Journal Pre-proof

Basic understanding of the color distinction of lignin and the

proper selection of lignin in color-depended utilizations

Hui Zhang, Shiyu Fu, Yuancai Chen

PII: S0141-8130(19)39992-1
DOI: https://doi.org/10.1016/j.ijbiomac.2020.01.105
Reference: BIOMAC 14409

To appear in: International Journal of Biological Macromolecules

Received date: 5 December 2019

Revised date: 10 January 2020
Accepted date: 10 January 2020

Please cite this article as: H. Zhang, S. Fu and Y. Chen, Basic understanding of the
color distinction of lignin and the proper selection of lignin in color-depended utilizations,
International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/
j.ijbiomac.2020.01.105

This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
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© 2018 Published by Elsevier.

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Basic understanding of the color distinction of lignin and the proper selection of

lignin in color-depended utilizations

Hui Zhanga, Shiyu Fua,* and Yuancai Chena,b

a
State Key Laboratory of Pulp and Paper Engineering, South China University of

Technology, Guangzhou 510640, Guangdong, China

b
Ministry of Education Key Laboratory of Pollution Control and Ecological

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Remediation for Industrial Agglomeration Area, College of Environment and Energy,

South China University of Technology, Guangzhou, 510006, China

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*Corresponding author, Tel.: +8613430364029; E-mail: shyfu@scut.edu.cn
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Abstract
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Lignin based materials and chemicals with outstanding sustainability have drawn
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increasingly attentions. However, the dark color of lignin limits the utilization in
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color-depended fields. In this work, the factors that influence the color of lignin were

investigated and mechanisms were illustrated by GPC, NBO, 2D HSQC, XPS, SEM,
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and visible light spectrum. It is found that the condensed structures were mainly
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separated at higher pH due to its high molecular weight and low solubility. The

condensation contributes to the conjugation and unsaturation, which resulted in the

dark-color of the lignin precipitated at high pH value. The oxidation is not crucial for

the color darkening of lignin in drying, it is the micro aggregation that dominantly

determined the color degree. The concentration of chromophore was decreased owing

to the decrease of bulk density (caused by the alleviation of aggregation), which

endowed lignin with the bright seeing macroscopically. Notably, the selection of
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light-colored lignin needs to be individually considered regarding different use, since

the dominating factors that influence the color at solid or solution are totally different.

In summary, this work offers guidance for acquiring light-colored lignin and helps

people select the light-colored lignin properly regarding utilizations.

Keywords: Kraft lignin; Color distinction; Lignin selection;

1. Introduction

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With the consumption of petroleum, alternatives are urgently needed for the

sustainable development of human being [1]. As one of the significant constituents of

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plant biomass and the most abundant aromatic biopolymer on earth [2,3], lignin
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becomes a promising substitute for petroleum-based phenolic polymers and gains
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growing interest owing to its renewability, biodegradability, and sustainability [4-6].

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At present, more than 50 million tons of industrial lignin are released each year as the
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byproduct of pulping and biorefinery, but it is used primarily as a low-cost fuel, and

only two 2% of the industrial lignin is used commercially, resulting in resource and
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energy waste, and even environmental pollution [7-9]. Efforts have been made to
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convert lignin into value added chemicals and materials [10-14], but the challenge

remains due to shortages of industrial lignin, such as the structural complexity,

heterogeneity, and low reactivity [15-17]. In addition of that, the dark color of

industrial lignin is also a serious obstacle for the development and application

extension of lignin-based products.

The color challenge was recognized as far back as the early 20th century when the

lignosulfonate, a byproduct of the sulfite pulping, was used as dispersant [18]. It is

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found that the dark colored lignin dispersant could not be used in the color-limited

fields like the dye-stuff dispersants. Because the dark colored lignin absorbs on the

surface of textiles and leads to the staining and color distortion [19]. The situation had

been getting worse as more and more sulfite pulping mills were shut down owing to

the quality limitation of the sulfite pulping, which led to the necessity of preparing the

dispersant by sulfonation with the darker-colored Kraft or soda lignin [20]. A patent

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application for isolating and manipulating lignin once stated that the color of lignin is

hard to be masked in a cost-efficient way with pigments, especially when the

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light-colored products are desired, and this has been the basis for the studies to
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acquire light or white lignin [21]. With the utilization extension, lignin is facing more
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challenge regarding the color issues. Based on the advantages of UV absorption and
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oxidation resistance [22], lignin has been evaluated as sunscreens, which pushes
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lignin into high-end field [23-25]. However, the dark color of industrial lignin causes

staining on skin and the market promotion of lignin-based sunscreens is totally

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hindered [26]. Besides, lignin is excellent biobased additive for improving the
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mechanical strength, thermostability, and biodegradability of polymer materials

[27,28], and it is also an outstanding surface modifier to construct superhydrophobic

surface and to enhance the anti-ultraviolet aging property of different kinds of

materials [29-31]. Nevertheless, the application field is restricted attributing to the

color issues. We summarized a range of industrial lignin and derivatives, and

identified some products for which the dark color can impede the utilization of

industrial lignin, including cosmetics, dye-stuff dispersants, pharmacy, plastics, paints,

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binders, resins and composite materials [32-34].

As early as 1980s, researchers realized the color issues and put forward a two-step

process to alter the color of sulfonated soda lignin and lignosulfonates, in which the

free phenoxyl and hydroxyl groups contained in a lignin was first blocked and

followed by an oxidation to destroy some other chromophores [18]. The

chromophores in lignin mainly include quinoids, catechols, aromatic ketones,

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stilbenes, conjugated carbonyls with phenolics, and metal complexes [35]. Besides,

there are some leucochromophores (potential chromophores) that become converted

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into chromophores upon oxidation, and some chromophores that derived from the
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remnant carbohydrates [21]. The formation and transformation of chromophores
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during pulping has been reported, but most of reports were from a papermaking
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perspective focusing on the bleaching of fibers with a low lignin content [36-38]. At
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present, the quantification of the chromophores with a high lignin content has not

been reported and the contribution rate of different kind of chromophores to the color
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of lignin is still unclear. The greatest challenge for chromophore quantification is that
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it is not feasible to distinguish and separate all the chromophores in lignin with

current technology, because of the extreme complexity of lignin structure and

structural variability depending on isolation techniques [39].

Recently, many exploitations have been devoted to lightening the color of lignin in

different ways [40]. Saritha and co-workers [41] isolated several native marine fungi

to investigate the biological decolorization ability. Their work showed that while

using synthetic lignin as culture substrates, the selected fungi can decrease the color
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of lignin by up to 70 ~ 80% after cultivation for 7 days, however the decolorization

performance using industrial lignin as culture substrates was not tested and the

biological decolorization mechanism was neither demonstrated. Wang and co-workers

[42] described a one-step color reduction method. Lignin was dissolved in

tetrahydrofuran and was irradiated under ultraviolet light. After at least 200 hours of

UV irradiation, the lignin was highly lightened, because the phenoxyl in lignin has

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been photo-oxidized into colorless aliphatic acid. These approaches primarily depend

on the digestion or oxidation of lignin, which inevitably caused the degradation of

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lignin macromolecules. It is hard to tell whether the color reduced product is suitable
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for the subsequent utilizations, as the properties that based on the macromolecules and
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aromatic structures have been destroyed. Zhang and co-workers [43] compared kinds
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of sulfonations and found that using butane sultone as sulfonating reagent can make
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the lignin sulfonated and hydroxyl blocked simultaneously. The macromolecular

structure was well maintained, at the same time the lignin presented a light color,
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which make it a good candidate for dye dispersant. Soon afterwards, Zhang and
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co-workers [44] discovered that the fractionation of lignin using methanol/water as a

solvent lead to color alteration, owing to the selectivity of methanol/water regarding

different kind of chromophore and auxochrome. Furthermore, Qian and co-workers

[45] whitened lignin to light yellow by blocking the free phenolic hydroxyl of lignin

and then self-assembling into colloidal spheres. Many steps need to be taken for the

color reduction of lignin in the above-mentioned studies. Therefore, we started to

think whether the color of lignin could be lightened from the very beginning, i.e., in
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the separation, purification and drying process. If so, the burden in the subsequent

color reduction treatment will be greatly decreased, meanwhile the following color

reductions will be more cost-efficient.

In this work, the color regulation of lignin in the separation, purification and drying

process was investigated. Lignin was acid precipitated and fractionated from the black

liquor at different pH values. The color degree was recorded and quantitatively

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compared. The reason that gave rise to the color distinction was analyzed from the

chemical structure aspect and related mechanism was demonstrated. The lignin

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precipitated by the same technique was dried in different manners, which resulted in
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different color performance. The dominating factor that influence the color of lignin
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in drying was summarized with the help of structure and aggregation investigations.
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In addition, it is found that the precise color of lignin needs to be evaluated by

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different techniques, since the dominating factors that influence the color at solid or

solution are totally different. In the present work, we provide fundamental

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understanding for the color reduction mechanisms in the separation and purification
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of lignin, and offer guidance for proper selection of the light-colored lignin based on

different applications.

2. Materials and methods

2.1. Materials

The black liquor derived from Kraft pulping using eucalyptus chips as feedstock was

obtained from a pulping mill in south China. The sulfuric acid (analytical grade) was

purchased from Guangzhou Chemical Reagent Factory. Dilute sulfuric acid with a
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mass ratio of 20% was prepared to adjust the pH value of the black liquor for the

precipitation of lignin. High purity nitrogen (N2) was purchased from Guangzhou

Yuejia Gases Co., Ltd.

2.2 Acid precipitation and fractionation of black liquor

The precipitation and fractionation procedure were illustrated in Fig. 1. Dilute sulfuric

acid with a mass ratio of 20% was dropwise added into black liquor under the

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mechanical agitation. When the pH value reached 6, the first filtration was carried out.

The intercepted solid content was well washed using dilute sulfuric acid with a same

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pH value of 6 to remove the dissolved impurities such as inorganic salts and degraded
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carbohydrates. Afterwards, the lignin cake was obtained and the color of it was
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recorded by the colorimeter. The black liquor through the first filter was subsequently
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adjusted to pH 4 and 2, the lignin solid obtained in each filtration was well washed
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using sulfuric acid solution with the corresponding pH value and the color of lignin

cake was recorded as aforementioned.

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2.3 Drying of lignin

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Different dying methods were used to investigated the impact on the color of solid

lignin. In the oven drying process, the lignin cake obtained in the filtration was

directly put in the oven with a pre-set temperature. Besides, different amount of

phosphorus pentoxide (P2O5) was used to control the drying rate in the vacuum drying

process. The air in the vacuum oven was exhausted and then N2 was charged. This

approach was repeated for several times to eliminate the oxidation interference of

oxygen. In the freeze drying, lignin was re-dispersed in water and was pre-frozen in a
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fridge before transferred into the vacuum tank for drying. As for spray drying, lignin

slurry or aqueous solution with a certain concentration was firstly prepared, afterward

the fluid was sprayed out through a nozzle at a pre-set temperature and flow velocity.

Eventually, the powder sample was collected in the dryer tank.

2.4 Gel permeation chromatography (GPC) analysis

The molecular weight distribution of lignin was characterized by GPC (Agilent,

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U.S.A.) equipped with PL1110-6300-6530 columns. Tetrahydrofuran was used as

mobile phase and the lignin was acetylated for a better solubility before test. The

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standard curve was built by polystyrene (molecular weight ranges from 2 to 100 k).
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20 μL of injection was used in each run and the testing temperature was kept at
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40±0.1 oC.
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2.5 Alkaline nitrobenzene oxidation (NBO)

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NBO of lignin was executed as follows. Each lignin sample together with NaOH

aqueous solution and nitrobenzene was mixed in a steel reactor and heated to 170 oC
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within 2.5 h. The oxidized product was extracted by chloroform for several times.
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After the first extraction, HCl solution was mixed to adjust the pH to 1. The collected

organic phase was evaporated and then reacted with N,O-Bis(trimethylsilyl)acetamide

(BSA) for 10 min at 100 oC. Afterwards, the sample was characterized by gas

chromatography (GC-14B, shimadzu, Japan) equipped with a fused silica capillary

column and flame ionization detector. Helium was used as carrier gas and the flow

rate was 2 mL/min. 3-ethoxy-4-hydroxybenzaldehyde was used as internal standard in

the quantification of the oxidation products.

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13
2.6 2D heteronuclear single quantum correlation (HSQC) and C nuclear magnetic

resonance (NMR) spectra analysis

Lignin was dissolved in dimethyl sulfoxide-d6 (DMSO-d6) in which

tetramethylsilane (TMS) has been previously added as internal standard. 2D HSQC

was recorded in NMR experiments (Bruker, Germany). The spectral widths of 13C and

1
H dimensions were 20000 and 5000 Hz, respectively. In the 1H dimension, the

number of collected complex points was 1024 with a recycle delay of 1.5 s. In the 13C

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dimension, the number of transients was 64, and 256 times increment was recorded.

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The original data was processed by Bruker Topspin-NMR software. The 13
C NMR
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spectra of lignin was characterized by an Avance-3-HD 400 MHz spectrometer
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(Bruker, Germany). 100 mg of lignin was dissolved in 0.5 mL of DMSO-d6 using

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TMS as internal reference. The scanning time is 10 h and the test was carried out at
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room temperature.

2.7 Optical evaluation

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The quantitative comparison of color of lignin was determined by CIE L*a*b* values
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using an L&W optical tester (L&W Elrepho 070, Sweden), the principle and

mechanism were illustrated in Fig. S1. In order to present a specific comparison of the

color distinction and avoid the chromatic aberration caused by the optical camera, the

color comparison figures were rebuilt according to the CIE L*a*b* values with the

help of ColorTell software. The brown index (BI) which was used to quantitatively

compare the color degree of lignin was calculated by the following equation (1):
(𝑎∗ +1.75𝐿∗ )𝑎∗
𝐵𝐼 = [5.645𝐿∗+𝑎∗−3.012𝑏∗ − 0.31] ÷ 0.17 eq (1)
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The visible light diffusion reflectance of lignin was investigated at solid state by a

Shimadzu UV-3600 spectrophotometer (Shimadzu, Japan) equipped with an

integrating sphere. BaSO4 standard sample was tableted and scanned to build the base

line. The diffusion reflectance of sample in the visible light region (400-760 nm) was

recorded with a step interval of 1 nm. The visible light absorbance of lignin in

aqueous solution was measured by an UV-2450 spectrophotometer (Shimadzu, Japan).

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Deionized water was scanned to build the base line and the visible light absorbance

between 400-760 nm was recorded.

2.8 Staining test

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The staining test was carried out according to the Chinese standard HG/T 3507-2008.
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Three kinds of textiles (cotton, dacron and chinlon) were used in the investigation. 2 g
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of cotton and dacron were added into the mixture of 50 mL of lignosulfonate solution
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and 110 mL of distilled water, respectively. Afterwards the mixture was heated to

130 °C using a high temperature dyeing autoclave and last for 1 h. The chinlon was
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stirred with 10 mL of lignosulfonate solution and 150 mL of distilled water at 95 °C

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for 15 min. Eventually, all the three textiles were well washed and oven dried to

compare the staining level.

2.9 XPS, SEM and bulk density analysis

X-ray photoelectron spectroscopy (XPS; Kratos Analytical, U.K.) equipped with a

monochromatic X-ray source (1486.6 eV) was conducted to collect the information of

lignin samples in the drying process. The morphology and aggregation state of lignin

in the microscopic scale was observed by a scanning electron microscopy (SEM;

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Zeiss, Germany). The bulk density of lignin was investigated as follow: A known

quantity of lignin was added into a small cylinder and glass rod was used to gently hit

the cylinder wall for 5 times. The bulk density was calculated by dividing the mass of

lignin by the recorded volume. Five tests were repeated for each sample to get an

average.

3. Results and discussion

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3.1 Color variation of lignin in acid precipitation

Acid precipitation is a facile and widely used method for the separation of lignin from

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black liquor. But the color of lignin was different at different precipitation pH value.
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As seen in Fig. 1, the black liquor was continuously adjusted by dilute H2SO4 solution
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and was filtrated, and the lignin fractions were cleanly washed at different pH values.
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It is obvious that the color of lignin was gradually lightened from black to brown as
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the decrease of pH value. Then, what gives raise to the color distinction of lignin

separated from the same black liquor.

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The structure was characterized to illustrate the color distinction. As shown in Fig. 2a
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the lignin precipitated at different pH has very different molecular weight distribution.

As the decrease of pH value, the molecular weight of lignin fraction decreased

gradually (Details in Table 1). At the pH of 6, three peaks (14487, 30293, 49276)

appeared in the curve and the molecular weight of the separated lignin fraction

distributed widely from 1000 to 100000 with an Mw of 18632 and a PDI of 2.406.

However, at the separation pH of 4 and 2, both lignin fractions presented single peak

(pH 4: 3785; pH 2: 2803) with the Mw of 5143 and 3746, respectively. Besides, the
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distribution curves became narrower compared with the curve at pH 6.

It is known that the Kraft lignin is soluble in black liquor because the original

macromolecule structure of lignin was degraded into small fractions and the phenolic

hydroxyl mainly derived from the cleavage of β-O-4 linkage was converted to

water-soluble phenolate in the alkaline conditions. In the acid precipitation, as the

decrease of pH, the phenolate was converted back to phenolic hydroxyl which is less

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soluble than the phenolate in aqueous solution, finally resulting in the phase

separation of lignin in the black liquor. In addition, the solubility of lignin also related

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to some other factors, such as the molecular weight distribution and condensation
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degree. Since the pulping was processed under harsh condition with high pressure and
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temperature, part of the original lignin was inevitably condensed in the pulping
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process [46]. The condensed structures that formed between lignin units or between
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lignin and hemicellulose increased the molecular weight distribution and presented a

lower solubility, which made it easy to be separated at a higher pH value. Besides, the
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condensed structures also enlarged the conjugation and unsaturation degree of lignin,
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which plays an important role in darkening the color of lignin separated at pH 6.

In a word, the condensed structure with a high molecular weight was first separated in

the acid precipitation at high pH stage, and it was highly suspected as the main factor

that response for the color distinction of lignin separated from the same black liquor.

In order to prove that, the NBOs of the lignin fraction separated at pH 6 and 2 were

carried out, since most of the condensed structures are not degradable by the NBO

process, and there will be a significant difference between the two fractions regarding
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the oxidation products. As shown in Fig. 2b, the oxidation products of the lignin

fractions obtained at pH 6 and 2 reached the mass ratio of 8.5% and 16.3% regarding

the syringyl unit, and reached 1.1% and 3.9% regarding the guaiacyl unit. As only

hardwood lignin was investigated in this experiment, and extremely weak signal of

the hydroxyphenyl unit was detected, the total yield of the oxidation products was

approximately calculated by the addition of syringyl and guaiacyl units. The oxidation

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product of lignin fraction separated at pH 6 reached 9.6%, which is only half of that of

the lignin separated at pH 2. The NBO result strongly proved that large amount of

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condensed structures was separated in the high pH precipitation stage. Besides, the
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fraction of pH 6 presents a higher S/G ratio, indicating the condensation mainly
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occurred on the guaiacyl unit [47].

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The lignin fraction that contains the most condensed structures also presents a dark
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color (Fig. 1). As aforementioned, the conjugation and unsaturation of lignin was

enlarged due to the presence of condensed structures, which can be well demonstrated
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in the 2D HSQC spectra. The lignin fractions separated at pH 6 and 2 were

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investigated. As seen in Fig. 3, δC/δH 0/0 ppm is the signal of TMS (internal

standard). The whole spectra were compared at a same signal intensity of TMS. In the

unoxidized aliphatic region (δC/δH 0~50/0~2.5 ppm), seldom signals can be found in

the lignin fraction of pH 6, indicating a high oxidation level. In contrast, the signals of

lignin separated at pH 2 in the same region are more abundant and stronger, which

indicates that more saturated side chains such as methyl and methylene exist in the

lignin structure [26]. δC/δH 50~95/2.5~6.0 ppm belongs to the oxygenated aliphatic
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region, where a variety of signals were detected in the lignin fraction separated at pH

6. As seen in the spectrum of pH 6, the signals of δC/δH (102.8/4.30; 72.9/3.12;

74.9/3.32; 76.2/3.58; 63.5/3.88) are derived from the carbons (C1,2,3,4,5) in the xylan

structure [48] and those signals present strong intensity. On the contrary, only weak

signals of C2, C3, and C4 of xylan are observed in the spectrum of pH 2, the rest

signals of xylan are totally absence. As was demonstrated in previous studies [49,50],

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part of the xylan was oxidized in the harsh pulping and covalently bonded with lignin

forming larger and stronger conjugations which is also known as the lignin

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carbohydrate composite (LCC). This structure shows a high molecular weight, low
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solubility and contributes to the dark color of lignin, as a result it was mainly
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separated from the black liquor with lignin fraction obtained at pH 6 and aggravated
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the color performance of this lignin fraction. Furthermore, the oxygenated region of
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δC/δH 80.9~89.3/4.25~4.81 ppm stands for the β-O-4 substructures with the

conjugated carbonyl or carboxyl group (shown as the insert in 2D HSQC spectrum of

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pH 2). Generally, independent carbonyl is colorless, but when it conjugated with the
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aromatic rings or carbon-carbon double bonds, the whole conjugation is enlarged and

strengthened, which is responsible for the darker color of lignin [42,51]. In this region,

the lignin fraction obtained at pH 6 presents stronger signals than that of the lignin

fraction separated at pH 2. The higher content of this kind of structure is also one of

the factors that make the color distinction between lignin fractions obtained at

different pH values.

The more popular and greener method of CO2 precipitation was also detected and it is
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found that the color of the separated lignin always presents a dark color. Based on the

illustrations above, it can be well explained because the carbonic acid derived from

the CO2 is just a weak acid, which is not capable of creating a low pH. Thus, the high

molecular weight fraction was mainly precipitated at high pH value, resulting in the

dark color of CO2 precipitated lignin. The details and related evidences are listed in

the Fig. S2 and S3.

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3.2 Color variation of lignin in drying

In addition of the acid precipitation influence, we found another fact in the experiment

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that the color of lignin varies greatly during drying process. The color of the
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above-mentioned lignin fraction precipitated at pH 4 was investigated as a
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representative. As seen in Fig. 4 a and b, the lignin was processed by oven drying at
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85 oC and air drying at ambient condition, respectively. The latter sample shows a
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brown color which is much lighter than that of the oven dried lignin. Then what gave

raise to this color difference. For most people, the first reason that come to mind is
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oxidation. As the presence of oxygen and a relative high temperature, lignin

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macromolecule should be oxidized to a dark color in the oven drying. But this

common knowledge was unverified, and it was proved incorrect in the experiment.

The lignin precipitated at pH 4 was divided into three part, and was dried by vacuum

drying. In order to eliminate the effect of oxidation in the air, the oxygen in the

vacuum drying tank was removed by the repeatedly charging and releasing of high

purity N2, and the lignin was dried at a low temperature of 40 oC. The lignin samples

were dried at almost same conditions except equipped with different amount of P2O5
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as moisture scavenger. As shown in Fig. 4c, d and e, the color of lignin was gradually

darkened even without oxygen. Apparently, the lignin that dried with the most amount

of P2O5 as moisture scavenger exhibits the darkest color which is as dark as the color

of lignin dried by the air oven at 85 oC (Fig. 4a).

The oxidation degree of the lignin samples in Fig. 4a and e can be seen from the

changes in the C (1s) and O (1s) XPS spectra. As shown in Fig. 5a and b, the fitted

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binding peaks around 287, 286, and 285 eV are originated from the C=O, C-O, and

C-C/C-H, respectively [52]. In the Fig. 5c and d, the fitted binding peaks around 534,

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533, and 532 eV are assigned to the C-O-C/Ph-O-C/Ph-O-Ph, C-O-H/O=C-O-C,
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C=O/O-C=O, respectively [53]. The C (1s) spectra in Fig. 5a present a broader
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binding energy curve compared with that of Fig. 5b, especially in the area where the
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binding energy is higher than 286 eV. An obvious increase is observed in the fitted
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C=O area, indicating a higher oxidation degree of the oven dried lignin at 85 oC. At

the same time, the C=O peak of the O (1s) that belongs to the oven dried lignin is well
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fitted in Fig. 5c. But in the same area, the characteristic peak of the C=O was hard to
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be isolated and only two peaks were fitted in the curve of the vacuum dried lignin at

40 oC (Fig. 5d) [54]. This evidence also proved that the oven dried lignin at 85 oC was

more oxidized and the vacuum dried lignin at 40 oC was less oxidized, which well

agrees with the result obtained in the C (1s) spectra. However, the color of lignin

looks the same and all are in black regardless of the oxidation degree. Additionally,

the vacuum dried lignin in Fig. 4e reaches 17.92/2.21/3.17 regarding the CIE L*a*b*

values (the L*a*b* values of the samples that appear in this work are detailed in Table
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S1), which is even slightly darker than that (18.56/2.13/3.57) of the oven dried lignin

in Fig. 4a. Based on this fact, we speculated that the oxidation dose exists, but it is not

a crucial factor that influence the color of lignin in the drying process. Then, what

give rise to the color distinctions.

The brown index which has taken all the L*a*b* values into account was calculated

with equation (1) and a higher brown index indicating a lighter color of lignin [21].

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The order of the lignin samples in Fig. 4 was rearranged in Fig. 6 according to the

color degree. It is found that the brown index of lignin was gradually decreased with

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the increase of the bulk density. Although the tendency of the brown index is in
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different direction, it presents a reasonable correlation with that of the bulk density.
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The five lignin samples are originated from the same black liquor and was separated
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by the same process, the most important difference between them is the dewatering
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rate resulted by different drying treatment. As seen in Fig. 6b, c and d, the increasing

amount of P2O5 led to the acceleration of dewatering rate in vacuum drying, which
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improved the shrink of lignin macromolecules and built a tighter connection between
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lignin clumps due to the hydrogen bonding interaction. As a result, the bulk density of

lignin increased, which lead to the concentration of the lignin chromophores in the

macroscopic scale. It is obvious that as the increase of P2O5 the brown index of lignin

gradually decreases, indicating that the color of lignin is getting darker. Similarly, the

oven dried lignin at 85 oC (Fig. 6e) also has experienced high dewatering rate forming

tightly connected structures, which as a result shows a high bulk density and dark

color. On the contrary, the lignin dried at ambient condition (Fig. 6a) and the lignin
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dried with few P2O5 have undergone mild dewatering rate. As consequence, they both

have lower bulk density and brighter color.

In fact, the dewatering rate is just an appearance influence on the color of lignin, and

the most essential factor that behind the dewatering rate is the aggregation state of

lignin, which was changed during different drying process. As shown in Fig. 7, the

aggregation state of the lignin fraction which was precipitated at pH 4 was

investigated. Large clumps were formed during the oven drying at 85 oC (Fig. 7a),

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and the surface of the lignin clump is flat and dense, indicating a tightly aggregated

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state of lignin macromolecules. While dried at the ambient conditions, the aggregation
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is alleviated and some gaps and humps can be seen in Fig. 7b, although the
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aggregation still exists due to the hydrogen bonding and Van der Waals interaction
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between lignin molecules [55]. Obviously, as the alleviation of the aggregated state,
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the color of lignin was lightened, which well agrees with the results obtained in Fig. 6.

Based on the aggregation regulation, the spray drying was carried out since lignin
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slurry can be scattered into micro-droplet forming lignin microsphere after flash
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evaporation (Fig. 7c). Similarly, the freeze drying which can maximally maintain the

separated structure of lignin after the precipitation was also processed (Fig. 7d). As

seen in the SEM, lignin clumps are totally absent after the spray and freeze drying.

The formation of lignin microsphere reduced the aggregation of lignin

macromolecules, consequently the apparent color of lignin was further lightened,

which is consistent with the results in Fig. 4 and 6. It is noteworthy that in order to

reduce the color of lignin, the freeze drying in Fig. 7d was carried out with a low
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concentration. While the concentration of lignin was increased, for example 45 g/L,

the stronger aggregation has appeared before freeze and it cannot be separated in the

freeze drying (Fig. 7e), therefore the obtained lignin came back to the dark color like

that of the strongly aggregated lignin in Fig. 7a. This phenomenon offers further

evidence that the aggregation state of lignin is exactly crucial to the color

performance of lignin.

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3.3 Proper selection of light-colored lignin regarding specific utilizations.

Based on the research discussed above, we can conclude that the color of lignin was

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determined by multi-factors which can be approximately summarized as structural
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and aggregation factors. No relationship was found yet between these two factors at
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present, but it is found that the two factors need to be particularly considered
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regarding specific utilizations. As seen in Fig. 8, the lignin fraction separated with the
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pH value higher than 5 and the fraction separated between the pH 5 and 2 were

obtained, subsequently they were sulfonated by a butane sultone method which was
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facile and well investigated in previous work [43]. The sulfonated lignin derived from
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the high pH fractionation was spray dried to solid powder and labeled as HLS. The

sulfonated lignin derived from the low pH fractionation was vacuum dried and labeled

as LLV. Apparently, the aqueous solution of HLS (Fig. 8A) exhibits a darker color

compared with that of the LLV (Fig. 8B) in a same concentration. The Fig. 8a also

proves the color distinction that the absorbance of HSL is always stronger than that of

the LLV in the visible light part (400-760 nm), indicating a darker color of the HSL.

On the contrary, the color of HLS (Fig. 8A’) is much lighter than that of the LLV (Fig.
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8B’) at the solid state. As seen in the visible light diffusion reflectance investigation

(Fig. 8b), the LLV absorb most of the incident light and little light scattered back into

eyes, resulting in the dark color of LLV at solid state. Meanwhile the reflection of

HLS is stronger than that of the LLV, especially at long wavelength area which

contributes more to the bright seeing of the HLS. Then what gives rise to the amazing

reversal of the color of lignin at different phase state. The reason that leads to the

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color distinction in the aqueous solution is mainly originated from the chemical

structural aspect. As was demonstrated above, more condensed structures were

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separated into the fractions obtained at a higher pH value due to its large molecular
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weight and poor solubility. The condensation enlarged the conjugation and
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unsaturation degree of lignin, which as a result gives rise to the dark colored
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performance of HLS in the solution. However, the HLS was spray dried to solid
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powder and lignin microspheres was formed in the spray drying (SEM in Fig. S4),

which created a lower bulk density than that of the vacuum dried LLV (0.235 and
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0.792 g/cm3, respectively) and decreased the chromophore concentration of lignin in

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the macroscopic aspect. Obviously, the macroscopic reduction of the concentration of

chromophore plays a more dominant role regarding the color performance of lignin in

the solid state. Therefore, the color of HSL was much more lightened than that of the

vacuum dried LLV, which well agrees with the advantages illustrated in the Fig. 6 and

7.

It is hard to say seeing is believing in that case since the color of lignin was highly

tunable with the change of structure and aggregation state, and it is hard to define
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which is the true color of lignin as was exhibited in Fig. 8. Then, how to choose the

lignin with a right color for the subsequent color-depended utilizations. The selection

of lignin needs to be separately considered regarding the using occasions. As for the

color-depended utilization of lignin at solid state, such as the preparation of

lignin-based sunscreens with a blending method, the aggregation adjustment is

efficient for the color reduction and the quantitative evaluation of L*a*b* values is

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effective in that case, since the lignin microsphere was not or seldom dissolved in the

matrix. As shown in Fig. 9, the sunscreens blended with different amount of lignin

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microspheres present regular and acceptable color performance. Similarly, it also
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applicative in the utilizations such as the manufacturing of biobased polymers using
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lignin microsphere to improve the mechanical strength, and some other fields using
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lignin microspheres as additives (related studies are not further illustrated here).
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Another major color-depended utilization of lignin is in solutions, such as the widely

used dyestuff dispersant. The staining is an import issue while using lignin as
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dispersant since the dark color of lignin causes color distortion of dyes. Therefore, the
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as prepared lignin sulfonate HLS and LLV were evaluated in the staining test. As seen

in Fig. 9 (right part), the HLS causes serious contamination on kinds of textiles, but

the LLV shows a very weak contamination which seems unchanged compared with

the blank samples. It is obvious that the color evaluation at solid state (Fig. 8 A’, B’

and b) is totally reverse with the staining test, instead, the visible light absorbance

(Fig. 8 a, A and B) keeps accordance with the staining result. On this condition, lignin

is dissolved and the influence caused by the aggregation is totally vanished. The
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structure distinctions become the dominant factor that influence the color of lignin in

solutions. This result indicates that the quantitative evaluation of color at solid state is

irrelevant with the utilization of lignin in the solutions and the investigation of visible

light absorbance become a reliable pathway for the selection of lignin with proper

color performance in solutions.

4. Conclusions

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The color of lignin varies greatly during the separation and purification process. The

factors that have impact on the color of lignin were investigated and related

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mechanisms were illustrated. Condensed structures were formed during the pulping
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process. They enlarged the conjugation and unsaturation degree of lignin, which as a
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result gives rise to the dark of lignin. Because of the high molecular weight and low
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solubility, the condensed structures were mainly separated at high pH value. It is the
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reason why the lignin fractions obtained with the pH value higher than 6 shows a

darker color. The oxidation does exist in the drying of lignin, but it is not a
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dominating factor that influence the color of lignin at solid. It is the aggregation
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difference which is determined by the drying techniques and parameters that

eventually leads to the color distinctions of lignin. The less aggregated lignin presents

a low bulk density, which decreases the chromophore concentration in the

macroscopic aspect, consequently lightening the apparent color of lignin at solid state.

The selection of lignin for the color-depended utilizations needs to be considered

separately. As for the lignin used at solid state or as microsphere additives, the

aggregation adjustment is an efficient way for the color reduction and the L*a*b*
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evaluation is effective. While used as dispersant or in solution state, the conjugation

and unsaturation become dominant, which means the selection of lignin needs to be

considered depending on the structural aspect.

Acknowledgements

We are grateful for support from the National Key Technologies R&D Program (Grant

No. 2017YFD0601003), the Project funded by China Postdoctoral Science

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Foundation (Grant No. 2019M662927), the Science and Technology Foundation of

Guangzhou City (Grant No. 201607020025), the Science and Technology Project of

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Sichuan province (Grant No. 2019YFN0029), and the National Natural Science
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Foundation of China (Grant No. 31570569).
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Conflict of interest
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The authors declare no conflict of interest.

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List of Tables and Figures

Table 1 Molecular weight distribution of lignin precipitated at different pH.

Fig. 1. Acid precipitation and fractionation procedure of lignin from black liquor and

the color performance of lignin fractions obtained under different pH value.

Fig. 2. (a) Molecular weight distribution and (b) alkaline nitrobenzene oxidation result

of lignin fractions obtained under different pH value. S, syringyl unit; G, guaiacyl

of
unit.

Fig. 3. 2D HSQC spectra of lignin separated at pH 6 and 2. The inserted chemical

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structures are xylan (top) and β-O-4 substructures with the conjugated carbonyl or
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carboxyl group (bottom).
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Fig. 4. The color of lignin at solid state. The lignin was obtained by (a) oven drying at
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85 oC; (b) air drying at ambient condition; (c)vacuum drying at 40 oC using 250 g of
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P2O5 as moisture scavenger; (d) vacuum drying at 40 oC with 500 g of P2O5; (e)

vacuum drying at 40 oC with 750 g of P2O5.

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Fig. 5. (a) C1s XPS spectra of the oven dried lignin under 85 oC; (b) C1s XPS spectra
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of the vacuum dried lignin under 40 oC; (c) O1s XPS spectra of the oven dried lignin

under 85 oC; (d) O1s XPS spectra of the vacuum dried lignin under 40 oC.

Fig. 6. The brown index and bulk density of (a) air dried lignin at ambient condition;

(b) vacuum dried lignin at 40 oC using 250 g of P2O5 as moisture scavenger; (d)

vacuum dried lignin at 40 oC with 500 g of P2O5; (d) vacuum dried lignin at 40 oC

with 750 g of P2O5; (e) oven dryied lignin at 85 oC.

Fig. 7. SEM observation and color comparison of (a) oven dried lignin under 85 oC;
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(b) air dried lignin at ambient condition; (c) spray dried lignin; (d) freeze dried lignin

with a concentration of 2 g/L; (e) freeze dried lignin with a concentration of 45 g/L.

Fig. 8. (a) The visible light absorbance of sulfonated lignin solutions with a same

concentration of 8 mg/L and (b) visible light reflectance of sulfonated lignin at solid

state. HLS: sulfonated lignin derived from the high pH fractionation and was dried by

spray drying. LLV: sulfonated lignin derived from the low pH fractionation and was

dried by vacuum drying. A and A’ are the HLS at solution and solid state, respectively.

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B and B’ are the LLV at solution and solid state, respectively.

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Fig. 9. Representatives of color-depended utilizations of lignin. Using lignin as
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sunscreen additive (left) and dye dispersant (right; staining test).
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Table 1 Molecular weight distribution of lignin precipitated at different pH.

Precipitation pH Peak No. Mp Mw Mn PDI

2 1 2803 3746 2250 1.665

4 1 3785 5143 3341 1.539

14487

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6 3 30293 18632 7744 2.406

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Credit Author Statement

Hui Zhang: Conceptualization, Methodology, Software, Data curation,

Writing-Original draft preparation, Visualization, Investigation, Resources,

Writing-Reviewing and Editing.

Shiyu Fu:. Validation, Supervision, Resources.

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Yuancai Chen: Supervision.

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Graphical abstract

Essential knowledge and mechanism were illustrated to reveal the true reason that

results in the color distinction of lignin.

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