introduction and detail theory about chromatography

1. Introduction to and Theory of
Chromatographic
Separations
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2. What is Chromatography?
 Chromatography is the separation of an
analyte from a complicated mixture of
similar constituents for qualitative or
quantitative identification
Applies to both organic and inorganic
compounds
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3. Basic Separation Principles and
Terms
 Compounds (analytes) are separated from a
mixture by passing them through a stationary
phase using a mobile phase carrier
mobile phase = solvent
stationary phase = column packing material
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4. Basic Separation Principles and
Terms
 Separations occur because analytes in the
mobile phase interact with the stationary
phase at different rates
Produces varying migration rates for analytes
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5. Basic
Separation
Principles
 Example: Column
elution
chromatography
 Introduce two solutes
(A & B) onto a
packed column
through which a mobil
phase (i.e. solvent) or
eluent is continuously
pumped
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6. Basic Separation Principles
 Separations depend on the extent to which
solutes partition between the mobile and
stationary phases
 We define this interaction with a partition
coefficient:
K = cs/cm
Where:
cs = stationary phase concentration (molar)
cm = mobile phase concentration (molar)
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7. Types of Chromatography
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Chromatography
Planar Column
Liquid Gas Supercritical Fluid
Partition
Bonded Phase
Adsorption
Ion Exchange
Size Exclusion
Gas - Liquid
Gas – Bonded Phase
Gas - Solid

8. Classification of Column
Chromatography Methods
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9. The Chromatogram
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Sample Mixture
Chromatogram
0 5 10 15 20
Time (minutes)
Abundance
A
B
C
D
E
Chromatograph
The time that a peak
appears (it’s retention
time) is diagnostic for a
given compound
The relative size of a
peak (area or height)
is proportional to the
relative abundance of
the compound in the
mixture

10. Chromatography Terms
 A number of terms can be defined from
the following chromatogram
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11. Chromatography Terms
 Retention Time (tR) = the time it takes after
injection for a solute to reach the detector
 Dead time (tM) = the time for an unretained
species to reach the detector
 Mobile phase velocity (u) = L/tM; the average
linear rate of movement of molecules in the
mobile phase
L = length of chromatographic column
 Linear Rate of solute migration ( ) = L/tR
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v

12. Chromatography Terms
 Retention Factor or Capacity Factor (k’A) = a
term used to describe the migration rate of
solutes (analytes) on columns
Where:
KA = is the distribution constant for species A
VS = volume of stationary phase
VM = volume of the mobile phase
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M
SA
A
V
VK
k ='

13. Chromatography Terms
 The retention factor can be determined
directly from a chromatogram using:
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M
MR
A
t
tt
k
−
='

14. Chromatography Terms
 Interpreting the retention factor
If k’A < 1; the elution is too rapid for accurate
determination of tR.
If k’A > approx. 10; the elution is too slow to
be practical
The preferred range for k’A is between 1 and
5
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15. Chromatography Terms
 Selectivity Factor (α) = KB /KA
Where KB is the distribution constant of the
more strongly retained species (so that
α >1)
 The selectivity factor can also be
defined in terms of retention factors and
retention times:
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MAR
MBR
A
B
tt
tt
k
k
−
−
==
)(
)(
'
'
α

16. Chromatography Terms
 Zone Broadening or Band Broadening
Refers to the shape of a peak as a solute
migrates through a chromatographic column; it
will “spread out” and “shorten in height” with
distance migrated
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17. Chromatography Terms
 Plate Height (H) and Theoretical Plates (N)
Terms used to quantitatively describe chromatographic
column efficiency
Column “efficiency” increases as N increases
N = L/H
Where:
N = the number of interactions (i.e. transitions between mobile and
stationary phases) that a solute has during its residence in the
column
H= the distance through the column a solute travels between
interactions (typically given in centimeters)
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18. Chromatography Terms
 Plate Theory – used to describe solute migration
through a column and the Gaussian shape of a
peak
If the shape of a chromatographic peak is assumed to be
Gaussian, then the plate height can be defined in
statistical terms involving standard deviation (σ)
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L
H
2
σ
=

19. Chromatography Terms
 Plate Theory (cont.)
H is defined as variance per unit length of
column
H is the length of column that contains the
fraction of analyte between L and L – σ
This is 34% of the analyte (1/2 of 1 std.
dev.)
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20. Calculation of N From a
Chromatogram
Where:
W = magnitude of the
base of the triangle
(in units of time)
tM = retention time of an
unretained solute
tR = retention time of the
solute
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2
16 





=
W
t
N R

21. Chromatography Terms
 Rate Theory (or Kinetic Theory) – used to explain (in
quantitative terms) the shapes of chromatographic
peaks and the effects of chromatographic variables
on peaks as a solute migrates through a column
The Gaussian shape of an peak can be attributed to the
additive combination of the random motions of individual
solute molecules
The migration of an individual molecule through a column
is irregular (based on “random walk” mechanism)
The time an individual molecule spends in a phase
depends upon (accidentally) gaining sufficient thermal
energy to change phases
Results in a symmetric spread of velocities around the
mean
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22. Chromatography Terms
 Column Resolution (RS)
is a quantitative measure
of the ability of a column
to separate two analytes:
 To increase resolution
increase column length
(L)
 Limitation: longer time and
broader bands
 Usually compromise
between resolution and
speed of analysis
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BA
ARBR
S
WW
tt
R
+
−
=
])()[(2

23. Summary of Important Terms
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24. Summary of Important Terms
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25. Variables Effecting Separation Efficiency in
Column Chromatography
1. Particle Size of Packing (as size ↓, N↑ and H↓)
2. Immobilized Film Thickness (as film thickness ↓, N↑ and
H↓) - due to faster diffusion rates in film
3. Viscosity of Mobil Phase (as viscosity ↓, N↑ and H↓)
4. Temperature (as T ↑, k' ↑, but α remains approximately
constant)
5. Linear Velocity of Mobil Phase; u = L/tM
(u is proportional to
1/tM
because L = constant)
6. Column Length (as L ↑, N↑, but H = constant, and
separation efficiency ↑)
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In general, Separation (or Column) EfficiencyIn general, Separation (or Column) Efficiency ↑↑ , as, as
NN ↑↑ and Hand H ↓↓

26. Variables Effecting Column Efficiency
 Mobile Phase Flow Rate
Optimum flow rate
corresponds to minimum H
H is much smaller for
HPLC than for GC (more
efficient), but in practice
GC columns are much
longer than for HPLC -
hence greater N for GC.
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Liquid Chromatography
Gas Chromatography

27. Variables Effecting Column Efficiency
 Particle Size of Column Packing
The smaller the packing particles, the greater the
column efficiency.
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28. The General Elution Problem
 It is often difficult to
find a set of
conditions which will
resolve all peaks to
the same degree and
also permit reliable
quantification
 Solutions
Multiple runs at
different conditions
Programmed elution
(i.e. change conditions
during the run)
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