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