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Course Outline

Liquid Solid Chromatography:


Plane chromatography.
High Performance Liquid Chromatography (HPLC)
Gas Chromatography (GC)
Ion Exchange,
Spectroscopy,
Basics of Visible and UV Spectroscopy,
Atomic Absorption Spectroscopy

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Recommended Books:
1. Inorganic Chemistry, Gary L. Miessler, Donald A. Tarr, Prentice-Hall, 2003
2. Analytical Chemistry, G.L. Hargis, Prentice Hall Inc. 2000.
3. Analytical Chemistry, G.D. Christian, J. Wiley 6th Ed. 2003
4. Fundamentals of Analytical Chemistry, D.A. Skoog, D.M. West, FJ. Holler 7th
Ed. Harcourt Asia 2001.
5. “Applied Colloid and Surface Chemistry”. Richard M Pashley; Marilyn E
Karaman. John Wiley and Sons, Ltd.2004

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RF

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Retention Factor (Rf)
In chromatography the retention factor, R, is the fraction of the
sample in the mobile phase at equilibrium, defined as.

Quantity of Substance in Mobil Phase


R = ----------------------------------------------------
Total Quantity of Substance in System

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Planar Chromatography (Rf)
“The 'Retention Factor' of a compound is a measure of how far it
has moved up a plate under certain conditions”.
it is can be use a quick way of identification.
Differences in Retention Factors are important when considering
solvent systems for Column Chromatography.

Migration Distance of substance


Rf = -----------------------------------------------------
Migration Distance of Solvent Front

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TLC
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Column Chromatography
“Column chromatography is a technique in which solid
stationary phase is packed in a column”.
 The classical preparative chromatography column, is a glass
tube with a diameter from 5 mm to 50 mm and a height of
5 cm to 2 m with a tap and some kind of a filter.
 The most common stationary phase for column
chromatography is silica gel (SiO2).
 There is an important ratio between the stationary phase
weight and the dry weight of the analyte mixture that can be
applied onto the column. 13
 The individual components are retained by the stationary phase
differently and separate from each other while they are running at
different speeds through the column with the eluent.
 At the end of the column they elute one at a time.
 During the entire chromatography process the eluent is collected in a
series of fractions.
 The composition of the eluent flow can be monitored and each
fraction is analyzed.
 Analytical chromatography, UV absorption, or fluorescence. Colored
compounds (or fluorescent compounds with the aid of an UV lamp)
can be seen through the glass wall as moving bands.
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Fast Running
Running Column Stopped Column Column

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A

B
C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 An

Pack.
Of Colm
A B C An 18
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High Performance Liquid Chromatography (HPLC)

 Highly improved form of column chromatography.


 Instead of a solvent being allowed to drip through a column
under gravity, it is forced through under high pressures of up to
400 atmospheres.
 Very much smaller particle size for the column packing material.
 Greater surface area for interactions between the stationary
phase and the molecules flowing past it.
 Much better separation of the components of the mixture.
 Detection methods can be used.
 These methods are highly automated and extremely sensitive.
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Injection Valve

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Columns
One of the most important components
 The separation of the sample components is achieved when
those components pass through the column.

Silica gel
 Its particle shape, surface properties, and pore structure help
to get a good separation.
 Silica is wetted by nearly every potential mobile phase, is inert
to most compounds.
 Has a high surface activity which can be modified easily with
water and other agents.
 Can be used to separate a wide variety of chemical
compounds.
 Predictable and reproducible.
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Normal Phase Column

 Stationary phase: high polar rigid silica, or silicabased


compositions.
 Mobile phases: relatively nonpolar solvent, hexane
methylene chloride, or mixtures of these.
 More polar solvent has higher eluent strength.
 The least polar component is eluted first.

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Reverse Phase Column

 Stationary phases: nonpolar hydrocarbons, waxy liquids,


or bonded hydrocarbons (such as C18, C8, etc.).
 Mobile phase: polar solvents or mixtures such as
methanol-water or acetonitrile-water.
 The most polar component is eluted first.
 Less polar solvent has higher eluent strength.
 Less sensitive to polar impurities in the eluent.

∇ Avoid to measure a sample that pH value is greater than 7.5


in a reversed –phase column, because of hydrolysis of the siloxane.
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Effect of Chain Length on Performance

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--- In a normal-phase column, decreasing the polarity of solvent will
increase
separation the components. In a reverse-phase column, the reverse is
true
--- In normal-phase column, less polar solute is eluted first; in a
reverse-phase 30

column, the reverse is true


Mobile Phase
 Water
 Methanol:
Higher viscous, poor miscibility with nonpolar solute
 Acetonitrile:
Less viscous, more compatible with non-polar solutes,
Shorter retention time under same conc.
 Solute Retention Time:
Decreased by increasing the conc. of organic solvent

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Distribution of Analytes Between Phases
The distribution of analytes between phases can often be described
quite simply. An analyte is in equilibrium between the two phases;
Amobile Astationary
The equilibrium constant, K, is termed the Partition Coefficient;
defined as the molar concentration of analyte in the stationary phase
divided by the molar concentration of the analyte in the mobile
phase.

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Theoretical Plate:
“A theoretical plate is a hypothetical zone or stage in which two
phases establish an equilibrium with each other.”

N = 16 (tr/w)2
N = Number of theoretical plates.
tr = retention time
W = width of peak at base line

 It is Important to Remember That The Plates Do Not Really Exist


 Helps us understand the processes at work in the column
 column efficiency
by Helps us understand the processes at work in the column
OR stating the plate height
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The Column

Theoretical
Plates

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Eluent
Isocratic Elution
 The eluent composition remains constant as it is pumped
through the column during the whole analysis.
 Single solvent or solvent mixture. (S # 22)
Gradient Elution: (S # 35)
 The eluent composition (and strength) is steadily changed
during the analysis.
 Increase separation efficiency
 Decrease the retention time
 Peak shape is improved (Less tailing) 35
Gradient Elution

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Two major types
• Gas-solid chromatography early
(stationary phase: solid)
• Gas-liquid chromatography important
(stationary phase: immobilized liquid)
• Gas – bonded phase “ relatively new

An estimated 200,000 GC in use worldwide

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 Gas chromatography - specifically gas-liquid chromatography
- involves a sample being vapourised and injected onto the
head of the chromatographic column.
 The sample is transported through the column by the flow of
inert, gaseous mobile phase.
 The column itself contains a liquid stationary phase which is
adsorbed onto the surface of an inert solid.

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Carrier gas: He (common), N2, H2
Pinlet 10-50 psig
F = 25-150 mL/min packed column
F = 1-25 mL/min open tubular column
Column: 2-50 m coiled stainless steel/glass/Teflon
Oven: 0-400 °C ~ average boiling point of
sample accurate to <1 °C
Detectors: FID, TCD, ECD, (MS)

GC GC
GC-MS
1 2

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What influences the separation?
1. Polarity of the stationary phase
2. Temperature
3. Carrier gas flow
4. Column length
5. Amount of material injected

Conclusion
High temperatures and high flow rates decrease the retention
time, but also deteriorate the quality of the separation.

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Instrumental Components
Carrier Gas
 The carrier gas must be chemically inert.
 Commonly used gases include nitrogen, helium, argon, and
carbon dioxide.
 The choice of carrier gas is often dependant upon the type of
detector which is used.
 The carrier gas system also contains a molecular sieve to
remove water and other impurities.

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Instrumental Components
Sample Injection Port
 For optimum column efficiency, the sample should not be too
large, and should be introduced onto the column as a "plug" of
vapour - slow injection of large samples causes band
broadening and loss of resolution.
 The most common injection method is where a microsyringe is
used to inject sample through a rubber septum into a flash
vapouriser port at the head of the column.
 The temperature of the sample port is usually about 50°C
higher than the boiling point of the least volatile component of
the sample.
 For packed columns, sample size ranges from tenths of a
microliter up to 20 microliters. Capillary columns, on the other
hand, need much less sample, typically around 10-3 µL.

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Instrumental Components

Rubber Septum
Septum Purge outlet
Carrier gas
inlet

Split Outlet
Heat metal block
Vapourisation chamber
Glass Liner

Column

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Split injection: routine method
0.1-1 % sample to column
remainder to waste

Splitless injection: all sample to column


best for quantitative analysis
only for trace analysis, low [sample]

On-column injection: for samples that decompose above boiling


point - no heated injection port
column at low temperature to condense
sample in narrow band
heating of column starts chromatography

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Columns
There are two general types of column,
Packed and Capillary (also known as open tubular).
Packed columns contain a finely divided, inert, solid
support material (commonly based on diatomaceous earth)
coated with liquid stationary phase.
Most packed columns are 1.5 - 10m in length and have an internal
diameter of 2 - 4mm.

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Capillary columns have an internal diameter of a few tenths of a
millimeter. They can be one of two types;
wall-coated open tubular (WCOT) or
support-coated open tubular (SCOT).

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Wall-coated columns consist of a capillary tube whose walls are
coated with liquid stationary phase.
support-coated columns, the inner wall of the capillary is lined
with a thin layer of support material, onto which the stationary
phase has been adsorbed.
 SCOT columns are generally less efficient than WCOT columns.
 Both types of capillary column are more efficient than packed
columns.

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a new type of WCOT column was devised - the Fused Silica Open
Tubular (FSOT) column;

Polyamide Coating
Fused Silica Tube
Chemically bonded stationary phase

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Which detectors are commonly used?
Mass Spectrometer (GC/MS)
Many GC instruments are coupled with a mass spectrometer,
which is a very good combination.
The GC separates the compounds from each other, while the mass
spectrometer helps to identify them based on their fragmentation
pattern.

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Flame Ionization Detector (FID)

 The detector is very sensitive towards organic molecules (10-


12 g/s) but relative insensitive to a few small molecules e.g. N2,
NOx, H2S, CO, CO2, H2O.
 If proper amounts of hydrogen/air are mixed, the combustion
does not afford any ions.
 If other components are introduced that contain carbon atoms
cations are produced in the effluent stream.
 The more carbon atoms are in the molecule, the more fragments
are formed and the more sensitive the detector is for this
compound (-- > response factor).

 However, due to the fact that the sample is burnt (pyrolysis), this
technique is not suitable for preparative GC.

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Ion-exchange chromatography

“Ion-exchange chromatography (or ion chromatography) is a


process that allows the separation of ions and polar molecules
based on their charge”.

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Principle:
 Ion-exchange chromatography retains analyte molecules on
the column based on coulombic (ionic) interactions.
 The stationary phase surface displays ionic functional groups
(R-X) that interact with analyte ions of opposite charge.
 This type of chromatography is further subdivided into cation
exchange chromatography and anion exchange
Chromatography.
 The ionic compound consisting of the cationic species M+ and
the anionic species B- can be retained by the stationary phase.

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Cation exchange chromatography retains positively charged cations
because the stationary phase displays a negatively charged
functional group:
-++- -++-
R
-
XC+
MB R
-
XM+
C
+B

Anion exchange chromatography retains anions using positively


charged functional group:
+- +
- +- +-
R
-
XA+
MB R
-
XB+
M+
A

 Equilibration
 Sample Application and Wash
 Elution
 Regeneration 56
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“Spectroscopy is a terms used to refer to the
measurement of radiation intensity as a
function of wavelength or frequency by
spectroscopic methods”

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 Historically, spectroscopy originated through the study of visible
light dispersed according to its wavelength, e.g., by a prism.
 Later the concept was expanded greatly to comprise any
interaction of mater with radiative energy as a function of its
wavelength or frequency.
 Spectroscopic data is often represented by a spectrum, a plot of
the response of interest as a function of wavelength or frequency

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THE ELECTROMAGNETIC SPECTRUM
high Frequency (n) low
high Energy low

X-RAY ULTRAVIOLET INFRARED MICRO- RADIO FREQUENCY


WAVE

Nuclear
Vibrational
Ultraviolet Visible magnetic
infrared
resonance
2.5 mm 15 mm 1m 5m
200 nm 400 nm 800 nm
BLUE RED

short Wavelength (l) long


Types of Energy Transitions in Each Region
of the Electromagnetic Spectrum

REGION ENERGY TRANSITIONS

X-ray Bond-breaking
UV/Visible Electronic
Infrared Vibrational
Microwave Rotational
Radio Frequency Nuclear and
(NMR) Electronic Spin
Nature of the Interaction
Types of spectroscopy can also be distinguished by the nature of the
interaction between the energy and the material.

 Absorption Spectroscopy
 Emission Spectroscopy
 Elastic Scattering Spectroscopy
 Impedance Spectroscopy
 Inelastic Scattering Spectroscopy
 Coherent or Resonance Spectroscopy

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 In quantum mechanical systems, the analogous resonance is a coupling
of two quantum mechanical stationary states of one system, such as an
atom, via an oscillatory source of energy such as a photon.
 The coupling of the two states is strongest when the energy of the
source matches the energy difference between the two states.
 The energy of a photon is related to its frequency by where is Planck's
constant, and so a spectrum of the system response vs. photon
frequency will peak at the resonant frequency or energy.

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Beer- Lambert Law: {Beer–Lambert–Bouguer law
(named after August Beer, Johann Heinrich Lambert, and
Pierre Bouguer)}
 The Lambert law states that absorption is
proportional to the light path length,
 The Beer law states that absorption is
proportional to the concentration of absorbing
species in the material.

Incombination
“Absorption is proportional to the light path length,
and the concentration of absorbing species in the
material”
UV-
T = I / I0 Spec.
A = - log10 I / I0
Al.c
A=.l.c 65
Ultraviolet-Visible Spectroscopy

Molecules containing π-electrons or non-bonding electrons (n-


electrons) can absorb the energy in the form of ultraviolet or visible
light to excite these electrons to higher anti-bonding molecular
orbitals.
σ* (anti-bonding)
n σ*
σ σ*
π σ*
π* (anti-bonding)
n π* σ π*
Energy

π π* n (non-bonding)

π (bonding)

σ (bonding)
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Chromophore Example Excitation λmax, nm ε Solvent
C=C Ethene π __> π* 171 15,000 hexane
C≡C 1-Hexyne π __> π* 180 10,000 hexane
n __> π* 290 15 hexane
C=O Ethanal __> π*
π 180 10,000 hexane
Nitromet n __> π* 275 17 ethanol
N=O __> π*
hane π 200 5,000 ethanol
Methyl
C-X X=Br bromide n __> σ* 205 200 hexane
X=I Methyl n __> σ* 255 360 hexane
Iodide

UV/Vis-
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Instument
http://winter.group.shef.ac.uk/orbitron/
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Factors of Column Efficiency

 Particle size of Packings


 Column Diameters
 Effect of Mobile-Phase Flow Rate.

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Course Outline
 Molecular Orbital Theory.
 Chemistry of Solutions:.
 Chemistry of Transition Metals.
 Coordination Compound and Radioactive Elements.
 Crystalline State of Metals and Lattice Structure.
 Industrial Inorganic Chemistry.
 Qualitative and Group Theory of Inorganic Chemistry.
 Electrochemistry, Including Fuel Cells.

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