Gelation of Na-alginate aqueous solution: A study of sodium ion dynamics via NMR relaxometry

Accepted Manuscript Title: Gelation of Na-Alginate Aqueous Solution: A Study of Sodium ion Dynamics via NMR Relaxometry Authors: Congxian Zhao, Chao Zhang, Hongliang Kang, Yanzhi Xia, Kunyan Sui, Ruigang Liu PII: DOI: Reference:

S0144-8617(17)30373-9 http://dx.doi.org/doi:10.1016/j.carbpol.2017.03.099 CARP 12188

To appear in: Received date: Revised date: Accepted date:

23-2-2017 30-3-2017 30-3-2017

Please cite this article as: Zhao, Congxian., Zhang, Chao., Kang, Hongliang., Xia, Yanzhi., Sui, Kunyan., & Liu, Ruigang., Gelation of Na-Alginate Aqueous Solution: A Study of Sodium ion Dynamics via NMR Relaxometry.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.03.099 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Gelation of Na-Alginate Aqueous Solution: A Study of Sodium ion Dynamics via NMR Relaxometry Congxian Zhaoa,b, Chao Zhangb, Hongliang Kangb, Yanzhi Xiaa,*, Kunyan Suia,*, Ruigang Liub,* a

Collaborative Innovation Center for Marine Biomass Fibers, Materials and Textiles of

Shandong Province, School of Materials Science and Engineering, Qingdao University, Qingdao 266071, China b

Sate Key Laboratory of Polymer Physics and Chemistry, Beijing National Laboratory of

Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China Corresponding

authors, E-mail: [email protected] (Xia), [email protected] (Sui),

[email protected] (Liu)

1

Highlights 

The gelation process of SA was investigated by using NMR relaxometry and PFG NMR diffusometry.



Dynamics of the Na+ in Na-alginate aqueous solution was investigated.



The distribution of the spin-spin relaxation time of Na nuclei identified the Na+ ions in different state.



The diffusion coefficient of Na+ ions increased with the crosslinking density.

ABSTRACT. Sodium alginate (SA) hydrogels have a wide range of applications including tissue engineering, drug delivery and formulations for preventing gastric reflux. The dynamics of sodium ions during the gelation process of SA solution is critical for clarification of the gelation procedure. In this work, nuclear magnetic resonance (NMR) relaxometry and pulsedfield-gradient (PFG) NMR diffusometry were used to investigate the dynamics of the sodium ions during the gelation of SA alginate. We find that sodium ions are in two different states with the addition of divalent calcium ions, corresponding to Ca2+ crosslinked and uncrosslinked regions in the hydrogels. The sodium ions within the un-crosslinked regions are those released from the alginate chains without Ca2+ crosslinking. The relative content of sodium ions within the Ca2+ crosslinked regions decreased with the increase in the content of calcium ions in the system. The relaxation time T2 of sodium ions within the Ca2+ crosslinked and un-crosslinked regions shift to shorter and longer relaxation time with the increase in concentration of calcium ion, which indicates the closer package of SA chains and the larger space for the diffusion of free sodium ions. This work clarifies the dynamics of calcium alginate gel at the equilibrium state.

Keywords: Sodium alginate; Gelation; NMR relaxometry; Dynamics

2

23

Na+ in a

1. Introduction Hydrogels, three-dimensional polymeric networks containing a mass of water that have a wide range of applications, have attracted increasing attention (Babensee et al., 1998; Bryant & Anseth, 2001; Lloyd et al., 2001; Lowman et al., 1999; Mongia et al., 1996; Tilakaratne et al., 2007). Hydrogel materials originating from natural polymers are more attractive due to the advantages of biocompatible, biodegradable and so on (Anseth et al., 1996; Bryant et al., 2000; Burdick et al., 2001). Sodium alginate (SA) is a natural polymer extracted from seaweed and consists of -D-mannuronic acid (M) and -L-guluronic acid (G), which has merits of biocompatibility, abundance, and low prices (Draget et al., 1990; Martinsen et al., 1989). SA is an ideal biomaterial for tissue engineering, drug delivery, and in some formulations for preventing gastric reflux (Kemp & Fryer, 2007; Kuo & Ma, 2001; Li et al., 2007). SA can be easily crosslinked into hydrogel by chelating divalent cations (Fang et al., 2008; Fang et al., 2007). The divalent cations such as calcium ions can collaboratively bind between the G-units of alginate chains to form ionic inter-chain crosslinks, which result the formation hydrogels (Grant et al., 1973; Stevens et al., 2004). Ca-alginate hydrogels have investigated comprehensively and have been used as the scaffold materials for tissue engineering recently (Kuo & Ma, 2001). However, the gelation process of SA is still not fully understood, especially the changes in the dynamics of sodium ions during the gelation. Nuclear magnetic resonance (NMR) relaxation is a powerful tool to study the structure and the dynamics properties of biopolymers in solution at an atomic level (Chen et al., 2013; Marincola et al., 2001; Mussel et al., 2015; Rijniers et al., 2004). The measurement parameters are mainly spin-spin relaxation time (T2), and the apparent diffusion coefficient (Callaghan, 1991). Spin-spin relaxation process of nuclei depends on the degree of confinement of the nuclei because of the interactions of the nuclei with other nuclei and internal field gradients, which has been widely utilized in the detection of both the surrounding and distribution of the

3

nuclei (Levitt, 2008), such as in the characterization of porous materials including rocks, silica, zeolites, cements, and soils (Chen et al., 2013; Kleinberg & Horsfield, 1990; Mitchell et al., 2014). The NMR relaxation of hydrogen nuclei has been successfully being used to determine the dynamics of water (Fundo et al., 2014; Geng et al., 2013; Patural et al., 2012; Peemoeller et al., 2013). The Brownian motion of water molecules can be characterized via the relaxation of 1

H NMR signal, which was proposed by Brownstein and Tarr (Barberon et al., 2003;

Brownstein & Tarr, 1979; Valckenborg et al., 2001). Besides, the interaction of polymeric DNA with counterions was also characterized by means of 23Na NMR spectroscopy (Marincola et al., 2001), such as the investigation of the interaction in DNA via the

23

Na and 7Li spin-lattice

relaxation measurements (Hald & Jacobsen, 1991). It’s well known that SA can chelate divalent cations to form egg-shell structure, which acts as the crosslinking points in the resultant hydrogels. During the gelation procedure, the exchange of sodium ions and divalent cations is one of the key points for the controlling the gelation process and the structure of the resultant SA hydrogels. However, few work has been paid attention to the dynamics of sodium ions during the gelation procedure of SA. In this work, 23

Na NMR relaxation was used to investigate the changes in the dynamics of sodium ion in SA

with the addition of divalent calcium ions. The diffusion coefficient of Na+ ions was measured by using pulsed-field-gradient (PFG) NMR for additional evidence for the dynamics of sodium ion of SA. Combined with the rheological properties of different Ca-alginate gel, the change of sodium ions during the gelation process of SA was discussed. This work demonstrates the dynamics of 23Na+ in a calcium alginate hydrogel at the equilibrium state, which is helpful in the understanding the gelation procedure of the SA and controlling the properties and structure of the SA hydrogels.

4

2. Experimental section 2.1. Materials Sodium alginates (SA) were supplied by Qingdao Hyzlin Biology Development Co.,Ltd. Qingdao Haizhilin Company, China. The SA is completely Na+ free except for the SA counterions and the guluronic (G) to mannuronic (M) ratio is G/M = 1 that provided by the supplier and confirmed by 1H NMR measurement (Fig. S1 in Supporting Information). The molecular weight of SA is Mw = 3.15 × 105 g/mol and Mw/Mn = 2.55, which was measured by gel permeation chromatography (GPC) on a Viscotek GPCmax (Malvern Instruments Ltd, UK) using A6000M GPC column. Poly(ethylene oxide) was used as standard for GPC calibration. NaNO3/H2O (0.1 M) solution was used as the solvent for SA and eluent for GPC measurement. Calcium carbonate (CaCO3) and D-glucono--lactone (GDL) were supplied by Sinopharm Chemical Reagent, China. All the materials were used without further purification. 2.2. Sample Preparation SA was first dissolved in deionized water at room temperature with constant stirring until complete dissolution. CaCO3 in combination with GDL was used as the source of calcium ions to initiate gelation. A CaCO3 to GDL molar ratio of 0.5 was always maintained to achieve a neutral pH value (Kuo & Ma, 2001). CaCO3 and GDL suspensions were added subsequently into SA solutions with continuous stirring. After homogeneous mixing, the mixed solutions were quickly injected into the 3 mm NMR tubes by syringe and allowed stable gelation in the NMR tubes for 24 hrs. Two series samples were prepared. One is the samples with the SA concentration in the range of 1–4 wt %, and the molar ratio of calcium ions to carboxyl groups of SA is kept at 0.18, which was designated as 1× (Kuo & Ma, 2001). The other are those samples with the SA concentration of 3 wt%, and the molar ratio of calcium ions to carboxyl groups of SA are 0.09 (0.5×), 0.18 (1×), 0.36 (2×), and 0.54 (3×), by which to adjust the crosslinking density of the SA gels.

5

2.3. NMR Experimental Procedures All measurements of the transverse relaxation process of 23Na spin of SA in the samples were carried out on a Bruker AVIII 500 spectrometer using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence at 25°C (Meiboom & Gill, 1958). The spin-spin relaxation time (T2) of the 23Na spin of SA was firstly estimated according to FID signal in the NMR spectrum. Then the parameters of the pulse sequence were optimized based on the estimated T2 for accurate detection of different T2 components (CPMG τ time of 0.25 ms and 41 data points was used). Multi-exponential decay transverse relaxation was de-convoluted into individual components by using the inverse Laplace transform (ILT) algorithm (Akin & Counts, 1969; Chen et al., 2013; Hansen, 1992; Hetnarski, 1975; Provencher, 1982; Salzer, 1953; Titus, 1955). This inversion method has been widely used in the systems of Castle-gate sandstone (Washburn & Callaghan, 2006), polymer coatings (Zhu et al., 2013), super-absorbing materials (Wu et al., 2006), cellulosic materials (Zhang et al., 2016), and so on. The data analysis was executed in MATLAB 8.2.0 (Math Works Inc., Natick, MA) with a homemade program. The details of the data processing refer to our previous work (Zhang et al., 2016). 2.4. PFG NMR Diffusometry PFG NMR experiments were performed on a Bruker AVIII 500 spectrometer. The robust and easily pulsed-gradient stimulated-echo sequence (PGSTE) was applied for all diffusion measurements at 25 °C. The PGSTE sequence used gradient pulse duration δ of 1−2 ms, diffusion time Δ of 10−20 ms, and the number of scans for each step was adjusted from 1 to 64 to ensure good signal-to-noise ratio (SNR). All parameters for the gradient have been calibrated and optimized according to previous reports (Lafitte et al., 2007; Li et al., 2009). The NMR signal attenuation due to diffusion follows Stejska–Tanner equation (Price, 1997). 𝐼 = 𝐼0 𝑒 −𝐷𝛾

2 𝐺 2 𝛿 2 (∆−𝛿/3)

(1)

where I and I0 refer to the spin–echo signal intensity and spin–echo signal intensity at zero

6

gradients, respectively. γ is the gyromagnetic ratio of the nucleus, G is the gradient strength, δ is the pulse duration, and Δ is the diffusion time. Thus, diffusion coefficients (D) of a desired nucleus can be obtained by fitting the experimental I vs G data using eq. 1. 2.5. Rheological Measurements The rheological measurements of all samples were carried out at room temperature with HAAKE MARS Rheometer equipped with a 20 mm standard steel parallel plate to determine the viscosity of samples.

3. Results and discussion In the SA solutions, the divalent calcium ions released from CaCO3-GDL can collaboratively link the G-units of alginate chains, which result the gelation of the SA solutions via Ca2+–Na+ ion exchange (Kuo & Ma, 2001). Therefore, the sodium ions in the system may have two states after the release of calcium ions. One could be the sodium ions in the Ca2+ crosslinked regions and the other is the sodium ions in the un-crosslinked regions. The dynamics of the sodium ions in SA solutions were first investigated. Fig. 1 shows the spin-spin relaxation of 23Na nuclei in the solutions with different SA concentration. The measuring parameters used in this work assured that the CPMG intensity can completely decay to 0 for the samples. Moreover, the redundant data points have no effect on the fitting results of T2. The spin-spin relaxation process is characterized by the speed of spin-spin relaxation (1/T2), the inverse of the spin-spin relaxation time (T2). The overall T2 of the

23

Na spins in the SA solutions was calculated by

fitting the raw relaxation data in Fig. 1 using a single exponential function (solid line) and the results are shown in Fig. 1e as a function of SA concentration. According to the results, the overall spin-spin relaxation time (T2) of 23Na reduces with the increase in the SA concentration in the samples. The result is due to that the SA chains become crowd with the increase in SA concentration in the system. Therefore, the vibration frequency of sodium ions accelerates with the increase in the chain entanglement and the decrease in the distance between the SA chains. 7

Inverse Laplace transformation calculation of the raw relaxation data (Fig. 1) results the relaxation time distribution spectra of 23Na spins in SA solutions with different concentration. Fig. 2 shows the NMR relaxation time distribution of 23Na spins. The results indicate that there is only one peak in the relaxation time distribution spectra of 23Na nuclei in the solutions with different SA concentration, which indicates that all the sodium ions in the SA solutions are in the same state. Moreover, the peak position shifts to smaller T2, which is similar to the overall T2 of 23Na spins obtained by single exponential fitting (Fig. 1e). Fig. 3a shows the spin-spin relaxation of 23Na nuclei in SA solution and SA solutions with the additives of CaCO3, GDL, and CaCO3–GDL. The intensity of the data was normalized via I/I0 for easy comparison. It was found that the decay curves of the 23Na nuclei in the solutions of SA, SA–CaCO3, and SA–GDL are substantially overlapped each other. While the decay curve of 23Na spin in SA–CaCO3–GDL system is different to the others. The results are due to that there is no divalent cations to crosslink the SA chains in the solutions of SA, SA–CaCO3, and SA–GDL. While in the SA–CaCO3–GDL solution, Ca2+ cations can release from the CaCO3–GDL system, which can exchange with the Na+ ions to form crosslinks via the interaction with the G-units of SA. Moreover, the plots of ln(I/I0) vs. t were also drawn (Fig. S2 in Supporting Information). The plots depicted that the data point are decay linearly at in the short decay time (< 10 ms). The spin-spin relaxation time (T2) distribution of 23Na nuclei was calculated by using inverse Laplace transformation from the raw relaxation data (Fig. 3a), and the results are shown in Fig. 3b. The results show that there is only one peak in the spin-spin relaxation time (T2) distribution of

23

Na nuclei in SA, SA–CaCO3, and SA–GDL solutions,

which indicates that the sodium ions have only one state in these samples. The addition of CaCO3 or GDL has no effect on the dynamics of sodium ion in the SA solutions. However, two peaks appear on the spin-spin relaxation time (T2) distribution curve of

23

Na nuclei with the

addition both of CaCO3 and GDL. The result indicates the existence of two states sodium ions

8

in SA–CaCO3–GDL system, corresponding to sodium ions in the Ca2+ crosslinked and uncrosslinked regions, respectively. The reason why this is that CaCO3 would release the Ca2+ ion in the case of the presence of GDL (Meiboom & Gill, 1958), and Ca2+ ions would replace part of the sodium ions of SA. The relaxation time T2 of the sodium ions within the Ca2+ crosslinked regions shifts to lower relaxation time T2, which is due to the formation of crosslinks among the SA chains. The relaxation time T2 of the sodium ions in un-crosslinked regions locates at around lgT2 = 0.75. Above results indicates that the addition of CaCO3–GDL could lead the crosslink of SA aqueous solutions via release of calcium ions from CaCO3–GDL. We further investigated the effects of the CaCO3–GDL content on the dynamic of sodium ions in the SA solutions. Fig. 4 shows the spin-spin relaxation of 23Na nuclei and the corresponding distribution of the relaxation time (T2) in SA solutions with different amount of CaCO3–GDL. The ln(I/I0) vs. t plots of Fig 4a show that the linear (single exponential) fittings can only describe the data point at lower decay time (Fig. S3, Supporting Information). The inverse Laplace transform calculation results indicate that there are two peaks on the curves of relaxation time distribution of 23Na nuclei (Fig. 4b). The peak on the left represents the relaxation time of the sodium ions in Ca2+ crosslinked regions, which is shorter than that of the sodium ions in uncrosslinked regions (Fig. 4b). The results show that the peak of the sodium ions in Ca2+ crosslinked regions shifts to shorter relaxation time T2 with the simultaneously decrease in relative intensity with the increase in the content of CaCO3–GDL in the system. The decrease in the relative intensity of the peak of the sodium ions in Ca2+ crosslinked regions is due to that the sodium ions in Ca2+ crosslinked regions were replaced by the calcium ions left shift of the T2 peak position is due to that the SA chains were crosslinked by the calcium ions and the mobility of sodium ions in Ca2+ crosslinked regions decreased. On the other hand, the peak of the sodium ions in un-crosslinked regions shifts to longer relaxation time T2 with the increase in the content of CaCO3–GDL in the system (Fig. 4b). The increase in the relative intensity of the peak of the sodium ions in un-crosslinked regions is due to that the sodium ions in the Ca2+ crosslinked regions were released into the un-crosslinked regions. The relaxation time T2 of the sodium ions in Ca2+ crosslinked and un-crosslinked regions shifts to shorter and longer relaxation time with the increase in the concentration of calcium ions, respectively (Fig. 4b). The results are due to that the increase in calcium ions result the higher crosslink density. The mobility of the SA segments decreased, which leads to the decrease in the relaxation time (T2) of the sodium ions in Ca2+ crosslinked regions. Moreover, the increase in the crosslink density also leads to the closer package of SA chains and the increase in the free space for the diffusion of free sodium ions in the hydrogel, which results the increase in the relaxation time (T2) of the sodium ions in un-crosslinked regions. Therefore, the mobility of the sodium ions in un-crosslinked regions increased, while the mobility of the sodium ions in Ca2+ crosslinked regions decreased. The diffusion of sodium ions in the SA solutions were investigated by using 23Na PEG NMR. Fig. 5a shows the normalized spin–echo signal intensity (I/I0) of 23Na nuclei as a

9

function of the square of the gradient strength G2 in the SA solutions (3wt%) with different amount of CaCO3–GDL. The data in Fig. 5a were plotted in ln(I/I0) vs. G2 and show a good linear relationship (Fig.S4 in Supporting Information), which indicates that the data can be fitted well by using eq. 1. The data of 23Na nuclei in 1M NaCl solution are also shown for comparison (Fig. 5a, ○).The overall diffusion coefficient of Na+ ions (𝐷Na+ ) in the SA– CaCO3–GDL system can be obtained by fitting the experimental data in Fig. 5a using eq. 1. Fig. 5b shows the diffusion coefficient of Na+ ions 𝐷Na+ as a function of the content of CaCO3–GDL. The results indicate that confirm that 𝐷Na+ increases with the increase in the content of CaCO3–GDL, gradually approaching the diffusion coefficient of 23Na nuclei in 1M NaCl solution measured in this work (Fig. 5b, ○), which is similar to that in literature (Wang et al., 2014). The results also confirm that the increase in 𝐷Na+ with crosslinking is consistent with the tighter package of alginate chains that are crosslinked, which increases the distance between chains (and thus volume fraction) in the un-crosslinked regions, which would results the increase in the mobility of Na+ ions. In order to diagnose the quality of T2 and diffusion experiments, The 1D 23Na spectra at pulse-acquire with short pulse length (8μs) and high power were also measured and the results is shown in Fig. S5 (Supporting Information). The results show the slight change of 23Na satellites as well as the central transition, which slightly move to the low field duo to electron cloud density decreasing with the addition of divalent calcium ions. Moreover, the 1D slice of the CPMG experiment with a 1 ms delay and the PGSTE experiment with the first gradient step were also checked and the results are shown in Fig. S6 (Supporting Information). The results show that the fraction of the central transition signal survives relative to the pulseacquire. The fractions of the total spins in the sample observing in the CPMG and PGSTE experiments were normal. When the amount of calcium ions increased, the fractions of the central transition signal survives become large corresponding with the result of the T2 and diffusion experiments. The above results indicate that the T2 and diffusion experiments are high quality. The above results and discussions indicate that the relaxation time of Na+ ions in the SA solutions relates to the distance between the SA chains and the degree of crosslinks among SA chains. For the further clarification of the dynamics of the sodium ions in the SA system, the rheological properties of SA solutions and the solutions with the addition of CaCO3–GDL were investigated. Fig. 6a shows the complex viscosity the pure SA solutions with different concentrations under the steady scanning at different shearing rate. All the SA solutions show a shear thinning behavior, and the viscosity of the solutions is gradually enhanced with the raising concentrations. Considering the viscosity of the SA solution in different concentration, the decrease in the relaxation time T2 of Na+ ions in SA solutions with the increase in the concentration of SA (Fig. 1e and Fig. 2) could be due to the increase in the viscosity of the SA solutions, in which the distance between the SA chains decreases and the degree of chain entanglement increases in the solution with the increase in SA concentration. As a result, the increase in SA concentration will cause Na+ to become coordinated with polymer-fixed ions on average for a greater fraction of the time and lowered T2 of the sodium ions electrostatically attached on the SA chains. Fig. 6b shows the complex viscosity of SA solutions (3 wt%) with the addition of different amount of CaCO3–GDL under the steady shearing rate. The results indicate that the viscosity is gradually increased with rising in the content of CaCO3–GDL, which corresponds to the reduction in the relaxation time (T2) of

10

sodium ions in Ca2+ crosslinked regions (Fig. 4b). The increase in the viscosity of SA solutions with increasing CaCO3–GDL content is due to the increase in the crosslink density correspondingly. The increase in the crosslink density leads to the close package of the SA chains, which cause the results the decrease in the mobility of the SA chains. Correspondingly, the mobility of sodium ions in Ca2+ crosslinked regions reduced and the spin-spin relaxation become fast (short T2). Based on the results and discussions above, the gelation process of SA can be depicted schematically as Fig. 7. In the pure SA solution, all sodium ions are in one state in the uncrosslinked samples. Therefore, there is only one peak on the curve of the spin-spin relaxation time (T2) distribution of 23Na nuclei (Fig. 2 and Fig. 3b). When the divalent calcium ions released from CaCO3-GDL, gelation occurred via the collaborative linkage of the G-units of alginate chains by the divalent calcium ions. The alginate chain segments are in two different regions in the hydrogels, say Ca2+ crosslinked regions and un-crosslinked regions. As a result, there are two different the spin-spin relaxation time (T2) of 23Na nuclei (Fig. 3b and Fig. 4b). With the increase of calcium ions concentration, the crosslink density of the hydrogels increased. The increase in the crosslink density lead to the decrease in the mobility of SA segments, which resulted the decrease in the relaxation time (T2) of the sodium ions in Ca2+ crosslinked regions. The size of the regions of heterogeneity in the gels can be estimated from the sodium ion relaxation value (Rijniers, 2004). The corresponding results are shown in Fig. S7 (Supporting Information). The results indicate that the Ca2+ crosslinked alginate hydrogels are inhomogeneous in structure with a length scale of tens nanometers to several micrometers (Fig. S7, Supporting Information). Simultaneously, the diffusion space for the sodium ions enlarged, which led the increase in the relaxation time (T2) of the sodium ions in uncrosslinked regions (Fig. 4b).

4. Conclusions The dynamics of sodium ions in a calcium gel at the equilibrium state were investigated by using NMR relaxometry and PFG NMR diffusometry. The NMR relaxometry results confirmed that the sodium ions are only in one state in SA solutions. The addition of CaCO3– GDL, which release calcium ions, led the crosslinking of SA via the chelation of divalent calcium ions with the G-block of SA chains. As a result, we find that sodium ions are in two different states in the Ca2+ crosslinked SA hydrogels, corresponding to those sodium ions in Ca2+ crosslinked regions and un-crosslinked regions. The characteristic relaxation time (T2) of sodium ions in Ca2+ crosslinked regions shifted to lower T2 value with the increase in the content of calcium ions in the system, which is due to the decrease in the mobility of SA segments. Simultaneously, the relaxation time (T2) of the sodium ions in un-crosslinked regions shifted to higher T2 value, which indicated that the free space for the diffusion of sodium ions enlarged with the increase in the crosslink density. PFG NMR diffusometry results consist with those of NMR relaxometry.

Acknowledgments Financial support from National Natural Science Foundation of China (21274154, 51473174, and 51573080), Program of Science and Technology in Qingdao City (14-2-4-122-jch), and

11

Taishan Scholar of Shandong Province are gratefully acknowledged.

References Akin, J. E., & Counts, J. (1969). On rational approximations to inverse laplace transform. Siam Journal on Applied Mathematics, 17(6), 1035-1040. Anseth, K. S., Bowman, C. N., & BrannonPeppas, L. (1996). Mechanical properties of hydrogels and their experimental determination. Biomaterials, 17(17), 1647-1657. Babensee, J. E., Cornelius, R. M., Brash, J. L., & Sefton, M. V. (1998). Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: Human serum proteins in vitro and rat proteins following implantation. Biomaterials, 19(7-9), 839-849. Barberon, F., Korb, J. P., Petit, D., Morin, V., & Bermejo, E. (2003). Probing the surface area of a cement-based material by nuclear magnetic relaxation dispersion. Physical review letters, 90(11), 116103. Brownstein, K. R., & Tarr, C. E. (1979). Importance of classical diffusion in NMR-studies of water in biological cells. Physical Review A, 19(6), 2446-2453. Bryant, S. J., & Anseth, K. S. (2001). The effects of scaffold thickness on tissue engineered cartilage in photocrosslinked poly(ethylene oxide) hydrogels. Biomaterials, 22(6), 619-626. Bryant, S. J., Nuttelman, C. R., & Anseth, K. S. (2000). Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro. Journal of Biomaterials Science-Polymer Edition, 11(5), 439-457. Burdick, J. A., Peterson, A. J., & Anseth, K. S. (2001). Conversion and temperature profiles during the photoinitiated polymerization of thick orthopaedic biomaterials. Biomaterials, 22(13), 1779-1786. Callaghan, P. T. (1991). Principles of Nuclear Magnetic Resonance Spectroscopy. Oxford: University Press,. Chen, J. J., Kong, X. Q., Sumida, K., Manumpil, M. A., Long, J. R., & Reimer, J. A. (2013). Ex Situ NMR Relaxometry of Metal-Organic Frameworks for Rapid Surface-Area Screening. Angewandte Chemie-International Edition, 52(46), 12043-12046. Draget, K. I., Ostgaard, K., & Smidsrod, O. (1990). Homogeneous alginate gels: A technical approach. Carbohydrate Polymers, 14(2), 159-178. Fang, Y., Al-Assaf, S., Phillips, G. O., Nishinari, K., Funami, T., & Williams, P. A. (2008). Binding behavior of calcium to polyuronates: Comparison of pectin with alginate. Carbohydrate Polymers, 72(2), 334-341. Fang, Y., Al-Assaf, S., Phillips, G. O., Nishinari, K., Funami, T., Williams, P. A., & Li, L. (2007). Multiple steps and critical behaviors of the binding of calcium to alginate. Journal of Physical Chemistry B, 111(10), 2456-2462. Fundo, J. F., Fernandes, R., Almeida, P. M., Carvalho, A., Feio, G., Silva, C. L. M., & Quintas, M. A. C. (2014). Molecular mobility, composition and structure analysis in glycerol plasticised chitosan films. Food Chemistry, 144, 2-8. Geng, Y., Almeida, P. L., Feio, G. M., Figueirinhas, J. L., & Godinho, M. H. (2013). Water-based cellulose liquid crystal system investigated by rheo-NMR. Macromolecules, 46(11), 4296-4302. Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C., & Thom, D. (1973). Biological interactions between polysaccharides and divalent cations: The egg-box model. FEBS Letters, 32(1), 195-198. Hald, M., & Jacobsen, J. P. (1991).

23

Na and 7Li NMR spin-lattice relaxation measurements in the study of

intercalation in DNA. Biophysical Chemistry, 41(2), 113-124. Hansen, P. C. (1992). Analysis of discrete ill-posed problems by means of the L-curve. SIAM Review, 34(4), 561580. Hetnarski, R. B. (1975). Algorithm for generating some inverse laplace transforms of exponential form. Zeitschrift Fur Angewandte Mathematik Und Physik, 26(2), 249-253.

12

Kemp, M. R., & Fryer, P. J. (2007). Enhancement of diffusion through foods using alternating electric fields. Innovative Food Science & Emerging Technologies, 8(1), 143-153. Kleinberg, R. L., & Horsfield, M. A. (1990). Transverse relaxation processes in porous sedimentary-rock. Journal of Magnetic Resonance, 88(1), 9-19. Kuo, C. K., & Ma, P. X. (2001). Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials, 22(6), 511-521. Lafitte, G., Soderman, O., Thuresson, K., & Davies, J. (2007). PFG-NMR diffusometry: A tool for investigating the structure and dynamics of noncommercial purified pig gastric mucin in a wide range of concentrations. Biopolymers, 86(2), 165-175. Levitt, M. H. (2008). Spin dynamics: Basics of nuclear magnetic resonance. John Wiley & Sons,Ltd. Li, J., Wilmsmeyer, K. G., & Madsen, L. A. (2009). Anisotropic diffusion and morphology in perfluorosulfonate ionomers investigated by NMR. Macromolecules, 42(1), 255-262. Li, T., Shi, X. W., Du, Y. M., & Tang, Y. F. (2007). Quaternized chitosan/alginate nanoparticles for protein delivery. Journal of Biomedical Materials Research Part A, 83A(2), 383-390. Lloyd, A. W., Faragher, R. G. A., & Denyer, S. P. (2001). Ocular biomaterials and implants. Biomaterials, 22(8), 769785. Lowman, A. M., Morishita, M., Kajita, M., Nagai, T., & Peppas, N. A. (1999). Oral delivery of insulin using pHresponsive complexation gels. Journal of Pharmaceutical Sciences, 88(9), 933-937. Marincola, F. C., Casu, M., Saba, G., & Lai, A. (2001). 23Na NMR relaxation studies of the Na-DNA/drug interaction. Chemphyschem, 2(10), 569-575. Martinsen, A., Skjakbraek, G., & Smidsrod, O. (1989). Alginate as immobilization material. 1. Correlation between chemical and physical-properties of alginate gel beads. Biotechnology and Bioengineering, 33(1), 79-89. Meiboom, S., & Gill, D. (1958). Modified spin-echo method for measuring nuclear relaxation times. Review of Scientific Instruments, 29(8), 688-691. Mitchell, J., Gladden, L. F., Chandrasekera, T. C., & Fordham, E. J. (2014). Low-field permanent magnets for industrial process and quality control. Progress in Nuclear Magnetic Resonance Spectroscopy, 76, 1-60. Mongia, N. K., Anseth, K. S., & Peppas, N. A. (1996). Mucoadhesive poly(vinyl alcohol) hydrogels produced by freezing/thawing processes: Applications in the development of wound healing systems. Journal of Biomaterials Science-Polymer Edition, 7(12), 1055-1064. Mussel, M., Wilczynski, E., Eliav, U., Gottesman, J., Wilk, M., & Nevo, U. (2015). Dynamics of water and sodium in gels under salt-induced phase transition. Journal of Polymer Science Part B-Polymer Physics, 53(22), 1620-1628. Patural, L., Korb, J. P., Govin, A., Grosseau, P., Ruot, B., & Deves, O. (2012). Nuclear magnetic relaxation dispersion investigations of water retention mechanism by cellulose ethers in mortars. Cement and Concrete Research, 42(10), 1371-1378. Peemoeller, H., Weglarz, W. P., Hinek, M. J., Holly, R., Lemaire, C., Teymoori, R., Liang, J. Z., Crone, J., Mansour, F. K., & Hartley, I. D. (2013). NMR detection of liquid-like wood polymer component in dry aspen wood. Polymer, 54(5), 1524-1529. Price, W. S. (1997). Pulsed-field gradient nuclear magnetic resonance as a tool for studying translational diffusion. 1. Basic theory. Concepts in Magnetic Resonance, 9(5), 299-336. Provencher, S. W. (1982). A constrained regularization method for inverting data represented by linear algebraic or integral-equations. Computer Physics Communications, 27(3), 213-227. Rijniers, L. A., Magusin, P., Huinink, H. P., Pel, L., & Kopinga, K. (2004). Sodium NMR relaxation in porous materials. Journal of Magnetic Resonance, 167(1), 25-30.

13

Rijniers, L. A. M., P.; Huinink, H. P.; Pel, L.; Kopinga, K. ( 2004). Sodium NMR relaxation in porous materials. J. Magn. Reson, 167(1), 25-30. Salzer, H. E. (1953). Orthogonal polynomials arising in the numerical evaluation of inverse laplace transforms. Bulletin of the American Mathematical Society, 59(6), 553-553. Stevens, M. M., Qanadilo, H. F., Langer, R., & Shastri, V. P. (2004). A rapid-curing alginate gel system: utility in periosteum-derived cartilage tissue engineering. Biomaterials, 25(5), 887-894. Tilakaratne, H. K., Hunter, S. K., Andracki, M. E., Benda, J. A., & Rodgers, V. G. J. (2007). Characterizing short-term release and neovascularization potential of multi-protein growth supplement delivered via alginate hollow fiber devices. Biomaterials, 28(1), 89-98. Titus, C. K. (1955). A general card-program for the evaluation of the inverse laplace transform. Journal of the Acm, 2(1), 18-27. Valckenborg, R. M. E., Pel, L., Hazrati, K., Kopinga, K., & Marchand, J. (2001). Pore water distribution in mortar during drying as determined by NMR. Materials and Structures, 34(244), 599-604. Wang, Y., Gao, J., Dingemans, T. J., & Madsen, L. A. (2014). Molecular Alignment and Ion Transport in Rigid Rod Polyelectrolyte Solutions. Macromolecules, 47(9), 2984-2992. Washburn, K. E., & Callaghan, P. T. (2006). Tracking pore to pore exchange using relaxation exchange spectroscopy. Physical review letters, 97(17). Wu, Z. Y., Bertram, H. C., Kohler, A., Bocker, U., Ofstad, R., & Andersen, H. J. (2006). Influence of aging and salting on protein secondary structures and water distribution in uncooked and cooked pork. A combined FT-IR microspectroscopy and 1H NMR relaxometry study. Journal of Agricultural and Food Chemistry, 54(22), 85898597. Zhang, C., Li, P. P., Zhang, Y. J., Lu, F., Li, W. W., Kang, H. L., Xiang, J. F., Huang, Y., & Liu, R. G. (2016). Hierarchical porous structures in cellulose: NMR relaxometry approach. Polymer, 98, 237-243. Zhu, H. J., Huinink, H. P., Adan, O. C. G., & Kopinga, K. (2013). NMR study of the microstructures and waterpolymer interactions in cross-linked polyurethane coatings. Macromolecules, 46(15), 6124-6131.

Norm.intensity

(b)

(e) 6.0

0.5 5.5

0.0

(c)

1.0

(d)

T2 (ms)

Norm.intensity

(a) 1.0

5.0

0.5 4.5

0.0 0

20

40

60

t (ms)

80 100

0

20

40

60

80 100

t (ms)



14

1

2

3

SA (wt%)

4

Fig. 1. The spin-spin relaxation of 23Na nuclei in SA solutions with the concentration of (a) 1 wt%, 2 wt%, (c) 3 wt%, and (d) 4 wt%. The lines are the fitting results using a single exponential function. (e) Overall T2 of 23Na spins as a function of SA concentration.

f(T2)

4%SA

3%SA

2%SA

1%SA

-0.5

0.0

0.5

1.0

1.5

2.0

lg(T2, ms)



Fig. 2. The distribution of the spin-spin relaxation time (T2) of 23Na nuclei in SA solutions with different SA concentration.

SA SA-CaCO3 SA-GDL SA-CaCO3-GDL

1.0

(b)

SA-CaCO3-GDL

0.6

f(T2)

Norm. intensity

0.8

(a)

SA-GDL

0.4 SA-CaCO3 0.2 SA

0.0 0

20

40

60

80

100

-0.5

t (ms)

0.0

0.5

1.0

1.5

2.0

lg (T2, ms)

Fig. 3. The (a) spin-spin relaxation and (b) the distribution of T2 of 23Na nuclei in SA solution with different additives. 15

Na+

(a)

1.0

-

(b)

+

-COO Na SA 0.5× 1× 2× 3×

0.6

3×

2×

f(T2)

Norm. intensity

0.8

0.4

1×

0.2

0.5×

0.0

SA 0

20

40

60

80

100

-0.5

0.0

0.5

t (ms)

1.0

1.5

2.0

lg (T2, ms)

Fig. 4. The (a) spin-spin relaxation and (b) the distribution of T2 of 23Na nuclei in SA solution with different content of CaCO3–GDL.

(a)

1.0

I / I0

0.6

0.4

1.5

DNa+ (10-9 m2/s)

SA 0.5X 1X 2X 3X NaCl

0.8

(b)

1M NaCl

1.6

1.4

1.3

0.2 1.2 0.0 1.1 0.0

0.5

1.0

1.5

2.0

2

5

2.5 2

3.0

3.5

4.0

0.0

2

G (10 G /cm )

0.5

1.0

1.5

Ca

2+

2.0

2.5

3.0

(X)

Fig. 5. The diffusion coefficient of 23Na nuclei. (a) The raw Na+ diffusion coefficient data in SA solution with different amount of CaCO3–GDL. (b) The characteristic diffusion coefficient of 23Na as function of the amount of CaCO3.

16

(a) 1% SA 2% SA 3% SA 4% SA

105

(b)

109 3% SA 0.5× 1× 2× 3×

108

(mPas)

(mPas)

107

104

106

105

104 3

10

103 10-2

10-1

100

.

101

102

10-2

10-1

100

.

(s )

101

102

(s )

-1

-1

Fig. 6. The complex viscosity of the SA solutions as a function of shearing rate. (a) Pure SA solutions with different content of SA; (b) SA solution (3 wt%) with different content of CaCO3–GDL.

Fig. 7. A schematic diagram of effect of calcium ions to sodium ions in SA solutions.

17