Lignosulfonate Separation Using Preparative Column

ARTICLE pubs.acs.org/IECR

Lignosulfonate Separation Using Preparative Column Chromatography Xinping Ouyang,† Pan Zhang,† Xueqing Qiu,*,† Yonghong Deng,† and Pu Chen*,‡ † ‡

School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, P.R. China, 510640 Department of Chemical Engineering, University of Waterloo, Waterloo, Canada, N2L3G1 ABSTRACT: A two-step chromatographic process was developed for the fractionation of lignosulfonate (LS), a main component of sulfite effluence. LS fractions with low polydispersity were obtained, which enables detailed investigation into the relationship between structure and property of LS with different molecular weight (Mw). Sephacryl S-100 was used for LS separation in the first step, and then Sephadex LH-20 and Sephacryl S-100 were chosen for further separation of LS fractions with Mw below and above 6000 Da, respectively. Although the mechanism of column chromatographic separation of LS was mainly dependent on its molecular size, 0.20 mol/L of NaCl or NaNO3 solution was needed in the mobile phase at pH 5.7 to minimize electrostatic interaction and polyelectrolyte effect. Compared with the polyelectrolyte effect, the electrostatic interaction was more important in the LS separation. Because the LS fractions of higher Mw had a characteristic of low negative charge, a mobile phase with high flow rate and high pH value was suitable for separating LS fractions with high Mw.

’ INTRODUCTION Currently, the chemical utilization of lignin derivatives is highly attractive because of environmental concerns, resource shortage, and the concept of sustainability for chemical industry.1,2 However, only a small amount of lignin derivatives (ca. 2%) are isolated from spent pulping liquor and commercialized as lignosulfonates or kraft lignin.3 Lignosulfonate (LS), the second most abundant lignin derivative after kraft lignin, possesses a certain degree of surface activity4 because of its hydrophobic hydrocarbon backbone of C6 C3 and hydrophilic sulfonic group, and hence can be used as dispersants for pesticides, dyestuff, coal-water slurries,5 concrete, and recirculating cooling water.6 In addition, LS is also used for producing vanillin and other high value-added phenolic compounds because it is considered to be constituted by three structural monomers: guayacryl, syringyl, and p-hydroxyphenyl.7 One of the key issues to higher efficiency utilization of LS is to understand the relationship between its structure and properties. However, derived from plants, the LS molecular structure is very complex with a broad molecular weight (Mw) distribution, and the properties of LS can vary depending on its Mw and Mw distribution.8 Moreover, higher polydispersity of LS (polydisperisty is defined as the ratio of weight-average to number-average molecular weight) is generally considered to hinder the investigation of the structure property relationship, and further prevents modification and industrial application of LS.9 Therefore, much effort has been focused on the separation and purification of LS. Ceramic membranes of different cut-offs (5, 10, and 15 kDa) were used to fractionate lignin. Different fractions obtained by ultrafiltration exhibited that the polydispersity of separated lignin varied from 1.87 to 3.10.10 Solvent extraction was also reported to effectively fractionate LS by polarity of solvent system. Isopropanol water solutions with isopropanol to water ratios at 100:0, 80:20, 60:40, 40:60, and 0:100 were used to extract ammonium lignosulfonate from a thimble in a Soxhlet apparatus at ambient temperature and yielded five LS fractions with different Mw.11 Because LS can be transferred into r 2011 American Chemical Society

Table 1. Molecular Weight and Content of Sulfur and Main Function Groups of Lignosulfonate molecular weight and

content of sulfur andmain

polydispersity

function groups (wt %)

Mw sample lignosulfonate

phenolic

(Da)

polydispersity

sulfur

carboxyl

hydroxyl

11048

4.554

6.991

7.731

2.200

lignosulfonic acid amine adducts with long-chain amines, dicyclohexylamine, and water were used to extract LS by liquid liquid extraction. The polydispersity of extracted LS can be 4.2 after double extraction.12 Another method for the separation and fractionation of LS was performed using a supported liquid membrane with fatty alcohol as solvent and amine as carrier. It was found that the fractionation was dependent on both solvent and the structure of the amine, and the fractionation could be controlled by choosing a suitable amine organic solvent combination.13,14 In general, these research studies focused on improving the property of separated LS rather than decreasing its polydispersity, and therefore the resulting fractions separated were still LS mixtures with diverse Mw. Gel chromatography is known to be efficient for investigation of Mw parameters of polymers. Currently, this powerful method is mostly limited to the analysis of LS to provide a qualitative description of Mw and its distribution.15 Sephadex is the most widely used column packing material in size exclusion chromatography, and a lot of research involves the separation of protein and peptide, in which the separation ranges for different types of Sephadex have been established.16 Only one report was found to Received: May 6, 2011 Accepted: August 9, 2011 Revised: August 8, 2011 Published: August 09, 2011 10792

dx.doi.org/10.1021/ie200975e | Ind. Eng. Chem. Res. 2011, 50, 10792–10799

Industrial & Engineering Chemistry Research

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Figure 1. Separation of LS with Sephadex LH-20 (a) and Sephacryl S-100 (b) as the stationary phase, respectively (0.20 mol/L NaNO3 solution with pH 5.7 as the mobile phase at 30 °C, 0.60 mL/min flow rate of elution, 2.0 mL injection volume).

Table 2. Mw and Polydispersity of LS Fractions after TwoStep Separation with Sephadex LH-20 As Stationary Phase

a

elution time/min

Mna/Da

Mw /Da

polydispersity

sample

8161

13066

1.601

0

8669

14024

10

8245

12904

20

7669

30 40

7311 6666

Table 4. Mw and Polydispersity of LS Fractions Obtained by Two-Step Chromatographic Separation with S-100 As Stationary Phase elution time/min

Mw /Da

Mna/Da

polydispersity

1.618

sample

45106

25643

1.759

1.565

0

54510

73114

1.341

11760

1.533

15

36248

45647

1.259

11193 9974

1.530 1.496

30

27935

34239

1.226

45

23035

27984

1.215

60

19065

22685

1.190

75

15234

18251

1.198

90 105

12907 10980

15153 12792

1.174 1.165

120

9280

10853

1.169

135

7916

9280

1.172

150

6301

7404

1.175

165

4376

5507

1.258

Mn represents number-average molecular weight.

Table 3. Mw and Polydispersity of LS Fractions Obtained by Two-Step Chromatographic Separation with LH-20 As Stationary Phase elution time/min

a

Mw /Da

Mna/Da

polydispersity

sample

2916

2637

1.106

0

3620

3070

1.179

10

3337

2828

1.180

15

3223

2898

1.112

20

3063

2820

1.086

25

2900

2715

1.068

30 35

2778 2691

2628 2562

1.057 1.050

’ EXPERIMENTAL SECTION

40

2568

2463

1.042

45

2517

2399

1.049

50

2465

2301

1.071

Materials. Lignosulfonate (LS), obtained from Huawei Youbang Chemical Ltd., China, was derived from byproduct of sulfite pulping. Before chromatographic separation, LS was treated by filtration to remove impurities and then by dialysis to remove inorganic salts. Deionized water and analytical grade inorganic salts were used in this work. The relative weight-average molecular weight (Mw), polydispersity, and the content of sulfur and main functional groups of LS are listed in Table 1. Gel Column Chromatographic Separation. The gel column chromatographic separation was conducted in an AKTA Prime chromatographic separation system (General Electric Company, USA). In brief, 2.0 6.0 mL of 20 wt % LS solution was injected into a 1.6 cm I.D. 70 cm long column packed with Sephadex LH-20 or Sephacryl S-100 gel (General Electric Company, USA), and was eluted with a mobile phase with pH of 3 9 at 20 30 °C and a flow rate at 0.20 0.60 mL/min. The mobile phase was 0.02 0.20 mol/L of NaNO3 solution, or a solution of NaCl, Na2SO4, or CaCl2 at 0.20 mol/L. Eluate was collected in


involve gel chromatographic separation of acetosolv lignin with a gel column packed with LH-20 gel and eluted with 90% dioxane solvent; lignin fractions with polydispersity from 1.16 to 1.29 were obtained.17 However, there are no fundamental data to guide the choice of the stationary phase for the column chromatographic separation of LS. In addition, little consideration has been given to the role of process parameters, especially in the context of practical semipreparative or preparative gel chromatographic method development. In the present work, the process variables of the separation of LS by preparative gel column chromatography were investigated to explore crucial factors affecting separation and obtaining LS fractions with low polydispersity.

a


The resulting LS fractions are expected to be suitable for further investigation of the relationship between structure and property of this complex natural polymer.

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dx.doi.org/10.1021/ie200975e |Ind. Eng. Chem. Res. 2011, 50, 10792–10799


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Figure 2. Influence of mobile phase on the separation of LS (Sephacryl S-100 as the stationary phase, 0.20 mL/min flow rate of elution, 2.0 mL injection volume, 5.7 pH of mobile phase at 30 °C).

Figure 3. Influence of concentration of NaNO3 on the zeta potential of Sephacryl S-100 gel (weight ratio of gel to water is 1:50).

test tubes (3.0 mL/tube) by a distribution collector, and time zero corresponded to the first drop of eluate flowing out. Determination of Relative Molecular Weight and Polydispersity. The Mw and polydispersity of LS after the gel column chromatographic separation were determined by a Waters 1515 gel permeation chromatograph instrument (Waters Corp., USA). The chromatographic column was made up in series by Ultrahydragel 120, Ultrahydragel 250, and Ultrahydragel 500 columns with pore sizes of 120, 250, and 500 Å, respectively. NaNO3 solution (0.10 mol/L, pH 8.0) was used as a mobile phase at a flow rate of 0.50 mL/min. The concentration of samples was 0.3 wt %, and the injection volume was 50 μL. The sample and eluant were filtered through 0.45 and 0.22 μm hydrophilic syringes prior to injection, respectively. The effluent was monitored at 280 nm with a Waters 2487 UV-detector, and the columns were calibrated with sodium polystyrene sulfonate in the 1260 78 000 Da range with a correlation coefficient (R2) at 0.9996. The signal detected was digitized at a frequency of 2 Hz, and the Mw and polydispersity were calculated from the recorded signal using normal GPC calculation procedures.18 Determination of Hydrodynamic Diameter of LS and Zeta Potential of Sephacryl S-100 Gel in Water. The hydrodynamic diameter of LS in water was determined using dynamic laser light scattering with a digital autocorrelator (Brookhaven Co., USA) at a scattering angle of 90°, a wavelength of 633 nm, and a temperature of 25 ( 0.1 °C. Each solution prior to the measurement was filtered with a 0.45-μm pore size syringe filter to remove dust particles.

Figure 4. Influence of concentration of NaNO3 on the hydrodynamic diameter of LS (concentration of LS is 0.5 wt %).

The zeta potential was determined using a ZetaPlus instrument (Brookhaven Co., USA) operated in electrophoretic light scattering mode. The weight ratio of Sephacryl S-100 gel to water (with and without addition of salt) is 1:50. The zeta potential was calculated automatically from the determined electrophoretic mobility via Smoluchowski’s equation.19 Sulfur Elemental Analysis of LS. After desalination via dialysis and drying under vacuum as well as with P2O5, the separated LS was subjected to elemental analysis on a PE 2400 Series II CHNS/O analyzer (Perkin-Elmer. Corp., USA) at the combustion temperature of 1150 °C. Approximately 3 mg of the sample was put on a tin foil and analyzed in air using oxygen as a combustion gas (at a flow rate of 25 mL min 1) and helium as a carrier gas (at a flow rate of 200 mL min 1).The resulting content of sulfur represented the content of sulfonic group.

’ RESULTS AND DISCUSSION Choice of Stationary Phase. The chromatographic separation with Sephadex LH-20 or Sephacryl S-100 as the stationary phase is presented in Figure 1. It can be seen from Figure 1a that LS fractions separated with Sephadex LH-20 as the stationary phase have a polydispersity of less than 1.2 when the Mw of LS ranges from 2000 to 6000 Da. When the Mw of LS is less than 2000 Da or more than 6000 Da, the polydispersity of the separated LS tends to increase, indicating that Sephadex LH-20 provides a good chromatographic selectivity for LS with Mw ranging from 2000 to 6000 Da. In contrast, as shown in Figure 1b, Sephacryl S-100 gel is suitable for 10794



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Figure 5. Influence of inorganic salt on the separation of LS (Sephacryl S-100 as the stationary phase, 0.20 mol/L of the concentration of mobile phase, 0.60 mL/min flow rate of elution, 2.0 mL injection volume, pH 5.7 of mobile phase at 30 °C).

separating a broader Mw range of LS fractions, giving polydispersity of around 1.2 for Mw from 2000 to 20 000 Da. Compared with Sephacryl S-100, Sephadex LH-20 is more suitable for separating LS fraction with low Mw, yielding LS fractions with slightly lower polydispersity. Lignosulfonate with Mw of 13 066 Da and polydispersity of 1.601 collected after one-step chromatographic separation is further separated once again using Sephadex LH-20 gel as the stationary under the same conditions, and the results are shown in Table 2. Table 2 presents that the polydispersity of the fractions collected with Mw ranging from 9974 to 12 904 Da varies from 1.496 to 1.565 after the two-step chromatographic separation. These results suggest that for the LS with large Mw, it is very difficult to obtain LS fractions with low polydispersity using Sephadex LH-20 as the stationary phase, even though repetitious separation is carried out. The reason is that the pore size of the Sephadex LH-20 gel is compatible with the molecular size of LS with the Mw ranging from 2000 to 6000 Da. While the Mw of LS is more than 6000 Da, total exclusion occurs, resulting in the separated LS possessing a larger polydispersity. However, when LS with Mw of 2916 Da (polydispersity of 1.106) and with Mw of 45 106 Da (polydispersity of 1.759) obtained by one-step chromatographic separation is separated for the second time with Sephadex LH-20 and Sephacryl S-100 as the stationary phase, respectively, optimal separation results are obtained. When Sephadex LH-20 is used for separation of LS with lower Mw, a nearly monodisperse LS fraction is obtained after the two-step chromatographic separation, and the lowest polydispersity is as low as 1.042 (Table 3). When Sephacryl S-100 is used to separate LS with larger Mw, the polydispersity of LS fractions after two-step separation decreases greatly (Table 4). As discussed above, Sephacryl S-100 is suitable for separation of LS within a wider Mw range, and Sephadex LH-20 is better applied for separating LS with low Mw. The polydisperisty of LS decreases greatly with two-step separation. To obtain the LS fractions with low polydispersity, Sephacryl S-100 can be used to separate LS for the first time, and then Sephadex LH-20 and Sephacryl S-100 can be chosen for further separation of LS fractions with Mw less than 6000 Da and more than 6000 Da obtained by one-step separation, respectively. Based on the results obtained from the stationary phase and its suitable fractionation range, it seems to deduce that the fractionation of LS is dependent on its molecular size: the small molecule permeation and the large molecule exclusion. However, it is

Table 5. Influence of Inorganic Salt (0.2 mol/L) on the Zeta Potential of Sephacryl S-100 Gel in Water

zeta potential (mV)

NaNO3

NaCl

CaCl2

Na2SO4

20.64

21.56

27.65

37.81

noted that nonsize exclusion commonly exists in the separation of water-soluble macromolecules and this effect may be minimized under certain separation conditions.20 Choice of Mobile Phase. The chromatographic separation using NaNO3 with different concentrations and deionized water as the mobile phase is depicted in Figure 2. Compared with deionized water as a mobile phase, the NaNO3 solution exhibits a better separation result. The Mw of LS fractions collected with the elution time ranging from 120 to 330 min varies from 14 100 to 1800 Da, and all the resulting LS fractions have the characteristic of low polydispersity when NaNO3 is presented in the mobile phase. When the elution time exceeds 330 min, the polydispersity of the separated fractions tends to increase. LS fractions with polydispersity ranging from 1.178 to 1.210 are obtained when 0.20 mol/L NaNO3 is used as the mobile phase. To explore the influence of NaNO3 solution on the separation, the zeta potential of Sephacryl S-100 gel with the addition of different concentrations of NaNO3 was determined, as shown in Figure 3. Figure 3 shows that the zeta potential of Sephacryl S-100 in deionized water is negative, implying that the Sephacryl S-100 in water is charged negatively. Therefore, electrostatic repulsion exists between Sephacryl S-100 gel and LS molecules containing negatively charged sulfonic groups. This ionic repulsion prevents LS molecules from diffusing into the pores of the Sephacryl S-100 gel. Consequently, LS molecules with lower Mw are eluted out too quickly and therefore the separation performance is reduced.21 With the increase in the concentration of NaNO3, the value of zeta potential of the Sephacryl S-100 gel increases and passes over the value of zero at 0.12 mol/L of NaNO3. After that point, the zeta potential changes to a positive value, where the electrostatic repulsion between Sephacryl S-100 gel and LS molecules changes to electrostatic attraction. This causes the adsorption of LS molecules in the gel and a decrease in separation performance. Theoretically, it can be deduced from Figure 3 that a better separation should be obtained when the concentration of NaNO3 is 0.12 mol/L. However, Figure 2 presents that better separation performance is obtained with 0.20 mol/L of NaNO3 10795



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Figure 6. Inflence of flow rate on the separation of LS (Sephacryl S-100 as the stationary phase, 0.20 mol/L of NaNO3 aqueous as the mobile phase, 2.0 mL injection volume, pH 5.7 for mobile phase at 30 °C).

as the mobile phase than with 0.10 mol/L of NaNO3. To figure out the reason behind this, the influence of NaNO3 concentration on the hydrodynamic diameter of LS molecule is investigated and the results are plotted in Figure 4. It can be seen that the size of LS molecule decreases at low NaNO3 concentration (0.10 mol/L) and then increases with the increase in the concentration of NaNO3 (0.20 to 0.50 mol/L). It is well recognized that LS is a nonlinear anionic polyelectrolyte, and hence there exists polyelectrolyte effect caused by Coulombic interactions between charged LS chains in the aqueous solution, which can be weakened by adding an inorganic salt.22 Therefore, an additional amount of NaNO3 is needed to eliminate this electrostatic effect. Consequently, 0.20 mol/L of NaNO3 as the mobile phase gives better separation performance. It should be noted that too high a concentration of NaNO3 will result in the oversalt effect.23 As the gel chromatographic separation is strongly influenced by ionic strength of a mobile phase, the influence of different inorganic salts on the separation effect is shown in Figure 5. Figure 5 presents that the existence of an inorganic salt in the mobile phase has a strong impact on the separation, where NaCl is comparable to NaNO3 at the same concentration. However, the separation performance decreases observably when Na2SO4 or CaCl2 is used. It is obvious that the ionic strength of Na2SO4 or CaCl2 is greater than that of NaCl or NaNO3 at the same concentration. The influence of the inorganic salt added on the zeta potential of Sephacryl S-100 gel is presented in Table 5, which shows that NaCl and NaNO3 have a similar impact on the zeta potential of the Sephacryl S-100 gel. In addition, the same cation at the same concentration results in the same ionic screening effect between NaCl and NaNO3. Therefore, NaCl and NaNO3 have the same influence on the separation of LS. At the same concentration, the ionic strength of the CaCl2 or Na2SO4 solution is three times that of NaCl or NaNO3 solution according to the ionic strength calculations.24 When CaCl2 or Na2SO4 is added into aqueous suspension of Sephacryl S-100 gel, the electric double layer between gel and water is contracted due to an increase in ionic strength and zeta potential of gel, resulting in retardation of elution time and hence a worse separation performance for LS. Theoretically, divalent Ca2+ should have a stronger electrostatic screening effect than monovalent Na+,25,26 but Figure 5 presents that the retardation behavior of elution for the addition of Na2SO4 into the mobile phase is stronger than that for the addition of CaCl2. Based on this phenomenon, it can be concluded that compared with polyelectrolyte effects, ionic exclusion or

Table 6. Content of Sulfur Element of Separated LS Fractions Mw /Da

polydispersity

content of sulfur element (wt %)

4700

1.129

8.605

9800

1.107

7.042

18 700

1.173

6.153

26 700

1.219

6.033

40 200

1.368

5.656

attraction is predominated during the gel column chromatographic separation of LS. Choice of Flow Rate. Considering that the collected elution time varies with the flow rate of elution, the tube numbers instead of elution time are used to discuss the influence of flow rate on the separation. The Mw and polydispersity of LS obtained by the chromatography under different flow rates are shown in Figure 6. Figure 6 shows that the flow rate, varying from 0.20 to 0.60 mL/min, does not make any significant difference for the separation of LS with Mw from 5000 to 16 000 Da, with the polydispersity of the separated LS fractions at about 1.2. However, a flow rate at 0.60 mL/min exhibits better separation performance for the fractions collected with Mw more than 16 000 Da. In contrast, a flow rate at 0.20 mL/min gives better separation efficiency for those with Mw less than 5000 Da (corresponding to the tube number of 19 or elution time of 95 min). These results suggest that a gradient elution, with a flow rate at 0.6 and 0.2 mL/min before and after 95 min, respectively, is necessary to obtain better separation performance. The sulfur element content of separated LS fractions with various Mw was determined and the results in Table 6 show that the content of sulfur element decreases with the increase in Mw of LS, indicating that LS fractions with higher Mw possess less negatively charged sulfonic groups. Therefore, LS molecules with higher Mw exhibit weaker polyelectrolyte effects compared to those with lower Mw due to the weaker electrostatic repulsion between charged LS chains, causing a decrease in the molecular size. Therefore, the LS molecules with higher Mw pass through the gel column relatively slowly compared to the pure size exclusion. As a result, a relatively higher flow rate is necessary during the separation process for LS with higher Mw. It should be pointed out that too high a flow rate leads to incomplete partitioning, whereas too low flow rates leads to unnecessary diffusion. Choice of Injection Volume. The results of Mw and polydispersity of LS fractions obtained by the chromatography under 10796



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Figure 7. Influence of injection volume on the separation of LS (Sephacryl S-100 as the stationary phase, 0.20 mol/L of NaCl aqueous as the mobile phase, 0.60 mL/min flow rate of elution, pH 5.7 for mobile phase at 30 °C).

Figure 8. Influence of temperature on the separation of LS (Sephacryl S-100 as the stationary phase, 0.20 mol/L NaCl aqueous as the mobile phase, 0.60 mL/min flow rate of elution, 2.0 mL injection volume, pH 5.7 for mobile phase).

different injection volumes are shown in Figure 7, which suggests that the polydispersity of separated LS fractions has a general trend of increase when the injection volume increases from 2.0 to 5.0 mL. It is well-known that the injection volume should be in proportion to the size of column used. The preferential fractions cannot be separated if the injection volume is too small. Although a larger injection volume is favorable for obtaining better throughput, it causes overload of the column, decreases column efficiency, and causes distortion of elution peaks.27 Influence of Temperature on the Column Chromatographic Separation. Considering that the chromatographic separation is usually conducted at room temperature, the effect of temperature in the range from 20 to 30 °C on the separation is investigated, and the results are shown in Figure 8. Figure 8 shows that when the temperature varies within a 10 °C range, the polydispersity of separated LS exhibits slight fluctuations, especially for the fractions with Mw less than 18 000 Da. The variation of temperature will affect the effective charge numbers of LS,28,29 which influences the interaction between LS and gel, and hence the separation. However, the charge numbers of LS decrease slightly with the variation of temperature in the range 10 30 °C according to Mafe et al.30 Although raising temperature reduces the plate height by reducing the viscosity of the eluent and the mass-transfer limitation via increasing diffusion coefficients, the low viscosity also allows a much higher flow rate of the eluent with reasonable backpressure to achieve fast separation.31,32 The results presented in this work imply that the variation of temperature between 20 and 30 °C does not change the mass transfer performance observably.

Influence of pH on the Column Chromatographic Separation. The influence of pH values on the separation of LS is shown

in Figure 9. It can be seen from Figure 9 that the separation of the LS fractions with Mw of 15 000 5000 Da is slightly affected by pH at the same concentration of salt. However, in the case of the LS fractions with Mw of more than 15 000 Da containing more phenolic hydroxyl groups, the elution profile is significantly affected by pH. The same pH effect is observed for the LS fractions with Mw of less than 5000 Da containing more sulfonic groups. These results suggest that the pH of 9.0 and 5.7 for the mobile phase can be used for fractionation of LS with Mw of above and below 15 000 Da, respectively. This interesting phenomenon is also found in fractionation of apple procyanidins using size exclusion chromatography by Yanagida,33 in which the fractionation is affected by pH for polymerized oligomers containing more phenolic hydroxyl groups, but not for monomeric and dimeric procyanidins. Unfortunately, there has been no further investigation into the mechanisms of this phenomenon. The influence of pH on the solution behavior of LS was reported earlier by our group,34 showing that a negative value of zeta potential and the size of LS increase with the increase of pH. This implies that the electrostatic repulsion between gel and LS increases with increasing pH of the mobile phase. Theoretically, the separation should be worse at higher pH without an addition of inorganic salt into the mobile phase.35 However, Table 6 shows that LS with higher Mw has low negative charge, due to a low content of sulfonic groups, and this decreases the electrostatic repulsion between LS and gel. It is worth noting 10797



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Figure 9. Influence of pH on the separation of LS (Sephacryl S-100 as the stationary phase, 0.20 mol/L of NaCl aqueous as the mobile phase, 0.60 mL/min flow rate of elution, injection volume 2.0 mL, 5.7 pH mobile phase at 30 °C).

that the inorganic salt and pH may jointly affect the separation of LS, and a further investigation is needed to understand this complex system.

’ CONCLUSIONS Gel column chromatography is an efficient method to separate LS into fine fractions with low polydispersity. Nearly monodisperse LS can be obtained after two-step chromatographic separation. Sephacryl S-100 is used for the separation of LS in the first step, and then Sephadex LH-20 and Sephacryl S-100 are chosen for further LS fractionation with Mw less than 6000 Da and more than 6000 Da, respectively, in the second step. Because Sephacryl S-100 is negatively charged, there is electrostatic repulsion between gel and LS, in addition to the polyelectrolyte effect of charged LS molecular chains in aqueous solution. To minimize the non size exclusion effect, 0.20 mol/L of NaCl or NaNO3 is needed in the mobile phase at pH of 5.7. Although the mechanism of column chromatographic separation of LS is mainly dependent on its molecular size, the electrostatic action and polyelectrolyte effect also affect the separation of LS. Because LS fractions with higher Mw possess lower content of negatively charged sulfonic groups, which results in a polyelectrolyte effect, high flow rate and high pH value for the mobile phase are beneficial to separation of LS fractions with high Mw. ’ AUTHOR INFORMATION Corresponding Author

*Tel.: 86-20-87114968. Fax: 86-20-87114721. E-mail: xueqingqiu66@ 163.com (X.Q.Q.). Tel.: 1-519-888-4567, x 35586. Fax: 1-519746-4979. E-mail: [email protected] (P.C.).

’ ACKNOWLEDGMENT This work is financially supported by the National Natural Science Fund of China (20876064), The China Excellent Young Scientist Fund (20925622), the Guangdong Provincial Natural Science Fund (9151064101000082), and the Guangdong Provincial International Cooperation Fund (2008B05010006). ’ REFERENCES (1) Voitl, T.; von Rohr, P. R. Demonstration of a Process for the Conversion of Kraft Lignin into Vanillin and Methyl Vanillate by Acidic Oxidation in Aqueous Methanol. Ind. Eng. Chem. Res. 2010, 49, 520.

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Lignosulfonate Separation Using Preparative Column

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