UNIVERSITI PUTRA MALAYSIA REACTION OF BETA·CAROTENE WITH METAL IONS - PRODUCTS ISOLATION AND CHARACTERISATION YII MEl WO FSAS 1998 21
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