2. Invented by M.Tswett a botanist in 1906
He used for separation of coloured compounds
Now chromatographic separation any given
mixture can be done.
Chromatography is greek word Chroma means
colour and graphy means writing
It is based on the difference in the rate at which
components of the mixture move through the
porous medium ( called stationary phase)
under the influence of solvent ( mobile phase)
3. Definition
Chromatography is a separation technique based on the different
interactions of compounds with two phases, a mobile phase and a
stationary phase, as the compounds travel through a supporting
medium.
Components:
mobile phase: a solvent that flows through the supporting medium
stationary phase: a layer or coating on the supporting medium that
interacts with the analytes
supporting medium: a solid surface on which the stationary phase is
bound or coated
4. The analytes interacting most
strongly with the stationary
phase will take longer to pass
through the system than those
with weaker interactions.
These interactions are usually
chemical in nature, but in some
cases physical interactions can
also be used.
5. Based on principle Classified as
Adsorption chromatography
Partition chromatography
Chromatography can be classified based on the type of
mobile phase, stationar phase and support material
6. 1.) The primary division of chromatographic techniques is based on the type of mobile phase
used in the system:
Type of Chromatography Type of Mobile Phase
1. Gas chromatography (GC) gas
2. Liquid chromatograph (LC) liquid
2.) Further divisions can be made based on the type of stationary phase used in the system:
Gas Chromatography
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support
7. Types of Chromatography
1. Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized support
Partition chromatography liquid-coated or derivatized support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support
Affinity chromatography support with immobilized ligand
8.
9. 3.) Chromatographic techniques may also be classified based on the type of support material
used in the system:
1. Packed bed (column) chromatograph
2. Open tubular (capillary) chromatography
3. Open bed (planar) chromatography
10. Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):
chromatogram - concentration versus elution time
Wh
Wb
Inject
Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
11. Note: The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
system)
Peak width & peak position
determine separation of peaks
A similar plot can be made in terms of elution volume instead of elution time. If volumes
are used, the volume of the mobile phase that it takes to elute a peak off of the column is
referred to as the retention volume (VR) and the amount of mobile phase that it takes to
elute a non-retained component is referred to as the void volume (VM).
12. 2.) Solute Retention:
A solute’s retention time or retention volume in chromatography is directly related to the
strength of the solute’s interaction with the mobile and stationary phases.
Retention on a given column pertain to the particulars of that system:
- size of the column
- flow rate of the mobile phase
Capacity factor (k’): more universal measure of retention, determined from tR or VR.
k’ = (tR –tM)/tM
or
k’ = (VR –VM)/VM
capacity factor is useful for comparing results obtained on different systems since it is
independent on column length and flow-rate.
13. The value of the capacity factor is useful in understanding the retention mechanisms for a
solute, since the fundamental definition of k’ is:
moles Astationary phase
k’ =
moles Amobile phase
k’ is directly related to the strength of the interaction between a solute with the stationary
and mobile phases.
Moles Astationary phase and moles Amobile phase represents the amount of solute present in each
phase at equilibrium.
Equilibrium is achieved or approached at the center of a chromatographic peak.
When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum
14. A simple example relating k’ to the interactions of a solute in a column is illustrated for
partition chromatography:
KD
A (mobile phase)
A (stationary phase)
where: KD = equilibrium constant for the distribution of A between the mobile
phase and stationary phase
Assuming local equilibrium at the center of the chromatographic peak:
[A]stationary phase Volumestationary phase
k’ =
[A]mobile phase Volumemobile phase
Volumestationary phase
k’ = KD
Volumemobile phase
As KD increases, interaction of the solute with the stationary phase becomes more
favorable and the solute’s retention (k’) increases
15. Volumestationary phase
k’ = KD
Volumemobile phase
Separation between two solutes requires different KD’s for their
interactions with the mobile and stationary phases
since G = -RT ln KD
peak separation also represents different changes in free energy
16. 4.) Measures of Solute Separation:
separation factor ( ) – parameter used to describe how well two solutes are separated by
a chromatographic system:
= k’2/k’1 k’ = (tR –tM)/tM
where:
k’1 = the capacity factor of the first solute
k’2 = the capacity factor of the second solute,
with k’2 $ k’1
A value of $1.1 is usually indicative of a good separation
Does not consider the effect of column efficiency or peak widths, only retention.
17. resolution (RS) – resolution between two peaks is a second measure of how well two
peaks are separated:
tr2 – tr1
RS =
(Wb2 + Wb1)/2
where:
tr1, Wb1 = retention time and baseline width for the
first eluting peak
tr2, Wb2 = retention time and baseline width for the
second eluting peak
Rs is preferred over since both
retention (tr) and column efficiency
(Wb) are considered in defining
peak separation.
Rs $ 1.5 represents baseline
resolution, or complete separation
of two neighboring solutes ideal
case.
Rs $ 1.0 considered adequate for
most separations.
18. Thin-layer chromatography and column
chromatography and are different types of
liquid chromatography.
The mobile (moving) phase is a liquid. The
stationary phase is usually silica or alumina.
This phase is very polar.
The principle of operation is the same!
19. simple equipments
Shorter developing time
Wide choice of stationary phase
Early recovery of separated components
Superior separation
Easy visualization of separated components
Sensitivity
Chemically inert stationary phase
21. Activation of Adsorbent
Sample application
Developing Tanks
Solvent Systems
Development methods
Detection of components
1. Visual Assessments
2. Determination of measuring the spot areas
3. Direct spectrophotometry
22. The surface of the plate consists of a very thin layer of silica on a plastic or
aluminum backing. The silica is very polar. This is the stationary phase. Spot
the material at the origin (bottom) of the TLC plate.
Place the plate into a glass jar with a small amount of a solvent in the glass jar.
This solvent acts as the moving phase.
Remove the plate from the bottle when the solvent is close to the top of the plate.
Visualize the spots.
Non-polar compounds will be less strongly attracted to the plate and will spend
more time in the moving phase. This compound will move faster and will appear
closer to the top of the plate.
Polar compounds will be more strongly attracted to the plate and will spend less
time in the moving phase and appear lower on the plate.
23. DEVELOPING
CHAMBER
SPOT
SOLVENT
Solvent
Front
1.1 cm
5.5 cm
Origin
Distance from starting origin to center of zone
Rf =
Distance from starting origin to solvent front
5.5 = 0.5
=
11
24. OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O
O O
O
Si
Si O
O
O O
O
25. O OH OH OH OH
Al Al Al Al Al
O O O O O O
Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
26. solvent front
component B Less polar!
solvent front
component B
component A More polar!
component A
origin mixture origin origin
solvent front
Increasing Development Time
27. The previous slide shows colored spots. Most
of the time, the spots won’t show unless they
are visualized!
Vizualization is a method that is used to render
the TLC spots visible.
A visualization method can be:
Ultraviolet light
Iodine vapors to stain spots
Colored reagents to stain spots
Reagents that selectively stain spots while leaving
others unaffected.
28. solvent front
Rf of component A =
dA
component B
dS
dS
dB
Rf of component B =
dB component A
dS dA
origin
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
30. To determine how many components there are
in a mixture (is it really pure?)
To determine the best solvent conditions for
separation on a column
To identify the substances being studied
To monitor the composition of fractions
collected from column chromatography
To monitor the progress of a reaction
31. The stationary phase (column packing) in the column is
very polar!
Polar compounds are going to be attracted to the polar
column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!
Non-polar compounds are going to come off the column
first, while the polar compounds are going to come off
the column last.
Usually, one starts will a less polar solvent to remove
the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
32.
33.
34.
35.
36.
37.
38.
39. The stationary phase is POLAR
The more polar component interacts more
strongly with the stationary phase
The more polar component moves more slowly.
The non-polar component moves more rapidly.
40. Silica is alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.
CH3
CH2
CH3 17 CH3
Si
CH2 SiCH3)3 CH3
17 CH3
SiCH3)3
SiCH3)3
O
Si O
CH3 O
O
O Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si
Si O
Si O
Si O O
Si O O
O O
O O
O
Si
Si O
O
O O
O
41. The stationary phase (column packing) is now
NON-POLAR
Non-polar compounds will move more slowly
because they are attracted to the column
packing.
The more polar component moves more
quickly down the column.
Polar solvents, such as water and methanol
are used in reverse phase chromatography
Used mainly in columns, such as HPLC
42. The column packing in the column is very polar!
Polar compounds are going to be attracted to the polar
column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!
Non-polar compounds are going to come off the column
first, while the polar compounds are going to come off
column last.
Usually, one starts will a less polar solvent to remove
the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
43. Partition
Chromatography
Fibers of paper is
stationary phase
RF value
46. Proper Developing solvents
Preparation of samples
Spotting
Drying the chromatography
Visualization
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