UNIVERSITI PUTRA MALAYSIA

REACTION OF BETA·CAROTENE WITH METAL IONS - PRODUCTS ISOLATION AND CHARACTERISATION

YII MEl WO

FSAS 1998 21

REACTION OF BETA·CAROTENE WITH METAL IONS - PRODUCTS ISOLATION AND CHARACTERISATION

By

YII MEl WO

Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science in the

Faculty of Science and Environmental Studies Universiti Putra Malaysia

June 1998

ACKNOWLEDGEMENTS

The author would like to take this opportunity to thank those who helped

him to finish his project especially his supervisor, Assoc. Prof Dr. Karen Badri

and co-supervisors Prof Dr. Badri Muhammad and Assoc. Prof Dr. M.T.H

Tarafder for their guidance, advice and their unlimited help.

He also will like to thank the laboratory officers Mr. Kamal Margona,

Mr. Abdul Wahab, Mr. Sri Jegan , Mr. Nazri Ahmad, Mr. Zainal Abidin for their

willingness to help in the analyses of the samples.

Besides, he would also like to thank his laboratory partners, Mdm.

Hazimah Abu Hassan, Mdm. Kamaliah Haji Sirat and Dr. Abdul Rahim Khan for

their help.

Thanks!

ii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS .......... ...... ............ ...................................... . ii LIST OF TABLES ....................................................... ......................... v LIST OF FIGURES ................ .............. ................ ........................ ........ vi LIST OF EQUATIONS ... ................... .............. ........................ .... ... ...... ix LIST OF ABBREVIATIONS ...... .......... ............ ............ ........................ x ABSTRACT .... .. ................. .......... ....... .. ........ ... ...... ........... ............ ........ xi ABSTRAK ................ ......... ..... .................................... ......................... xiii

CHAPTER

I INTRODUCTION ..... . .................................................... 1 Carotene and Carotenoids ....................... ..... . . . ........... ....... 1 Natural Occurrence of Carotenoids ..... .............................. 3 Physical and Chemical Properties of �-Carotene ........ ......... 5 Biological Functions, Actions and Uses of Carotenoids ........ 9

Colour and Coloration ...... ........ ......... ............ ........ .... 10 Photosynthesis and Photoprotection . ..... .......... .......... II Nutrition and Health ........... ...................................... 12

Carotene in Palm Oil .. .......... ........ ......... ........... ...... .......... 13 �-Carotene Complexes ...................................................... 14 Iron and Its Compounds ...................................... ............... 14

The Element ................................ .............................. 14 Complexes of Iron ... ........ .............................. ............ 15 Organometallic Compounds of Iron ............ ........ ....... 18

Tin and Its Compounds . ........................ ............................ 19 The Element ...... ................ ........... ................. ..... ....... 19 Compounds of Tin .................................................... 20 Organometallic Compounds of Tin ......... ................... 22

Objectives . .. . . . . . ......... ... . . ................................................ . . 24

II MATERIALS AND METHODS .................... ............ ...... 25 Chemicals .............................. .......................................... 25 Preparation and Decomposition of Complexes .................. 27

Preparation of Ferric-�-Carotene Complex . ...... ........ 27 Decomposition of Ferric-�-Carotene Complex ........... 28 Preparation of Tin-�-Carotene Complex .............. ...... 28 Tin-�-Carotene Complexes with Mixed Ligands . . . . . . . . . 29

Physical measurements and Elemental Analyses .. . . ............ 29 Determination of Iron ..... ................. .......................... 29

iii

Page

Determination of Tin . . . . . . . . . .. . . .. . . . . .. . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . 30 Thermogravimetric Analysis (TGA) . . . . . . . . ... . . . . . . . . . . . . . .. . . 30 CIIN . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ....... ... . . . . . . . . .. . .. . . .. . . . . . . . .. 30 Infrared Spectra (IR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Thin Layer Chromatography (TLC) . . .. . . . . .. . . .. . . . . .. . . . . . ... .

Ultra-VioletIVisible Spectrophotometry (UV Nis) . . .. . . . .

31 31 31

III RESULTS AN D DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Part I : Reaction of Iron With �-Carotene . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 3 3

lron-�-Carotene Complex . . . . ... . . . . . . . . . . . . ... . . . . . . . . . . . . . . . 33 Decomposition Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 Kinetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ... . . . .. .. . . . 44

Part II : Reaction of Tin With �-Carotene ... .. . .. ... . .. . . . ... . . .. . .... 59 Tin-�-Carotene Complex . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . 59 Mixed Li gands Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Kinetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 64

IV CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . ..... . ... . . . .. . . . . . . . . . . . . . . .. . . . . 7 6

REFERENCES . . . . . . . . . . . . . . . . .. . . . . . . . .... .. . . . . . .... . . . . . . . . . . . . . . . . ... . . .. . . . . . . . . . . . .. . . . . .. . . . . . . . . 78

APPENDI X A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Table Al : C HN Analysis Data .... . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 8 2

APPENDIX B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 Figure A l : TG Spectrum for Pure �-Carotene . . . . . . . . . ... . . . . . . . . . . . . . . .. . .. . . . . 8 3 Figure A2 : IR Spectra for Two Powder Forms of Deco mposition Products . . 84 Figure A3 : TLC for Some Solvent Systems . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 8 5 Figure A4 : UV Spectrum for Pure �-Carotene . . . .. . . . . . . . . . . . . . .. . .. . ... . . . .. . 8 6 Figure A5 : UV Repetitive Scanning for Pure � -Carotene at 60°C ... . . . . . . . . . 87 Figure A6 : UV Repetitive Scanning for Pure Ferric Nitrate at 60°C . . . . . . . . . 87 Figure A7 : IR Spectrum for Tin(lI) Chloride, SnCh ... . . .. . .. . . . . . .. . . . . . . . . . . 88 Fi gure A8 : UV Spectrum for Tin (II) Chloride in Ethanol . . . . . . ... . .. . . .. . . .. . 8 8

APPENDIX C ... . . .. . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . .. . . . . .. . . . . . . .. . . .. . . .. . . . . . . . 8 9 Determination ofIron . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 9 Determination ofEa, A, .1S*, Mf* . . .... . .. . . . ... . . . . . . . . ... . . .. . . . . . . . . . . . . . . . . . . . 90

iv

LIST OF TABLES

Table Page

1 Elemental Analysis of Complex I ......... ......... ........... . . . .. .... ... .... . . . .. . . 33

2 Time and Absorbance Data for Formation Reaction ..................... 46

3 Time and Absorbance Data for Decomposition Reaction at 25 °C ..... 5 0

4 Time and Absorbance Data for Decomposition Reaction at 3 0°C ..... 5 1

5 Time and Absorbance Data for Decomposition Reaction at 35 °C ..... 5 2

6 Time and Absorbance Data for Decomposition Reaction at 4 0°C ..... 53

7 Time and Absorbance Data for Decomposition Reaction at 45 °C ..... 54

8 Time and Absorbance Data for Decomposition Reaction at 5 0°C. ... 55

9 Rate Constants at Various Temperatures... . . . . .. . ..... . . .. . .. . .. . .. ... ... 57

10 The Values of Ea, A, ilS *, ilH* for Decomposition Reaction ......... 58

1 1 Time and Absorbance Data for Decomposition of �-carotene at 55 °C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

12 Time and Absorbance Data for Formation Reaction at 4 0°C......... 68

13 Time and Absorbance Data for Formation Reaction at 45 °C ... . ... .. 69

14 Time and Absorbance Data for Formation Reaction at 5 0°C. . . . .. .. . 7 1

15 Time and Absorbance Data for Formation Reaction at 55 °C. .. ... ... 7 2

16 Rate Constant at Various Temperatures ................................ 73

17 The Values of Ea, A, L1S*, L1H* for Decomposition Reaction........ 74

Al CHN Analysis Data ... ... ... .. . .. .... ... .. . ... .. . ... . .. .. . . .. . .. . .. .. ... .... 82

v

LIST OF FIGURES

Figure Page

1 . 1 a-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1 .2 �-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1 .3 y-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1 .4 Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . 4

1 .5 Lutein . . . .. . . . . . . . . . . . . . . . . ... .. . . .. ... . . . . . . . . . ..... . . . . . . . . . . . . . .. . . . . ... . . . ... . .... . . ... . . .. . 4

1 .6 Cryptoxanthin . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . 5

1 . 7 Mechanism for the Fragmentation of Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1 .8 Bixin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

1 .9 Crocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1

1 . 1 0 Zeaxanthin . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1

1 . 1 1 Haem . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7

1 . 12 Ferrocene . .. . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .... . . . . . .... . . . 1 8

3. 1 IR Spectrum for Fe(N03)3 . 9H20 . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . 35

3 .2 IR Spectrum for Pure �-Carotene . . . . .. . . . . . . . . .. . . . . ..... . . . . . .. . .... . . 36

3 . 3 IR Spectrum for Fe(N03)3 .C40H s6 .2CH2Ch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6

3.4 IR Spectrum for Fe(N03)3 .C40H s 6 .2CH2Ch a fter Storage in Open Atmosphere at Room Temperature . . . . . . .. . . . ... .. . . . . . . . .. . . 3 8

3.6 IR Spectrum for Fe(N03k C40H s 6 .2CH2Ch after Heating to 200°C . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . . , . . . . . . . .. . . . . . . ' " . . . . . . . . . . . . . . . . . 40

3.7 IR Spectrum for Fe(N03)3 .C4oH s6 .2CH2Ch after Heating to 500°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3 . 8 IR Spectrum for Fe(N03k C4oH s6 .2CH2Ch after Heating to 1 000° C . . ...... . ..... ... ......... ..... . ... . .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

vi

Figure Page

3 .9 IR Spectrum for Decomposition Product in Powder Form 42

3. 1 0 IR spectrum for Decomposition Product in Liquid Form . .. . . . . . . . . . 43

3 . 1 1 Repetitive Scanning for Reaction between Fe and �-carotene at 20°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3 . 12 Time Scan for Formation Reaction at 450 nm at 20°C . . . . . . . . . . . . . . . 45

3. 1 3 In (At-Ain) versus Time for Formation Reaction . . . . . . . . . . . . . . . . . . . . . . 47

3. 14 Repetitive Scanning for Product Decomposition at 60°C

3 . 1 5 Time Scan at 325 nm at Various Temperatures for

49

Decomposition Reaction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

3 . 16 In (At-Ain) versus Time for Decomposition Reaction at 25°C .. . . . . . . . . 50

3 . 1 7 In (At-Ain) versus Time for Decomposition Reaction at 30°C . . . . . . . . . . 5 1

3 . 1 8 In (At-Ain) versus Time for Decomposition Reaction at 3 5°C . . . . . . . . . . 52

3 . 1 9 In (At-Ain) versus Time for Decomposition Reaction at 40°C . . . . . . . . . . 53

3 .20 In (At-Ain) versus Time for Decomposition Reaction at 45°C . . . . . . . . . . 54

3 .2 1 In (At-An) versus Time for Decomposition Reaction at 50°C . . . . . . . . . . 5 5

3 .22 In k versus l iT for Decomposition Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7

3 .23 I n (kiT) versus liT for Decomposition Reaction . . . . . . . . . . . . . . . . . . . . . . . . 58

3 .24 IR Spectrum for SnCh + Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1

3 .25 IR Spectrum for SnCh + Pyridine . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1

3.26 IR Spectrum for SnCh + Aniline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 62

3 .27 IR Spectrum for SnCh + Pyridine-N-oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

3 .28 IR Spectrum for SnCh + o-Phenanthroline Monohydrate . . . . . . . . . . . . 63

3 .29 IR Spectrum for SnC lz + 2, 2' -Bipyridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

3 . 30 Repetitive Scanning for Tin(II) with �-carotene (Increasing) . . . . . . . . . . 64

vii

Figure Page

3 . 3 1 Repetitive Scanning for Tin(II) with �-carotene (Decreasing) . . . . . . . . . 65

3 .32 Repetitive Scanning for Tin(II) Chloride in Acetone . . . . . . . . . . . . . . . . . . . 65

3 .3 3 Repetitive scanning for �-carotene i n acetone . . . . . . . .. . . . . . . . . . . . . . . . . . . 66

3 . 34 In (At-Ain) versus Time for Decomposition of �-carotene at 5 5°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

3 . 3 5 In (At-Ain) versus Time for Formation Reaction at 40°C . . . . . . . . . . . . . . . . 69

3 .3 6 I n (At-Ain) versus Time for Formation Reaction at 45°C . . . . . . . . . . . . . . . . 70

3 .3 7 In (At-Ain) versus Time for Formation Reaction at 50°C . . . . . . . . . . . . . . . 72

3.38 In (At-Ain) versus Time for Formation Reaction at 5 5°C . . . . . . . . . . . . . . . 73

3 . 39 In k versus l iT for Formation Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

3 .40 In (kiT) versus liT for Formation Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Al TG Spectrum for Pure �-Carotene . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 83

A2 IR Spectra for Two Powder Forms of Decomposition Products . . . . . . 84

A3 TLC for Some Solvent Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

A4 UV Spectrum for Pure �-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

A5 UV Repetitive Scanning for Pure �-Carotene at 60°C . . . . . . . . . . . . . . . . 87

A6 UV Repetitive Scanning for Pure Ferric Nitrate at 60°C . . . . . . . . . . . . . 87

A7 IR Spectrum for Tin(II) Chloride, SnCh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

A8 UV Spectrum for Tin(II) Chloride in Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . 88

viii

Equation

1

2

LIST OF EQUATIONS

Page

In (At-Au) = -lct + In Ao . . . . .. . ........ ............. .......... ...... ..... 47

k= A exp (-Ea/RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

3 k = KBT/h exp (LiS*IR) exp (-Llli*IRT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

ix

CRN

HPLC

IR

PORIM

ppm

TGA

TLC

VV-Vis

LIST OF ABBREVIATIONS

-- Carbon, Hydrogen, Nitrogen Analyses

-- High Performance Liquid Chromatography

-- Infra-Red

-- Palm Oil Research Institute of Malaysia

-- parts per million

-- Thermogravimetric Analysis

-- Thin Layer Chromatography

-- Ultra violet - Visible

x

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of requirements for the degree of Master of Science.

REACTION OF BETA-CAROTENE WITH METAL IONS - PRODUCTS ISOLATION AND CHARACTERISATION

By

YIIMEIWO

June 1998

Chairman : Associate Professor Karen Badri, Ph.D.

Faculty: Science and Environmental Studies

Reaction of �-carotene with ferric nitrate nonahydrate and anhydrous

stannous chloride has been carried out. A 1t-complex of iron-�-carotene,

isolated from the reaction between stannous chloride and �-carotene. The

structure of the iron-�-carotene complex was proposed on the basis of IR and

UV -Vis spectra and elemental analyses.

Kinetic studies of the reaction of �-carotene with stannous chloride and

stannic chloride were carried out in acetone and ethanol and that of ferric nitrate

in 2: 1 ethanol-CCl4 mixture. Kinetic studies for the reaction of �-carotene with

SnCh failed. The decomposition reaction of ferric nitrate and �-carotene and the

xi

formation reaction of stannic chloride and �-carotene were found to be pseudo­

first order with respect to �-carotene.

Thermodynamic parameters (viz. Mf* and �S*) for the both reactions

were determined. The negative value of �S * indicates that the transition state

was more ordered and has less degree of freedom than the initial state.

The reaction between �-carotene and metal ions do not follow the

Pearson's Universal Soft Acid-Soft Base Theories.

xii

Abstrak tesis yang dikemukan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains.

TINDAK BALAS BETA-KAROTENA DENGAN ION LOGAM - PENGASINGAN BASIL DAN PENCIRIAN

Oleh

YTIMEIWO

Jun 1998

Pengerusi: Profesor Madya Karen Badri, Ph.D.

Fakulti: Sains dan Pengajian Alam Sekitar

Tindak balas bagi �-karotena dengan besi(III) nitrat nonahidrat dan

timah(II) klorida kontang telah dilakukan. Satu kompleks 1t bagi besi-�-

karotena, Fe(N03)3.C40H56.2CH2Ch, telah disintesis dan pencirian dibuat. Tetapi

tiada tindak balas berlaku di antara timah(II) klorida dengan �-karotena.

Struktur bagi kompleks besi-�-karotena dicadangkan dengan berpandu kepada

data-data dari IR, UV dan analisis unsur.

Kajian kinetik bagi tindak balas �-karotena dengan timah(II) klorida dan

timah(IV) klorida telah dilakukan dalam aseton dan etanol dan bagi besi(III)

nitrat dalam campuran 2 : 1 etanol-CCI4. Kajian kinetik timah(II) klorida dengan

�-karotena telah gaga!. Tindak balas penguraian bagi besi(I lI) nitrat dengan �-

xiii

karotena dan tindak balas pembentukan bagi timah(IV) klorida dengan�­

karotena didapati ialah tindak balas tertib satu pseudo (palsu) terhadap �­

karotena.

Parameter termodinamik seperti L1H* dan L1S* bagi kedua-dua

tindakbalas telah ditentukan. Nilai negatif L1S * menunjukkan bahawa keadaan

peralihan adalah lebih teratur dan kurang darjah kebebasan berbanding keadaan

awal.

Tindak balas di antara �-karotena dengan ion logam tidak mematuhi

Teori Universal Asid Lembut - Bes Lembut Pearson.

xiv

CHAPTER I

INTRODUCTION

Carotene and Carotenoids

Carotene, a red crystalline substance, was first isolated in 1 83 1 by

Wackenroder55 from carrots. Willstater and Meig5? ( 1 907) established that the

carotenoids were the derivatives of isoprenoids. Zechmeister60 ( 1 928) realized

that carotenoids were polyenes and these polyenes were shown to conjugate

among themselves by Kuhn31 in 1 93 1 . Kuhn and Brockmann30 were able to

isolate three isomers which are, 1 ) a-carotene (Figure 1 . 1 ), m.p 1 88°C, 2) �­

carotene (Figure 1 .2), m.p 1 84°C and 3 ) y-carotene (Figure 1 . 3), m.p 1 78°C

using a technique of applied column chromatography discovered by Tswett in

1 906.53,54

Figure 1 . 1 : a-Carotene

1

2

Figure 1 .2 : �-Carotene

Figure 1 . 3 : y-Carotene

Carotenoids are of importance in mammalian nutrition as pro-vitamin

A. 18 Although they are highly valued nutritional products, carotenoids, in

particular the carotenes, are easily destroyed and bleached during their refining

and processing. Carotenoids are the only naturally occurring tetraterpenes

consisting of eight isoprenoid residues.

The physical and chemical properties of carotene determine its use in

various industries such as in medicines, cosmetics, food colorants, vitamin A

preparations and animal feeds.22 p-Carotene was found to stimulate the body's

immune defense mechanism by way of increased capacity of macrophages to

kill tumor cells and to increase the production of tumor necrosis factor. 47

In mammals, �-carotene readily undergoes oxidative cleavage at the

central double bond, C9, to give two equivalents of an aldehyde which is known

as retina1.24 Von Euler15 ( 1 929) showed that crystall ine carotene possesses high

3

vitamin A activity, and a pioneering English biochemist, Moore36 ( 1 930)

demonstrated that, in the rat, absorbed carotene is converted to vitamin A which

is stored in the liver.

B-Carotene exists m several different configurations (isomers) .

Synthetic B-carotene is almost 1 00% trans-B-carotene. Meanwhile, B-carotene

found in fruits and vegetables contains about 1 0% cis-isomers and B-carotene

derived from algae contains about 50% of the 9-cis isomer.4, 16 Therefore, this

shows that B-carotene appears mostly in the trans-form.48,49

Most carotenoids, including B-carotene, easily undergo decomposition

under light and oxygen. This implies that studies should be carried out in an

inert atmosphere, in subdued light and with highly purified solvents. Therefore,

during an experiment, heating of the carotenoid compounds in solution should

be done carefully, to prevent the carotenoids being destroyed before they react.

The carotenoids should be stored in the dark, under an inert gas atmosphere and

refrigerated. 13,20

Natural Occurrence of Carotenoids

Carotenoids are widely distributed in higher plants and in some

microorganisms. In higher plants, the carotenoids are found in the green leaves,

together with chlorophyll . They are found in great abundance in dark yellow­

orange vegetables (eg. carrots)34 and dark green vegetables (eg. kale, broccoli

4

and spinach), and also in potatoes, fiuits (eg. apricots, cantaloupe, peaches,

papayas)46 and many others. It is found that carotenoids play an important role

in coloration of flowers. Carotenoids are also found in the chloroplasts of many

organisms such as red and green algae, yeast, fungi and photosynthetic

bacteria. 19

Carotenoids can be classified into two major groups,

a) Carotenes - hydrocarbons which are highly soluble in petroleum ether and

carbon tetrachloride but only slightly soluble in ethanol and other polar

solvents. e.g., a-carotene, �-carotene, y-carotene and lycopene (Figure

1 .4).5

b) Xanthophylls - oxygenated derivatives of the carotenes which are quite

soluble in ethanol, methanol and petroleum ether. e.g. , lutein (Figure 1 . 5)

and cryptoxanthin (Figure 1 .6i

Figure 1 .4 : Lycopene

.. ' I,OH

Figure 1 . 5 : Lutein

5

OH

Figure 1.6 : Cryptoxanthin

Carotenoids, as noted above, are highly unsaturated polyenes and in

some cases, like carotene, they contain eleven conjugated double bonds. The

long chain of carbon atoms and the double-bonds which are located

alternatively keep the molecule inflexible and elongated.

Physical and Chemical Properties of �·Carotene

As was indicated above, carotenes are insoluble in water, slightly

soluble in fats, moderately soluble in aliphatic hydrocarbons, and very soluble

in aromatic and chlorinated hydrocarbons such as benzene and carbon

tetrachloride.28 Their melting points are fairly high (around 200°C) and

increase with increasing molecular weight and number of functional groups.

When they are oxidised, carotenes lose their red-orange colour and turn

colourless. 19 The highly unsaturated structure of �-carotene enables this

compound to undergo oxidation, isomerisation and thermal degradation very

easily.6

The structure of the carotenoids which are made up of eight isoprenoid

structures, formed by head to tail condensation of four isoprenoid units that

joined tail to tail was shown by Zechmeister ( 1928) . 59

6

For molecules containing conjugated double bonds, the ultra-violet or

visible spectra give an indication of the nature of the polyene systems. The

conjugated double bond system gives colour to the molecule. As example, p­

carotene has three absorption maxima at 425 nm, 45 1 nm and 482 nm. 18 All

these three bands are located in the visible region. The first band has intense

absorption and gives the colour characteristic of this class. There is another

weak band at about 220 nm in the ultra-violet which indicates the presence of

the long conjugated chain . The wavelengths of the three absorption bands in the

visible spectrum increase with each extension to the conjugated system.37 This

is important for the analysis of the carotenes13 since the absorption of samples of

the same concentration will give different values depending on the number of

conjugated double bonds present. The solvent in which the absorption spectrum

of a carotenoid is measured also has a marked effect on the position of the

maxima and on the molecular absorbance of the compound. 20

Advances in chromatographic techniques, leading to a marked

improvement in sensitivity and resolution, have paved the way for great

development in the identification of carotenoids. Through working with the

counter current extraction techniques applied to solutions containing

carotenoids and chlorophylls, Tswett ( 1 906) first isolated carotenoids and then

adopted the application of the column chromatographic technique to separate

the components. 53 ,54

7

However it was not until 1 93 1 , that the separation of isomeric �-

carotene and a_carotene30,3 1 and the enrichment of vitamin A26 were achieved.

Chromatography on various forms of paper for separation of mixed compounds

was first introduced around 1 950, and was followed by the more efficient thin-

layer chromatography (TLC). Later, with the introduction of specialized

uniform stationary phases and refined instrumentation, column chromatography

evolved into High Performance Liquid Chromatography (HPLC), which permits

the highly efficient separation of mixtures of carotenoids that are of very similar

polarity, and their sensitive detection and accurate quantitative analysis.

A useful tool for structural identification of carotenoids IS Mass

Spectroscopy.37 Schweiter43 proposed a mechanism to explain that the

elimination of M-92 and M-I06 ions recorded in the mass spectrum is a result

of the fragmentation of the carotenoids (like �-carotene) actually corresponded

to toluene (C7Hs) and m-xylene (C12H1S) respectively.

+. +.

+

M-106

8

+. +.

+

M-92

Figure 1 .7 : Mechanism for the Fragmentation of Carotene

The above reaction occurs by the coiling of the polyene ehain and the

expulsion of the fragment. 33

Infra-red Spectroscopy has been widely applied to carotenoids. The

main peaks in the spectrum of pure p-carotene are

a) above 3000 cm-I , due to the stretching ofC-H bond in C=CH groups.

b) around 2900 cm-I, resulting from the stretching of C-H single bonds in

C-C-H groups.

c) near 970 em-I, due to the bending of the C-H bond of the C=CH grouping.8

Because of the presence of acyclic conjugated units, reactions

involving hydrogenation, oxidation, methyl group migration and chain

elongation or shortening may occur. Sometimes, antioxidants like tocopherol

and butylated hydroxytoluene (BHT) are used to enhance the stability against

oxidation.

The oxidation of p-carotene by strong reagents normally gives several

derivatives.

9

For instance,

a) potassium permanganate oxidizes �-carotene to beta-apo-8' -carotenal

and beta-apo- 12 ' -carotenal. 25

b) iodine reacts with it to form 5 , 5 ' ,6,6' -tetraiodo beta-carotene. 20

c) peracids such as peracetic acid combine with �-carotene form 5,6-

d· 'd b 20 lepoXI e eta-carotene.

Meanwhile, the normal trans �-carotene may undergo cis-trans

isomerization under the following conditions,28

a) exposure to light,

b) heating in hydrocarbon solvents,

c) melting briefly in vacuo,

d) prolonged contact with an active surface, e.g. AhO),

e) treatment with acids.

Thermal degradation of �-carotene in solution gives several simple

aromatic compounds such as 2,6-dimethylnapthalene, ionene, toluene and

xylene. I7

Biological Functions, Actions and Uses of Carotenoids

Most of the functions, applications and uses of carotenoids, are a

consequence of the light-absorbing properties of the polyene chromophore.

10

Natural roles in coloration, photosynthesis and photoprotection are well

established and are of major biological importance. The great importance of

carotenoids in human and animal nutrition and health, however, is not based

upon the light-absorbing properties of the molecules.

Colour and Coloration

Carotenoids, particularly carotenes are responsible for the colours of

some parts of living organisms.32 They are harmless colorants which can be

used in foods and cosmetics.9 Some of them have been used for centuries,

especially bixin (Figure 1.8), crocin (Figure 1 .9) and lycopene (Figure 1 .4 )

which can be found easily in annatto, saffron and tomato, respectively. Even

though nature-identical carotenoids are now produced synthetically and are

used on a large scale as food colorants, the demand for the natural extract

carotenoids is increasing greatly . Carotenes are used as additives to animal

feed, for the purpose of imparting a desired colour either to animal tissues or to

derived products (e.g. �-carotene, Figure 1.2, to cattle for cream or fat

coloration; lutein, Figu're 1.5, and zeaxanthin, Figure 1. 10, to chickens for egg

yolk and skin coloration) or to provide adequate supplies of vitamin A.7

HOOC

COOCH3

Figure 1.8 : Bixin