Capstone Project - Do Quoc Khanh Bui Duc Khang

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY
CHEMICAL ENGINEERING
FOOD TECHNOLOGY

CAPSTONE PROJECT
TOPIC:
RESEARCH ON CITRULLINE EXTRACTION

FROM WATERMELON RIND
BY ION-EXCHANGE METHOD
STUDENT: Đỗ Quốc Khánh – 1952070

Bùi Đức Khang – 1952756
CLASS: CC19HTP2
SUPERVISOR: Assoc. Prof. Lại Quốc Đạt
CAPSTONE PROJECT INSTRUCTOR: ASSOC. PROF. LAI QUOC DAT
TRƯỜNG ĐẠI HỌC BÁCH KHOA CỘNG HÒA XÃ HỘI CHỦ NGHĨA VIỆT NAM
KHOA KỸ THUẬT HÓA HỌC Độc Lập – Tự Do – Hạnh Phúc
Số: ____ /ĐHBK-

NHIỆM VỤ LUẬN VĂN TỐT NGHIỆP
Bộ môn: CÔNG NGHỆ
THỰC PHẨM (Chú ý: sinh viên phải dán tờ này vào trang thứ nhất của bản thuyết minh)
HỌ VÀ TÊN: Đỗ Quốc Khánh MSSV: 1952070

Bùi Đức Khang 1952756
NGÀNH: Food Technology LỚP: CC19HTP2
1. Đầu đề luận văn:

Nghiên cứu trích xuất Citrulline từ vỏ dưa hấu bằng phương pháp trao đổi ion
Research on Citrulline extraction from watermelon rind by ion-exchange method
Số liệu:
- Citrulline adsorption and elution process of ion-exchange resin
2. Nhiệm vụ:
- Research the L-citrulline and D-glucose adsorption capacity of ion-exchange resins
- Research of L-citrulline adsorption and elution efficiency of ion-exchange resins
- Research L-citrulline elution efficiency of different pH value
- Research L-citrulline elution efficiency with Potassium in solution
- Số bản vẽ dự kiến: -
3. Ngày giao nhiệm vụ luận văn: 01 / 01 / 2023
4. Ngày hoàn thành nhiệm vụ: 20 / 05 / 2023
5. Họ và tên người hướng dẫn: Phần hướng dẫn:
- Lại Quốc Đạt , BM Công nghệ thực phẩm 100%
Nội dung và yêu cầu LVTN đã được thông qua Bộ môn.
Ngày 22 tháng 05 năm 2023

CHỦ NHIỆM BỘ MÔN NGƯỜI HƯỚNG DẪN CHÍNH
(Ký và ghi rõ họ tên) (Ký và ghi rõ họ tên)
PHẦN DÀNH CHO KHOA, BỘ MÔN

Người duyệt (chấm sơ bộ):
Đơn vị:
Ngày bảo vệ:
Điểm tổng kết:
Nơi lưu trữ luận án:
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INSTRUCTOR’S COMMENT
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TP. HCM, date … month … year 2023
Signature of instructor
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COMMENT OF REVIEWER
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TP. HCM, date … month … year 2023
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ACKNOWLEDGEMENT
With the deepest gratitude, we would like to thank the teachers in the Department
of Food Technology - Ho Chi Minh City University of Science and Technology for
wholeheartedly teaching and equipping us with knowledge. necessary for the duration of
the lecture period. This valuable knowledge will serve as a solid foundation for us on our
future journey.
We would like to thank Mr. Lai Quoc Dat has enthusiastically supported, helped
and taught us a lot of knowledge during the thesis work with his valuable experience.
We would like to thank our family and friends who have always been there to
encourage and support us throughout the years.
Although we have tried to complete the thesis within the scope and ability, we will
inevitably have shortcomings. We look forward to receiving your guidance and
understanding to improve the thesis.
We sincerely grateful!
i
ABSTRACT
The purpose of the project "Study on the dynamic adsorption of L - citrulline
from watermelon rind by ion-exchange resins and research elution method" is
investigation of the dynamic adsorption behaviour of L-citrulline from watermelon rind
extract using ion-exchange resins. L-citrulline, an important bioactive compound with
numerous health benefits, is found in significant quantities in watermelon rinds. The aim
of this research is to develop an effective method for the extraction and purification of L-
citrulline from watermelon rind, utilizing ion-exchange resins and optimizing the elution
process. The results of this research provide valuable insights into the dynamic adsorption
behaviour of L-citrulline from watermelon rind using ion-exchange resins. The optimized
elution method ensures efficient recovery of L-citrulline with high purity. These findings
contribute to the development of a cost-effective and environmentally friendly process for
extracting and purifying L-citrulline, which can be utilized in various applications,
including pharmaceuticals, functional foods, …
The topics covered in the thesis include:
- Overview on Citrulline properties and Citrulline in watermelon rind; ion-
exchange resins and adsorption isotherm model.
- Research content includes:
+ Research L-citrulline adsorption efficiency of ion-exchange resins.
+ Establishing L – citrulline adsorption isotherm model of ion-exchange resins
+ Research L-citrulline elution efficiency at different pH
+ Research L-citrulline elution efficiency at different time
+ Research L-citrulline elution efficiency with Potassium in stimulation solution
ii
TABLE OF CONTENT
ACKNOWLEDGEMENT ......................................................................................................... i
ABSTRACT ................................................................................................................................. ii
TABLE OF CONTENT ...........................................................................................................iii
LIST OF FIGURES .................................................................................................................. vi
LIST OF TABLES ...................................................................................................................vii
LIST OF ACRONYMS ......................................................................................................... viii
CHAPTER 1: INTRODUCTION ........................................................................................... 1
CHAPTER 2: OVERVIEW ..................................................................................................... 3
2.1. CITRULLINE OVERVIEW ........................................................................................ 3
2.1.1. Physical properties ..................................................................................................... 3
2.1.2. Chemical properties.................................................................................................... 3
2.1.2.1. Acid-base calculation.......................................................................................... 3
2.1.2.2. Urea functional group......................................................................................... 4
2.1.3. Function of L-citrulline.............................................................................................. 5
2.1.3.1. Synthesis and metabolism of L-citrulline ......................................................... 5
2.1.3.2. Biological activity................................................................................................ 5
2.1.3.3. Dosage .................................................................................................................. 6
2.1.4. L-citrulline production ............................................................................................... 7
2.1.5. L-citrulline in watermelon ......................................................................................... 7
2.2. ION-EXCHANGE OVERVIEW ................................................................................. 8
2.2.1. Introduction ................................................................................................................. 8
2.2.2. Ion-exchange materials .............................................................................................. 9
2.2.3. Ion-exchange selectivity .......................................................................................... 10
2.2.4. Mechanism of ion-exchange ................................................................................... 11
2.2.5. Model of adsorption isotherm ................................................................................. 12
2.2.6. Factors affecting ....................................................................................................... 13
CHAPTER 3: MATERIALS, CONTENTS AND RESEARCH METHODS ............ 15
3.1. MATERIALS ................................................................................................................. 15
3.1.1. Watermelon rind simulation solution..................................................................... 15
iii
3.1.2. Ion-exchange resin ................................................................................................... 15

3.1.2.1. Na + ions resin .................................................................................................... 15
3.1.2.2. Storage and operating conditions ................................................................... 16
3.2. CHEMICALS................................................................................................................. 17
3.3. RESEARCH CONTENT............................................................................................. 17
3.3.1. Research L-citrulline adsorption efficiency of ion-exchange resins.................. 18
3.3.2. Establishing L – citrulline adsorption isotherm model of ion-exchange resins 19
3.3.3. Research L-citrulline elution efficiency at different pH...................................... 19
3.3.4. Research L-citrulline elution efficiency at different time ................................... 20
3.3.5. Research L-citrulline elution efficiency with Potassium in stimulation solution
............................................................................................................................................... 21
3.4. ANALYSIS METHODS .............................................................................................. 21
3.4.1. Determination of adsorption capacity .................................................................... 21
3.4.2. Determination of adsorption efficiency ................................................................. 22
3.4.3. Modelling adsorption isotherm............................................................................... 22
3.4.3.1. Langmuir's equation.......................................................................................... 22
3.4.3.2. Freundlich's equation ....................................................................................... 23
3.4.4. Determination of elution efficiency ....................................................................... 23
3.5. DATA ANALYSIS METHODS................................................................................. 23
CHAPTER 4: RESULTS AND DISCUSSION .................................................................. 24
4.1. Research L-citrulline adsorption efficiency of ion-exchange resins .................. 24
4.2. Establishing L – citrulline adsorption isotherm model of ion-exchange resins
................................................................................................................................................... 25
4.3. Research L-citrulline elution efficiency at different pH....................................... 28
4.4. Research L-citrulline elution efficiency at different time .................................... 29
4.5. Research L-citrulline elution efficiency with Potassium in stimulation solution
................................................................................................................................................... 30
CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS .................................. 32
5.1. CONCLUSIONS ........................................................................................................... 32
5.2. RECOMMENDATIONS ............................................................................................. 32
CHAPTER 6: REFERERNCES ........................................................................................... 33
iv
APPENDIX ................................................................................................................................ 35
APPENDIX A: ANALYSIS METHODS......................................................................... 35
A.1. Analysis of Citrulline content ................................................................................... 35
APPENDIX B: STANDARD CURVE ............................................................................. 38
B.1. Standard curve L – citrulline ..................................................................................... 38
APPENDIX C: EXPERIMENTAL DATA ..................................................................... 39
C.1. Research L-citrulline adsorption efficiency of ion-exchange resins .................... 39
C.2. Establishing L – citrulline adsorption isotherm model of ion-exchange resins .. 40
C.3. Research L-citrulline elution efficiency at different pH ........................................ 42
C.4. Research L-citrulline elution efficiency at different time ...................................... 42
C.5. Research L-citrulline elution efficiency with Potassium in stimulation solution43
v
LIST OF FIGURES
Figure 2.1. The molecular structure of L-citrulline................................................................. 3
Figure 2.2. Amino acid ion conversion reaction. .................................................................... 4
Figure 2.3. Metabolism of Citrulline.. ...................................................................................... 5
Figure 2.4. Chemical synthesis of L-citrulline from arginine ................................................ 7
Figure 2.5. Ion-exchange process .............................................................................................. 8
Figure 2.6. Classification of ion-exchangers............................................................................ 9
Figure 2.7. Ion-exchange reaction ........................................................................................... 10
Figure 2.8. Mechanism of ion-exchange process .................................................................. 12
Figure 3.1. Research content. .................................................................................................. 18
Figure 4.1. L-citrulline adsorption efficiency of resins ...................................................... 24
Figure 4.2. One-Way-ANOVA ................................................................................................ 25
Figure 4.3. Linear graph showing isotherm according to Langmuir model ...................... 27
Figure 4.4. Linear graph showing isotherm according to Freundlich model.................... 27
Figure 4.5. L-citrulline elution efficiency at different pH .................................................. 28
Figure 4.6. L-citrulline elution efficiency at different time ............................................... 29
Figure 4.7. L-citrulline elution efficiency with Potassium in stimulation solution ........ 30
Figure B.1. Standard curve of L – citrulline ......................................................................... 38
vi
LIST OF TABLES
Table 2.1. Citrulline content of watermelon rind and intestines in different varieties . ..... 8
Table 3.1. Information of Purolite® C100 resin. .................................................................. 16
Table 3.2. Chemicals used in the thesis. ................................................................................ 17
Table 4.1. Value of adsorption isotherm model.................................................................... 26
Table 4.2. RL parameter values at different initial concentrations of L - citrulline ........ 26
Table B.1. Absorbance of L -citrulline at different concentration .................................... 38
Table C.1. L-citrulline adsorption capacity of ion-exchange resins ................................. 39
Table C.2.1. L-citrulline adsorption capacity ....................................................................... 40
Table C.2.2. L-citrulline adsorption efficiency .................................................................... 41
Table C.3. L-citrulline elution efficiency at different pH................................................... 42
Table C.4. L-citrulline elution efficiency at different time ................................................ 42
Table C.5. L-citrulline elution efficiency with Potassium stimulation solution ............. 43
vii
LIST OF ACRONYMS
Cit+: HOOC-CH(R)-NH3+ – Citrulline is in the protonated state.
NO: Nitric oxide.
SP: Sulfoxyl (-SO3H).
CM: Carboxymetyl (-CH2-COOH).
viii
CHAPTER 1: INTRODUCTION
For many people, participating in sports, exercising, and maintaining good health
has become a way of life. Since then, supplements encourage physical activity, such as
sports nutrition drinks, tablets, and powders rich in amino acids, etc., have been paid more
attention. These goods are now recommended to a wide range of consumers outside just
athletes. Citrulline is being utilized more frequently in sports drinks in addition to minerals,
vitamins, and amino acids like taurine and lysine, particularly in t he category of pre-
exercise nutrition. Watermelon rinds contain a variety of nutrients, including vitamins,
minerals, and especially having a rich amount of L-citrulline. Exploring the potential of
extracting L-citrulline from watermelon rinds lead to the development of value-added
products or ingredients with potential health benefits, such as functional foods or dietary
supplements.
L-citrulline has gained significant attention in recent years due to its various health-
promoting properties. It is naturally abundant in watermelon rind, making it an attractive
source for the extraction and production of L-citrulline. However, the efficient and
selective separation of L-citrulline from complex matrices such as watermelon rind poses
a significant challenge.
The adsorption behavior of L-citrulline using ion-exchange resins has been explored
in previous studies, but in static adsorption method. Therefore, this research aims to
investigate the dynamic adsorption of L-citrulline from watermelon rind extract using
commercially available ion-exchange resins. The specific objectives include evaluating
different ion-exchange resins for their adsorption capacity, selectivity, and kinetics, as well
as optimizing the elution process to obtain a purified L-citrulline product.
Vietnam is one of the top watermelon producers in the world. As a result,

watermelon rinds are readily available in large quantities, especially during the harvest
season. Using a locally abundant material for research can be cost -effective and
environmentally sustainable.
ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 1

Watermelon rinds are typically discarded as waste after the fruit is consumed. By
utilizing watermelon rinds for research purposes, you can contribute to waste reduction
efforts and promote a circular economy by finding alternative uses for this agricultural
byproduct.
We made the decision to carry out the project "Research on Citrulline extraction
from watermelon rind by ion-exchange method" based on the current situation as well as
the advantages and future development potential of L - citrulline. The goal of the study is
to use the ion-exchange technology to gather L-citrulline from watermelon rind and explore
the process of adsorption.

CHAPTER 2: OVERVIEW
2.1. CITRULLINE OVERVIEW
L-citrulline is a neutral, non-essential -amino acid which can be synthesized by
body endogenously. It has an important role in urea cycle in the liver and kidneys as a
precursor of arginine, a substrate for nitric oxide synthase (NOS). Therefore, L-citrulline
is an effective L-arginine and NO supplies having large potential for enhancing sport
performance.
2.1.1. Physical properties

Citrulline has molecular formula C6H13 N3O3 (Figure 2.1) and 175.188 g/mol in
molecular weight.
Figure 2.1. The molecular structure of L-citrulline.
At normal conditions, citrulline has appearance of white and odorless crystalline

powder with melting point at 214 oC. Citrulline is soluble slightly in water and sparingly in
aqueous acid but insoluble in methanol and ethanol. [1]
In the body, liver and intestines, Citrulline exists in two different forms: (i) free L-
citrulline and (ii) citrullinated protein produced when the enzyme PADIs deaminated
arginine. In food products, citrulline is added as free or (iii) citrulline malate when L-
citrulline is mixed with malic acid. [2]
2.1.2. Chemical properties

2.1.2.1. Acid-base calculation
Zwitterionic form of L-citrulline having an anionic carboxy group and a protonated
amino group; a neutral compound with pH = 7.3 and the isoelectric point of citrulline pI =
5.91. [1]

Citrulline has two acid-base radicals: a carboxylic acid group (pK 1 ~ 2.4) and an
amine group (pK2 ~ 9.4), being able to absorb two protons. The ion conversion reaction
between citrulline and neutral amino acids is demonstrated in Figure 2.2. The figure shows
that the dipole form remains electrically neutral, whereas the acidic form acquires a
positive charge and the base form takes a negative charge. This observation is consistent
with the fundamental principles of ionization chemistry. [2]
Figure 2.2. Amino acid ion conversion reaction. [17]
2.1.2.2. Urea functional group

Citrulline can be distinguished from other amino acids by the presence of a urea
functional group within its carbon chain, as depicted in Figure 2.1. The biological activity
of Citrulline is contingent upon the chemical processes that take place at its functional
group, particularly those that involve arginine, a closely interactive molecule.
The reaction under consideration involves the ureide functional group, in which the
electrophilic carbon is crucial due to its considerable electron affinity towards the nitrogen
and oxygen atoms. Subsequently, the carbon in question may engage with nucleophilic
species to yield the precarious N2C(R)O- configuration. Upon separation of NH3, R-NH2
(yielding ornithine), or water from the reaction output, a state of stability is attained. All of
the aforementioned chemical reactions can only occur in the presence of either proton
donors or acceptors, specifically water or, in a majority of instan ces, appropriate amino
acids located within the active site of the enzymes.

2.1.3. Function of L-citrulline

2.1.3.1. Synthesis and metabolism of L-citrulline
Urea cycle (90%): Figure 2.3. shows the urea cycle in liver, detoxifies ammonia
from nitrogen from enternal sources (dietary protein) and muscle, thus metabolizing it into
urea while Citrulline will transform back to Arginine in kidneys by enzymes ASS and ASL
in Arginine synthesis process.
Figure 2.3. Metabolism of citrulline.
(NO: Nitric oxide; NOS: Nitric oxide synthase; ADMA: Asymmetric dimethyl
arginine; ASS: Argininosuccinate synthase; ASL: Argininosuccinate lyase. [3] )
The blood vessel's nitric oxide cycle (10%): Nitric oxide (NO) can be synthesized
endogenously in two ways: by a reduction of inorganic nitrate or through NOS enzyme
action from Arginine, producing Citrulline as a byproduct (Figure 2.3). These processes
will make NO enter the bloodstream, improving cardiovascular health.
2.1.3.2. Biological activity

- Antioxidant:

L-citrulline may serve as antioxidant, interacting directly with hydroxyl radicals via
-amino acids in the protonated state NH3, to prevents DNA damage-induced oxidative
stress caused by the production of hydroxyl radicals. [4]
- Anti-inflammatory:
Citrulline cooperatively exerts an anti-inflammatory effect on synovial cells with

glucosamine and N-acetylglucosamine [5] . Moreover, Citrulline also indirectly improve
vascular system by decreasing low-grade chronic inflammation.
- Anti-hypertensive:
According to NO production, Citrulline indirectly reduce arterial blood pressure

and boost patient health. Citrulline has been stated the ability to effectively reduce blood
pressure in both prehypertensive and hypertensive patients. [6]
- Strengthening of skeletal muscles:
Citrulline is able to stimulate muscle protein synthesis through an increase in

skeletal muscle weight, which is derived from an improvement in NO synthesis in
endothelial cells. [7]
- Improve erectile dysfunction:
L-citrulline supplementation has been proved to be safe and psychologically well

accepted by patientsm, an alternative treatment for mild to moderate ED. Nitric oxide
(NO) acts as a neurotransmitter within the nerve fibers of the penis and operates as a
vasodilator on the smooth muscle cells present in the penile artery. Consequently, NO
represents a critical physiological signal for the initiation of penile erection . [8]
2.1.3.3. Dosage
Citrulline is safe and very well absorbed when used briefly. The most commonly
employed dose of CM is a single acute 8 g dose which reflect early work observing
performance benefits during resistance exercise using this dose [9] . However, at high dose

(>10g/dose) Citrulline can cause gastrointestinal side effects. This might be because high
amounts of arginine cause diarrhea by saturating intestinal arginine absorption .
2.1.4. L-citrulline production

Citrulline is frequently produced in industrial scale by microbial fermentation (using
certain microbes such as Corynebacterium glutamicum from glucose [10] ) and chemical
synthesis from Arginine such as in Figure 2.4.
Figure 2.4. Chemical synthesis of L-citrulline from arginine .[17]
With the abundance of natural L-citrulline, watermelon has been a rich source of L-
citrulline particularly when utilizing the white rind waste from other businesses. Until now,
many ways have been researched to extract L-citrulline from watermelons, including:
- Extraction: strict conditions (HCl 6M, 145 oC, time 4h) [11]
- Ion-exchange and crystallization (can be combined with extraction or membrane

use): the product is crystal with high purity (up to 99.8%) and is not harmful to the
environment [12].
2.1.5. L-citrulline in watermelon

The most abundant source of Citrulline in nature is watermelon. Citrulline content
ranged from 7.9 to 28.5 mg/g dry weight (dwt) and was similar between seeded and
seedless types (16.6 and 20.3 mg/g dwt, respectively). Red flesh watermelons had slightly
less citrulline than the yellow or orange flesh watermelons (7.4, 28.5 and 14.2 mg/g dwt,
respectively). Rind contained more citrulline than flesh on a dry weight basis (24.7 and
16.7 mg/g dwt, respectively) but a little less on a fresh weight (fwt) basis (1.3 and 1.9 mg/g
fwt, respectively). These results indicate that watermelon rind, an underutilized agricultural
waste, offers a source of natural citrulline. [13]

Table 2.1. Citrulline content of watermelon rind and intestines in different varieties
Color (mg/g dwt) Rind (mg/g Flesh (mg/g Rind (mg/g

dwt) fwt) fwt)
Red 7.9 15.6 1.0 0.8
Yellow 28.5 29.4 3.5 1.5
Orange 14.2 28.2 1.8 1.5
With a watermelon fruit is edible, consisting of about 70% flesh and 30% rind,
goes to waste [14]. From this study, it is apparent that the rind contains citrulline in high
quantities. This study shows that the watermelon rind is a rich source of an important
amino acid and may yield a useful product from an agricultural waste.
2.2. ION-EXCHANGE OVERVIEW

2.2.1. Introduction
Ion-exchange is when some ions in a solid are swapped for different ions in a
liquid. The exchanger needs to have a structure that is open and can carry ions through it.
It can be made of organic or inorganic material. An ion-exchanger is a substance that can
swap its charged particles with other similar charged particles in something it touches,
but it can't be dissolved in water. The meaning of this also illustrate how ions are
exchanged. Basically, it means that the exchanger and the substance being exchanged
each other. The fact that ions are exchanged implies that the exchanger must be ionized,
but only one of the ions in the exchanger is soluble
For example, the ionized ion-exchanger A+B− with M+ being the soluble ion and
placed in a solution of the salt NY, which ionizes to give the ions C+ and D−, the
exchange reaction as follows:
A+B- + C+ + D- → C+B- + A+ + D-
Figure 2.5. Ion-exchange process

2.2.2. Ion-exchange materials

According to electrolytic polymeric form of ion-exchangers having mobile ion-
exchange groups connected to an inactive support, they are divided into 3 categories: (i)
natural, (ii) modified natural and (iii) synthetic
Figure 2.6. Classification of ion-exchangers [15]
- Natural:
+ Inorganic: Vermiculite, Zeolites, Clays, …
Materials with mineral compounds has natural ability to exchange ion, easy
workability.
+ Organic: Polysaccharide, Protein, Carbonaceous materials, …
Plant and animal cells act as ion-exchangers by the presence of the carboxyl groups (-
CO2 H) after being given simple chemical treatment
- Modified natural: Some naturally occurring organic ion-exchangers are modified

to improve exchange capacity and selectivity
- Synthetic:
+ Inorganic: Zeolites, Titanates and silicotitanate, ...

Categorizing into Synthetic zeolite (aluminosilicate), Hydrated oxides of metals,

Poly acid salts, Hydrous oxides, … with different properties.
+ Organic: Polystyrene divinyl, Phenolic, Acrylic, …
Ion-exchange is the product of the lattice polymerization of styrene or acrylic with

divinylbenzene. The functionally attached groups can be acidic (carboxyl-COOH,
sulphonic -SO3H or phenolic -OH) or alkaline (amine, –NH2, N-substituted amine)
Synthetic organic exchangers also can be classified into 2 categories by the charge
properties: (i) cation-exchangers and (ii) anion-exchangers. Figure 2.7 below demonstrates
the exchange reaction of cation and anion-exchangers.
Figure 2.7. Ion-exchange reaction [15]
(a) Cation-exchange reaction; (b) Anion-exchange reaction
Cation-exchange: chemically attached substances capable of exchanging cations,

such as sufonic acid or carboxylic groups. The active group of the cat ion-exchangers has
negative charge such as -SO3H, -PO3H, -COOH..., its counter-ion has positive charge. This
resin will perform exchange with positive ions. This cation-exchangers are often produced
with Na + ions form.
Anion-exchange: opposite to cation-exchangers, the anion-exchangers have the

positive charge in the active group such as tertiary amines -NR2 or the quaternary
ammonium -N+R3, its anti-ion has a negative charge. Resins are usually produced with their
ionic antagonists Cl - and OH-.
2.2.3. Ion-exchange selectivity

To select the appropriate exchangers, the properties should be considered as
following target:

+ Total capacity: the maximum amount ion-exchangers can adsorb, (eq/g or eq/ml)
+ Effective capacity: the maximum amount ion-exchangers can adsorb under

specific conditions and always smaller than the total capacity, depending on pH, ion
concentration to be exchanged, etc.
+ Dynamic capacity: usually used in case of ion-exchange with columns operating

with continuous flow.
+ Thermal stability: this parameter indicates the durability of resin in a certain

temperature range. The cation-exchangers of all ions are stable in water up to 120°C.
Anion-exchangers are less stable. [16]
2.2.4. Mechanism of ion-exchange

A basic ion-exchange process consists of an ion-exchange material (resin) that has
swelled to water and the surrounding liquid solution. In addition to these two phases, there
is a solution-formed film at the surface of the particle called the Nernst film. The formation
of this film is inevitable and it has different properties from the surrounding solution.
Continuous stirring can reduce the thickness of the film but cannot destroy it.
The mechanism of the ion-exchange process is depicted in Figure 2.8, the details of
this process are as follows:
The molecules in solution dissociate ion pairs into cations and anions as soon as it’s
soluble to solution (1). Ions continue to diffuse in solution (2), then an ion diffuse in film
and in exchanger (3, 4). When ions went through film, it formulated with ion of resin create
a new ion pair (5). Immediately, another ion pair on surface of resin will be dissociate (6),
then dissociate in exchanger (7), through film (8) and in solution (9) and finally formulates
with other ion which left at (1) step completing the exchange process.

Figure 2.8. Mechanism of ion-exchange process [15]
2.2.5. Model of adsorption isotherm

Ion-exchange may be perceived as an adsorption phenomenon due to their same
characteristics. Hence, it is reasonable that the ion-exchange information can be justified
for the Langmuir or Freundlich adsorption models based on the balance of ion-exchange.
The Langmuir model is founded on the exchange onto surface between

concentration of adsorbate in liquid phase and capacity of adsorbent in solid phase, both in
the equilibrium point and at specific temperature.
Langmuir isotherm adsorption equation:
(2.1)

Where: KL: Langmuir adsorption constant
qe: adsorption capacity at equilibrium (mg/g)
qmax : Maximum adsorption capacity of adsorbent (mg/g)
Ce: Concentration of adsorbent in solution at equilibrium (mg/L)
As same as Langmuir model, but different in phase of adsorbate, Freundlich model

shows the exchange onto surface between concentration of adsorbate in gas phase and
capacity of adsorbent in solid phase, both in the equilibrium point and at specific
temperature.
Freundlich isotherm adsorption equation:
(2.2)
Where: qe: adsorption capacity at equilibrium point (mg/g)
Ce: Concentration of adsorbent in solution at equilibrium (mg/L)

1
KF and : Freundlich adsorption constant
𝑛
2.2.6. Factors affecting

- The nature of ions: affects the stability, adsorption capacity, type of ion adsorbed
yes, operating conditions, etc.
- Nature of the sample material: the composition of the charged components and the
charge present in the sample, the values of the charges, pH, ionic strength, etc.
- Technological factors:
+ Temperature: the temperature change leading to the change of the diffusion of

components in the solution; otherwise at high temperatures undesirable reactions can
happened and increase costs.

+ pH: most important for substances whose charge changes with different pH such
as amino acids, proteins, etc.
+ Time: industrial companies conduct continuous method in ion-exchange processes

making the sample flow rate through the ion column or retention time or combine them
with filtration technique, showing the importance.

CHAPTER 3: MATERIALS, CONTENTS AND RESEARCH

METHODS
3.1. MATERIALS
3.1.1. Watermelon rind simulation solution
The simulation solution consists of 1000mg/L of L - citrulline 98%, 60g/L of
reducing sugar D - glucose, with or without potassium in varying concentrations (100, 200,
300 mg/L). These ingredients are based on previous research project on watermelon rind
of Diem Quynh, Quynh Loan, 2022 [17]. The reason why stimulation solution not the real
watermelon rind juice is to remove the undesirable factors such as minerals, metals, … and
research the highest amount Citrulline can be extracted in ideal environment.
D – glucose is available in a large amount in watermelon rind and has familiar

properties like Citrulline (soluble in water and similar molecular mass) but doesn’t carry
electricity whereas Citrulline has different charge value at different pH levels. This
difference makes ion-exchange process to separate Citrulline from a sugar-rich solution
like watermelon rind feasible. Another purpose to add D-glucose to stimulation solution is
to research the ability to extract Citrulline from solution with dissolved substance like D-
glucose or mineral elements which other methods such as nanofiltration (NF),
ultrafiltration (UF), …cannot separate. The filtration techniques are often used to remove
suspended solids to obtain a semi-finished product having identical properties as
stimulation solution, and in combination with ion – exchanger, Citrulline is separated
potentially.
3.1.2. Ion-exchange resin

3.1.2.1. Na + ions resin
The type of ion-exchange resin used is the strongly acidic cation-exchanger
Purolite® C100. The details of this grain are shown in the table below.

Table 3.1. Information of Purolite® C100 resins [18]
Properties Details
Polymer structure Polystyrene gel cross-linked with

divinylbenzene
Shape Spherical
Active group -SO3- (Acid sulfonic)
Ion Na+
Capacity 2.0 eq/L or 4.5 eq/kg
Moisture content 44-48%
Size 300 – 1200 µm (< 300 µm, max 1%)
Volume 800-840 g/L
Temperature 120 oC
pH operating range 6-10
Before being put into use, newly resins must be expanded. The activated treatment
is necessary, the resin is washing several times with distilled water to avoid impurities and
contaminants until waste water clear; then swelling with saturated NaCl for 4 -6 hours to
maximize capacity and to avoid small cracks caused by quick expand by water. After that,
to be able to using, the resin will be washed again with distilled water.
3.1.2.2. Storage and operating conditions

Ion-exchange resin must be kept in swollen or partially swollen condition in NaCl
cause if not, it will shrink and its surface which contains ion to perform exchange process
will dry out and unable to be used anymore because when reabsorbing water, dried resins
will break easily due to the loss of elasticity.

Operating conditions of ion-exchangers must be free of impurities such as sludge,

soil, pigments, colloids, microorganisms, oil, suspended solid, … and not in emulsion or
suspension state. If impurities stick on the surface of resins, it will prevent ions of adsorbate
to be exchanged with active group of ion-exchanger.
3.2. CHEMICALS
The chemicals used in the thesis are presented in Table 3.2 below.
Table 3.2. Chemicals used in the thesis.
Chemical Origin Purpose
L - citrulline Germany Prepare the pseudo-sample solution.

L-citrulline quantification calibration standard.
D - glucose China Prepare the pseudo-sample solution.
Reducing sugar quantification calibration standard.
KCl VN Prepare the pseudo-sample solution.
HCl and NaOH VN Adjust pH value
H3PO4 China Medium for the quantitative reaction of L-
citrulline.
H2SO4 VN Medium for the quantitative reaction of L-
citrulline.
Sample inorganicization for total protein
quantification.
Diacetyl monoxime Germany Coloring reagent for the quantitative analysis of L-
(DAMO) citrulline.

3.3. RESEARCH CONTENT
Figure 3.1. Research content
3.3.1. Research L-citrulline adsorption efficiency of ion-exchange resins

3.3.1.1. Purpose
Determine the L - citrulline adsorption efficiency of ion-exchange resins
3.3.1.2. Methods
Prepare the sample:

The initial solution used is a simulation solution with a concentration of L - citrulline

of 1000 mg/L, reducing sugar D - glucose of 60 g/L, adjusted to pH = 3 with standard 0.1
N HCl solution [12].
Procedure:
Adding 30g of activated resins into a burette, cotton at the bottom, connected with
infusion bag containing stimulation solution and set flowrate stable.
Analytical metrics:
- Content of L - citrulline solution initially and at the end of the experiment.
3.3.2. Establishing L – citrulline adsorption isotherm model of ion-exchange resins

3.3.2.1. Purpose
Determine the L-citrulline adsorption capacity and efficiency of ion-exchange resins
Establish an adsorption isotherm model.
3.3.2.2. Methods
Prepare samples
The initial solution used is a simulation solution with several L-citrulline

concentration of 400; 600; 800; 1000; 1200; 1400 mg/L, D-glucose 60 g/L, adjusted to pH
= 3 with standard 0.1N HCl solution [12] .
Procedure
Adding 30g of activated resins into a burette, cotton at the bottom, connected with
infusion bag containing stimulation solution and set flowrate stable.
Analytical metrics
- Content of L – citrulline solution initially and at the end of the experiment.
3.3.3. Research L-citrulline elution efficiency at different pH

3.3.3.1. Purpose
Determine L - citrulline elution efficiency at different pH

3.3.3.2. Methods
Prepare the sample:
The initial solution used was a stimulation solution with a concentration of L-

citrulline of 1000 mg/L, a concentration of reducing sugar D-glucose of 60 g/L, adjusted
to pH = 3 with standard 0.1N HCl solution [12] . Prepare eluent solution with NaOH and
distilled water at different pH = 8, pH = 9, pH = 10, pH = 11.
Procedure:
Use 1000mL the eluent solution with pH = 8, pH = 9, pH = 10, pH = 11. Add 4

eluent solutions respectively to 4 resins columns after adding stimulation solution.
Analytical metrics:
- Content of L - citrulline solution at the end of the experiment.
3.3.4. Research L-citrulline elution efficiency at different time

3.3.4.1. Purpose
Determine L - citrulline elution efficiency at different time
3.3.4.2. Methods
Prepare the sample:
The initial solution used was a stimulation solution with a concentration of L-

citrulline of 1000 mg/L, a concentration of reducing sugar D-glucose of 60 g/L, adjusted
to pH = 3 with standard 0.1N HCl solution [12] . Prepare eluent solution with NaOH and
distilled water, pH=10.
Procedure:
Add 1000mL eluent solution to resins column after adding stimulation solution, set
flowrate stable. Take sample to test at different time.
Analytical metrics:

3.3.5. Research L-citrulline elution efficiency with Potassium in stimulation solution

3.3.5.1. Purpose
Determine L - citrulline elution efficiency with different concentrations of
Potassium in the stimulation solution.
3.3.5.2. Methods
Prepare the sample:
The initial solution used is a simulation solution with a concentration of L - citrulline

1000 mg/L, reducing sugar D - glucose 60 g/L. Adding to stimulation solution different
Potassium concentration 100 mg/L, 200 mg/L, 300 mg/L, adjusted to pH = 3 with standard
0.1N HCl solution [12]. Prepare eluent solution of NaOH and distilled water, pH=10.
Procedure:
Add the eluent solution to resins column after adding stimulation solution, set
flowrate stable.
Analytical metrics:
3.4. ANALYSIS METHODS

3.4.1. Determination of adsorption capacity
Adsorption capacity of resins is the amount of L-citrulline adsorbed per unit dry
mass of adsorbent (resins). The adsorption capacity at time t and at the equilibrium point
was calculated according to equations (3.1) and (3.2).
(𝐶0 −𝐶𝑡 )×𝑉

𝑞𝑡 = (3.1)
𝑚
(𝐶0 − 𝐶𝑒 )×𝑉
𝑞𝑒 = (3.2)
𝑚

Where:
q t and q e: Adsorption capacity at time t and at equilibrium (mg/g)
V: Volume of solution (L)
Co, Ct, Ce: Initial L - citrulline concentration, at time t and at equilibrium (mg/L)
m: Weight of dry resin (g)
3.4.2. Determination of adsorption efficiency

H adsorption efficiency (%) indicates the ratio of L - citrulline adsorbed into the
resin mass compared to the concentration in the original solution.
𝐶0 − 𝐶𝑒
𝐻 (%) = × 100% (3.3)
𝐶0
3.4.3. Modelling adsorption isotherm

3.4.3.1. Langmuir's equation
To research the model based on experimental data, we use the linear form of
Langmuir adsorption isotherm equation:
1 1 1 1
= × + (3.4)
𝑞𝑒 𝑞𝑚𝑎𝑥 𝐾𝐿 𝐶𝑒 𝑞𝑚𝑎𝑥
Where:
KL: Langmuir adsorption constant
q max : Maximum adsorption capacity of adsorbent (mg/g)
From q e and Ce experimental data, we can find q max and KL after plotting the method
linear equation 3.4 with a vertical axis of 1/q e and a horizontal axis of 1/Ce.
Another factor to evaluate the compatibility of the Langmuir model is the

dimensionless decomposition constant RL.
1
𝑅𝐿 = (3.5)
1+𝐶0 𝐾𝐿
If RL = 0, the adsorption is irreversible.

0 < RL < 1, compatible models.
RL=1, linear model.
RL > 1, model is not compatible.
3.4.3.2. Freundlich's equation

To research the model based on experimental data, we use the linear form of
Freundlich adsorption isotherm equation:
1
𝑙𝑛𝑞𝑒 = . 𝑙𝑛𝐶𝑒 + 𝑙𝑛𝐾F (3.6)
𝑛
1
Where: KF and : Freundlich adsorption constant [46]
𝑛
1
From the experimental data q e and Ce, we can find and KF after plotting the method
𝑛
linear equation 3.6 with vertical axis lnq e and horizontal axis lnCe.
3.4.4. Determination of elution efficiency

H elution efficiency (%) indicates the ratio of L – citrulline eluted from resins
compared to the concentration of L-citrulline being adsorbed before.
𝐶𝑒
𝐻 (%) = × 100% (3.7)
𝐶𝑎𝑏𝑠𝑜𝑟𝑏𝑒𝑑
3.5. DATA ANALYSIS METHODS

The experiments in the subject were repeated no less than 3 times. The results are
presented as mean ± standard deviation. Experimental results were processed by Analysis
of Variance (ANOVA) with Statgraphics 18. Pairwise comparison of experimental results
was performed by Multiple Range Tests posteriorly using LSD (p ≤ 0.05). Use the Excel
software used to conduct the above analysis.

CHAPTER 4: RESULTS AND DISCUSSION

4.1. Research L-citrulline adsorption efficiency of ion-exchange resins
50
L-citrulline adsorption efficiency (%) 45
40
35
30
25
20
15
10
5
0
0 50 100 150 200 250
Time (minutes)
Figure 4.1. L-citrulline adsorption efficiency of resins
According to the Figure 4.1, the adsorbed L-citrulline reach the peak at 200 minutes
with 67.064% adsorption efficiency and capacity is 44.709 mg L-citrulline/g resins. This
phenomenon can be explained that pH of stimulation solution is 3 while pI of L-citrulline
is 5.9, making L-citrulline positively charged being able for ion-exchange process with
ions Na + on the surface of resins.
At this time reaction (4.1) happened, ion Na + connected with active group of resins
RSO3− was replaced by ion Cit + from stimulation solution, making L – citrulline
concentration in stimulation solution to decrease gradually. However, reaction (4.1) may
proceed in the reverse direction and L - citrulline is desorbed and returned to stimulation
solution. The reaction continued happening until it reached the equilibrium point, that’s
why it can’t completely adsorbed Citrulline from stimulation solution.
𝑅𝑆𝑂3 − − 𝑁𝑎 + + 𝐶𝑖𝑡+ ↔ 𝑅𝑆𝑂3 − − 𝐶𝑖𝑡+ + 𝑁𝑎+ (4.1)

4.2. Establishing L – citrulline adsorption isotherm model of ion-exchange resins

In order to determine the equilibrium point, the 1-way-ANOVA has been processed
to show the difference in statistics of adsorption capacity of L-citrulline. Figure 4.2 has
shown the result:
Figure 4.2. One-Way-ANOVA
According to Figure 4.2, at 160 minutes and 180 minutes, there is no difference in
statistics, so the equilibrium can be considered at this point.
1 1
The figure showing the relationship between and for the Langmuir model is
𝑞𝑒 𝐶𝑒
shown in Figure 4.3 and the graph showing the relationship between lnq e and lnCe for the
Freundlich model is shown in Figure 4.4.
The values of Langmuir and Freundlich isotherm equations are presented in Table
4.1 and RL parameter at different initial concentration of L-citrulline are presented in Table
4.2.

Table 4.1. Value of adsorption isotherm model
Langmuir Freundlich
KL qmax R2 KF 1/n R2
0.0005 195.941 0.989 0.044 1.173 0.981
Table 4.2. RL parameter values at different initial concentrations of L - citrulline
Concentration 400 600 800 1000 1200 1400

(mg/L)
RL 0.833 0.769 0.714 0.667 0.625 0.588
The results from Table 4.1 show that the values of the parameters q max and KL
according to the Langmuir equation are 195.941 mg/g and 0.0005 respectively with the
correlation coefficient R2 of 0.989, the RL values according to the initial L - citrulline
concentration starting from 400 to 1400 mg/L ranges from 0.588 to 0.833 (Table 4.2). For
the Freundlich model, the correlation coefficient R 2 is 0.951, the Freundlich constant KF is
0.044 and the value of 1/n is 1.173. From that, it can be seen that the Langmuir and
Freundlich adsorption isotherm models have high reliability coefficients R 2 (R2 = 0.951 for
Freundlich, R2 = 0.963 for Langmuir). Besides, the equilibrium parameter values R L for
different concentrations of L - citrulline in the initial solution (Table 4.2) for the Langmuir
model are in the range 0 < RL < 1 but the value 1/n = 1.173 for the Freundlich model is not
in the range from 0 to 1. From here, we can assume that the L - citrulline adsorption process
on the cation resin follows Langmuir adsorption isotherm models.
Mechanism of Langmuir is monomolecular exchange theory happening on the

surface of resins in solid phase, where exists adsorption centres and is homogeneous in
terms of energy. In this situation, the reaction (4.1) happened, ion Na + connected with
active group of resins RSO3 − was replaced by ion Cit + from stimulation solution, causing
L – citrulline concentration in stimulation solution to gradually decrease.

Figure 4.3. Linear graph showing isotherm according to Langmuir model
Figure 4.4. Linear graph showing isotherm according to Freundlich model

4.3. Research L-citrulline elution efficiency at different pH

For ion-exchange process, pH is mostly important factor. Figure 4.5, show the
influence of the eluent solution pH on the elution efficiency and L - citrulline eluent
capacity of the Na+ ion resin when the reaction has reached equilibrium. Using NaOH with
another purpose for regenerating the resin after exchanging ion.
100
90
80
Elution efficiency (%)
70
60
50
40
30
20
10
0
8 9 10 11
pH
Figure 4.5. L-citrulline elution efficiency at different pH
When pH value rising higher than pI of L-citrulline (pI=5.9), capacity of Cit+ ions
in output solution increase, due to Na + in eluent solution bonded to resins and release Cit +
ions when reactions (4.2) take place.
𝑅𝑆𝑂3 − − 𝐶𝑖𝑡+ + 𝑂𝐻− + 𝑁𝑎 + ↔ 𝑅𝑆𝑂3 − − 𝑁𝑎+ + 𝐶𝑖𝑡+ + 𝑂𝐻− (4.2)
Results show that elution efficiency and capacity of L-citrulline has uptrend when
pH rising from 8.0-10.0 with pH = 8.0 (67.134%), pH = 9.0 (79.839%) and pH =10.0 with
the highest records (90.155 %). But at pH=11.0, both capacity and efficiency suffered a
downfall (82.880%). This can be explained that according to the properties of resins
Purolite® C100, resin performs well in range of pH 6-10 which did match experiments
results, and obviously at pH=11 the performance of resin is decreasing in efficiency. The

reaction (4.2) still happens but somehow reduce the amount of resins interacted due to the
decrease in function caused by high pH = 11.
In summary, the initial solution pH of 10.0 is optimal to carry out the cat ion-
exchange process with L – citrulline. Therefore, the pH 10.0 will be fixed for the following
elution experiments.
4.4. Research L-citrulline elution efficiency at different time

100
90
80
Elution efficiency (%)
70
60
50
40
30
20
10
0
0 50 100 150 200 250 300
Time (minutes)
Figure 4.6. L-citrulline elution efficiency at different time
Due to previous experiment, the optimal condition for eluting L-citrulline is with
pH=10.0. According to Figure 4.6, at first 10 minutes, the elution efficiency of L-citrulline
is 20.551%. It continues to increase gradually each 10 minutes and reach the peak at final
point 240 minutes with efficiency 89.286% at. From 200 minutes, the graph increased
slowly, easily notified that the reaction was about to reach the equilibrium point. It can be
explained that, when adding eluent solution causing reactions (4.2), this reaction happens
in 2 ways till solution reach equilibrium and from that L-citrulline content do not change,
components in solution is balance.

In summary, the optimal time for eluting would get the highest amount of extracted
L-citrulline is about 240 minutes. That is the time when the ion-exchange has reached the
equilibrium point and solution is balance.
4.5. Research L-citrulline elution efficiency with Potassium in stimulation solution

To investigate the potential impact of unwanted variables, potassium was introduced
into the stimulation solution to evaluate its effect on the elution efficiency of L-citrulline
as a KCl salt. The reason for selecting this representative is its prevalence in the mineral of
watermelon rind.
The elution efficiency of L - citrulline when the simulation solution has Potassium
is shown in Figures 4.7
100
90
L-citrulline elution efficiency (%)
80
70
60
50
40
30
20
10
0
0 100 200 300
Potassium concentration (mg/L)
Figure 4.7. L-citrulline elution efficiency with Potassium in stimulation solution
As the concentration of potassium (K) was increased from 0 to 300 mg/L, there was
a gradual decline observed in the elution efficiency of L-citrulline. Firstly, in the range of
K concentration from 0 to 100 mg/L, a substantial decrease in efficiency is observed
(89.11% at 0mg K/L to 50.521% at 100mgK/L). This results in the graph exhibiting a steep
decline. Similarly, at other K concentrations of 200 and 300 mg/L, there was no substantial
variance observed in the elution efficiency of L-citrulline, and the reduction experienced

was deemed negligible (44.983% at 200mgK/L and 40.205% at 300mgK/L). Adding

Potassium into adsorption solution caused reaction (4.3) in which K+ competes with Cit +,
largely prevent Cit+ from getting carried by resins instead that is K+, obviously leading to
decline in the amount of L-citrulline extracted.
𝑅𝑆𝑂3 − − 𝐶𝑖𝑡+ + 𝐾 + ↔ 𝑅𝑆𝑂3 − − 𝐾 + + 𝐶𝑖𝑡+ (4.3)
Reasons for negligible reduce at higher K concentration range, from 200 to 300
mg/L, is that the reaction (4.3) approached the equilibrium point. Both ions are not
competing each other anymore, components between elution solution and resins almost
balance.
In summary, with the presence of Potassium, resins cannot interact with Citrulline,
causing a decrease in elution efficiency. In order to get the highest amount of L-citrulline
extracted, Potassium should be removed at the beginning; so that elution process without
Potassium will be more effective.

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS

5.1. CONCLUSIONS
The highly acidic cation-exchange resin Purolite® C100 can be used for the
adsorption of L - citrulline in liquid solution, the Na+ ion form with adsorption efficiency
of resins to L-citrulline is high at 67.064% and adsorption capacity is 44.709 mg L-
citrulline/g resins. The optimal condition for eluted solution is pH = 10.0 and optimal time
for maximum adsorption is 200 minutes and for maximum amount of Citrulline to be
separated from resins is 240 minutes. The result also shows that elution process without
the presence of Potassium have better outcome, with higher capacity and efficiency. The
obtained results illustrated that the application of ion-exchange technique to separate L -
citrulline from the solution is highly feasible.
Experiments on stimulation solution gave better results than real watermelon rind
water due to the absence of undesirable factors or reactions. Therefore, L - citrulline
adsorption isotherm (Langmuir) were investigated on the simulation solution. More studies
and researches about the effects undesirable factors or reactions in watermelon rind on
efficiency of extracting Citrulline need to be examined.
5.2. RECOMMENDATIONS
To apply in real life extracting Citrulline from watermelon rind, more stud ies and
researches should be done, these are some follow-up research contents:
- Applying this ion-exchange method with real watermelon rind to research and study
if there are any undesirable factors affect the process or efficiency
- Studying method to remove Potassium and other sediments before starting ion-
exchange process in order to extract the highest amount of L-citrulline
- Investigate the filtration technique to remove suspended solids in watermelon rind,
in combination with ion-exchange for the purest amount of extracted L-citrulline
- Apply and combine research results into actual production processes.

CHAPTER 6: REFERERNCES
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Summary for CID 9750, Citrulline. Retrieved May 26, 2023 from
https://pubchem.ncbi.nlm.nih.gov/compound/Citrulline.
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Physiol. Endocrinol. Metab. 1981, 241, E473-E480.
[3] Aguayo, E.; MartínezSánchez, A.; Fernández- Lobato, B.; Alacid, F. L-Citrulline:
A Non-Essential Amino Acid with Important Roles in Human Health. Appl. Sci. 2021, 11,
3293.
[4] Laurentius, Andrea & Wikanendra, Gregorius & Arozal, Wawaimuli & Juniantito,
Vetnizah & Instiaty, Instiaty & Cong, Tzeto. L-citrulline as Potential Supplementation in
Protecting against Cardiovascular Disease. Proceedings for Annual Meeting of The
Japanese Pharmacological Society. 2018
[5] Yamagishi Y, Someya A, Nagaoka I. Citrulline cooperatively exerts an anti-
inflammatory effect on synovial cells with glucosamine and N-acetylglucosamine.
Biomed Rep. 2020 ;13(1):37-42.
[6] Yang, HH., Li, XL., Zhang, WG. et al. Effect of oral L-citrulline on brachial and
aortic blood pressure defined by resting status: evidence from randomized controlled
trials. Nutr Metab. 2019.
[7] Villareal MO, Matsukawa T, Isoda H. L-Citrulline Supplementation-Increased
Skeletal Muscle PGC-1α Expression Is Associated with Exercise Performance and
Increased Skeletal Muscle Weight. Mol Nutr Food Res. 2018.
[8] Cormio L, De Siati M, Lorusso F, Selvaggio O, Mirabella L, Sanguedolce F,
Carrieri G. Oral L-citrulline supplementation improves erection hardness in men with
mild erectile dysfunction. Urology. 2011;77(1):119-22.
[9] Gough, Lewis & Sparks, Andy & Mcnaughton, Lars & Higgins, Matthew &
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citrulline malate supplementation and exercise performance. European Journal of
Applied Physiology. 2021.

[10] Eberhardt D, Jensen JV, Wendisch VF. L-citrulline production by metabolically

engineered Corynebacterium glutamicum from glucose and alternative carbon sources .
AMB Express. 2014; 4(1):85.
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[12] Wayne W. Fish, Bartlesville. Process for the production of L-citrulline from
watermelon flesh and rind. U.S. Patent. 2012.
[13] Rimando AM, Perkins-Veazie PM. Determination of citrulline in watermelon
rind. J Chromatogr A. 2005 Jun 17;1078(1-2):196-200.
[14] US Department of Agriculture, Agricultural Research Service, USDA National
Nutrient Database for Standard Reference. 2004
[15] Sanjeev Kumar and Sapna Jain. History, Introduction, and Kinetics of Ion-
exchange Materials. Journal of Chemistry. 2013.
[16] A.S. Gokhale, P.K. Mathur, K.S. Venkateswarlu, Ion-exchange Resins For Water
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hấu bằng phương pháp trao đổi ion. Capstone Project, 2022.
[18] Purolite C-100 Strong Acid Cation Exchange Resin – Technical Data. The
Purolite Company.

APPENDIX
APPENDIX A: ANALYSIS METHODS
A.1. Analysis of Citrulline content
Equipment:
- UV-VIS spectrophotometer.
- Vortex shaker.
- Electric stove or thermostatic tank
Chemicals:
- L - standard citrulline (purity 98%)
- H3PO4 85%.
- H2SO4 95 - 98%.
- Diacetyl monoxime DAMO (98%)
- Activated carbon
Procedure:
Step 1: Prepare DAMO. reagent solution
- Weigh 3g of DAMO dissolved in 100 ml of distilled water to obtain a DAMO

reagent solution with a concentration of 30 g/L. Store the solution in a brown bottle,
cover with foil, protect from light and store at 10oC.
Step 2: Prepare the acid solution mixture
- Measure 300 ml of H2SO4 solution into a 500ml beaker, accurately measure 100
ml of H3PO4 solution into the upper beaker. We get 400 ml of H2SO4:H3PO4
solution (3:1)
Step 3: Prepare standard L - citrulline solution
- Weigh 0.1g of standard L-citrulline powder and make up to 100ml with distilled
water. We get a standard solution of L - citrulline 1000 mg/L.

- Dilute to the concentrations of 200, 400, 600, 800 mg/L according to the following
ratio.
Sample 1 2 3 4 5 6
Standard L - 0 0.2 0.4 0.6 0.8 1
citrulline solution
(ml)
Distilled water (ml) 1 0.8 0.6 0.4 0.2 0
L-citrulline 0 200 400 600 800 1000
concentration
(mg/L)
Step 4: Build the standard curve
- Pipette 1ml of L - citrulline solution at the above concentrations into test tubes (6
test tubes). This 1 ml was further diluted with 7 ml of distilled water and passed
through a vortex machine for homogenization.
- Then draw 1 ml of solution from the test tubes into 6 other test tubes, add to each
tube in turn:
+ 4 ml of distilled water.
+ 2ml acid solution H2SO4:H3PO4 (3:1).
+ 0.25 ml of DAMO solution (30g/L).
- Shake the test tubes well in the dark.

- The above test tubes are heated at 100℃ for 30 minutes with electric
stove/thermostat. Then cool to room temperature.
- Measure the absorbance at 490 nm of the above 6 tubes and build a standard curve.
Determine the concentration of L - citrulline in the diluent x (g/L) by interpolation method

from the standard curve equation.

Calculate the result:

From the standard curve equation, we can calculate the L - Citrulline content of the sample
after dilution x (g/L). The content of L - Citrulline X (g/L) in the original sample is:
X (g/L) = x* f
where f is the sample dilution factor.

APPENDIX B: STANDARD CURVE

B.1. Standard curve L – citrulline
Figure B.1. Standard curve of L – citrulline
Table B.1. Absorbance of L -citrulline at different concentration
L – citrulline concentration 0 200 400 600 800 1000

(mg/L)
Absorbance (OD) 0 0.301 0.515 0.869 1.153 1.328

APPENDIX C: EXPERIMENTAL DATA

C.1. Research L-citrulline adsorption efficiency of ion-exchange resins
Table C.1. L-citrulline adsorption capacity of ion-exchange resins
Time Adsorption capacity (mg L- Adsorption efficiency of L-

(minutes) Citrulline/L resin) Citrulline (%)
10 5.24±1.82ᵃ 7.86±2.73ᵃ
20 9.33±0.24ᵇ 14±0.36ᵇ
30 10.97±0.76ᵇ 16.45±1.14ᵇ
40 11.84±0.41ᵇᶜ 17.76±0.62ᵇᶜ
50 14.01±0.96ᶜ 21.02±1.44ᶜ
60 20.55±2.82ᵈ 30.82±4.23ᵈ
70 25.21±1.75ᵉ 37.81±2.62ᵉ
80 28.87±1.09 f 43.3±1.63 f
90 31.7±1.56 g 47.55±2.34 g
100 33.22±1.26 g 49.83±1.89 g
120 39.95±3.06 i 59.93±4.59 i
140 41.65±2.19 i 62.48±3.28 i
160 42.95±1.04 j 64.42±1.56 j
180 44.13±0.26 j 66.19±0.39 j
200 44.79±0.28 k 67.19±0.41 k
(In the same column, different lowercase letters (a to k) indicate a statistically significant
difference (α = 0.05))

C.2. Establishing L – citrulline adsorption isotherm model of ion-exchange resins

Table C.2.1. L-citrulline adsorption capacity
Time L-citrulline concentration (mg/L)

(minutes) 400 600 800 1000 1200 1400
10 0.66 ± 0.36ᵃ 0.92±0.35ᵃ 3.82 ± 1.52ᵃ 5.24 ± 1.82ᵃ 7.64 ± 1.03ᵃ 12.6 ± 3.08ᵃ
20 1.89 ± 0.5ᵇ 3.81±1.27ᵇ 5.75 ± 0.81ᵃᵇ 9.33 ± 0.24ᵇ 14.87 ± 3.47ᵇ 17.85 ± 0.94ᵇ
30 4.29 ± 0.29ᶜ 7.41±0.74ᶜ 7.58 ± 0.48ᵇᶜ 10.97 ± 0.76ᵇ 18.19 ± 2.65ᵇ 23.46 ± 0.98ᶜ
40 6.07 ± 0.44ᵈ 9.88±1.97ᵈ 9.85 ± 0.88ᶜ 11.84 ± 0.41ᵇᶜ 22.73 ± 1.15ᶜ 26.54 ± 0.73ᵈ
50 8.08 ± 0.96ᵉ 12.56±0.84ᵉ 13 ± 1.09ᵈ 14.01 ± 0.96ᶜ 24.67 ± 4.84ᶜ 29.55 ± 1.98ᵉ
60 9.82 ± 0.96 f 14.83±0.76 f 15.94 ± 1.92ᵉ 20.55 ± 2.82ᵈ 29.11 ± 3.83ᵈ 32.17 ± 1.13f
70 11.95 ± 0.48 g 16.62±0.86 g 19.21 ± 1.89 f 25.21 ± 1.75ᵉ 31.93 ± 1.42ᵈ 35.78 ± 1.34 g
80 13.34 ± 0.36 h 18.82±0.99 h 23.25 ± 2.68 g 28.87 ± 1.09 f 35.74 ± 0.59ᵉ 39.19 ± 1.36 h
90 14.26 ± 0.05 h 19.97±1.11 h 26.38 ± 1.98 h 31.7 ± 1.56 g 41.51 ± 0.09 f 43.25 ± 1.25 i
100 15.12 ± 0.2 i 21.85±1.54 i 29.06 ± 0.97 i 33.22 ± 1.26 g 45.79 ± 0.97 g 50.36 ± 1.77 j
120 15.7 ± 0.3 j 23.36±0.98 i 30.58 ± 0.81 i 39.95 ± 3.06 i 48.18 ± 0.98 g 53.8 ± 0.5 k
140 16.1 ± 0.74 j 24.71±0.85 j 31.46 ± 0.95 j 41.65 ± 2.19 i 50.01 ± 1.57 h 57.27 ± 0.58 l
160 16.46 ± 0.79 k 25.34±0.85 k 31.69 ± 0.86 j 42.95 ± 1.04 j 52.62 ± 1.11 i 59.94 ± 0.99 m
180 16.75 ± 0.66 l 25.51±0.81 k 32.12 ± 0.73 j 44.13 ± 0.26 j 53.7 ± 0.6 j 61.41 ± 0.32 m
200 17.05 ± 0.8 l 25.84±0.61 k 32.71 ± 1.38 j 44.79 ± 0.28 k 54.18 ± 0.66 j 61.85 ± 0.14 m
(In the same column, different lowercase letters (a to m) indicate a statistically significant difference (α = 0.05)

Table C.2.2. L-citrulline adsorption efficiency
Time L-citrulline concentration (mg/L)

(minutes) 400 600 800 1000 1200 1400
10 2.47 ± 1.36ᵃ 2.29 ± 0.87ᵃ 7.17 ± 2.85ᵃ 7.86 ± 2.73ᵃ 9.55 ± 1.28ᵃ 13.5 ± 3.3ᵃ
20 7.1± 1.88ᵇ 9.52 ± 3.17ᵇ 10.78 ± 1.51ᵃᵇ 14 ± 0.36ᵇ 18.59 ± 4.34ᵇ 19.13 ± 1.01ᵇ
30 16.09 ± 1.1ᶜ 18.52 ± 1.86ᶜ 14.21 ± 0.9ᵇᶜ 16.45 ± 1.14ᵇ 22.74 ± 3.31ᵇ 25.13 ± 1.05ᶜ
40 22.75 ± 1.66ᵈ 24.7 ± 4.92ᵈ 18.47 ± 1.65ᶜ 17.76 ± 0.62ᵇᶜ 28.42 ± 1.43ᶜ 28.43 ± 0.78ᵈ
50 30.29 ± 3.61ᵉ 31.39 ± 2.1ᵉ 24.38 ± 2.04ᵈ 21.02 ± 1.44ᶜ 30.84 ± 6.05ᶜ 31.66 ± 2.12ᵉ
60 36.82 ± 3.61f 37.07 ± 1.91 f 29.89 ± 3.6ᵉ 30.82 ± 4.23ᵈ 36.39 ± 4.78ᵈ 34.47 ± 1.21f
70 44.81 ± 1.79 g 41.55 ± 2.15 g 36.02 ± 3.55 f 37.81 ± 2.62ᵉ 39.91 ± 1.77ᵈ 38.34 ± 1.44 g
80 50.04 ± 1.36 h 47.04 ± 2.48 h 43.58 ± 5.03 g 43.3 ± 1.63 f 44.68 ± 0.74ᵉ 41.99 ± 1.45 h
90 53.49 ± 0.17 h 49.91 ± 2.78 h 49.46 ± 3.71 h 47.55 ± 2.34 g 51.89 ± 0.12 f 46.34 ± 1.34 i
100 56.7 ± 0.74 i 54.63 ± 3.86 i 54.48 ± 1.82 i 49.83 ± 1.89 g 57.24 ± 1.21 g 53.95 ± 1.89 j
120 58.88 ± 1.13j 58.41 ± 2.44 i 57.33 ± 1.52 i 59.93 ± 4.59 i 60.22 ± 1.23 g 57.65 ± 0.53 k
140 60.37 ± 2.78 j 61.77 ± 2.13 j 59 ± 1.78 j 62.48 ± 3.28 i 62.51 ± 1.96 h 61.36 ± 0.62 l
160 61.71 ± 2.95 k 63.35 ± 2.13 k 59.42 ± 1.61 j 64.42 ± 1.56 j 65.78 ± 1.38 i 64.23 ± 1.07 m
180 62.81 ± 2.46 l 63.76 ± 2.02 k 60.22 ± 1.36 j 66.19 ± 0.39 j 67.13 ± 0.75 j 65.8 ± 0.34 m
200 63.93 ± 3 l 64.59 ± 1.52 k 61.33 ± 2.59 j 67.19 ± 0.41 k 67.72 ± 0.82 j 66.27 ± 0.16 m
(In the same column, different lowercase letters (a to m) indicate a statistically significant difference (α = 0.05))

C.3. Research L-citrulline elution efficiency at different pH

Table C.3. L-citrulline elution efficiency at different pH
Eluent capacity (mg L-Citrulline/L Elution efficiency of L-

pH
resin) Citrulline (%)
8 29.66±1.11ᵃ 66.34±2.49ᵃ
9 35.96±0.67ᵇ 80.42±1.5ᵇ
10 39.94±0.86ᶜ 89.32±1.93ᶜ
11 36.86±1.08ᵇ 82.45±2.42ᵇ
(In the same column, different lowercase letters (a to c) indicate a statistically significant
C.4. Research L-citrulline elution efficiency at different time

Table C.4. L-citrulline elution efficiency at different time
Elution capacity (mg L- Elution efficiency of L-

Time
Citrulline/resin) Citrulline (%)
10 9.2 ± 0.32ᵃ 20.59 ± 0.71ᵃ
20 11.05 ± 0.41ᵃᵇ 24.71 ± 0.92ᵃᵇ
30 12.81 ± 0.66ᵇᶜ 28.66 ± 1.48ᵇᶜ
40 14.58 ± 1.02ᶜ 32.6 ± 2.28ᶜ
50 16.6 ± 0.91ᵈ 37.13 ± 2.04ᵈ
60 19.89 ± 1.18ᵉ 44.48 ± 2.64ᵉ
70 21.84 ± 1f 48.86 ± 2.23 f
80 24.81 ± 1.73 g 55.48 ± 3.88 g
90 27.28 ± 1.42 h 61.02 ± 3.18 h
100 30.02 ± 1.43 i 67.13 ± 3.21 i
120 32.25 ± 1.28 j 72.13 ± 2.85 j
140 34.45 ± 1.39 k 77.05 ± 3.1k
160 36.46 ± 1.52 l 81.54 ± 3.41 l
180 38.4 ± 1.67 m 85.88 ± 3.73 m
200 39.21 ± 0.99 m 87.69 ± 2.2 m
220 39.68 ± 0.34 m 88.74 ± 0.76 m
240 39.84 ± 0.37 m 89.11 ± 0.83 m
(In the same column, different lowercase letters (a to m) indicate a statistically significant
difference (α = 0.05)

C.5. Research L-citrulline elution efficiency with Potassium in stimulation solution

Table C.5. L-citrulline elution efficiency with Potassium in stimulation solution
Potassium
Elution capacity (mg L- Elution efficiency of L-
concentration
Citrulline/L resin) Citrulline (%)
(mg/L)
0 39.84 ± 0.37 a 89.11 ± 0.83 a
100 22.587 ± 0.45 b 50.521 ± 1.09 b
200 20.111 ± 0.29ᶜ 44.983 ± 1.12ᶜ
300 17.975 ± 0.59ᵈ 40.205 ± 1.01 d
(In the same column, different lowercase letters (a to d) indicate a statistically significant

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HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

RESEARCH ON CITRULLINE EXTRACTION

STUDENT: Đỗ Quốc Khánh – 1952070

Số: ____ /ĐHBK-

HỌ VÀ TÊN: Đỗ Quốc Khánh MSSV: 1952070

1. Đầu đề luận văn:

Nội dung và yêu cầu LVTN đã được thông qua Bộ môn.

Ngày 22 tháng 05 năm 2023

PHẦN DÀNH CHO KHOA, BỘ MÔN

TP. HCM, date … month … year 2023

TP. HCM, date … month … year 2023

3.1.2. Ion-exchange resin ................................................................................................... 15

Figure 2.1. The molecular structure of L-citrulline................................................................. 3

Figure 2.2. Amino acid ion conversion reaction. .................................................................... 4

Figure 2.3. Metabolism of Citrulline.. ...................................................................................... 5

Figure 2.4. Chemical synthesis of L-citrulline from arginine ................................................ 7

Figure 2.5. Ion-exchange process .............................................................................................. 8

Figure 2.6. Classification of ion-exchangers............................................................................ 9

Figure 2.7. Ion-exchange reaction ........................................................................................... 10

Figure 2.8. Mechanism of ion-exchange process .................................................................. 12

Figure 3.1. Research content. .................................................................................................. 18

Figure 4.1. L-citrulline adsorption efficiency of resins ...................................................... 24

Figure 4.2. One-Way-ANOVA ................................................................................................ 25

Figure 4.4. Linear graph showing isotherm according to Freundlich model.................... 27

Figure 4.5. L-citrulline elution efficiency at different pH .................................................. 28

Figure 4.6. L-citrulline elution efficiency at different time ............................................... 29

Figure B.1. Standard curve of L – citrulline ......................................................................... 38

Table 3.1. Information of Purolite® C100 resin. .................................................................. 16

Table 3.2. Chemicals used in the thesis. ................................................................................ 17

Table 4.1. Value of adsorption isotherm model.................................................................... 26

Table 4.2. RL parameter values at different initial concentrations of L - citrulline ........ 26

Table B.1. Absorbance of L -citrulline at different concentration .................................... 38

Table C.1. L-citrulline adsorption capacity of ion-exchange resins ................................. 39

Table C.2.1. L-citrulline adsorption capacity ....................................................................... 40

Table C.2.2. L-citrulline adsorption efficiency .................................................................... 41

Table C.3. L-citrulline elution efficiency at different pH................................................... 42

Table C.4. L-citrulline elution efficiency at different time ................................................ 42

Cit+: HOOC-CH(R)-NH3+ – Citrulline is in the protonated state.

NO: Nitric oxide.

SP: Sulfoxyl (-SO3H).

CM: Carboxymetyl (-CH2-COOH).

Vietnam is one of the top watermelon producers in the world. As a result,

ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 1

ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 2

2.1.1. Physical properties

Figure 2.1. The molecular structure of L-citrulline.

At normal conditions, citrulline has appearance of white and odorless crystalline

2.1.2. Chemical properties

ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 3

Figure 2.2. Amino acid ion conversion reaction. [17]

2.1.2.2. Urea functional group

ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 4

2.1.3. Function of L-citrulline

Figure 2.3. Metabolism of citrulline.

2.1.3.2. Biological activity

ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 5

Citrulline cooperatively exerts an anti-inflammatory effect on synovial cells with

According to NO production, Citrulline indirectly reduce arterial blood pressure

- Strengthening of skeletal muscles:

Citrulline is able to stimulate muscle protein synthesis through an increase in

- Improve erectile dysfunction:

L-citrulline supplementation has been proved to be safe and psychologically well

ĐỖ QUỐC KHÁNH – BÙI ĐỨC KHANG PAGE 6