**2.5. Gel permeation chromatography**

The heterogeneity and the molecular weight polydispersity of polysaccharide fractions can be analyzed by gel permeation chromatography (GPC) [6,37,56]. This analytical technique is also called size-exclusion chromatography because the fractionation occurs according to the molecular size, V (not molecular weight!), of the polymer which easily or not permeates pores of a suitable dimension of the gel matrix packed in the column which has no specific, or weak, interactions with eluted polymers.

Unpublished data (R. Urbani) of exopolysaccharides obtained from mucilage marine samples are presented in Figure 3. The purified polysaccharides were dissolved in 0.05M NaCl and solutions were filtered on 0.45 µm filters and injected in a conditioned GPC column (1.5 m length, 310 mL internal volume), packed with Sepharose CL6B (104-106 Da). The chromatographic apparatus was pre-equilibrated using 0.05M NaCl at 25°C at a flow rate of 10 mL·h-1 and the column was calibrated with standard dextran solutions (Mw = 2·104 Da and Mw = 5·105 Da). A differential refractive index instrument was used as detector.

**Figure 3.** GPC of purified exopolysaccharides from Adriatic mucilage.

350 The Complex World of Polysaccharides

*decipiens* culture.

solution [54,55].

linear chain (5.6% of 2-Man and 6-Man).

**2.5. Gel permeation chromatography** 

or weak, interactions with eluted polymers.

**Methylation derivative Linkage %** 2,3,4-Me3 Rha t-Rha 21.4 2,3,4-Me3 Fuc t-Fuc 10.7 2,3,4,6-Me4 Man t-Man*p* 3.2 3,4-Me2 Rha 2-Rha 8.3 2,3,4,6-Me4 Glc t-Glc*p* 4.0 2,3,4,6-Me4 Gal t-Gal*p* 3.2 3,4-Me2 Fuc 2-Fuc 2.7 3,4,6-Me3 Man 2-Man*p* 2.3 2,3,4-Me3 Man 6-Man*p* 3.3 3,5-Me2 Xyl 2-Xyl*f* 1.0 Fuc 2,3,4-Fuc 5.4 3,4,6-Me3 Gal 2-Gal*p* 13.4 2,3,6- Me3 Gal 4-Galp 10.5 2,3,6-Me3 Glc 4-Glc*p* 2.4 3-Me Xyl 2,4-Xyl*p* 1.8 3,6-Me2 Man 2,4-Man*p* 1.9 4,6-Me2 Man 2,3-Man*p* 1.0

**Table 2.** Methylation derivatives of monosaccharidic units of exopolysaccharide from *Chaetoceros* 

represented branched residues (2,3,4-Fuc). Galactose residues which were linked at carbon 2 and 4 (23.9% of 2-Galp and 4-Galp) resided predominantly in the backbone, while mannose was both a branched residue (2.9% of 2,4-Man and 2,3-Man) and mono-substituted in a

The relatively high percentage of galactose which could be present as or as anomeric configuration in the chain backbone suggested a possible extended and rigid chain conformation of the *D. decipiens* polysaccharide as also found for model polysaccharides in

The heterogeneity and the molecular weight polydispersity of polysaccharide fractions can be analyzed by gel permeation chromatography (GPC) [6,37,56]. This analytical technique is also called size-exclusion chromatography because the fractionation occurs according to the molecular size, V (not molecular weight!), of the polymer which easily or not permeates pores of a suitable dimension of the gel matrix packed in the column which has no specific,

Unpublished data (R. Urbani) of exopolysaccharides obtained from mucilage marine samples are presented in Figure 3. The purified polysaccharides were dissolved in 0.05M NaCl and solutions were filtered on 0.45 µm filters and injected in a conditioned GPC The small difference in the retention time (volumes) between peak 1 of M1 and M2 samples (tR about 15 hours) is related to different stage (or age) of the mucilage aggregates with different degraded state [20]. The other peaks (tR about 23 hours) very close to the (total) permeation volume, Vp, correspond to the low molecular weight fractions, very likely derived from hydrolytic activities of the bacterial pool on the native aggregates. High molecular weight fractions (peak 1 of M1 and M2) were collected, purified and derivatized for gas-chromatographic analysis. Their monosaccharidic patterns shown in Table 3 are quite similar suggesting the same origin of the high molecular weight polysaccharides even for aggregates of different degraded state [20].


**Table 3.** Relative monosaccharide composition (%) of unfractionated exopolysaccharide and corresponding fraction at high molecular weight.

A rapid and powerful non-preparative technique for the characterization of molecular dimensions of marine dissolved organic matter and exopolymeric material from diatoms cultures is the high performance size-exclusion chromatography (HPSEC) [57]. By using a hydrogel column system (cut-off 50.000-1.000.000 Da) HPSEC experiments were performed on purified exopolysaccharides from *Chaetoceros decipiens* cultures [44] and results are shown in Figure 4.

**Figure 4.** HPSEC chromatogram of purified exopolysaccharide from cultured *Chaetoceros decipiens.*

Two distinct peaks were observed: at lower (V=16-20 mL) and at higher retention volume (V=25-30 mL) which corresponded to about 1700 kDa and 20 kDa molecular weight substances, respectively. The low molecular size peaks were near the permeation volume (Vp) very likely due to oligosaccharides with an average number of monosaccharidic residues of about 110 units. The authors (R. Urbani, P. Sist) hypothesized that *Chaetoceros decipiens* produces a high molecular weight exopolysaccharide and some oligomers at low molecular weight probably derived from the release of storage carbohydrates by cultured diatom in given culture condition [44].

## **3. Solution and aggregation properties**

In the macromolecular science it is well documented that going from flexible polysaccharides toward more rigid and extended chains, as well as with the presence of an ordered sequence in the primary structure, there is a general propensity of polymer chains to form chain associations/aggregations or multiple helical structures. Even if that sequence specificity were present, the absence of pronounced amphiphilic character in the sugar building blocks is not favorable for the formation of some kind of globular folding that occurs in many proteins and some nucleic acids [58]. In other words such globular structures are unknown in polysaccharides. Nonetheless, ordered conformations were proposed to represent the structure of many polysaccharides, both in solid state and in solution. In some cases the functionality is most closely associated with the occurrence of a randomly coiling polymeric character, that is, a propensity for the chain to move continuously through a vast range of nearly equally energetic conformations.

The presence of charged groups on the polysaccharidic chain confers peculiar properties to the macromolecules favoring, for example, the solubility or the association/dissociation processes in solution. On the polymer side the presence of the charged groups influences strongly all the conformational properties by enhancing the chain dimensions and increasing the hydrodynamic volume. Knowledge of polysaccharidic chain structures, from single chain up to the three-dimensional molecular shape and chain association, is essential to understand their capability to form supra-molecular structures including physical gels [54].

#### **3.1. Viscometry and light scattering characterization**

352 The Complex World of Polysaccharides

diatom in given culture condition [44].

**3. Solution and aggregation properties** 

**Figure 4.** HPSEC chromatogram of purified exopolysaccharide from cultured *Chaetoceros decipiens.*

Two distinct peaks were observed: at lower (V=16-20 mL) and at higher retention volume (V=25-30 mL) which corresponded to about 1700 kDa and 20 kDa molecular weight substances, respectively. The low molecular size peaks were near the permeation volume (Vp) very likely due to oligosaccharides with an average number of monosaccharidic residues of about 110 units. The authors (R. Urbani, P. Sist) hypothesized that *Chaetoceros decipiens* produces a high molecular weight exopolysaccharide and some oligomers at low molecular weight probably derived from the release of storage carbohydrates by cultured

In the macromolecular science it is well documented that going from flexible polysaccharides toward more rigid and extended chains, as well as with the presence of an ordered sequence in the primary structure, there is a general propensity of polymer chains to form chain associations/aggregations or multiple helical structures. Even if that sequence specificity were present, the absence of pronounced amphiphilic character in the sugar building blocks is not favorable for the formation of some kind of globular folding that occurs in many proteins and some nucleic acids [58]. In other words such globular structures are unknown in polysaccharides. Nonetheless, ordered conformations were proposed to represent the structure of many polysaccharides, both in solid state and in solution. In some cases the functionality is most closely associated with the occurrence of a randomly coiling polymeric character, that is, a propensity for the chain to move

The presence of charged groups on the polysaccharidic chain confers peculiar properties to the macromolecules favoring, for example, the solubility or the association/dissociation processes in solution. On the polymer side the presence of the charged groups influences

continuously through a vast range of nearly equally energetic conformations.

Physico-chemical techniques are widely used in order to estimate the biopolymer features such as chain stiffness and chain dimensions which are strictly related to the polymer propensity to give aggregation and highly structured systems like gels. The Smidsrød-Haug parameter B [59] measured by using capillary viscosity techniques as a function of ionic strength is related to chain stiffness: the more flexible the chain, the higher the response of the intrinsic viscosity on the ionic strength variation and the higher the B value.

Intrinsic viscosities, [], of exopolysaccharides from Adriatic mucilage (M1 and M2) were measured in Cannon-Ubbelohde suspended-level capillary viscometers at different ionic strength and obtained by linear regression of reduced specific viscosities, sp/C (dL·g-1), as a function of polymer concentrations. By plotting the intrinsic viscosity (the polymer hydrodynamic volume) as a function of the inverse of the root of ionic strength (Figure 5), both exopolysaccharides show a linear reduction of [] at increasing of ionic strength due to screening effect of the salt on polymer charges. From the Smidsrød-Haug equations [59]:

$$
\left[\eta \mathbf{\bar{\eta}}\right] = \mathbf{A} + \mathbf{S}^{\cdot} \underline{\mathbf{k}}^{-1} \approx \mathbf{A} + \mathbf{S} \left(\sqrt{\mathbf{I}}\right)^{-1}
$$

the stiffness parameter B is obtained where []0.1 is the intrinsic viscosity at 0.1 M ionic strength:

$$\mathbb{B} = \frac{\mathbb{S}}{\left(\left[\eta\right]\_{0.1}\right)^{1.3}}$$

It is interesting to compare the B stiffness parameter of exopolysaccharides with those reported in the literature for other polysaccharides of similar molecular weight. This is shown in Table 4 where dextran sulphate and polyphosphate represent rather flexible polymers with a high B value and, on the other hand, DNA and xanthan represent typical stiff polymers exhibiting a low B parameter. The M1/M2 exopolysaccharides exhibited a value similar to that of alginate considered as a semiflexible polymer.

Static and dynamic laser light scattering (SLLS and DLLS, respectively) are techniques widely used for polymer characterization, measuring average chain properties and thermodynamic quantities in solution. These properties are related to the propensity of the polymer system to give elongated and stiff chain: the radius of gyration (RG), the weight-average molecular weight (Mw), the second virial coefficient (A2) and the hydrodynamic diameter (dh).

**Figure 5.** Plot of intrinsic viscosity *vs.* inverse of the square root of ionic strength for .M1 and M2 polysaccharides.


**Table 4.** Stiffness parameters (B) of different model polymers.

Measurement of the scattering intensity at many angles allows the evaluation of the radius of gyration RG, while by measuring the scattering intensity for many samples of various concentrations, the coefficient A2 is obtained. Simultaneous linear least squares fits to both the angular and concentration dependence of scattering intensities are employed for the properties determination.

In Figures 6a and 6b the two Zimm plots of the M2 exopolysaccharide in 0.3M and 0.7M NaCl solutions, respectively, are presented. The results show a constancy of RG (and MW) at different salt concentrations, while A2 becomes more negative for higher salt concentration as a consequence of the screening effect on polymer charges which may lead to an extensive degree of aggregation. In general, with respect to the thermodynamic stability of the polymer solution, negative A2 values are a clear indication of the tendency of polysaccharide solution to undergo a phase separation to form a amorphous carbohydrate solid phase or a gel-like structure.

With comparison to model polysaccharides of different stiffness having the same Mw (Table 5), M2 polysaccharide possess higher RG value than the flexible and coiled pullulan but also than the semi-rigid chain of the wellan and alginate biopolymers.

**Figure 6.** Zimm plot of M2 at two ionic strength (a) 0.3M and (b) 0.7M NaCl.


a in 0.3M NaCl

354 The Complex World of Polysaccharides

polysaccharides.

properties determination.

gel-like structure.

**Figure 5.** Plot of intrinsic viscosity *vs.* inverse of the square root of ionic strength for .M1 and M2

**Polymer B** Polyphosphate 0.44 Dextran sulphate 0.23 Hyaluronic acid 0.065 Alginate (mannuronic rich) 0.040 M1, M2 0.036 Alginate (guluronic rich) 0.031 DNA 0.0055 Xanthan 0.0053

Measurement of the scattering intensity at many angles allows the evaluation of the radius of gyration RG, while by measuring the scattering intensity for many samples of various concentrations, the coefficient A2 is obtained. Simultaneous linear least squares fits to both the angular and concentration dependence of scattering intensities are employed for the

In Figures 6a and 6b the two Zimm plots of the M2 exopolysaccharide in 0.3M and 0.7M NaCl solutions, respectively, are presented. The results show a constancy of RG (and MW) at different salt concentrations, while A2 becomes more negative for higher salt concentration as a consequence of the screening effect on polymer charges which may lead to an extensive degree of aggregation. In general, with respect to the thermodynamic stability of the polymer solution, negative A2 values are a clear indication of the tendency of polysaccharide solution to undergo a phase separation to form a amorphous carbohydrate solid phase or a

With comparison to model polysaccharides of different stiffness having the same Mw (Table 5), M2 polysaccharide possess higher RG value than the flexible and coiled pullulan but also

than the semi-rigid chain of the wellan and alginate biopolymers.

**Table 4.** Stiffness parameters (B) of different model polymers.

**Table 5.** Radius of gyration of purified polysaccharides M1 and M2 compared to model polysaccharides with Mw=220,000 dalton in 0.1M NaCl.

Finally, taking into account all these results one may say that the M2 polysaccharide shows a marked polyelectrolytic behavior and an intrinsic chain stiffness, favoring at higher salt concentrations chain-to-chain association and/or gel formation. Thus, coupled to other favorable environmental conditions, the salinity gradient of halocline in the seawater column could play an important role in the first stage of aggregation of dissolved biopolymers.

Dynamic laser light scattering (DLLS), also called photon correlation spectroscopy, is a technique for the determination of hydrodynamic diameter of macromolecules and particulate matter in solution. In DLLS the fluctuation of the scattered light due to the Brownian motion of the molecules is detected by a photon counting detector. This technique is used nowadays to measure particle sizes at the nanometer scale and to follow the kinetics of particle formation from dissolved EPS material. As reported by Verdugo [60,61] marine aggregates may self-assemble from free DOM biopolymers and this process was easily followed by DLLS.

Following the same procedure the authors (R. Urbani, P. Sist) measured hydrodynamic diameters using Adriatic seawater samples collected during a three-years monitoring activity (1999-2001). Polymer assembly was monitored for 8 days by analyzing the scattering fluctuations detected at a 45° scattering angle. The autocorrelation function of the scattering intensity fluctuations was averaged over a 1-min sampling time and the particle size distribution calculated by the CONTIN method. Figure 7 shows an example of time dependence of hydrodynamic diameters in three seawater samples taken at different depths.

Although the aggregation kinetics was the same (asymptotic shaped curve in Figure 7) the highest value (1 µm) of hydrodynamic diameter was significantly lower than values reported by Verdugo group (several micrometers) for oceanic samples [60,61] and cultured diatoms, as well [62].

**Figure 7.** Hydrodynamic diameter in dependence on time for Adriatic seawater samples.
