Chinese Medicine, 2010, 1, 28-29
doi:10.4236/cm.2010.11005 Published Online June 2010 (http://www.SciRP.org/journal/cm)
Non-Exclusion Effects in Aqueous Size-Exclusion
Chromatography of Polysaccharides
Azamat Solievich Boymirzaev1, Abbaskhan Sabirkhanovich Turaev2
1Department of Mechanical-Technology, Namangan Engineering-Economic Institute, Namangan, Uzbekistan
2O.Sadikov Institute of Bioorganic Chemistry Uzbek Academy of Sciences, Tashkent, Uzbekistan
Received January 10, 2010; revised April 29, 2010; accepted May 10, 2010
Abstract
This paper concerned to investigation of aggregate formation processes in aqueous Size-exclusion chroma-
tography (SEC) of Na-carboxymethylcellulose (Na-CMC).
Keywords: SEC, Na Carboxymethylcellulose, Aggregate Formation
1. Introduction
Size-exclusion chromatography is one of the powerful
methods for determination and investigation of molecu-
lar weight distribution of polymers [1]. In aqueous SEC
of polymers [2], the understanding of the separation
mechanism demands much more attention due to the
enthalpy interactions distorting a pure size-exclusion
separation mechanism [1]. Because of the presence of
polar, and often anionic, groups in the stationary phases
used in SEC, the mobile phase must be carefully chosen
to repress polymer-gel and intermolecular interactions.
This is particularly important in SEC of polyelectrolytes
and polar molecules such as carbohydrates [3]. Suppres-
sion of interactions, such as polyelectrolyte expansion,
ion-exclusion, molecular adsorption and aggregate for-
mation depends on nature of electrolyte, optimal value of
pH and ionic strength of eluent.
The aim of this paper is to investigate of aggregate
formation process in SEC of Na-CMC in order to deter-
mine of the suitable aqueous eluent for true size-exclu-
sion separation mechanism of macromolecules.
2. Materials and Methods
SEC was performed on the liquid chromatograph, con-
sisting from syringe pump Merk-Hitachi L-6000A model,
Shodex RI-101 refractive index detector, multiangle laser
light scattering detector DAWN NSP (Watt technology),
manual sample injector Rheodine 2104, degasser of elu-
ent and two chromatographic columns PL Aquagel-OH
Mixed termostated at 25˚C and connected in series. Syn-
thesis of Na-CMC was described in [4]. SEC analysis
were performed using two types of eluent: NaCl and
NaNO3 in the water with concentration 0.1 mol/L.
3. Results and Discussion
Many of hydrophilic polymers are polyelectrolytes and,
therefore, their elution properties in SEC is complicated
by various non-exclusion effects, such as ion exclusion,
polyelectrolyte expansion, molecular adsorption, and
aggregate formation, which distort the normal SEC
separation mechanism. These effects can be eliminated
by increasing the ionic strength and changing the pH of
the eluent so as to decrease the degree of dissociation of
ionic groups both in the macromolecular chain and on
the sorbent surface [5]. Physicochemical properties such
as structure, molecular weight and shape or conformation
are primary factors controlling their functional properties.
A typical molar mass sensitive detector is a multi angle
laser light scattering (MALLS). This detector has the
advantage of providing structural information in addition
to the molar masses. Analysis of CMC by SEC in 0.1 М
NaNO3 solutions were complicated by presence of the low
amount associates forming due to intermolecular interac-
tions [6,7]. To avoid of the aggregates of macromolecules
Hoogendam C.W. [7] demonstrated that the solutes
Na-CMC in first step were prepared in pure water, after
0.1 M NaNO3 were added to sample solution. We have
received bimodal chromatograms of CMC from MALLS
detector in SEC analysis when used of water consisting
NaNO3 with concentration 0.1 mol/L (Figure 1(a)). Same
result was occurring, when we used 0.1 M NaNO3 in
water as eluent. But when 0.1 M NaCl was used first
peak in the chromatogram is disappeared indicating that

G. JEPPESEN ET AL.29
(a)
(b)
Figure 1. Gel chromatograms of the Na-CMC (Mw = 2.18 × 104)
received from MALLS (a) and RI detector (b) in 0.1 M
NaNO3
(a)
(b)
Figure 2. Gel chromatograms of the Na-CMC (Mw = 2.18 × 104)
received from MALLS (a) and RI detector (b) in 0.1 M
NaCl.
formation of molecular aggregates not realized and further
investigations on SEC of cellulose derivatives were con-
ducted using 0.1M NaCl in water (Figure 2(a)). Second,
the presence of microgels as a result of small but signifi-
cant amounts of very high molecular weight CMC was
detected using a MALLS detector as first peak in chro-
matogram. However, the microgel was detected by the
refractive index detector (Figures 1 and 2(b)) as very
small peak, suggesting that this detector responds as if to
a true polymeric solution.
4. Conclusions
Specific polymer-solvent and intermolecular interactions
in aqueous SEC can lead to formation of aggregates of
Na-CMC in 0.1 M NaNO3. Dual detection in SEC allows
determining and evaluating of degree of formation of the
aggregates. In SEC of Na-CMC low amount of aggre-
gates in 0.1 M NaNO3 was detected. To eliminate of ag-
gregates and realize pure SEC separation mechanism of
Na-CMC we are recommend use of 0.1 M NaCl as elu-
ent.
5. References
[1] S. Mori and H. Barth, “Size Exclusion Chromatography,”
Springer Verlag, Berlin, Heidelberg, 1999, p. 234.
[2] J. Desbrieres, J. Mazet and M. Rinaudo, “Gel Permeation
Chromatography on Polyelectrolytes in Aqueous Solu-
tion,” European Polymer Journal, Vol. 18, No. 3, 1982,
pp. 269-272.
[3] S. C. Churms, “Modern Size-Exclusion Chromatography
of Carbohydrates and Gly-Coconjugates,” Journal of
Chromatography, Vol. 66, 2002, pp. 267-303.
[4] S. Shomuratov, E. A. Murodov and A. S. Turaev, “Syn-
thesis and Investigation of Prolonged Preparation on the
Basis of Carboxymethylcellulose,” Journal of Pharmacia,
Vol. 4, 2005, p. 61.
[5] M. Rinaudo and B. J. Tinland, “Some Problems in Aque-
ous Size-Exclusion Chromatography of Synthetic Poly-
mer and Biopolymer Characterization,” Journal of Ap-
plied Polymer Science. Applied Polymer Symposium, Vol.
48, 1991, p. 19.
[6] L. Picton, L. Merle and Muller, “Solution Behavior of
Hydrofobically Accociating Cellulose Derivatives,” In-
ternational Journal of Polymer Analysis and Characteri-
zation, Vol. 2, No. 2, 1996, pp. 103-113.
[7] C. W. Hoogendam, A. de Keizer, M. A. C. Stuart, B. H.
Bijsterbosch, J. A. M. Smit, J. A. P. P. van Dijk, P. M.
van der Horst and J. G. Batelaan, “Persistence Length of
Carboxymethyl Cellulose as Evaluated from Size Exclu-
sion Chromatography and Potentiometric Titrations,”
Macromolecules, Vol. 31, No. 18, 1998, pp. 6297-6309.