2. Chromatography
Is a technique used to separate and identify the
components of a mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
mobile and a stationary phase.
mobile phase = solvent or gas
stationary phase = column packing material
2
5. How separation occur?
Chromatography is a powerful separation
method that is used to separate and
identify the components of complex
mixtures.
Works by allowing the molecules present in
the mixture to distribute themselves
between a stationary and a mobile phase to
varying degrees.
Those components that are strongly
retained by the stationary phase move
slowly with the flow of mobile phase.
5
6. How separation occur?
In contrast, components that are weakly
held by the stationary phase travel rapidly
(fast).
As a consequence of these differences in
mobility, sample components separate into
discrete bands that can be analyzed
qualitatively and/or quantitatively.
6
7. Classification of
Chromatographic Methods
1. Based on physical means
The way stationary and mobile phases are brought into
contact.
2. Based on the types of mobile phase
Either gas, liquid or supercritical fluid.
1. Based on the kinds of equilibria involved in the in
solute transfer between the phases
Interaction of analyte between stationary and mobile
phases.
7
8. Chromatographic Methods
based on physical means
Column Planar
chromatography chromatography
stationary phase is stationary phase is
held in narrow tube; supported on a flat plate
mobile phase moves by or in the interstices of a
pressure or gravity paper; mobile phase moves
through capillary action
or gravity
Example: Example:
Gas chromatography (GC) Thin-layer chromatography
Supercritical-fluid (TLC)
chromatography (SFC) Paper chromatography (PC)
8
9. Chromatography based on
types of mobile phase
Mobile
Phase
Gas (Supercritical
Gas Chromatography fluid)
Supercritical-fluid
Chromatography
(Liquid)
Liquid Chromatography
9
10. Chromatography based on
interaction of the analyte
with stationary phase
1. Adsorption - of solute on surface of stationary
phase; for polar non-ionic compounds.
2. Ion Exchange - attraction of ions of opposite
charges; for ionic compounds.
1. Anion - analyte is anion; bonded phase has
positive charge.
2. Cation – analyte is cation; bonded phase has
negative charge.
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11. Chromatography based on
interaction of the analyte
with stationary phase
3.Partition - based on the relative solubility of
analyte in mobile and stationary phases.
a. Normal phase – stationary phase polar, the
mobile phase nonpolar.
b. Reverse phase– stationary phase nonpolar,
the mobile phase polar.
3.Size Exclusion – separate molecules by size;
sieving- stationary phase is a porous matrix.
11
15. Adsorption
Chromatography
Components of the mixture selectively adsorb
(stick) on the surface of a finely divided solid
stationary phase.
As mobile phase (gas/liquid) carries the
mixture through the stationary phase, the
components of the mixture stick to its surface
with varying degrees of strength and thus
separate.
Stationary phase: solid
Mobile phase: gas or liquid 15
18. Partition
Chromatography
Accomplished by selective and continuous
transfer of the components of the mixture
back and forth between a liquid stationary
phase and a liquid mobile phase as the
mobile phase liquid passes through the
stationary phase liquid.
Stationary phase: liquid
Mobile phase: liquid or gas
18
19. Partition
Chromatography
Partitioning is a distribution (by dissolving) of the
components between 2 immiscible phases.
Seperations of the components will be based on
relative solubilities of the components in the mobile
and stationary phase.
Example of partitioning using polar stationary
phase.
Polar components will retain longer than the non-polar
components.
Non-polar components will move quickly through
stationary phase and will elute first before the polar
components and vice-versa. 19
20. Partition
Chromatography
The stationary phase actually consists of a thin
film adsorbed (stuck) on or chemically bonded to
the surface of a finely divided solid particles.
If the mobile phase is gas, the volatility (vapor
pressure) and solubility in stationary phase plays
an important role.
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23. Ion exchange
Chromatography
Method for separating mixture of ions.
Sample is aqueous solution of inorganic ions or
organic ions
Stationary phase are small polymer resin “beads”
usually packed in a glass tube.
a. These beads have ionic bonding sites on their surfaces
which selectively exchange ions with certain mobile
phase compositions as the mobile phase penetrates
through it.
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24. Ion exchange
Chromatography
Ions that bond to the charged site on the
resin bead are separated from organic or
inorganic ions aqueous solution.
The process is repeated several times by
changing of the mobile phase composition.
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25. Ion exchange
Chromatography
The process begin with initially running the
analysis using a mobile phase with all the ions in
the mixture.
The mobile phase is then change for several
times in a stepwise fashion so that one kind of
ion at a time is removed.
The process is repeated until complete
separation achieved.
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28. Size exclusion
Chromatography
Also called gel permeation chromatography.
Separation technique of dissolved species is
based on the size of the components.
Stationary phase: porous polymer resin
particles (molecular sieves).
The components to be separated enter the
pores of these particles and are slowed down
from progressing through this stationary
phase.
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29. Size exclusion
Chromatography
Separation depends on the sizes of the pores
relative to the sizes of the molecules to be
separated.
Small particles are retarded to a greater extent
than large particles (some of which may not
enter the pores at all) and separation occurs.
Particles with size bigger than the pore size will
be eluted first from the column.
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32. Elution
A process in which species are washed through a
chromatographic column by addition of fresh solvent.
Mobile phase
Is one that moves over or through an immobilized phase
that is fixed in place in a column or on the surface of flat
plate.
Stationary phase
A solid or liquid that is fixed in place. A mobile phase
then passes over or through the stationary phase.
Retention time
Time required for the sample to travel from the injection port
through the column to the detector.
32
33. Migration Rates of
Solutes
Distribution constant, K
Retention time, tR
Capacity factor, k’
Selectivity factor, α
33
34. Distribution Constant
In chromatography, the distribution equilibrium of
analytes between the mobile and stationary phases
can often be described quite simple.
Let say, we have analyte A. The distribution
equilibrium is written as:
Therefore, the equilibrium constant K is called
distribution constant and is defined as:
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35. Retention Time
Time required for the sample to travel from the
injection port through the column to the detector.
A
A typical chromatogram for a two-component mixture.
The small peak on the left represents a species that is not retained on the
column & so reaches the detector almost immediately after elution is
started.
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36. Dead Time, tM
Defined as time taken for the unretained
species to reach the detector.
Rate of migration of the unretained species is
SIMILAR as the average rate of motion of
mobile phase molecules.
So, tM also can be expressed as the time
required for an average molecule of the
mobile phase to pass through the
column.
36
37. Capacity factor, k’
Term used to measure the migration rates of
analytes in columns.
Also known as Retention Factor.
37
38. Selectivity factor
is defined as:
distribution constants
A measure of the relative migration rates of species A and B
with a stationary phase material in chromatography.
38
40. Column Efficiency
Two related terms widely used as quantitative
measures of chromatographic column efficiency are
A.Plate height, H
B.Number of theoretical plates, N
40
41. Column Efficiency
The relationship between H and N is given by the
formula Column length
Number of
theoretical
plates
Plate height
The efficiency of chromatographic
columns increases as the number of plates
becomes greater and plate height become
smaller.
41
42. Experimentally, H and N can be approximated
from the width of the base of the chromatographic
peak.
The equation:
N can be calculated using tR and W.
To obtain H, the length of the column must be
known. 42
43. Another method for approximating N is to
determine W1/2, the width of the peak at
half its maximum height.
2
N = 5.54 tR
W1/2
43
44. Resolution, Rs
a measure of the separation of two
chromatographic peaks.
Baseline resolution is achieved when Rs = 1.5
44
46. Rs values of less than 1.0 are considered unresolved
peaks.
46
47. Effect of Selectivity and
Capacity Factor on Resolution
Relationship btw the resolution of a column and
the capacity factor k’, selectivity factor α and the
number of plates N is given by this equation:
Rs = √N α–1 k’
4 α 1 + k’
Simplified: Rs = √ N
47
48. Effect of Resolution on
Retention Time
Relationship btw the resolution of a column and
retention time:
2 3
tR = 16Rs H
2
α 1 + k’
u α–1 (k’)2
Simplified: tR = Rs2
48
49. Example
17.63 min
Length of column:
16.40 min 30 cm
Peak widths for
1.30 min
A = 1.11 min
B = 1.21 min.
Calculate:
i) column resolution, Rs
ii) the average number of plates, N
iii) the average plate height, H
iv) length of column to achieve Rs 1.5
49
50. i)
Rs = 2 (17.63 min – 16.40 min)
(1.11 min + 1.21 min)
= 1.06
ii)
2
N = 16 16.40 min = 3.49 x 103 Therefore, calculate
the N average
1.11 min
Nave = 3.44 x 103
2
N = 16 17.63 min = 3.40 x 103
1.21 min
50
51. Variables Affecting
Column Efficiency
1. Mobile phase flow rate
2. Particle size
3. Diameter of column
4. Film thickness
51
52. Effect of mobile phase
flow
(a) refer to liquid chromatography (b) refer to gas chromatography
From both the plots for LC and GC, we can see that
both show a minimum in H at low linear flow rates.
H increases as the mobile phase flow rate
increases.
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53. Effect of particle size
Effect of particle size on plate height for a packed GC column.
The numbers to the right is the particle diameters.
The smaller the particle size, the more uniform the
column packing, then the more tolerant to the
change in mobile-phase velocity.
H increases as the particle size increases.
53
54. Effect of diameter of
the column
For packed column, the most important variable
that affect column efficiency is the diameter of the
particles that making up the packing.
While for open tubular column, the diameter of the
column itself is an important variable.
The mobile-phase mass-transfer coefficient is
known to be inversely proportional to the diffusion
coefficient of the analyte in the mobile phase.
54
55. Effect of diameter of
the column
Mass transfer coefficient is proportional to the
square of the particle diameter of the packing
material, d2p (packed column).
Mass transfer coefficient is proportional to the
square of the column diameter, d2c (open tubular
column).
As a conclusion, the bigger the column diameter,
the smaller the diffusion coefficient. Therefore, we
can say that increase in column diameter will
increase the plate height.
55
56. Effect of film thickness
When stationary phase is an immobilized liquid,
the mass-transfer coefficient is directly
proportional to the square of the thickness of the
film on the support particles d2f and inversely
proportional to the diffusion coefficient of the
solute in the film.
With thick films and smaller diffusion
coefficient, analyte molecule travel slower.
As a result, thick films will reduce the mass
transfer rate and increase in plate height.
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58. Qualitative and
quantitative analysis
25.6 min
Retention time tell as about
compound identity = qualitative
Peak Area or height tell us how
much of compound is there =
quantitative
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59. Qualitative analysis
Based on retention time
Provided the sample produce the peak at the
same retention time as a standard under
identical conditions.
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60. Quantitative analysis
1. Analysis based on Peak Height
The height of chromatographic peak is
obtained by connecting the base lines on
either side of the peak by a straight line and
measuring the perpendicular distance from
this line to the peak.
2. Analysis based on Peak Area
Peak areas are usually the preferred method
of quantitation since peak areas are
independent of broadening effects.
Most modern chromatographic instruments
are equipped with computer or digital
electronic integrator that permit precise
estimation of peak areas.
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61. 3. Calibration method (also known as external
method)
Involve preparation of series of standard
solutions that approximate the composition of
the unknown.
The peak heights or areas are plotted as a
function of concentration.
The concentration of the component(s) to be
analysed is determined by comparing the
response(s) (peak(s)) obtained with the
standard solutions.
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62. 4. Internal Standard Method
Equal amounts of an internal standard
substance is introduced into each standard and
sample.
The internal standard should not react with the
substance to be examined; it must be stable and
must not contain impurities. The retention time
must be similar to that of the substance to be
examined.
The concentration of the substance to be
examined is determined by comparing the ratio
of the peak areas (or heights) due to the
substance to be examined and the internal
standard in the test solution with the ratio of the
peak areas (or heights) due to the substance to
be examined and the internal standard in the
standard solution.
62
63. 5. Area Normalization Method
In the normalization method, the areas of all
eluted peaks is normalized.
The percentage content of one or more
components of the substance to be examined
is calculated by determining the area of the
peak(s) as a percentage of the total area of all
the peaks, excluding those due to solvents or
any added reagents and those below the
disregard limit.
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64. Tailing and fronting
A common cause of tailing and fronting is a
distribution constant that varies with
concentration.
Fronting also arises when the amount of sample
introduced onto a column is too large.
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