Instrumental Analytical
Techniques
An Overview
of
Chromatography and Spectroscopy
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Chromatographic Techniques
-Thin layer and column chromatography
-Gas Chromatography (GC)
-High Performance Liquid Chromatography
(HPLC)
Spectroscopic Techniques
- Atomic Absorption Spectroscopy(AAS)
- Colorimetry
- UV-Visible Spectroscopy (UV-Vis)
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Chromatographic
• Chromatography is the term used to describe
a separation technique in which a mobile
phase carrying a mixture is caused to move in
contact with a selectively absorbent stationary
phase.
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Types of Chromatography
Paper Chromatography and Thin Layer Chromatography (TLC)
Column Chromatography
Gas Liquid Chromatography (GLC)
High Performance Liquid Chromatography (HPLC)
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Paper Chromatography and Thin Layer
Chromatography (TLC)
1. the mobile phase is also a solvent, and the
stationary phase is a thin of finely divided solid,
such as silica gel or alumina, supported on glass
or aluminium.
2. Thin layer chromatography is similar to paper
chromatography
Thin Layer (and Paper) Chromatography
 Thin layer chromatography in that it involves spotting the
mixture on the plate and the solvent (mobile phase) rises
up the plate in the chromatography tank
 Apply a concentrated drop of sample
(•) with a capillary or dropping tube
to bottom of plate (origin pencil line)
• Stand plate in a sealed vessel.
• carefully add solvent (keep solvent level
below sample).
•
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• Allow solvent to adsorb up the plate,
drawing the sample with it.
Thin Layer and Paper Chromatography
The components of the mixture move up the paper with the
solvent at different rates due to their differing interactions with
the stationary and mobile phases.
Mixed
standards
standards
•
•
•
A
B
C
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Unknown +
standards
•
•
•
A+B+C
A+B+C ?
Thin Layer (and Paper) Chromatography
The ratio of distance travelled by the component (from origin) compared with the
distance travelled by the solvent front (from origin) is called the Rf value.
x
Solvent front
a
b
•
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c
Rf of
= a/x
Rf of
= b/x
Rf of
= c/x
Rf = Distance the solute moves
Distance the solvent front moves
Column Chromatography
In column chromatography, the mobile phase is again a solvent, and
the stationary phase is a finely divided solid, such as silica gel or
alumina. Chromatography columns vary in size and polarity.
if the sample was too soluble the mobile phase (solvent) would
move the solutes too quickly, resulting in the non-separation of
the different constituents
Column Chromatography
A mixture is applied to a solid support in a
chromatography column, and eluted by a solvent.
Elute with solvent
1
2
3
Absorbent
medium
tap
Cotton wool
plug
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4
Gas Chromatography
• A gas is the mobile phase and the stationary
phase can be either a solid or a non- volatile
liquid.
Gas Liquid Chromatography
A mixture is injected into a very thin“steel-jacketed”
chromatography column. Inject sample as gas or liquid. A solid
component can be dissolved in solvent but a solvent peak will
also be seen.
Inject sample
Gas mobile phase dense liquid (on solid) SP
Column in oven up to approx. 300 C.
Substance must be able to vaporise and not decompose
Elute with inert gas
Extremely
sensitive
FID detector
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High Performance Liquid Chromatography
HPLC
A mixture is injected into a “steel-jacketed”
chromatography column and eluted with solvent at high
pressure (4000psi or approx 130 atm).
Inject sample as gas or liquid.
A solid component can be dissolved in solvent but a solvent
peak will also be seen.
Elute with solvent
UV detector
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Spectroscopy
 Spectroscopy may also be defined as the study of the
interaction between light and matter.
• UV-Visible Spectroscopy (UV-Vis)
 Qualitative Analysis by UV-Visible Spectroscopy (UV-Vis)
UV-VIS spectroscopy studies the electronic transitions of
molecules as they absorb light in the UV and visible regions of
the electromagnetic spectrum. The data is used to produce
absorbance spectra.
1. prism used to analysis the light to seven light
2. The function of the monochromator is to select a single
wavelength or frequency from the light source.
3. sample cells composed of basic salts, such sodium chloride or
potassium bromide, are commonly used because they will not
absorb light
4. The detector measures the quantity of radiation that passes
thru the sample by converting it to an electrical signal
The optics of the light source in UV-visible spectroscopy
allow either visible [approx. 400nm (blue end) to 750nm
(red end) ] or ultraviolet (below 400nm) to be directed at
the sample under analysis.
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Spectroscopy
Utilises the
Absorption and Emission
of electromagnetic radiation by atoms
Absorption:
Low energy electrons absorb energy to move to higher energy level
Emission:
Excited electrons return to lower energy states
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Absorption v. Emission
Energy is emitted by
electrons returning
to lower energy
levels
Excited
States
3rd
2nd
1st
Energy is absorbed
as electrons jump to
higher energy levels
Ground State
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photo
Electronic Transitions:
1. Transitions involving π, σ, and n electrons
2. Transitions involving charge-transfer electrons
3. Transitions involving d and f electrons
1. Transitions involving π, σ, and n electrons
σ → σ* Transitions:
* The energy required for an electron in a bonding σ orbital to get excited to
the corresponding anti bonding orbital is large. For example, methane (which
has only C-H bonds, and can only undergo (σ → σ* transitions) shows an
absorbance maximum at 125 nm and are thus not seen in typical UV-VIS.
spectra (200 - 780 nm).
σ*
Methane (125nm)
σ
Methane (125nm)
n → σ* Transitions:
* The energy required for an electron in a bonding n orbital to get excited to the
corresponding anti bonding orbital is large. For example, alcohols, amines (which
has only C-OH bonds and NH2, and can only undergo (n → σ* transitions)
compounds absorb light having wavelength in the range 150 - 250 nm and are
thus not seen in typical UV-VIS. spectra (200 - 780 nm).
σ*
Methane (125nm)
n
Methane (125nm)
π → π* Transitions:
* The energy required for an electron in a bonding π orbital to get excited to
the corresponding anti bonding orbital is large. For example, alkenes absorb at
170-190 nm
π*
Methane (125nm)
π
Methane (125nm)
n → π* Transitions
The energy required for an electron in a bonding n orbital to get excited to the
corresponding anti bonding orbital is large. For example, ketone(which has only
c=o bonds aliphatic Ketones absorb at 280nm
π*
n
Quantitative UV/vis Analysis
Quantitative UV/vis is used to determine the concentration of an
analyte, which can absorb in this region, in a solution. Beer's Law,
which gives a linear relationship between absorbance and
concentration for dilute solutions, is used. A calibration plot is
formed by measuring the absorbance of a series of analyte solutions
with different known concentrations and the concentration of the
target analyte in the sample under study is determined from the
plot
The Beer-Lambert Law
A=ebc
1. (e ) is the molar absorptivity with units of L mol-1 cm-1
2. b is the path length of the sample
We will express this measurement in centimetres.
3. c is the concentration of the compound in solution,
expressed in mol L-1
A=ebc
Absorbance
1.00
A=ebc
0.80
0.60
0.40
0.20
0
0
0.10
0.20
0.30 0.40
Concentration in mol / Litre
0.50
Atomic Absorption Spectrometry
• measures small concentrations of metal ions
in solution
– Al, As, Au, B, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ge, K, Li, Mg, Mn, Mo, Na, Ni,
Pb, Si, Sr, Ti, V, W and Zn
• used by industry
– analysis of ores for metal content
– quality control of metals in steel
– testing water for metals ions
– analysing food and pharmaceuticals for metal ions
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Advantages of using AAS
• very sensitive:
can detect concentrations as small as a few parts to
g / Litre (parts per billion)
• generally very specific:
set wavelength is strongly absorbed by the
particular metal ion being analysed (and not by
other components)
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An introduction to Colorimetry
Colorimetry is a quantitative technique which makes use of the
intensity in colour of a solution is directly related to the concentration
of the coloured species in it.
Colorimetry can be used if the substance to be analysed is coloured, or
if it can be made coloured by a chemical reaction.
The concentration of the unknown solution can be estimated by the
naked eye by comparing its colour to those of a series of standard
solutions prepared by successive dilution. However at low
concentrations, colour may not be detected.
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