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
2
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
3
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
4
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
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
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
11
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:
13
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
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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Editor's Notes
Overview of what a GC can do. ~ microliter injection volume is example only - sould not be construed as definitive