV and Visible Spectrophotometry
The Nature of Electronic Transitions
The Ultra-Violet(UV) and Visible regions of the electromagnetic spectrum are
associated with a large enough Kinetic Energy that the energy that is absorbed
will affect the energy states of electrons occupying the molecular orbitals
within the molecule. If the energy of the radiation is equal to or greater than
the the energy of transition for an electron to be promoted to the next available
molecular orbital then energy will be absorbed by that electron and be
promoted to the higher energy molecular orbital. This absorption of energy
will occur when energy from the UV or Visible regions are supplied. Infrared
radiation is not energetic enough to cause electronic transitions within
molecules.
UV and Visible Spectrophotometry
p to p * Transitions
For molecules that possess p bonding as in alkenes, alkynes, aromatics, acyl
compounds or nitriles, energy that is available can promote electrons from a p
Bonding molecular orbital to a p Antibonding molecular orbital. This is called a p
---> p * transition. The energy difference for such a transition to occur will depend
upon the atoms p bonded to each other, other atoms attached as well as the
relationship between two or more p bonds within the molecule. p bonds between
two carbon atoms will have a different a p ---> p * transition compared to p bonds
between a carbon and an Oxygen atom (a carbonyl) or a p bond between a carbon
atom and a nitrogen atom (a nitrile). This is because there will be a different
energy gap between the p Bonding and p Antibonding molecular orbital energy
states. Other atoms such as Hydrogen( as in an aldehyde) or another SP3 carbon(
as in a ketone) that would be bonded to one of the p bonded atoms in the molecule
would also cause the energy of transition to vary. The greater the energy of
transition the shorter the wavelength of UV or visible radiation will have to be for
electrons to be promoted from the bonding to the antibonding state.
UV and Visible Spectrophotometry
Every group of atoms with p bonding will have a different wavelength where
maximum abosrption will take place. This is called the “lMax", the wavelength
where maximum absorption takes place, and the group of atoms with the p
bonding is called a "chromaphore". Each chromaphore will have a different
energy of transition between the bonding and antibonding molecular orbitals
for which the electron transition takes place. For example, alkenes and nonconjugated polyenes will have lamda max absorbances that are below 200
nanometers(nm). Such a short wavelength which indicates a larger energy of
transition is because such chromaphore molecules have only p ---> p *
transitions.
UV and Visible Spectrophotometry
n to p * Transitions
Even lone pairs that exist on Oxygen atoms and Nitrogen atoms may be promoted
from their non-bonding molecular orbital to a p antibonding molecular orbital
within the molecule. This is called an n---> p * transition and requires less
energy(longer wavelength) compared to a p ---> p * transition within the same
chromaphore .
UV and Visible Spectrophotometry
Acyl compounds containing the carbonyl C=O will have a lamda max at
longer wavelengths above 200 nm compared to non-conjugated
alkenes and alkynes. For example, ethene has a lamda max of 171 nm
whereas acetone, CH3-CO-CH3 having a C=O has a lamda max of 280
nm, 109 nm longer. A longer wavelength indicates a shorter energy
gap between molecular orbitals for the electron to be propelled to.
UV and Visible Spectrophotometry
The Conjugation Effect on Lamda Max
Conjugated polyenes will have lamda max that are higher than 200 nm. This
would indicate that the Pi--->Pi* transition involves a smaller amount of
energy. If we compare the molecular orbital levels in a non-conjugated alkene
with the molecular orbitals of a conjugated diene, we find that for a conjugated
diene there are two Pi bonding and two Pi antibonding molecular orbitals in
the diene compared to one each in the alkene. (See Fig 2 below)
UV and Visible Spectrophotometry
The Conjugation Effect and Color Changes During Chemical Reactions
During a chemical reaction it is possible to go from a colorless starting material
where the lamda max is well within the colorless UV region to a colored
product where the lamda max has shifted into the Visible region of the
spectrum. For example in the Benzoin Condensation of Benzaldehyde the
colorless liquid, Benzaldehyde is converted to light yellow Benzoin. If we
compared the extent of conjugation between the reactant and the product we
would see that the degree of conjugation roughly doubled. This doubling of the
conjugation shifted the lamda max of absorption from the colorless UV region
into the Blue end of the Visible region. Since Benzoin absorbs nearer the blue
end of the visible spectrum it reflects radiation nearer the red end hence the
reason for it appearing yellow. The conjugation effect can explain the color
change in most cases where chemical change results in the change in color.
UV and Visible Spectrophotometry
The Conjugation Effect and Color Changes During Chemical Reactions
During a chemical reaction it is possible to go from a colorless starting material
where the lamda max is well within the colorless UV region to a colored
product where the lamda max has shifted into the Visible region of the
spectrum. For example in the Benzoin Condensation of Benzaldehyde the
colorless liquid, Benzaldehyde is converted to light yellow Benzoin. If we
compared the extent of conjugation between the reactant and the product we
would see that the degree of conjugation roughly doubled. This doubling of the
conjugation shifted the lamda max of absorption from the colorless UV region
into the Blue end of the Visible region. Since Benzoin absorbs nearer the blue
end of the visible spectrum it reflects radiation nearer the red end hence the
reason for it appearing yellow. The conjugation effect can explain the color
change in most cases where chemical change results in the change in color.