Effect of Sodium Chloride on Structure of Collagen Fiber Network in Pickling and Tanning

A work by Xinxin Li, Ya-nan Wang, Jing Li and Bi Shi National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, P. R. China, Foshan Shunde Longting New Material Co., Ltd., Foshan 528305, P. R. China

Abstract
Tannery wastewater usually contains a high salinity due to the use of sodium chloride (NaCl) in curing and pickling. Although some no-pickle tanning and salt-free pickling technologies were developed, few of them have been widely used due to relatively poor mechanical and bulk properties of the resultant leathers. Therefore, the role of NaCl in pickling and tanning should be investigated in the first place. In this study, bated pelts were pickled by salt-free pickling and conventional salt-assisted pickling processes, respectively, and then tanned by chrome tanning agent. The hierarchical structures of collagen fiber network of the pickled pelts and leathers were observed by optical microscope and SEM, while the porosity of leathers was measured. The results showed that the fiber bundles of the pelt pickled in the presence of NaCl were more sufficiently dispersed compared with those of salt-free pickled pelt. Both of the chrome tanned leathers had a regular arrangement of collagen fibers, but the leather with salt-assisted pickling presented remarkably higher degree of fiber dispersion, as well as larger porosity. Moreover, the role of NaCl in organic tanning using an amphoteric organic tanning agent was investigated. The results also showed that the presence of NaCl in tanning could improve the opening up of collagen fiber network and the porosity of the leather. In general, NaCl used in leather processing presented a positive effect in consideration of leather quality.

Introduction
Sodium chloride (NaCl) is one of the commonest chemicals used in leather industry. It is mainly used in curing, soaking and pickling processes. However, with the increase of people’s environmental awareness, chloride is regarded as one of the most concerned pollutants in tannery wastewater. It is reported that the content of chloride in effluent could be reduced by 30%-40% when salt-free pickling technology was used. Therefore, some salt-free or salt-reduced pickling processes and no-pickle tanning techniques have been developed to minimize chloride impact from the origin of leather making. However, few of them have been widely used due to the fact that these technologies produce leathers with relatively poor mechanical and bulk properties in comparison with the conventional process. NaCl is regarded to dehydrate and prevent swelling of pelt during pickling in classical theories of leather manufacture, but none have mentioned its effect on leather properties. Therefore, to understand the reason of the phenomenon above, the role of NaCl in pickling and tanning processes should be investigated in the first place. We know that the hierarchical structure of leather is described as a network in three dimensions that is woven by collagen fiber bundles (20-200 μm). The bundles are assembled by elemental fibers (10 μm), and the elemental fibers are regularly arrayed by fibrils (0.01-0.5 μm), which are self-assembled by collagen molecules with a parallel staggering. Some research indicated that a good quality of leather will be achieved when the collagen fiber bundles were dispersed adequately in beamhouse processes.This means that fibrous network structure has an important influence on the mechanical and morphological properties of leather. In our preceding work, the role of neutral salt in the assembly behaviors of collagen molecules was investigated, and it was found that NaCl dehydrated collagen molecules and induced collagen molecules to assemble into collagen fibers. On the basis of these observations, we speculated that NaCl would lead to formation of wider spaces between collagen fibers during pickling and tanning, and therefore affect fibrous network structure of resultant leather. However, this assumption was based the research on collagen molecules rather than leather. In this work, the effect of NaCl on fibrous network of pelt/leather in pickling and tanning (chrome tanning and amphoteric organic tanning) was investigated. The hierarchical structures of fibrous network were observed by optical microscope and scanning electron microscope, and the porosity of leather was measured to explore the role of NaCl in pickling and tanning processes.

Experimental Materials
Raw materials used in experiments were cowhides from Queensland, Australia. They were soaked, limed, split (thickness of upper layer 2 mm), weighed, delimed and bated conventionally. The chemicals used for leather processing were of commercial grade, and the chemicals used for analysis were of analytical grade. Salt-free pickling auxiliary mainly composed of sulfone sulfonic acid was from Dowell Science & Technology Inc. (Sichuan, China). Chrome tanning agent (Cr2O3 content 24%, basicity 33%) was from Minfeng Chemical Co., Ltd. (Chongqing, China). Amphoteric organic tanning agent (TWT) was supplied by Tingjiang New Material Co., Ltd. (Sichuan, China).

Preparation of Pickled Pelts
A piece of bated pelt (approx. 2 kg, on the back of the pelt) was cut along the backbone in halves and pickled using salt-free process and conventional process, respectively (TABLE I). Next day ran for 30 min and collected the samples from matched areas near the backbone for further analyses.

table1

Preparation of Tanned Leathers
Chrome Tanned Leathers
The pickled pelts obtained above were tanned using 6% chrome tanning agent in 50% pickle liquor (based on weight of limed pelts, the same below) for 2 h, respectively. Then they were basified to pH 3.8-4.0 using sodium bicarbonate. 200% water was added and ran for another 2 h at 40°C, then left overnight. Next day ran for 30 min and collected the samples from matched areas near the backbone for further analyses.

TWT Tanned Leathers
The pH of a piece of bated pelt (approx. 1 kg, on the back of the pelt) was adjusted to 6.0 using 0.1% formic acid in 50% water. Then the pelt was cut along the backbone and divided into two groups. Group 1 was directly tanned by 5% TWT for 4 h using no-pickle tanning and then basified to pH 8.0 using sodium carbonate. 200% water was added and ran for another 2 h at 40oC, then left overnight. Next day ran for 30 min and collected the samples from matched areas near the backbone for further analyses.
Group 2 was treated with 6% NaCl in 50% water for 3 h and left overnight (12 h).
Next day it was tanned with TWT in the same conditions as group 1.In addition, a piece of conventional pickled pelt (approx. 1 kg, on the back of the pelt) was depickled to pH 6.0 with sodium carbonate in 50% pickle liquor. Then it was also cut along the backbone and divided into group 3 and 4. Group 3 was immediately tanned with TWT in 50% depickling liquor in the same conditions as group 1. Group 4 was washed 6 times with 200% water for 10 min to remove neutral salts, and then tanned with TWT in the same conditions as group 1.

Histological Staining
Samples (1 cm × 1 cm) from pickled pelts, chrome tanned leathers and TWT tanned leathers were cut into 20μm thickness cross sections by freeze microtome (CM1950, Leica, Germany). It should be noted that the pickled pelts were fixed in 10% neutral buffered formalin for 24 h before cutting to prevent from acid swelling. The sections were stained with hematoxylin and then counterstained with eosin. The stained sections were observed by biological microscope (CX41, Olympus, Japan).

Scanning Electron Microscopy
Samples from pickled pelts, chrome tanned leathers and TWT tanned leathers were lyophilized by vacuum freeze dryer (LGJ-30F, XinYi, China). The cross sections of the samples were observed by Scanning Electron Microscope (SEM, Phenom Pro, Phenom-world, Netherlands) at low vacuum with an accelerating voltage of 5/10KV.

BET Surface Area Analysis
Samples from pickled pelts, chrome tanned leathers and TWT tanned leathers were lyophilized by vacuum freeze dryer and cut into 3 mm × 3 mm pieces. Then they were vacuum dried for 10 h at different temperatures (40oC for pickled pelts, 90°C for chrome tanned leathers and 60oC for TWT tanned leathers). The specific surface areas of the samples were determined using surface area and porosity analyzer (TriStar 3000, Micromeritics, USA).

Capillary Flow Porometry
Samples from pickled pelts, chrome tanned leathers and TWT tanned leathers were lyophilized by vacuum freeze dryer and then cut into F 25 mm circular pieces. The thicknesses of the samples were determined using a thickness gauge with 100 g
pressure. Porometry was performed using a capillary flow porometer (POROLUXTM 1000, POROMETER, Germany). A sample was first soaked in Porofil wetting liquid of low vapor pressure (0.4 kPa), low reactivity and low interfacial tension (0.016 N/m) that can be assumed to fully wet the samples. Then it was placed in the sample chamber and sealed. Pressure was increased from 0 to 2.5 bar to obtain two gas flow curves (wet curve and dry curve) as a function of pressure. The mean flow pore size of the sample could be calculated according to the curves. In addition, the linear dry curve can be expressed as:

Image-1

where F is the gas flow rate (m3/s), A is the area of the sample (m2), u is the thickness of the sample (m), K is the air permeability coefficient of the sample (m2), h is the dynamic viscosity of gas flow (Pa·s), and Dr is the pressure differential between the two sides of the sample.
Darcy air permeability (K—h, m2/(Pa·s)), which is related to the porosity and pore size distribution of the sample, could be obtained according to the dry curve. The bigger value of Darcy air permeability indicates better air permeability of the sample.

Measurement of Porosity of Pelt/leather
Samples from pickled pelts, chrome tanned leathers and TWT tanned leathers were lyophilized by vacuum freeze dryer and then cut into 20 mm × 3 mm pieces. The porosities of the samples were measured as follows. Firstly, over 100 mL of benzyl alcohol was added to a special volumetric flask (the neck of the flask has 0-10 mL tick marks), and the volume (mL) was recorded as V1. Secondly, pelt/leather pieces were added into the flask and soaked for 48 h, and the volume (mL) was recorded as V2. Then the pelt/leather pieces and benzyl alcohol were all poured out of the flask, and the surfaces of the pelt/leather pieces were slightly dried by filter paper. Over 100 mL of fresh benzyl alcohol was once again added into the flask. The volume (mL) was recorded as V3. Finally, the pelt/leather pieces were added into the flask, and the volume (mL) was recorded as V4. The porosity of pelt/leather was calculated as:

Image-2

Results and Discussion
Effect of NaCl on the Fibrous Network in Pickling
Bated pelts were pickled using salt-free and conventional salt-assisted processes, respectively. Figure 1 shows the histological stained cross sections of the pickled pelts. It can be seen from Figure 1a that collagen fiber bundles of salt-free pickled pelt, particularly those in reticular layer, were mainly in the form of thick bundles due to the lack of opening up. However, the thick collagen fiber bundles were separated into thin bundles in the reticular layer of conventional pickled pelt (Figure 1b), which suggested that the fibrous network was more sufficiently dispersed in the presence of NaCl. In order to observe finer fibrous structure, SEM analysis of the cross sections of pickled pelts was performed, as shown in Figure 2. It is known that in the hierarchical structures of hide/leather, fiber bundles (20-200 μm) are composed of elemental fibers (10 μm), which can be further divided into even finer fibrils (0.01-0.5 μm). Figure 2a shows that the fiber bundles of salt-free pickled pelt were cemented together. It was even difficult to observe them clearly as the weave of fibers was relatively disordered. Furthermore, the collagen fibers in the magnified reticular layer (Figure 2a) were stuck together in clumps. On the contrary, Figure 2b displays that fiber bundles were well distinguished and arranged in order when pickled with NaCl. What’s more, they were divided into individual elemental fibers that were separated from each other and quite distinct in the reticular layer. This phenomenon implies the increase of gaps and pores in fibrous network. The surface area measurement will give the concrete proof for fiber dispersion. Unsurprisingly, the BET surface area of the conventional pickled pelt (TABLE II) was much larger, indicating better fiber dispersion performance than that of salt-free pickled pelt. Moreover, lower mean flow pore size, larger value of Darcy air permeability and higher porosity was found for pelt pickled using conventional salt-assisted pickling process (TABLE II). These results suggested that the presence of NaCl benefits the opening up of fibrous network, which is in accordance with the microscopic observation. To confirm this hypothesis, more investigations were carried out in the following sections.

tab_2

Effect of NaCl on the Fibrous Network in Chrome Tanning
Chrome tanning was conducted in pickling bath, where the presence of NaCl may still influence the fibrous structure of leather during chrome tanning process. Figure 3 shows the histologically stained fibrous structure of chrome tanned leathers. With salt-free pickling, most of the collagen fibers in leather were adhered to each other and presented as thick bundles (Figure 3a). However, as for pickling with salt, the fibrous network of leather were dispersed better and displayed as thinner bundles (Figure 3b). Moreover, the fiber bundles in Figure 3 were denser and more robust compared with those in Figure 1, which should be due to the crosslinking/tanning effect of chrome tanning agent among the collagen fibers.
On a more microscopic scale, the cross sections of chrome tanned leathers with different pickling processes were observed by SEM. Figure 4a shows the chrome tanned leather using salt-free pickling process, where most of the elemental fibers were adhered and assembled into fiber bundles. However, Figure 4b shows that the elemental fibers in the leather using conventional salt-assisted pickling process were not only assembled into fiber bundles, but also well separated from each other. This fact was also demonstrated by the porosity properties of chrome tanned leather (Table III). The BET surface area of chrome tanned leather using conventional pickling process was larger than that of leather using salt-free pickling process. The air permeability of conventional chrome tanned leather was better. The porosity of the chrome tanned leather using conventional pickling process was approx. 3% higher than that of using salt-free pickling process. In combination of the results in Section 3.1, we can infer that NaCl plays an important role in the dispersion of elemental fibers in fiber bundles during pickling and tanning processes.
Collagen has a large number of hydrophilic amino acid residues that combine with water through hydrogen bonds. NaCl can break the hydrogen bonds among collagen and water, leading to the damage to the hydrated layer and the dehydration of collagen. Therefore, in conventional pickling and tanning, NaCl is added to prevent pelt from absorbing an excessive amount of water so as to avoid the defect of acid plumping. The damage to the hydrated layer would improve the exposure and ionization of charged groups in collagen. The electrostatic interactions of these charged groups may result in better dispersion of collagen fibers. In addition, Hofmeister ion effects on protein stability arise repeatedly in protein research. Na+ and Cl- are believed as kosmotropes which lead to stabilization of protein by salting-out effect. It has been found that NaCl can induce collagen molecules to assemble into collagen fibers and stabilize the fibrous network structure. It is likely that this would be another reason for the orderly arrangement of fiber bundles in the presence of NaCl.

tab_3

Effect of NaCl on the Fibrous Network in Tanning with Amphoteric Organic Tanning Agent
Four groups of tanning trials using amphoteric organic agent (TWT) were performed according to Section 2.3.2. The histologically stained cross sections of all the TWT tanned leathers are shown in Figure 5. The fiber bundles of leathers using no-pickle tanning process (group 1 and 2) were thick and tightly woven together (Figure 5a and 5b). Even though group 2 was tanned in the presence of 6% NaCl, the dispersion extent of fiber bundles was not sufficient (Figure 5b). This fact was also confirmed by SEM images shown in Figure 6a and 6b. The elemental fibers were cemented together in grain layer and assembled into thick bundles with insufficient separation in reticular layer. Both groups did not exhibit any differences in the arrangement of fibrous network. It was found in histological staining and SEM images that the fiber bundles of leathers using pickling-depickling-tanning process (group 3 and 4) illustrated better dispersing performance (Figure 5c, 5d, 6c and 6d) than those of group 1 and 2 (Figure 5a, 5b, 6a and 6b).
The fiber bundles were separated into elemental fibers both in grain and reticular layers, and there were more gaps and pores in the fibrous network, particularly for the leather tanned in the presence of NaCl (group 3). We know from the results above that the presence of NaCl benefits the separation of fiber bundles. Meanwhile, acid used in pickling process had broken some chemical bonds among collagen fibers, and this action contributed to the opening up of the collagen fibers as well.Furthermore, the crosslink action of TWT tanning agent seemed to tighten the fiber bundles. Therefore, under the influence of these factors, the porosity properties of TWT tanned leather illustrated some interesting rules, as shown in TABLE IV. As expected, the BET surface area and porosity of leather tanned without pickling and in the absence of NaCl (group 1) was the lowest among all the groups. Leathers treated with NaCl (group 2 and 3) showed higher BET surface area and porosity compared to group 1. Meanwhile, it was found that leathers of these two groups had better air permeability. This fact once again demonstrates that NaCl plays an important role in the opening up of fibrous network of leather.

tab_4

Conclusion
The presence of NaCl in pickling and tanning processes improves the dispersion of elemental fibers, orderly arrangement of fiber bundles and formation of a porous fiber network due to the dehydration and salting-out effects of NaCl. This should be an important factor for acquisition of excellent mechanical and organoleptic properties of leather made by using salt-assisted pickling process. Correct understanding of the role of NaCl in leather manufacture might be favorable for us to further develop novel chemicals and practical technologies aiming at reducing chloride pollution as well as guaranteeing leather quality.

Acknowledgement
This project is financially supported by South Wisdom Valley Innovative Research Team Program and National Natural Science Foundation of China (21476149, 21506129).

INSIGHTS

Chao Wu, Wenhua Zhang, Xuepin Liao, Yunhang Zeng and Bi Shi, Transposition of chrome tanning in leather making, JALCA 109, 176-183, 2014
To avoid the release of chrome from leather into post tanning effluents and the generation of chrome shavings, an inverse chrome tanning technology based on wet white was investigated. Conventional bated pelt was firstly tanned using an amphoteric organic tanning agent (Tingjiang white tanning agent, TWT) without pickling. Then, the TWT tanned wet white was directly processed with conventional post tanning processes. Chrome tanning was transposed to the end of the post tanning. The wet white had a shrinkage temperature (Ts) around 85°C that met the needs of shaving operation, and did not generate chrome shavings. The Ts and Cr2O3 content of the leather, by using this inverse chrome tanning technology, were higher than those of the conventional chrome tanned leather. With this inverse technology, the chrome output was reduced by 48%, mainly because no chrome was released from leather in post tanning processes. Meanwhile, the volume of chromium-containing wastewater discharged from the inverse processes was barely 31% of that from the conventional processes, which makes it much easier to collect and recover chromium from the effluents. Additionally, the tensile strength, tear strength and general appearances of the leather produced by the inverse technology were comparable to those of the conventional chrome tanned leather.

Wei X. Y., Zhang W. H., Shi B., Effect of neutral salts on pickling and tanning. A study based on assembly behavior of collagen, JSLTC, 98, 30-34, 2014
The roles of neutral salt in pickling and tanning processes were investigated by observing the assembly behaviours of collagen in the presences of NaCl and tanning agents. The results indicated that NaCl dehydrated collagen molecules and thus induced collagen to assemble into fibres. This fibre assembly led to the close approach of collagens into fibres and generated wide spaces between collagen fibres. Accordingly, the presence of NaCl could promote the penetration of tanning agents and favour the tanning reaction. Importantly, the dehydration and fibre-forming functions of NaCl resulted in a porous and orderly fibrous microstructure of collagens, which might contribute to the satisfactory mechanical and aesthetic properties of leathers. This underlying action mechanism of NaCl revealed in this research might be useful for us to optimize traditional pickling and tanning techniques and develop practical salt free or low-salt pickling and tanning techniques.

Cheng-Kung Liu, Nicholas P. Latona, Maryann M. Taylor, and Renée J. Latona,
Effects of bating, pickling and crosslinking treatments on the characteristics of fibrous networks from un-tanned hides, JALCA 108, 79-85, 2013
The U.S. hides and leather industries are facing many challenges today, such as overcoming relatively high U.S. energy and labor costs; meeting environmental imperatives; quantifying, maintaining, and improving current hides and leather product quality; developing new processes and products; and improving utilization of waste. One of our efforts to address these new challenges is to develop new uses and novel biobased products from hides to improve prospective markets and to secure a viable future for the hides and leather industries. We hypothesize collagen fiber networks derived from un-tanned hides can be utilized to prepare high performance green composites and air filters, of which both have a great market potential. This study focused on understanding the effects of processing steps such as bating, pickling and crosslinking treatments on the morphology and physical properties of the fiber networks derived from un-tanned hides, which will be the starting material for constructing air filters and green composites. Results showed that glutaraldehyde treatment yielded a highly open structure, in which the fibers are well separated from each other. This could be attributed to the action of acids during the pickling step.
El Zahar, K., Mounir, S., Allaf, T., et al., Fundamental modeling, functional attributes, porosity, cohesivity index (Hausner ratio) and compressibility
of expanded-granule powder of Egyptian Ras pure cheese. LWT-Food Sci. Technology, 64, 297-307, 2015
Food industries and restaurants are increasingly soliciting high functionality cheese dried powder. Spray-drying is usually inadequate for cheese. Grinding of airflow dried cheese is very difficult because the product is compact and too hard. Instant controlled pressure drop DIC texturing assisted Swell-Drying SD usually allows the product to be more appropriated to obtain crispy snacks, leading to an innovative concept of cheese powder. The present work aimed at defining and optimizing this operation for the well-known Egyptian Ras Cheese. Relative expansion ratio, porosity, compressibility, and cohesivity were measured. Compared to Hot-Air Dried HAD, they were up to 210%, 120%, 856%, and 165%, respectively; all depending on DIC operating parameters. This was confirmed by scanning electron microscopy observations. Thanks to the instant autovaporization, vapor is generated within the product developing structural modification with vacuoles and cavities. Swell-Drying-grinding process allowed obtaining cheese powder with a well-controlled quality depending on consumer requirements.

Xinxin Li
Received her Bachelor’s Degree in Light Chemical Engineering at Sichuan University in the year 2013. Now she is undertaking her Master’s degree in Leather Chemistry and Engineering at Sichuan University. Her main research in in the field of tanning chemistry.
Yanan Wang
Received his Ph.D. degree in Leather Chemistry and Engineering from Sichuan University, China in the year 2013. After graduating, he joined Sichuan University as faculty member. His research interests include tanning chemistry and clean technology for leather manufacturing.
Jing Li
Received his Ph.D. degree in Leather Chemistry and Engineering from Sichuan University, China in the year 2012. After graduating he joined Fosan Shunde Longting New Material Co., Ltd. As a researcher. His research focuses on synthesis of tanning and retanning agents.
Bi Shi
Born in 1958, professor SHI Bi has been engaged as the doctoral supervisor of Light Textile and Food College, and he is the director of national engineering laboratory for clean technology of leather manufacture and key laboratory of ministry of education. All the honorary title of Prof. Shi contain National “Cheung Kong scholars to associate professor”, winner of National Outstanding Youth Fund, Academic Degree Committee members of the Council Disciplines, chairman of International Union of Leather Technologists and Chemists (IULTCS), president of the 10th National People’s Congress, the 15th National Union Representative, the gainer of the title of Model Teacher and National Labor Medal. As a well-known expert, Prof. Shi devoted himself to leather cleaning technology, reclamation of leather wastes, deep processing of plant tannins, and he has completed more than 20 items of countries and provincial research projects, 1 item of national scientific and technological progress pride of 2nd, 1 item of national state invention second prizes award, 6 items of advanced science and technology awards of provincial or ministerial. Regarding to the academic achievement, a total of 268 all classes of research papers were published, including SCI index 59, EI index 34, international conference paper 29 (ISTP 11), published books 5. Prof. SHI had cultured 14 post doctorates, 17 doctors and 25 masters, during them 1 post doctorate was a foreigner, and 1 person won national 100 excellent doctoral dissertations, another one entry prize award.