LECTURE 4

Fractionation
 Fractionation-Introduction
• Chemical species present in a given sample are
not stable enough to be directly determined.

• During the measurement partitioning of the

element among its species may be changed.

• This behavior can be caused by;

a) Change in pH during the analytical procedure,
b) Procedures of methods may affect
equilibrium between species.
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 Fractionation-Introduction…
• Partitioning may be affected by;

a) when analyte must be transformed into a

complex to enable measured or

b) The analyte must be separated from the

sample matrix.

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 Fractionation-Introduction…
• Even when the complexation reaction is highly
selective, errors can occur if the sample contains
other species that can be transformed into the target
analyte within the analysis process.

• The direct determination of labile species at their

natural levels requires non invasive methods such as;
a) Direct spectroscopic or
b) Potentiometric measurements.

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 Fractionation-Introduction…
• Thus identification of various classes of species of an
element and determination of sum of its
concentrations in each class is of need.

• Such fractionations can be based on many different

properties of the chemical species, such as; size,
solubility, affinity, charge, and hydrophobicity.

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 Fractionation-Introduction…
• Fractionation may involve an actual physical separation
such as;
a) Filtration, or
b) Size-exclusion chromatography.
• Fractionation may be refined by supplementary
speciation analysis.

• For example, for copper in natural waters a

fractionation can be performed to determine organic
and inorganic copper concentrations.
• With further analyses and calculations the inorganic
fraction can be subdivided into individual species.
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 Fractionation-Introduction…
• It is also desirable to measure the total
concentration of the element in order to verify
the mass balance.
• Many existing methods are claimed to give
concentrations for free or available elements.

• Most of these methods are based on electrochemical

and/or kinetic discrimination.
• In order to produce information on the chemical
speciation of the elements, the measured fraction is
factored into chemical species concentrations using
an equilibrium model.
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 Fractionation-Introduction…
• These attempts at indirect measurements of
speciation suffer the same shortcomings as do
fractionations according to size.

• Further, the equilibrium model applied is based

on a choice of complexes and their stability
constants, which makes the results questionable.

• Although a direct determination of the speciation of

an element is often not attainable, the available
methods can still provide much useful
information.
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 Fractionation-Introduction…
• An evaluation of the environmental impact of an
element may sometimes be made without
determining its speciation and even though the
fractions are only operationally defined.

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 Methodological approaches for
fractionation
• A typical example of a fractionation procedure is that
for aluminum in natural waters.
• The method is based on kinetic discrimination, by
measuring only those aluminum species that react
within the timeframe defined by the analytical
procedure.

• In order to achieve consistency between results

obtained using different methods and by different
analysts it is important to validate procedures.

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 Methodological approaches for
fractionation…
• The procedure for reactive aluminum can be
validated based on the interaction between
aluminum and fluoride in synthetic solutions.

• Another example is fractionation using membranes

of different pore sizes.
• The most obvious fractions are of dissolved species
and particulates.
• Fractionations according to molecular size are
also possible using appropriate membranes.
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 Methodological approaches for
fractionation…
• The distinction between dissolved and particulate
fractions is arbitrary and results obtained with a
given set-up are critically dependent on operating
conditions.

• For instance, the retention of iron colloid

particles on a standard 0.45 µm membrane filter
is strongly dependent on the filter load.
• When filter clogs it starts retaining much smaller
particles as well.

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 Methodological approaches for
fractionation…
• Further, the molar mass cut-off given for a certain type of membrane
is based on measurements using a certain set of calibrants.
• Calibrants with globular shape, and this cut-off does not necessarily
apply to all the chemical species fractionated.

• Another way of fractionating is to use field-flow fractionation (FFF).

• FFF is a separation technique where a field is applied to the fluid
suspension or solution pumped through a long narrow channel
perpendicular to the direction of the flow.

• The separation is caused by difference in mobility induced by the

field/force.

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 Methodological approaches for
fractionation…
• The fields involved in field-flow fractionation include;
a) asymmetric flow through semipermeable
membrane,
b) gravitational,
c) centrifugal,
d) thermal gradient,
e) electrical,
f) magnetic etc.

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 Methodological approaches for
fractionation…
• Using on-channel pre-concentration, the cross-flow
version of FFF coupled on-line with ICP-MS has
been applied to the study of element
distributions in colloids.
• Sedimentation FFF has also been used on-line
with ICP-MS for the characterization of larger
particles.
• These techniques are of great help in studies of
element transport in rivers, estuaries, and
aquifers.

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 Methodological approaches for
fractionation…
• Many research groups have worked on the design and
study of extraction schemes aiming at;
a) the sequential solubilization of metals bound to the
specific substrates making up sediments,
b) metals bound to the specific substrates known to
undergo changes in the yearly life cycle of lakes or rivers.
• Typical substrates are;
a) carbonates of calcium,
b) magnesium and iron, which dissolve upon
decrease in pH,

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 Methodological approaches for
fractionation…
c) iron and manganese compounds present in the
sediments and changing their adsorption capacities
drastically according to the redox conditions,

d) organic matter present in sediments undergoing slow

degradation, thus setting free the incorporated metals,

e) silicates and other refractory minerals, which might

contain high metal concentration, but will not, under any
environmental conditions, release them to the aquatic
environment.
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 Methodological approaches for
fractionation…
• Harmonization of methods is necessary because the
wide variety of procedures led to incomparability of
results even for single aquatic systems.

• A collaborative study led to the development of a

three-step sequential extraction procedure, yielding
information on four metal fractions;

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 Methodological approaches for
fractionation…
1) exchangeable and associated with carbonates,
2) associated with easily and oderately reducible iron
and manganese compounds,
3) associated with organic matter and sulfidic phases,
4) residual fraction.

• This information is essentially based on the Tessier

procedure which was later modified by Förstner and
co-workers.

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 SELECTED TECHNIQUE FOR
FRACTIONATION

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 Chromatographic Techniques

• Historically, the word chromatography was used by

Tswett in 1903.
• It described the separation of plant pigments by
percolating a petroleum-ether extract through a glass
column packed with powdered calcium carbonate.
• Colored zones were produced by the various pigments
migrating through the column at different rates.
• Components being isolated by extrusion and sectioning
of the calcium carbonate packing.

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 Chromatographic Techniques…
• Modern chromatographic techniques are more
complex and are used for a wide variety of
separations frequently involving colorless substances,
but the original term is retained.
• All the techniques depend upon the same basic
principle;
a) variation in the rate at which different
components migrate through a stationary phase,
b) influence of a mobile phase.
• Rates of migration vary because of differences in
distribution ratios.
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 Chromatographic Techniques…
• Note that;
a) The stationary phase must be continuous,
b) The mobile phase is allowed to move
continuously.

• The liquid stationary phase is coated onto an inert,

granular or powdered solid support which is either
packed into a column or spread on a supporting
sheet in the form of a thin layer.

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 Chromatographic Techniques…
• The solid stationary phases used in some
chromatographic techniques have no need of a
support if packed into a column but still require a
supporting sheet for thin-layer operation.
• As the distributing components of a mixture are
moved down a column or across a surface by the
mobile phase, they assume a Gaussian concentration
profile.
• Because both phases are continuous, diffusion and
kinetic effects play a significant role in determining
the width of the profile.

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 Chromatographic Mechanisms
• During a chromatographic separation, solute molecules
are continually moving back and forth between the
stationary and mobile phases.

• While they are in the mobile phase, they are carried

forward with it but remain virtually stationary during
the time they spend in the stationary phase.

• The rate of migration of each solute is therefore

determined by the proportion of time it spends in the
mobile phase.
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 Chromatographic Mechanisms…

• The process hereby a solute is transferred from a

mobile to a stationary phase is called sorption.
•
• Chromatographic techniques are based on four
different sorption mechanisms namely;
a) surface adsorption,
b) partition,
c) ion- exchange and
d) exclusion.

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 Chromatographic Mechanisms…
• The original method employed by Tswett involved
surface adsorption.
• In this case polarities of solute and solid stationary
phase determine the rate of movement of that solute
through a column or cross a surface.

• If a liquid is coated onto the surface of an inert solid

support, the sorption process is one of partition.
• In that case, movement of the solute is determined
by its relative solubility in the two phases or by its
volatility if the mobile phase is a gas
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 Chromatographic Mechanisms…
• Both adsorption and partition may occur
simultaneously, and the distribution of each is
determined by;
a) the nature of the mobile and stationary
phases,
b) solid support and solute.
• For example, a stationary phase of aluminium oxide
is highly polar and normally exhibits strong
adsorptive properties.

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 Chromatographic Mechanisms…
• However, these may be modified by the presence of
adsorbed water which introduces a degree of
partition into the overall sorption process by acting
as a liquid stationary phase.

• Conversely, paper (cellulose) is relatively non-polar

and retains a large amount of water which functions
as a partition medium.
• Nevertheless, residual polar groups in the structure
of the paper can lead to adsorptive effects.

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 Chromatographic Mechanisms…
• In the ion-exchange the stationary phase is a
permeable polymeric solid containing fixed charged
groups.
• Its mobile contain counter-ions which can exchange
with the ions of a solute as the mobile phase carries
them through the structure.
• The exclusion mechanism is not a true sorption
process as the separating solutes remain in the
mobile phase throughout.

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 Chromatographic Mechanisms…
• Separations occur because of variations in the extent
to which the solute molecules can diffuse through an
inert but porous stationary phase.
• This is normally a gel structure which has a small
pore size and into which small molecules of a certain
critical size can diffuse.
• Molecules larger than the critical size are excluded
from the gel and move unhindered through the
column whilst smaller ones are retarded to an extent
dependent on molecular size.

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 Chromatographic Mechanisms…
• Note that, one of the four mechanisms
predominates, but it should be emphasized that two
or more may be involved simultaneously.

• Partition and adsorption frequently occur together

and in paper chromatography.

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 ------------------------END----------------------

1ST TEST
23-12-2018

LECTURE 5
28-12-2018
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