‫بسم هللا الرحمن الرحيم‬
Marine Analytical Chemistry
MC 361
(Chem 312)
Contents
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Introduction to Spectroscopy
Concept of Spectroscopy
Infrared Absorption
Ultraviolet Molecular Absorption Spectroscopy
Colorimetry (Spectrophotometry)
Emission Spectrography (Flame Photometery)
Total Organic Carbon (TOC)
Chapter 1
1-Introduction to Spectroscopy
• The Interaction between energy and matter:
• In case of visible light
• In general
Wavelength range
What is Radiant Energy?
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Wave??
Or
Small Particles??
Properties: Wave like character
λ = wavelength
γ = frequency
Energy E = hγ
h=Planck constant (6.6256 x 10-27 erg/sec)
γ= C/λ
C= speed of light
E = h C/λ
What is Matter?
Matter:
Atoms or Molecules
Motion of Molecules
Gases (alone)
Rotational
Liquid (agglomerates)
Vibrational
Solid
(crystal or random)
Translational
• Both chemical structures and the
arrangement of the molecules affect the
way in which any given material interacts
with energy.
How does radiant energy interact with matter?
• A given molecule can
absorb only radiation
of a certain definite
frequency.
• Red, blue and yellow
light not just a single
wavelength or
frequency.
• Given molecule can
exist only in certain
well-defined energy
state.
• Energy levels are not continuous.
• Quantized? The energy difference
between two defined energy levels is
fixed.
• The molecules or atoms of same chemical
species absorb same frequency.
• The molecules or atoms of different
chemical species absorb different
frequency
• The uniqueness of the frequencies at
which a given molecular species absorbs
is the basis of absorption spectroscopy.
The Absorption of Energy by Atoms
Collision of Bodies
Elastic collision
Inelastic collision
No exchange of energy between bodies
Exchange of energy between bodies
The Absorption of Energy by Molecules
Molecular Motion
Rotation
Vibration
Transition
Vibration of Molecules
Absorption Caused by Electronic
Transistors in Molecules
• The vibrational and rotational energies of the
molecules are added to the electronic energies.
• The difference in vibrational or rotational energy
leads to the absorption of energy over wide
frequency range.
The Effect of the Absorption of Energy
Electromagnetic spectrum
UV and Visible
Electronic excitation
Unsaturated organic
Aromatic ,olefins, X,N
Nondestructive.
Very short wavelength
Penetrate the nuclei
of atom.
Change to different element
Basis of nuclear science.
Long wavelength IR
Rotational and Vibrational
Functional groups
Organic molecules
Nondestructive.
X-ray
Remove e from inner shell
e of outer shell fill up inner
shell ------ X-ray
Dimensions of crystals
Elemental analysis.
The Emission of Radiant Energy by
Atoms and Molecules
In the case of molecules
• Not all molecules fluorescence, and very few
phosphorescence.
• Fluorescence intensity is very high.
• Can detect very small conc. of certain compound
Methods of Excitation of Atoms
• 1-Electronic Discharge
• Putting the sample in an
electric discharge
between two electrodes.
• The sample breaks
down into excited atoms
• All different atoms emit
their emission spectrum.
2-Flame Excitation
• This is the basis of flame photometry.
• Flame energy is lower than that of an electrical
discharge
• Fewer transitions are possible-Fewer spectrum
lines.
3-Excitation by Radiation
• Sample absorb uv and
reemit fluorescence.
• This the bases of Molecular
Fluorescence (RF).
• Phosphorescence:
• If the e changes its direction
of spin before returning to
the G.S. emission will be
difficult (forbidden).
• The radiation takes place
over a long period of time.
Absorption Laws
1-Lambert‫׳‬s Law
• A=ab
• A = absorbance
• a = absorptivity of the liquid
• b = optical path length
• a b = log I0/I1
• I1 = I0 10 -ab
• I0 = I1 10 ab
• Relationship between absorbance and optical
path length
2- Beer΄s Law
• A=ac
• c = concentration of
solution.
• a c = log I0/I1
• I1 = I0 10 -ac
• I0 = I1 10 ac
• Relationship between
the absorbance and
the concentration of
the solution
3-Beer-Lambert Law
• A=abc
• I0 = I1 10 abc
• Linear relationship between A and c if b is
constant and the radiation wavelength
constant.
• By measuring I1/I0 we can measure A, and
we can calculate c.
• Valid for low conc. But deviations are
common at higher concentrations.
4-Deviation from Beer‫׳‬s Law
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1-Impurities.
2-Chemical equilibrium.
3-Optical slit.
4-Dimerization.
5- Interaction with solvent
Calibration curve will
eliminate all the
deviations.
Calibration Curves
• Series of solutions with
known concentration.
• When a, b, I0 are constant
• c ά –logI1
• Base line disturbed by an
interfering compounds.
• Can solve this problem :
• 1-Mathmatically
• 2-Double beam
instrument (reference cell)
2-Standard Addition Method
• This method used if:
• 1-No suitable calibration
curve.
• 2-Time delay.
• 3-No sufficient information
on the solvent in the
sample.
• 4-Very low concentration.
• Known conc. are added to
the sample
Chapter 2
Concepts of Spectroscopy
Spectroscopy
Emission
Absorption
It is important to know
1- The wavelength at which the sample emits or absorbs radiation.
2- The intensity of radiation or the degree of absorption.
The basic design of all instruments:
1- Source of radiation (absorption) or excitation (emission).
2- Monochromatic (selecting the wavelength).
3- Sample holder.
4- Detector (measure the intensity of radiation).
Spectroscopy instruments
Single-Beam Optics
• 1- single beam optics:
Double beam Optics
a- Radiation Source
• 1-Emite radiation of wavelength in the
range to be studied (x-ray, infrared,
ultraviolet).
• 2-The Intensity in all range should be high.
• 3-The intensity should not vary
significantly at different wavelengths.
• 4-The intensity should not fluctuate over
long time intervals.
b-Monochromator
• Function: disperse the
radiation according to the
wavelength.
• 1-Prism Monochromators:
• The prism bends longerwavelengths (red end)
radiation less than it does
shorter-wavelength radiation
(blue end)?.
• The refractive index of
prism is greater for shortwavelength light than it is for
long-wavelength light.
2-Grating Monochromator:
• More popular than prism.
• A series of parallel lines cut
into a plane surface.
• From 15000-30000 groves per
square inch.
• The more lines per square
inch, the shorter λ of radiation
that the grating can disperse
and the greater the dispersing
power.
• Separation of light occurs
because light of different λ is
dispersed at different angles.
3-Resolution of a Monochromator
• The ability to disperse radiation is called
resolving power (dispersive power).
• The resolving power of a prism increase
with the thickness of the prism.
• The resolving power of a prism increase
when the material used is improved.
• The resolving power of a grating increase
with increase the line groves.
c-Slits
• Used to select the light beam after it
has been dispersed by the
monochromator.
• The entrance slit selects a beam of
light from the source.
• The exit slit allow radiation from the
monochromator to proceed to the
sample and detector.
• Only a selected λ range is permitted
through the slit.
• Other radiation is blocked and
prevented from passing further.
• The slits are kept as narrow as
possible to ensure optimum resolution.
d-Detector
• Measure the intensity of the radiation that
falls on it.
• Radiation energy is turned into electrical
energy.
• The amount of energy produced usually
low and must be amplified.
• Amplifying the signal from the detector
increase its sensitivity.
• If the signal is amplified too much, it
becomes erratic and unsteady (noisy).
e- Uses for Single-Beam Optics
• Single-beam optics are used for all
spectroscopic emission methods.
• The method allows the emission intensity and
wavelength to be measured accurately and
rapidly.
• In spectroscopic absorption studies the intensity
before and after passing the sample must be
measured.
• Any variation in the intensity lead to analytical
error. (that is why double beam developed)
2-The Double-Beam System.
• Used for spectroscopic
absorption studies.
• One very important
difference from single
beam:
• The radiation from the
source is split into two
beams with equal intensity
(reference beam and
sample beam).
• Any variation in the
intensity of source I0
simultaneously decreases
I1 but does not change the
ratio I1/I0.
Chapter 3
Infrared Absorption.
• The wavelength (λ) of infrared (ir) radiation
falls in the range 750 nm- 4500 nm.
• The frequency (u) range:
2.2x1014-7.5x1015 cps.
• Infrared radiation has less energy than
visible radiation but more than radio
waves.
A-Requirements for Infrared
Absorption
• 1-Correct Wavelength of Radiation:
• Molecules absorb radiation when some part of
the molecule (atom, or group) vibrates at the
same frequency as the incident radiant energy.
• After absorbing radiation, the molecule vibrate at
an increased rate.
• Atoms can vibrate in several ways.
• The rate of vibration is quantized and can take
place only at well defined frequencies that are
characteristic of the atom.
2-Electric Dipole
• For a molecule to be able to
absorb ir, it must have a
changeable electric dipole.
• The dipole must change as a
result of the vibrational
transition resulting from ir
absorption.
• If the rate of change of the
dipole during vibration is fast,
the absorption of radiation is
intense (and vice versa).
Electric dipole:
a slight positive
and a slight
negative electric
charge on the atoms
B-Movements of Molecules:
• The total radiant energy absorbed by
molecules = (the molecule's vibrational
energy + the molecule's rotational energy)
• Rotational energy levels are very small
compared to vibrational energy levels.
1- Vibrational Movement
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•
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u : frequency of vibration
K : constant.
f : binding strength of the spring
m : reduced mass.
• Modes of vibration of C and
H in methane molecule
including:
• a-symmetrical stretching.
• b- asymmetrical stretching.
• c- scissoring.
• d- rocking.
• e- wagging.
• f- twisting.
• Each of the modes
vibration absorb
radiation at different
wavelength.
2- Rotational Movement
• At the same time that the parts of a
molecule vibrate toward each other, the
molecule as a whole may rotate (spin).
• The energy involved in spinning a
molecule is very small compared to the
energy required to cause it to vibrate.
C-Equipment (double beam system)
• 1- Radiation Source:
Both sources fulfill important requirements:
1-steady intensity.
2-intensity constant over long periods of time.
3-wide wavelength range.
But the intensity of the radiation from them is not the
same at all frequencies.
2- Monochromators
• Select desired frequency from source, and
eliminate the radiation at other frequencies.
• a- Prism Monochromator:
• Material must be:
• 1- Transparent to IR radiation (not glass, not
quartz).
• 2-Smooth (prevent random scattering).
• 3-High quality crystal.
• 4-Dry all time (use heater to keep dry).
• 5-The machine must be in air condition room.
• Material used can be single large crystal metal
salt : NaCl, (KBr,CsBr), or CaF2.
B-Grating Monochromators:
• Recently it is more popular in IR
spectroscopy.
• Material : Aluminum.
• Advantages of grating:
• 1- Stable in the atmosphere and are not
attacked by moisture.
• 2- Can be used over considerable
wavelength range.
3- Slit Systems:
• Allow small section of radiation beam to
pass through exit slit to the detector.
• 4- Detectors:
b-Thermocouples:
• C- Thermistors:
• Made of fused mixture of metal oxide.
• As their temperature increase, their resistance
decrease (as bolometers).
5- sample Cell:
• IR spectrum can be used for the characterization
of solid, liquid, or gas samples.
• The material used to contain the sample must
always be transparent to ir radiation.
• a- solid samples:
• Mixing ground solid sample with powdered KBr.
• Press the mixture under very high pressure
(small disk 1cm diameter and 1-2 mm thickness)
• (called KBr pellet method).
b-Cells for Liquid Samples
• The easiest samples to handle are liquid
samples.
• Cells made of rectangular of NaCl, or KBr.
• All cells must be protected from water
because they are water soluble.
• Organic liquid samples should be dried.
• c-Gas samples:
• The gas sample cell is similar to liquid
samples (NaCl, or KBr), but longer than
liquid samples (about 10 cm long).
D- Analytical Applications:
• 1- Qualitative Analysis:
• Function groups in organic compounds
(methyl, aldehyde, ketone, alcohol, atc.).
• Information about the geometry of
molecule.
• 2-Quantitive Analysis:
• By using a solution with known
concentration, we could measure the
concentration of unknown sample using
Beer's law.
3- Analysis Carried Out by IR Spectroscopy:
• 1-Detection of paraffins, aromatic, olefins,
acetylenes, alcohols, ketones, carboxylic
acids, phenols, esters, ethers, amines,
sulfur compounds, and haldies.
• 2-Distinguish one polymer from another.
• 3-Identify atmospheric pollutants.
• 4-Examine the old painting and artifacts.
• 5-Determine the make and year of the car.
• 6-Determine the impurities in raw material.
Absorption IR Spectrum
Methyl Ethanoate ir spectrum
Chapter 4
Ultraviolet Molecular Absorption Spectroscopy
• Wavelength range : 200 – 400 nm.
• Visible light act in the same way as uv light
(consider as a part of the uv range).
Function
Analytical field
Analytical
Application
Atomic uv
Absorption of uv
Atomic Absorption
Quantitative analysis
Emission of uv
Flame photometry
Quantitative analysis:
Alkali metals, alkaline
metal earths
Absorption of uv
uv absorption
Qualitative and
quantitative for
aromatics
Emission of uv
Molecular
fluorescence
Detection of small
quantities of aromatics
Emission of uv
Molecular
phosphorescence
Limited application
Molecular uv
1-Electronic Excitation
• Three types of electrons are involved in organic
molecules
Saturated
Unsaturated
Non-bonding
Symbol
s
p
n
Example
Paraffinic
compounds
(C-H)
Conjugated
double bond
(Aromatics)
Organic
compounds
contain O, S, Cl.
Absorbance of uv
Can‘t absorb uv
(need higher
energy)
Absorb uv
Absorb uv
2-The Shape of UV Absorption Curves.
Electronic
transition
Molecular
vibration
Molecular
rotation
Time
10-15 sec
(fsec.)
10-12 sec
10-10 sec
Energy
UV
IR
Micro
High
energy>
Low energy>
Very low energy
• The electronic excitation line is split into many
sublevels by vibrational energy.
• Each sublevel is split by rotational energy.
• The gross effect is to produce an absorption
band rather than an absorption line.
UV Spectrum of DNA
1- General Optical System
• a- Single beam system:
• Single beam problems:
• 1-Measure the total light absorbed, rather
than the percentage absorbed.
• 2-Light may be lost by reflecting surfaces.
• 3-Light may be absorbed by solvent used.
• 4-The source intensity may vary with
changes in line voltage.
• 5-The sensitivity of the detector varies
significantly with the wavelength.
B- Double-beam System:
• All the previous problems can be largely
overcome by using the double-beam
system.
• The source radiation is split into two
beams of equal intensity.
• One beam to the reference cell and the
other to the sample cell.
• The difference in intensity of the two
beams should be a direct measure of the
absorption of sample.
2- Components of the Equipment:
• a- Radiation source:
• 1- Tungsten lamp: heated electrically to
white heat, stable, common, easy to use,
but the intensity at short wavelengths is
small.
• 2- Hydrogen lamp: hydrogen gas in high
pressure with electrical discharge.H2
molecules excited and emit uv radiation.
• 3- Deuterium lamp: D2 is used instead H2.
Emission intensity is increased three
times.
• 4- Mercury discharge lamp: Hg vapor in the
discharge lamp is under high pressure.
• 5- Xenon lamp: Like H2 lamps. They provide very
high radiation intensity.
• B- Monochromators:
• Prism and grating are used in uv spectroscopy.
• 1- Glass: has the highest resolving power, but it
is not transparent to radiation between 350-200
nm.
• 2-Fused silica: more transparent to short
wavelengths, but very expensive.
• 3-Quartz:
used
extensively
in
uv
spectrophotometers.
C- Detectors:
• 1- Photocell:
• Consists of a metal
surface (cathode) that is
sensitive to light.
• When light falls upon it,
the surface gives off
electrons, which attracted
and collected by an
anode.
• Current created measure
the intensity of light.
2-Photomultiplier:
• Work in a similar way like
photocell.
• Electrons attracted to +ve
dynode,
that
cause
several electrons to emit.
• This process repeated
several times until a
shower of electrons arrive
at a collector.
• A single photon may
generate many electrons
and give a high signal.
3- Sample cell:
•
•
•
•
The cells used in uv absorption must be:
a- Transparent to uv radiation.
b- Chemically inert.
Quartz or fused silica are most common
material used.
C-Analytical Applications
• 1- Qualitative analysis:
• Nonbonding electrons and p electrons
absorbed over similar wavelength range.
• This makes it difficult to identify the
presence of any particular group (function
groups).
• UV is very useful in detecting aromatic
compounds and conjugated olefins.
2- Quantitative Analysis:
• Powerful tool for quantitative determination
of compounds that absorb uv (nonbonding
electrons and conjugated p bond
compounds).
• Using Beer's law and calibration curve can
calculate the concentration of unknown
sample that absorb uv.
• The technique is quite sensitive as low as
1 ppm.
3- Applications:
•
•
•
•
•
•
•
•
•
Determination of:
1-Polynuclear compounds.
2-Natural products.
3-Dye-stuff.
4-Vitamines.
5-Impuirties in organic samples.
In the field of agriculture can determine:
1-Pesticides on plants.
2-Polluted rivers, and in fish and animals.
• In the medical field can be used for the
analysis of:
• 1-Enzymes, vitamins, hormones, steroids,
alkaloids, and barbiturates.
• 2-Diagnosis of diabetes, kidney damage.
• In pharmacy: purity of drugs.
• Measure the kinetics of chemical reactions
• In HPLC as detector.
Chapter 5
Spectrophotometry
• Measure how much light is absorbed by
sample solution (light intensity).
• There are single-beam and double-beam
spectrometers.
• Electronic
transition
(excitation)
of
electrons in the last orbital (absorb light in
the visible range 400-850 nm).
A-Spectrophotometeric equipment
• 1- Source:
• Tungsten lamp: heated electrically to white
heat (400 – 850 nm).
• The signal must be constant over a long
periods of time.
• The signal intensity is not exactly equal at
different wavelengths (double beam
system solve this problem).
• 2-Monochromator:
• a- Light filter:
• Allows light of the required wavelength to
pass, but absorbs light of other
wavelengths.
• Several filters can be used for different
analysis.
• b- Glass prism:
• Easier than filters as any required
wavelength may be chosen.
• 3- sample cell:
• For visible part of the spectrum cells are from
glass.
• Quartz cells used if the studies involving both uv
and visible regions.
• 4- Detectors:
• Most common are photomultipliers or photocell.
• Convert the radiant energy to electrical energy.
• Double-beam instrumentation gives more
accurate absorption spectra and more accurate
quantitative measurements.
2- Analytical applications:
• Spectrophotometry is very widely used
method of quantitative analysis.
• Whenever the sample is color we can use
spectrophotometry.
• If the sample is colorless we can color it
using special reagent (ex. NO2-, PO4-3,..).
• This technique is sensitive and accurate
but time consuming.
• Requirements for this process:
• 1- Selective reagent react only with a
specific ion.
• 2- Must undergo color change.
• 3- Intensity of the color should be related
to the concentration of the ions in the
sample.
Chapter 6
Emission Spectrography
• Emission spectrography: is the study of
the radiation emitted by a sample when it
is introduced into an electrical discharge.
• Since each element emits a different
spectrum, it is possible to determine what
element are present in the sample.
• Most important technique for elemental
qualitative analysis for: all metallic
elements, metalloids (liq. or solid), halides,
and inert gases.
• Quantitative analysis: concentration
levels as low as 1 ppm.
A-Origin of Spectra
• Using thermal energy: Flame photometry.
• Using spectral energy: Atomic fluorescence.
• Electrical discharge: Produces more energy and
therefore causes brighter spectra than the other
forms.
B-Equipment:
• The sample introduced to electrical discharge,
where it is excited.
• Excited sample emits radiation, which is
detected and measured by the detector.
1- Electrical source
DC arc (spark)
AC arc (spark)
Voltage
50-300 v
10-50 v (low)
Intensity
High temperature
intense radiation
Application
Low intensity than
DC but more
reproducible.
Qualitative
Useful for
analysis of trace
quantitative
components when
analysis.
high sensitivity is
required
2-Sample Holder
• a- Solid Sample:
• Solid sample reducing to powder, then
loading the powder to carbon sample
holder which act as one electrode used in
the discharge.
• Animal tissue and plant materials change
to ash then mix with carbon or alumina to
avoid sudden emission.
• Metallic samples (alloys or pure metals)
can use as it is after clean and shape it to
be one electrode.
• b- Liquid samples:
• Liquid samples may be analyzed directly
by the following method:
• 1-Cup container.
• 2-Rotating disk.
• Both methods gives steady rate into the
electrical discharge.
• Organic solutions tend to ignite in the
discharge and cause erratic emission.
3-Monochromator:
• The function of the monochromator is to
separate the various lines of a sample's
emission spectrum.
• a-Prism monochromators:
• Quartz or fused silica suitable for
transparent to uv radiation.
• Polarization: the act of separating a light
beam into two beams vibrating at right
angles to each other.
• Quartz prisms split light
into two beams of light that
are
polarized
perpendicularly to each
other.
• Beam splitting causes the
loss of half the lights
intensity, making both
qualitative and quantitative
analysis difficult.
• This
problem
can
overcome by using two
half prisms. The first splits
the light into two beams;
the second recombines
them.
b- Grating monochromators:
• It gives better resolution than prisms.
• Concave gratings can be used to conserve
energy.
• A given wavelength that falls on the
concave grating to focus at a given point.
• This relationship helps us to locate the
correct position for placing either
photographic film detector or a series of
photomultipliers.
4-Slits:
• Two parallel metal strips.
• Slits might be 1 cm high and 0.1 mm wide,
the width can be varied for resolution
requirements.
• Entrance slits: keep out stray light and
permit only the light from the sample to
enter the optical path.
• Exit slits: placed after the monochromator
block out all but the desired wavelength
range from reaching the detector.
5- Detectors:
• a- Photographic plates: or films used for all qualitative
analysis.
• For quantitative analysis the intensity of one emission
line from each element is measured, which correlated
with the concentration of the related element in the
sample.
• b- The photomultiplier:
• The photomultiplier
is used only for
quantitative work.
• The
immediate
response and ease
of
interpretation
make it the most
desirable detector.
C-Analytical Applications
• Emission spectrograph used for elemental
qualitative and quantitative analysis. But
gives very little direct information on the
molecular form.
• The sample is destroyed by the electrical
discharge.
1-Qualitative Analysis
• a-Raies Ultima:
• When the metal excited, it emits complex
spectrum that consists of many lines,
some strong, some weak.
• By dilution of the sample, the weaker lines
disappear. Further dilution less strong
lines disappear until only a few are visible.
• The lines that are left called raies ultima or
RU lines (three lines should be detected).
b- Metals
• Most elements can be detected at low
concentrations with a high degree of
confidence?? Because the emission
spectra for most elements in the periodic
table
are
intense,
moreover
the
background
interference
is
greatly
reduced.
• Unknown sample is taken together with
the spectrum of iron. By comparison with
the iron spectra, the elements in the
sample can be identified.
2-Quantitative analysis:
• It carried out by measuring the intensity of one
emission line of the spectrum of each element to be
determined.
• The choice of the line depend on the concentration
of the element.
• For small concentrations of the element, an intense
line must be used. With a larger concentration, a
weaker line would be measured.
• New machines contain more than photomultiplier
detectors can measure up to 60 element
simultaneously.
• Calibration curves must be prepared for each
element to be determined.
Specific Applications
• Metallurgy: the presence of iron and steel of
many elements like Ni, Cr, Si, Mg, Mo, Cu, Al,
As, Sn, Co, V, Pb, and P can be determined by
emission spectrography.
• Alloys: the percentage of different metals can
determined by emission spectrography.
• Oil industry: the amount of different metals in the
oil which the degree of purity of oil.
• Soil samples, animal tissue samples, and plant
roots have been analyzed for many elements.
Chapter 7
Examples of Absorption and
Emission Instruments.
• A-Emission Instruments:
• 1-Flame photometry (atoms):
• Flame photometry is an atomic emission method for the
routine detection of metal salts, principally Na, K, Li, Ca,
and Ba.
• The low temperature of the natural gas and air flame,
compared to other excitation methods such as arcs,
sparks, and rare gas plasmas, limit the method to easily
ionized metals.
• Since the temperature isn't high enough to excite
transition metals, the method is selective toward
detection of alkali and alkali earth metals.
• Quantitative analysis of
these
species
is
performed by measuring
the flame emission of
solutions containing the
metal salts.
• Solutions are aspirated
into the flame.
• The
hot
flame
evaporates the solvent,
atomizes the metal, and
excites
a
valence
electron to an upper
state.
• Light is emitted at characteristic wavelengths for
each metal as the electron returns to the ground
state.
• Optical filters are used to select the emission
wavelength monitored for the analyte species.
• Comparison of emission intensities of
unknowns to the standard solutions,
allows quantitative analysis of the analyte
metal in the sample solution.
• Advantages:
• Flame photometry is a simple, relatively
inexpensive method used for clinical,
biological, and environmental analysis.
• Disadvantages:
• The low temperatures of this method lead to
certain disadvantages:
• 1- Most of them related to interference and the
stability of the flame and aspiration conditions.
• 2- Fuel and oxidant flow rates and purity of fuel.
• 3- Aspiration rates, solution viscosity of samples.
• It is therefore very important to measure the
emission of the standard and unknown solutions
under conditions that are as nearly identical as
possible.
• 2- Spectrofluorimetry (Molecules):
• Measurement of fluorescence - a form of
light emitted by a substance after irradiation
at other wavelengths.
• Origin of Photoluminescence
• Absorption of visible or UV radiation raises
molecule to an excited state.
• Electron absorbs quantum of energy and
jumps to a higher energy orbital.
• When electron drops back to the ground
state, excitation energy can be liberated by:
• 1- QUENCHING (or RADIATIONLESS
TRANSFER)
- Most common
Energy temporarily increases vibrational
and rotational energy of bonds in the
molecule - ultimately dissipated as heat in
surrounding solvent.
• 2- RE-EMISSION OF RADIATION,
- Less common
Gives rise to... FLUORESCENCE and/or
PHOSPHORESCENCE
(two forms of PHOTOLUMINESCENCE)
• Stokes' Law Of Fluorescence:
• ENERGY JUMP UP
(from ground to excited electronic state) is
larger than ENERGY JUMP DOWN
(for the reverse transition).
• The wavelength of light absorbed for
excitation will be shorter than the
wavelength emitted during de-excitation.
(Stokes' law)
Fluorescence vs Phosphorescence
• FLUORESCENCE: if it occurs, is within
nanoseconds (10-9 sec) of the excitation.
• PHOSPHORESCENCE : is caused by
electron becoming transferred into a triplet
state. (Electrons of the same spin in the
one orbital). It is much slower (vary from
milliseconds to weeks) (and rarer)
process.
Instrumentation : The Spectrofluorimeter
A Fluorescent Species Has Three Spectra
• Not every absorption peak gives rise to
fluorescence.
• However peaks in a fluorescence excitation
spectrum usually correspond closely in
wavelength to absorption peaks.
• Fluorescence emission spectrum has
maximum at higher wavelength than
excitation spectrum (Stokes' law).
• Emission spectrum is usually simpler usually only a single broad peak.
• Fluorescence is not measured relative to a
blank.
• Quantitative Spectrofluorimetry
• Linear response, Fluorescence vs Mass of
Analyte, only at LOW CONCENTRATIONS.
• Advantages and Disadvantages of
Spectrofluorimetric Assay
• Major advantage is high sensitivity.
(better than spectrophotometer).
• A potential advantage is improved
selectivity.
• Requirement to set two wavelengths in
spectrofluorimetry
(excitation
and
emission) hence unlikely that an impurity
is being co-measured - it would have to
absorb and emit at the same two
wavelengths.
• Disadvantages :a very exacting technique,
requiring careful attention to experimental
detail, including purity of reagents.
• What Compounds Fluoresce?
• 1- Most fluorescent species are
compounds that absorb UV light.
• 2- An aromatic ring is the most common
structural requirement.
• 3-Additional requirement for fluorescence
is that quenching must not occur while
the molecule is in the excited state.
• 4- In aromatic molecules,
electron withdrawing groups
tend to produce quenching.
eg
-NO2,
or
-COOH
substituents on the aromatic
ring tend to decrease the
chances of fluorescence.
• 5- Electron rich groups inhibit
quenching. eg -NH2 or -OH
are likely to enhance the
fluorescence.
• Product has 4 electron donor
groups. Highly fluorescent at
530 nm (360 nm excitation).
B- Absorption Instruments
Total Organic Carbon (TOC)
• There are two types of TOC measurement
methods, one is the differential method
(TC-IC = OC) and the other is the direct
method (NPOC).
• In differential method both TC (Total
Carbon) and IC (Inorganic Carbon)
determined separately .
• This method is suitable for samples in
which IC is less than TOC, or at least of
similar size.
• In the direct method : first IC is removed from
a sample by purging the acidified sample
with a purified gas, and then TOC may be
determined by means of TC (TC = TOC).
• This method is also called as NPOC .
• The direct method is suitable for surface
water, ground water and drinking water
(negligible amount of POC in these
samples).
Scheme for TOC machine.
TC measurement:
• TC is measured by injecting of tens micro
liter, of the sample into a heated
combustion tube packed with an oxidation
catalyst. The water is vaporized and TC, is
converted to carbon dioxide (CO2).
• The carbon dioxide is carried with the
carrier gas stream from the combustion
tube to a NDIR ( non-dispersive infrared
gas analyzer) and concentration of carbon
dioxide is measured.
IC measurement:
• IC is measured by injecting a portion of the
sample into an IC reaction chamber filled with
phosphoric acid solution.
• All IC is converted to carbon dioxide and
concentration of carbon dioxide is measured
with a NDIR.
• TOC may be obtained as the difference of TC
and IC.
• In the direct method the sample from
which IC was removed previously, injected
into the combustion tube and TOC
(NPOC) is measured directly.
Total Nitrogen Analyzer
• Total nitrogen analyzer based on catalytic
thermal decomposition. Chemiluminescence
method.
• First, nitrogen (N) compound is oxidized at
high temperature (600 to 900°C) by the
catalytic thermal decomposition method to
generate nitrogen monoxide (NO).
• Next, this nitrogen monoxide (NO) is
reacted with ozone (O*) to form NO2*.
• Nitrogen dioxide (NO2*) excited in
metastable
state
then
generates
chemiluminescence when it becomes
stable nitrogen dioxide (NO2).
• Intensity of this chemiluminescence is
proportional to the nitrogen concentration.
TOC solid module
• For detection of TOC in
solid samples.
• TOC measurement
method is the differential
method (TC-IC = OC).
• Both TC (Total Carbon)
and IC (Inorganic Carbon)
determined separately.
•TC (solid) introduce
directly to oven at 700 C
• IC (solid)
subjected to:
• 1 ml H3PO4 then
introduce to
oven at 200 C
only.