Académique Documents
Professionnel Documents
Culture Documents
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
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
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
t texcl
,
tperm texcl
(1)
KSEC =
(1 )m
if 0 1
if > 1
(3)
rG
rp
(4)
54
(5)
(6)
(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)
() = exp
np
1
1
1 ip
(9)
f (t) =
np t/p np
e
I1
tp
4np t
p
(10)
k = ik
dk ()
dt
(11)
=0
(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)
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].
55
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
(, rp ) = exp
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)
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
(21)
erfc
k 2 + ln
2
(25)
56
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
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).
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
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
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
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.
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.
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.
References
[1] F. Gritti, I. Leonardis, J. Abia, G. Guiochon, J. Chromatogr. A 1217 (2010)
3819.
[2] B.M. Wagner, S.A. Schuster, B.E. Boyes, J.J. Kirkland, J. Chromatogr. A 1264 (2012)
22.
[3] J.H. Knox, H.P. Scott, J. Chromatogr. 316 (1984) 311.
[4] J.H. Knox, H.J. Ritchie, J. Chromatogr. 387 (1987) 65.
[5] Y. Yao, A.M. Lenhoff, J. Chromatogr. A 1037 (2004) 273.
[6] M. Kubin, J. Chromatogr. 108 (1975) 1.
[7] I. Halsz, K. Martin, Angew. Chem. Int. Ed. Engl. 17 (1978) 901.
[8] J.C. Giddings, H. Eyring, J. Phys. Chem. 59 (1955) 416.
[9] F. Dondi, M. Remelli, J. Phys. Chem. 90 (1986) 1885.
[10] F. Dondi, A. Cavazzini, M. Remelli, A. Felinger, M. Martin, J. Chromatogr. A 943
(2002) 185.
[11] L. Pasti, F. Dondi, M. Van Hulst, P.J. Schoenmakers, M. Martin, A. Felinger, Chromatographia 57 (2003) S171.
[12] A. Felinger, L. Pasti, F. Dondi, M. van Hulst, P.J. Schoenmakers, M. Martin, Anal.
Chem. 77 (2005) 3138.
[13] A. Felinger, J. Chromatogr. A 1184 (2008) 20.
[14] I. Teraoka, Macromolecules 37 (2004) 6632.
[15] W.W. Yau, J.J. Kirkland, D.D. Bly, Modern Size-Exclusion Liquid Chromatography, Wiley, New York, 1979.
[16] J.B. Carmichael, J. Polym. Sci. A-2 6 (1968) 517.
[17] J.B. Carmichael, Macromolecules 1 (1968) 526.
60
[18]
[19]
[20]
[21]