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Enrica Alasonati1 Maria-Anna Benincasa1, 2 Vera I. Slaveykova1 1
Environmental Biophysical Chemistry, ISTE-ENAC, Ecole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland 2 Department of Chemistry, University of Rome “La Sapienza”, P.le A. Moro, Rome, Italy
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Original Paper Asymmetrical flow field-flow fractionation coupled to multiangle laser light scattering detector: Optimization of crossflow rate, carrier characteristics, and injected mass in alginate separation The coupling of the flow field-flow fractionation (FlFFF) to differential refractive index (DRI) and multiangle laser light scattering (LS) detectors is a powerful tool for characterizing charged polysaccharides such as alginate. However, the correct interpretation of the experimental results and extrapolation of meaningful molecular parameters by using an analytical tool with such a level of complexity requires improvement of the knowledge of the alginate behavior in the channel and careful optimization of the operating conditions. Therefore, the influence of the critical operating parameters, such as crossflow rate, carrier composition and concentration, and sample load, on the alginate retention was carefully evaluated. Combined information obtained simultaneously by DRI and LS detectors over the wide range of the crossflow rate, carrier liquid concentration, and injected amount, allowed to set the appropriate combination of optimal parameters. It was found that the crossflow rate of 0.25 mL/min, carrier solution containing 5610 – 2 mol/L ammonium or sodium chloride, and 50 – 100 lg of injected sample mass were necessary to achieve complete separation and determination of the meaningful molecular characteristics. The values of the weight-average hydrodynamic radius (RHw), radius of gyration (RG), and molar mass (M), obtained under the optimal conditions were in good agreement to those found for alginates in the literature. Keywords: Alginate / Asymmetrical flow field-flow fractionation / Differential refractive index / Light scattering / Molar mass distribution / Received: May 16, 2007; revised: June 12, 2007; accepted: June 13, 2007 DOI 10.1002/jssc.200700211
1 Introduction Alginates are naturally occurring linear polysaccharides composed of b-D-mannuronic and a-L-guluronic acid, found in marine macroalgae and produced by different bacteria [1, 2]. Their rheological and mechanical properties led to wide applications in the food industry and pharmaceutics as well as in biotechnology [2, 3]. More recently, the finding that alginates have the required chemical affinity to sequester metals has attracted much Correspondence: Professor Vera I. Slaveykova, Environmental Biophysical Chemistry, ISTE-ENAC, Ecole Polytechnique Fdrale de Lausanne, Station 2, Lausanne CH-1015, Switzerland E-mail:
[email protected] Fax: +41-21-693-37-39 Abbreviations: aFlFFF, asymmetrical flow field-flow fractionation; DRI, differential refractive index; FFF, field-flow fractionation; FlFFF, flow field-flow fractionation; LS, multiangle laser light scattering; PDI, polydispersity index
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attention on these biopolymers as potential biosorbents in wastewater decontamination [4]. As for most of the polysaccharides, alginate composition, structure, and molecular size are highly variable and dependent on the source [5, 6] and the season of harvest [7]. Studies aimed at elucidating in detail structure – function relationships have shown that the alginate affinity for multivalent cations is dependent on the content of polyguluronate fraction [1, 2, 4, 5]. Since properties may depend on the composition and molar mass, it appears that knowledge at a detailed level of the behavior of narrow molar mass fractions rather average characteristics can strongly contribute to their implementation in high-value technological applications. Flow field-flow fractionation (FlFFF) is one of the bestsuited field-flow fractionation (FFF) subtechniques that find increasing application for the separation of polymeric samples of different origin [8 – 10]. This versatile tool allows to overcome some of the shortcomings that www.jss-journal.com
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limit the use of other separation techniques such as size exclusion chromatography [11, 12]. The combination of a molar mass selective separation technique with analytical systems capable of revealing specific molecular features allows simultaneous collection of independent data and hence determination of the polymer functionality as a function of molar mass (M). Thus hyphenation of an asymmetrical flow field-flow fractionation (aFlFFF) channel and a multiangle laser light scattering (LS) and differential refractive index (DRI) detectors has provided extensive information on a large number of polymers [13 – 18] without the need for calibration. However, correct interpretation of the experimental results and extraction of meaningful molecular parameters for polydisperse samples, by using a hyphenated tool with such a level of complexity requires extensive knowledge of the mechanisms underlying each analytical technique. Both FlFFF and LS theories were worked out for systems under specific (often idealized) conditions. Therefore, ways to extend the applicability of the theoretically derived relationships to real systems have been explored [19]. This may be performed experimentally by studying the results obtained under working conditions varied consistently. The results of such a study done on alginate polysaccharide isolated from macroalgae Macrocystis pyrifera are reported here. The specific objective of the present work was to find the optimal set of parameters that will ensure the efficient alginate fractionation with minimal sample losses. Crossflow rate, salt concentration of carrier liquid, and the injected mass were chosen as key parameters in alginate separation and were varied systematically. Alginate recovery, equivalent hydrodynamic radius (RH), radius of gyration (RG), number-average (Mn) and weight-average (Mw) molar mass, and polydispersity index (PDI) evolution were followed from the DRI and LS signals over the large range of the separation conditions.
2 Theory 2.1 FlFFF FlFFF theory was widely exposed elsewhere [3, 19 – 21]. As in chromatography, the characterization of retention is done by estimating the velocity of an eluting species relative to that of the carrier liquid. The retention time, tr, of a given species is related to its effective hydrodynamic radius RH by Eq. (1) (e. g., ref. [19]) tr ¼
t0 2 pg RH w Vc kT V0
ð1Þ
where t0 is the eluent average residence time, V0 the void volume, V_ c the crossflow rate, g the eluent viscosity, w the total channel thickness, k the Boltzmann constant, and T is the absolute temperature. RH corresponds to the
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radius of the sphere of equivalent hydrodynamic behavior. Even though RH does not strictly represent any geometrical parameter it depends on the effective size of the macromolecules. In practice, however, a number of factors have been shown to affect the polymer retention in the FlFFF and to cause the deviation from the ideal elution behavior. In the case of polyelectrolytes in aqueous solutions of low ionic strength, intermolecular charge repulsion is by far the predominant effect capable to break any correlation of retention to molecular size [10, 22]. Injected amount of the sample can also influence the polyelectrolyte elution. For example, too large a quantity of sample can cause interference between particles. This can occur either by a steric effect or by attraction and aggregation of the particles [19]. The extent of such interferences depends on experimental parameters such as the polymer – solvent system or the flow conditions [23 – 26]. Therefore, careful optimization of the crossflow rate, carrier salt concentration, and injected mass is required to diminish or avoid nonideal elution behavior.
2.2 LS The theory of light scattering has been treated in detail in a number of publications [27, 28]. The basic equation of LS relates the intensity of the light scattered by particles with size smaller than the incident light wavelength [27] Kc 1 þ 2A2 c ¼ Rh PðhÞM PðhÞ ¼ 1
16p2 2 2 h R sin 2 3k20 G
ð2Þ
ð3Þ
where c is the sample concentration, Rh the Rayleigh ratio which depends on the scattered intensity, M the weight-average molar mass, and h is the angle between incident and scattered beam. K is the instrumental constant depending on the refractive index of the medium, the wavelength of the incident light in the vacuum, and the incremental refractive index of the sample. A2 is the second viral coefficient, assumed to be negligible because the high sample dilution occurring in FFF makes these concentration-dependent terms very small. From the above equation, molar mass and radius of gyration RG (root mean-square radius) can be calculated for each elution time. The accuracy in M and RG determination is, however, dependent on a number of factors spanning from the absence of spurious signals from extraneous materials (impurities) to the method applied for data reduction [29]. It is implicit in the above discussion that the exact values of different constants in Eqs. (2) and (3) are well known and effectively invariable at least under the experimental conditions used. www.jss-journal.com
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3 Experimental The stock solutions of 2 g/L of alginate isolated from M. pyrifera (Sigma) were prepared in Milli-Q water. Stock solution was filtered on a 0.22 lm pore size filter of PVDF (Millipore) and stored at 48C for a maximum of 10 days. The experimental solutions containing between 0.25 and 1 g/L were prepared by further dilution.
3.1 aFlFFF – DRI – LS setup Experiments were carried out with an aFlFFF system (AF2000 Focus, PostNova Analytics) coupled online to seven-angle laser light scattering (PostNova Analyitics) and DRI (Shodex RI 101) detectors. System control as well as data collection and treatment were performed using the AF2000 Control software (version 1.1.011, PostNova Analytics). aFlFFF channel dimensions were: tip-to-tip length of 27.5 cm, inlet triangle breadth and length of 2.0 and 3.4 cm, respectively; and outlet triangle breadth and length of 0.5 and 1.0 cm, respectively. The spacer nominal thickness of 350 lm decreased to 302 lm when measured from the retention time of BSA using tabulated values of its diffusion coefficient. The effective spacer thickness yielded the value of 0.98 mL for the channel void volume. Calibration of the DRI detector and 908 angle LS detectors was performed with BSA (weightaverage molar mass Mw = 66 kg/mol) dissolved in the carrier, following the procedure recommended by the manufacturer. A solution of polystyrene sulfonate sodium salt (Mw = 43.3 kg/mol), which has isotropic light scattering behavior, was used to normalize the other angles of the LS.
3.2 Experimental conditions One hundred microgram of alginate were injected in the aFlFFF channel by using a 100 lL sample loop, if not otherwise stated. Focusing conditions were: focusing time tfoc = 6.5 min, inlet flow rate V_ in = 0.2 mL/min, focus flow rate V_ foc = 2.8 mL/min, crossflow rate V_ c = 2 mL/min, and outlet flow rate V_ out = 1 mL/min. Elution conditions were: crossflow rate V_ c = 0.25 mL/min, inlet flow rate V_ in = 1.25 mL/min, and outlet flow rate V_ out = 1 mL/min. A regenerated cellulose membrane with 10 kDa molecular weight cutoff (PostNova Analytics) was used as accumulation wall. To test the effect of the operating conditions on the alginate fractionation, different sets of experiments were run by varying systematically a given operating parameter while keeping the other parameters constant. (i) The effect of the field strength on the separation was tested by varying the crossflow rate from 0.1 to 1 mL/min for a constant outlet flow rate V_ out of 1 mL/min; (ii) the role of carrier ionic strength was studied by increase in
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salt concentration from 10 – 3 to 10–1 mol/L NH4Cl (pH = 8); (iii) aqueous solutions of sodium and ammonium chlorides at concentration of 5610 – 2 mol/L were compared as carrier liquid; and (iv) to verify under what conditions sample overloading can affect the alginate separation and characterization, the injected amount was increased from 25 to 200 lg. Two series of experiments were performed. In the first one, the injection loop volume was kept constant at 100 lL and the alginate solution concentration was increased from 0.25 to 2 g/L. In the second one, a constant alginate concentration of 1 g/L was used and the injection loop volume was increased from 20 to 2000 lL.
3.3 Data handling To compare the DRI and LS signals obtained under different separation conditions, the signals were normalized to the peak height. For each experimental condition, the equivalent hydrodynamic radius was determined from Eq. (1) for every point of the distribution, by employing the void time corresponding to each crossflow (calculated according to the FFF theory for asymmetrical systems and trapezoidal channels [30]). The weight-average hydrodynamic radius, RHw, was calculated as follows: RHw ¼
RRHi ci Rci
ð4Þ
where RHi is the hydrodynamic radius obtained from Eq. (1) and ci is the sample concentration for a slice i of the corresponding distribution. The radius of gyration and the molar mass were obtained from the slope and intercept of the Zimm plot, representing Kc=Rh as a function of sin2 (h/2) in Eqs. (2) and (3), by using the AF2000 software (version 1.1.011, PostNova Analytics). Mean value of 0.150 for the incremental refractive index of the sample, qn/qc, found in the literature for alginates [31] was employed. Number-average (Mn) and weight-average (Mw) molar mass, PDI as well as the weight-average radius of gyration (RGw) were determined from the respective distributions obtained from light scattering measurement according to X ci Mi M Mn ¼ X ic ð5Þ i Mi Mw ¼
X ci Mi Rci
ð6Þ
RGw ¼
Rci Ri Rci
ð7Þ
PDI ¼
Mw Mn
ð8Þ
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where ci, Mi, and Ri are the sample concentration, the molar mass, and the radius of gyration for a slice i of the corresponding distributions. The alginate recovery was determined as the ratio of the sample mass eluted (measured by DRI) over the injected mass.
4 Results and discussion 4.1 Effect of the crossflow rate on alginate separation When increasing the crossflow rate V_ c from 0.1 to 1.0 mL/min for a constant outlet flow rate, the alginate retention time shifted to larger values (Fig. 1), as expected from Eq. (1). It is noted here that the dependence of t0 on the inlet and crossflow rate (Fig. 1, inset) in the aFlFFF system [32] smoothed slightly the effect of V_ c on the retention time for high values of crossflow rate. Data presented in Fig. 1 were collected until no signal was detected by the DRI even though some larger size material was still eluting out of the channel after 40 min as evidenced by the LS signal. As the crossflow rate increased, alginate macromolecules migrated closer to the accumulation wall, which resulted in a longer retention in the channel and larger retention times, as evidenced by the signal in both DRI and LS detectors. A pronounced peak broadening, in particular at the high molar mass fraction was obtained at crossflow rates equal to or greater than 0.5 mL/min. Under these conditions, the LS signal corresponding to the higher retention times does not attain the baseline within 40 min. Since data cannot be processed in the absence of the concentration detector signal, LS longer acquisition time would have been useless. The shift of the LS signal toward larger sizes clearly suggests that the lower sample mass detected by refractive index detector is compensated in the LS signal, because of the more sensitive response of LS detector at higher molar mass fraction. The resolution of alginate from the void peak improved significantly with increase in the field strength, V_ c , thus confirming a major role of the crossflow on efficient fractionation. As a matter of fact the general expression for resolution in chromatography becomes in flow FFF a higher order function of V_ c [33]. Therefore, much higher resolution might be achieved at higher crossflow velocity, but this often occurs on penalty of reduced sample recovery [24, 34]. Particular attention should be paid to the fact that in this instance proportionate recovery [35] may not be obtained for larger molar mass species since they are less recovered than smaller ones. This phenomenon is of particular importance in the characterization of polydisperse samples since it affects size distribution, hence also the average value of the obtained molecular characteristics. There-
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Figure 1. Normalized DRI (a) and 908 LS (b) signals as well as molar mass distributions (c) versus elution time for crossflow rates increasing from 0.1 to 1.0 mL/min; alginate injected mass 100 lg; outlet flow rate V_ out = 1 mL/min; carrier liquid concentration 5610 – 2 mol/L NH4Cl at pH = 8. DRI and LS signals were normalized to the peak maximum.
fore, insufficient resolution at inadequate crossflow rates and loss of some species not proportionate to their original content may also affect the measured polydispersity. Unresolved components or lost fractions that are originally present in a broadly distributed sample do not contribute or only partially determine the PDI. Both these effects might yield to an underestimation of the sample polydispersity imputable to higher crossflow rates. Examination of the distributions and average molar mass, average radius of gyration and hydrodynamic radius, sample recovery, and polydispersity obtained from fractograms in Fig. 1a and b validated the previous general considerations. For example, molar masses determined for the crossflow rates of 0.1 and 0.25 mL/min were largely distributed in the range from 50 to 800 kg/ mol (Fig. 1c). Molar mass increased steadily with the elution time as expected for the normal mode of FFF operation. Moreover, a selective loss of larger fraction, specially noticeable at V_ c = 1 mL/min is clearly seen in the www.jss-journal.com
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4.2 Effect of carrier liquid concentration and composition on the alginate separation
Figure 2. Effect of crossflow rate on average molar mass Mw, average radius of gyration RGw, average hydrodynamic radius RHw, polydispersity, and relative recovery obtained from the runs presented in Fig. 1.
molar mass distributions. This may account for both the decreased average molar mass (Fig. 2a) and the lower relative recovery (Fig. 2e). Similarly, lower values of the weight-average radius of gyration are found at elevated crossflow velocities (Fig. 2b). An additional indication of loss of higher molar mass polymers was given by the release of material observed when V_ c was set to 0 at the end of the run (data not shown). The low amount of sample below the DRI LOD did not yield a response by this detector but LS molecular size sensitivity allowed registering this material. On the ground of the sample recovery results one could expect a lower polydispersity measured at higher crossflow rates. By contrast, the polydispersity increases as crossflow V_ c rose from 0.1 to 0.5 mL/min (Fig. 2d). This is probably accounted by a higher estimation of the better resolved larger macromolecules. At V_ c = 1 mL/min one can hypothesize that alginate macromolecules are adsorbed to the membrane which results in an incomplete recovery of the higher molar mass species and a decrease in the determined PDI. In addition, crossflow rates greater than 0.5 mL/min could induce the formation of alginate aggregate, which probably did not elute completely from the channel, thus leading to incomplete recovery and biased alginate characteristics. The increased noise in LS signal observed at V_ c greater than 0.5 mL/min gives a support of this hypothesis.
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The behavior of highly charged polyelectrolytes, such as alginate, in flow FFF (in the absence of other effects) depends on the molecular volume resulting in intramolecular and intermolecular repulsions [10, 35, 36] (and references therein). Consequently, the ionic strength of the carrier liquid determined from the concentration of the salt solution is of utmost importance in the highly charged polyelectrolytes as the alginate. For instance, when the carrier liquid concentration is not sufficient to screen completely the polyelectrolyte charged moieties of alginate, different phenomena may interfere with the alginate elution, thus interfering with the determination of the meaningful macromolecular parameters. Indeed, at lower carrier liquid concentrations, corresponding to lower ionic strength, the electric double layer on the membrane become more diffuse, thus increasing the equilibrium height of the alginate in the channel and resulting in faster elution. It is evident that at the lowest concentration studied (10 – 3 mol/L), the preponderant volume exclusion effects, emphasized by the alginate stiff conformation in this medium, hamper retention so that most of the sample is not resolved from the void peak. This is well demonstrated in the narrow and biased molar mass distribution at 10 – 3 mol/L NH4Cl (Fig. 3c). An increase of the carrier liquid concentration to 5610 – 3 and 10 – 2 mol/L improved the alginate retention; however, a salt concentration of 5610 – 2 mol/L was necessary to obtain a well-retained peak, almost invariant even when carrier concentration was raised to 10–1 mol/L. The above observation is in good agreement with the experimental results for the other charged macromolecules such as sodium hyaluronate [18]. In contrast, electrostatic double layer at the membrane surface decreased with increase in the carrier concentration, allowing alginate to migrate closer to the accumulation wall and to be retained in the channel longer. Indeed, the alginate retention time extended significantly to the longer time scale as carrier concentration increased from 10 – 3 to 10 – 1 mol/L (Fig. 3). Moreover, the elution profiles in both the DRI and the LS detectors broadened for the carrier concentration equal to or greater than 5610 – 2 mol/L, with important shift corresponding to the increase in the amount of the eluted larger molecules. Although a comparative analysis of alginate molecular characteristics obtained from LS under different concentrations would be very informative, it cannot be done correctly for this polysaccharide at the lower carrier concentrations, because Eqs. (2) and (3) are invalid for polyelectrolyte in solutions of low-salt concentration. Under these conditions, corrections to account for interactions arising from charge fluctuations should be introduced
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Figure 4. Comparison of the normalized DRI (a) and 908 LS (b) elution profiles for 5610 – 2 mol/L ammonium and sodium chlorides as carrier liquids; alginate injected mass 100 lg; outlet flow rate V_ out = 1 mL/min; crossflow rate V_ c = 0.25 mL/ min. DRI and LS signals were normalized to the peak maximum. Figure 3. Normalized DRI (a) and 908 LS (b) elution profiles as well as molar mass distributions (c) as a function of the carrier concentration; alginate injected mass 100 lg; outlet flow rate V_ out = 1 mL/min; carrier liquid concentrations NH4Cl at pH = 8; crossflow rate V_ c = 0.25 mL/min. DRI and LS signals were normalized to the peak maximum.
[28]. Furthermore, the lack of efficient separation between different alginate fractions and the void peak at very low retention levels (observed at carrier concentration range of 10 – 3 – 10 – 2 mol/L) would result in biased average hydrodynamic radius and radius of gyration, molar mass distributions, and average values. It is worth mentioning that almost total sample recovery (97%) was obtained at 10 – 3 mol/L NH4Cl as a carrier, since for an unretained sample, the subtractive interactions such as adsorption on the accumulation wall are very unlikely to occur. A noticeable decrease in the amount of recovered sample to l79% was registered, when the concentration rose to 5610 – 2 mol/L. To test the effect of a salt used to prepare carrier solution, and in particular that of the type and charge of the salt cation, alginate fractogram obtained in 5610 – 2 mol/ L NH4Cl was compared to that obtained in 5610 – 2 mol/L NaCl as a carrier (Fig. 4). No significant difference in the elution profiles and alginate characteristics was found. Furthermore, alginate elution was strongly affected
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when CaCl2 with concentration of 5610 – 2 mol/L was employed as a carrier. In fact, no sample was released from the channel even at 0 crossflow, or sample eluted out in the void peak. This observation is consistent with the formation of a gel-like phase known to occur in the presence of high concentration of Ca2+ ions [5].
4.3 Effect of injected sample mass As in all separation techniques, the amount of sample to inject in the FlFFF separation channel is a trade off the need to work under ideal conditions and the requirements for a measurable detector signal. The latter is chiefly strengthened in LS detection which requires relatively high amount of sample, in particular when dealing with samples of low molar mass. The study of the effects of injected mass is therefore particularly important when a FFF system is coupled to a LS detector. aFlFFF experiments with increase in the sample load were carried out by injecting the same volume (100 lL) of sample solutions at increasing concentration. Such experimental design allowed to keep constant band broadening due to injection volume and time. In theory, the retention of the eluting particles or molecules in normal mode FlFFF should dependent only on www.jss-journal.com
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Figure 6. Alginate retention time versus injected mass for increasing carrier liquid concentrations: (0) 10 – 2, (9) 3610 – 2, (j) 5610 – 2 mol/L NH4Cl at pH = 8; outlet flow rate V_ out = 1 mL/min; crossflow rate V_ c = 0.25 mL/min. Error bars are given when bigger than the symbol size.
Figure 5. DRI (a) and 908 LS (b) elution profiles, and molar mass distributions (c) for increasing injected mass of alginate. Crossflow rate over elution time is given; loop volume 100 lL; carrier liquid concentration 5610 – 2 mol/L NH4Cl at pH = 8; outlet flow rate V_ out = 1 mL/min.
their diffusion coefficients [37] and is invariant with sample concentration and injected mass. In practice, the influence of the particle effective volume was recognized very early. Peak distortion and variation in elution profiles with the increase in the sample load were reported for various samples [23, 24, 26, 36, 38]. This effect termed overloading was found to depend on sample characteristics as well as on the composition of the carrier solution. For samples in aqueous solutions, a clear difference between polyelectrolytes and neutral polymers was found: the former eluting earlier [18] as sample concentration increased and the latter following an opposite pattern [26]. Inspection of the DRI profiles (Fig. 5a) reveals a general slight shift of tr toward higher values with increase in the injected mass, a behavior similar to that observed for the neutral polymers such as pullulan [26]. Although a polyelectrolyte, at the salt concentration of 5610 – 2 mol/L
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used in these experimental series, alginate charges seem to be completely shielded. To explore further the hypothesis that alginate behaves as a neutral polymer, the variation of the retention time with the injected mass obtained in the 5610 – 2 mol/L of ammonium chloride as carrier liquid was compared to that obtained in 10 – 2 and 3610 – 2 mol/L. In the case of carrier liquid concentration lower than 3610 – 2 mol/L, a shift of the retention times toward lower values was observed when increasing the injected mass (Fig. 6), as could be expected for the charged macromolecules. The opposite tendency was found for 5610 – 2 mol/L NH4Cl as a carrier. Therefore, a complete shielding of the alginate charges seems to occur when the concentration of the carrier liquid is higher than 5610 – 2 mol/L. The difficulty in analyzing the effect of sample load on the sample retention and decoupling the various mechanisms that may contribute to it, appears from the data on alginate recovery, increasing for higher sample load (data not shown). Assuming adsorption of alginate on the membrane as the main reason for incomplete recovery, the higher relative recovery observed for larger injected amounts could be explained by the formation of a thin layer of molecules adsorbed on the accumulation wall, which might prevent further adsorption of the sample [39]. The adsorption of alginate on accumulation membrane was evidenced by the release of a small amount of high molar mass material from the channel detected by the LS detector, when the crossflow was set to 0 (Fig. 5). These small peaks were superimposed when normalizing fractograms (graphics not shown), demonstrating that always the same percentage of sample was released after reducing the field strength to 0. Hence the appearance of these small peaks can be related to crossflow rate applied, rather than mass loading. Although a good resolution obtained with low alginate load, difficulty in detection due to the low signal www.jss-journal.com
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and incomplete recovery may bias evaluation of the radius of gyration and average molar mass for small sample amounts. Indeed, too low average radius of gyration (approx. 30 nm) was obtained for an injected mass of 25 lg. For injected mass of 100 lg or higher, RGw increased to approximately 55 nm and became independent of the sample load. The lack of significant overloading in the sample mass range can be seen in the fairly good overlay of molar mass distributions (Fig. 5c). S/N was calculated as the ratio of the alginate peak maximum and the SD of the baseline signal for both DRI and LS detectors. Under the working conditions, S/N for the DRI was always greater than 100, for injected mass between 25 and 200 lg. However, the S/N of the LS signal was lower than 10, considered as the quantification limit, when 25 lg of alginate were injected. Therefore, injected masses of 100 lg are preferred to ensure sufficiently high S/N for the calculation of correct molecular parameters by LS. In other experiments performed at constant injected mass (100 lg), an increasing of the injected volume from 250 to 1000 lL resulted in overlapped fractograms. This demonstrates that no aggregation phenomena due to increased alginate concentration took place either in the solution or during the focusing time (data not shown) as well as that the peak broadening due to the increased injection time was negligible. Overall, following the systematic variation of the elution parameters, a crossflow rate of 0.25 mL/min, carrier solution containing 5610 – 2 mol/L ammonium or sodium chloride, and 100 lg of injected mass of alginate were found as optimal. Alginate molecular characteristics obtained from the aFlFFF-DRI-LS under the optimal conditions were Mw = 180 kg/mol, RGw = 50 nm, RHw = 28 nm, PDI = 1.4, and recovery = 77%. The obtained values were in good agreement with the available literature data [31, 40, 41] as well as our previous work on alginate performed in the absence and presence of Pb or Cd [42]. Finally, the detailed knowledge of the alginate behavior in the aFlFFF is of primary importance not only for establishing the correct experimental conditions, but also for better understanding of the dynamics of the separation and detection, with the aim to manage the system even in situations which impose working on the extremes of the ranges of variation.
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nate, the crossflow rate, salt concentration of carrier solution, and injected mass were varied systematically. Combined information from DRI and LS detector signals, obtained over the wide range of the experimental conditions, allowed to improve our knowledge on the behavior of the alginate in the aFlFFF channel and to set a combination of optimal parameters. It was found that among the studied parameters, the crossflow rate and the salt concentration of the carrier liquid were critical for the efficient separation and extraction of meaningful polysaccharide characteristics. It was found that the crossflow rate of 0.25 mL/min, carrier salt concentration of 5610 – 2 mol/L ammonium or sodium chloride, and 100 lg of injected amount in the channel were necessary to ensure sufficient retention and at the same time to avoid the loss of some fractions not proportionate to the original content. The weight-average hydrodynamic radius, radius of gyration, and molar masses, obtained under optimized conditions were in accordance to those found for alginates in the literature. Warm thanks are extended to the Swiss National Science Foundation (grant PP002-102640) for providing funds directly related to the present work. M. A. B. thanks the EPFL for providing funds for her academic visit.
6 References [1] Moe, S., Draget, K., Skjakbraek, G., Smidsrod, O., Carbohydr. Polym. 1992, 19, 279 – 284. [2] Khotimchenko, Y. S., Kovalev, V. V., Savchenko, O. V., Ziganshina, O. A., Russ. J. Mar. Biol. 2001, V27, S53 – S64. [3] Fraunhofer, W., Winter, G., Eur. J. Pharm. Biopharm. 2004, 58, 369 – 383. [4] Davis, T., Volesky, B., Mucci, A., Water Res. 2003, 37, 4311 – 4330. [5] Stokke, B., Draget, K., Smidsrod, O., Yuguchi, Y., Urakawa, H., Kajiwara, K., Macromolecules 2000, 33, 1853 – 1863. [6] Larsen, B., Salem, D., Sallam, M., Mishrikey, M., Beltagy, A., Carbohydr. Res. 2003, 338, 2325 – 2336. [7] Apoya, M., Ogawa, H., Nanba, N., Bot. Mar. 2002, 45, 445 – 452. [8] Hassellv, M., Von Der Kammer, F., Beckett, R., in: J. R. Lead, K. J. Wilkinson (Eds.), Environmental Colloids: Behaviour, Structure and Characterization, John Wiley & Sons, Ltd, Chichester 2007, pp. 223 – 276. [9] Kowalkowski, T., Buszewski, B., Cantado, C., Dondi, F., Crit. Rev. Anal. Chem. 2006, 36, 129 – 135. [10] Benincasa, M., Cartoni, G., Delle Fratte, C., J. Chromatogr. A 2002, 967, 219 – 234. [11] Picton, L., Bataille, I., Muller, G., Carbohydr. Polym. 2000, 42, 23 – 31.
5 Concluding remarks The capabilities of an aFlFFF coupled online with DRI and LS detectors as a tool for separation and characterization of the linear charged polysaccharide alginate were examined. To achieve a reliable fractionation and determination of the meaningful characteristics of the algi-
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