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Journal of Chromatography A, 1331 (2014) 5260

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Molecular theory of size exclusion chromatography for wide


pore size distributions
Annamria Sepsey a , Ivett Bacskay b , Attila Felinger a,b,
a
b

MTAPTE Molecular Interactions in Separation Science Research Group, Ifjsg tja 6, H-7624 Pcs, Hungary
Department of Analytical and Environmental Chemistry and Szentgothai Research Center, University of Pcs, Ifjsg tja 6, H-7624 Pcs, Hungary

a r t i c l e

i n f o

Article history:
Received 14 November 2013
Received in revised form 7 January 2014
Accepted 9 January 2014
Available online 16 January 2014
Keywords:
Pore size distribution
Stochastic theory
Size exclusion chromatography

a b s t r a c t
Chromatographic processes can conveniently be modeled at a microscopic level using the molecular
theory of chromatography. This molecular or microscopic theory is completely general; therefore it can
be used for any chromatographic process such as adsorption, partition, ion-exchange or size exclusion
chromatography. The molecular theory of chromatography allows taking into account the kinetics of the
pore ingress and egress processes, the heterogeneity of the pore sizes and polymer polydispersion. In
this work, we assume that the pore size in the stationary phase of chromatographic columns is governed
by a wide lognormal distribution. This property is integrated into the molecular model of size exclusion
chromatography and the moments of the elution proles were calculated for several kinds of pore structure. Our results demonstrate that wide pore size distributions have strong inuence on the retention
properties (retention time, peak width, and peak shape) of macromolecules. The novel model allows us
to estimate the real pore size distribution of commonly used HPLC stationary phases, and the effect of
this distribution on the size exclusion process.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Modern porous or coreshell stationary phases may exhibit a
momentous pore size distribution. Experimental data conrm that
the size of mesopores can cover a rather wide range [1,2]. The
nature and the breadth of pore size distributions have signicant
impact on the mass-transfer properties of stationary phases. The
separation of macromolecules is particularly inuenced by the pore
size distribution, since their hindered diffusion in the pore network
gives a critical contribution to band broadening.
Size exclusion chromatography (SEC) is one of the most widely
used techniques to determine the molecular size distribution of
polymers of any kind. The separation mechanism relies on the size
and shape of sample molecules relative to the size and shape of the
pores in the stationary phase particles. Because we do not exactly
know the structure and the dimensions of the porous media, the
determination of the molecular mass relies on a calibration step
based on the behavior of well-known monodisperse polymers in
the columns containing porous stationary phase particles.
The study of the pore structure of the stationary phases
used in liquid chromatography has been of great interest among

Corresponding author at: Department of Analytical and Environmental Chemistry and Szentgothai Research Center, University of Pcs, Ifjsg tja 6, H-7624
Pcs, Hungary. Tel.: +36 72 501500x24582; fax: +36 72 501518.
E-mail address: felinger@ttk.pte.hu (A. Felinger).
0021-9673/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2014.01.017

chromatographers in the last decades [36]. Kubn has modeled


pore size irregularity with a diffusion model, assuming that
molecules can penetrate into the porous particles to a distance that
depends on the size of the molecules [6].
There are a number of methods for determining relevant information about the porous media such as low-temperature nitrogen
adsorption, mercury intrusion, microscopy and solute exclusion.
These techniques are either too expensive and/or they destroy the
chromatographic column (so they cannot be used for any further
analysis), or they do not give relevant information about all ne
details.
The inuence of pore size distribution on separation efciency
can conveniently be studied with inverse size exclusion chromatography. Inverse size exclusion chromatography is used to derive
information about the structure of the pores of the packing material
from the retention data of a series of known analytes, for instance,
polymers of narrow molecular mass distribution and known average molecular mass [7].
In this study, we develop a model that integrates the pore size
distribution into the microscopic theory of size exclusion chromatography. With this model one is able to determine the inuence
of the breadth of pore size distribution on retention properties and
efciency.
The molecular, or stochastic theory of chromatography is a
microscopic model introduced by Giddings and Eyring in 1955
[8]. That theory uses random variables and probabilistic terms to
describe the migration of the molecules along the chromatographic

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260

column. The stochastic approaches may become very complex to


work with for situations except the most simple case of adsorption,
so the use of this theory was not convenient until the characteristic
function (CF) approach was introduced to this eld [9]. The use of
characteristic functions made the stochastic theory of chromatography simpler even for complicated cases such as heterogeneous
adsorption chromatography.
The stochastic theory seems rather suitable for describing size
exclusion chromatography among the basic theories of chromatography. Size exclusion chromatography is indeed based on the
random migration of molecules, where randomly occurring entrapment and release of molecules in the mesopores of the stationary
phase builds up the separation process [1013].

2. Theory
2.1. Classical size exclusion considerations
Size exclusion chromatography (SEC) has been investigated
from many point of views, which led to an asystematic nomenclature. To eliminate the misapprehensions, all techniques that are
founded on the size exclusion separation mechanism, such as gel
ltration, molecular sieve chromatography, gel chromatography
and gel-permeation, are parts of SEC. However the only difference
between the individual ones is in the samples and solutions used.
Size exclusion chromatography is a well-known separation
method where the main retention mechanism is the size exclusion
effect. However there are a lot of other mechanisms responsible for the migration of the molecules along the column, such as
hydrodynamic and stress-induced diffusion, the polarization effect,
multipath, enthalpic and soft-body interactions and the so-called
wall effect, they can be ignored in almost every case. In an ideal case
there is no interaction between the molecules and the stationary
phase particles or the mobile phase particles.
In SEC, the sample components are separated according to their
size and molecular mass. The separation is governed by entropy
only; the retention depends on the relative penetration of the sample molecules to the pores. The stationary phase of a SEC column
is always a mechanically stable porous media that can be built up
on a rigid carrier such as silica or the whole stationary phase is
made from this porous material. The molecules traveling along the
column in the mobile phase can enter the pores if the size of the
pore is larger than that of the dimensions of the molecule. However it is still uncertain which exact size parameter determines the
separation [14]. In general it is accepted that the gyration radius
or diameter of the molecule is used to determine whether or not
the molecule can enter the pore. The mobile phase in the pores
is stagnant; the molecules can migrate in the pores only by diffusion.
Two molecule sizes have special signicance in SEC: the size
of the completely permeable particle and the size of the barely
excluded particle. The completely permeable particle (indicated by
a subscript perm in the equations) is small enough to visit all the
pores so that both the stagnant mobile phase in the pores of the stationary phase and the moving zone of the mobile phase between
the stationary phase particles is completely accessible for it. If the
molecule is too large to enter the pores, it will be excluded (indicated by a subscript excl in the equations). These molecules can
only wander in the moving mobile phase and have access only to
the interstitial volume of the mobile phase between the stationary
phase particles. The excluded molecules elute at the void volume
(V0 ). According to this mechanism, we can obtain information of
the size, shape, aggregation state or kinetics of the ligandpolymer
binding of the molecules investigated.

53

The partition coefcient can easily be calculated by means of the


retention times of the above mentioned and the unknown particles
using the following equation:
KSEC =

t texcl
,
tperm texcl

(1)

where the numerator stands for time spent by the investigated


molecule in the pores of the stationary phase particles and the
denominator indicates the residence time spent by the completely
permeable particle in the pores. The partition coefcient can be
rewritten as
tp
KSEC =
(2)
tp,perm
where the subscript p indicates the time spent in the pores by the
molecules.
The partition coefcient strongly depends on the ratio of the
size of the migrating particle to the size of the pore. The partition
coefcient is in a widely used retention model [10,11,15] dened
using the size of the molecule investigated and the size of the pore
of the stationary phase used as follows:

KSEC =

(1 )m

if 0  1

if  > 1

(3)

where m is a constant whose value depends on the pore shape,


and  is the size of the molecule relative to the pore size. The size
parameter  can be dened as
=

rG
rp

(4)

where rG stands for the gyration radius of the molecule while rp


indicates the radius of the pore opening. The retention depends on
the hydrodynamic radius or the gyration radius of the molecules,
which can be changed by the hydration state and the shape.
2.2. Stochastic theory of SEC
The stochastic theory of chromatography, in which the chromatographic process is modeled at a molecular level was developed
in 1955 by Giddings and Eyring for adsorption chromatography
[8]. The theory assumes that, if we ignore the axial dispersion,
the number of adsorption and desorption steps is determined by
a Poisson process and the time that a molecule spends bound to
the stationary phase (residence or sojourn time) is determined by
an exponential distribution. The stochastic theory is completely
independent of the physicalchemical mechanisms responsible for
the retention; therefore it can be used in any eld of chromatography. Accordingly, the model has been extended and improved
for several chromatographic methods such as adsorption, partition, ion-exchange or size exclusion chromatography (adapted to
SEC by Carmichael [1619]). The real breakthrough in developing the theory was the introduction of the characteristic function
approach. Later the effects of the mobile phase dispersion were
introduced (stochastic-dispersive model) and they involved an
increasing number of parameters specied in the description of the
system. A detailed description of the stochastic theory of SEC via the
characteristic function method was introduced by Dondi et al. [10].
The simplest theory of size exclusion chromatography assumes
that a molecule of a certain size enters and leaves the pores n times
on average during the migration along the column and spends  p
time on average in a single pore. After leaving a pore, the molecule
spends  m time on average in the mobile phase before entering
another pore. All these variables are random quantities, therefore
each molecule has an individual path while migrating along the
column. However, the molecules of the same size behave in a similar manner, because of the anomalies in the retention paths of the

54

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260

molecules we observe a nearly Gaussian curve as chromatographic


peak. The observed retention time of a peak is the mean of the distribution of the individual retention times and the width of the peak
is described by the standard deviation.
The average number of the pore ingress steps (if  takes a value
between 0 and 1) can be written as
np = nperm (1 )me ,

(5)

and the residence time in the pores will be:


p = perm (1 )mp ,

(6)

where me and mp are constants depending on the ingress and the


egress processes, respectively. Both np and  p take the value 0 if
 > 1.
As Dondi et al. showed earlier [10], parameters me , mp and m
are related as
m = me + mp

(7)

Thus it can be seen that both the pore ingress and egress processes affect the selectivity of SEC. The relationship between m, me ,
and mp can be re-expressed when we introduce parameter to
characterize the relative contribution of the pore egress process to
the overall size exclusion effect [12]:
mp
=
.
m

(8)

The characteristic function is the main equation of the stochastic


theory. It is the Fourier transform of the elution prole and it contains all the information about the separation process. In this simple
case the following characteristic function describes the system the
best:

() = exp

np



1
1
1 ip

(9)

where i is the imaginary unit, np is the average number of the pore


ingress and egress steps,  p is the average time spent by a molecule
in a single pore and is an auxiliary real variable (frequency). In
this case the probability density function (the time-domain signal)
can be written as the inverse Fourier transform of the characteristic
function as:

f (t) =

np t/p np
e
I1
tp



4np t
p

(10)

where I1 is a modied Bessel function of the rst kind and rst


order.
There is a simple relationship between the characteristic function and the moments about the origin. The kth moment of the
chromatographic peak can be calculated from the characteristic
function (Eq. (9)) by the moment theorem of the Fourier transform
as

k = ik

dk ()
dt

(11)

=0

It is well-known that the rst moment is the mean residence


time and the second central moment is the variance of the observed
chromatographic peak. By calculating these moments using the
equation above we obtain:
1 = np p

(12)

and
2 = 2np p2 .

(13)

The third central moment gives information about the peak symmetry:
3 = 6np p3 .

(14)

The above equations are only valid if we assume that the pore
size in the stationary phase particles is uniform.
For heterogeneous kinetics, the simplest stochastic model cannot be employed. Cavazzini et al. extended the stochastic model of
chromatography to the case where the stationary phase consists of
more than one types of adsorption sites and for the case when the
adsorption energy of the sites is determined by a distribution [20].
It was assumed that if there are several types of adsorption sites in
the column, the molecules bind with a certain probability to each
of them. Thus, the probability density function describing the peak
shape can be obtained as the probability-weighted convolution of
the probability density functions of the different sites. For example, for two different adsorption sites the following characteristic
function was obtained:
() = exp

n1

1
1
1 i1

exp

n2

1
1
1 i2

(15)

The corresponding peak shape is:


f (t) =

n1 t/1 n1
e
I1
t1





n2 t/2 n2
e
I1
t2


4n1 t
1




4n2 t
2

(16)

where the subscripts refer to the respective sites and the sign *
stands for convolution. The calculation of the moments and that of
the peak prole is difcult in time domain if it is possible at all,
so it cannot practically be used in this form.
3. Experiments
All experiments were carried out with the software package
Mathematica 9 (Wolfram Research). The elution proles were
obtained via numerical inverse Fourier transform using 1024
points. Except for the case where we illustrate the effect of parameter , in all other cases  perm , nperm and rp,0 were arbitrarily set
to 1 s, 2000 and 12.434 nm, respectively. To illustrate the effect of
changing the parameter  on the elution proles we used  perm = 0.1
s.
4. Results and discussion
In size exclusion chromatography, the most important factor is
the pore size of the stationary phase, since the separation is based
on the size of the analyte molecules relative to the pore size.
In ideal SEC, because there is no physico-chemical interaction
between the sample molecules and the stationary phase surface,
the type of the silica and the chemical modication has no effect
on the retention and on the selectivity. The size of the stationary
phase particles of the modern HPLC columns varies in a quite thin
range, and it has been recently demonstrated that there is no evident correlation between the particle size distribution and column
efciency [21]. The effect of the structure of the stationary phase
particle (e.g. non-porous, fully porous particles, coreshell) on the
separation efciency is quite signicant, because diffusion within
the particles has a strong impact on the brand broadening in all
modes of HPLC. The size exclusion effect on non-porous particles
is nonexistent, only the hydrodynamic effect is present. The fullyporous particles have a large pore volume where the molecules can
diffuse and macromolecules may spend long time there, thus slow
pore diffusion gives rise to band broadening of the observed peaks.
In the coreshell particles the pore volume is more limited and the
diffusion times are shorter [22].

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260

55

Results obtained with low-temperature nitrogen adsorption


conrmed that the presently commercially available HPLC columns
do not have a uniform pore size, but contain pores in a relatively
wide pore size range. A packing material which is marketed as a
200-A pore size stationary phase will denitely contain pores of 100
and of 300 A as well. This is an important aspect, because molecules
of a given size are not equally likely to enter each pore. Molecules
that are small enough to enter the pores of 200 A may be excluded
This ultimately leads to the distortion in
from the pores of 100 A.
the peak shapes in practice.
The pore size distribution (PSD) of the porous stationary phase
particles has been already investigated and modeled in the early
1980s by Knox and Scott [3].
The stochastic theory describes the chromatographic process
at the molecular level so it is obvious to introduce the pore size
distribution into that in order to obtain more relevant information
about the retention properties. The concept introduced by Eq. (15)
can be extended to multisite heterogeneous adsorption and the
following characteristic function is obtained when m type of sites
are present, each with a relative abundance of pj , (j = 1, . . ., m) [20]:

Eq. (21) is the characteristic function of the peak shape for lognormal pore size distribution. It is important to note that both the
number of pore entries and the individual sojourn times of the
molecules in a pore depend on the size of the molecule relative
to the size of the pore. The respective relationships are given by
Eqs. (5) and (6).
The above characteristic function describes the peak shape in
Fourier domain for size exclusion chromatography when the pore
sizes are not uniform. The peak shape itself can be calculated as the
inverse Fourier transform of ().
Unfortunately, Eq. (21) cannot be evaluated analytically for the
general case. Nevertheless, the calculation of the moments is possible. An analytical expression of the moments can only be obtained
if parameters me and mp are both integers. Intuition suggests and
extensive data processing of SEC data conrms [12] that for the
ingress process me > 0 in Eq. (5) and for the egress process mp < 0 in
Eq. (6).
The rst absolute moment as well as the second and the third
central moments of the elution prole will be obtained from Eq.
(21) using Eq. (11) as


1

() = exp n 1 +
pj
1 ij

1 = nperm perm a,

(22)

2
2 = 2nperm perm
a,

(23)

3
a,
3 = 6nperm perm

(24)

(17)

j=1

This equation can be extended for a continuous distribution of


sites. When size exclusion chromatography is modeled and both
the pore ingress and egress processes are inuenced by the pore
size distribution, the following characteristic function is obtained:



(, rp ) = exp

(rp , rp,0 , )np


rG

1
1
1 ip


dr p

 

(rp , rp,0 , ) =
exp
2 rp

ln rp ln rp,0

(18)

2 

2 2

(19)

where rp,0 and  represents the maximum and width of the lognormal distribution, respectively. One should note that the true rst
absolute and second central moments of the lognormal distribution
(Eq. (19)) are
1,lognorm = rp,0 e

2 /2

2
2,lognorm = rp,0
e

e 1

(20)

For the sake of simplicity, however, further on we refer to rp,0


and  as the mean and the standard deviation of the lognormal
distribution, respectively.
We obtain the characteristic function in the case of lognormal
pore size distribution when we combine Eqs. (18) and (19):
() = exp

1
2 

rG

np (rp )
exp
rp

1
1
1 ip (rp )

 

ln rp ln rp,0

2 

2 2


dr p

k2  2
1
=
()k e 2
2

a = KSEC

k=0

If all the pores were of the same size, one would obtain the characteristic function and the moments of the band prole as written
by Eqs. (9)(11). However, when the pore size is governed by a
distribution, the probability density function of the pore size distribution, (rp , rp,0 , ), is included in the model. As a consequence,
we replace the term rp in Eq. (4) by a probability density function
(PDF). If we assume a lognormal distribution, which is shown by the
experimental evidence to describe the pore size distribution of the
HPLC packing materials, the following probability density function
is used:
1

where parameter a is actually equal to the KSEC .

(21)

erfc

k 2 + ln 

2

(25)

Parameter a and thus KSEC strongly depends on the pore


shape. For the calculation of 1 ,
= me + mp should be used and if

= 1, the pore is slit shaped, if


= 2 it is cylindrical and if
= 3, the
pore is either conical or spherical. The second central moment can
be calculated using
= me + 2mp , and the third central moment by
using
= me + 3mp .
Parameter a can only be calculated by Eq. (25) when
> 0. For
instance, when mp = 3 and me = 6, and thus m = 3, in the calculation
of the third central moment
= 3, and the analytical calculation
of that moment is not possible with the equations written above,
nevertheless, the rst and the second moments can be still evaluated. The rst three moments can be calculated in all the cases
when > 0.5. If < 0.5, for the calculation of the third moment
one would get
< 0 and it is not possible to use Eqs. (22)(25).
One can always obtain the elution prole with the inverse
Fourier transform of Eq. (21) and calculate the moments by numerically integrating the peak prole.
The equations above demonstrate that pore size distribution
will have important consequences on retention time and peak
shape. By plotting parameter a when m = 0 against  and  we
obtain the relative pore accessibility (see Fig. 1). One can see in
that gure that in the case of monopores ( = 0), there is a sharp
distinction between the molecules that visit the pores and the ones
that are excluded. All the molecules that are smaller than  = 1, i.e.
molecules for which rG < rp can visit all the pores. On the other hand,
every molecule for which  > 1 is excluded from all the pores. However, when a range of pore sizes are present in the stationary phase,
( > 0), there are pores that are accessible for the molecules larger
than rp,0 too. The broader the pore size distribution, the smoother
is the transition between inclusion and exclusion. For the effect of
 and  on the partition coefcient in case of m=1 (slit shaped pore
geometry), m=2 (cylindrical pore geometry) and m=3 (conical or
spherical pore geometry), see Fig. 2.
To exploit the effect of the pore size distribution on the elution prole, we calculated various chromatograms by changing the

56

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260

Fig. 1. Relative pore accessibility. The effect of the breadth of the PSD () and the
effect of the size parameter () on the size-exclusion process.

standard deviation of the pore size distribution and the size of the
solute molecule relative to the pore size.
The effect of increasing the breadth of the distribution () on the
chromatograms can be seen in Fig. 3 for a relatively large molecule,
when the relative sizing parameter is  = 0.95. This gure illustrates the positive effect of the PSD on the chromatographic process,
where the solid lines represent  = 0.1, the long-dashed lines represent  = 0.5 and the short-dashed lines stand for  = 1. Depending
on the pore shape, the retention times of the proles vary in a quite
wide range. However, it can be seen in all cases, that the retention
time increases and the skew of the elution prole decreases as 
increases. If the sample molecules are very large, they can only in
the rarest case get inside a pore. However once they enter a pore,
they cannot escape from it and the molecule remains trapped at
the same position as time passes (n is very small and  is very

1.0

1.0

a.
=1

0.6
0.4

0
0.2
0.4
0.6
0.8
1

0.2

=1

0.4
0.2

0.5

1.0

1.5

2.0

1.0

2.5

0
0.2
0.4
0.6
0.8
1

0.6
=1

0.4
0.2

=1

0.5

1.0

1.5

2.0

1.0

2.5

d.

0.8

KSEC

KSEC

0.0
0.0

c.

0.8

0.0
0.0

0
0.2
0.4
0.6
0.8
1

0.6

=0

0.0
0.0

b.

0.8

KSEC

0.8

KSEC

large). Most of the molecules, however, cannot enter a pore and


for this reason they are unretained and elute at the void time (t0 ).
This is best illustrated by the green solid line that stands for conical or spherical pore shapes (m = 3) and relatively small variance
( = 0.1) in Fig. 3. This is also demonstrated for the case of cylindrical pores, too, where we obtain an exponential decreasing line as
the elution prole when the pore size distribution is rather narrow.
As the standard deviation of the pore size is increased, the elution
prole becomes a Gaussian peak and if  = 1 one obtains the most
symmetric prole for every kind of pore shape.
For smaller molecules ( = 0.10.5), the trends when  is
increased are rather different depending on the pore shapes. In
case of slit shaped pores,  has a more dominant effect on the KSEC
value than in the other two cases (cylindrical or conical pores) and
it was also demonstrated in Fig. 2. For example if the particular
molecular size is  = 0.5 the retention time increases when m = 2
or 3 and decreases when m = 1 as the value of  is increasing. The
chromatograms become more asymmetrical when  increases. The
increase of asymmetry is most signicant for m = 3. For smaller 
values (such as  = 0.2) the retention time will decrease in the case
of all kind of pore shapes.
The effect of the relative size of the molecules, , on the elution
prole is of course most important for the retention time, i.e. for the
rst moment. The larger the , the less included is the molecule. By
calculating the skew of the peak proles, we can see that the peaks
become more asymmetrical while  increases. Chromatograms are
plotted in Fig. 4 for different pore shapes and molecular sizes. The
retention times of the observed chromatogram changes considerably. We can conclude that the pore size distribution has little
inuence on the behavior of the small molecules while its effect on
the large molecules ( > 0.5) is intensive. The larger the molecule,
the harder it can enter the pores, but once it enters a pore it gets
stuck there, remains at the same position as time passes and will
not emerge from the pore for a wile. That is why the peaks of large

0
0.2
0.4
0.6
0.8
1

0.6
=1

0.4
0.2

=0

0.5

1.0

1.5

2.0

2.5

0.0
0.0

=0

0.5

1.0

1.5

2.0

2.5

Fig. 2. The inuence of the pore shape parameter (m) and the size of the molecule relative to the pore size () on the partition coefcient when (a) the pore geometry is not
included (m = 0 i.e. relative pore accessibility), (b) slit shaped pores (m = 1), (c) cylindrical pores (m = 2) and (d) conical or spherical pores (m = 3).

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260

57

20
m=3

15

m=3

Intensity

m=2

m=2

0.4

0.6

10

0.8

m=2

m=2

0.10
0.05

m=1

0.2

m=3

m=1

0.15

1.0
0.5

m=2

0.20

m=3

1.5

Skew

2.0

1.0

0.00

m=3

0.2

0.4

0.6

0.8

m=1

m=1
m=1

m
m
m
m
m
m
m
m
m

0
0.0

0.1

0.2
Normalized retention time

1,
1,
1,
2,
2,
2,
3,
3,
3,

0.1
0.5
1
0.1
0.5
1
0.1
0.5
1

0.3

0.4

Fig. 3. The effect of the breadth of the PSD () on the calculated chromatograms for slit shaped pores, for cylindrical pores and for spherical or conical pores when the size
parameter is  = 0.95. In the inserts the skew and the rst absolute moment of the chromatograms are plotted.

m=3

0.8

m=2

Skew

60

m=3

0.6

40
m=2

150

20

Intensity

100

m=1

m=3

0.2

m=1

0.2

0.4

m=2

0.6

0.8

m=1

m=1
m=2

0.4
m=3

1.0

0.2

m=3 m=2

0.4

0.8

m
m
m
m
m
m
m
m
m

0. 2

0. 4

0.6

1.0

m=1

50

0
0. 0

0.6

0.8

1. 0

1,
1,
1,
2,
2,
2,
3,
3,
3,

0.1
0.5
1
0.1
0.5
1
0.1
0.5
1

1.2

1.4

Normalized retention time


Fig. 4. The effect of the size parameter () on the calculated chromatograms for slit shaped pores, for cylindrical pores and for spherical or conical pores when the breadth
of the PSD is  = 0.5. In the inserts the skew and the rst absolute moment of the chromatograms are plotted.

molecules (i.e. with high  values) become extremely broad and


skewed.
Parameter expresses the relation of the pore ingress and egress
processes (see Eq. (8)). We calculated elution proles for various
values and for the mentioned pore shapes. The observed proles
are demonstrated in Fig. 5 where the relative size parameter ()
was set to 0.95 and the variance of the PSD was  = 0.5. In this case
the size of the sample molecules is very close to the pore size, however the effects of changing the parameter is very meaningful.
As it could be expected, the retention time (the rst moment) does
not depend on . The second central moment and the skew of the
observed proles increase as decreases, and this is demonstrated
in the subgures of Fig. 5. In the case of slit shaped pores (m = 1), the
relation of the ingress and egress processes does not affect the peak
symmetry as much as in the other cases where the peaks become
more asymmetrical as increases. The same tendency could be

Table 1
The effect of the relative contribution of the pore egress process to the overall size
exclusion effect () on the value of the pore ingress and egress depending constants
(mp and me ) and on the value of the calculated rst absolute, second and third central
moments (1 , 2 and 3 ) in case of slit shaped pores (m = 1).
m

mp

me

me + mp

me + 2mp

me + 3mp

1
1
1
1
1
1
1
1
1
1

0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2

1
1
1
1
1
1
1
1
1
1

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

0.8
0.6
0.4
0.2
0
0.2
0.4
0.6
0.8
1

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260

m=3

Skew

30

25

m=2

3.5
3.0
2.5
2.0
1.5
1.0
0.5

m=3

58

m=2
m=1

1.0

0.8

Intensity

20

0.6

0.4

0.0005
0.0004
0.0003
0.0002
0.0001

m=1
m=2
m=3

1.0

0.2

0.8

0.6

m=1

m
m
m
m
m
m
m
m
m

15

10

0.4

1,
1,
1,
2,
2,
2,
3,
3,
3,

0.2

0.1
0.5
1
0.1
0.5
1
0.1
0.5
1

0
0.00

0.05

0.10

0.15

0.2 0

0.25

Normalized retention time


Fig. 5. The effect of the relative contribution of the pore egress process to the overall size exclusion effect () on the calculated chromatograms for slit shaped pores, for
cylindrical pores and for spherical or conical pores when the size parameter is  = 0.95, and the breadth of the PSD is  = 0.5. In the inserts the skew and the second central
moment of the chromatograms are plotted.

observed for all value of : the proles become more asymmetrical


as decreases.
From the results presented in Figs. 25 we can conclude that the
effect of the parameters studied (i.e. , , ) is the most intensive
in case of conical or spherical pores and the least intensive in case
of slit shaped pores.
We investigated how the peak resolution and the efciency of
the separation are affected by the pore size distribution. The analytical calculation of the relative resolution is only possible if the rst
absolute and the second central moments of the peak are calculated
for integer me and mp values. There are only a few situations this
calculation can be done. Table 1 helps the reader to consider

1 0.2, 2 0.3
1 0.5, 2 0.6
1 0.8, 2 0.9

Relative resolution

Relative resolution

1 0.1, 2 0.2
1 0.4, 2 0.5
1 0.7, 2 0.8

a.
1. 2
1. 0
0. 8
0. 6
0. 4
0.0

whether the moments and the resolution can analytically be calculated, or not.
The change of the resolution and the number of theoretical
plates with the breadth of pore size distribution are reported in
Figs. 6 and 7.
Several important conclusions can be drawn from the data presented in the gures. The curves can be divided into two cases
based on the molecule size. In the rst one, the molecules are
small enough to separate them by size exclusion chromatography
and neither the relative resolution nor the number of theoretical
plates is affected by the pore size distribution. This can be seen in
Figs. 6 and 7, where the plots referring to the small molecules hardly

0. 2

0. 4

0.6

0. 8

1.0

1 0.3, 2 0.4
1 0.6, 2 0.7
1 0.9, 2 0.95

b.

0.9
0.8
0.7
0.6

1.0

0. 0

0.2

0.4

2. 0 c.
1. 8
1. 6
1. 4
1. 2
1. 0
0. 8
0. 6
0. 0

0. 2

0. 4

0.6

0. 8

1.0

0.6

0.8

1. 0

Relative resolution

Relative resolution

0. 6

0. 8

1.0

12 d.
10
8
6
4
2
0
0.0

0.2

0.4

Fig. 6. Relative resolution for calculated chromatograms of differing size parameters (). The pore shape parameter (m) and the relative contribution of the pore egress
process to the overall size exclusion effect () were varied as (a) m = 1 and = 1, (b) m = 2 and = 0.5, (c) m = 2 and = 1 and (d) m = 3 and = 1.

A. Sepsey et al. / J. Chromatogr. A 1331 (2014) 5260


2
2
2
2
2

a.

1500

500
0
0. 0

0. 2

0.4

0.6

0. 8

1. 0

c.

1200

1400
1200
1000
800
600
400
200
0
0. 0

b.

1000

80 0

800

60 0

600

0. 4

0.6

0.8

1.0

d.

40 0

400

20 0

200

0
0. 0

0.2

1000

0.2
0.4
0.6
0.8
1

2
2
2
2
2

1000

0.1
0.3
0.5
0.7
0.9

59

0.2

0.4

0.6

0. 8

1. 0

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 7. Number of theoretical plates for calculated chromatograms of differing size parameters (). The pore shape parameter (m) and the relative contribution of the pore
egress process to the overall size exclusion effect () were varied as (a); m = 1 and = 1, (b); m = 2 and = 0.5, (c); m = 2 and = 1 and (d); m = 3 and = 1.

show any change as  increases, while the relative resolution and


the number of theoretical plates for the larger and especially for the
largest molecules signicantly increase as  increases. The effect is
stronger as the value of m increases (i.e. in the case of cylindrical
and spherical or conical pores relative to slit shaped pores).
This observation drives us to more general conclusions in the
eld of chromatography, because the size exclusion effect is not
only in size exclusion chromatography important, but also in other
chromatographic methods, such as reversed phase or HILIC separations. In those cases, the hindered pore diffusion of macromolecules
is very important, and the size exclusion process becomes more
important than the other effects of the retention.

5. Conclusions
The stochastic theory of size exclusion chromatography was
extended to wide pore size distribution for a number of various
pore geometries (slit shaped, cylindrical, and conical or spherical).
The statistical moments of the peak proles can easily be calculated
by assuming a pore shape. The calculation of the chromatograms is
feasible by inverse Fourier transform of the characteristic function.
The trend we observe for the calculated chromatograms emphasizes the signicance of pore size distribution: the PSD has strong
inuence on the retention properties (retention time, peak width,
and peak shape) of macromolecules.
Eq. (25) summarizes how pore size distribution affects the partition coefcient in size exclusion chromatography. That equation
can serve as the basis for the experimental determination of the
width of pore size distribution when KSEC is plotted against the
gyration radius of polymer molecules. This is going to be exploited
in a forthcoming study [23].
However, inverse size exclusion chromatography (ISEC) is used
to derive information about the pore structure of the packing material from the retention data of series of known analytes, is was
shown that it does not give appropriate characterization of the
irregularly shaped porous materials [24]. We truly believe that our
model which contains information about both the pore geometry
and the distribution of the pore sizes is usable to develop ISEC and

so to obtain relevant information from the pore structure by nondestructive ISEC measurements. The experimental chromatograms
may be characterized by pore geometry contributions and so the
effect of different types of pores could be investigated.
For the separation of macromolecules, the wide pore size distribution will increase retention and efciency. Therefore in all
modes of liquid chromatography, the efcient separation of macromolecules calls for a broad pore size distribution.
Acknowledgements
This research was realized in the frames of TMOP 4.2.4. A/211-1-2012-0001 National Excellence Program Elaborating and
operating an inland student and researcher personal support system. The project was subsidized by the European Union and
co-nanced by the European Social Fund.
The work was supported in part by the grants TMOP-4.2.2. A11/1/KONV-2012-0065 and OTKA K 106044.
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