EXTRACTION OF ORGANIC CHEMICALS FROM MESQUITE by SHOU-JEN R. CHEN, B . S . A THESIS IN CHEMISTRY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved /Dekn of/ciJfe GrAfiuate School December, 1981
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EXTRACTIOn of Organic Chemicals of Prosopis Juliflora
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EXTRACTION OF ORGANIC CHEMICALS
FROM MESQUITE
by
SHOU-JEN R. CHEN, B . S .
A THESIS
IN
CHEMISTRY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
/Dekn of/ciJfe GrAfiuate School
December, 1981
)Jd>, l?'?'^ ACKNOWLEDGMENTS
I would like to express my special thanks to my research advisor
and committee chairman. Dr. Richard A. Bartsch, for his encouragement,
valuable guidance and assistance. Without his generous help this
research would have been impossible. I would also like to thank
Dr. John N. Marx for the helpful suggestions and advice during the
research period, and to Dr. John A. Anderson for serving as member
of my committee and spending time in analyzing and evaluating the
thesis.
I want to extend my thanks to Mr. Alan Croft for helping me
correct the manuscript. Finally, I would like to express my sincerest
thanks to my wife Feng-Ying A. Chen for her patience, understanding
and most importantly her continued faith in me.
11
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
CHAPTER I. GENERAL INTRODUCTION 1
Background 1
Literature and Previous Mesquite Research 2
Long Term Proj ect Goals 5
CHAPTER II. EXPERIMENTAL 6
General Methods 6
Proton Magnetic Resonance
Spectroscopy 6
Infrared Spectroscopy 6
Ultraviolet Spectroscopy 7
High Pressure Liquid Chromatography 7
Column Chromatography 7
Gas Chromatography 7
Thin Layer Chromatography 9
Preparative Thin Layer
Chromatography 9 Extraction of Organic Compounds
from Mesquite Plants 9
Source of Mesquite Plants 9
Preparation of Segregated
Mesquite Plant Parts 10 Extraction of Different Parts of the Mesquite Plant n
iii
Page
Column Chromatography 12
Preliminary Separation 12
Gradient Solvent Separation 13
Final Separation 13
Properties of the Isolated Compound 1 15
Decomposition of Compound 1_ 15
Treatment of Compound _1 with Base 18
Structural Determination of Compound 1_ 19
Formation of Trimethylsilyl
Ether Derivative 19
Formation of Ester Derivative 20
Shift Reagnet Experiment 20
CHAPTER III. RESULT AND DISCUSSION 22
Extraction and Spectral Analysis
of the Crude Extracts 22
Heartwood 22
Seasonal Changes 26
Heartwood from Chemically
Defoliated Mesquite 27
Other Parts of the Mesquite Plant 28
Chromatography and Spectral Analysis
of the Separated Material 30
Properties of the Isolated Compound 1 35
Decomposition of Compound 1_ 35
Treatment of Compound 1 with Base 39
Structural Determination of Compound 1 40 IV
Page
Silylation of Compound 1_ 40
Esterification of Compound 1 42
Shift Reagent Experiment 44
Proposed Structure of Compound 1_ 45
CHAPTER IV. SUMMARY AND SUGGESTIONS FOR FURTHER RESEARCH 52
Summary 52
Suggestions for Future Research 54
LIST OF REFERENCES 55
APPENDIX A
B
D
HPLC Chromatograms 56
Proton Magnetic Resonance Spectra 75
Infrared Spectra.
Ultraviolet Spectra,
89
96
LIST OF TABLES
Page
1. Extraction of Honey Mesquite 2
2. Chromatographic Conditions for HPLC Analysis 8
3. Gradient Solvent System 14
4. Extraction of Chipped Mesquite Heartwood 23
5. Extraction of Chipped Mesquite Sapwood, Chipped Bark,
and Shredded Leaves 29
6. Solubility of Compound 1_ in Various Solvents 36
7. Effect of Shift Reagent upon the Chemical Shifts of Absorptions in the PMR Spectrum of Acetylated _1 46
8. Shift in PMR Absorptions Cause by the Addition of Shift Reagent 47
VI
LIST OF FIGURES Page
1. Soxhlet Extraction Apparatus 11
2. Flow Scheme A 16
3. Flow Scheme B 17
4. Flow Scheme C 18
5. Gradient Solvent Separation 33
6. Trimethylsilyl Ether Derivative of (+)-Catechin and Its
PMR Spectrum 49
7. Proposed Structure of Compound 1 and Its Derivatives 50
8. Possible Stereo Structures of Compound 1^ 51
9. 3,3;4;7,8-Pentahydroxyflavan 53
10. HPLC-1, Crude Mesquite Heartwood Extract 57 11. HPLC-2, Fraction B from Gradient Solvent Column
Chromatography 58
12. HPLC-3, Fraction D from Gradient Solvent Column Chromatography 59
13. HPLC-4, Major Portion of Fraction C from Gradient Solvent Column Chromatography 60
14. HPLC-5, HPLC Chromatogram of Compound 2 61
15. HPLC-6, Photodecomposition of Compound 3 (1) 62
16. HPLC-7, Photodecomposition of Compound 2.(2) 63
17. HPLC-8, Photodecomposition of Compound j (3) 64
18. HPLC-9, Photodecomposition of Compound ] (4) 65
19. HPLC-10, Photodecomposition of Compound 1 (5) 66
20. HPLC-11, Compound 1^ before Irradiation with the Light Source 67
21. HPLC-12, Compound 1^ after Irradiation with the Light Source for 24 Hours 68
vii
Page
22. HPLC-13, Compound 1^ after Irradiation with the Light Source for 48 Hours 69
23. HPLC-14, Compound 1^ after Irradiation with the Light Source for 72 Hours 70
24. HPLC-15, Chromatogram of Sample 3 71
25. HPLC-16, Sample 3 after Being Kept in the Dark for 72 Hours 72
26. HPLC-17, Sample 3 after Irradiation with the Light Source for 72 Hours 73
27. HPLC-18, Sample 3 after Adding One Drop of
Concentrated HCl 74
28. PMR-1, Crude Mesquite Heartwood Extract 76
29. PMR-2, Non-Polar Fraction of Mesquite Heartwood Extract.... 77
30. PMR-3, Polar Fraction of Mesquite Heartwood Extract 78
31. PMR-4, PMR Spectrum of Compound 1^ 79
32. PMR-5, PMR Spectrum of Mesquite Sapwood Extract 80
33. PMR-6, Trimethylsilyl Ether Derivative of Compound 1_ 81
34. PMR-7, Acetate Derivative of Compound 1_ 82 35. PMR-8, First Addition of Shift Reagent to Acetate
Derivative of Compound 1^ 83
36. PMR-9, Second Addition of Shift Reagent to Acetate Derivative of Compound 1^ 84
37. PMR-10, Third Addition of Shift Reagent to Acetate Derivative of Compound _1 85
38. PMR-11, Fourth Addition of Shift Reagent to Acetate Derivative of Compound 1^ 86
39. PMR-12, Fifth Addition of Shift Reagent to Acetate Derivative of Compound 1^ 87
40. PMR-13, Sixth Addition of Shift Reagent to Acetate Derivative of Compound _1 88
viii
Page
41. IR-1, Crude Mesquite Heartwood Extract
IR-2, Non-Polar Fraction of Mesquite Heartwood Extract 90
42. IR-3, Polar Fraction of Mesquite Heartwood Extract 91
43. IR-4, IR Spectrum of Compound J 92
44. IR-5, IR Spectrum of Mesquite Sapwood Extract 93
45. IR-6, Trimethylsilyl Ether Derivative of Compound ] 94
46. IR-7, Acetate Derivative of Compound 1^ 95
47. UV-1, UV Spectrum of Compound 1. 97
48. UV-2, Compound 1^ after Irradiation with the Light Source for 24 Hours 98
49. UV-3, Compound 1 after Irradiation with the Light Source for 48 Hours " 99
50. UV-4, Compound 1_ after Irradiation with the
Light Source for 72 Hours 100
51. UV-5, Base and Acid Treatment of Sample 4 101
52. UV-6, Acid Treatment of Sample 4 102
53. UV-7, Base Treatment of Sample 4 103
IX
CHAPTER I
GENERAL INTRODUCTION
Background
The control of mesquite proliferation is a major problem in the
West Texas area. Destruction and removal of mesquite from rangeland
and farmland are expensive if the only objective is to destroy the
undesirable brush. However, potential uses of mesquite are many and
varied. Presently, mesquite is being used primarily as a source of
fuel. Occasionally mesquite has been used for fenceposts, but there
does not appear to be any significant commercial utilization of
mesquite for this purpose at this time. If economically valuable
products can be obtained from mesquite, its removal would be consider
ably more attractive.
An almost totally unexplored area of potential mesquite utili
zation involves the isolation and use of the organic compounds, other
than carbohydrates, that are present in the mesquite plant. Although
mesquite contains a relatively high amount of material which may be
extracted with solvents. little is known concerning the composition
of the organic compounds which make up such extracts.
Currently, the feasibility of utilizing treated mesquite for
animal feed is being evaluated by the Chemical Engineering Department
at Texas Tech University. In connection with these investigations.
it would be very beneficial to identify compounds present in the
mesquite plant or produced by chemical treatment of mesquite which
may be potential digestion inhibitors.
Literature and Previous Mesquite Research
The first report of mesquite wood extraction was published in
2
1922. G. J. Ritter and L. C. Fleck performed the extraction on un
specified proportions of mesquite sapwood and heartwood. The extrac
tion results have been cited numerous times between 1922 and 1972.
In 1972, I. S. Goldstein and A. Villarreal published a paper
entitled "Chemical Composition and Accessibility to Cellulose of
3
Mesquite Wood." In this paper, the relative amounts of chemicals
which may be extracted from mesquite sapwood and heartwood were
reported for the first time.
Table 1. Extraction of Honey Mesquite
Extraction Solvent
Water
Benzene-ethanol
Benzene-ethanol-water
Extraction Yield, Percent of Air-Dry Sample
1% NaOH
From Sapwo
6.0%
4.4%
10.4%
20.5%
od Fn om Heartwood
5.8%
12.2%
18.0%
28.9%
The mesquite wood was extracted according to the procedures in
4 Browning,, but the chemical composition of the extracts was not
determined.
"In 1975, the State of Texas appropriated funds to the College
of Agricultural Sciences at Texas Tech University to investigate
methods for commercial use of mesquite which had been harvested from
ranchlands." For the potential use of mesquite as roughage in
animal feed, the Chemical Engineering Department at Texas Tech Univer
sity investigated a number of thermochemical pretreatments with the
goal of improving the digestibility of harvested mesquite. These
included the treatment of mesquite with sulfur dioxide, sulfuric acid,
elemental sulfur and methanol. The sulfur dioxide treatment has
shown the most promise for increasing the J^ vitro digestibility of
mesquite wood. This significant increase in the _in vitro digesti
bility may allow treated mesquite wood to be used as a roughage sub
stitute in animal feed.
Isolated reports of mesquite wood extraction using various
solvents demonstrate that appreciable amounts of organic compounds
2 3 may be removed. * However, in no instance has the identity of the
extracted organic compounds been determined.
During the summer of 1979, Gaul and Bartsch prepared a report
entitled "Survey of the Literature Pertaining to the Extraction of
Organic Chemicals from Mesquite". Since information up to 1969 was
Q
contained in the book Literature on Mesquite (edited by Joseph L.
Shuster), this survey treated only the post-1969 literature on the
subject. The goal of the report was to summarize the existing data
4
concerning organic compounds which can be extracted from mesquite
(Prosopis juliflora).
A few references dealing with the isolation of certain compounds
or classes of compounds such as tannins, waxes, and flavonoids from
specific mesquite plant parts or unspecified mesquite sources were
located. However, the data was found to be extremely fragmentary and
provided little basis for a judgement concerning whether or not
economically attractive non-carbohydrate organic chemicals could be
extracted from mesquite, in general, or, specifically, the heartwood
of mesquite.
Gaul and Bartsch also performed an exploratory study of the
extraction and identification of non-carbohydrate compounds from
mesquite. In this initial effort, shredded whole mesquite plants
(Prosopis juliflora) from the same source utilized in the chemical
treatment of mesquite for animal feed were used. With a varity of
solvents, organic materials were extracted from the shredded whole
mesquite and were shown to be mixtures of mostly waxes and carbohy
drates by spectroscopic methods.
It was also found that the amount of extracted organic compound
was very sensitive to the weathering which the mesquite had experienced.
From this observation, it was concluded that the source of mesquite
used in these preliminary extraction studies was unsuitable because
potentially interesting organic compounds had probably disappeared
during the weathering process.
Long Term Project Goals
The specific objective of this research is to continue the
investigation of non-carbohydrate organic chemicals which may be
extracted from mesquite. Attention is to be focused upon freshly
harvested mesquite plants. In addition, separated plant parts
(particularly the heartwood) is to be used rather than the entire
mesquite plant.
Long term project goals are centered around two main subjects.
The first objective is an assessment of the feasibility of obtaining
useful non-carbohydrate organic compounds from mesquite. If unique
or rare organic compounds are found to be present in reasonable
amounts, economical extraction methods will be explored. The second
goal is a search for non-carbohydrate organic compounds in mesquite
which might have an adverse effects (ie. digestion inhibitors) upon
the use of untreated or treated mesquite as a source of roughage in
animal feed.
CHAPTER II
EXPERIMENTAL
General Methods
Proton Magnetic Resonance Spectroscopy
Proton magnetic resonance (PMR) spectra were measured with a
Varian EM 360 nuclear magnetic resonance spectrometer. Samples were
dissolved in various deuterated solvents:
Acetone-d, (CD^COCD-, Aldrich Chemical Co.)
Chloroform-d^ (CDC1-, Norell Chemical Co., Inc.)
Carbon tetrachloride (CCl,, Norell Chemical Co., Inc.)
All PMR spectral data are reported using the 5 scale (parts per
million, ppm) with tetramethylsilane (Norell Chemical Co., Inc.) as
an internal standard (0.0 ppm).
Infrared Spectroscopy
The samples were examined using a Perkin-Elmer Model 457 infrared
spectrophotometer or a Beckman Acculab 8 infrared spectrophotometer.
The majority of the samples were prepared as thin films between NaCl
plates. In a few cases, solid samples were prepared as KBr pellets.
All infrared (IR) spectral data are reported in wavenumbers (cm ).
Ultraviolet Spectroscopy
Ultraviolet spectra (UV) were recorded with a Gary Model 17
ultraviolet-visible spectrophotometer. In all cases, methanol
(anhydrous, MCB, Omnisolv) was used as the solvent. All UV spectral
data are reported in wavelength units (nm).
High Pressure Liquid Chromatography
High pressure liquid chromatography (HPLC) was performed on a
Waters Associates Model 244 high pressure liquid chromatograph
equipped with a Model 440 ultraviolet absorbance detector
( \= 254 nm) and a Waters Associates Model 660 solvent programmer.
HPLC analysis utilized a Waters Associates y-Bondapak C-18 column.
The chromatographic conditions are given in Table 2. In all cases,
solutions of samples and standards were prepared using methanol
(anhydrous, MCB, Omnisolv) as the solvent.
Column Chromatography
All column chromatography was performed using silica gel
^ After each addition of shift reagent, the PMR sample tube was shaken vigorously and the PMR spectrum was taken after all of the shift reagent had dissolved.
tical with that of Compound 1 some IR spectra of flavonoid compounds
showed types of absorption in the regions of 3750 to 2800 cm and
1800 to 1200 cm which were similar to those in the IR spectrum of
Compound ] ,
A number of literature references regarding flavonoid compounds
were then searched. » After substantial time and effort had been
expended, a report published in 1963 by A. C. Waiss and co-workers
12 concerning the study of flavonoid compounds was found. In this
study of various trimethylsilyl ethers of flavonoid compounds, some
PMR spectra of derivatives were presented. It was noted immediately J
that the PMR spectrum of the trimethylsilyl ether derivative of 5l
(+)-catechin (Figure 6) showed great similarity to the PMR spectrum "H
n
of the trimethylsilyl ether derivative of Compound 1 (PMR-6) . Jlj
The only difference observed between the PMR spectra of trimethyl- •»«
silyl derivatives of (+)-catechin and Compound 1 is that the signals ai II ()
at 6.1 ppm and 5.9 ppm shown in the PMR spectrum of the catechin
derivative are not present in the PMR spectrum of the derivative of
Compound 1. Instead the latter shows a singlet at 6.33 ppm. This
suggests that Compound 1 and (+)-catechin are structurally quite similar.
With all the spectral data in hand, a possible structure of
Compound _1 is proposed in Figure 7.
In the proposed structure, protons at positions 5, and 6 are
in similar chemical environments. Thus they are assigned to the
almost identical absorptions at 6.40 ppm in PMR-4; and 6.33 ppm in
PMR-6. In the acetate derivative the proton at position 6 is more
49
.Sppm
5.9 ppm OR "Q'2pDm H H ^ ' )
V / 3.9 ppm
2.8 ppm
.8 ppm
CH-
R = -S i -CH3
CH.
f\ '^{
n m
'«! -'i <l ^1
II •I
25:6'
8 6
Jjiiiu IV
10 8 4 0 ppm
Figure 6. Trimethylsilyl Ether Derivative of (+)-Catechin and Its PMR Spectrum
50
COMPOUND 1:
R=H
DERIVATIVES:
R'= or
R'=
9 -C-CH
-Si-CHq I ^ CHo
Figure 7. Proposed Structure of Compound 1 and Its Derivatives
n
•n
:%l ill
strongly affected by the acetoxy group than that at position 5.
Therefore they show an AB pattern centered at 6.83 ppm.
The proposed structure has a molecular formula of C^-H^.O^. 15 14 6
Elemental analysis data were obtained for the compound. Calculation
for C, H,,0.: C, 62.07; H, 4.83. Found: C, 58.17: H, 5.18. Since 15 14 D
many flavonoid compounds have been reported to occur in the hydrated form , percentages were calculated for C, .H , O^-H^O, C, c-H, , 0^*2H^0,
and C,^H,,0,*3H„0. It was found that the values for C,^H..0,-H^O: 15 14 0 2 15 14 D z
C, 58.44; H, 5.19, best reproduce the elemental analysis result.
Thus, Compound ] may well exist as the C j-H ,0^ monohydrate, with
molecular weight equal to 308.
II
An attempt to determine the stereochemistry at carbons 2 and 3
51
of the Compound 1_ was also made by measuring the coupling constant
for the hydrogens attached to these positions in the Compound 1_
(PMR-4) and trimethylsilyl ether derivative of Compound 1 (PMR-6).
It was found that J^^ = 8 Hz. Therefore, according to the Karplus
rule, the dihedral angle between 2H and 3H is either 180° or 0**.
13 Based on the report by King and co-workers that 2H and 3H have
trans-configuration in (+)-catechin and have cis-configuration in
(-)-epicatechin (an isome of (+)-catechin, differing only with respect
to 2H and 3H), two possible structures _A and B have been proposed for
Compound 1.
OH
41
•I f\ %\ CI
II •!
B
Figure 8. Possible Stereo Structures of Compound _1
It is expected that in trans-2,3-flavan derivatives, the confor
mation in which both the 2- and the 3-substituent are quasi-equatorial
14 15 (structure A ) will be highly favored at room temperature. '
52
CHAPTER IV
SUMMARY AND SUGGESTIONS FOR FURTHER RESERACH
Summary
During the research period, attention has been focused pri
marily upon the reddish-brown heartwood of mesquite, Prosopis
juliflora. Effects of varying the extracting organic solvent upon •*!
the amount of material extracted from the heartwood and the overall i
composition of the extracted material were assessed. The amounts ,
of non-carbohydrate organic chemicals which can be extracted from ^
the heartwood of mesquite were found to depend upon the solvent and
the season of the year when the mesquite was harvested. ['
ii
In order to separate the heartwood extract of mesquite into •!
individual components or fractions of components, portions of the
heartwood extract were submitted to various chromatographic
methods, such as: column chromatography, thin layer chromatography,
preparative thin layer chromatography, gas chromatography, and high
pressure liquid chromatography. Among these chromatographic methods,
column chromatography proved to be the most effective for separating
the components of the heartwood extract in useful amounts.
By column chromatography, the heartwood extract was readily
separable into non-polar fraction (about 10%) and polar fraction
(about 90%). The non-polar fraction was shown to be a mixture of
53
at least twelve components. Many of these components are probably
phenolic compounds,
A high pressure liquid chromatographic system was developed for
analyzing the individual components of the polar fraction of the
mesquite heartwood extract. This analytical technique was used to
monitor the effectiveness of the column chromatographic separation of
individual compounds in amounts which allow for their identification.
The polar extract of mesquite heartwood was separated by column
chromatography into fractions of components. The major component
of the polar extract. Compound 1_, was isolated from one of these Jj
41 fractions.
The isolated Compound 1_ was examined by infrared, proton magnetic 'H 1 «i
resonance, and ultraviolet spectroscopy. The trimethylsilyl ether ;||
and acetate derivatives of Compound 1 were prepared and examined by „
spectroscopic methods. Shift reagent experiments were conducted ., il
upon the acetate derivative of Compound 1 .
From the accumulated data, the structure of Compound 1 is pro
posed to be:
(MONOHYDRATE)
Figure 9. 3,3,'4 J 7 ,8-Pentahydroxyf lavan
54
Suggestion for Future Research
It is believed that with a proper modification of the techniques
developed in this research, more components of the mesquite heartwood
may be separated. Thus, the composition of the mesquite heartwood
extract may be more clearly identified.
Although all the spectral data obtained form Compound jL and its
derivatives supported the proposed structure, the actual mass spec
troscopic analysis of Compound 1 , its trimethylsilyl ether and ace
tate derivatives would provide most convincing evidence for the J
proposed structure. li
Compound _1 was found to be sensitive to both light and base. •••
%l The UV spectra of both the irradiated sample and the base treated z\
sample showed similar types of decomposition. Among these decom- -.i
'\\ position products, some appear to be the same as some components in ji
II
the polar fraction of the heartwood extract. It would be useful if
the composition of the decomposed samples could be identified.
Due to the known anti-bacterial properties of phenolic compounds,
it is thought that the digestion inhibitors in freshly harvested
mesquite may be compounds such as Compound 1_ in the heartwood. More
investigation of this possibility is necessary.
LIST OF REFERENCES
1. Mesquite: Growth and Development, Management, Economics, Control, Uses." Texas A & M University, The Texas Agricultural Experiment Station, Research Monograph 1, Nov. 1973, p. 20.
2. Ritter, G. J., Fleck, L. C , Ind. Eng. Chem. 1922, 1^, p. 1050.
3. Goldstein, I. S., and Villarreal, A., Wood Science, 1972, 5, p. 15. ~
4. Browning, B. L., "Methods of Wood Chemistry," Vol. 1, Inter-science, New York, 1967, p. 79-82, 87.
5. Fahle, D. W., "Processing Mesquite as a Cattle Feed," Texas Tech University, Lubbock, Texas. 1978, p. 1.
6. Vernor, T. E., "Processing of Mesquite for Cattle Feeding," Texas Tech University, Lubbock, Texas. 1977. Jf
^t 7. Gaul, D. F., and Bartsch, R. A., "Survey of the Literature J[
Pertaining to Extraction of Organic Compounds from Mesquite," Mesquite Utilization Program, College of Agriculture, Texas J!j Tech University, 1979. ^
SI 8. Schuster, J. L., "Literature on the Mesquite (Prosopis L.) of
North America," Texas Tech University, 1969. 2 'l
9. Pierce, A. E., "Silylation of Organic Compounds." Pierce -• Chemical Co., 1968, p. 33-39, 72-154.
10. Geissman, T. A., "The Chemistry of Flavonoid Compounds." The Mamillan Company. 1962, p. 70.
11. Richard, J. H., and Hendeichson, J. B., "The Biosynthesis of Steroids, Terpenes, and Acetogenins." W. A. Benjamin, INc, 1964, p. 50-61.
12. Waiss, A. C. Jr., Lundin, R. E., and Stern, D. J., Tetrahedron Letter, 1964, p. 513.
13. King, F. E., Clark-Lewis, J. W., and Forbes, W. F., J. Chem. Soc. 1955, p. 1338.
14. Clark-Lewis, J. W., and Jackman, L. M,, Proceedings Chemical Society, 1961, p. 165.
15. Weinges, K., and Paulus, E., Liebigs Ann. Chem., 1965, 681, p. 154.
55
II
APPENDIX A
*l n 0» m
High Pressure Liquid Chromatograms 5
II
II
56
57
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1 ' I
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T 1 ' '
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1 1 IT-I I 1 1 M Hi ' ' ,=F • M l
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M r ' ^ i' r ' M 1 1 l l ! i 1 ! M 1 1 I ' 1 ! ' ' ' 1 1 ' ' 1 1 1
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