Liu 

and Liu Heritage Science (2022) 10:110

[Link]

RESEARCH Open Access

Reproducing ancient Chinese ink depending

on gelatin/chitosan and modern experimental
methodology
Zhen Liu1,2* and Kun Liu3 

Abstract 
Chinese ink has very special significance for presentation of artistic effects and preservation of works of art; however,
wasted nonrenewable resources, potential toxicity, and complex and inefficient production technologies have limited
the development of inks for traditional Chinese culture. Herein, environmentally friendly, practical and antibacterial
Chinese ink was prepared by facile heating-stirring of gelatin and the natural polysaccharide chitosan. The internal
composition, viscosity, morphology, particle size and antibacterial properties of the ink were characterized by Fourier
transform-infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and bacteriostatic
zone tests. The results showed that commercial ink (CM) spread easily on Xuan paper and led to imperfect artwork,
while the chitosan and gelatin ink (CG) showed good adhesion and stability on Xuan paper. Based on this study, we
believe that the good performance of CG ink should be attributed to restrictions arising from its internal chitosan
and gelatin network, which restrict diffusion. Finally, the author used CG ink to display traditional Chinese calligra-
phy and landscape painting and believes that it has significant application prospects and will be used in large-scale
production.
Keywords:  Chinese culture, Chinese ink, Chitosan, Gelatin, Calligraphy

Introduction artists to express their feelings. Therefore, we hoped to

The history of ink can be traced back for 3000 years to avoid harmful substances and use modern technology to
ancient China. The earliest written record was prepared prepare a practical ink for calligraphy.
in the Western Zhou Dynasty, and the ink-making pro- The person who specialized in making ink in ancient
cess had reached maturity by the Eastern Han Dynasty China was called the ink official, which was a low-level
[1]. During the Wei and Jin Dynasties, the “Mix-ink official position. This was mainly due to the contempt
method” mentioned in Qi Min Yao Shu was written by Jia held for workers in ancient times; the status of handi-
Sixie in the Northern Wei Dynasty and revealed the tech- craftsmen in society was relatively humble, which also
nology and formula of ink [2]. Compared with brush and limited the development of ink fabrication processes
paper, ink has very special significance for presentation of and materials. The complex and inefficient technology
artistic effects and preservation of works of art. It is not used for production of ancient ink was one of the dis-
only the result of brush, technique and artistic style but advantages of ancient ink. In addition, the popularity of
also an important medium allowing calligraphy and paint modern industrial ink has made studies of ancient ink
relatively rare. However, modern industrial ink often
contains potentially toxic substances, such as isopro-
*Correspondence: 76013659@[Link]
panol, ethyl acetate, butyl acetate and phenol, which are
2
Calligraphy Research Institute, Jinan University, Guangzhou 510632, People’s corrosive to the human body [3].
Republic of China
Full list of author information is available at the end of the article

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Liu and Liu Heritage Science (2022) 10:110 Page 2 of 12

Fig. 1  Schematic diagram of preparation of CG ink

In ancient times, incomplete combustion was used to paper and provide suitable inks for calligraphy and paint-
extract smoke and dust from pine branches or grease to ing by adjusting the ratio of CS and gelatin. In addition,
prepare pine smoke and oil fume, respectively. During some natural products were also introduced into our ink,
combustion, the fire temperature, tuyere and smoke col- such as tannic acid extracted from plants and borneol
lection time were strictly controlled to ensure soot black- extracted from borneol resin, which may have affected
ness, fineness, and oil and ash contents [4]. First, the glue corrosion resistance and incense enhancement. Moreo-
was enclosed by a gentle fire, pigment raw materials and ver, commercial (CM) ink was used as the control group
raw materials were added, the mixture was fully stirred, for comparison with the properties of our CG ink. In par-
and a pestle was used to pound the mixture evenly [5–7]. ticular, rare ink from the Qing Dynasty was also used to
It was said that the mixture should be hammered repeat- investigate the differences among inks. Based on these
edly with “100,000 pestles” so that the smoke and binder studies, we went further back into ancient Chinese cul-
reached a delicate and uniform degree and could be ture to understand the special cultural roles of Chinese
made into blanks [1]. In production of ancient ink, ani- ink.
mal tissue was needed to make collagen [4, 8]. The addi-
tion of animal collagen improved the viscosity of the ink Methods
and enhanced the experience of writing with the ink, Materials and reagents
but sources of animal collagen were very limited, espe- CS (Mw =100,000) and gelatin were obtained from
cially for some precious wild animals. Gelatin is a form Shanghai Macklin Biochemical Co., Ltd. Ethanol and
of hydrolyzed collagen and is essentially a product of other reagents were of analytical grade and purchased
collagen decomposition; it is similar to collagen in com- from Guangzhou Chemical Reagent Plant. The carbon
position and structure and is expected to replace animal used was water-soluble carbon black with high pigment
collagen when added to the ink to improve the writing (N330). Other ingredients (tannic acid, borneol and glyc-
experience [9, 10]. Chitosan (CS) is a partially deacety- erol) were obtained from Aladdin Chemical Co., Ltd.
lated product of the natural polysaccharide chitin, which (Shanghai, China).
has many functions, such as biodegradability, biocom-
patibility, bacteriostasis, and enhancement of immu- Preparation of CS‑gelatin ink
nity [11–13]. It is widely used in food additives, textiles, The formulas of the inks are listed in Table 1. The main
agriculture, environmental protection, beauty and health components of the inks were CS, gelatin and carbon pow-
care, cosmetics, antimicrobial agents, biomedical fields ders. In detail, as shown in Fig. 1 and 2% CS/acetic acid
and drug development and in many other fields and other solution and gelatin aqueous solution were prepared first
chemical industries. and stirred for 1 h at 60 °C to mix them well. Then, the
Hence, CS and gelatin were used in this study to carbon powders were added to the precursor and stirred
develop proper formulas for producing Chinese ink for another 3 h. Subsequently, ingredients such as tannic
(named CG ink). Combined application of CS and gela- acid, borneol and glycerol were introduced into the pre-
tin in inks was expected to enhance adhesion to Xuan cursor. After 6 h, the CS-gelatin (CG) ink was obtained.
 Liu and Liu Heritage Science (2022) 10:110 Page 3 of 12

Table 1  The formula of inks Bacteriostatic cycle experiments

wt% CS (2%) Gelatin (20%) Carbon H2O ingredients
Escherichia coli (E. coli, BNCC352086) on the slant of
powder each test tube were picked up with the inoculation ring
in aseptic water with glass beads, the spores were dis-
A 20 0 50 20 10
persed by hand for several minutes, and a mixed spore
B 15 5 50 20 10
suspension was made after filtration. Then, 0.5 mL of
C 10 10 50 20 10
a mixed spore suspension was injected into each Petri
D 5 15 50 20 10
dish with a sterile straw. Molten agar medium, 15–20
E 0 20 50 20 10
mL, was injected into each Petri dish (approximately
45 ℃), and the bacterial liquid was mixed with the
medium evenly and allowed to cool. Finally, tweezers
were used to soak discs of filter paper in different con-
Characterization
centrations of anti-mildewing agent, remove it, place
The morphologies of the inks and calligraphic works
it in the center of the medium plate with bacteria, and
were observed through a field emission scanning elec-
cover it with the lid. After culturing for 2–3 days at a
tron microscope (FESEM, ULTRA 55, Carl Zeiss,
suitable temperature, the sizes of the bacteriostatic cir-
Germany). In detail, the samples were first dried and
cles around the ink filter paper disks were observed.
dehydrated and then glued to the aluminum table with
The bacteriostatic effect was quantified by calculat-
conductive tape for observation.
ing the distance from the filter paper to the outermost
The rheological properties of the inks were inves-
periphery of the bacteriostatic zone, and the distance
tigated at 25 ℃ with a rotary rheometer (TA Instru-
was calculated with ImageJ software.
ments, New Castle, DE, USA). The viscosities of the
inks were tested with shear frequencies in the range
0.001–1000  ­s−1. The viscoelasticities were tested at a Results and discussion
strain of 0.5% and with frequencies ranging from 0.1 to Analyses of the properties of gelatin, chitosan and inks
10 rad/s. Chitosan and gelatin were the main components of the
X-ray photoelectron spectroscopy (XPS) with an alu- ink used in this study. As raw materials, their physical
minum (mono) ­Kα source (1486  eV) (Thermo Scien- and chemical properties directly affected the experience
tific @ K-Alpha, USA) was employed to investigate the of writing with the final ink, so we conducted detailed
chemical compositions of membrane surfaces. studies on the functional group compositions of raw
Fourier transform-infrared spectra of CS, gelatin and materials and inks. As shown in Fig.  2A, the charac-
inks were acquired using a Bruker EQUINOX55 Fou- teristic functional groups of chitosan were obtained
rier transform-infrared spectrophotometer (Germany) by total reflection infrared spectrometry. Because
in the range 400–4000 ­cm−1. The KBr pelleting method the number of amino groups in the chitosan polysac-
was used for sample preparation. charide chains began to increase during chitin dea-
Dependent upon how the paper is produced, the cetylation [14, 15], more amino functional groups were
“Xuan Paper” can be categorized into three types: raw detected on chitosan. The amine vibrations resulted in
Xuan, semi-processed paper (PiMade Xuan) and pro- sharp characteristic peaks at 3200–3500 ­cm−1 [16, 17].
cessed paper (Sized Xuan). In particular, PiMade Xuan Due to partial deacetylation, amide groups were still
owns the suitable water absorption and is commonly retained on the chain segments and showed carbonyl
used in writing. All papers used during the writing tests vibrational peaks at 1720  ­cm−1 in the infrared spec-
were made of PiMade Xuan paper, and commercial trum. The above results showed that the chitosan had
(CM) ink and ancient ink refer to Guhuan ink and Qing good purity and many amino functional groups. As
Dynasty ink. shown in Fig. 2B, gelatin is composed of various protein
Liu and Liu Heritage Science (2022) 10:110 Page 4 of 12

Fig. 2  Fourier transform-infrared spectra of CS, gelatin and inks (A–C). The schematic diagram of interaction of CS and gelatin in CG ink (D)

segments, so it contained many of the common func- compared the main functional groups of modern com-
tional groups of proteins, such as amino groups, car- mercial ink and antique ink (Fig.  2C). On the whole,
boxyl groups and even disulfide bonds [18, 19]. Similar the antique ink we prepared showed some similarities
to chitosan, there were typical amino peaks in the range with commercial ink; for example, there was an obvi-
3200–3500  ­cm−1, but there were large numbers of ous broad peak at approximately 3300 ­cm−1, which was
imine groups in gelatin, so there was no obvious peak attributed to the hydroxyl and amino groups in ink [20,
similar to those of chitosan. In addition, there was a 21]. However, the difference was that the antique ink
strong peak at 1720  ­cm−1, which confirmed that there showed two peaks in the low wavenumber region, while
were more carbonyl groups in gelatin [19]. Then, we the commercial ink had only one. This was attributed
 Liu and Liu Heritage Science (2022) 10:110 Page 5 of 12

Fig. 3  The schematic diagram of interaction between the brush and CG ink (A). The viscosity analysis of inks with different formulas (B)

to addition of gelatin and chitosan to the antique ink, reduced, ink in the brush will not be retained effectively.
especially the presence of more carboxyl groups in the Therefore, we needed to prepare ink with a viscosity that
gelatin. As shown in Fig.  2D, we simulated the main facilitated direct writing. The viscosity changes result-
functional groups of chitosan and gelatin molecules in ing from different proportions of inks were determined
antique ink. A large number of amino groups in chi- with rheological tests. As shown in Fig. 3B, it was obvious
tosan and carboxyl groups in gelatin produced strong that the gelatin content was directly related to the ink vis-
electrostatic interactions, which directly affected the cosity, and the viscosity of Group A was the lowest; this
viscosity of the antique ink. Additionally, the brush, viscosity was similar to that of water and was not condu-
which was made from animal hair, was also a protein cive to enveloping and retaining ink. On the other hand,
and had a composition similar to that of the gelatin Groups B and C had similar viscosities and maintained
[22, 23], which made it easy to produce physical bonds approximately 10  Pa.s. The viscosities of Groups D and
involving electrostatic interactions and hydrogen bond- E were higher; these inks had considerable viscosity after
ing between the ink and the brush upon addition of chi- contact with the Xuan paper, which was not conducive to
tosan and collagen; these were firmly absorbed by the writing. In conclusion, Groups B and C were considered
brush and enabled the brush to retain the ink. to have the proper viscosity, and Group C was selected
for further testing.
Analyses of rheological properties for inks with different
formulas Morphological and elemental analyses
The viscosity of an ink is directly related to the writing The particle sizes and dispersion of materials in ink are
experience. As shown in Fig. 3A, we hoped that ink would also very important properties. As shown in Fig.  4A
be retained after the brush was dipped and then would and B, we determined the particle sizes and dispersion
flow out smoothly during writing, which was determined levels of CM and CG inks. The sizes of the particles in
by interactions between the ink and brush. For example, the CM and CG inks were relatively similar, approxi-
when the interaction between ink and brush is strong, the mately 5–10  μm, while the difference was that the CG
brush is able to retain ink very strongly, which is reflected ink showed better dispersion and the particle dispersions
macroscopically by a sense of stickiness felt during brush were not concentrated. This meant that the CG ink in
writing; when the interaction is reduced, the brush ena- this experiment was comparable to the CM ink in terms
bles smooth writing, but when the interaction is further of particle sizes and slightly better in dispersibility. This
Liu and Liu Heritage Science (2022) 10:110 Page 6 of 12

Fig. 4  The FESEM images (A, B) and element analysis (C, D) of CM and CG inks

should be attributed to the highly beneficial properties Elemental analyses of CM, CG and Qing inks on paper
of CS in ink, which promoted dispersion of toner par- XPS was employed to determine the surface elemental
ticles. On the other hand, we also carried out elemental compositions from survey spectra, C 1s, O 1s and N 1s
analyses, and these results showed that there were differ- core-level scans of CM, CG and Qing inks on paper, as
ences in the compositions of the two inks (Fig. 4C and D). shown in Fig.  5. The XPS survey spectrum showed that
There was no elemental N in the CM ink, which meant there was no N in the CM ink, which was consistent
that collagen was not present in the formula and it dif- with the EDS results. With the CG and Qing ancient ink,
fered greatly from that of the CG ink. Furthermore, large the peaks for N were very obvious. These results indi-
amounts of calcium salts were present in the CM inks, cated that animal collagen may have been employed in
while only 0.2% calcium salts were detected in CG ink Qing ancient ink. Furthermore, the composition of CG
due to the limited use of calcium salts. ink was also proven to be similar to that of ancient ink
 Liu and Liu Heritage Science (2022) 10:110 Page 7 of 12

Fig. 5  XPS survey spectra, C1s, O1s and N1s core-level scan of CM, CG and Qing inks on paper

(11.1%), and the proportion of N was approximately 9.2%. character “中 (zhong)” is the first position where the
Moreover, the C 1s spectra indicated that the peak shapes brush touched the paper; the brush contained more ink,
were very similar, and there were peaks at 287.2 eV and and it was easily spread. As shown in Fig. 6C, the diffu-
285.3 eV. The above results revealed that the CG ink was sion area seen on the paper for CM ink was filled with
similar to Qing ancient ink in terms of elemental content. toner, which made it impossible to determine the origi-
nal trajectory. For CG ink, although there was diffusion,
Stabilities of inks on Xuan paper the diffusion area did not contain toner, but diffusion of
Inks with different compositions will show different water would not lead to unclear ink. Subsequently, we
spreading behaviors on paper; hence, the diffusivities used the dripping method to explore the stabilities of inks
of the as-prepared inks required further investigation. on paper. After a drop of water was dropped at the top of
One of the important factors affecting writing is diffu- the Chinese character, the CM ink was impregnated, and
sion of the carbon powder in ink, which makes it difficult a large amount of toner was spread and finally formed a
to show the edges and details of calligraphy. Therefore, stained area. In contrast, the CG ink only produced water
to compare the differences in ink diffusivity for CG and stains and did not exhibit ink diffusion. The above results
CM, we used the same kind of paper to produce calligra- showed that there was good interfacial adhesion between
phy. As shown in Fig. 6A and B, the head of the Chinese the CG ink and the Xuan paper used in this study.
Liu and Liu Heritage Science (2022) 10:110 Page 8 of 12

Fig. 6  The ink written on Xuan paper shows different traces and diffusion states before (A–C) and after (D) dropping water

We also verified the combination of ink and materi- dotted line); at the same time, even after larger magni-
als microscopically through FESEM. As shown in Fig. 7, fication, the boundary was still clear. This result verified
ink samples were taken from the edge and center of the that CG ink showed excellent interfacial bonding with-
paper. In Fig.  7A, there is no obvious boundary at the out any diffusion. Subsequently, we observed the central
junction of the CM ink and the Xuan paper, which may positions of the inks. It was clearly seen that the molec-
be because the edge was the weakest part of the ink mark ular networks of chitosan and gelatin and the fibers of
due to diffusion of the gradient. CG ink in the Xuan the Xuan paper cross-fused with each other and formed
paper appeared with a very clear dividing line (yellow a solid adhesion layer. However, these phenomena were
 Liu and Liu Heritage Science (2022) 10:110 Page 9 of 12

Fig. 7  The FESEM images of Xuan paper with ink (A, B). The schematic diagram of the interaction among the CS, gelatin and Xuan paper fibers (C)

not observed for the components of the CM ink. The Bacteriostatic properties of inks
above results showed that the network of chitosan and During long-term preservation, ink is easily attacked by
gelatin restricted the flow of toner effectively, and after bacteria, fungi and other microorganisms, which can
contact with Xuan paper, chitosan and gelatin crossed result in ink failure. Therefore, we introduced the anti-
deeply into the fibers [24] so they were not diffused by bacterial components chitosan and tannic acid into the
water. This is of great practical significance for preserva- ink. Chitosan is positively charged, while tannic acid has
tion and dissemination of calligraphic works. many phenolic hydroxyl groups, which have a strong
Liu and Liu Heritage Science (2022) 10:110 Page 10 of 12

were created, which was intended to test the black and

light effects of the CG ink. The stone was mostly done on
the side in order to test the ink adsorption capacities in
different parts of the brush tip, belly, root and so on. On
the whole, the performance of the CG ink was similar to
those of mainstream inks, but the effects of CG ink on dif-
ferent papers remain to be studied further.

Conclusions
Fig. 8  The bacteriostatic property of CM and CG inks using the E. coli. Although the process for preparation of ancient ink
The existence (a) and size (b) of the bacteriostatic circle around the
has been handed down to this day, it is so hard to use
inks filter paper disk (n = 5). The quantitative results were exhibited
as mean ± standard deviation, and one-way analysis of variance was ancient technology to make ink today due to the harsh
employed to make the statistical analysis. When p < 0.05, it means a preparation and preservation conditions required for
significant difference (*); when p < 0.01, it represents a very significant ancient Chinese ink. Therefore, to promote the spread
difference (**); when p < 0.001, it indicates a very high significant of this intangible cultural heritage of mankind based on
difference (***)
ancient ink, such as in Chinese calligraphy, it is neces-
sary to explore new technology and a material system
for Chinese ink used in calligraphy and painting. In the
antibacterial effect. In this study, the bacteriostatic prop- laboratory, an environmentally friendly, practical and
erties of CM ink and CG ink were tested with prelimi- antibacterial Chinese ink was prepared with a facile
nary bacteriostatic zone experiments. As shown in Fig. 8, heating-stirring process based on gelatin and the natu-
growth of bacteria around the CG ink were inhibited and ral polysaccharide chitosan. We found that the ink had
showed a clear culture medium, while there was some the proper viscosity when the ratio of chitosan to gelatin
decomposition of the bacterial community around the was 1:1. Then, we observed the micromorphologies and
industrial ink but no obvious bacteriostatic circle. These elemental compositions of the two inks with SEM and
results showed that the antique ink we prepared had found that although there were great differences in the
good antibacterial properties and is expected to be pre- elemental compositions of the CG ink and CM ink, the
served for a long time. particle sizes of the CG ink were comparable to those of
modern industrial inks. We further analyzed this differ-
Display of traditional chinese calligraphy and painting ence through XPS, and the results showed that the ele-
The author employed the CG ink to copy the representa- ments of CG ink were very similar to those of ancient ink.
tive work Zijin Research Post in Fig.  9. Originating from Then, the performance of each ink was evaluated by writ-
the author’s understanding of the copied object, the ing on Xuan paper and with SEM analysis. The results
author mainly tried to realize a perfect combination of showed that CM ink spread easily on Xuan paper, while
likeness and spiritual resemblance during the process of the CG ink showed good adhesion and stability on Xuan
copying. According to this work (Fig.  9C), CG ink pre- paper. Based on this study, we believe that the good per-
sented a good writing process, which showed the edge formance of CG ink should be attributed to restriction of
and turning well. Moreover, a strong ink color and rich toner by its internal CS and gelatin network, which limit
sense of layers also appeared. Furthermore, we applied diffusion. Finally, the author used CG ink to display tra-
CG ink to create landscape paintings, as shown in Fig. 9D. ditional Chinese calligraphy and landscape painting and
Compared with calligraphy, painting has loftier require- believes that it has significant application prospects and
ments for the descriptions of objects and the presentation will be used in large-scale production.
of ink. In the painting, the brush moved slowly when trees
 Liu and Liu Heritage Science (2022) 10:110 Page 11 of 12

Fig. 9  Prof. Liu took a group photo with his calligraphy work (A). The image of Zijin Research Post of Mi Fu on Song Dynasty (B) [25], and the
imitation of the Paste created by Prof. Liu with CG ink (C). Prof. Liu applied CG ink to create landscape paintings (D)

Acknowledgements software, validation, formal analysis, writing—original draft, writing—review

Thanks for the necessary experimental conditions provided by College of Art and editing. All authors read and approved the final manuscript.
and College of Chemistry and Materials of Jinan University.
Funding
Author contributions This work was supported by the Major Key Projects of the Ministry of Educa-
ZL: conceptualization, methodology, software, validation, formal analysis, tion of China (21JZD044), and Special Project for Unpopular Research of
writing—original draft, supervision. KL: conceptualization, methodology, Guangdong, China (GD21LMZYS02).
Liu and Liu Heritage Science (2022) 10:110 Page 12 of 12

Availability of data and materials 17. Liu Y-W, Zhou Y, Huang G-Q, Guo L-P, Li X-D, Xiao J-X. Fabrication of
The datasets used or analyzed during the current study are available from the lipase-loaded particles by coacervation with chitosan. Food Chem.
corresponding author on reasonable request. 2022;385:132689.
18. Chen T, Song Z, Liu H, Zhou C, Hong P, Deng C. Physicochemical proper-
ties of gelatin produced from Nile tilapia skin using chemical and fermen-
Declarations tation pretreatments. Food Biosci. 2022;47:101650.
19. Zhu Y, Chen S, Zhang C, Ikoma T, Guo H, Zhang X, Li X, Chen W. Novel
Competing interests microsphere-packing synthesis, microstructure, formation mechanism
There are no competing interests to declare. and in vitro biocompatibility of porous gelatin/hydroxyapatite micro-
sphere scaffolds. Ceram Int. 2021;47:32187–94.
Author details 20. Jamwal R, Amit S, Kumari S, Sharma S, Kelly A, Cannavan DK, Singh.
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Guangzhou 510632, People’s Republic of China. 3 Department of Material Sci- 2021;113:103222.
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and Materials, Jinan University, Guangzhou 510632, People’s Republic of China. imaging for the analysis of biopharmaceuticals. Spectrochim Acta Part A
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