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EVALUATION OF TANNIN FROM RHIZOPHORA
APICULATA AS RUST CONVERTER
.. by·" :• . •
HENNY SUMILO SANTOSO
Thesis submitted in fulfillment of the requirements
for the degree of
Master of Science
March 2005
DECLARATION
Saya mengisytiharkan bahawa kandungan yang dibentang]s:an di dalam tesis ini adalah ' ' .
hasil kerja saya sendiri dan telah dijalankan di Universiti Sains Malaysia. Tesis ini
juga tidak pemah diserahkan untuk ijazah yang lain sebelum ini.
I declare that the content which is presented in this thesis is my own work which was
done at Universiti Sains Malaysia. The thesis has not been previously submitted for
any other degree.
-----------~-:ll~----------------------Tandatangan Calon:
Signature ofCcindidate:
Nama Calon: Henny Sumilo Santoso
Name ofCandidate:
KIP I Passport No: M569995
Disaksikan oleh:
Witnessed by:
Tandatangan Saksi:
Signature ofWitness:
Nama Saksi: Prof. Madya Dr. Mohd. Jain Noordin Mohd. Kassim
Name a/Witness:
KIP I Passport No: 540905-10-56_57
ACKNOWLEDGEMENTS
I would like to take this opportunity to acknowledge in_any individuals who have
helped me throughout my research. First and foremost, I would like to express my
gratitude to my supervisor, Assoc. Prof. Dr. Mohd. Jain. Noordin Mohd. Kassim for
his superb guidance, advice, inspiration, support and help.
My thanks also go to Pn. Afidah Abdul Rahim, for her generous advice and assistance
in some of my experimental. I would like to acknowledge the technical staff of the '• I
School of Chemical Science, in particular Mr. Ali for his help throughout my work in
laboratory, Mr. Sobri for his guidance in HPLC analysis and Mr. Aw Yeong for his
assistance in FTIR study.
My gratitude also goes to all of my friends and colleagues for their help and
encouragement. And last but not least, my sincere appreciation to my family back in
Indonesia for giving me this chance to complete postgraduate study and for their
encouragement and support.
Henny Sumilo Santoso
March 2005
11
TABLE OF CONTENTS
Page
Acknowledgements 11
Table of Contents 11l
List of Tables Vll
List of Figures Vlll
Abstrak Xl
Abstract xu
CHAPTER 1 : INTRODUCTION
1.1 Corrosion 1
1.1.1 Corrosion of Iron 1
1.1.2 Corrosion Protection 3
1.2 Rust Converter 4
1.3 Tannin 6
1.3.1 Hydrolysable tannin 7
1.3 .1.1 Gallo tannin 8
1.3.1.2 Ellagitannin 9
1.3.2 Condensed Tannin 10
1.3.2.1 Flavan-3-ol 12
1.3.2.2 Flavan-3,4-diol 13
1.3.2.3 Flavan-4-ol 14
1.3.3 Complex Tannin 15
1.4 Reaction of Tannin with Iron 16
111
1.5 Reaction of Phosphoric Acid with Iron 19
1.6 Objective 21
CHAPTER 2 : MATERIALS AND METHODS
2.1 Tannin Extraction 22
2.2 Assays for The Quantification of Tannin 24
2.2.1 Prussian Blue Assay for Total Phenols 24
2.2.1.1 Preparation of Reagents 24
2.2.1.2 Assay Procedure 24
2.2 .1.3 Preparation of Standard Curve 25
2.2.2 Vanillin Assay 25
2.2.2.1 Preparation of Reagents 25
2.2.2.2 Preparation of Working Reagents 26
2.2.2.3 Assay Procedure 26
2.2.2.4 Preparation of Standard Curve 26
2.3 Stiasny Test 27
2.4 Identification ofth~.Monomer of Condensed Tannin by Reverse 27 Phase HPLC
2.4.1 Preparation of Reagents and Solvents 27
2.4.2 Phloroglucinol Degradation 28
2.4.3 HPLC Elution System 28
2.5 Infrared Spectroscopy Analysis 29
2.6 Preparation of Ferric Tannate 29
2.7 Preparation of Rusted Steel 29
2.8 Reaction of Rust Powder with Tannin 30
lV
2.9 Rust Converter Formulations 30
2.9.1 Single Pack Rust Converter 30
2.9.2 Two Pack Rust Converter 31
CHAPTER 3 : RESULT AND DISCUSSION
3.1 Tannin Extraction 33
3.2 Assays for the Quantification of Tannin 35
3.2.1 Prussian Blue Assay for Total Phenols 35
3.2.1.1 Visual Estimation of the Tannin Quantity 36
3.2.1.2 Choice of Standard 36
3.2.2 Vanillin Assay 39
3.3 Stiasny Test 43
3.4 Identification of the Monomer of Condensed Tannin by Reverse 44 Phase HPLC
3.5 Infrared (FTIR) Spectroscopy Study 48
3.5.1 FTIR of Rhizophora apiculata Tannins 48
3.5.2 FTIR of Ferric Phosphate 52
3.5.3 Infrared Spectroscopy of Ferric Tannate 54
3.5.4 Infrared Spectroscopy of standards goethite (a-FeOOH), 62 lepidocrocite (y-FeOOH) and magnetite (Fe304)
3.5.5 Infrared Spectroscopy of Rust Powder 66
3.6 Reaction of Rust Powder with Tannin 68
3.6.1 Reaction of Rust Powder with 3% Tannin 68
3.6.2 Reaction of Rust Powder with 6% Tannin 69
3.6.3 Reaction of Rust Powder with 9 % Tannin 69
3.7 Rust Converter Formulations 74
v
3.7.1 Single Pack Rust Converter 74
3.7.1.1 Rusted Steel Treated with 5 % Phosphoric Acid. 74
3.7.1.2 Rusted steel treated with 3% Tannin+ 5% 76 . Phosphoric Acid
3.7.1.3 Rusted steel treated with 6 % Tannin + 5 % 77 Phosphoric Acid
3.7.1.4 . Rusted steel treated with 9% Tannin+ 5% 78 Phosphoric Acid
3.7.2 Two Pack Rust Converter 82
3.7.2.1 Rust Converter Formulations Performance after 83 One Day of Exposure
3.1 0.2.2 Rust Converter Formulations Performance after 83 Four Days of Exposure
3.7.2.3 Rust Converter Formulations Performance after 86 Seven Days of Exposure
3.7.2.4 Rust Converter Formulations Performance after 86 Ten Days of Exposure
3.7.2.5. Rust Converter Formulations Performance after 89 14 Days of Exposure
3.7.2.6 Rust Converter Formulations Performance after 89 21 Days of Exposure
CHAPTER 4 : CONCLUSION 94
CHAPTER 5: RECOMMENDATION FOR FUTURE RESEARCH 96
CHAPTER 6 : REFERENCES 97
PUBLICATION 101
vi
LIST OF TABLES
Page
Table 2.1 Rust converter formulations for part A 32 -...
Table 2.2 Rust converter formulations for part B 32
Table 3.1 Average yields of extract from Rhizophora apiculata bark 33
Table 3.2 The absorbance (A720) of gallic acid as standard with different 37 concentration in Prussian blue assay
Table 3.3 The absorbance (A720) of tannins in Prussian blue assay 37
Table 3.4 _The percentages of tannins in the term of gallic acid equivalent 38
Table 3.5 The absorbance (A720) of catechin as standard with different 41 concentration in vanillin assay
Table 3.6 The absorbance (A720) of tannins in vanillin assay 41
Table 3.7 The percentages for tannins in the term of cathecin equivalent 42
Table 3.8 The percentages of Stiasny reaction precipitates formed by tannins 44
Table 3.9 The retention time (tR) of (-)-catechin, (-)-epicatechin and 47 (-)-epigallocatechin gallate as standards
Table3.10 The retention time (tR) of condensed tannin fractions 47
Table 3.11 Infrared absorption of raw tannin, low molecular weight tannin 49 and mixed tannin
Table 3.12 Infrared absorption of hydrolysable tannin and condensed tannin 49
Table 3.13 Infrared absorption of ferric tannate with different pH 56
Table 3.14 The colors of the rust converter formulations 83
Table 3.15 Qualitative description of performance rating 93
vii
LIST OF FIGURES
Page
Figure 1.1 Reaction of tannin with protein 7
Figure 1.2 The structure of tannic acid, the best-known hydro1ysab1e tannin 8
Figure 1.3 Example ~f despide bond which is formed between the phenolic 9 group of the upper and the acid group ofthe lower gallic acid units
-~· ., .
Figure 1.4 The structure of the simplest ellagitannin, hexahydroxydiphenic 10 acid and its stable dilactone, ellagic acid
Figure 1.5 The heterocyclic ring system of condensed tannin 11
Figure 1.6 The structures of(a) anthocyanidin, (b) flavanone, (c) flavone and 11 (d) flavonol
Figure 1.7 Model structure for a condensed tannin. The 4~6 linkage (dotted 12 line) is an alternative interflavan bond. The terminal unit is at the bottom of such a multi-unit structure
Figure 1.8 The structures of flavan-3-ol( +)-catechin and (-)-epicatechin 13
Figure 1.9 The structures of ( + )-gallocatechin and (-)-epigallocatechin 13
Figure 1.10 The structure offlavan-3,4-diol 14
Figure 1.11 The structures of cyanidin and delphinidin that are yielded from 14 flavan-3,4-diol
Figure 1.12 The structure of flavan-4-ol 14
Figure 1.13 Ferric tannate chelate where R represents the rest of the tannin 18 molecule
Figure 2.1 Procedure in tannin extraction from Rhizophora apiculata bark 23
Figure 3.1 The percentages of tannin extraction from Rhizophora apiculata 34 bark
Figure 3.2 Absorbance (A720) for Prussian blue method vs. concentration of 38 gallic acid
Figure 3.3 The underlying reaction ofvanillin with condensed tannin. The 40 arrowhead points to a second potentially reactive site
Vlll
I Figure 3.4 Absorbance (Asoo) for vanillin assay vs. concentration of catechin 42 ; '
~--~- Figure 3.5 The reaction of phenol with formaldehyde 43 ~-;~· ... ~· ... 1.:· Figure 3.6 HPLC chromatogram of condensed tannin fractions 45 ~-
Figure 3.7 HPLC chromatogram of (-)-catechin 45
Figure 3.8 HPLC clu:omatogram of (-)-epicatechin 46
Figure 3.9 HPLC chromatogram of (-)-epigallocatechin gallate 46
Figure 3.10 The infrared spectra comparison of (a) raw tannin, (b) low 50 molecular weight tannin and (c) mixed tannin
Figure 3.11 The infrared spectra comparison of (a) hydrolysable tannin and 51 (b) condensed tannin
Figure 3.12 The infrared spectrum of ferric phosphate 53
Figure 3.13 The infrared spectrum of ferric tannate at pH 1.17 55
Figure 3.14 The infrared spectra of ferric tannate at (a) pH 2, (b) pH 3 and 60 (c) pH 4
Figure 3.15 The infrared spectra of ferric tannate at (a) pH 5, (b) pH 6 and 61 (c) pH 7
Figure 3.16 The infrared spectrum of goethite (a-FeOOH) 63
Figure 3.17 The infrared spectrum of lepidocrocite (y-FeOOH) 64
Figure 3.18 The infrared spectrum of magnetite (F e304) 65
Figure 3.19 The infrared spectrum of rust, G = Goethite, L = Lepidocrocite 67 and M = Magnetite
Figure 3.20 The infrared spectra of (a) rust powder, treatment with 71 3% tannin solution for (b) one day, (c) one week, (d) two weeks and (e) one month
Figure 3.21 The infrared spectra of (a) rust powder, treatment with 72 6% tannin solution for (b) one day, (c) one week, (d) two weeks and (e) one month
lX
Figure 3.22 The infrared spectra of (a) rust powder, treatment with 73 9% tannin solution for (b) one day, (c) one week, (d) two weeks and (e) one month
Figure 3.23 The infrared spectra of (a) rust powder, treatl!lent of rusted 75 steel with 5% phosphoric acid for (b) one day, (c) one week, (d) two weeks and (e) one month
Figure 3.24 The infrared spectra of (a) rust powder, treatment of rusted 77 steel with 3% tannin+ 5% phosphoric acid for (b) one day, (c) one week, (d) two weeks and (e) one month
Figure 3.25 The infrared spectra of (a) rust powder, treatment of rusted, 79 steel with 6% tannin+ 5% phosphoric acid for (b) one day, (c) one week, (d) two weeks and (e) one month
Figure 3.26 The infrared spectra of (a) rust powder, treatment of rusted 80 '.,.::..'. steel with 9% tannin+ 5% phosphoric acid for (b) one day,
(c) one week, (d) two weeks and (e) one month
Figure 3.27 Rust converter formulations performance after one day: 84 (a) control; (b) formula I; (c) formula 2; (d) formula 3; (e) formula 4; (f) formula 5
Figure 3.28 Rust converter formulations performance after four days: 85 (a) control; (b) formula 1; (c) formula 2; (d).formula 3; (e) formula 4; (f) formula 5
Figure 3.29 Rust converter formulations performance after seven days: 87 (a) control; (b) formula 1; (c) formula 2; (d) formula 3; (e) formula 4; (f) formula 5
Figure 3.30 Rust converter formulations performance after ten days: 88 (a) control; (b) formula 1; (c) formula 2; (d) formula 3; (e) formula 4; (f) formula 5
Figure 3.31 Rust converter formulations performance after 14 days: 90 (a) control; (b) formula 1; (c) formula 2; (d) formula 3; (e) formula 4; (f) formula 5
Figure 3.32 Rust converter formulations performance after 21 days: 91 (a) control; (b) formula 1; (c) formula 2; (d) formula 3; (e) formula 4; (f) formula 5
Figure 3.33 Rating of general and scribe corrosion performance on steel 93 panels after 21 days of exposure of various rust converter formulations
X
PENILAIAN TERHADAP TANIN RHIZOPHORA APICULATA
SEBAGAI PENUKAR KARAT
ABSTRAK
Tujuan utama daripada kajian ini adalah memformulasikan penukar karat berasaskan
tanin dan mengkaji keberkesanannya sebagai penukar karat. Tanin yang digunakan
dalam kajian ini diekstrak daripada kulit kayu bakau (Rhizophora apiculata) yang
merupakan bahan buangan dalam industri arang kayu. Pengekstrakan daripada kulit
kayu bakau dengan 70 % akueus aseton meghasilkan 23.6 % tanin campuran, yang
mengandungi 12.3 % tanin terhidrolisis dan 8.7 % tanin terkondensasi. Beberapa
pengujian dilakukan untuk mengkaji tanin, seperti ujian Prussian blue untuk
penentuan fenol total, ujian vanillin dilakukan untuk menganggarkan tanin
terkondensasi, ujian Stiasny untuk menganggarkan peratus flavanoid yang bertindak
balas dengan formaldehid. Pengenalpastian monomer yang terkandung dalam tanin
terkondensasi dilakukan dengan HPLC. Rawatan serbuk karat dengan pelbagai
peratusan larutan tanin menghasilkan penukaran hasil karat besi kepada ferik tanat
atau hasil fasa besi lainnya. Dalam formulasi penukar karat satu pek berasaskan tanin,
kajian FTIR menunjukkan bahawa campuran asid fosforik dan tanin lebih efektif
daripada tanin atau asid fosforik sahaja dalam menukarkan karat. Penukar karat yang
mengandungi 9 % tanin menunjukkan keupayaan untuk menukar hasil karat besi
sepenuhnya. Formula penukar karat berasaskan resin polivinil-butiral (PVBR) yang
mengandungi gabungan tanin dan zink fosfat dengan nisbah 1 : 1 mempunyai potensi
yang besar sebagai penukar karat. Penukar karat yang hanya mengandungi tanin atau
zink fosfat sahaja tidak menunjukkan keberkesanannya sebagai penukar karat.
XI
ABSTRACT
The main objective of this research is to formulate rust C<?nverter formulations based
on tannin and to evaluate their rust conversion performance. The tannins that were
used in this study were extracted from mangrove (Rhizophora apiculata) barks which
are the waste product from charcoal industry. The extraction of mangrove bark with
70 % aqueous acetone produced 23.6 % mixed tannin, which contained 12.3 %
hydrolysable tannin and 8.7 % condensed tannin. The quantifications of tannin have
been carried out by several assays, such as Prussian blue assay for total phenolic
determination; vanillin assay to estimate condensed tannin, Stiasny test to provide the
estimation of the total flavanoid that react with formaldehyde. The identification of
the tannin monomer of condensed tannin was carried out by reverse phase HPLC.
Treatment of rust powder with various percentage of tannin solution resulted in the
transformation of iron rust products into ferric tannate or other iron phase products. In
single pack rust converter formulations based on tannin, the FTIR study showed that a
mixture of phosphoric acid and tannin is more effective than the tannin or phosphoric
acid alone in converting or transforming the iron nist products. Rust converter which
contained 9 % of tannin had been showed to be able to convert completely all the iron
rust products. Rust converter based on a polyvinyl-butyral resin (PVBR) containing
tannin in combination with zinc phosphate in the ratio of 1 : 1 had great potential as
rust converter. Using tannin or zinc phosphate alone was not effective as rust
converter.
xii
1.0 INTRODUCTION
1.1 Corrosion
Corrosion may be defined as the destruction or deterioration of metal or metal alloy
by chemical or electrochemical reactions with its environment [1]. The serious
consequences of the corrosion process have become a problem of world wide
significance. In addition to our everyday encounters with form of degradation,
corrosion causes plant shutdown, waste of valuable resources, loss or contamination
of products, reduction in efficiency, costly maintenance, and expensive overdesign. It
can also jeopardize safety and inhibit technology progress. It is estimated that roughly
3% of the annual production of steel is lost by corrosion [2] and approximately 5% of
an industrial nation's income is spent on corrosion preventions and maintenance or
replacement of products lost or contaminated as a result of corrosion reactions [3].
The corrosiOn characteristics are influenced by many variables including relative
humidity, pollutant, temperature, sulfur dioxide content, hydrogen sulfide content,
chloride content, amount of rainfall, dew formation, dust and even the position of the
exposed metal. Geographic location also plays an important part. All types of
corrosion may take place depending on the specific contaminants present and the
material of construction [ 4].
1.1.1 Corrosion of Iron
Iron corrodes in reaction with the environment because of the thermodynamically
unstable condition of iron after it has been extracted from its ores. Corrosion is
essentially the conversion of iron into a hydrated form of iron oxide, i.e. rust. The
1
driving force of the reaction is the tendency of iron to combine with oxygen [2]. Iron
rusts owing to the presence of anodic and cathodic sites on its surface. During
corrosion, more than one oxidation and one reduction react_ion may occur.
In the anodic zone, the iron ionizes:
Fe~ Fe2+ + 2e- ......................................... (1.1)
In the presence of water and oxygen, hydroxyl ions are produced in the cathodic zone:
H20 + 1/202 + 2e- ~ 20K .......................................... (1.2)
If no other oxidation or reduction reaction occurs, the total electrochemical reaction is
just the sum of reaction (1.1) and (1.2) to form ferrous hydroxide:
Fe2+ + 20K ~ Fe(OH)2 .......................................... (1.3)
In the presence of atmospheric oxygen, the Fe(OH)2 is rapidly oxidized to reddish
brown color rust which is generally written as FeO.OH or hydrated iron (III) oxide
(Fe203.H20):
4Fe(OH)2 + 0 2 ~ 4y-FeO.OH + 2H20 ............................... (1.4)
Lepidocrocite (y-FeO.OH) can be transformed into the more stable goethite
(a-FeO.OH) or, in deficiency of oxygen, reduced to magnetite (Fe304).
Fe(OH)2 + 2y-FeO.OH ~ Fe304 + 2H20 .............................. (1.5)
Later, the magnetite produced is re-oxidized in air by oxygen in the presence of water
to regenerate y-FeOOH:
Fe304 + 1/402 + 3/z HzO ~ 3 y-FeOOH ............................... (1.6)
From the reactions above, it serves to illustrate that the electrochemical nature of
rusting and the essential parts played by moisture and oxygen [2]. However, in the
presence of electrolyte such as sodium chloride, the reactions are modified, ferrous
2
chloride is formed at the anode and sodium hydroxide is formed at the cathode [2].
These two compounds are very soluble and not easily oxidized, so that they diffuse
away from the sites of formation and react at a distance.from the metal surface to
form ferrous hydroxide, or a basic salt, which then combines with oxygen to form rust
with the regeneration of sodium chloride:
FeCh + 2NaOH--+ Fe(OH)2 + 2NaCl ............................... (1.7)
4Fe(OH)2 + 0 2--+ 4Fe00H+ 2H20 ................................ (1.8)
Goethite (y-FeOOH), lepidocrocite (y-FeO.OH) and magnetite (Fe304) are the main
constituents of atmospheric corrosion of steel. The proportions of each depend on pH,
temperature, the atmosphere in which the rust forms and the presence or absence of
atmospheric pollutants [5]. Akaganeite (~-FeOOH) is often observed as a corrosion
product of iron in chloride-containing environments, such as marine atmospheres [6].
1.1.2 Corrosion Protection
While corrosion will always remain as a threat, there are many ways that can be
deployed to protect a structural metal against the environment corrosion severity.
Basically, there are five essential ways of corrosion control; they are based on the
application of good design practice, a sensitive approach to materials selection, the
use of inhibitors, the application of protective coating and finally using anodic or
cathodic protection [7]. Most of the structural steels are protected from corrosion by
surface coating, principally by painting schemes. Paint coating will provide long-term
. protection only if applied to well prepared substrate. Thus, proper surface preparation
is the key to obtain maximum protection performance by a surface coating.
3
The mam function of a protective coating is to isolate structural metal from
environmental corrosives. A coating must provide to a substrate a continuous barrier,
any imperfection can become a focal point for degradation and corrosion of the
substrate. A paint system often comprising at least three coats of paints viz. a primer,
an undercoat and a topcoat. The primer is a universal component of all anticorrosive
coatings and is considered to be one of the most important elements of a protective
system. A good primer generally provides the ability to retard the spread of corrosion
and adhere well to the base metal. Generally, the primer consists of anticorrosive
pigment such as lead and chromates or sacrificially metallic pigment which is more
base than the metallic structure being protected [5,8].
The major problem in preparing a steel surface to be painted is the removal of all mill
scale, rust and associated contaminants from the substrate [9]. It is either technically
impossible or economically undesirable to achieve surface preparation to bright steel
finish [10]. The excessive cost and inconvenience associated with the mechanical
methods such as hand tool, power tool, and sandblasting cleaning have incited efforts
to develop alternative surface preparation methods, which can be applied to
marginally prepared surfaces. The efforts have centered on rust conversion coating or
so-called rust converter that either impregnate rust, convert rust to inactivate soluble
salt, or convert iron oxides to other products [1 0].
1.2 Rust Converter
Rust converters are water-based coatings that are applied to corroded surfaces· to
convert rust into a water-insoluble, coherent and protective layer over which further
paint coatings or topcoat may be applied, which provide protection against corrosion
4
[ 11]. The rust converter convert corrosion products of iron into iron compounds that
are more stable and corrosion resistant than . oxides and oxyhydroxides of iron,
therefore do not further react with corrosive agents and impede steel corrosion. If rust
is stabilized so that it can no longer redox between Fe(II) and Fe(III) states, the
cathodic cycle in the atmospheric corrosion process will cease and corrosion should
be prevented [12]. These rust converter formulations could be applied on partially
rusted substrate, reducing the effort and cost needed for cleaning the surface by
sandblasting or other methods.
The results on ·protection efficiency of rust converters are controversial due to several
factors include finding the appropriate components and their optimal concentrations,
the proper time of reaction of the converters with the rust and the application method.
The type and concentration of alcohol used as solvent is another important factor.
However, the nature of the rust layer (e.g. thickness and composition) and its time of
formation at the corroded steel surface are other factors influencing the converter
protection efficiency. Younger rust reacts better with the converters than the rust with
considerable time of formation, because of its high reactivity [11,13].
The vast majority of commercial rust converters incorporate some type of
polyhydroxylated or tannin-like compound as the pigments [9]. In relation to the
composition, it is well known that the most common prime candidates to be used in
rust converters are based on tannin and phosphoric acids and their derivatives. The
dramatic transformation of rusty steel to the typical blue-black coating, which occitrs
after the application of the product, has been attributed to the complexatio1_1 of the
polyphenolic moiety of the tannin resin to the iron oxide and hydroxides in rust. The
5
blue-black coating that is formed is knovvn as ferric tannate complex. This complex
formation can be used to disturb the kinetic of reactions involved in corrosion.
1.3 Tannin
The term "tannins" have a long and well-established usage in the scientific literature
which relate specifically to the application of certain plant extracts [14]. They are very
complex group of secondary metabolism of plants [15]. Tannins are the most
widespread polyphenols in plants after lignin [16]. They are found in approximately
80% of woody, 15% of herbaceous dicotyledonous species and in nearly every
species of higher plants, both gymnosperms and angiosperms, and can occur at high
levels in some forages, animal feeds and human foods of plant origin, leaves, fruits
and galls [17, 18]. They are absent or only found in small quantities in lower plants
(algae, mosses, lichens, fungi, ferns) and comparatively rare in monocotyledons [19].
In a few families, many species contain tannin in large quantities, e.g. Rhizophoraceae
and Combretaceae [19].
Tannins are water-soluble phenolic compounds with molecular weight approximately
between 500 and 3000 Daltons [20]. However, this definition does not include all
tannins, since, more recently, molecules with molar mass up to 20000 Daltons have
been isolated, and that should also be classified as tannins on the basis of their
molecular structures [21]. Besides giving the usual phenolic reaction, tannins contain
sufficient phenolic hydroxyl groups that permit them to have special properties such
as capable of forming stable cross-linking and/or precipitating proteins, collagens,
alkaloids, gelatins and minerals (Figure 1.1) [22,23].
6
protein
tannin
Figure 1.1: Reaction of tannin with protein [24].
Chemically the extracts of tannins fall into two main categories, on the basis of their
structural types and their reactivity toward hydrolytic agents, particularly acids; which
are largely unrelated apart from the fact that both contain polyhydric phenols within
their structures. They are classified as hydrolysable tannins and condensed tannins
[25,26]. The latter present complex structures such as dimmers, trimes, oligomers and
polymerics [20]. Condensed tannins are widely distributed in higher plants, whereas
hydrolysable tannins are of limited distributed in nature [23].
1.3.1 Hydrolysable tannin
Hydrolysable tannins, being polyester, are readily hydrolyzed by acids, alkalis or
enzymes into sugars or related polyhydric alcohols and phenol-carboxylic acids [27].
Hydrolysable tannins are further subdivided on the basis of the phenol-carboxylic
acids yielded on hydrolysis. The two major groups of hydrolysable tannins are
gallotannins, which are derived from gallic acid and ellagitannins, which are derived
from hexahydroxydiphenic acid. The structural variation amongst these compounds is
7
caused by oxidative coupling of neighboring gallic acid units or by oxidation of
aromatic rings [28].
1.3.1.1 Gallotannin
The gallotannins are the simplest hydrolysable tannins [18]. Gallotannins consist of a
central polyol (generally glucose) as a central core, which is surrounded by several
gallic acid units (3,4,5-trihydroxyl benzoic acid). The prototypical gallotannin is
pentagalloyl glucose, which is the central point for many complex tannin structures
[22]. The best-known and the most widely used hydrolysable tannins are tannic acid,
which is a gallotannin that has five identical gallic acid units which involve aliphatic
hydroxyl groups of the core sugar (Figure 1.2) [27]. Gallic acid may be further
esterified or oxidatively cross linked to yield more complex hydrolysable tannins.
Further gallic acid units can be attached through a meta- or para- despide bond
(Figure 1.3) [18].
R represents the structure:
HO
RO HO--- " ·-- C· OH
H H 0
HO OR H gallic acid
Figure 1.2: The structure of tannic acid, the best-known hydrolysable tannin [26].
8
sugar"' 0
0
Despide bond
OH
OH
upper gallic acid
lower gallic acid
Figure 1.3: Example of despide bond which is formed between the phenolic group of
the upper and the acid group ofthe lower gallic acid units [18].
1.3.1.2 Ellagitannin
In contrast to the rather limited occurrence of gallotannins, ellagitannins are widely
distributed and have been found to occur in many plant families and have a strong
tendency to combine to higher aggregates [29]. Ellagitannins are derived
biosynthetically from pentagalloyl glucose (gallotannin) by oxidative reactions
between at least two gallic acid units [20]. The simplest ellagitannins are esters of
hexahydroxydiphenic acid. Hexahydroxydiphenic acid spontaneously lactonizes to
ellagic acid in aqueous solution (Figure 1.4).
9
OH OH
\ I c=o o=c
HO HO OH ---.
HO HO OH HO OH
hexahydroxydiphenic acid ellagic acid
Figure 1.4: The structure of the simplest ellagitannin, hexahydroxydiphenic acid and
its stable dilactone, ellagic acid [25].
1.3.2 Condensed Tannin
OH
Condensed tannins are high-molecular-weight polymers [27]. Condensed tannins, or
commonly referred to as proanthocyanidins [23], are flavanoid polymers, with
carbon-carbon bonds joining the individual flavanoids monomers [30]. Condensed
tannins are not polyester, therefore are not hydrolyzed by acids, they contain only
small amount of carbohydrate and polymerize under the action of acids [30].
The polyflavanoid of condensed tannin consist essentially 2-50 monoflavanoid units
linked as a straight chain or with branching [31]. The monoflavanoid is a diverse
group of metabolites based on the heterocyclic ring system (C-ring, containing one
oxygen atom) derived from two benzene rings, which are phenylalanine (B-ring) and
polyketide biosynthesis (A-ring) (Figure 1.5) (32,33]. The basic structures of
condensed tannins can be represented by the anthocyanidin, flavanone, flavone and
flavonol (Figure 1.6) [22]. The monoflavanoid units of condensed tannin are bound
together to form a polymeric chain (Figure 1.7) through the C4-C6 inter-flavanoid
10
bonds or if a hydroxyl group is present on the C5 position, the crosslinking occurs at
the carbon atoms in the 4 and 8 positions; other hydroxylation pattern occur, but there
are much less common [34,35].
5 4
Figure 1.5: The heterocyclic ring system of condensed tannin [33,36].
anthocyanidin
(a)
flavone
(c)
HO
flavanone
(b)
OH
flavonol
(d)
0
OH
Figure 1.6: The structures of (a) anthocyanidin, (b) flavanone, (c) flavone and
(d) flavonol [3 7].
11
R
HO~ N,,,,···
HO
OH
~ I
HO
-·· -·· -··
OH
OH Extender unit NOH
-·· ..
H .... )l)~R
OH
Terminal unit
OH
Figure 1. 7: Model structure for a condensed tannin. The 4---+6 linkage (dotted line) is
an alternative interflavan bond. The terminal unit is at the bottom of such
a multi-unit structure [34].
1.3.2.1 Flavan-3-ol
The chemistry of flavan-3-ol is intimately linked to progress in the understanding of
the oligomeric proanthocyanidin [33]. Common classes of proanthocyanidin are the
procyanidin which consist of chains of the flavan-3-ol(+)-catechin and (-)-epicatechin
(Figure 1.8). The catechin and epicatechin monomer has asymmetric centers at
position 2 and 3 in ring C [38]. The stereochemistry of (+)-gallocatechin and
(-)-epigallocatechin were subsequently related to that of catechin (Figure 1.9) [34].
Flavan-3-ols have two hydroxyl groups on the A-ring and two (catechin and
epicatechin) or three (gallocatechin or epigallocatechin) hydroxyl groups· on the
B-ring:
12
n
H
OH
catechin
OH _NoH
.... ,,,,,u H
OH
epicatechin
Figure 1.8: The structures offlavan-3-ol(+)-catechin and (-)-epicatechin [39].
OH OH
cXH &H H H
···'''''I "" ···'''''I "" OH OH
'OH 'OH OH OH
gallocatechin epigallocatechin
Figure 1.9: The structures of(+)-gallocatechin and (-)-epigallocatechin [40].
1.3.2.2 Flavan-3,4-diol
Other important units involved in proanthocyanidin structure are the flavan-3,4-diols
(Figure 1.1 0) or leucoanthocyanidins (Figure 1.11 ). Leucoanthocyanidins are
monomeric flavanoids which yield cyanidin and delphinidin by cleavage of a C-0
bond upon heating with mineral acid [33].
13
R NOH .... ,,,,,u"'
R'
R = H, R' = OH, leucocyanidin
H R = R' = H, Ieucopelargonidin .
R = R' = OH, leucodelphinidin
OH OH
Figure l.l 0: The structure offlavan-3,4-diol [22,33].
OH I i
.l /OH
HO 0+
H
OH
OH
cyanidin
HO
:I i!
I
) 'OH
OH
delphinidin
OH
OH
Figure 1.11: The structures of cyanidin and delphinidin that are yielded from
flavan-3,4-diol [37].
1.3.2.3 Flavan-4-ol
The flavan-4-ols (Figure 1.12) are also leucoanthocyanidins, but they are unique in
their lability. They may be chemically, and presumably also biosynthetically, derived
by a single reduction step from a flavanone. They yield anthocyanidins upon
treatment with alcoholic acid at room temperature [33].
H
OH
···"''ex R
OH OH
Figure 1.12: The structure offlavan-4-ol [33].
14
1.3.3 Complex Tannin
Recent accumulated data of tannin structural studies have shown that they may be
classified into three major groups based mainly on the strU~tural features rather than
on chemical properties [28]. The term "complex tannin" appears to be established as a
descriptor for the class of polyphenols that contain both hydrolysable and condensed
tannin [33]. They are often difficult to recognize in their sometimes highly modified
structures.
From their structural features, they are further divided into three groups. The most
commonly occurring is flavano-ellagitannin, in which a flavan-3-ol (catechin,
epicatechin or gallocatechin), representing a constituent unit of the condensed tannins,
is connected to the glucose portion of the ellagitannin, which representing
hydrolysable tannin, through a carbon-carbon linkage [33]. The second is
procyanidino-ellagitannin that contains both proanthocyanidins and ellagitannins, and
the third is the flavono-ellagitannin in which a flavonoid glucoside is linked to an
ellagitannin moiety through a carbon-carbon bond [28].
Originally tannins were used by humankind over several thousand years for tanning
leather, as an ingredient in the synthesis of wood adhesives and in several industrial
applications, such as in the production of paint, paper, glue, ink, cosmetic and in food
industry as natural colorant and preservative [ 41]. Interest is now based on the use of
tannin as anti-corrosive pigment in rust converter for the protection of steel structures
as an alternative to toxic anti-corrosive inhibitor such as lead or chromate [ 42].
Tannins are polyphenols ofvegetal origin, therefore they are environmentally safe and
15
are obtained from renewable sources [43]. The proximity of hydroxyl groups on the
aromatic rings makes them able to form chelate with iron ions.
1.4 Reaction ofTannin with Iron
Tannin and phosphoric acid and their derivatives are the most common prime
candidates to be used in rust converter [11]. Tannin and phosphoric acid treatments
are useful on rusted steel where it is impossible or undesirable to remove all rust.
They will delay the onset of rusting and thereby retard the breakdown of subsequent
paint films [ 44].
The polyphenolic groups of the tannins confer them the ability to form complexes
( chelates) with iron and other metallic cations. This complex formation can be used to
disturb the kinetic of reactions involved in corrosion [12,42]. The mechanism of the
reaction of tannin with rust is characterized by three general steps: (1) absorption of
tannin to the rusty surface, (2) complexation of ferric ions or complexation of surface
iron hydroxides/oxides followed by dissolution, and (3) partial or complete
readsorption of the iron-tannate complexes to the substrate [9].
Tannin reacts with iron or rusty steel to form an intense blue-black ferric tannate
complex, which is the basis of the corrosion protective coating [ 1 0]. The ferric
tannates are highly insoluble and act as electric insulators between cathodic and
anodic sites on the metal surfaces [12,43].Tannins with low molecular weights have
lower reactivity with iron and lower capacity to absorb oxygen [8]. As tanni:n
molecules are generally polymeric in nature with flavanoid structure, each molecule
could react with a number of ferric ions to form a network structure [44]. Seavell [45]
16
described that the reaction with ferric ions involve the C3 and C4 adjacent hydroxyl
groups of tannin, this reaction is common to all flavanoid units. Reaction between ion
Fe3\aq) and the phenolic hydroxyl groups gives an insol~ble bis-chelate (Figure
1.13). With chelation giving oxonium ions, reaction is formed under conditions which
reduce acidity. The tris-chelate is not likely to form, except under alkaline conditions
[45].
It might be expected that the Fe3+ would complex as the follow structure:
R---11
, OH/
OH
However, despite the high pKa value for the hydroxyl group at the C4 position of the
singly ionized anion, the greater stability of the doubly ionized chelate leads to the
formation of the ortho-diphenol complex:
R
OH
17
The his-chelate that is formed between Fe3+(aq) and tannin:
R--(j"OH
f"oH OH
+
H20
H20~! /H20
H2o/je~H20 H20
!
+ H)y <!? I R
H ~
OH
Figure 1.13: Ferric tannate chelate where R represents the rest ofthe tannin molecule
[45,46].
The tannin chelates with Fe3\aq) but not with Fe2+(aq). Although Fe2\aq) ions do not
chelate with phenolic hydroxy groups, they are readily oxidized to Fe3+(aq), especially
if the reaction is carried out under conditions when there is ready access of air to
supply oxygen for the oxidation [45]:
4Fe2+ + 2H20 + 0 2 ~ 4Fe3+ + 40H" .......................................... (1.9)
Work done by Seavell [12] showed that the tannin functions by reacting with the
Fe3\aq) ion and not by direct reaction with either iron or rust. The reactions of tannin
and Fe3+(aq) ion were proposed as follows [8]:
18
Fe+ 3H+ + 3/402 ---+ Fe3+ + 3/2H20 ......... ; ...... ~ ........... : ........... (1.10)
Fe+ 2H+ + 1/202 ---+ Fe2+ + H20 ........................................ (1.11)
Fe2+ +
1/402 + H+ ---+ Fe
3+ +
1/2H20 ··················:······················ (1.12)
Tannin-OH + Fe3+---+ (Tannin-0- Fe3+i+ + H+ ............................ (1.13)
Tannin-20H + Fe3+---+ (Tannin-20- Fe3+)+ + 2H+ ........................... (1.14)
Tannin-20H + (Taimin-20- Fe3+t---+ (Tannin-40- Fe3+r + 2H+ ....... (1.15)
If the steel presented oxides in its surface (i.e., lepidocrocite, FeOOH and/or
magnetite, Fe304), the tannin primer reacted with ferrous or ferric ions, according to
the following reactions:
FeOOH + 3H+ ---+ Fe3+ + 2H20 ................................ (1.16)
Fe304 + 8H+ ---+ 2Fe3+ + Fe2+ + 4H20 ................................. (1.17)
............................... (1.18)
Ferric ions formed in Reactions ( 1.16) and ( 1.17), in tum, reacted with tannin
according to Reactions (1.13) and (1.14), generating iron tannates that contain
corrosion inhibitor characteristics. A study done by Deslauriers [9] showed that the
performances of rust converter depend more on the barrier properties of the entire
paint system and the amount of the rust present on the surface than on its ability to
form ferric tannate chelates.
1.5 Reaction of Phosphoric Acid with Iron
Phosphoric acid alone is not recommended as rust converter because of their
reputation of poor performance [47]. However, the addition of phosphoric a~id to a
tannin solution produced a coating which showed better corrosion resistance than that
19
formed by either of the individual constituents alone [43]. Nigam et a/. [48]
concluded that the rust conversion performance usmg phosphoric acid solution
depends strongly on the concentration of the acid and the condition of the surface. It is
only above 8 .M concentrations that ferric phosphate is formed. Below this
concentration level, the transformation is dependant on the initial corroding medium
[43].
In formulation containing phosphoric acid alone, it was found to migrate and react
with the surface of the steel. This resulted in the formation of metal phosphates such
as Fe(H2P04)2, FeHP04, Fe3(P04)2, FeP04, etc. These compounds are thought to
passivate the substrate and provide the proper functionalities for the condensation
reactions with binder [43]. When reacts with metallic iron and magnetite,
concentrated phosphoric acid form a mixture of FeP04.4H20 and Fe3(P04)2.8H20,
whereas with oxyhydroxides like goethite and lepidocrocite, it forms acid ferric
phosphate Fe3(P04)2.5H20 that exhibits a relatively high stability [49]. A test carried
out by Nasrazadani [50] concluded that the reaction of phosphoric acid is fastest with
lepidocrocite, a little slower with magnetite, and slowest with goethite.
Phosphoric acid solution with low concentration reacts with iron corrosion product to
form iron phosphate precipitation, which act as corrosion protection, according to
Reactions (1.18) and (1.19) [47]. However, according to Gust [51], ifphosphoric acid
with medium and high concentration were applied, the iron seems to be unable to
allow the iron phosphate precipitation.
6H3P04 + 3Fe ----jo 3Fe(H2P04)2 + 3H2
3Fe(H2P04)2 ----jo Fe3(P04)2! + 4 H3P04
.. ........................... (1.18)
............................. (1.19)
20
.; ..
Tannin, as well as phosphoric acid, reacts with phase components of rust, converting
them into ferric tannates and ferric phosphates, respectively. While agent containing
both tannin and phosphoric acid, they convert the phase corp.ponents into mixtures of
original compounds, which are ferric tannates and ferric phosphates [51].
1.6 Objective
It is well known that tannin and phosphoric acid are the most prime candidates to be
used in rust converter due to their ability to convert corrosion products of iron into
more stable and corrosion resistant iron compounds. Therefore, the objectives of this
study are:
1. To extract and quantify tannin from mangrove (Rhizophora apiculata) bark
with different types of solvent.
2. To evaluate the chemical and physical properties of the tannins that are
extracted by means of infrared spectroscopy (FTIR), UV-VIS and several
tannin assays such as Prussian blue assay, vanillin assay and Stiasny test.
3. To identify the tannin components (monomer) by means of reverse phase
HPLC .
4. To optimize the condition of the reaction between Fe(III) ion with tannin and
phosphoric acid using infrared spectroscopy.
5. To formulate and evaluate the rust converter formulations based on
Rhizophora apiculata tannins by salt spray test (ASTM B 117).
21
2.0 MATERIALS AND METHODS
The mangrove barks (Rhizophora apiculata) were collectecl.from charcoal industry at
Larut Matang, Perak, Malaysia. The barks were dried under the sun until constant
weight and then were ground into powder form with the mesh size of 150 IJ.m. The
ground barks were kept ·in closed container at room temperature for subsequently
extraction. All the chemicals and solvents used in this study were AR grade and been
used without further purification.
2.1 Tannin Extraction
A 150.0 g of ground Rhizophora apiculata bark was wrapped with cotton cloth and
immersed in 600 mL of 70 % aqueous acetone for 24 hours with occasionally stirred
at ambient temperature. The immersion was repeated with new solvent for three
times. Acetone extracts were reduced to the aqueous phase by rotary evaporation
under reduced pressure and the resulting aqueous solution was freeze-dried to obtain
raw tannin powder.
The raw tannin (1.5 g) that was obtained by extraction of 70 % acetone was
redissolved in 150 mL water and extracted with 50 mL of n-hexane using separating
funnel to remove lipid. The residue was then extracted with 50 mL ethyl acetate to
remove the non-tannin containing extract include low molecular weight tannin. Traces
of ethyl acetate were removed from the ethyl-acetate soluble fraction (upper phase) by
rotary evaporation to obtain low molecular weight tannin. The ethyl acetate was also
removed from the remaining aqueous fraction (lower phase) to obtain mixed tannin
. (mixtureofhydrolysable and condensed tannin).
22
Mixed tannin (1 g) was diluted in 1 00 mL of 50 % aqueous methanol and applied to a
2.5 em x 20 em Sephadex LH-20 column. The column was first eluted with 50 %
aqueous methanol until the eluate was clear to recover· pydrolysable tannin and
condensed tannin was eluted from the column with 50 % aqueous acetone. The type
of tannin that was fully applied in the whole experimental was mixed tannin, unless
stated in the experimentaL'
150 g Bark Powder
.1600 mL of 70 % aqueous acetone
3 x 24 hours
Raw Tannin
l 13 x 50 mL of n-hexane
Lipid
Low Molecular
Weight Tannin
l Raw Tannin
I 3 x 50 mL of ethyl acetate
l Mixed Tannin
2.5 em x 20 em Sephadex LH-20 column
50 % aqueous methanol 50% aqueous acetone
Hydrolysable Tannin Condensed Tannin
Figure 2.1: Procedure in tannin extraction from Rhizophora apiculata bark.
23
2.2 Assays for The Quantification of Tannin
2.2.1 Prussian Blue Assay for Total Phenols [52)
Gallic acid was obtained from Sigma-Aldrich and \YaS used without further
purification. All other reagents were of analytical grade.
2.2.1.1 Preparation of Reagents
(I) 0.10 M FeNH4(S04)2 in 0.10 M HCl
Concentrated HCl was diluted to 0.10 M by bringing 8.3 mL of the
concentrated acid to 1 L with distilled water. A 48.2 g of dodecahydrate salt
(FeNH4(S04)2.l2H20) was dissolved in 1 L of 0.10 M HCl to obtain a pale
yellow solution of FeNH4(S04)2.
(2) 0.008 M K3Fe(CN)6
A yellow solution of 0.008 M K3Fe(CN)6 was prepared by dissolving 2.63 g
potassium ferricyanide in 1 L distilled water.
2.2.1.2 Assay Procedure
Tannin sample (0.01 g) was dissolved in 10 mL methanol. A 0.10 mL of the sample
was dispensed into a 125 mL Erlenmeyer flask, followed by adding 50.0 mL distilled
water. Three millimeters of 0.10 M FeNH4(S04)2 was added and swirled. Exactly 20
minutes after the addition of 0.10 M FeNH4(S04)2, three millimeters of 0.008 M
K3Fe(CN)6 was added and swirled. Absorbance at 720 nm was read 20 minutes after
the addition of 0.008 M K3Fe(CN)6. Solvent-only blank was prepared in the same
manner. The absorbance of the blank was subtracted from the absorbance obtained for
each sample. All determinations were done in' duplicate. The absorbance was read
with U 2000, Hitachi, Japan UV-Vis spectrophotometer. -· .~ -~-· --· -- -
24