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Introduction to chromatographic separation
 Chromatography is a widely used method that allows the separation,
identification, and determination of the chemical components in
complex mixtures
...
The history of
chromatography spans from the mid-19th century to the 21st
...
He continued to work
with chromatography in the first decade of the 20th century, primarily
for the separation of plant pigments such as chlorophyll (which is
green) and carotenoids (which are orange and yellow)
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Principles of Chromatography
In all chromatography there is a mobile phase and a stationary
phase
...
The mobile phase moves
through the stationary phase picking up the compounds to be tested
...
The
solutes in a mobile phase go through a stationary phase
...
As the solutes
move through the stationary phase they separate
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Types of Chromatography
 Chromatography can be classified into different types on the basis
of;
1
...
Planar chromatography (Paper and Thin layer chromatography)
II
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The Nature of the stationary phase
I
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Partition chromatography
III
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Molecular Exclusion chromatography(size exclusion)
V
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The nature of mobile phase
I
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Liquid chromatography
III
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Here the
mobile phase moves through the stationary phase by capillary action
sometimes assisted by gravity or an electrical potential
...

 In column chromatography, the stationary phase is placed in a
narrow column through which the mobile phase moves under the
influence of gravity or pressure
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On the basis of the Nature of the stationary phase
 In Adsorption chromatography, solutes separate based on their
ability to adsorb to a solid stationary phase
...
Separation is based on a
difference in the equilibrium partitioning of solutes between the liquid
stationary phase and the mobile phase
...
g
...
g
...
Ionic solutes are separate based
on a difference in the electrostatic interaction with the stationary phase
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Separation is due to differences in the size of the
solutes
...
A drop of a solution of a mixture of dyes or inks is
placed on a piece of chromatography paper and allowed to dry
...
Filter
paper and blotting paper are frequently substituted for chromatography
paper if precision is not required
...

 Paper chromatography works by the partition of solutes between
water in the paper fibres (stationary phase) and the solvent (mobile
phase)
...
Mixtures of solvents are also used, including
aqueous solutions, and solvent systems with a range of polarities can
be made
...
This fraction is variously
called the retardation factor or the retention ratio, and is given the
symbol Rf:

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 NB; The final chromatogram can be compared with other known
mixture chromatograms to identify sample mixes using the Rf values,
because Rf values are standards for a given compound, known Rf

values can be used to identify an unknown substance in the experiment
...
In such a case a
second run is carried out by a different solvents system in a direction

perpendicular to the first run
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 NB; Rf values, often depends on the temperature, solvent and type
of paper used in the experiment, the most effective way to identify a
compound is to spot known substance next to unknown substance on

the same chromatogram
...
An impure sample will often develop as two or more
spots, while a pure sample will show only one spot
...

 The mixture is ‘spotted’ at the bottom of the TLC plate and allowed
to dry
...

 TLC has advantages over paper chromatography in that its results
are more reproducible, and that separations are very efficient because
of the much smaller particle size of the stationary phase
...

 This technique is usually done in a closed vessel to ensure that the
atmosphere is saturated with solvent vapour and that evaporation from
the plate is minimised before the run is complete
...


 In paper and thin-layer chromatography the mobile phase is the
solvent
...
In thin-layer
chromatography the stationary phase is the thin-layer cell
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Principles of Thin layer chromatography (TLC)
 In thin layer chromatography, a solid phase, the adsorbent, is coated
onto a solid support as a thin layer (about 0
...
In many
cases, a small amount of a binder such as plaster of Paris is mixed with
the absorbent to facilitate the coating
...
The mixture (A plus B) to be separated is
dissolved in a solvent and the resulting solution is spotted onto the thin
layer plate near the bottom
...

 A substance that is strongly adsorbed (say, A) will have a greater
fraction of its molecules adsorbed at any one time and thus any one
molecule of A will spend more time sitting still and less time moving
...
Thus,
the more weakly a substance is adsorbed, the farther up the plate it will
move
...
TLC consists of three steps - spotting, development,
and visualization
...
Spotting the TLC plate
 One advantage TLC has over other separation methods is that it is
truly a micro-scale technique
...
Once the
sample is prepared, a spotting capillary must be used to add the sample
to the plate
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Figure 1
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The solvent should evaporate quickly leaving
your mixture behind on the plate
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If too much solute is added to the plate, a poor
separation will result
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B
...
Just like paper chromatography, the
solvent must be in contact with the stationary phase
...

 In addition, a piece of filter paper is put in the bottle to help create
an atmosphere saturated with solvent
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If the spots are
submerged in the solvent, they are washed off the plate and lost
...
Using a pencil, immediately draw a line across the plate
where the solvent front can be seen
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C
...
If you are fortunate enough
to be separating organic molecules that are colored such as dyes, inks
or indicators, then visualizing the separated spots is easy
...

 In most cases observing the separated spots by UV light works well
...

Compounds that absorb UV light will quench the green fluorescence
yielding dark purple or bluish spots on the plate
...

Lightly circle the spots, so that you will have a permanent record of
their location for later calculations
...

Iodine sublimes and will absorb to organic molecules in the vapor
phase
...
Also circle these observed spots, since the color stain will
eventually fade from the plate
...
Some compounds
are not “UV active”, that is, they do not absorb light at the wavelength
of 254 nm
...

D
...
The Rf value is expressed as a decimal fraction
...
6 cm from the
base line while the solvent had travelled 12
...
80 cm
Example 2; Compound X has Rf value of 0
...
How far will
compound X have travelled from the origin when the eluent front has
traveled 4 cm?
Solution;
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Rf =

0
...
25 cm)
X = 1 cm

 If two spots travel the same distance or have the same Rf value then it
might be concluded that the two components are the same molecule
...
These conditions include the stationary
phase, mobile phase, and temperature
...
Additional information must be
obtained before this conclusion can be made
...

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Figure 1
...

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 The RF Value of the substance depends upon a number of factors
...
The solvent employed

2
...
e
...
The nature of the mixture
4
...
The size of the vessel in which the operation is carried out
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Column Chromatography
 Of the two methods for bringing the stationary and mobile phases

into contact, the most important is column chromatography
...
With appropriate modifications, this theory
also can be applied to planar chromatography
...
Although the figure depicts a liquid–solid chromatographic
experiment similar to that first used by Tswett, the design of the
column and the physical state of the stationary and mobile phases may
vary
...
3: (a) Elution process in Column Chromatography (b)
chromatogram
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 The sample is introduced at the top of the column as a narrow band
...
As the
sample moves down the column the solutes begin to separate, and the
individual solute bands begin to broaden and develop a Gaussian
profile
...
The progress of a chromatographic
separation is monitored with a suitable detector situated at the end of
the column
...
A
chromatographic peak may be characterized in many ways, two of
which are the retention time and the base line width
...
4: Chromatogram detector signal vs time
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Baseline width, w, is determined by
the intersection with the baseline of tangent lines drawn through the
inflection points on either side of the chromatographic peak
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Basic Chromatographic terminology
 Chromatograph: Instrument employed for a chromatography
...
Can
be a particular solid or gel-based packing (LC) or a highly viscous
liquid coated on the inside of the column (GC) or stationary phase is
the extracting phase that remains in a fixed position
...

 Eluent: Fluid entering a column
...
It moves the analytes through the chromatograph
...

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 Eluate: Fluid exiting the column
...
It specifically includes both the
analytes and solutes passing through the column
...

 Chromatogram: Graph showing detector response as a function of
a time or volume
...

 Linear velocity: Distance passed by mobile phase per 1 min in the
column (cm/min)
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 Retention volume: The volume of mobile phase needed to move a
solute from its point of injection to the detector (Vr)
...


 Distribution constant:

Where K is distribution constant, CS is concentration of stationary phase
and CM is concentration of mobile phase
...
If k’ is less than 1, determination of fast tR is
inaccurate
...


where, nS is mole in stationary phase and nM is mole in mobile phase
...

 Resolution is a quantitative measure of the degree of separation
between two chromatographic peaks, A and B, and is defined as
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Example In a chromatographic analysis of lemon oil a peak for
limonene has a retention time of 8
...
96
min
...
54 min, with a baseline width of 0
...

What is the resolution between the two peaks?
answer
= 1
...
For two peaks of equal size, a
resolution of 1
...
13%
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Figure 1
...


 From the equation above it is clear that resolution may be improved
either by increasing Δtr or by decreasing WA or WB
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Migration Rate of Solutes
 The effectiveness of a chromatographic column in separating two
solutes depends in part on the relative rates at which the two species
are eluted
...
For the solute species A, the
equilibrium involved is described by the equation
Sm
KD

Ss and its associated partition coefficient, KD, and
distribution ratio, D,
D=

 Where the subscripts m and s refer to the mobile phase and
stationary phase, respectively
...
Conservation of mass
requires that the total moles of solute remain constant throughout the
separation, thus;
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(moles S)tot = (moles S)m + (moles S)s
D=
Where Vm and Vs are the volumes of the mobile and stationary phases
...

By the same reasoning, the solute’s average linear
velocity, v, is

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 The solute can only move through the column when it is in the
mobile phase
...


v = ufm
=

(

)

Finally, solving this equation for k’ gives;
K’ =
Example; In a chromatographic analysis of low-molecular-weight acids,
butyric acid elutes with a retention time of 7
...
The column’s void
time is 0
...
Calculate the capacity factor for butyric acid
...
6
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Column Selectivity
The relative selectivity of a chromatographic column for a pair of
solutes is given by the selectivity factor, α, which is defined as

The identities of the solutes are defined such that solute A always has
the smaller retention time
...

Example; In the same chromatographic analysis for low-molecularweight acids considered in Example above, the retention time for
isobutyric acid is 5
...
What is the selectivity factor for isobutyric
acid and butyric acid?
Solution;
First we must calculate the capacity factor for isobutyric acid
...
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Chromatographic theories
Plate theory (1941) by Martin & Synge who won the Nobel Prize in
Chemistry in 1952 for it: Thermodynamic theory with the assumption
of equilibrium
...


Plate theory

Separation of two solutes depends on the rates at which the two
species are eluted
...
The theory is based on an analogy with distillation
and countercurrent extraction
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 Deals with general relationships such as
1)
2)
3)
4)
5)

Distribution constant
Retention time
Relationship between distribution constant and retention time
Capacity factor
Selectivity factor

 The plate theory successfully accounts for the Gaussian shape of
chromatographic peaks and their rates of movement down a column
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Figure 1
...

Does not account for the mechanism causing peak broadening
No indication of other parameters effects
No indication for adjusting experimental parameters

Rate theory
Band broadening
A part from specific characteristics of solute that cause differential
migration, average migration rates for molecules of the same solute are
not identical
...
These are;
H =A+

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+ (Cs + CM) u

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Multiple pathways/Eddy diffusion (A)
Eddy diffusion band broadening process results from different path
lengths passed by solutes
...

A = 2dp where dp is diameter of packing  is constant that depends
on the quality of the packing
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Longitudinal diffusion (B/u)
 Molecules tend to diffuse in all directions because these are always
present in concentration zone as compared to other parts of the column
...
The longitudinal diffusion effect on H is
inversely proportional to u because the solute residence time is shorter
at high u and the extent of diffusion is less
...

 DM is smaller in liquids (even smaller in more viscous mobile phase)
than in gases, so

 B/u is less pronounced in Liquid Chromatography (LC) than in Gas
Chromatography (GC)
...
e
...
6)
than for an unpacked (capillary) column (i
...
 = 1)
...
So analyte molecules at the
front of a band are swept ahead without equilibrating with the
stationary phase and those at the tailing edge are left behind for a
longer time in the stationary phase
...

CSu is less if the liquid stationary phase film thickness, df, is smaller,
or the solute diffusion coefficient in the stationary phase, DS is larger
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Column Efficiency
 At the beginning of a chromatographic separation the solute
occupies a narrow band of finite width
...
Column efficiency provides a quantitative
measure of the extent of band broadening
...
In their original theoretical model of
chromatography, Martin and Synge treated the chromatographic
column as though it consists of discrete sections at which partitioning
of the solute between the stationary and mobile phases occur
...
Therefore the plate height can be defined as the variance per
unit length of the column

H=
The width of the peak at the baseline, is related to t by the relation W =
4t and the peak width can also be represented interms of time, t
...
354t

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Assignment; Express Rs interms of (N, K’ and ) or show the
following expression

Hint; Let tRB = tm (1 + k’B)
tRA = tm (1 + k’A)
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