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PartII

Chromatographic
Theory
O Partitionbetweenmobilephase/stationaryphase:
S
m
S
s

S
m
:thesoluteinthemobilephase
S
s
:thesoluteinthestationaryphase.

SeparationprocessElution
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O Twoapproachesexplaintheseparationprocess:

PlatetheoryMartinandSynge(1941),basedonan
analogywithdistillationandcountercurrent
extraction.
RatetheoryvanDeemter(1956),accountsforthe
dynamicsofaseparation.

o Advantages
o Limitations
Theories
O partition coefficient (equilibrium constant), K

K =
Solutc
mobilc

pbosc
Solutc
stotionory

pbosc


K is assumed to be independent of concentration.
SoluteRetentionFactor
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O Capacity ratio, k

k = K
I
s
I
m

k =
t
R
-tM
t
M
=
t
|
R
t
M


t
R
- the adjusted retention time

SoluteRetentionFactor
Retentiontime
O retention time (t
R
): time between sample
injection and an analyte peak reaching a
detector at the end of the column
O The time taken for the mobile phase to pass
through the column is called t
M
.

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O Solute fractions
mobilc pbosc, m =
1
1 + k


stotionory pbosc, s =
k
1 + k


If constant mobile phase velocity:
m
=
t
m
t
R


SoluteRetentionFactor
Terms: retention factor, k, the capacity factor, the capacity ratio, and
the partition ratio, and is sometimes given the symbol k.
Example1
In a chromatographic analysis of low molecular weight acids,
butyric acid elutes with a retention time of 7.63 min. The
columns void time is 0.31 min. Calculate the retention factor
for butyric acid.

Solution

K
butyric
= (t
r
t
m
) / t
m
= (7.63 min 0.31 min) / 0.31 min = 23.6
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Chromatogram for a two-component mixture. Determine the
retention factor for each solute, sample injected time t = 0.

Relationship between
elution time and distance
is proportional, we can
measure t
m
, t
r,1
, and t
r,2

using a ruler.
The measurements are
7.8 mm, 40.2 mm, and
51.5 mm, respectively.
Solution: The retention factors for solute A and solute B are:
k
1
= (t
r
t
m
) / t
m
= (40.2 mm 7.8 mm) / 7.8 mm = 4.15
k
2
=(t
r
t
m
) / t
m
= (51.5 mm 7.8 mm) / 7.8 mm = 5.60
Example2
Retentiontimeandvolume
O Retention volume, V
R
volume of mobile
phase required to elute a solute to a maximum
from a column.
O Retention time, t
R
time required to reach the
same maximum at constant flow.
I
R
= tR low rotc
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Retentiontimeandvolume
O For a constant column length:

t
R
t
m
=
F
R
F
m
= 1 +k

O Retention time for mobile phase, t
M

t
R
= tH(1 +k)
Componentseparation
O If solutes 1 and 2 have capacity factor of k
1
and k
2
then
their retention times, t
R1
and t
R2
:

t
R1
= tM 1 +k
1

t
R2
= tM(1 +k
2
)

O Peak separation:

t
R2
-
t
R1
=
tH(k
2
-k
1
)
t
R2
> t
R1
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O selectivity factor,
the separation of two species (A and B) on the
column;



species A elutes faster than species B: >1.

Selectivityfactor
Example3
In the chromatographic analysis for low molecular
weight acids, the columns void time is 0.31 min. The
retention time for isobutyric acid is 5.98 min. What is
the selectivity factor for isobutyric acid and butyric acid
(retention time of 7.63 min)?

Solution

Calculate the retention factor for isobutyric acid and butyric acid.
k
iso
= (t
r
t
m
) / t
m
= (5.98 min 0.31 min) / 0.31 min = 18.3

The selectivity factor, therefore, is
= k
but
/ k
iso
=23.6 / 18.3 = 1.29
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Example4
O Determine the selectivity factor
The selectivity
factor is
= k
2
/ k
1

= 5.60 / 4.15
= 1.35
ColumnEfficiency
Band broadening
Sharp, symmetrical chromatographic peaks
Efficiency of the column
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PlatetheoryofChromatography
O A chromatographic column is mathematically
divided into theoretical plates (N).
O There is an equilibrium partitioning of the
solute between the stationary phase and the
mobile phase.

O The analyte moves down the column by transfer
of equilibrated mobile phase from one plate to
the next.
N = L / H
Column efficiency in terms of the number of theoretical
plates, N,


L - columns length; H plate height.

Note: more theoretical plates (the better) Column efficiency
improves chromatographic peaks become narrower.
Height Equivalent to a Theoretical Plate

HETP = L / N

the smaller the better.
DeterminationofN
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The solutes average linear velocity is the distance it travels, L,
divided by its retention time, t
r
.
If peak broadening: H is the variance per unit length of the
column (.



- standard deviation, sec or min,
Time of elution, T ( /solutes average linear velocity, ).
H =
2
/ L
= / = t
r
/ L
DeterminationofN
w = 4
H = Lw
2
/ 16t
r
2

For a Gaussian peak shape, the width at the
baseline, w, is four times its standard deviation, .


Height of a theoretical plate in terms of t
r
and w:
N = 16(t
r
2
/ w
2
)
Number of theoretical plates:
DeterminationofN
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Since it is difficult to accurately measure the beginning
and end of a peak, it is common to use the width at half
height and assume the peak is Gaussian.
DeterminationofN
Example5
A chromatographic analysis for the chlorinated pesticide Dieldrin
gives a peak with a retention time of 8.68 min and a baseline
width of 0.29 min. What is the number of theoretical plates?
Given that the column is 2.0 m long, what is the height of a
theoretical plate in mm?

Solution

The number of theoretical plates:
N = 16 t
r
2
/w
2
= N = 16(8.68 min)
2
/ (0.29 min)
2
= 14300 plates

The average height of a theoretical plate:

H = L / N = (2.0 m / 14300 plates) (1000 mm / m) = 0.14
mm/plate
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Resolution
O Resolution, R
s
, how well two neighboring peaks
completely separated from each other.
O The resolution of two peaks A and B:




O controlling the capacity factor, k, separations
greatly improved:
O by changing the temperature (in GC) or the
composition of the mobile phase (in LC).

Resolution
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Example6
In a chromatographic analysis of lemon oil a peak for
limonene has a retention time of 8.36 min with a baseline
width of 0.96 min. -Terpinene elutes at 9.54 min with a
baseline width of 0.64 min. What is the resolution between
the two peaks? Please give your comments of the results.

Solution

The resolution is
R
AB
= 2t
r
/ (w
B
+ w
A
) = 2(9.54 min 8.36 min) / (1.64 min +
0.96 min) = 1.48
Example7

O This Figure shows the
separation of a two
component mixture. What is
the resolution between the
two components? Use a
ruler to measure t
r
, w
A
, and
w
B
in millimeters.

Measurements are 8.5 mm for t
r
,
and 12.0 mm each for w
A
and w
B

Using these values, the resolution is
R
AB
= 2t
r
/ (w
A
+ w
B
) = 2(8.5 mm) / (12.0 mm + 12.0 mm) = 0.70
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PeakCapacity
n
c
= 1 + ((N)/ 4)ln(V
max
/ V
min
)
o A measure of the number of solutes that can
be separated, n
c
.
o Based on isothermal/isocratic conditions.



n
c
- columns peak capacity
V
min
and V
max
- the smallest and the largest
volumes of mobile phase in which we can elute
and detect a solute.
Example8
O A column with 10 000 theoretical plates can resolve no
more than:
n
c
= 1 + ((10000) / 4)ln(30 mL / 1 mL)
= 86 solutes
assumed V
min
=1 mL and V
max
= 30 mL.

consideration a column enough theoretical plates to
separate a complex mixture.

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AsymmetricPeaks
O Poisson distribution
O Gaussian distribution
Optimizing Chromatographic
Separations
R
AB
= (t
r,B
t
r,A
) / (0.5(w
B
+w
A
)) (t
r,B
t
r,A
)/(0.5(2w
B
)) = (t
r,B
t
r,A
)/w
B

Define the effects of
solute retention factor
selectivity,
column efficiency
resolution of two closely eluting peaks.

If the two peaks (A and B) have similar retention times, it is
reasonable to assume that their peak widths are nearly
identical:



R
AB
= ((N) / 4) (t
r,B
t
r,A
) / t
r,B

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R
AB
= ((N) / 4) ((1) / ) (k
B
/ (1 + k
B
))
Reretention times of solutes A and B.
t
r,A
= k
A
t
M
+ t
M
t
r,B
= k
B
t
M
+ t
M


R
AB
= ((N) / 4) ((k
B
- k
A
) / (1 + k
B
))

t
r,B
= (16R
AB
2
H / u) ( / ( 1))
2
((1 + k
B
)
3
/ k
B
2
)
u - mobile phases velocity
Optimizing Chromatographic
Separations
UsingtheRetentionfactorto
OptimizeResolution
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UsingSelectivityto
OptimizeResolution
UsingColumnEfficiencyto
OptimizeResolution
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RateTheoryofChromatography
O Plate theory neglects the concepts of solute diffusion and
flow paths.
O Rate theory accounts for these and can be used to predict
the effect on column performance factors such as:
O Phase properties
O Phase thickness
O Solute diffusivities
O Support size
O Partition coefficients
O Support porosity
O Phase velocity
O Flow rates
RateTheoryofChromatography
O Rate theory (inside a column): time for the solute to
equilibrate between the stationary and mobile phase.
O band shape of chromatographic peak is affected by:
O the rate of elution
O the different paths available to solute molecules as they
travel between particles of stationary phase.
O band broadening:
O variations in paths length
O longitudinal diffusion
O mass transfer in the stationary phase, and
O mass transfer in the mobile phase.


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Equationofparameters
H = H
p
+ H
d
+ H
s
+ H
m

The height of a theoretical plate is a summation
of the contributions:



An alternative form of this equation is the van
Deemter equation, accounting for the dynamics of
the separation process.

VanDeemterequation
factor characteristic of packing
d
P
particle diameter
factor for irregularity of interparticle spaces
D
g
diffusion coefficient of compound in gas
D
l
diffusion coefficient of compound in liquid
u linear gas velocity
k capacity ratio
d
f
liquid phase effective film thickness
H height of theoretical plate
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VanDeemterequation
The equation consists of three basic terms.

Packing related term


Gas (mobile phase) term


Liquid (stationary phase) term
O A - Eddy diffusion/multipath
O B - Longitudinal diffusion/molecular diffusion
O C - Resistance to mass transfer
HETP = A + B / u + C u

u - average velocity of the mobile phase.
VanDeemterequation
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Plot showing the relationship between the height of a theoretical plate, H,
and the mobile phases velocity, u, based on the van Deemter equation.
VanDeemterplots
Otherequations
H = (B / u)+ (C
s
+ C
m
)u
C
s
and C
m
- mass transfer for the stationary phase
and the mobile phase

H = Au
1/3
+ (B / u) + Cu