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Chromatography.
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CHAPTER 4CHAPTER 4(AN INTRODUCTION TO (AN INTRODUCTION TO CHROMATOGRAPHY SEPARATION)CHROMATOGRAPHY SEPARATION) 1
4.1 GENERAL DISCRIPTION OF CHROMOTOGRAPHY
Method Basis of Method
Mechanical phase separation• Precipitation and filtration• Distillation• Extraction
• Ion exchange
Difference in solubility of compounds formedDifference in volatility of compoundsDifference in solubility in two immiscible liquids
Difference in interaction of reactants with ion-
exchange resin
Chromatography Difference in rate of movement of a solute through a stationary phase
Electrophoresis Difference in migration rate of charged species in an electric field
Field-flow fractionation Difference in interaction with a field or gradient applied perpendicular to transport direction
2
4.1.1 Definition of Chromatographic Terms
a. Chromatography
» A technique
in which the components of a mixture are separated
based on differences in the rates
at which they are carried
through a fixed or stationary phase
by a gaseous or liquid mobile
phase.
b. Stationary Phase
» A solid or an immobilized liquid
on which analyte
species are
partitioned
during passage of a mobile phase.
c. Mobile Phase
» A liquid or a gas
that carries analytes
through a liquid or solid
stationary phase. 3
4.1.2
Classification of Chromatography• Chromatography methods are of two basic types :
Column Chromatography
The stationary phase
is held in
a narrow tube called column,
and the mobile phase
is forced
through the tube under pressure
or by gravity.
4
Planar Chromatography
The stationary phase
is supported on a flat plate or in the pores
of a paper. The mobile phase
moves through the stationary
phase by capillary action or under the influence of gravity.
5
• Classification of chromatography column
6
V
VV
VV
V
VV
V V
V
V
Elution
V
VV
V
V
V
XX
XA
A
A
A
A
A
X
XX
X
X
X A
AA
A
A A
X
X
XX
XX
A
A
AA
A
A
Injection
Interaction
7
4.1.3 Elution of Chromatography4.1.3 (a) Terms • Elution :
A process in which solutes are washed through a stationary
phase
by the movement of a mobile phase.
• Eluate :
The mobile phase
that exits the column.
• Eluent :
A fresh solvent
used to carry the components of a mixture
through a stationary phase.
4.1.3 (b) Mechanism• Initially, a solution of a mixture of A and B in the mobile phase is
introduced at the head of the column at time to
.
• The two components separate themselves between the mobile
phase and stationary phase.8
• Elution then occurs
by forcing the sample components through the
column by continuously adding fresh mobile phase.
• With the first introduction of fresh mobile fresh, the eluent
moves
down the column, where further partitioning between the mobile
phase and the stationary phase takes occurs (time t1
).
• Further additions of solvent carry solute molecules down
the
column in a continuous series of transfers between the two phases.
• Because solute movement can occur only in the mobile phase, the
average rate at which a solute migrates depends on the fraction of
time it spends in that phase.
9
• This fraction is small
for solutes that are strongly retained
by the
stationary phase (component B), and large
when retention in the
mobile phase is more likely
(component A).
• The resulting differences in rates
cause the components in a
mixture into bands or zones
along the length of the column.
• Isolation of the separated species
is then accomplished by passing a
sufficient quantity of mobile phase
through the column to cause the
individual bands to pass out the end, where they can be collected or
detected
(times t3
and t4
).
10
a)
Diagram showing the separation of a mixture of components A and B by elution chromatography
b)
The detector signal at the various stages of elution shown in a
11
12
4.1.3 (c) Chromatograms• If a detector that responds to solute concentration is placed at the
end of the column during elution and its signal is plotted as a
function of time
(or of volume of added mobile phase), a series of
peaks is obtained.
• Such a plot is called a chromatograms, which is useful for both
qualitative and quantitative analysis.
• The positions of the peaks on the time axis
can be used to identify
the components of the sample.
13
14S
S
• On a chromatogram, the perfect elution
would have the same as the
graphical representation
of the law of Normal distribution of
random errors a (Gaussian curve).
• Gaussian Distribution
: a theoretical bell-shaped distribution
of
results obtained for replicate measurements that are affected by
random errors.
• In keeping the classical notation, μ, would correspond to the
retention time of the eluting peak while σ
to the standard deviation of
the peak ( represents the variance
σ2). y
represents the signal as a
function of time, x, from the detector located at the outlet of the
column.
15
4.1.4 Methods of Improving Column Performance•
By changing experimental conditions, non-separated bands can be separated : (a) Adjust migration rates for A and B
(increase band separation)(b) Adjust zone broadening
(decrease band spread)
16
4.2 MIGRATION RATES OF SOLUTE•
The effectiveness of a chromatographic column in separating two
solutes
depends on the relative rates at which the two species are
eluted.
•
These rates in turned are determined by the ratios of the solute
concentrations in each of the two phases.
•
Other factors:
Distribution constant
Retention times
Retention factor
Selectivity factor
17
4.2.1 Distribution Constant• Distribution constant for a solute in chromatography is equal to the
ratio of its molar concentration in the stationary phase (cs) to its
molar concentration in the mobile phase (cm).
• Analyte
A in equilibrium with two phases :
where K
is distribution constant
• K
is constant over a wide range
of solute concentration; that is Cs
is
directly proportional to Cm. 18
mobile stationary
stationary
mobile
A A
CK
C
↔
=
4.2.2 Retention Time• In chromatograms, the small peak
on the left is for a species that is
not retained by the stationary phase.
• Time tM
between sample injection and the appearance
of this peak is
called dead time or void time.
• Dead time/ void time
: time it takes for an unretained
species to pass
through a chromatographic column.
19
• The larger peak
on the right is that of an analyte
species.
• The time required for this zone to reach the detector after sample
injection is called the retention time, tR
.
• The analyte
has been retained because it spends a time tS
in the
stationary phase.
The retention time is then :
• Retention time :
is the time between injection of a sample and the
appearance
of a solute peak at the detector of a chromatographic
column In chromatograms.
20
R M St t t= +
• Average linear rate of solute migration :
where L
= length of column packing
• Average linear rate of mobile-phase migration :
R
Lt
υ =
M
Lut
=
21
4.2.3 Retention Factor• The retention factor is an important experimental parameter that is
widely used to compare the migration rates of solutes on columns.
• Retention factor for a solute A:
• Retention factor
: Retention factor kA
for a solute A is related to the
rate at which A migrates through a column. It is the amount of time a
solute spends in the stationary phase relative to the time it spends
in the mobile phase.
M
S
M
MRA t
ttttk =
−=
22
•
Value of
kA
:
when kA is ≤ 1.0 » separation is poor
when kA is > 30 » separation is slow
when kA is = 2 – 10 » separation is optimum
4.2.4 Selectivity Factor•
Selectivity factor :
the ratio of the distribution constant of the more
strongly retained solute
(B) to the distribution constant for the
less strongly held solute
(A).
•
Selectivity factor of a column for the two solutes is defined as
:
A
B
KK
=α23
where KB
is the distribution constant for the more strongly retained
species B.
KA
is the constant for the less strongly held or more rapidly
eluted species A.
•
α
is always greater than unity
and larger α
»
better separation
•
Selectivity factor can also be described with retention factor.
:
where kB
and kA
are the retention factors for B and A.
A
B
kk
=α
24
4.3 THE EFFICIENCY OF CHROMATOGRAPHIC COLUMN
•
The efficiency of a chromatographic column
is affected by the amount of band broadening
that occurs as a compound passes through the column.
25
4.3.1 Quantitative Description of Column Efficiency•
Two related terms are widely used as quantitative measures of chromatographic column efficiency :
Plate height HPlate count or number of theoretical plates N
•
The two are related by the equation :
where L
is length (in cm) of column packing.
•
The efficiency of chromatographic columns
increases
as the plate
count N becomes greater
and as the plate H becomes smaller.
HLN =
26
•
Thus, H is needed to be as small as possible »
more plates can be fitted into a column.
27
HLN =
5 Plates
10 Plates(Larger H) (Smaller H)“Thicker”
“Thinner”
L, columnlength
Plate
↓
H ↑ No of Plate ↑ Efficiency
•
Because chromatographic bands are usually Gaussian
and because
the efficiency of a column is reflected in the breadth of
chromatographic peaks, the variance per unit length of column is
used by chromatographers as a measure of column efficiency.
•
That is the column efficiency
is defined by :
•
Efficient column has small plate height
»
less zone broadening.
•
Thus, unit H is in cm.
LH
2σ=
28
(a)
Gaussian distribution of sample molecules
(b)
Column length as the distance from the sample entrance point to the detector 29
•
In simple relationship, N can be approximated from the width, W (unit in time) of the base of the chromatographic peak :
30
2
16 R
B
tNW
⎛ ⎞= ⎜ ⎟
⎝ ⎠
4.3.2 Other Variables That Affect Columns Efficiency•
Band broadening reflects a loss of column efficiency.
•
The slower the rate of mass transfer processes occurring while a
solute migrates through a column, the broader the band at the column exit.
•
Some of the variables that affect mass-transfer rates are controllable can improve the separation.
4.3.2 (a)
The Effect of Mobile-Phase Flow Rate
•
Higher mobile phase velocity, less time in column »
less zone broadening.
•
However, plate height, H, can changes with flow rate. The plot of H versus u
»
Van Deemter
plot. 31
where
H is the plate height, u
is the average linear velocity of the
mobile phase, and A, B, and C
are (positive) constants determined
by various physical properties of the mobile and stationary phases.
CuuBAH ++= CuuBAH ++=
CuuBAH ++=
32
33
34
i. Multipath
Term/ Eddy Diffusion (A)•
Eddy diffusion, which is caused by non-uniform packing of chromatographic columns.
35
•
Molecules moves through different paths.
•
Larger difference in pathlengths
for a larger particles.
•
At low flow rates, diffusion allows particles to switch between paths
quickly and reduces variation in transit time.
36
ii. Longitudinal Diffusion Term (B)
37
•
Longitudinal diffusion, which is caused by the tendency of a solute species to diffuse from regions of high concentration
(such as the center of a chromatographic band) to regions of low concentration(such as the leading or trailing edge of a chromatographic band).
•
Proportional to mobile phase diffusion coefficient.•
Inversely proportional to flow rate
»
high flow rate, less time for diffusion.
38
iii. Mass Transfer Coefficients (C)
39
•
Mass transfer in the form of partitioning
of the solute into the stationary phase, which does not occur instantaneously and depends on the solute’s partition and diffusion coefficients.
•
All species need a certain amount of time to equilibrate between
the stationary and mobile phases.
•
High flow rates » species with strong affinity for stationary phase will be slowed
and the molecules with no affinity to the stationary phase move ahead which causes band broadening.
• Cs
is rate for adsorption onto stationary phase
• CM
is rate for analyte
to desorb
from stationary phase.
•
Effect proportional to flow rate
−
at high flowrate
» less time to approach equilibrium.
( )uCCuBAH MS +++=
( )uCCuBAH MS +++=
4.4 COLUMN RESOLUTION•
The resolution Rs
of a column »
how far apart two bands are relative to their widths.
•
The resolution provides a quantitative measure of the ability of the
column to separate two analytes.
•
The chromatogram consists species A and B on three columns with
different resolving powers.
•
Resolution :
0.75 = no separation occur
1.0 = zone A contain ~ 4% B and zone B contain ~ 4% A
1.5 = complete separation of A and B (overlap 0.3%)
•
The resolution for a given stationary can be improved by lengthening column »increasing the no of plates.
41
•
42
( )( ) ( )
2
2
SA B
R B R A
A B
ZRW W
t tW W
Δ=
+
−=
+
4.4.1 Effect of Retention and Selectivity Factors on Resolution
•
Resolution equation
of a column that relates to the
no of plates with the retention time and selectivity
factors of a pair of solutes
on the column :
where kB
= retention factor of the slower moving
species and α
= the selectivity factor
⎟⎟⎠
⎞⎜⎜⎝
⎛+
⎟⎠⎞
⎜⎝⎛ −
=B
Bs k
kNR1
14 α
α
43
4.4.2 Effect of Resolution on Retention Time•
The goal of chromatography
is the highest possible resolution in the shortest possible elapsed time.
•
Unfortunately, these goals tend to be incompatible, and a
compromise between the two usually necessary.
•
The time (tR
)B
required to elute the two species with a resolution Rs
is given by:
where u
is the linear velocity of the mobile phase
•
The resolution will increase
if the u
is low.
( ) ( )( )2
322 11
16
B
BSBR k
kuHRt +
⎟⎠⎞
⎜⎝⎛
−=
αα
44
4.5 APPLICATION OF CHROMATGOGRAPHY•
Gas Chromatography (GC)•
High Liquid Performance Chromatography (HPLC)
45