Citation
A study of the relationships between chromatographic behavior and structure of steroid hormones

Material Information

Title:
A study of the relationships between chromatographic behavior and structure of steroid hormones
Creator:
Hartman, Iclal Sirel, 1930-
Place of Publication:
Gainesville
Publisher:
[s.n.]
Publication Date:
Language:
English
Physical Description:
ix, 155 l. : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Acetates ( jstor )
Activating transcription factors ( jstor )
Awe ( jstor )
Bees ( jstor )
Cholestanes ( jstor )
Ethers ( jstor )
Functional groups ( jstor )
Seas ( jstor )
Steroids ( jstor )
Toes ( jstor )
Androstane ( lcsh )
Chromatographic analysis ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: l. 148-154.
General Note:
Manuscript copy.
General Note:
Vita.
Statement of Responsibility:
by Iclal Sirel Hartman.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
000423960 ( ALEPH )
ACH2365 ( NOTIS )
11045751 ( OCLC )
AA00004950_00001 ( sobekcm )

Downloads

This item has the following downloads:

studyofrelations00hart.pdf

studyofrelations00hart_0120.txt

studyofrelations00hart_0012.txt

studyofrelations00hart_0055.txt

studyofrelations00hart_0080.txt

studyofrelations00hart_0078.txt

studyofrelations00hart_0023.txt

studyofrelations00hart_0010.txt

studyofrelations00hart_0009.txt

studyofrelations00hart_0089.txt

studyofrelations00hart_0091.txt

studyofrelations00hart_0152.txt

studyofrelations00hart_0164.txt

studyofrelations00hart_0045.txt

studyofrelations00hart_0135.txt

studyofrelations00hart_0084.txt

studyofrelations00hart_0112.txt

studyofrelations00hart_0037.txt

studyofrelations00hart_0146.txt

studyofrelations00hart_0116.txt

studyofrelations00hart_0015.txt

studyofrelations00hart_0044.txt

studyofrelations00hart_0018.txt

studyofrelations00hart_0070.txt

studyofrelations00hart_0006.txt

studyofrelations00hart_0019.txt

studyofrelations00hart_0113.txt

studyofrelations00hart_0082.txt

studyofrelations00hart_0128.txt

studyofrelations00hart_0125.txt

studyofrelations00hart_0132.txt

studyofrelations00hart_0002.txt

studyofrelations00hart_0140.txt

studyofrelations00hart_0127.txt

studyofrelations00hart_0139.txt

studyofrelations00hart_0064.txt

studyofrelations00hart_0100.txt

studyofrelations00hart_0098.txt

studyofrelations00hart_0095.txt

studyofrelations00hart_0165.txt

studyofrelations00hart_0029.txt

studyofrelations00hart_0000.txt

studyofrelations00hart_0056.txt

studyofrelations00hart_0004.txt

studyofrelations00hart_0061.txt

studyofrelations00hart_0050.txt

AA00004950_00001.pdf

studyofrelations00hart_0133.txt

studyofrelations00hart_0025.txt

studyofrelations00hart_0053.txt

studyofrelations00hart_0031.txt

studyofrelations00hart_0141.txt

studyofrelations00hart_0157.txt

studyofrelations00hart_0054.txt

studyofrelations00hart_0123.txt

AA00004950_00001_pdf.txt

studyofrelations00hart_0028.txt

studyofrelations00hart_0076.txt

studyofrelations00hart_0163.txt

studyofrelations00hart_0007.txt

studyofrelations00hart_0144.txt

studyofrelations00hart_0068.txt

studyofrelations00hart_0047.txt

studyofrelations00hart_0069.txt

studyofrelations00hart_0101.txt

studyofrelations00hart_0119.txt

studyofrelations00hart_0077.txt

studyofrelations00hart_0049.txt

studyofrelations00hart_0117.txt

studyofrelations00hart_0058.txt

studyofrelations00hart_0072.txt

studyofrelations00hart_0134.txt

studyofrelations00hart_0108.txt

studyofrelations00hart_0065.txt

studyofrelations00hart_0085.txt

studyofrelations00hart_0131.txt

studyofrelations00hart_0063.txt

studyofrelations00hart_0036.txt

studyofrelations00hart_0162.txt

studyofrelations00hart_0150.txt

studyofrelations00hart_0106.txt

studyofrelations00hart_0097.txt

studyofrelations00hart_0008.txt

studyofrelations00hart_0052.txt

studyofrelations00hart_0099.txt

studyofrelations00hart_0071.txt

studyofrelations00hart_0147.txt

studyofrelations00hart_0166.txt

studyofrelations00hart_0038.txt

studyofrelations00hart_0046.txt

studyofrelations00hart_0030.txt

studyofrelations00hart_0014.txt

studyofrelations00hart_0130.txt

studyofrelations00hart_0034.txt

studyofrelations00hart_0062.txt

studyofrelations00hart_0103.txt

studyofrelations00hart_0033.txt

studyofrelations00hart_0151.txt

studyofrelations00hart_0043.txt

studyofrelations00hart_0129.txt

studyofrelations00hart_0093.txt

studyofrelations00hart_0126.txt

studyofrelations00hart_0066.txt

studyofrelations00hart_0111.txt

studyofrelations00hart_0143.txt

studyofrelations00hart_0057.txt

studyofrelations00hart_0142.txt

studyofrelations00hart_0136.txt

studyofrelations00hart_0115.txt

studyofrelations00hart_0001.txt

studyofrelations00hart_0051.txt

studyofrelations00hart_0122.txt

studyofrelations00hart_0110.txt

studyofrelations00hart_0022.txt

studyofrelations00hart_0090.txt

studyofrelations00hart_0017.txt

studyofrelations00hart_0156.txt

studyofrelations00hart_0021.txt

studyofrelations00hart_0016.txt

studyofrelations00hart_0088.txt

studyofrelations00hart_0003.txt

studyofrelations00hart_0160.txt

studyofrelations00hart_0020.txt

studyofrelations00hart_0048.txt

studyofrelations00hart_0011.txt

studyofrelations00hart_0161.txt

studyofrelations00hart_0145.txt

studyofrelations00hart_0073.txt

studyofrelations00hart_0109.txt

studyofrelations00hart_0013.txt

studyofrelations00hart_0027.txt

studyofrelations00hart_0124.txt

studyofrelations00hart_0096.txt

studyofrelations00hart_0035.txt

studyofrelations00hart_0158.txt

studyofrelations00hart_0074.txt

studyofrelations00hart_0107.txt

studyofrelations00hart_0137.txt

studyofrelations00hart_0148.txt

studyofrelations00hart_0087.txt

studyofrelations00hart_pdf.txt

studyofrelations00hart_0138.txt

studyofrelations00hart_0042.txt

studyofrelations00hart_0041.txt

studyofrelations00hart_0032.txt

studyofrelations00hart_0024.txt

studyofrelations00hart_0092.txt

studyofrelations00hart_0102.txt

studyofrelations00hart_0094.txt

studyofrelations00hart_0121.txt

studyofrelations00hart_0060.txt

studyofrelations00hart_0075.txt

studyofrelations00hart_0114.txt

studyofrelations00hart_0005.txt

studyofrelations00hart_0040.txt

studyofrelations00hart_0026.txt

studyofrelations00hart_0059.txt

studyofrelations00hart_0118.txt

studyofrelations00hart_0104.txt

studyofrelations00hart_0086.txt

studyofrelations00hart_0149.txt

studyofrelations00hart_0154.txt

studyofrelations00hart_0081.txt

studyofrelations00hart_0155.txt

studyofrelations00hart_0153.txt

studyofrelations00hart_0105.txt

studyofrelations00hart_0159.txt

studyofrelations00hart_0083.txt

studyofrelations00hart_0079.txt

studyofrelations00hart_0067.txt

studyofrelations00hart_0039.txt


Full Text







A STUDY OF THE RELATIONSHIPS BETWEEN
CHROMATOGRAPHIC BEHAVIOR
AND STRUCTURE OF STEROID HORMONES







By
ICLAL SIREL HARTMAN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY







UNIVERSITY OF FLORIDA
April, 1963


I




A STUDY OF THE RELATIONSHIPS BETWEEN
CHROMATOGRAPHIC BEHAVIOR
AND STRUCTURE OF STEROID HORMONES
By
ICLAL SIREL HARTMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
April, 1963


This dissertation is dedicated to ay husband


ACKNOWLEDGEMENTS
The guidance and encouragement of Dr. Herbert H. Wotis and
Dr. Thomas W. Stearns were of the utmost value to the author as a
constant source of Inspiration and support and are most gratefully
acknowledged.
Particular thanks are also due to Dr. Armin H. Gropp for his
advice and Inspiration throughout the graduate training of the
author.
The aid of Dr. Herman E. Carr,Jr. In the criticism and
correction of the original manuscript Is deeply appreciated.
The author also wishes to acknowledge the competent assistance
of Mrs. L. Joyce Smith In the computation of some of the data and In
the typing of this dissertation.
Ill


TABLE OF CONTENTS
Paga
DEDICATION 11
ACKNOWLEDGEMENTS HI
LIST OF TABLES vll
LIST OF FIGURES lx
INTRODUCTION 1
Statement af Purpese 1
Chromatography 2
Historical Background 2
Gas Chromatographic Methods 6
Principles of Gas Chromatography 8
Basic Gas Chromatographic Apparatus 16
Androstanes 18
Conformations of Androstane Ring System 18
Isomerism In Androstanes 24
Androstane Isomers Possessing Hormonal Activity 25
Methods of Assay for Androgens and 17-Ketostarolds 27
Chemical and Physical Methods of Characterization
of Steroids 28
Nomenclature 30
EXPERIMENTAL 32
Materials and Methods 32
Compounds Studied 32
lv


Page
Preparation of Derivatives 34
Studies on the Free Steroids and their Derivatives 35
Reference Standard 35
Solvents and Reagents 35
Column Supports 37
Stationary Phase 37
Preparation of Columns 39
Equipment 40
Preliminary Experiments 42
SE-30 Methyl Silicone Polymer (SE-30) 42
Mixed Phases Containing SE-30 44
SE-52 Methyl Phenyl Silicone Polymer (SE-52) 51
Dow Corning High Vacuum Silicone Grease 51
Ethyleneglycol Succinate Polyester (EGS) 51
Neopentylglycol Adipate Polyester (NGAd) 51
Neopentylglycol Sebacate Polyester (NGSeb) 53
Silicone Nitrile Fluids XF-1150 and XF-1105 53
QF-1-0065 Fluoroalfcyl Polymer (QF-1-0065) 56
Discussion of Preliminary Experiments 58
Comparative Experiments 62
Discussion of Comparative Experiments 93
Concentration of the Stationary Phase and Mesh Sise 107
of Support
v


Separation of Urinary 17-KetoBteroids 113
Known Mixtures 113
Preparation of Urine Sample 118
Discussion of the Separation of Urinary
17-Ketosterolds 118
Study of the Dependence of Relative Retention Time on
Temperature and on the Nature of the Reference Standard 121
Discussion of Factors Affecting Retention Time Values 121
DISCUSSION
Relationship of Chromatographic Behavior to Chemical
Structure 125
Retention Patterns 125
Effect of Structure on Retention Data 128
Theoretical Aspects of Partition Chromatography:
Relationship of Structure to Chromatographic
Mobility 131
The Application of Gas-liquid Chromatography to the
Isolation and Measurement of Urinary 17-Ketosteroids 141
A Method for the Simultaneous Separation of C}g02
and CigOj 17-Ketosterolds and Progesterone
Metabolites 141
Summary 146
BIBLIOGRAPHY 148
BIOGRAPHICAL SKETCH 155
Vi


LIST OF TABLES
Tabla
1 Variation of Retention Tina with Changes In Temperature
and Pressure on 6Z SB-30 Column 43
2 Variation of Separation Factor with Temperature and
Pressure on 3Z SB-30 Column 45
3 Retention Times on 3Z SB-30 Column 46
4 Retention Times and Separation Factors on 3Z SB-30 Column 47
5 Comparison of Separation Factors on SB-30 and Ethylene
glycol Ieophtalate (EGIP) Columns 49
6 Comparison of Retention Times on SB-30 and Bis-(m-phanoxy-
phanyl)-ether Columns 50
7 Retention Times on 3X SB-52 Colum 52
8 Isomeric Retention Times on 3Z Neopentylglycol Adlpete
Column 54
9 Retention Times of 17-Retosterold Acetates on 31
Neopentylglycol Adipate Column 55
10 Retention Times and Separation Factors on 3% XF-1105
Column 57
11 A Comparison of the Relative Retention Times of
Substituted Androstenes 64
12 Comparison of Retention Date of Isomeric Pairs:
Substituent Effects 71
13 Calculated Group Retention Factors
(log r log rn + log k, + log fcj,) 77
14 Comparison of Calculated and Observed Retention Time
Values Relative to Cholestane 83
15 Ratio of Relative Retention Times of Derivatives to
Free Steroids 84
vil


c
T*bU Page
16 The Average Change In Retention Tine Occurring in
Derivative Formation (r^^/r^* x 100 r\Z) 90
17 Values of T at 260 Based on Measureaents Obtained
with 3% SE-30 and 3X XE-60 Columns
(T txB-60 t'gg.30 / t'gg.30) 91
18 Comparison of Selective Retention of the Four Phases
for Ketone and Hydroxyl Groups 92
19 The Change in log r Contribution from Hydroxyl to
Ketone Transitions (LogAr OH-one) 94
20 Isomeric Separation Factors (5a / 5f3) 100
21 Separation of Principal 17-Ketosterolds and Progesterone
Metabolites as Trlmethylsllyl Ethers 114
22 The Effect of the Reference Standard on the Variation
of Relative Retention Time with Temperature on IX
SE-30 Column 122
viii


LIST OF FIGURES
Figure Pag*
I Distribution Isotherms 4
2. A method for calculating number of theoretical plates 11
3 Influence of carrier gas velocity on column efficiency 15
4 Steroid nucleus 19
5 Conformations of cyclohexane 19
6 A-Equatorial bonds. B-Axial bonds. 19
7 Cholestanol 22
I
8 5a-Androstane skeleton 22
9 Gas-liquid chromatogram of a mixture of known steroid
trlaethy1stly1 ethers on 3X XE-60 column 115
10 Gas-liquid chromatogram of a mixture of known steroid
trlmethylsllyl ethers on IX Hl-Bff 8B column 116
11 Gas-liquid chromatogram of a mixture of known steroid
trlmethylsllyl ethers on IX NCSeb column 117
. A < ;
12 Gas-liquid chromatogram of urinary components as
trlmethylsllyl ethers on 3X XE-60 column 119
lx


INTRODUCTION
Statement of Purpose
This study had two main purposes:
ttie investigation of the gas-liquid chromatographic behavior
of a series of isomeric androstaes with the ala of correlating chemi
cal structure to chromatographic mobility.
Through the knowledge and insight gained in the course of the
above study to apply the gas-liquid chromatographic method as an
analytical technique in the Isolation and measurement of C-^ steroids
of importance in man. The ultimate objective was the actual measure
ment of these steroids in biological fluids.
In the first part of the study twenty-eight isomeric androstsnes
were utilised. These were chosen on the basis of their particular
structural relationships. The structures ranged from the unsubstituted
androstane nucleus to the dlsubstituted steroid. The substituent was
an oxygen function in all coses.
For the separation study of biologically significant androstsnes,
two pregnane Isomers were Included which occur together with the C^
steroids in biological fluids.
- 1 -


Chromatography
Historical Background
Tswett la generally given credit for the Invention of the
method of chromatography, although In 1850 a German dye chemist,
P. F. Bunge, had described a separation process which might be
classified as paper chromatography (1). In 1903 Tsvett (2) sepa
rated the pigments of green leaves Into several components on an
adsorbent column of calcium carbonata. Since this separation by
selective adsorption Involved the formation of colored bands on the
column, Tsvett named the process chromatography. The process has
since been extensively applied to colorless materials, thus the term
chromatography Is a misnomer.
The purpose of chrosmtography is the separation of closely
similar substances. Most separations depend upon the distribution of
substances between two phases. One phase Is fixed and may be liquid
or solid and the other phase Is mobile and may be liquid or gas. The
phases must be selected so as to enhance the msximum difference between
the distribution of the desired substance and that of the other materi
als. The particularly distinguishing feature of chromatography Is the
amplification of the distribution process. This amplification Is
accomplished by the Intimate contact of each element of the moving
phase with each element of the very finely divided fixed phase. The
2


process of continuous and successive contacts results in the distri
bution of the solute between the elements of the two phases and the
attainment of equilibrium in a very small length of time.
The two basic types of chromatography are adsorption and
partition and are distinguished by the nature of the process of distri
bution of the solute between the phases. (See Figure 1.) In ad
sorption systems, the distribution is dependent on the concentration
of the substance that is being distributed. At any one temperature,
the adsorption: isotherm, represented as the function of the quantity
of solute in one phase and the total amount in the system is a curve.
The adsorption isotherm is effected by the presence of other substances
in the system. This is probably due to the limited surface area of
the solid phase. In contrast to the adsorption isotherms, the Isotherms
for the partition systems (liquid-gas or liquid-liquid) are linear for
a wide range of concentration of the solute as well as for moderate
concentrations of other substances in the system. This is the major
distinguishing feature of partition system and accounts for their tre
mendous success (3).
With the exception of the work of Bunge, early phases of the
development of chromatography were limited to column chromatography.
In the steroid field both adsorption and partition methods found wide
application.


A B
FIGURE 1
Distribution isotherms:
A Adsorption isotherm.
B Partition isotherm.
4


Two types of adsorption methods were employed by the early
investigators. The first type Involved the use of large columns for
large samples and were primarily preparative in purpose (4,5). The
second type was for separation and estimation of small quantities of
sasiples on small columns. This method found application in the
separation and estimation of urinary 17-ketosteroids (6,7) and of
adrenal steroids and metabolites (8). Alumina, magnesia and silica
gel were used siost frequently for column packing.
The flrat successful partition methods were developed by
Butt, Morris and Morris (9,10) who utilized Cellte columns. Shortly
after the publication of these methods, intensive research was di
rected towards partition chromatography techniques. Hie problem of
the low solubility of steroids in systems based on water as the
stationary phase was overcome by the development of organic systems
and a whole new field of partition chromatography of steroids became
available. Among the pioneers in this field were Morris and his group
(9,10), Zaffaronl and his associates (11), Heftmann(12), Bush (13,14)
and Savard (15) to name a few.
The systems of partition chromatography consisted of three
components: a major component of the mobile phase which was relatively
non-polar, a component of the stationary phase more or less polar in
character to enhance the solubility of the steroid and a third com
ponent of a small amount of water. The water was later omitted from
systems using impregnated paper and from certain column methods. In
- 5 -


reversed-phase partition chromatography systems the mobile phase was
more polar than the stationary phase and the support vas hydrophobic.
The principles of liquid-liquid and gas-liquid partition chroma
tography were described by Martin and Synge in 1941 (16) but the concept
of a gaseous moving phase was not applied until 1952 when James and
Martin published their now classical paper (17). Since that time
extensive research on gas chromatography has been carried out in labo
ratories throughout the world for many types of problems. The great
Interest in various forms of gas chromatography is due to its wide
range of application speed of analysis and the sensitivity of de
tection for micro-quantities of sample.
Gas Chromatographic Methods
Gas chromatography is the process by which a mixture is sepa
rated into its individual components by a gaseous mobile phase. If
the stationary phase is liquid the method is called gas-liquid
chromatography and the separation is due to the differential solubility
of the materials between the liquid and gas. If the stationary phase
is a solid the method is called gas-solid chromatography and the
separation is due to the adsorption of the sample components on the
solid surface.
The two types of gas chromatographic methods are further dis
tinguished by the manner in which the sample moves through the column.
6


Elution, displacement and frontal analyses are three techniques
which nay be used In gas-solid chromatography. The elution
technique Is used almost exclusively In gas-liquid chromatography.
Frontal analysis Is the passage of a continuous flow of
carrier gas containing the sample mixture through a column of
adsorbent. The Idea of using a continuous stream of the sample
Itself as a displacer where each solute attains Its own competitive
adsorption equilibrium was the contribution of Tlsellua (18). Sepa
ration Is achieved only for that component which emerges first from
the column, consequently frontal analysis la of limited use as a
method of separation.
Displacement analysis, which was elaborated by Claesson (19),
Is the method of displacing the sample already on the column by means
of passing a continuous flow of a carrier gas which contains the
vapor of a substance called the displacer. The displacer Is so
chosen that it is more strongly adsorbed onto the column than any
component of the sample mixture. The component displaced by the
displacer in turn displaces another component of the sample that is
less strongly adsorbed. Thus the components of a sample move along
the column at the same rate that the displacer la saturating the
column behind the sample.
Elution chromatography Involves the continuous passage of an
unadsorbed gas or liquid through the chromatographic system. A
volatilized sample la introduced into this stream of gas and la carried
- 7 -


down the column. Components of the sample distribute themselves
between the mobile gaseous phase and the stationary phase. Each
component eventually emerges from the column after the passage of
a characteristic volume of carrier gas. The unique feature of
elution technique, as compared to frontal and dlsplac sent analysis,
Is the mergence of the solute as a peak. The retention time t&,
defined as the length of time between the Introduction of the sample
Into the chromatographic system and the emergence of the peak maxi
mum, is a means of qualitative Identification of the compound. The
peak area can usually be related directly to the concentration of the
Principles of gas Chromatography
A distinguishing feature of gas-chromatography la the utilisation of
high temperatures In order to maintain the solutes In the vapor state.
The vide selection of liquid phases enhances the applicability of gas-
liquid chromatography methods to a vide range of analyses. The tech
nique of gas-liquid chromatography Is similar to liquid-liquid partition
and the gas-llquld column la analogous to a distillation column.
However, there is one major difference between the two systems, which
Is of great practical Importance. In systems of gas-llquld chroma
tography there Is no Interference with separation due to azeotropy.
At the Inlet of a gas-llquld chromatography column there might occur
an Interaction between the components of the sample but over the whole
length of the column, the Interaction of these components with the
8


liquid phase far outweigh any mutual effects (20). Thus each
component is carried through the column independently of any other
component.
A theoretical plate treatment is applicable to both liquid*
liquid and gas*liquid columns. A theoretical plate is defined as
a section of the column in which the vapor leaving the section has
the composition that would be in equilibrium with the average con
centration of liquid solution within that section. The most im
portant assumption underlying the theoretical description of the
chromatographic process is that equilibrium between the two phases
is obtained after each minute movement of the mobile phase.
The separation per plate (separation factor) and the total
number of plates (N) existing in a column determine the over-all
separation achieved. The temperature of the column and the nature
of the stationary phase of the components to be analyzed all determine
the separation factor. This factor is defined as the ratio of the
retention times of two components to each other. Separations can be
improved by the use of a liquid phase with specific characteristics,
such as selective retention of compounds containing double bonds or
ketone groups.
The length of column in which equilibrium occurs is defined
as the height of an effective theoretical plate (16). The number of
theoretical plates exhibited on a column for a specific liquid phase,


solute and temperature can be calculated In a number of ways all of
which are a measure of the degree of peak spread relative to its
residence in the column. The length and diameter of the column,
the diffusion constant of the solute in the two phases, the amount
of stationary phase, uniformity of column packing, the rate of flow,
temperature and the nature of the carrier gas all are variables
affecting the value of N. In general, N is Increased by an Increase
in length of column and a decrease in its diameter.
"he equation commonly used for the calculations of N is
M. ( CR >2
A t
where tR is the retention time measured from the introduction of the
sample and At is the width of peak base. (See Figure 2.) The units
may be time or length provided both tR and 4 t are in the same units.
This equation for N assumes that the component being studied is in
equilibrium with the column. N calculated in this manner is signifi
cant only for symmetrical peaks since the above equation is based on
probability theory (21).
The height equivalent to a theoretical plate is given by the
equation
H L / N
where L is the length of the column, usually in millimeters. The
value of H is generally low for columns operating at high efficiency
and decreases with Increases in retention time. The plate theory
provides a useful means of evaluating column efficiency. The
10 -


Response
FIGURE 2
A method for calculating number of theoretical plates.


relationship of various terms which determine the value of H are
given in the van Deeater equation (22).
S .'jU, + 2Ta + sk- 4
" Vd + k')2 DUq
where \ is a quantity characteristic of the packing, dp Is the
average particle diameter, y Is a correction factor for the
tortuosity of the path of the gas In the interparticle space, D
Is the diffusion coefficient of the solute In the gaseous phase,
u Is the linear gas velocity, k1 is the ratio of the fraction of
sample in the liquid phase to the fraction In the vapor phase, d
is the thickness of the liquid film, and Dj^ is the diffusion co
efficient of the solute in the liquid phase.
In the simplified van Deemter equation
H A + B/u + Cu
A represents the eddy diffusion term, also called the multiple path
effect, B/u term is the molecular diffusion and Cu Is the term that
describes the resistance to mass transfer. The eddy diffusion Is
the result of the Irregular path which the gas follows through the
peeked column. The irregularities In this path contribute to a broaden
ing of the chromatographic band. A reduction in particle size (dp),
uniform packing and uniform particle size tend to decrease the contri
bution to H of this term. The second factor Is the diffusion of the
sample molecules in the carrier gas, which results in band broadening.
The lower the molecular weight of the carrier gas, the greater Is this
- 12


factor. Diffusion increases with Increase in temperature. An
increase in pressure, on the other hand, by increasing the density
of the gas, tends to decrease the diffusion. For this reason,
high temperatures are employed together with high pressures of the
carrier gas.
The term Cu, resistance to mass transfer, Includes several
parameters. The value of k' is dependent on the partition coef
ficient as well as the relative volumes of the liquid and the gaseous
phase in the column. k' is generally greater than one because the
solubility of the solute in the liquid phase is usually greater than
in the gaseous phase. Thus any increase in the solubility of the
sample in the liquid phase will decrease H by a small amount. The d^
term, defined as the effective thickness of the liquid film, is raised
to the second power in the van Deemter equation. Its contribution ,
will therefore be appreciable. A reduction in df results in Increased
column efficiency, which may be counteracted by a simultaneous decrease
in k* if the amount of liquid phase is reduced. The liquid diffusion
coefficient (Diiq) is dependent on the viscosity of the liquid phase.
An Increase in temperature might be expected to reduce the viscosity
but since Increased temperatures affect k' and Dgaa adversely, the net
result is difficult to assess.
The last term in the van Deemter equation is the rate of flow
of the carrier gas. Theoretically eddy diffusion is Independent of
the rate of flow. The contribution to H of molecular diffusion is in
inverse ratio to the rate of flow. Resistance to mass transfer,
- 13


however, varies directly with the velocity of the gas. If all
other variables are kept constant, there will exist an optima rate
of flow for the most efficient operation of a colman as Indicated
In Figure 3. At flow rates less than the optlsum value, the contri
bution of no locular diffusion to H will be appreciable. Above optima
flow rate resistance to ness transfer becomes Important. However,
this part of the curve Is relatively flat and operations above optimum
velocity will not have too deleterious an effect on H. The molecular
diffusion term la large for carrier gases of high dlffuslblllty*
Resistance to mass transfer will be dominant for liquids of high
viscosity and for higfr concentrations of stationary phase. Qualitative
and quantitative predictions of the effect of H of changes In the
parameters of the van Deemter equation are summarised by Patton (23),
Bohemen and Purnell (24) and Purnell (25).
For any packed column there exists a pressure drop between
the Inlet and the outlet ends of the column. An Increase In the
absolute pressure at either end of the column or the use of a tapered
column with a widening diameter towards the outlet end are possible
solutions to minimising the pressure drop. A correction factor for
the pressure drop across the column has been derived by Jams and
Martin (17,26). The retention volume defined as
VR Fc*R
where Fe Is the volumetric flow rate of gas, can he corrected to give
the limiting retention volume which Is Independent of pressure (27).
14


H
Molcula1
diffusion
Resistance to mass
transfer
Eddy diffusion
Gas velocity, cm/sec.
FIGURE 3
Influence of carrier gas velocity on column efficiency.
15


Po / P
where VR Is the Uniting retention volume et zero pressure drop
scross the column, VR is the retention volume, pQ Is the coition
pressure et the outlet end and f Is the everege column pressure.
Various experiments have been devised to test the applies*
blllty of the van Deemter theory and will be considered in the
Discussion section. The theory has been useful In attaining
high column efficiencies.
Basic Gas Chromatographic Apparatus
The apparatus for any gas chromatographic separation consists
of a carrier gas supply, a flow regulator, a pressure gauge, a
recorder, sample port id a plpet for Introducing the sample, a
column, a detector and heating units for the last three items.
Carrier gas is supplied from a compressed source and Its
flow rate through the column Is regulated by a pressure valve. The
gas passes through the detector and proceeds to the sample port. The
sample is Introduced Into the sample port where it Is heated to effect
volatilization. The carrier gas carries the sample vapor Into the
column where the distribution process takes place. The separated
components of the sample In the carrier gas enter the detector and
pass out to atmosphere or to a collection apparatus for further study.
Tesiperature control of the apparatus components is an Important con
sideration In gas chromatography. The minimum detector temperature
16


should be thst of the column to prevent condensation of solutes
which would result in change in concentration. The sample port
tesperatures should be adjustable to permit the development of
temperatures sufficiently high for the rapid vaporization of
solutes.
17


Androstones
Androstaes belong to the brood group of biologlcolly
important organic compounds colled the steroids which possess the
cyclopentanoperhydrophenanthrene nucleus contelning 19 corbon atoms.
(This nomenclature Includes members previously designated as etio-
cholanes.) The nucleus contains 17 corbon atoms which moke up
the condensed system of four rings (Figure 4). The rings are
designated os A, B, C and D and the corbon atoms ore numbered os
Indicated in the above structure. The two angular methyl groups
(designated os carbon atoms 13 and 19) project to the front of the
steroid swlecule. This projection is described as ^-oriented end
is represented by a solid line. Similarly, ony projection to the
rear of the ring system is designated os a-oriented and is represented
by o dotted line*
Conformations of Androstone Ring System
The term conformation describes the different spatial arrange
ments of the atoms in a single classical structure or configuration.
Transformations from one arrangement to another take place by the
simple rotation of a single bond. The best known examples are the
chair and boat forms of the cyclohexane model (Figure 5). Vie differ
ences in energy which are primarily due to differences in the
18 -


18
FIGURE 5
Conformations of cyclohexane: A chair, B boat.
FIGURE 6
A Equatorial bonds. B Axial bonds.


repulsion* between closely approaching atoms among various confor
mations determine the stability of the particular conformation. In
general, it may be stated that even though reactions often proceed
through unstable forms, the unexcited molecules exist in the confor
mation of lowest energy and therefore of highest stability (28).
The bonds of the carbon atoms in a cyclohexane molecule are
of two types (Figure 6):
1. Those more or less In the plane of the ring are
called equatorial.
2. Those perpendicular to the plane of the ring are
called axial. These were formerly designated
as polar bonds.
The stability relationship can be inferred by consideration of
non-bonded H:H interactions ( van der Waals overlap )* Since these
interactions contribute to instability to an extant which Increases
with decreasing interatomic distance, this factor can be used to
estimate the relative stabilities of different conformations (29). In
the boat form the 2,3 and 5,6 groups are closer to each other than in
the chair form. Therefore, the chair is the more stable conformation.
In addition to van der Waals overlap, electron density in or near axial
bonds has been reported to be a factor in sterlc interactions (30). The
interatomic distances between any pair of axial groups are smaller than
those between equatorial groups, therefore, axial groups exhibit great
er repulsion towards each other. Thus, a compound carrying axial
20


substituents is less stable than the corresponding compound with
equatorial substituents, where non-bonded repulsions are at a
minimum.
The generalization that can be made for condensed ring
systems made up of six membered rings such as the steroids is that
the stable structures are chair conformations with as many as
possible of the substituents in the equatorial positions. As a
representative structure of the steroid nucleus, the cholestanol
molecule (Figure 7) may be examined. In this molecule rings B
and C are locked in the chair conformation by trans-fusion to rings
A and D. Ring A is free to assume the boat or chair forms. But
the instability associated with the boat form of the cyclohexane
molecule is augmented by the interaction between the methyl group
on C-10 and the hydroxyl group on C-3* Similarly, in cholestane
the 3p-hydrogen and the C-10 methyl group oppose the boat form (29).
In the androstane molecule (Figure 8) the ring system of
cholestane is present but the side chain at 0-17 is lacking. The
androstane molecule is described as having three six-membered chair
rings and one five-membered ring. The axial hydrogens at position 1,
3, 5, 7, 9 and 14 are in a line parallel to one another. The carbon
pairs 2-3, 5-10 and 7-8 lie in one plane and carbon atoms 1, 9, 11,
13 and 14 lie in a second plane parallel to the first (29). For
mation of ring D produces a slightly puckered ring due to the bending
of the bonds. The hydrogens attached to C-15 and C-16 are eclipsed
21 -




and the equatorial and axial concept is not applicable. The bonds
at 015 and 017 have quasi-axial (a') and quasi-equatorlal (s')
relationships with respect to ring C.
Useful generalisations regarding stability and reactivity
of equatorial and axial substituents have been developed (31-34) and
a few of the pertinent ones say be summarised as follows:
1. A substituent is generally more stable in the
equatorial than in the axial orientation. Thus,
if a substituent can be equilibrated in different
orientations, the equatorial will predominate in
equilibrium mixtures.
2. An equatorial hydroxyl group is more accessible
than the axial and therefore reacts more rapidly
than the axial group in astarlfleetion or hydrolysis
type reactions.
3. In chromatography on paper or on alumina an equa
torial alcohol is more strongly adsorbed than its
axial Isomer.
4. The equatorial or axial orientation of a substituent
is often reflected in Its Infrared absorption spectrum.
23


Iaorna risa In Androstae
Orientation of substituents at specific positions are
suanarized below (35)
Position
1
2
3
4
5
10
6
7
8
9
11
12
13
14
15
16
17
5G-Serles
Q-confia
a
e
B -confia
e
a
a(AB)
a
a
as
(AB)
5a- and 5B-
Series
e
a
a
. *
a
a
e
a
a
e
a
a
m
e'
a<
a*
e 1
5p-Series
q-confia b-confia
(A)e(B)
(A)*(B)
24 -


The spatial orientation of a substituent group Is desig
nated with reference to the angular methyl groups which are
^-oriented. Thus a group projecting in front of the nucleus is
referred to as being els to the methyl groups and a group project
ing to the rear Is trans to them.
The two major classas of Isomeric androstanes are desig
nated as 5a- and 5f?-andrstane and differ in the spatial configu
ration of the hydrogen on C-5. In the 5a-conflguration the rings
A and B trans to each other and In the 5f5-serlea they are els.
Similarly, the 5a-hydrogen is trans to the angular methyl groups
and the 5f3-hydrogen Is els to them. Substitution of any ring
hydrogan further results In the production of stereolsomerlc pairs.
Thus 5a-androstan-3a-ol and 5a-androstan-3p-ol differ only In the
orientation of the hydroxyl group on C-3.
Androstane Isomers Possessing Hormonal Activity
The Isolation and structural elucidation of androstanes which
exhibit hormonal activity posed a problem to the early Investigators.
The amounts of steroid hormones which can be isolated from testis or
adrenal cortex la very small; however, larger amounts of the steroid
hormone or their metabolites are normally found In urine. Functions
characterising the testicular hormone Include development of secondary
sex characteristics. Among the early methods of bloassay of hormonal
activity, the most satisfactory one was the method based on the ability
25


of the hormone to promote comb growth In cepons (36-37). Butenandt
(38,39) using urine as the source of the hormone, was the first to
isolate and identify one of the principal metabolites of the mala
hormone. The Isolated substance exhibited biological activity.
Chemical characterization indicated it to be a sterol-like ketone
and Butenandt named the hormone androsterone. The structure of
androsterone was further elucidated by partial synthesis from
cholesterol by Ruzlcka and his associates (40-42). The synthetic
compound was identical to that Isolated from urine by Butenandt,
both chemically and physiologically, and its structure was shown to
be 3o-hydroxy-5a-androstan-J.7-one. Two other biologically active
isomers of androstan were synthesized and identified shortly there
after. These were 3p -hydroxy-5p-androstan-17-one and 3p-hydroxy-
Sa-androstan-17-one (40-43), both of which have since been Isolated
from human urine (44,45).
Among the C^Oj andr os taes the first to be Isolated was 3a,
l7p-dlhydroxy-5a-androstan-17-one, which was followed by the isolation
of 3a-hydroxy-5a-androstane-ll,17-dlone, 3a-hydroxy-5p-androstane-
11,17-dione and 3a,ll£-dlhydraxy-5p-androstan-17-one (46-48).
Androgenic activity was demonstrated for 3a,17p-dihydroxy-5a-androstan-
17 -one (49).
Human urine also contains steroids having a double bond be
tween carbons 5 and 6. Butendandt and his co-workers (50,51)
Isolated and identified 3p-hydroxyandrost-5-en-17-one.
26


Lieberman and hit group (52) reported the isolation of 5a-
androstane-3,17-dione and 5f-andro8tane-3,17-dione from normal male
and female urines.
During the last decade the complete synthesis of the steroid
nucleus and of almost all of the naturally occurring androgens have
been accomplished (53-56).
Methods of Assay for Androgens and 17-Ketostarolda
Androgens extracted from urine or tissues were first studied
(38,39) by bloassay methods to determine biological activity. By
definition only those androstane Isomers exhibiting biological
activity as measured by comb growth of capons can be called androgens.
Urine has been the major source of steroids to the investigator
and since most of the urinary steroids which exhibit androgenic
activity also contain 17-ketone group, the development of chemical
methods have been directed toward specific reactions of this group.
The procedure most widely used for the determination of 17-ketosteroids
is the colorimetric measurement based on the Zlmnermann reaction (57).
Treatment of a 17-ketone with m-dinitrobenzene in the presence of
alkali gives a transient reaction product of violet color but of un
known structure. The colored compound has an absorption maximum at
520 mfi and is sufficiently stable to permit reproducible readings.
However, the ketone group on carbon 3 can Interfere with the results.
Modifications of the above procedure have been reviewed by Zlmnermann(58).
27


Chemical and Physical Methods of Characterization of Steroids
The chemical properties of a steroid like those of any other
organic compound should be considered as the reactions of the
functional groups of the steroid molecule. It may be assumed that
an Isolated functional group undergoes reactions with little or no
Interference from other groups. In cases where two groups are
sufficiently close to Influence each others properties, their
reactions must be considered together.
The relative reactivities of axial and equatorial substituents
have already been mentioned. In general, the hydroxyl group on the
steroid nucleus will undergo the common reactions known for this
group, i.e., esterification, ether formation, epoxide formation,
replacement by a halogen, etc. These reactions have been utilized
in the isolation and analysis of steroids. In the present investiga-
tlon two such methods employed were esterification in the preparation
of acetyl and trlfluoroacetyl derivatives and ether formation in the
preparation of trlmsthylsilyl ethers of the steroids.
Physical methods employed in the characterization of steroids
include ultraviolet and infrared absorption spectra, rotatory dispersion,
X-ray crystallography and chromatographic analysis.
Ultraviolet absorption was Introduced into the steroid field
in the early 1930's and became extremely popular as a tool for the
detection and determination of trace amounts of compounds with
28


chromophorlc groups. With the accumulation of data, correlation of
structure and ultraviolet absorption afforded a powerful new method
for the elucidation of the structure of a large variety of steroids.
The field has been reviewed by Dorfman (59). More recently Jones
and his co-workers (60) Investigated the infrared absorption charac
teristics of steroids. The elucidation of axial and equatorial
orientation Is In part due to Infrared spectral analysis. The vari
ation of optical activity with the wave length of the light was known
and used by early Investigators until 1860 whan Bunsen developed his
sodium lamp and a monochromatic light source became available. The
application of rotatory dispersion methods to the steroid field was
pioneered by Djerassl and his associates (61,62). The absorption
bands In rotatory dispersion measurements are sensitive to confor
mational changes and have been useful in establishing the validity
of certain structural relationships. X-ray diffraction pattern of
steroids have been useful In supplementing Infrared analysis and melt
ing point determinations (63).
With the advent of chromatographic techniques a valuable tool
was added to steroid analysis. Adsorption chromatography was applied
to the purification and separation of steroids (4). Solvent systems
of different polarities were designed for successful separations by
partition chromatography (9,11,64). Savard (15,65) investigated the
behavior of a wide range of C19 and C21 ketosteroids and established
the applicability of the llgroln-propylene glycol system to the
resolution of these ketosteroids. Certain correlations between
chromatographic mobility and structure began to emerge. The mobility
- 29


of the steroid molecule was observed to decrease with Increasing
number of oxygen functions. The less than expected retardation
in the mobility of a steroid with a C-21 hydroxyl substituent was
interpreted as an Indication of Intramolecular hydrogen bonding. The
general pattern of mobilities observed by Savard was consistent with
the concept developed by Barton that steroids possessing equatorial
hydroxyl groups exhibit lower mobilities than the corresponding
Isomers with an axial hydroxyl groups (31,66). Furthermore, the
correlations between mobility, number and nature of oxygen function,
position and orientation of hydroxyl groups together with the relation*
ship of mobilities between 5a- and 5p-isomers provided generalizations
which were In agreement with the observations of Lleberman and his
group (52,67). From the relationship of chromatographic elution
time to structure, certain clues to the nature of unknown compounds
were obtained (68). Chromatography before and after acetylation
enabled the Investigator to assess the number of acetylable hydroxyl
groups (69,70).
Nomenclature
Steroid nomenclature la based on the rules defined at the 1950
Clba Foundation Conference In London (71,72). These rules have been
provisionally approved by the International Onion of Pure and Applied
Chemistry. Since the nomenclature system Is of recent origin and
since many Investigators still employ common names, a brief summary
of the approved nomenclature will be given.
30 -


According to the new system, the neme of the steroid Is
based on Its hydrocarbon nucleus to which prefixes and suffixes
are added to Indicate the nature of the substituent. The position
of these substituents are indicated by the number of the carbon
atom to which they are attached. The projections of the substi
tuents to the front and to the rear of the hydrocarbon skeleton are
designated as p and a, respectively.
The parent compounds are androstane, pregnane, cholestane,
etc,, and with each name, the configuration at 05 Is Indicated by
the prefix 5a or 5{3. Unsaturation Is Indicated by the suffix 'ene1,
e.g. 5-ene. The rules for the substituents state that only one
kind of substituent in each compound Is Indicated by a suffix, the
remaining substituents are indicated by prefixes. The substituent
to be designated by a suffix Is chosen according to the following
order of priority: carboxylic acid or derivative Including esters,
carbonyl, alcohol, amine, ether, halogen.
31


EXPERIMENTAL
Materials and Methods
Compounds studied
Steroids Investigated In the course of this study were
chosen for their particular structural features and biological
importance. All except two were androstane isomers, the
remaining two were progesterone metabolites. The isomeric
androstaes employed were:
Sa-Serias. 5a-andros tane, 5a-androstan-3p-ol, 5a-
endros tan-17f3 ol, 5a-androstan-3-one, 5a-androstan-17-one,
5a-androstane-3a,17p-dlol, 5a-androstane-3p,17p-diol, 3a-hydroxy-
5a-androstan-17-one, 3a-hydroxy-5a-androstan-17-one, 17f-hydroxy-
5a-androstan-3-one, 17a-hydroxy-5a-androstan-3-one, 5a-androstane-
3,17-dione, 3a, 1 lp-dihydroxy-5a-andros tan-17-one, 3a-hydroxy-5a-
endrostene-ll,17-dlone.
32


56Serle. The 50-isouers of ell the compound listed
above were studied simultaneously with the 5a-cowpound. The
nomenclature Is exactly the same as stated above except that the
50-d esIgnatIon will be substituted for 5a In each Instance.
The only unsaturated androstane isomer investigated was
30-hydroxyandrost-5-en-l7-one, which was included because of its
biological importance.
The two pregnane isomers were 5j3-pregnane-3ac,20a-diol end
3a-hydroxy-5f -pregnen-20-one.
In the section of this work involving the separation of
17-ketosteroids, reference will be made to compounds for which
common names are still widely used in the literature especially in
the area of biological applications. The cosnon and the approved
names of these compounds are summarised below.
Androsterone
Ktlocholanolone
Dehydroeplandrosterone
11-Ketoetlocholaolone
110 -Hydroxyandrosterone
Pregnanedlol
Pregnenolone
3a-Hydroxy-5a-androstan-17-one
3a-Hydroxy-50-androstan-17-one
30-Hydroxyandrost-5-en-17-one
3a-Hydroxy-50-androetane-ll, 17-dlone
3a, ll0-Dihydroxy-5a-endrostan-17-one
50-Pregnane-3a,2fla-dlol
3a-Hydroxy-5f -pregnan-20-one
33 -


In the tables suamarlzlng the resulte obtained In this
study, an abbreviated form of naming is used and since one purpose
of this investigation was the comparison of 5a/5p isomeric pairs,
the orientation at 05 Is written as the prefix in these abbreviated
forms. For example, 3a-hydroxy-5a-androstan-17-one Is abbreviated
as 5a-A-3a-ol,17-one and 3p-hydroxy-5p-androstan-17-ons Is abbrevi
ated es 5p-A-3p-ol,17-one.
The steroids were obtained from commercial sources and were
of sufficiently high purity for direct use. Some, however, showed
minute amounts of Impurities when examined by gas chromatography.
Three of the 17-katosterolds (3a-hydroxy-5a-androstan-17-one, 3a-
hydroxy-5p-androstan-17-one and 3p-hydroxyandrost-5-en-17-one)
when analysed by paper chromatography exhibited no Impurities.
Preparation of PerIvetIves
The derivatives of the steroids were prepared by the following
procedures.
Acetates. The steroid (2-5 mg) was allowed to react with
acetic anhydride (0.5 ml) and pyridine (0.2 ml) at 67 In a dry bath
for two hours. The solvents were evaporated under nitrogen and
the steroid acetate was dissolved In acetone.
34


Trlfluoroacetates (TFA). The steroid (2-5 mg) vas
allowed to react with trlfluoroaeetlc anhydride (0.5 ml) and
pyridine (0.2 1). The reaction was cooplets in a few minutes
at room temperature (73). The solvents were evaporated under
nitrogen and the derivative was dissolved In acetone.
Trlmethylsllyl ethers (TMS1). The steroid (1-3 mg)
was allowed to react with hexamethyldlsllazane (0.5 ml) and a
few drops of triaethylchlorosllane as catalyst for 6-8 hours at
room temperature (74). The reaction mixture was centrifuged and
the supernatant solvents were evaporated under nitrogen. The
residue was triturated with hexane and recentrifuged. The super
natant was again evaporated under nitrogen and the trlmethylsllyl
ether of the steroid was dissolved In tetrahydrofuran (75).
Studies on the Free Steroids and their Derivatives
The free steroids, acetates and trlfluoroacetates were chro
matographed from acetone solutions, the trlmethylsllyl ethers were
chromatographed from tetrahydrofuran solutions. All concentrations
were 2 pg/pl. Volumes of 0.2 to 1 ul were used for Injections. All
measurements were mede in duplicate.
35


One sample of each derivative was analyzed by infrared
spectroscopy to ascertain the substitution of the desired group
on the steroid nucleus. Some of the derivatives shoved small
amounts of the free steroid, vhich were easily identified by their
characteristic peaks on the chromatograms.
Reference Standard
Cholestane was used as the reference standard for dally
measurements. A saturated solution of cholestane in acetone was
Injected into the column each day before any series of runs were
carried out and was repeated every two or three hours. The retention
times of the samples were then related to the retention time of
cholestane and were expressed as relative retention times.
Solvents and Reagents
Spectral grade acetone (Fisher Scientific Co.) and reagent
grade tetrahydrofuran (Eastman Organic Chemicals), chloroform,
pyridine, hexane and acetic anhydride (all from Fisher Scientific
Co.) were employed. Pyridine and acetic anhydride were freshly
distilled before use. Hexamethyldlsilazane, trimethylchlorosliana
and trlfluoroacetlc anhydride were obtained K + K Laboratories and
were used without further purification.
- 36 -


Column Supports
Causaretally obtained acid-washed ( and in the case of the
last two, siliconized) diatomaceous earths were used as coluan
supports. These were:
(Applied Science Laboratories,State College,Pa.)
(Burrell Corp., Pittsburg, Pa.)
(Analabs, Hamden, Connecticut)
(F + M Scientific Corp., Avondale,Pa.)
Chromosorb W
Kromat CE
Anakrom ABS
Diataport S
Different mesh sizes employed are indicated under individual
coluan preparations.
Stationary Phase
The following commercially obtained polymers were utilised
as the stationary phases:
Silicone Rubber Gums:
SE-30 methyl silicone polymer
SE-52 methyl phenyl silicone polymer
Silicone Fluids:
XF-1150 nitrile silicone polymer
(50 mole per cent cyanoethyl)
XF-1105 nitrile silicone polymer
(5 mole per cent cyanoethyl)
The above polymers ware obtained from General Electric
Company, Waterford, New York.
37


Silicon* Copolymer
XE-60 silicone polymer made up of 501 dlmethylslloxane
end 5OX cyanoethylmethyl siloxane. This was obtained
from F + H Scientific Corp., Avondale, Penns lyvania
Fluorlnated Silicone Polymer
QF-1-0065 fluoroalkyl silicone polymer.
Silicone Crease
Do Corning high vacuum silicone grease (DC-silieone)
(ethyl ecetate extract). The above two mere obtained
from Dow Corning Corp., Midland, Michigan.
Polyesters
Ethyleneglycol Isophtlete (ECXP)
Ethyleneglycol succinate (E6S)
Neopentylglycol edlpate (NGAd)
Neopentylglyco1 sebacate (NGSeb)
These were obtained from Analabs, Hamden, Connecticut.
Hi*Eff 8B Dlmethylcyc lohexylsuccinate
This was obtained from Applied Science Laboratories,
State College, Pennsylvania.
38


Bla -fe-phenoxypheny1)-ether
( C6H50C6H4)20
This compound was obtained from Eastman Organic
Chemicals.
Preparation of Columns
The desired amount of liquid phase was weighed and dissolved
In a small amount of chloroform (for silicone polymers) or acetone
(for all other phases). The solution was transferred, with rinsings,
to a beaker containing the weighed support suspended in the same
solvent as used for dissolving the liquid polymer. The mixture was
heated gently with constant stirring until the solvent had evapo
rated completely and a free flowing uniformly coated support was
obtained.
This method of deposition of the liquid phase onto the support
was reliable and reproducible and was preferable to the filtration
method (76)* In the latter method, a slurry of liquid phase, solvent
and support was filtered and then dried with stirring* The concen
tration of the liquid phase deposited on the support was thus de
pendent on the mesh size of the support as well as on the volume of
solvent used.
39


The coated support was packed, with gentle tapping, Into
a copper or stainless steel column one end of which was plugged
with glass wool. The open end of the column was flared for ease
of packing. A vibrator as an aid In packing was found to be un
desirable as It resulted In fragmentation of the support and also
produced tightly packed columns. The open end of the column was
plugged with glass wool and the column was bent Into a U shape,
taking care not to constrict the diameter of the tube at the curve.
The column was then suspended in the gas chromatograph and cured for
24-48 hours at the desired temperature under 10 psl of pressure of
carrier gas. During the curing process the outlet end of the
column was not connected In order to prevent the possible accumu
lation of volatile substances In the detector. After the column
was cured, connections to the detector were made and the column was
saturated with a 1-3 pg quantity of the reference standard cholestana.
The so called 'priming' or saturation process was necessary on those
columns where a certain amount of sample bss, attributable to irre
versible adsorption was observed. This phenomenon varied from
column to column and was dependent on the nature of the sample. For
example, on the XB-60 column, there was no observable loss of androstanes
while estrogens were adsorbed irreversibly.
Equipment
Measurements were made on two gas chromatographic instruments,
Model 600 from Research Specialties Company, Richmond, California, one
equipped with a ^-Ionization detector (77) and the other with a flame
40


ionization detector (78). Slightly higher sensitivities were
observed with the flame ionization unit. It has been reported
(79) that multiple hydroxyl groups tend to depress the sensitivi
ty of the p-ionization detector.
Both instruments were equipped with an injection system
designed for Tenney and Harris type microdipper injectors (80).
In the author's experience this type of injector was more convenient
to use and gave more reproducible results than syringe type injectors.
The column temperature was controlled by a proportional
controller. A fan inside the heating cabinet was employed to
insure uniform distribution of heat throughout the column.
The carrier gases used were argon for the p-ionization
detector unit and nitrogen for the flame unit. In the latter unit,
compressed air and hydrogen were employed for the flame.
- 41 -


Preliminary Experiments
Results of preliminary experiments will be reported briefly
and only for a few representative cases. All measurements were
made on the chromatograph equipped with the p-lonizatlon detector
unless indicated otherwise. Columns were packed and cured as de
scribed. Retention times (tR) are reported in minutes. All inject
ions were made with a 2 pi aliquots of 1 pg / pi solutions. The
p-lonization detector was operated at 1500 volts. Detector temper
ature 245-250. Vaporizer temperature 290-310.
SE-30 Methyl Silicone Polymer (SE-30)
Three columns differing in concentration of SB-30, mesh size
of support or the length or diameter of the column were employed.
1* A column of 67, SE-30 coated on 30-60 mesh Chromosorb W
in a 5-foot 3/16" (o.d.) copper tube was prepared.
Variations of retention time with temperature of the
column and pressure of the carrier gas are given in
Table 1.
2. A column of 37 SE-30 coated on 30-60 mesh Chromosorb W
in a 5-foot 3/16" (o.d.) copper tube was prepared.
- 42 -


TABLE 1
Variation of Retention Time with Changes in Temperature and Pressure on 67, SE-30 Column
Conditions Steroids
Temp.C
Pres, in psi
5a-Androstan.-
¡
3B-ol,17-one
3a-ol,17-one
3-one, 17|3ol
3,17-dione
5|3-A-3a-ol-17-one
192
11.0
18.6
16.8
17.8
j
18.5
16.1
210
10.5
10.8
9.8
-
-
210
11.0
10.0
9.2
-
-
210
11.5
9.4
8.9
-
-
210
13.0
8.2
7.7
-
-
f
210
14.0
8.0
7.4
7.6
8.1
7.0

224
15.0
3.8
3.7
3.8
4.0 |
3.4
In minutes.


Separation factors for androatane ismera under
different conditions of temperature and pressure
were measured (Table 2). Acetate derivatives of
the steroids were prepared with the aim of improv
ing separation factors. Comparative retention
time values of free steroids and steroid acetates
are given In Table 3. Column conditions:
T 195, P 18 psl.
3. A column of 3% SB-30 coated on 50-80 mesh Kromat CE
In a 5.5-foot 1/4" (o.d.) copper tube was prepared.
Retention values and separation factors of three
17-keto8terolds and the respective acetates are
given In Table 4. Column conditions: T 200,
P 20 psl.
Individual components could not be resolved when a mixture
containing the three principal urinary 17-ketosterolds was chromato
graphed. Acetylation of the steroids did not result in improved
separation. (See sections 2 and 3 above.)
Mixed Phases Containing SE-30
1. SE-30 and DC-si11cone
A column coated with 3% SB-30 and 10% DC-si11cone was found
unsatisfactory. Results were not reproducible. The column
was discarded.
44


TABLE 2
Variation of Separation Factor with Temperature
and Pressure on 3% SE-30 Column
Steroids
Conditions
224, 15 psi
210, 14 psi
192, 11 psi
5p-A-3a-ol,17-one
1.00
1.00
1.00
5a-A-3a-ol, 17-one
1.08
1.06
1.04
5a-A-3|3-ol, 17-one
1.11
1.14
1.16
5a-A-3-one,17p-ol
1.11
1.09
1.11
5a-A-3,17-dione
1.18
1.16
1.16
45


TABLE 3
a
Retention Times on 3% SE-30 Column
Steroids
Free
Acetate
5p-A-3a-ol,17-one
8.9
-
5a-A-3a-ol,17-one
9.2
12.5
5a-A-3-one,17(3-ol
9.8
15.9
5f3-A-3-one,17|3-ol
10.2
14.0
5a-A-3p-ol,17-one
10.1
-
5C-A-3,17 -dione
10.4
-
5a-A-3a,17(3-dio 1
10.6
17.5
In minutes.
- 46 -
\


TABLE 4
a
Retention Times and Separation Factors on 37o SE-30 Column
Steroids Free Acetate
tR
Sep.fac.
CR
Sep.fac.
5p-A-3a-ol,17-one
6.3
1.00
20.5
1.00
5a-A-3a-ol,17-one
6.5
1.03
20.3
0.99
A-5-en-3(3-ol, 17-one
7.1
1.14
21.2
1.03
In minutes.
47 -


2. SE-30 and Ethyleneglycol Isopti tlete (EGIP)
Three coluana vera prepared
a. 1.5X SE-30 + 0.5X EGIP
b. 0.5X EGIP
e. 1.5X SE-30
Comparative results obtained on the three columns
are given In Table 5, where the Individual contri
butions of each phase la visible, e.g., 2a vs. 2b.
3. SE-30 and Bis-(a-phenoxypheny1)-ether Mixed Phase
Three columns were prepared:
a. 3X Bis-(m-phenoxyphany1)-ether (Abbreviated
as phenoxy In Table 6.)
b. 3X Bis-(m-phenoxypheny1)-ether + 3X SE-30 (1:1 ratio)
c* 3X Bis-(m-phenoxypheny1)-ether + 3X SE-30 (1:2 ratio)
Comparison of the retention times are given In Table 6*
Column conditions: T 245, P 30 psi
The dependence of the chromatographic pattern of the steroids
on the concentration of each component of the stationary phase
was noteworthy (3b and 3c above). Separations could not be
affected due to large peak areas and some skewing of the peaks.
After a short period of use, Irregular bizarre patterns Indica
tive of phase decomposition were observed on this column and
further work was not continued.
48


TABLE 5
Comparison of Separation Factors on SE-30 and
Ethyleneglycol Isophtalate (EGIP) Columns
1.5% SE-30 +
FREE
0.57 EGIP
0.5% EGIP
1.5% SE-30
5(3-A-3a-ol, 17-one
1.03
1.00
0.70
5a-A-3a-ol,17-onea
1.00
1.00
1.00
5p-A-3-one,17f3-ol
1.33
1.12
1.30
5a-A-3-one, 17f3-ol
1.18
1.12
0.87
5a-A-3p-o1,17-one
1.17
1.03
0.83
A-5-en-3p-ol,17-one
1.08
-
0.87
5a-A-3,17-dione
1.36
1.32
0.80
5a-A-3a,17p-diol
1.10
1.10
0.87
ACETATES
5f3-A-3a-ol, 17-one
1.06
1.17
1.25
a
5a-A-3a-ol, 17-one
1.00
1.00
1.00
5p-A-3-one,17(3-ol
1.34
1.75
1.25
5a-A-3-one,17p-o1
1.57
2.00
1.05
A-5-en-3(3-ol,17-one
1.43
-
1.40
5a-A-3a,17p-diol
1.20
0.96
1.20
a
The values of these compounds were
for the calculation of separation
the acetates, respectively.
used as the point
factors of the free
of reference
steroids and
- 49 -


TABLE 6
a
Comparison of Retention Times on SE-30 and
Bis-(m-phenoxyphenyl)-ether Columns
Steroid Acetates
37o Phenoxy
37
SE-30 and
37o Phenoxy
ratio)
(2:
1 ratio)
R
Sep.fac.
CR
Sep.fac.
Sep.fac.
5a-A-3a-ol,17-one
17.6
4.4
21.5
1.36
12.5
0.54
5(3-A-3a-ol, 17-one
12.0
3.0
23.0
1.45
15.8
0.69
A-5-en-3(3-ol, 17-one
8.0
2.0
22.5
1.43
14.0
0.61
Cholestane
4.0
1.0
15.8
1.00
23.0
1.00
a
In minutes.
S

- 50 -


SE-52 Methyl Phenyl Silicone Polymer (SB-52)
A column of 3% SE-52 coated on 100-110 mesh Anakrom ABS In a
4-foot 3/16" (o.d.) copper tube was prepared. Retention time values
are given In Table 7. Column conditions: T 212, p 25 psl
and flow rate 2 ml/mln.
* #
There was no observable advantage to be gained by the use of
SE-52 polymer as compared to SS-30 phase. The fine mesh support
caused a considerable decrease In flow rate and It was decided to per
form subsequent measurements on coarser than 100 mesh supports*
. i. K i i ,i
Dow Corning High Vacuum Silicone grease
' \ ' 1 '* / '* .
A column of 30% DC-silieone coated on 40-50 mesh Anakrom ABS
In a 5-foot 3/16" (o.d.) copper tube was prepared. Column was too
retentive even after It was cut down to a length of 3 feet.
Bthyleneglycol Succinate Polyester (EGS)
A column of 12% EGS coated on 30-60 mesh Chromosorb W In 2.5-
foot 3/16" (o.d.) copper column was prepared. Column was very
retentive; no peaks were observed.
Neopentylglycol Adipate Polyester (NGAd)
A column of 3% NGAd coated on 30-60 mesh Chromosorb W in a
4-foot, 3/16" (o.d.) copper tube was prepared. Hie column required
51


TABLE 7
Retention Times on 3% SE-52 Column
Steroid
fcR
Rel. t.
Cholestane
14.6
1.00
5|3-A-17-one
2.9
0.20
5p-A-3-one,17p-ol
6.7
0.46
5(3-A-3a, 17(3-diol
6.2
0.42
5a-A-3a, 17f3-diol
6.5
0.44
5a-A-3p, 17p-diol
6.7
0.46
5f3-A-3,17-dione
6.7
0.46
In minutes.
52


daily priming with eholeataae. laonerlc rttwtiov tinea arc glwaa
t* Tabla 8. Colma condition* T 220*, P JO pci, flow rata <
100 ml/aia.
Iha rctentioa of aolutes cm the 4-foot BOM colon was wary
taw. Therefore, a colon of ft WBAd coated am 30*60 meah Chrenoaorb V
la a 6foot 3/16" (o.d.) copper tuba waa prepared. The retention data
on three 17-katoetaroid acetates are reported la Table 9. Colana
conditions: T 230*, t 20 pal, flew rate 54 nl/ala.
A colean of ft TOSeh coated on 30*40 mash Chroaoaorb W in a
4-foot 1/U" (o.d.) copper tube waa prepared, iha remita appeared
aatlafactory bet the colum waa subjected te high temperaturca overnight
by e breakdown la the traperature control unit. It uae discarded.
A colum of 31 XF-1150 coated on 30-40 much Promt Cl in
4-foot 3/16 (o.d.) copper tuba was prepared. The flow rate wea
extremely clow eed leca ef phase wee detected. The eelum wee
discarded.
-53-


TABLE 8
a
Isomeric Retention Times
on 3% Neopentylglycol Adipate
l
Column
Steroid
tT?
Rel.t,,
j^p-A-3,17 -dione
5.8
3.06
v5a-A-3,17-dione
6.5
3.32
^5o:-A-3p, 17p-diol
7.7
4.05
l 5CC-A-3Q:, 17p-diol
6.7
3.53
( 5a-A-3a,17(3-diol
J
6.7
3.53
C 5p-A-3a,17p-diol
6.7
3.53
pa-A-3a-ol, 17-one
5.4
2.84
^5p-A-3a:-ol, 17-one
5.5
2.89
' 5a-A-3a-ol, 17-one
5.4
2.84
^ 5CC-A-3(3-ol, 17-one
6.1
3.21
rA-5-en-3p-ol,17-one
<
6.4
3.37
v5a-A-3p-ol,17-one
6.1
3.21
r5p-A-3,17-dione
/
5.8
3.06
5p-A-3-one, 17f3-ol
6.7
3.53
'5p-A-3-one,17(3-ol
6.7
3.53
^5p-A-3a-ol,17p-diol
6.7
3.53
^5a-A-17-ol
1.0
0.53
\
VCholestane
1.9
1.00
In minutes.
54 -


TABLE 9
Retention Times of 17-Ketosteroid Acetates on
3% Neopentylglycol Adipate Column
Steroid Acetates^Rel.tp
5a-A-3a-ol, 17-one
23.5
1.6
5(3-A-3a-ol,17-one
25.0
1.7
* A-5-en-3(3-ol, 17-one
27.5
1.8
Cholestane
15.0
1.0
a
In minutes.
55


A column of 37, XF-1105 coated on 50-60 mesh Kromat CE in a
6-foot 3/16" (o.d.) copper tube was prepared. Retention data and
separation factors are given in Table 10. Column conditions:
T 250, P 35 psi, flow rate 20 ml/min. The flow rate was
maximal.
Even though the separation factors indicated theoretical
separations, actual resolutions of the components of the mixture were
not accomplished. The wide peak area, presence of some skewing and
the high retentivlty of the column in spite of the high temperature
of operation were major drawbacks.
OF-1-0065 Fluoroaklyl Polymer (OF-1-0065)
A column of 3% QF-1-0065 coated on 70-80 mesh Anakrom in a
6-foot 1/8" (o.d.) stainless steel tube was prepared. Measurements
were made on the chromatograph equipped with the flame ionisation
detector. Column conditions: T 238, P 14 psi. It was
hoped that selective retention of this column for ketones could be
used to advantage in effecting separations. Loss of phase and
Irregular chromatographic patterns were observed and in spite of
numerous efforts, no reliable data could be obtained. One interpre
tation of the data was that the phase had reacted with the glass wool
plug.
- 56


TABLE 10
a
Retention Times and Separation Factors on 3% XF-1105 Column
Steroid
fcR
Rel.
Sep.fac.
5f3-A-3a-ol, 17-one
13.8
0.63
1.00
5a-A-3a-ol,17-one
16.8
0.76
1.22
A-5-en-3p-ol,17-one
24.0
-
1.09
1.74
5|3-A-3a-ol, 11,17-dione
25.0
1.14
1.81
5a-A-3a,11£-dio1,17-one
37.8
1.72
0.74
Cholestane
22.0
1.00
1.59
a
In minutes.
- 57


Discussion of Preliminary Kxperiaents
The choice of the proper solvent for the stationery phase vas
the first consideration for this study. The extended ring system of
the steroid molecule provides a fairly wide area of hydrocarbon
skeleton for interactions with solvent molecules. Because of the
relative rigidity of the skeleton end the short range effectiveness
of the ran der Weals forces of attraction betvaan the solute and the
solvent molecules, these interactions are sensitive to sterlc factors
(3). The interactions between the parts of the solute end the
solvent molecules conpete with Interactions between the solvent mole
cules themselves. The solution of a polar substance in a polar
solvent may therefore be considered to depend upon the ability of the
polar groups of the solute to attract parts of the solvent molecule
with a force comparable to or greater than that among the solvent
molecules themselves. A non-polar solute, which cannot exert such a
force of attraction is squeezed out of the polar solvent end the normal
attractive forces in the pure solvent ere re-established. Non-polar
solvents, on the other hand, exhibit far less short range attractive
forces than do polar solvents. The molecules of non-pplar solvents
are attracted to one another by relatively weak dispersion forces
and a non-polar solute may exhibit the same weak attractions as do
the solvent molecules themselves. A polar solute Introduced into a
non-polar solvent is expected to have very little or no interaction
with the solvent.
58 ~


In highly unsaturated molecules, stronger Interactions nay
occur due to delocalisation of electrons and result In an Increase
In the dispersion forces (81). The theoretical basis of such Inter
actions Is still Incompleta, but empirically It Is known that un
saturated molecules tend to attract one another more strongly than
they attract saturated molecules. Similarly, unsaturated groups
attract the molecules of polar substances much more strongly than
do saturated groups. Unsaturated groups may thus be assumed to be
po larisable.
In the present investigation the liquid solvent phases selected,
In addition to the chemical property considerations mentioned above,
had to meet as many of the following requirements as possible at the
temperature of operations! low volatility, thermal stability, low
viscosity and chemical Inertness. For temperatures above 200, as
employed in this study, the choice of phases was limited. Satisfactory
phases at these higher temperatures Included silicone gums and poly-
t . ) 1
esters of low volatility.
Since oxygen substituted steroids of relatively high polarity
were to be studied simultaneously with their derivatives of higher or
lower polarity two basic types of liquid phases were Indicated. A
polar stationary phase would enhance the Interaction of the polar groups
of the steroid molecule with the phase and a non-polar phase would be
expected preferrentlally to attract the non-polar functions.
59


SB-30 polymer was found to be a satisfactory non-selectiva
phase. Bis-(m-phenoxyphenyl)-ether was interesting in the unique
retention pattern of the 17-fcetosteroid acetates on this stationary
phase.(Table 6). However the compound was not stable at elevated
temperatures. The polyester phases exhibited the desired polarity
but the choice was narrowed to those which were thermostable. The
separation factors of the androstane Isomers on XF-1105 phase was
promising, but here again a phase combining the selective properties
of the silicone nitrile fluids and the thermel stability of silicone
polymers was indicated.
After preliminary investigations with different silicone
elastomers, silicone nitrile polymers and various polyester phases,
both singly and in mixtures, four stationary phases which differed in
degrees of polarity and selectivity were chosen: the methyl substituted
silicone gum SK-30 (non-selective, heat stable), the silicone nitrile
polymer XB-60 (similar to silicone nitrile fluids but more stable at
elevated temperatures) and neopentylglyeol sebacate and Hi-gff 8B
(heat stable, polar) polyester phases.
The effect of varying the chemical composition of the stationary
phase for the separation of a wide range of steroids had been reported
(82-86). Lipsky and Landowne (83) had reported the thermal stability
and polar properties of neopentylglyeol sebacate* SB-30 silicone
polymer had found wide use as stationary phase since the early appli
cations of the gas-liquid chromatographic methods to steroid analyses.
60


XE-60 and Hi-Eff 8B were two new polymer* developed specifically for
high tamperature uses.
- 61 -


Comparative Exper traen te
All of the androataes and the derivatives listed under
Materials and Methods were utilised In this portion of the study. The
concentration of the solutions were 2 jug/pi and 0.2 to 1 jul aliquots
were Injected. The detector and vaporiser temperatures were the same
as those given In the preliminary experiments. The gas chromatograph
equipped with the p-ionlration detector (operated at 1500 volts) was
employed unless Indicated otherwise.
Following columns were used:
SB-30. A column of 3X SE-30 coated on 40-50 mesh Anakrom ABS
In a 5-foot 3/16" (o.d.) copper tube was prepared. The chromatograph
equipped with the flame Ionisation detector was employed. Column
conditions : T 260, F 10 psl* Retention time for cholestane *
12-14 minutes.
XK-60. A column of 3X XE-60 coated on 80-100 mesh Dlatoport S
In a 6-foot 1/8" (o.d.) stainless steel tube was prepared. The chro
matograph equipped with the flame Ionisation detector was employed.
Column conditions: T 260-262, P m 14 psl. Retention time of
cholestane 6-9 minutes.
62


Hl-Bff 8B. A column of 3X Hi-Eff 8B coatad on 40-50 mash
Anakrom ABS in a 5-foot 3/16" (o.d.) copper tuba was prepared.
Column conditions: T 238, P 15 psi and flow rata 39 ml.
Retention time for choleatane 6-8 minutes.
A column of IX Hi-Eff 8B coated on 80-100 mesh Diataport S
in a 6-foot 1/8" (o.d.) stainless steel tube was prepared. Column
conditions: T 242, P 33 psi and flow rate 24 ml/min.
Retention time for cbolestane 5-7 minutes.
NGSeb. A column of 3X NGSeb on 30-60 mesh Chromosorb V in a
6-foot 1/8" (o.d.) stainless steel tube ms prepared. Coltasn
conditions: T 232, P 25 psi and flow rate 74 ml/mln.
Retention time for choleatane 8-9 minutes for the measurements
of the free steroids and the steroid acetates and 11 minutes for all
other compounds.
A column of IX NGSeb coated on 80-90 mesh Anakrom ABS in a
6-foot 1/8" (o.d.) stainless steel tube was prepared. Column conditions:
T 231, P 30 pal and flow rate 24 ml/mln. Retention time for
cholestane 10 minutes.
The relative retention time values with respect to cholestane
observed on the six columns under the conditions described above are
susmarlzed in Table 11.
63 -


TABLE 11 SE-30 3%*
A Comparison of the Relative Retention Times of Substituted Androstanes
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.21
-
-
-
5|3-Androstane
0.20
mm
m
--
5a-A-17p-ol
0.32
0.39
0.33
-
5p-A-17p-ol
0.41
0.52
0.49
-
5a-A-17-one
0.32
-
-
-
5p-A-17-one
0.42
-
-
5a-A-3p-ol
0.31
0.39
0.34
0.24
5p-A-3p-ol
0.29
0.35
0.31
0.22
5a-A-3-one
0.33
-
-
5(3-A-3-one
0.32
-
-
-
5a-A-3a,17|3-diol
0.54
0.89
0.58
0.28
5(3-A-3a,17p-diol
0.51
0.87
0.61
0.32
5a-A-3p,17p-diol
0.59
0.98
0.69
0.36
5p-A-3p,17(3-diol
0.49
%
0.86
0.56
-
5a-A-3a-ol,17-one
0.51
0.58
0.47
-
5p-A-3a-ol,17-one
0.47
0.56
0.46
0.28
5a-A-3p-ol,17-one
0.55
0.66
0.55
0.30
5p-A-3p-ol,17-one

-
-
-
5a-A-3-one,17p-ol
0.59
0.76
0.64
0.35
5p-A-3-one,17f3-ol
0.54
0.69
0.59
0.33
5a-A-3-one,17a-ol
0.56
0.75
0.60
-
5(3-A-3-one,17a-ol
0.53
0.61
0.47
0.33
5a-A-3,17-dione
0.58
-
-
-
5p-A-3,17-dione
0.50
a.
-
-
A-5-en-3p-ol,17-one
0.61
0.64
0.55
0.33


TABLE 11 XE-60 3%b
(cont.)
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.27
-
-
-
5(3-Andr stane
0.25
-
-
-
5a-A-17p-ol
0.60
0.71
0.38
-
5(3-A-17(3-ol
0.89
1.04
0.52
5a-A-17-one
0.65
-
-
5{3-A-17-one
0.95
-
-
-
5a-A-3p-ol
0.65
0.70
0.39
-
5p-A-3p-ol
0.53
0.59
0.32
-
5a-A-3-one
0.77
-
am
-
5¡3-A-3-one
0.68
-
-
-
5a-A-3a, 17(3-diol
1.67
2.05
0.49
-
5(3-A-3a,17p-diol
1.68
1.95
0.50
-
5a-A-3(3,17(3-diol
1.89
2.21
0.66
-
5(3-A-3(3,17(3-diol
1.56
1.90
0.51
-
5a-A-3a-ol,17-one
2.00
2.14
0.87
-
5p-A-3a-ol,17-one
1.90
2.15
0.94
-
5a-A-3(3-ol,17-one
2.10
2.44
1.16
-
5(3-A-3(3-ol,17-one
1.98
2.98
0.86
-
5a-A-3-one,17(3-ol
2.58
2.81
1.32
-
5(3-A-3-one,17(3-ol
%
2.44
2.53
1.18
--
5a-A-3-one,17a-ol
2.03
2.32
1.16
5p-A-3-one,17a-ol
2.14
2.14
0.91
-
5a-A-3,17-dione
2.64
-

-
5(3-A-3,17 -dione
2.48
-
m
mm
A-5-en-3(3-ol,17-one
2.01
- 65 -
2.38
1.10
*


TABLE 11 Hi-Eff 3%C
(cont.)
Steroids Free Acetate TMSi.XFA
5a-Androstane
0.13
-
-
-
5(3-Androstane
0.12
-
m
-
5a-A-17(3ol
0.71
0.58
0.22
0.16
5p-A-17p-ol
1.48
1.27
-
-
5a-A-17-one
0.61
-
-
-
5(3-A-17-one
1.15
-
-
-
5a-A-3p-ol
0.75
0.71
0.24
0.20
5p-A-3p-ol
0.61
0.57
-
0.15
5a-A-3-one
0.73
m
-
-
5(3-A-3-one
0.67
-
-
-
5a-A-3a,17(3-diol
3.94
2.48
0.28
0.27
5p-A-3a,17(3-diol
3.85
2.82
0.37
0.30
5a-A-3(3,17|3-diol
4.19
3.18
0.42
0.24
5(3-A-3(3, 17(3-diol
3.50
2.42
0.28
0.23
5a-A-3a-ol,17-one
3.73
2.62
0.74
0.80
5(3-A-3a-ol,17-one
3.58
2.86
1.03
0.61
5a-A-3p-ol,17-one
3.97
3.44
1.20
0.67
5p-A-3p-ol,17-one
-
-
-
-
5a-A-3-one,17p-ol
4.61
3.88
1.27
1.24
5p-A-3-one,17p-ol
4.18
3.44
1.13
1.14
5a-A-3-one,17a-ol
4.36
3.31
1.09
-
5p-A-3-one,17a-ol
3.97
2.91
0.81
0.78
5a-A-3,17-dione
4.00
-
-
-
5(3-A-3,17-dione
3.64
-
-
-
A-5-en-3p-ol,17-one
4.00
- 66 -
3.38
1.16
1.02


TABLE 11 Hi-Eff l%d
(cont.)
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.27
-
-
-
5(3-Andros tane
0.26
-
a*
-
5a-A-17(3-ol
0.74
0.65
0.29
0.16
5(3-A-17(3-ol
1.32
1.04
0.41
-
5a-A-17-one
0.63
-
-
-
5(3-A-17-one
1.14
-
-
-
5a-A-3(3-ol
0.75
0.67
0.32
0.20
5(3-A-3(3-ol
0.63
0.57
0.25
0.15
5a-A-3-one
0.78
-
-
-
5(3-A-3-one
0.70
-
-
-
5a-A3a,17(3-diol
3.23
3.27
0.88
0.27
5(3-A-3a, 17(3-diol
3.20
2.52
1.07
0.30
5a-A-3(3,17(3-diol
3.60
2.86
1.18
0.24
5(3-A-3(3,17(3-diol
2.93
2.23
0.86
0.23
5a-A-3a-ol,17-one
2.88
2.43
0.78
0.80
5(3-A-3a-o1,17-one
2.92
2.61
0.99
0.61
5a-A-3(3-ol, 17-one
3.24
2.95
1.15
0.67
5(3-A-3(3-ol,17-one
-
-
-
-
5a-A-3-one,17p-ol
3.84
3.28
1.24
1.24
5p-A-3-one,17p-ol
3.49
3.04
1.13
1.14
5a-A-3-one,17a-ol
3.18
2.92
1.11
-
5p-A-3-one,17a-ol
3.26
2.58
0.85
0.78
5a-A-3,17-dione
3.63
-
-
-
5p-A-3,17-dione
3.32
-
-
-
A-5-en-3(3-ol, 17-one
3.32
- 67 -
2.92
1.14
.1.02


TABLE 11 NGSeb 37,
(cont.)
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.09
-
-
-
5f3-Androstane
0.08
-
-
-
5a-A-17(3-ol
0.47
0.44
0.20
0.16
5p-A-17p-ol
0.84
0.80
-
0.32
5a-A-17-one
0.39
-
-
-
5(3-A-17-one
0.71
-
-
-
5a-A-3(3-ol
0.47
0.47
0.21
0.18
5p-A-3(3-ol
0.38
0.37
-
0.13
5a-A-3-one
0.49
-
-
5p-A-3-one
0.42
-
-
-
5a-A-3a,17(}-diol
2.27
1.69
0.31
0.29
5(3-A-3a,17p-diol
2.08
1.89
0.35
0.34
5a-A-3p,17(3-diol
2.44
2.20
0.44
0.41
5p-A-3p,17p-diol
1.93
1.66
0.28
0.28
5a-A-3a-ol,17-one
1.83
1.61
0.58
0.67
5f3-A-3a-ol,17-one
1.80
1.76
0.72
0.73
5a-A-3p-ol,17-one
2.10
2.00
0.91
0.94
5(3-A-3f3-ol,17-one
1.88
-
-
-
5a-A-3-one, 17|3-ol
2.61
2.38
1.07
1.18
5p-A-3-one,17p-ol
2.20
1.98
0.91
0.97
5a-A-3-one,17a-ol
2.22
2.12
0.94
-
5(3-A-3-one, 17a-ol
2.05
1.76
0.62
0.50
5a-A-3,17-dione
2.27
-
-
-
5p-A-3,17-dione
1.98
m
-
-
A-5-en-3p-ol,17-one
2.09
1.99
0.90
0.84
- 68 -


f
TABLE 11 NGSeb 17
(cont.)
Steroids
Free
Acetate
TMSi TFA
5a-Androstane
0.14
-
-
5|3-Androstane
0.12
-
-
5a-A-17p-ol
0.46
0.45
0.24
5f3-A-17f3-ol
0.80
0.80
0.39
5a-A-17-one
0.39
-
-
5(3-A-17-one
0.68
-
-
5a-A-3(3-ol
0.48
0.49
0.26
5(3-A-3(3-ol
0.39
0.41
0.40
5a-A-3-one
0.48
-
-
5(3-A-3-one
0.42
-
-
5 a-A-3a,17(3-diol
1.88
0.73
0.36
5(3-A-3a,17(3-diol
1.82
0.84
0.40
5a-A-3(3,17p-diol
2.14
0.82
0.50
5(3-A-3(3,17(3-diol
1.70
0.65
0.35
5a-A-3a-ol,17-one
1.64
1.50
0.60
5p-A-3a-ol,17-one
1.59
1.57
0.73
5a-A-3(3-ol,17-one
1.85
1.88
0.88
5(3-A-3(3-ol,17-one
1.53
1.48
0.74
5a-A-3-one,17(3-ol
2.24
2.10
0.96
5p-A-3-one,17(3-ol
1.96
1.91
0.87
5a-A-3-one, 17a-ol
-
-
-
5(3-A-3-one,17a-ol
1.82
1.61
0.63
5a-A-3,17-dione
2.00
-
-
5(3-A-3,17-dione
1.78
-
-
Ati5-en-3(3-ol, 17-one
1.86
a b
1.78
c e
0.84
f
Retention time of Cholestane:
12-14, 6-9,
6-8, 9-10,
10 minutes.
6?


Elution pattern of the leomerlc free steroids on the six
columns ere given In Table 12.
The calculated group retention factors for the hydroxyl,
ketone, acetyl and trlmethylsllyl groups are given In Table 13. A
comparison of observed and calculated retention time values based
on average log k values are reported In Table 14.
The changes In retention time upon derivative formation are
calculated according to the equation.
^ 'derivative ^ 'free steroid^ 100 r* *
The results are summarised In Table 15.
The average change In retention time consequent to derivative
formation and the dependence of this change upon the number of
substituent groups are shown In Table 16.
Table 17 gives the values of the T term calculated according
to the following equation
t* t'
T XE-60 SB-30
SB-30
A comparison of the ketone selective properties of the four
column pheses Is given In Table 18.
70


TABLE 12 SE-30 3%
Comparison of Retention Data of
Isomeric Pairs: Substituent Effects
Functional Group
5a
. 5e
No Substitution
0.21
0.20
170-ol
0.32
0.41
30-ol
0.31
0.29
3a,170-diol
0.54
0.51
30,170-diol
0.59
0.49
17-one
0.32
0.42
3-one
0.33
0.32
3,17-dione
0.58
0.50
3a-ol,17-one
0.51
0.47
30-ol,17-one
0.55
-
3-one,170-ol
0.59
0.54
3-one,17a-ol
0.56
0.53
71


TABLE 12 XE-60 3%
( cont. )
Functional Group
5a
No Substitution
170-ol
3^-oi
3a,170-diol
30,170-diol
17-one
3-one
3,17-dione
3a-ol,17-one
30-ol,17-one
3-one,170-ol
3-one,17a-ol
0.27
0.25
0.60
0.89
0.65
0.53
1.67
1.68
1.89
1.56
0.65
0.95
0.77
0.68
J
2.64
2.48
2.00
1.90
2.10
1.98
2.58
2.44
2.09
2.14
72 -


TABLE 12 Hi-Eff 3%
( cont. )
Functional Group
5a
5P
No Substitution
0.13
0.12
17p-ol
0.71
1.48
3£-ol
0.75
0.61
3a, 17^-diol
3.94
3.85
3£,17p-diol
4.19
3.18
17-one
0.61
1.15
3-one
0.73
0.67
3,17-dione
4.00
3.64
3a-ol,17-one
3.73
3.58
3£-ol,17-one
3.97
-
3-one,17¡3-ol
4.61
4.18
3-one, 17a-ol-
4.36
3.97
73


TABLE 12 Hi-Eff 1%
( -cont. )
Functional Group
5a
50
No Substitution
0.27
0.26
170-ol
0.74
1.32
3p-ol
0. /5
0.63
3a,17p-diol
\
3.23
3.20
3p,17£-diol
3.60
2.93
17-one
0.63
1.14
3-one
0.78
0.70
3,17-dione
3.63
3.32
3a-ol,17-one
2.88
2.92
*
3p-ol,17-one
3.24
-
3-one,17f3-ol
3.84
3.49
3-one,17a-ol
3.18
3.26
.
-
- 74


TABLE 12 NGSeb 37
( cont. )
Functional Group
5a
53
No Substitution
0.09
0.08
170-ol
0.47
0.84
30-ol
0.47
0.38
3a,170-diol
2.27
2.08
30,170-diol
2.44
1.93
17-one
0.39
0.71
3-one
0.49
0.42
3,17-dione
2.27
1.98
3a-ol,17-one
1.83
1.80
30-ol,17-one
2.10
1.88
3-one,170-ol
2.61
2.20
3-one,17a-ol
2.22
2.05
75


TABLE 12 NGSeb 1%
( cont. )
Fue tiona1 Group
5a
5P
No Substitution
0.13
0.12
17p-ol
0.46
0.80
3p-ol
0.48
0.39
3a,17^-diol
1.88
1.82
3p,17g-diol
2.14
1.70
17-one
0.39
0.68
3-one
0.48
0.42
3,17-dione
2.00
1.78
3a-ol,17-one
1.64
1.59
3j3-ol, 17-one
1.85
1.53
3-one,17p-ol
2.25
1.96
3-one,17a-ol
-
1.82


TABLE 13 SE-30 3%
Calculated Group Retention Factors
(log r = log rn + log ka + log kb)
Functional 5a 5p
Group Free Acet TMSi Free Acet TMSi
176-ol
17(3-ol / Androstane
0.19
0.28
0.20
0.31
0.41
3(3,17(3-diol / 3p-ol
0.27
0.40
0.31
0.23
0.39
3-one,17p-ol / 3-one
0.25
0.36
0.29
0.23
0.34
17a-ol
3-one,17a-ol / 3-one
0.23
0.36
0.26
0.22
0.28
36-ol
3(3-ol / Androstane
0.18
0.28
0.22
0.16
0.25
3(3,17(3-diol / 17(3-ol
0.26
0.40
0.32
0.08
0.22
3(3-ol, 17-one / 17-one
0.24
0.31
0.23
-
-
3a-ol
3a,17p-diol / 17p-ol '
0.22
0.35
0.25
0.10
0.23
3a-ol,17-one / 17-one
0.21
0.26
0.17
0.05
0.13
17-one
17-one / Androstane
0.19
-
-
0.32
0.32
3p-ol, 17-one / 3(3-ol
0.25
0.22
0.20

_
3,17-dione / 3-one
0.24
-
-
0.20
-
3-one
3-one / Androstane
0.20
-
-
0.20
_
3-one, 17(3-ol / 17(3-ol
0.26
0.29
0.29
0.12
0.13
3,17-dione / 17-one
0.26
-
-
0.08
-


TABLE 13
C. cont.
- XE-60 37.
5 a
56
Grout)
Free
Acet
TMSi
Free
Acet
TMSi
176-ol
17p-ol / Androstane
0.35
0.42
0.14
0.55
0.62
0.32
36,176-diol / 3g-ol
0.41
0.50
0.23
0.47'
0.51
0.20
3-one, 17f}-ol / 3-one
0.51
0.63
0.23
0.55
0.57
0.24
17a-ol
3-ona, 17a-ol / 3-one .
0.42
0.48
0.18
0.50
0.50
0.13
3p-ol
3g-ol / Androstane
0.38
0.41
0.16
0.33
0.37
0.11
3p,17^-diol / 176-ol
0.45
0.49
0.25
0.24
0.26
-0.01
36-ol,17-one / 17-one
0.51
0.57
0.25
0.32
0.50
-0.04
3a-ol
3a,176-diol / 176~ol
0.44
0.46
0.12
0.28
0.27
-0.02
3a-ol,17-one / 17-one
0.49
0.47
0.07
0.30
0.35
0.00
17-one
17-one / Androstane
0.38
.
0.58
36~ol,17-one / 36-ol
0.51
0.54
0.47
0.57
0.70
0.43
3,17-dione / 3-one
0.53
-
-
0.56
-
-
3-one
3-one / Androstane
0.45
-
-
0.43
-
-
3-one,176_ol / 176_ol
0.62
0.60
0.55
0.44
0.38
0.36
3,17-dione / 17-one
0.61
-
-
0.42
-
-
p
-
78 -


TABLE 13 Hi-Eff 3%
( cont. )
5a '5p
runcLiunai
Grouo
Free
Acet
TMSi
Free
Acet .
TMSi.
176-Ql
U3-0I / Androstane
0.74
0.66
0.23
1.09
1.02
-
33,17p-dio1 / 33-ol
0.75
0.65
0.26
0.76
0.63
-
3-one, U3-0I / 3-one
0.80
0.73
0.24
0.79
0.71
0.23
17a-ol
3-one,17a-ol / 3-one
0.78
0.66
0.18
0.77
0.64
0.08
3f3-o 1
33-ol / Androstene
0.77
0.74
0.27
0.70
0.67
-
33,173-diol / 173-ol
0.77
0.74
0.28
0.37
0.28
-
33-01,17-one / 17-one
0.81
0.75
-0.70
-
-
-
3a-ol
3a, 173-diol / 173-0I
0.74
0.63
0.10
0.41
0.35
-
3a-ol,17-one / 17-one
0.79
0.63
0.08
0.49
0.40
-0.05
17-one
17-one / Androstane
0.68
-
-
0.98
-
-
33-0I,17-one / 33-0I
0.72
0.68
-0.30
-
-
-
3,17-dione / 3-one
0.74
-
-
0.73
-
3-one
3-one / Androstane
0.76
-
-
0.74
-
-
3-one,I73-0I / I73-0I
0.81
0.82
0.76
0.45
0.43
-
3,17-dione / 17-one
0.82
0.50
\
79


TABLE 13 Hi-Eff 1%
( cont. )
5a 5g
Functional ^
Group Free Acet TMSi,Free Acet TMSi
178-ol
17(3-ol / Androstane
0.44
0.38
0.03
0.71
0.61
0.20
3£,17£-diol / 3p-ol
0.68
0.63
0.57
0.67
0.59
0.54
3-one,17p-ol / 3-one
0.69
0.62
0.20
0.70
0.64
0.21
17a-ol
3-one,17a-ol / 3-one .
0.61
0.57
0.15
0.67
0.57
Jh08
3s-ol
3(3-ol / Androstane
0.45
0.40
0.08
0.39
0.35
-0.01
3£,17p-diol / 170-ol
0.69
0.64
0.61
0.35
0.33
0.45
30-ol,17-one / 17-one
0.71
0.67
0.26
0.48
0.50
-0.18
3a-ol
3a, 170-diol / 170-ol
0.64
0.54
0.48
0.38
0.38
0.42
3a-ol,17-one / 17-one
0.66
0.59
0.09
0.40
0.36
-0.06
17-one
17-one / Androstane
0.37
-
-
0.65
-
-
3p-ol,17-one / 3p-ol
0.64
0.64
0.56
0.74
0.80
0.48
3,17-dione / 3-one
0.67
-
-
0.67
-
-
3-one
3-one / Androstane
0.46
0.43
3-one,17p-ol / 17p-ol
0.71
0.70
0.63
0.42
0.47
0.44
3,17-dione / 17-one
0.76
-
-
0.46
-
-
- 80 -


TABLE 13-NGSeb 3%
(,'cont. )
5a 53
r Liuuci jl
Group
Free
AceC
TMSi
Free
AceC
TMSi
/
178-01
17¡3-ol / Andros cane
0.72
0.69
0.35
1.02
1.00
33,173-diol / 3p-ol
0.71
0.67
0.32
0.71
0.65
3-one,17p-ol / 3-one
0.73
0.69
0.34
0.72
0.67
0.34
17a-ol
3-one,17a-ol / 3-one
0.66
0.64
0.28
0.69
0.62
0.17
3p-ol
33-ol / AndrosCane
0.72
0.72
0.37
0.68
0.66
--
33,173-dio 1 / 173-01
0.71
0.70
0.34
0.36
0.32
--
33~ol,17-one / 17-one
0.73
0.71
0.37
0.42
--
--
3a-ol
3a,173-dio 1 / 173-ol
0.68
0.58
0.19
0.39
0.37
--
3a-ol,17-one / 17-one
0.67
0.62
0.17
0.40
0.39
0
17-one
17-one / Androscane
0.64
--

0.94


38-ol,17-one / 33-0I
0.65
0.63
0.64
0.69


3,17-dione / 3-one
0.67


0.67

- -
3-one
3-one / Androscane
0.73


0.72
--

3-one,I73-0I / I73-0I
0.74
0.73
0.73
0.42
0.39

3,17-dione / 17-one
0.76

--
0.44
--
81


TABLE 13 NGSeb 1%
( cont. )
5a 5p
Functional ,
Group Free Acet TMSi Free Acet TMSi
176-Ql
17p-ol / Androstane
0.53
0.52
0.25
0.82
0.82
0.51
3g,17B-diol / 3p-ol
0.65
0.22
0.28
0.64

-.06
3-one, 17f}-ol / 3-one
0.67
0.64
0.30
0.67
0.66
0.32
17a-ol
3-one,17a-ol / 3-one
-
-
-
.64
.58
.18
3b-o1
33-0I / Androstane
0*55
0.56
0.28
0.51
0.53
0.52
3^, 17f3-diol / 17B-ol
0.67
0.26
0.32
0.33
-
-.05
3f3-ol, 17-one /17-one
0.68
0.68
0.35
0.35
0.34
0.03
3a-ol
3a, 17f3-diol / 17£-ol
0.61
0.21
0.18
0.36
0.02
0.01
3a-ol,17-one / 17-one
0.62
0.58
0.19
0.37
0.36
0.03
17-one
17-one / Androstane
0.46
--
--
0.75


33-01,17-one / 3j3-ol
0.58,
0.58
0.53
0.59
0.5,6
0.27
3,17-dione / 3-one
0.61
""
0.63
3-one
3-one / Androstane
0.55
. --
0.54
-

3-one,17p-ol / 17g-ol
0.69
0.67
0.60
0.39
0.38
0.35
3,17-dione / 17-one
0.71
-
-
- 0.42

82


TABLE 14
a
Comparison of .Calculated and Observed Retention Time Values
Relative to Cholestane
Steroids
3% SE-
-30
37
XE-60
37 Hi
-Eff 8B
37o
NGSeb
FREE
5CC-A-3,17-dione
Caled.
0.60
Obsd.
0.58
Caled,
2.88
, Obsd.
2.64
Caled.
4.17
Obsd.
4.00
Caled.
2.19
Obsd.
2.27
5p-A-3,17-dione
0.50
0.50
2.52
2.48
3.17
3.64
1.59
1.98
5a-A-3-one, 17(3-ol
0.63
0.59
2.58
2.58
4.68
4.61
2.57
2.61
5(3-A-3-one, 17(3-ol
0.50
0.54
2.24
2.44
3.32
4.18
1.74
2.20
ACETATES
5a-A-3p,17p-diol
1.00
0.98
2.75
2.21
3.39
3.18
2.19 2.20
5p-A-3p,17p-diol
0.81
0.86
2.24
1.90
_
2.42
1.66
TMSi Ether
5a-A-3p,17p-diol
0.69
0.69
0.71
0.66
-
0.42
0.40
0.44
5p-A-3(3,17p-diol
-
-
0.47
0.51
0.28
0.28
Based on average log k values


TABLE
15 SE-30 3%3
Ratio of Relative
Retention
Times of Derivatives
to Free
Steroids
Steroids
Acetate
TMSi
TFA
5a-Androstane
-
-
-
5p-Andrstane

-
-
5a-A-17(3-oi
1.22
1.02
-
5p-A-17p-ol
1.27
1.19
-
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3p-ol
1.25
1.09
0.75
5p-A-3p-ol
1.22
1.09
0.75
5a-A-3-one
-
-
-
5p-A-3-one
-
-

5a-A-3a,17¡3-diol
1.65
1.09
0.52
5p-A-3a,17p-diol
1.69
1.09
0.63
5a-A-3p,17¡3-diol
1.68
1.17
0.62
5p-A-3p,17(3-diol
1.75
1.14
-
5a-A-3a-ol,17-one
1.13
0.91
-
5p-A-3a-ol,17-one
1.20
0.98
0.60
5a-A-3p-ol,17-one
1.89
0.99
0.54
5p-A-3p-ol,17-one
-
-
-
5a-A-3-one,17p-ol
1.30
1.09
0.60
5p-A-3-one,17p-ol
1.28
1.09
0.61
5a-A-3-one,17a-ol
1.32
1.07
-
5p-A-3-one,17a-ol
1.14
0.90
0.62
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
84


TABLE 15 XE-60 3%b
(Cont.)
Steroids
Acetate
TMSi
5a-Androstane
5(3-Androstane
-
5a-A-17p-ol
1.18
0.63
5p-A-17p-ol
1.17
0.58
5a-A-17-one
-

5p-A-17-one
-
-
5a-A-3p-ol
1.08
0.59
5p-A-3p-ol
1.11
0.60
5a-A-3-one
-
-
5p-A-3-one
-
-
r
5a-A-3a,17p-diol
1.23
0.29
5p-A-3a,17p-diol
1.16
0.30
5a-A-3p,17p-diol
1.17
0.30
5p-A-3p,17p-diol
1.22
0.33
5a-A-3a-ol,17-one
1.07
0.44
5p-A-3a-ol,17-one
1.13
0.50
5a-A-3p-ol,17-one
1.16
0.55
5(3-A-3(3-ol,17-one
1.51
0.43
5a-A-3-one,17(3-ol
1.09
0.51
5(3-A-3-one,17p-ol
1.04
0.48
5a-A-3-one,17a-ol
1.14
0.57
50-A-3-one,17a-ol
1.00
0.43
5a-A-3,17-dione
-
-
5p-A-3,17-dione

- 85


I
TABLE 15
(Cont.)
- Hi-Eff 3%c
Steroids
Acetate
TMSi
TFA
5a-Androstane
-
-
-
5^-Androstane
-
-
5a-A-17p-ol
0.82
0.31
0.23
5p-A-17p-ol
0.86
-
-
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3p-ol
0.95
0.32
0.27
5(3-A-3j3-ol
0.93
-
0.25
5a-A-3-one
-
-
-
5(3-A-3-one
-
-
-
5a-A-3a,17p-diol
0.63
0.07
0.07
5p-A-3a,17f3-diol
0.73
0.10
0.08
5a-A-3p,17p-diol
0.76
0.10
0.06
5p-A-3p,17p-diol
0.69
0.08
0.07
5a-A-3a-ol,17-one
0.70
0.20
0.21
5(3-A-3a-ol,17-one
0.80
0.29
0.17
5a-A-3p-ol,17-one
0.87
0.30
0.17
5f}-A-3p-ol, 17-one
-
-
-
5a-A-3-one,17p-ol
0.84
0.28
0.27
5p-A-3-one, 17f3-ol
0.82
0.27
0.27
5a-A-3-one,17a-ol
0.76
0.25
-
5p-A-3-one,17a-ol
0.73
0.20
0.20
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
_
mm
86


TABLE 15
(Cont.)
- Hi-Eff 17od
Steroid
Acetate
TMSi
TFA
5a-Androstane
-
-

5(3-Androstane
-
-
-
5a-A-17p-ol
0.88
0.39
0.23
5f3-A-17f3-ol
0.79
0.31
-
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3j3-ol
0.89
0.43
0.27
5p-A-3p-ol
0.90
0.40
0.25
5a-A-3-one
-
-
-
5p-A-3-one
-
-
-
5a-A-3a, 17p-diol
0.70
0.27
0.07
5p-A-3a,17p-diol
0.79
0.33
0.08
5a-A-3p,17p-diol
0.79
0.33
0.05
5(3-A-3f3, 17¡3-diol
0.76
0.29
0.06
5a-A-3a-ol,17-one
0.84
0.27
0.21
5p-A-3a-ol,17-one
0.89
0.34
0.17
5a-A-3p-ol,17-one
0.91
0.35
0.17
5f3-A-3(3-ol,17-one
-
-
-
5a-A-3-one, 17(3-ol
0.85
0.32
0.27
5p-A-3-one,17f3-ol
0.87
0.32
0.27
5a-A-3-one,17a-ol
0.92
0.35
-
5(3-A-3-one, 17a-ol
0.79
0.26
0.20
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
_
87


TABLE 15 NGSeb 3%e
(Cont.)
Steroids
Acetate
TMSi
TFA
5a-Androstane
-
-
-
5(3-Androstane
-
-
-
5a-A-17(3-ol
0.94
0.43
0.34
5p-A-17f}-ol
0.95
-
0.38
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3p-ol
1.00
0.45
0.38
5p-A-3p-ol
0.97
-
0.34
5a-A-3-one
-
-
-
5p-A-3-one
-
-
-
5a-A-3a,17p-diol
0.74
0.14
0.13
5(3-A-3a,17f3-diol
0.91
0.17
0.16
5a-A-3p,17p-diol
0.91
0.18
0.17
5f3-A-3¡3,17p-diol
0.86
0.15
0.15
5a-A-3a-ol,17-one
0.88
0.32
0.37
5p-A-3a-ol,17-one
0.98
0.40
0.41
5a-A-3j3-ol,17-one
0.95
0.43
0.45
5f3-A-3j3-ol,17-one
-
-
-
5a-A-3-one,17p-ol
0.91
0.41
0.45
5p-A-3-one,17p-ol
0.90
0.41
0.44
5a-A-3-one,17a-ol
0.95
0.42
-
5p-A-3-one,17a-ol
0.86
0.30
0.24
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
m
88


c
TABLE 15 NGSeb 17.f
Steroids Acetate TMSi
5a-Andros tane
5(3-Androstane
5a-A-17£-ol
5p-A-17p-ol
5a-A-17-one
5p-A-17-one
5a-A-3p-ol
5p-A-3p-ol
5a-A-3-one
5p-A-3-one
5a-A-3a,17p-diol
5f3-A-3a,17(3-diol
5a-A-3p,17p-diol
5f3-A-3(3,17p-diol
5a-A-3a-ol,17-one
5|3-A-3a-ol, 17-one
5a-A-3(3-ol, 17-one
5|3-A-3(3-ol,17-one
5a-A-3-one,17p-ol
5(3-A-3-one, 17(3-ol
5a-A-3-one,17a-ol
5p-A-3-one,17a-ol
5a-A-3,17-dione
5p-A-3,17-dione
Retention time of Cholestane: a12-14
0.98
0.52
0.10
0.49
1.02
0.54
1.05
0.39
0.19
0.46
0.22
0.38
0.23
-
0.21
0.92
0.37
0.99
0.46
1.02
0.48
0.97
0.48
0.93
0.43
0.97
0.44
0.89
0.35
b6-9, C6-8, d5-7, e9-ll, f10 minutes
89


TABLE -16
The Average Change in Retention Time Occurring in Derivative Formation
( rderiv/ rfree x 100 = r'>4 )
Column
Acetate
TMSi
TFA
Mono
Di
Mono
Di
Mono
Di
SE-30 3%
123
169
102
115
69
59
XE-60 37o
110
120
53
30


Hi-Eff 8B 37o
83
70
27
9
23
7
NGSeb 37o
93
85
41
16
40
15
- 90


Full Text
A STUDY OF THE RELATIONSHIPS BETWEEN
CHROMATOGRAPHIC BEHAVIOR
AND STRUCTURE OF STEROID HORMONES
By
ICLAL SIREL HARTMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
April, 1963

This dissertation is dedicated to ay husband

ACKNOWLEDGEMENTS
The guidance end encouragement of Dr. Herbert H. Wot is and
Dr. Thomas w. Stearns were of the utmost value to the author as a
constant source of Inspiration and support and are most gratefully
acknowledged.
Particular thanks are also due to Dr. Arndn H. Gropp for his
advice and Inspiration throughout the graduate training of the
author.
The aid of Dr. Herman E. Carr,Jr. In the criticism and
correction of the original manuscript Is deeply appreciated.
The author also wishes to acknowledge the competent assistance
of Mrs. L. Joyce Smith In the computation of some of the data and In
the typing of this dissertation.
Ill

TABU OF CONTENTS
Page
DEDICATION 11
ACKNOWLEDGEMENTS HI
LIST OF TABUS vll
LIST OF FIGURES lx
INTRODUCTION 1
Statement ef Purpose 1
Chromatography 2
Historical Background 2
Gas Chromatographic Methods 6
Principles of Gas Chromatography 8
Basic Gas Chromatographic Apparatus 16
Androstanes 18
Conformations of Androstane Ring System 18
Isomerism In Androstanes 24
Androstane Isomers Possessing Hormonal Activity 25
Methods of Assay for Androgens and 17-Ketostarolds 27
Chemical and Physical Methods of Characterization
of Steroids 28
Nomenclature 30
EXPERIMENTAL 32
Materials and Methods 32
Compounds Studied 32
lv

Page
Preparation of Derivatives 34
Studies on the Free Steroids and their Derivatives 35
Reference Standard 35
Solvents and Reagents 36
Colimo Supports 37
Stationary Phase 37
Preparation of Columns 39
Equipment 40
Preliminary Experiments 42
SE-30 Methyl Silicone Polymer (SE-30) 42
Mixed Phases Containing SE-30 44
SE-52 Methyl Phenyl Silicone Polymer (SE-52) 51
Dow Corning High Vacuum Silicone Grease 51
Ethyleneglycol Succinate Polyester (EGS) 51
Neopentylglycol Adipate Polyester (NGAd) 51
Neopentylglycol Sebacate Polyester (NGSeb) 53
Silicone Nitrile Fluids XF-1150 and XF-1105 53
QF-1-0065 Fluoroalfcyl Polymer (QF-1-0065) 56
Discussion of Preliminary Experiments 58
Comparative Experiments 62
Discussion of Comparative Experiments 93
Concentration of the Stationary Phase and Mesh Sise 107
of Support
v

Separation of Urinary 17-KetoBteroids 113
Known Mixtures 113
Preparation of Urine Sample 118
Discussion of the Separation of Urinary
17-Ketosterolds 118
Study of the Dependence of Relative Retention Time on
Temperature and on the Nature of the Reference Standard 121
Discussion of Factors Affecting Retention Time Values 121
DISCUSSION
Relationship of Chromatographic Behavior to Chemical
Structure 125
Retention Patterns 125
Effect of Structure on Retention Data 128
Theoretical Aspects of Partition Chromatography:
Relationship of Structure to Chromatographic
Mobility 131
The Application of Gas-liquid Chromatography to the
Isolation and Measurement of Urinary 17-Ketosteroids 141
A Method for the Simultaneous Separation of C}g02
and CigOj 17-Ketosterolds and Progesterone
Metabolites 141
Summary 146
BIBLIOGRAPHY 148
BIOGRAPHICAL SKETCH 155
Vi

LIST OF TABLES
Tabla
1 Variation of Retention Tina with Changes In Temperature
and Pressure on 6Z SB-30 Column 43
2 Variation of Separation Factor vlth Temperature and
Pressure on 3Z SB-30 Column 45
3 Retention Times on 31 SB-30 Column 46
4 Retention Times and Separation Factors on 3Z SB-30 Column 47
5 Comparison of Separation Factors on SB-30 and Ethylene¬
glycol Ieophtalate (EGIP) Columns 49
6 Comparison of Retention Times on SB-30 and Bis-(m-phanoxy-
phanyl)-ether Columns 50
7 Retention Times on 31 SB-52 Column 52
8 Isomeric Retention Timas on 3Z Neopentylglycol Adlpete
Column 54
9 Retention Times of 17-Betosterold Acetates on 31
Neopentylglycol Adipate Column 55
10 Retention Times and Separation Factors on 3% XF-1105
Column 57
11 A Comparison of the Relative Retention Times of
Substituted Androstones 64
12 Comparison of Retention Date of Isomeric Pairs:
Substituent Effects 71
13 Calculated Group Retention Factors
(log r • log rn + log k, + log 1%) 77
14 Comparison of Calculated and Observed Retention Time
Values Relative to Cholestane 83
15 Ratio of Relative Retention Times of Derivatives to
Free Steroids 84
vil

c
T*bU Page
16 The Average Change In Retention Tine Occurring In
Derivative Formation x 100 - r'.X) 90
17 Values of T at 260° Based on Measurements obtained
with 3% SE-30 and 3X XE-60 Columns
(T - t‘xR-60 - t'gg-30 / t'gg.30) 91
18 Comparison of Selective Retention of the Four Phases
for Ketone and Hydroxyl Groups 92
19 The Change In log r Contribution from Hydroxyl to
Ketone Transitions (LogAr OH-one) 94
20 Isomeric Separation Factors (5a / 5fs) 100
21 Separation of Principal 17-Ketosterolds and Progesterone
Metabolites as Trlmethylsllyl Ethers 114
22 The Effect of the Reference Standard on the Variation
of Relative Retention Time with Temperature on IX
SE-30 Column 122
viii

LIST OF FIGURES
Figure Pag*
I Distribution Isotherms 4
2. A method for calculating number of theoretical plates 11
3 Influence of carrier gas velocity on column efficiency 15
4 Steroid nucleus 19
5 Conformations of cyclohexane 19
6 A-Equatorial bonds. B-Axial bonds. 19
7 Cholestanol 22
I
8 5a-Androstane skeleton 22
9 Gas-liquid chromatogram of a mixture of known steroid
triaethy1stly1 ethers on 3X XE-60 column 115
10 Gas-liquid chromatogram of a mixture of known steroid
trlmethylsllyl ethers on IX Hl-Eff 8B column 116
11 Gas-liquid chromatogram of a mixture of known steroid
trlmethylsllyl ethers on IX NCSeb column 117
12 Gas-liquid chromatogram of urinary components as
trlmethylsllyl ethers on 3X XE-60 column 119
lx

INTRODUCTION
Statement of Purpose
This study had two main purposes:
ttie investigation of the gas-liquid chromatographic behavior
of a series of isomeric androstañes with the ala of correlating chemi¬
cal structure to chromatographic mobility.
Through the knowledge and insight gained in the course of the
above study to apply the gas-liquid chromatographic method as an
analytical technique in the Isolation and measurement of C-^ steroids
of importance in man. The ultimate objective was the actual measure¬
ment of these steroids in biological fluids.
In the first part of the study twenty-eight isomeric androstsnes
were utilised. These were chosen on the basis of their particular
structural relationships. The structures ranged from the unsubstituted
androstane nucleus to the dlsubstituted steroid. The substituent was
an oxygen function in all cases.
For the separation study of biologically significant androstsnes,
two pregnane Isomers were Included which occur together with the C^
steroids in biological fluids.
- 1 -

Chromatography
Historical Background
Tswett la generally given credit for the Invention of the
method of chromatography, although In 1850 a German dye chemist,
P. F. Bunge, had described a separation process which might be
classified as paper chromatography (1). In 1903 Tsvett (2) sepa¬
rated the pigments of green leaves Into several components on an
adsorbent column of calcium carbonata. Since this separation by
selective adsorption Involved the formation of colored bands on the
column, Tsvett named the process chromatography. The process has
since been extensively applied to colorless materials, thus the term
chromatography Is a misnomer.
The purpose of chromatography is the separation of closely
similar substances. Most separations depend upon the distribution of
substances between two phases. One phase Is fixed and may be liquid
or solid and the other phase Is mobile and may be liquid or gas. The
phases must be selected so as to enhance the nexinum difference between
the distribution of the desired substance and that of the other materi¬
als. The particularly distinguishing feature of chromatography Is the
amplification of the distribution process. This amplification Is
accomplished by the Intimate contact of each element of the moving
phase with each element of the very finely divided fixed phase. The
2

process of continuous and successive contacts results in the distri¬
bution of the solute between the elements of the two phases and the
attainment of equilibrium in a very small length of time.
The two basic types of chromatography are adsorption and
partition and are distinguished by the nature of the process of distri¬
bution of the solute between the phases. (See Figure 1.) In ad¬
sorption systems, the distribution is dependent on the concentration
of the substance that is being distributed. At any one temperature,
the adsorption: isotherm, represented as the function of the quantity
of solute in one phase and the total amount in the system is a curve.
The adsorption isotherm is effected by the presence of other substances
in the system. This is probably due to the limited surface area of
the solid phase. In contrast to the adsorption isotherms, the Isotherms
for the partition systems (liquid-gas or liquid-liquid) are linear for
a wide range of concentration of the solute as well as for moderate
concentrations of other substances in the system. This is the major
distinguishing feature of partition system and accounts for their tre¬
mendous success (3).
With the exception of the work of Bunge, early phases of the
development of chromatography were limited to column chromatography.
In the steroid field both adsorption and partition methods found wide
application.

A B
FIGURE 1
Distribution isotherms:
A Adsorption isotherm.
B Partition isotherm.

Two types of adsorption methods were employed by the early
investigators. The first type Involved the use of large columns for
large samples and were primarily preparative in purpose (4,5). The
second type was for separation and estimation of small quantities of
samples on small columns. This method found application in the
separation and estimation of urinary 17-ketosteroids (6,7) and of
adrenal steroids and metabolites (8). Alumina, magnesia and silica
gel were used siost frequently for column packing.
The flrat successful partition methods were developed by
Butt, Morris and Morris (9,10) who utilized Cellte columns. Shortly
after the publication of these methods, intensive research was di¬
rected towards partition chromatography techniques. Hie problem of
the low solubility of steroids in systems based on water as the
stationary phase was overcome by the development of organic systems
and a whole new field of partition chromatography of steroids becas»
available. Among the pioneers in this field were Morris and his group
(9,10), Zaffaronl and his associates (11), Heftmann(12), Bush (13,14)
and Savard (15) to name a few.
The systems of partition chromatography consisted of three
components: a major component of the mobile phase which was relatively
non-polar, a component of the stationary phase more or less polar in
character to enhance the solubility of the steroid and a third com¬
ponent of a small amount of water. The water was later omitted from
systems using impregnated paper and from certain column methods. In
- 5 -

reversed-phase partition chromatography systems» the mobile phase was
more polar than the stationary phase and the support vas hydrophobic.
The principles of liquid-liquid and gas-liquid partition chroma¬
tography were described by Martin and Synge in 1941 (16) but the concept
of a gaseous moving phase was not applied until 1952 when James and
Martin published their now classical paper (17). Since that time»
extensive research on gas chromatography has been carried out in labo¬
ratories throughout the world for many types of problems. The great
Interest in various forms of gas chromatography is due to its wide
range of application» speed of analysis and the sensitivity of de¬
tection for micro-quantities of sample.
Gas Chromatographic Methods
Gas chromatography is the process by which a mixture is sepa¬
rated into its individual components by a gaseous mobile phase. If
the stationary phase is liquid» the method is called gas-liquid
chromatography and the separation is due to the differential solubility
of the materials between the liquid and gas. If the stationary phase
is a solid» the method is called gas-solid chromatography and the
separation is due to the adsorption of the sample components on the
solid surface.
The two types of gas chromatographic methods are further dis¬
tinguished by the manner in which the sample moves through the column.
6

Elution, displacement and frontal analyses ara three techniques
which may be used In gas-solid chromatography. The elution
technique Is used almost exclusively In gas*liquid chromatography.
Frontal analysis Is the passage of a continuous flow of
carrier gas containing the sample mixture through a column of
adsorbent. The Idea of using a continuous stream of the sample
Itself as a displacer where each solute attains Its own competitive
adsorption equilibrium was the contribution of Tlsellus (18). Sepa¬
ration Is achieved only for that component which emerges first from
the column, consequently frontal analysis la of limited use as a
method of separation.
Displacement analysis, which was elaborated by Claesson (19),
Is the method of displacing the sample already on the column by means
of passing a continuous flow of a carrier gas which contains the
vapor of a substance called the displacer. The displacer Is so
chosen that it is more strongly adsorbed onto the column than any
component of the sample mixture. The component displaced by the
displacer in turn displaces another component of the sample that is
less strongly adsorbed. Thus the components of a sample move along
the column at the same rate that the displacer la saturating the
column behind the sample.
Elution chromatography Involves the continuous passage of an
unadsorbed gas or liquid through the chromatographic system. A
volatilized sample la introduced into this stream of gas and la carried
- 7 -

down the column. Components of the sample distribute themselves
between the mobile gaseous phase and the stationary phase. Each
component eventually emerges from the column after the passage of
a characteristic volume of carrier gas. The unique feature of
elution technique, as compared to frontal and dlsplac «sent analysis,
Is the emergence of the solute as a peak. The retention time t&,
defined as the length of time between the Introduction of the sample
into the chromatographic system and the emergence of the peak maxi¬
mum, is a means of qualitative Identification of the compound. The
peak area can usually be related directly to the concentration of the
Principles of gas Chromatography
A distinguishing feature of gas-chromatography Is the utilisation of
high temperatures In order to maintain the solutes In the vapor state.
The vide selection of liquid phases enhances the applicability of gas-
liquid chromatography methods to a vide range of analyses. The tech¬
nique of gas-liquid chromatography is similar to liquid-liquid partition
and the gas-liquid column is analogous to a distillation column.
However, there is one major difference between the two systems, which
is of great practical importance. In systems of gas-liquid chroma¬
tography there is no interference with separation due to azeotropy.
At the inlet of a gas-liquid chromatography column there might occur
an interaction between the components of the sample but over the whole
length of the column, the interaction of these components with the
8

liquid phase far outweigh any mutual effects (20). Thus each
component is carried through the column independently of any other
component.
A theoretical plate treatment is applicable to both liquid*
liquid and gas*liquid columns. A theoretical plate is defined as
a section of the colusn in which the vapor leaving the section has
the composition that would be in equilibrium with the average con¬
centration of liquid solution within that section. The most im¬
portant assumption underlying the theoretical description of the
chromatographic process is that equilibrium between the two phases
is obtained after each minute movement of the mobile phase.
The separation per plate (separation factor) and the total
number of plates (N) existing in a column determine the over-all
separation achieved. The temperature of the colusn and the nature
of the stationary phase of the components to be analyzed all determine
the separation factor. This factor is defined as the ratio of the
retention times of two components to each other. Separations can be
improved by the use of a liquid phase with specific characteristics,
such as selective retention of compounds containing double bonds or
ketone groups.
The length of colusn in which equilibrium occurs is defined
as the height of an effective theoretical plate (16). The number of
theoretical plates exhibited on a column for a specific liquid phase,

solute and temperature can be calculated In a number of ways all of
which are a measure of the degree of peak spread relative to its
residence in the column. The length and diameter of the column,
the diffusion constant of the solute in the two phases, the amount
of stationary phase, uniformity of column packing, the rate of flow,
temperature and the nature of the carrier gas all are variables
affecting the value of N. In general, N is Increased by an Increase
in length of column and a decrease in its diameter.
"he equation commonly used for the calculations of N is
M. ( CR >2
A t
where tR is the retention time measured from the introduction of the
sample and At is the width of peak base. (See Figure 2.) The units
may be time or length provided both tR and 4 t are in the same units.
This equation for N assumes that the component being studied is in
equilibrium with the column. N calculated in this manner is signifi¬
cant only for symmetrical peaks since the above equation is based on
probability theory(21).
The height equivalent to a theoretical plate is given by the
equation
H - L / N
where L is the length of the column, usually in millimeters. The
value of H is generally low for columns operating at high efficiency
and decreases with Increases in retention time. The plate theory
provides a useful means of evaluating column efficiency. The
10 -

Response
FIGURE 2
A method for calculating number of theoretical plates.

relationship of various terms which determine the value of H are
given in the van Deeater equation (22).
S ■' 2Ad + 2T°a«« + „ 8k' 4
" 7^(1 + k')2 DUq
where \ is a quantity characteristic of the packing, dp Is the
average particle diameter, y Is a correction factor for the
tortuosity of the path of the gas In the interparticle space, D
is the diffusion coefficient of the solute In the gaseous phase,
u Is the linear gas velocity, k1 is the ratio of the fraction of
sample in the liquid phase to the fraction In the vapor phase, d¿
is the thickness of the liquid film, and Is the diffusion co¬
efficient of the solute In the liquid phase.
In the simplified van Deemter equation
H - A + B/u + Cu
A represents the eddy diffusion term, also called the multiple path
effect, B/u term is the molecular diffusion and Cu Is the term that
describes the resistance to mass transfer. The eddy diffusion Is
the result of the Irregular path which the gas follows through the
peeked column. The irregularities In this path contribute to a broaden¬
ing of the chromatographic band. A reduction in particle size (dp),
uniform packing and uniform particle size tend to decrease the contri¬
bution to H of this term. The second factor Is the diffusion of the
sample molecules in the carrier gas, which results in band broadening.
The lower the molecular weight of the carrier gas, the greater Is this
- 12

factor. Diffusion increases with Increase in temperature. An
increase in pressure, on the other hand, by increasing the density
of the gas, tends to decrease the diffusion. For this reason,
high temperatures are employed together with high pressures of the
carrier gas.
The term Cu, resistance to mass transfer, Includes several
parameters. The value of k' is dependent on the partition coef¬
ficient as well as the relative volumes of the liquid and the gaseous
phase in the column. k' is generally greater than one because the
solubility of the solute in the liquid phase is usually greater than
in the gaseous phase. Thus any increase in the solubility of the
sample in the liquid phase will decrease H by a small amount. The d^
term, defined as the effective thickness of the liquid film, is raised
to the second power in the van Deemter equation. Its contribution ,
will therefore be appreciable. A reduction in df results in Increased
column efficiency, which may be counteracted by a simultaneous decrease
in k* if the amount of liquid phase is reduced. The liquid diffusion
coefficient (Diiq) is dependent on the viscosity of the liquid phase.
An Increase in temperature might be expected to reduce the viscosity
but since Increased temperatures affect k' and Dgaa adversely, the net
result is difficult to assess.
The last term in the van Deemter equation is the rate of flow
of the carrier gas. Theoretically eddy diffusion is Independent of
the rate of flow. The contribution to H of molecular diffusion is in
inverse ratio to the rate of flow. Resistance to mass transfer,
- 13

however, varies directly with the velocity of the gas. If all
other variables are kept constant, there will exist an optima rate
of flow for the most efficient operation of a colman as Indicated
In Figure 3. At flow rates less than the optlsum value, the contri¬
bution of isolecular diffusion to H will be appreciable. Above opt ¿tama
flow rate resistance to ness transfer becomes Important. However,
this part of the curve Is relatively flat and operations above optlama
velocity will not have too deleterious an effect on H. The aiolecular
diffusion tern la large for carrier gases of high dlffuslblllty*
Resistance to mss transfer will be dominant for liquids of high
viscosity and for high concentrations of stationary phase. Qualitative
and quantitative predictions of the effect of H of changes In the
parameters of the van Deeater equation are smnaarlsed by Patton (23),
Bohemen and Purnell (24) and Purnell (25).
For any packed column there exists a pressure drop between
the Inlet and the outlet ends of the coluan. An Increase In the
absolute pressure at either end of the column or the use of a tapered
coluan with a widening diameter towards the outlet end are possible
solutions to minimising the pressure drop. A correction factor for
the pressure drop across the coluan has been derived by Jams and
Martin (17,26). The retention volume defined as
VR " Fc*R
where Fe Is the volumetric flow rate of gas, can he corrected to give
the limiting retention volume which Is Independent of pressure (27).
14

FIGURE 3
Influence of carrier gas velocity on column efficiency.
- 15

Po / P
where VR Is the Uniting retention volume et zero pressure drop
scross the column, VR is the retention volume, pQ Is the coition
pressure et the outlet end and f Is the everege column pressure.
Various experiments have been devised to test the applies*
blllty of the van Deemter theory and will be considered in the
Discussion section. The theory has been useful In attaining
high column efficiencies.
Basic Gas Chromatographic Apparatus
The apparatus for any gas chromatographic separation consists
of a carrier gas supply, a flow regulator, a pressure gauge, a
recorder, sample port and a plpet for Introducing the sample, a
column, a detector and heating units for the last three items.
Carrier gas is supplied from a compressed source and Its
flow rate through the column Is regulated by a pressure valve. The
gas passes through the detector and proceeds to the sample port. The
sample is Introduced Into the sample port where it Is heated to effect
volatilization. The carrier gas carries the sample vapor Into the
column where the distribution process takes place. The separated
components of the sample In the carrier gas enter the detector and
pass out to atmosphere or to a collection apparatus for further study.
Tesiperature control of the apparatus components is an Important con¬
sideration In gas chromatography. The minimum detector temperature
16

should be thet of the column to prevent condensation of solutes
which would result In change In concentration. The sample port
temperatures should be adjustable to permit the development of
temperatures sufficiently high for the rapid vaporization of
solutes.
17

Androstones
Androstañes belong to the brood group of biologically
important organic compounds colled the steroids which possess the
cyclopentonoperhydrophenonthrene nucleus containing 19 carbon atoa».
(This nomenclature Includes members previously designated as etio-
cholanes.) The nucleus contains 17 carbon atoms which make up
the condensed system of four rings (Figure 4). The rings are
designated as A, B, C and D and the carbon atoms are numbered as
indicated in the above structure. The two angular methyl groups
(designated as carbon atoms 13 and 19) project to the front of the
steroid molecule. This projection is described as ^-oriented and
is represented by a solid line. Similarly» any projection to the
rear of the ring system is designated as a-oriented and is represented
by a dotted line*
Conformations of Androstane Ring System
The term conformation describes the different spatial arrange¬
ments of the atoms in a single classical structure or configuration.
Transformations from one arrangement to another take place by the
simple rotation of a single bond. The best known examples are the
chair and boat forms of the cyclohexane model (Figure 5). The differ¬
ences in energy which are primarily due to differences in the
18 -

18
FIGURE 5
Conformations of cyclohexane: A chair, B boat.
FIGURE 6
A “ Equatorial bonds. B - Axial bonds.

repulsion* between closely approaching atoas among various confor¬
mations determine the stability of the particular conformation. In
general, it nay be stated that even though reactions often proceed
through unstable forms, the unexcited molecules exist in the confor¬
mation of lowest energy and therefore of highest stability (28).
The bonds of the carbon atona in a cyclohexane molecule are
of two types (Figure 6):
1. Those more or less In the plane of the ring are
called equatorial.
2. Those perpendicular to the plane of the ring are
called axial. These were formerly designated
as polar bonds.
The stability relationship can be inferred by consideration of
non-bonded H:H interactions ( van der Waals overlap ). Since these
interactions contribute to instability to an extant which Increases
with decreasing interatomic distance, this factor can be used to
estimate the relative stabilities of different conformations (29). In
the boat form tha 2,3 and 5,6 groups are closer to each other than in
the chair form. Therefore, the chair is the more stable conformation.
In addition to van dar Waals overlap, electron density in or near axial
bonds has been reported to be a factor in sterlc interactions (30). The
interatomic distances between any pair of axial groups are smaller than
those between equatorial groups, therefore, axial grotqps exhibit great¬
er repulsion towards each other. Thus, a compound carrying axial
20

substituents is less stable than the corresponding compound with
equatorial substituents, where non-bonded repulsions are at a
minimum.
The generalization that can be made for condensed ring
systems made up of six membered rings such as the steroids is that
the stable structures are chair conformations with as many as
possible of the substituents in the equatorial positions. As a
representative structure of the steroid nucleus, the cholestanol
molecule (Figure 7) may be examined. In this molecule rings B
and C are locked in the chair conformation by trans-fusion to rings
A and D. Ring A is free to assume the boat or chair forms. But
the instability associated with the boat form of the cyclohexane
molecule is augmented by the interaction between the methyl group
on C-10 and the hydroxyl group on C-3* Similarly, in cholestane
the 3p-hydrogen and the C-10 methyl group oppose the boat form (29).
In the androstane molecule (Figure 8) the ring system of
cholestane is present but the side chain at 0-17 is lacking. The
androstane molecule is described as having three six-membered chair
rings and one five-membered ring. The axial hydrogens at position 1,
3, 5, 7, 9 and 14 are in a line parallel to one another. The carbon
pairs 2-3, 5-10 and 7-8 lie in one plane and carbon atoms 1, 9, 11,
13 and 14 lie in a second plane parallel to the first (29). For¬
mation of ring D produces a slightly puckered ring due to the bending
of the bonds. The hydrogens attached to C-15 and C-16 are eclipsed
21 -

FIGURE 8
5a-Androstane skeleton; a-bonds p-bonds
The equatorial bonds have been omitted for clarity.
22

and the equatorial and axial concept is not applicable. The bonds
at 015 and 017 have quasi-axial (a') and quasi-equatorlal (a1)
relationships with respect to ring C.
Useful generalisations regarding stability and reactivity
of equatorial and axial substituents have been developed (31-34) and
a few of the pertinent ones say be summarised as follows:
1. A substituent is generally more stable in the
equatorial than in the axial orientation. Thus,
if a substituent can be equilibrated in different
orientations, the equatorial will predominate in
equilibrium mixtures.
2. An equatorial hydroxyl group is more accessible
than the axial and therefore reacts more rapidly
than the axial group in astarlfleetion or hydrolysis
type reactions.
3. In chromatography on paper or on alumina an equa¬
torial alcohol is more strongly adsorbed than its
axial Isomer.
4. The equatorial or axial orientation of a substituent
is often reflected in Its Infrared absorption spectrum.
23

Isomería» In Androstanas
Orientation of substituents at specific positions are
suanarlzed below (35)
Position
1
2
3
4
5
10
5a-Series
5p-Series
Q-conflg 6-con fig a-conflg B-con fig
e
a
a
*(AB)
e
e
a a
as m
a(AB)
(A)e(B)
(A)«(B)
6
7
8
9
11
12
13
14
15
16
17
5Q- and 5p-Series
e a
a e
24 -

The spatial orientation of a substituent group is desig¬
nated with reference to the angular methyl groups which are
^-oriented. Thus a group projecting in front of the nucleus is
referred to as being els to the methyl groups and a group project¬
ing to the rear Is trans to them.
The two major classes of Isomeric androstanes are desig¬
nated as 5a- and 5p-andróstane and differ in the spatial configu¬
ration of the hydrogen on C-5. In the 5a-conflguration the rings
A and B trans to each other and In the 5f3-serlea they are els.
Similarly, the 5a-hydrogen is trans to the angular methyl groups
and the 5f3-hydrogen Is els to them. Substitution of any ring
hydrogen further results In the production of stereoisomerlc pairs.
Thus 5a-androstan-3a-ol and 5a-androstan-3p-ol differ only In the
orientation of the hydroxyl group on C-3.
Androstane Isomers Possessing Hormonal Activity
The Isolation and structural elucidation of androstanes which
exhibit hormonal activity posed a problem to the early Investigators.
The amounts of steroid hormones which can be isolated from testis or
adrenal cortex Is very small; however, larger amounts of the steroid
hormone or their metabolites are normally found in urine. Functions
characterising the testicular hormone Include development of secondary
sex characteristics. Among the early methods of bloassay of hormonal
activity, the most satisfactory one was the method based on the ability
25

of the hor«one to promote comb growth In capone (36-37). Butenandt
(38,39) using urine as the source of the hormone, was the first to
isolate and identify one of the principal metabolites of the mala
hormone. The Isolated substance exhibited biological activity.
Chemical characterization indicated it to be a sterol-like ketone
and Butenandt named the hormone androsterone. The structure of
androsterone was further elucidated by partial synthesis from
cholesterol by Ruzlcka and his associates (40-42). The synthetic
compound was identical to that Isolated from urine by Butenandt,
both chemically and physiologically, and its structure was shown to
be 3o-hydroxy-5a-androstan-J.7-one. Two other biologically active
isomers of androstan were synthesized and identified shortly there¬
after. These were 3p-hydroxy-5^-androstan-17-one and 3p-hydroxy-
Sa-androstan-17-one (40-43), both of which have since been Isolated
from human urine (44,45).
Among the C^Oj andr os tañes the first to be Isolated was 3a,
l7p-dlhydroxy-5a-androstan-17-one, which was followed by the isolation
of 3a-hydroxy-5a-androstane-ll,17-dlone, 3a-hydroxy-5p-androstane-
11,17-dione and 3a,ll£-dlhydraxy-5p-androstan-17-one (46-48).
Androgenic activity was demonstrated for 3a,17p-dihydroxy-5a-androstan-
17 -one (49).
Human urine also contains steroids having a double bond be¬
tween carbons 5 and 6. Butendandt and his co-workers (50,51)
Isolated and identified 3p-hydroxyandrost-5-en-17-one.
26

Lieberman and hit group (52) reported the isolation of 5a-
androstane-3,17-dione and 5f-androstane-3,17-dione from normal male
and female urines.
During the last decade the complete synthesis of the steroid
nucleus and of almost all of the naturally occurring androgens have
been accomplished (53-56).
Methods of Assay for Androgens and 17-Ketosterolda
Androgens extracted from urine or tissues were first studied
(38,39) by bioassay methods to determine biological activity. By
definition only those androstane Isomers exhibiting biological
activity as measured by comb growth of capons can be called androgens.
Urine has been the major source of steroids to the investigator
and since most of the urinary steroids which exhibit androgenic
activity also contain 17-ketone group, the development of chemical
methods have been directed toward specific reactions of this group.
The procedure most widely used for the determination of 17-ketosteroids
is the colorimetric measurement based on the Zimeermann reaction (57).
Treatment of a 17-ketone with m-dinitrobenzene in the presence of
alkali gives a transient reaction product of violet color but of un¬
known structure. The colored compound has an absorption maximum at
520 mfi and is sufficiently stable to permit reproducible readings.
However, the ketone group on carbon 3 can Interfere with the results.
Modifications of the above procedure have been reviewed by Zimmermann(58).
27

Chemical and Physical Methods of Characterization of Steroids
The chemical properties of a steroid like those of any other
organic compound should be considered as the reactions of the
functional groups of the steroid molecule. It may be assumed that
an Isolated functional group undergoes reactions with little or no
Interference from other groups. In cases where two groups are
sufficiently close to Influence each others properties, their
reactions must be considered together.
The relative reactivities of axial and equatorial substituents
have already been mentioned. In general, the hydroxyl group on the
steroid nucleus will undergo the common reactions known for this
group, i.e., esterification, ether formation, epoxide formation,
replacement by a halogen, etc. These reactions have been utilized
in the isolation and analysis of steroids. In the present investiga-
tlon two such methods employed were esterification in the preparation
of acetyl and trlfluoroacetyl derivatives and ether formation in the
preparation of trlmethylsilyl ethers of the steroids.
Physical methods employed in the characterization of steroids
include ultraviolet and infrared absorption spectra, rotatory dispersion,
X-ray crystallography and chromatographic analysis.
Ultraviolet absorption was Introduced into the steroid field
in the early 1930's and became extremely popular as a tool for the
detection and determination of trace amounts of compounds with
28

chromophorlc groups. With the accumulation of data, correlation of
structure and ultraviolet absorption afforded a powerful new method
for the elucidation of the structure of a large variety of steroids.
The field has been reviewed by Dorfman (59). More recently Jones
and his co-workers (60) Investigated the infrared absorption charac¬
teristics of steroids. The elucidation of axial and equatorial
orientation Is In part due to Infrared spectral analysis. The vari¬
ation of optical activity with the wave length of the light was known
and used by early Investigators until 1860 whan Bunsen developed his
sodium lamp and a monochromatic light source became available. The
application of rotatory dispersion methods to the steroid field was
pioneered by Djerassl and his associates (61,62). The absorption
bands In rotatory dispersion measurements are sensitive to confor¬
mational changes and have been useful in establishing the validity
of certain structural relationships. X-ray diffraction pattern of
steroids have been useful In supplementing Infrared analysis and melt¬
ing point determinations (63).
With the advent of chromatographic techniques a valuable tool
was added to steroid analysis. Adsorption chromatography was applied
to the purification and separation of steroids (4). Solvent systems
of different polarities were designed for successful separations by
partition chromatography (9,11,64). Savard (15,65) investigated the
behavior of a wide range of C19 and C21 ketosteroids and established
the applicability of the llgroln-propylene glycol system to the
resolution of these ketosteroids. Certain correlations between
chromatographic mobility and structure began to emerge. The mobility
- 29

of the steroid molecule wes observed to decrease with Increasing
number of oxygen functions. The less than expected retardation
In the mobility of a steroid with a 0*21 hydroxyl substituent was
Interpreted as an Indication of Intramolecular hydrogen bonding. The
general pattern of mobilities observed by Savard was consistent with
the concept developed by Barton that steroids possessing equatorial
hydroxyl groups exhibit lower mobilities than the corresponding
Isomers with an axial hydroxyl groups (31,66). Furthermore, the
correlations between mobility, number and nature of oxygen function,
position and orientation of hydroxyl groups together with the relation*
ship of mobilities between 5a* and 5p-isomers provided generalizations
which were In agreement with the observations of Lleberman and his
group (52,67). From the relationship of chromatographic elution
time to structure, certain clues to the nature of unknown compounds
were obtained (68). Chromatography before and after acetylation
enabled the Investigator to assess the number of acetylable hydroxyl
groups (69,70).
nomenclature
Steroid nomenclature la based on the rules defined at the 1950
Clba Foundation Conference In London (71,72). These rules have been
provisionally approved by the International Union of Pure and Applied
Chemistry. Since the nomenclature system la of recent origin and
since many Investigators still employ common names, a brief summary
of the approved nomenclature will be given.
30 -

According to the new system, the neme of the steroid Is
based on Its hydrocarbon nucleus to which prefixes and suffixes
are added to Indicate the nature of the substituent. The position
of these substituents are indicated by the number of the carbon
atom to which they are attached. The projections of the substi¬
tuents to the front and to the rear of the hydrocarbon skeleton are
designated as p and a, respectively.
The parent compounds are androstane, pregnane, cholestane,
etc,, and with each name, the configuration at 05 Is Indicated by
the prefix 5a or 5{3. Unsaturation Is Indicated by the suffix 'ene1,
e.g. 5-ene. The rules for the substituents state that only one
kind of substituent in each compound Is Indicated by a suffix, the
remaining substituents are Indicated by prefixes. The substituent
to be designated by a suffix Is chosen according to the following
order of priority: carboxylic acid or derivative Including esters,
carbonyl, alcohol, amine, ether, halogen.
31

EXPERIMENTAL
Materials and Methods
Compounds Studied
Steroids Investigated In the course of this study were
chosen for their particular structural features and biological
Importanee. All except two were androatane Isomers, the
remaining two were progesterone aetabolites. The isomeric
androstenes employed were:
5a-Series. 5a-androstane, 5a-androstan-3p-ol, 5a-
sndros tan-17p *ol, 5a-androstan-3-one, 5a-androstan-17-one,
5a-androstane-3a,17p-dlol, 5a-androstane-3£,17p-dlol, 3a-hydroxy-
5a-androstan-17-one, 3a-hydroxy-5a-andros tan-17-one, 17p-hydroxy-
5a-androstan-3-one, 17a-hydroxy-5a-androstan-3-one, 5a-androstane-
3,17-dlone, 3a, 11$ -dihydroxy-5a-andros tan-17-one, 3a-hydroxy-5a-
androstane-11,17-dlone.
- 32

56-Serie». The 50-isomers of ell the compound* listed
above were studied simultaneously with the 5a-compounds. The
nomenclature is exactly the same as stated above except that the
50-designation will be substituted for 5a in each instance.
The only unsaturated androstane isomer investigated was
3p-hydroxyandro»t-5-en-l7-one, which was included because of its
biological Importance.
The two pregnane Isomers were 50-pregnane-3a,20a-diol end
3a-hydroxy-50 -pregnen-20-one.
In the section of this work involving the separation of
17-ketosteroids, reference will be made to compounds for which
common names are still widely used in the literature» especially in
the area of biological applications. The common and the approved
names of these compounds are summarised below.
Androsterone
Stlocholanolone
Dehydroeplandrosterone
11-Ketoetlocholañolone
110-Hydroxyandrosterone
Pregnanedlol
Pregnenolone
3a-Hydroxy-5a-andros tan-17 -one
3a-Hydroxy-50-androstan-17-one
30-Hydroxyandrost-5-en-17-one
3a-Hydroxy-50-androstane-ll, 17-dlone
3a, ll0-Dlhydroxy-5a-endrostan-17-one
50-Pregnane-3a,2fla-dlol
3a-Hydroxy-50 -pregnen-20-one
33 -

In the tables summarizing the resulte obtained In this
study, an abbreviated form of naming is used and since one purpose
of this investigation was the comparison of 5a/5p isomeric pairs,
the orientation at 05 is written as the prefix in these abbreviated
forms. For example, 3a>hydroxy-5a-androstan-17-one is abbreviated
as 5a-A-3a-ol,17-one end 3p-hydroxy-5p-androstan-17-one is abbrevi¬
ated as 5p-A-3p-ol,17-one.
The steroids were obtained from commercial sources and were
of sufficiently high purity for direct use. Some, however, showed
minute amounts of impurities when examined by gas chromatography.
Three of the 17-ketosterolds (3a-hydroxy-5a-androstan-17-one, 3a-
hydroxy-5p-androstan-17-one and 3p-hydroxyandrost-5-en-17-one)
when analysed by paper chromatography exhibited no impurities.
Preparation of Derivatives
The derivatives of the steroids were prepared by the following
procedures.
Acetates. The steroid (2-5 mg) was allowed to react with
acetic anhydride (0.5 ml) and pyridine (0.2 ml) at 67° in a dry bath
for two hours. The solvents were evaporated under nitrogen and
the steroid acetate was dissolved in acetone.
34

Trlfluoroacetates (TFA). The steroid (2-5 mg) was
allowed to react with trlfluoroaeetlc anhydride (0.5 ml) and
pyridine (0.2 «1). The reaction was complete in a few minutes
at room temperature (73). The solvents were evaporated under
nitrogen and the derivative was dissolved In acetone.
Trlmethylsllyl ethers (TMS1). The steroid (1-3 mg)
was allowed to react with hexamethyldlsllazane (0.5 ml) and a
few drops of trinethylchlorosllane as catalyst for 6-8 hours at
room temperature (74). The reaction mixture was centrifuged and
the supernatant solvents were evaporated under nitrogen. The
residue was triturated with hexane and recentrifuged. The super¬
natant was again evaporated under nitrogen and the trlmethylsllyl
ether of the steroid was dissolved In tetrahydrofuran (75).
Studies on the Free Steroids and their Derivatives
The free steroids, acetates and trlfluoroacetatea were chro¬
matographed from acetone solutions, the trlmethylsllyl ethers were
chromatographed from tetrahydrofuran solutions. All concentrations
were 2 pg/pl. Volumes of 0.2 to 1 ul were used for Injections. All
measurements were mede in duplicate.
35

One sample of each derivative vas analyzed by infrared
spectroscopy to ascertain the substitution of the desired group
on the steroid nucleus. Some of the derivatives shoved small
amounts of the free steroid, vhich were easily identified by their
characteristic peaks on the chromatograms.
Reference Standard
Cholestane was used as the reference standard for dally
measurements. A saturated solution of cholestane in acetone was
Injected into the column each day before any series of runs were
carried out and was repeated every two or three hours. The retention
times of the samples were then related to the retention time of
cholestane and were expressed as relative retention times.
Solvents and Reagents
Spectral grade acetone (Fisher Scientific Co.) and reagent
grade tetrahydrofuran (Eastman Organic Chemicals), chloroform,
pyridine, hexane and acetic anhydride (all from Fisher Scientific
Co.) were employed. Pyridine and acetic anhydride were freshly
distilled before use. Hexamethyldlsilazane, trimethylchlorosilane
and trlfluoroacetlc anhydride were obtained K + K Laboratories and
were used without further purification.
- 36 -

Column Supports
Comearetally obtained acid-washed ( and in the case of the
last two, siliconized) diatomaceous earths were used as coluan
supports. These were:
Chromosorb W
Kroraat CE
Anakrom ABS
Diataport S
(Applied Science Laboratories.State College,Pa.)
(Burrell Corp., Pittsburg, Pa.)
(Analabs, Hamden, Connecticut)
(F + M Scientific Corp., Avondale,Pa.)
Different mesh sizes employed are Indicated under Individual
coluan preparations.
Stationary Phase
The following commercially obtained polymers were utilised
as the stationary phases:
Silicone Rubber Gums:
SE-30 methyl silicone polymer
SE-52 methyl phenyl silicone polymer
Silicone Fluids:
XF-1150 nitrile silicone polymer
(50 mole per cent cyanoethyl)
XF-1105 nitrile silicone polymer
(5 mole per cent cyanoethyl)
The above polymers ware obtained from General Electric
Company, Waterford, New York.
37

Silicons Copolymer
XE-60 silicone polymer made up of 501 dlmethylslloxane
end 5OX cyanoethylmethyl siloxane. This ves obtained
from F + H Scientific Corp., Avondale, Penns lyvania
Fluorlnatad Silicone Polymer
QF-1-0065 fluoroelkyl silicone polymer.
Silicone Creese
Dow Corning high vacuum silicone grease (DC-silieone)
(ethyl acetate extract). The above two were obtained
from Dow Corning Corp., Midland, Michigan.
Polyesters
Ethyleneglycol Isophtálete (SOI?)
Ethyleneglycol succinate (EGS)
Naopentylglycol adipate (NGAd)
Neopentylglycol sebacate (NGSeb)
These were obtained from Analabs, Hamden, Connecticut.
Hi*»Eff 8B - Dlmethylcyc lohexylsuccInate
This was obtained from Applied Science Laboratories,
State College, Pennsylvania.
38

Bla -fe-phenoxypheny1)-ether
( C6H50C6H4)20
This compound was obtained from Eastman Organic
Chemicals.
Preparation of Columns
The desired amount of liquid phase was weighed and dissolved
In a small amount of chloroform (for silicone polymers) or acetone
(for all other phases). The solution was transferred, with rinsings,
to a beaker containing the weighed support suspended in the same
solvent as used for dissolving the liquid polymer. The mixture was
heated gently with constant stirring until the solvent had evapo¬
rated completely and a free flowing uniformly coated support was
obtained.
This method of deposition of the liquid phase onto the support
was reliable and reproducible and was preferable to the filtration
method (76)* In the latter method, a slurry of liquid phase, solvent
and support was filtered and then dried with stirring* The concen¬
tration of the liquid phase deposited on the support was thus de¬
pendent on the mesh size of the support as well as on the volume of
solvent used*
39

The coated support was packed, with gentle tapping, Into
a copper or stainless steel column one end of which was plugged
with glass wool. The open end of the coition was flared for ease
of packing. A vibrator as an aid In packing was found to be un¬
desirable as It resulted In fragmentation of the support and also
produced tightly packed columns. The open end of the column was
plugged with glass wool and the column was bent Into a U shape,
taking care not to constrict the diameter of the tube at the curve.
The column was then suspended in the gas chromatograph and cured for
24-48 hours at the desired temperature under 10 psl of pressure of
carrier gas. During the curing process the outlet end of the
column was not connected In order to prevent the possible accumu¬
lation of volatile substances In the detector. After the column
was cured, connections to the detector were made and the column was
saturated with a 1-3 pg quantity of the reference standard cholestana.
The so called 'priming' or saturation process was necessary on those
columns where a certain amount of sample bss, attributable to irre¬
versible adsorption was observed. This phenomenon varied from
column to column and was dependent on the nature of the sample. For
example, on the XB-60 column, there was no observable loss of androstanes
while estrogens were adsorbed irreversibly.
Equipment
Measurements were made on two gas chromatographic instruments,
Model 600 from Research Specialties Company, Richmond, California, one
equipped with a ^-Ionization detector (77) and the other with a flame
40

ionization detector (78). Slightly higher sensitivities were
observed with the flame ionization unit. It has been reported
(79) that multiple hydroxyl groups tend to depress the sensitivi¬
ty of the p-ionization detector.
Both instruments were equipped with an injection system
designed for Tenney and Harris type microdipper Injectors (80).
In the author's experience this type of Injector was more convenient
to use and gave more reproducible results than syringe type Injectors.
The column temperature was controlled by a proportional
controller. A fan inside the heating cabinet was employed to
insure uniform distribution of heat throughout the column.
The carrier gases used were argon for the p-ionizatlon
detector unit and nitrogen for the flame unit. In the latter unit,
compressed air and hydrogen were employed for the flame.
- 41 -

Preliminary Experiments
Results of preliminary experiments will be reported briefly
and only for a few representative cases. All measurements were
made on the chromatograph equipped with the p-lonizatlon detector
unless indicated otherwise. Columns were packed and cured as de¬
scribed. Retention times (tR) are reported in minutes. All inject¬
ions were made with a 2 pi aliquots of 1 pg / pi solutions. The
p-ionization detector was operated at 1500 volts. Detector temper¬
ature » 245-250°. Vaporizer temperature • 290-310°.
SE-30 Methyl Silicone Polymer (SE-30)
Three columns differing in concentration of SB-30, mesh size
of support or the length or diameter of the column were employed.
1* A column of 67, SE-30 coated on 30-60 mesh Chromosorb W
in a 5-foot 3/16" (o.d.) copper tube was prepared.
Variations of retention time with temperature of the
column and pressure of the carrier gas are given in
Table 1.
2. A column of 37 SE-30 coated on 30-60 mesh Chromosorb W
in a 5-foot 3/16" (o.d.) copper tube was prepared.
- 42 -

TABLE 1
Variation of Retention Time with Changes in Temperature and Pressure on 67, SE-30 Column
Conditions Steroids
Temp.°C
Pres, in psi
5a-Androstan.-“
¡
,r
3B-ol,17-one
3a-ol,17-one
3-one, 17|3“ol
3,17-dione
5[3-A-3a-ol-17-one
192
11.0
18.6
16.8
17.8
j
18.5
16.1
210
10.5
10.8
9.8
-
-
210
11.0
10.0
9.2
-
-
210
11.5
9.4
8.9
-
-
210
13.0
8.2
7.7
-
-
f•
210
14.0
8.0
7.4
7.6
8.1
7.0
•
224
15.0
3.8
3.7
3.8
4.0 |
3.4
In minutes.

Separation factors for androstane Isomers under
different conditions of temperature and pressure
were measured (Table 2). Acetate derivatives of
the steroids were prepared with the aim of improv¬
ing separation factors. Comparative retention
time values of free steroids and steroid acetates
are given In Table 3. Column conditions:
T • 195°, P - 18 psl.
3. A column of 3% SE-30 coated on 50-80 mesh Kromat CE
In a 5.5-foot 1/4" (o.d.) copper tube was prepared.
Retention values and separation factors of three
17-keto8terolds and the respective acetates are
given In Table 4. Column conditions: T » 200°,
P â–  20 psl.
Individual components could not be resolved when a mixture
containing the three principal urinary 17-ketosterolds was chromato¬
graphed. Acetylation of the steroids did not result In improved
separation. (See sections 2 and 3 above.)
Mixed Phases Containing SE-30
1. SE-30 and DC-silieone
A column coated with 3% SE-30 and 10X DC-si11cone was found
unsatisfactory. Results were not reproducible. The column
was discarded.
44

TABLE 2
Variation of Separation Factor with Temperature
and Pressure on 3% SE-30 Column
Steroids
Conditions
224°, 15 psi
210°, 14 psi
192°, 11 psi
5p-A-3a-ol,17-one
1.00
1.00
1.00
5a-A-3a-ol, 17-one
1.08
1.06
1.04
5a-A-3|3-ol, 17-one
1.11
1.14
1.16
5a-A-3-one,17p-ol
1.11
1.09
1.11
5a-A-3,17-dione
1.18
1.16
1.16
45

TABLE 3
a
Retention Times on 3% SE-30 Column
Steroids
Free
Acetate
5p-A-3a-ol,17-one
8.9
-
5a-A-3a-ol,17-one
9.2
12.5
5a-A-3-one,17(3-ol
9.8
15.9
5{3-A-3-one,17|3-ol
10.2
14.0
5Cü-A-3p-ol,17-one
10.1
-
5CÜ-A-3,17 -dione
10.4
-
5a-A-3a,17(3-dio 1
10.6
17.5
In minutes.
- 46 -

TABLE 4
a
Retention Times and Separation Factors on 37o SE-30 Column
Steroids Free Acetate
tR
Sep.fac.
CR
Sep.fac.
5p-A-3a-ol,17-one
6.3
1.00
20.5
1.00
5a-A-3a-ol,17-one
6.5
1.03
20.3
0.99
A-5-en-3|3-ol, 17-one
7.1
1.14
21.2
1.03
In minutes.
47 -

2. SE-30 and Ethyleneglycol Isoptitálete (EGIP)
Three coloans were prepared
a. 1.5X SE-30 + 0.5X EGIP
b. 0.5X EGIP
c. 1.5X SE-30
Comparative results obtained on the three columns
are given In Table 5, where the Individual contri¬
butions of each phase la visible, e.g., 2a vs. 2b.
3. SE-30 and Bis-(n-phenoxypheny1)-ether Mixed Phase
Three columns were prepared:
e. 3X Bis-(m-phenoxypheny1)-ether (Abbreviated
as phenoxy In Teble 6.)
b. 3X Bis-(m-phenoxyphenyl)-ether + 3X SE-30 (1:1 ratio)
c* 3X Bis-(m-phenoxypheny1)-ether + 3X SE-30 (1:2 ratio)
Comparison of the retention times are given In Table 6*
Column conditions: T • 245°, P « 30 psi«
The dependence of the chromatographic pattern of the steroids
on the concentration of each component of the stationary phase
was noteworthy (3b and 3c above). Separations could not be
affected due to large peak areas and some skewing of the peaks.
After a short period of use, Irregular bizarre patterns Indica¬
tive of phase decomposition were observed on this column and
further work was not continued.
48

TABLE 5
Comparison of Separation Factors on SE-30 and
Ethyleneglycol Isophtalate (EGIP) Columns
1.5% SE-30 +
FREE
0.57„ EGIP
0.5% EGIP
1.5% SE-30
5(3-A-3a-ol, 17-one
1.03
1.00
0.70
5a-A-3a-ol,17-onea
1.00
1.00
1.00
5p-A-3-one,17f3-ol
1.33
1.12
1.30
5a-A-3-one, 17f3-ol
1.18
1.12
0.87
5a-A-3p-o1,17-one
1.17
1.03
0.83
A-5-en-3p-ol,17-one
1.08
-
0.87
5a-A-3,17-dione
1.36
1.32
0.80
5a-A-3a,17p-diol
1.10
1.10
0.87
ACETATES
5f3-A-3a-ol, 17-one
1.06
1.17
1.25
a
5a-A-3a-ol, 17-one
1.00
1.00
1.00
5p-A-3-one,17(3-ol
1.34
1.75
1.25
5a-A-3-one,17p-o1
1.57
2.00
1.05
A-5-en-3(3-ol, 17-one
1.43
-
1.40
5a-A-3a,17p-diol
1.20
0.96
1.20
a
The values of these compounds were
for the calculation of separation
the acetates, respectively.
used as the point
factors of the free
of reference
steroids and
- 49 -

TABLE 6
a
Comparison of Retention Times on SE-30 and
Bis-(m-phenoxyphenyl)-ether Columns
Steroid Acetates
37o Phenoxy
3%
SE-30 and
37o Phenoxy
d:l
ratio)
(2:
1 ratio)
-R
Sep.fac.
CR
Sep.fac.
Sep.fac.
5a-A-3a-ol,17-one
17.6
4.4
21.5
1.36
12.5
0.54
5(3-A-3a-ol, 17-one
12.0
3.0
23.0
1.45
15.8
0.69
A-5-en-3(3-ol, 17-one
8.0
2.0
22.5
1.43
14.0
0.61
Cholestane
4.0
1.0
15.8
1.00
23.0
1.00
a
In minutes.
S
«
- 50 -

SB-52 Methyl Phenyl Silicone Polymer (SB-52)
A column of 3% SE-52 coated on 100-110 mesh Anakrom ABS In a
4-foot 3/16" (o.d.) copper tube was prepared. Retention time values
are given In Table 7. Column conditions: T ■ 212°, p • 25 psl
and flow rate â–  2 ml/mln.
«• - ¥
There was no observable advantage to be gained by the use of
SE-52 polymer as compared to SB-30 phase. The fine mesh support
caused a considerable decrease In flow rate and It was decided to per¬
form subsequent measurements on coarser than 100 mesh supports*
, K i i , j ,, i * -
Dow Corning High Vacuum Silicone grease
‘ * * \ • ' ' * '* ✓ '* ■■ .
A column of 30% DC-silieone coated on 40-50 mesh Anakrom ABS
In a 5-foot 3/16" (o.d.) copper tube was prepared. Column was too
retentive even after It was cut down to a length of 3 feet.
Bthyleneglycol Succinate Polyester (BGS)
A column of 12% B6S coated on 30-60 mesh Chromosorb V In 2.5-
foot 3/16" (o.d.) copper column was prepared* Column was very
retentive; no peaks were observed.
Neopentylglycol Adipate Polyester (NGAd)
A coliman of 3% NGAd coated on 30-60 mesh Chromosorb W in a
4-foot, 3/16" (o.d.) copper tube was prepared. Hie column required
51

TABLE 7
Retention Times on 3% SE-52 Column
Steroid
fcR
Rel. t.
Cholestane
14.6
1.00
5|3-A-17-one
2.9
0.20
5p-A-3-one,17p-ol
6.7
0.46
5(3-A-3a, 17(3-diol
6.2
0.42
5a-A-3a, 17f3-diol
6.5
0.44
5 a-A-3(3, 17p-diol
6.7
0.46
5f3-A-3,17-dione
6.7
0.46
In minutes.
52

daily priming with abólseteme. Isomeric retention time are given
in Table 8. Calven condition* T • 220*, P * 30 pel, flow rate ■
100 ml/via.
The retention of eolutee on the 4-foot MM colon me very
taw. Therefore, n column of 3* MAd coated on 30-60 neah Chronoeerh V
in n 6-foot 3/16" (o.d.) copper tobe wee prepared. The retention date
on three 17-ketoeterold acetates ere reported in Table 9. Colme
conditions: f • 230*, P • 20 pal, flew rata • 54 al/aln.
A colean of 31 PSSch coated en 30-60 nesh cturaaoaorh V Is t
6-foet 3/16" (o.d.) copper tube na prepared. The remite appeared
antIsfactory but the colman wee subjected te high temperaturas overnight
by e breakdown la the fay stature control unit. It wee discarded.
A colme ef 31 XT-1150 coated oe 50-60 neck Kroaet Ct is «
6-foot 3/16" (o.d.) copper tube wee prepared. The flew rate wee
extremely slew and lees ef phase me detested. The column wee
dicearded.
-33-

TABLE 8
a
Isomeric Retention Times
on 3% Neopentylglycol Adipate
!
Column
Steroid
tT?
Rel.t,,
j^p-A-3,17 -dione
5.8
3.06
v5a-A-3,17-dione
6.5
3.32
^5o:-A-3p, 17p-diol
7.7
4.05
l 5a-A-3a, 17(3-diol
6.7
3.53
/5a-A-3a,17(3-diol
J
6.7
3.53
C 5[3-A-3a, 17p-diol
6.7
3.53
pa-A-3a-ol, 17-one
5.4
2.84
^-5p-A-3a-ol, 17-one
5.5
2.89
' 5a-A-3a-ol, 17-one
5.4
2.84
^ 5a-A-3p-ol,17-one
6.1
3.21
rA-5-en-3p-ol,17-one
j
6.4
3.37
v5a-A-3p-ol,17-one
6.1
3.21
(5f3-A-3,17-dione
/
5.8
3.06
5f}-A-3-one, 17f3-ol
6.7
3.53
'5p-A-3-one,17(3-ol
6.7
3.53
^ 5(3-A-3a-ol, 17p-diol
6.7
3.53
^5a-A-17-ol
1.0
0.53
\
VCholestane
1.9
1.00
In minutes.
54 -

TABLE 9
Retention Times of 17-Ketosteroid Acetates on
3% Neopentylglycol Adipate Column
Steroid Acetates^Rel.tp
5a-A-3a-ol, 17-one
23.5
1.6
5¡3-A-3a-ol,17-one
25.0
1.7
® A-5-en-3(3-ol, 17-one
27.5
1.8
Cholestane
15.0
1.0
a
In minutes.
55

A column of 3% XF-1105 coated on 50-60 mesh Kromat CE in a
6-foot 3/16" (o.d.) copper tube was prepared. Retention data and
separation factors are given in Table 10. Column conditions:
T ■ 250°, P ■ 35 psi, flow rate ■ 20 ml/min. The flow rate was
maximal.
Even though the separation factors indicated theoretical
separations, actual resolutions of the components of the mixture were
not accomplished. The wide peak area, presence of some skewing and
the high retentivlty of the column in spite of the high temperature
of operation were major drawbacks.
OF-1-0065 Fluoroaklyl Polymer (OF-1-0065)
A column of 3% QF-1-0065 coated on 70-80 mesh Anakrom in a
6-foot 1/8" (o.d.) stainless steel tube was prepared. Measurements
were made on the chromatograph equipped with the flame ionisation
detector. Column conditions: T ■ 238°, P ■ 14 psi. It was
hoped that selective retention of this column for ketones could be
used to advantage in effecting separations. Loss of phase and
Irregular chromatographic patterns were observed and in spite of
numerous efforts, no reliable data could be obtained. One interpre¬
tation of the data was that the phase had reacted with the glass wool
plug.
- 56

TABLE 10
a
Retention Times and Separation Factors on 3% XF-1105 Column
Steroid
fcR
Rel.
Sep.fac.
5f3-A-3a-ol, 17-one
13.8
0.63
1.00
5a-A-3a-ol,17-one
16.8
0.76
1.22
A-5-en-3p-ol,17-one
24.0
-
1.09
1.74
5|3-A-3a-ol, 11,17-dione
25.0
1.14
1.81
5a-A-3a,11£-dio1,17-one
37.8
1.72
0.74
Cholestane
22.0
1.00
1.59
a
In minutes.
- 57

Discussion of Preliminary Kxperiaents
The choice of the proper solvent for the stationery phase ves
the first consideration for this study. The extended ring system of
the steroid aolecule provides e fairly wide eras of hydrocarbon
skeleton for Interactions with solvent molecules. Because of the
relative rigidity of the skeleton and the short range effectiveness
of the ran der Weals forces of attraction between the solute and the
solvent molecules, these Interactions are sensitive to sterlc factors
(3). The Interactions between the parts of the solute end the
solvent molecules compete with Interactions between the solvent mole¬
cules themselves. The solution of a polar substance in a polar
solvent may therefore be considered to depend upon the ability of the
polar groups of the solute to attract parts of the solvent molecule
with a force comparable to or greater than that among the solvent
molecules themselves. A non-polar solute, which cannot exert such a
force of attraction Is squeezed out of the polar solvent and the normal
attractive forces In the pure solvent ere re-established. Non-polar
solvents, on the other hand, exhibit far less short rengo attractive
forces than do polar solvents. The molecules of non-pplar solvents
are attracted to one another by relatively weak dispersion forces
and a non-polar solute may exhibit the same weak attractions as do
the solvent molecules themselves. A polar solute Introduced Into a
non-polar solvent Is expected to have very little or no Interaction
with the solvent.
58 ~

In highly unsaturated molecules, stronger Interactions may
occur due to delocalisation of electrons and result In an Increase
In the dispersion forces (81). The theoretical basis of such Inter¬
actions Is still Incomplete, but empirically It Is known that un¬
saturated molecules tend to attract one another more strongly than
they attract saturated molecules. Similarly, unsaturated groups
attract the molecules of polar substances much more strongly than
do saturated groups. Unsaturated groups may thus be assumed to be
po larisable.
In the present investigation the liquid solvent phases selected,
In addition to the chemical property considerations mentioned above,
had to meet as many of the following requirements as possible at the
temperature of operations! low volatility, thermal stability, low
viscosity and chemical Inertness. For temperatures above 200°, as
employed in this study, the choice of phases was limited. Satisfactory
phases at these higher temperatures Included silicone gums and poly-
♦ • • • -V • f 1
esters of low volatility.
Since oxygen substituted steroids of relatively high polarity
were to be studied simultaneously with their derivatives of higher or
lower polarity two basic types of liquid phases were Indicated. A
polar stationary phase would enhance the interaction of the polar groups
of the steroid molecule with the phase and a non-polar phase would be
expected preferentially to attract the non-polar functions.
59

SB-30 polymer was found to be a satisfactory non-selective
phase. Bis-(m-phenoxyphenyl)-ether was interesting in the unique
retention pattern of the 17-fcetosteroid acetates on this stationary
phase.(Table 6). However the compound was not stable at elevated
temperatures. The polyester phases exhibited the desired polarity
but the choice was narrowed to those which were thermostable. The
separation factors of the androstane Isomers on XF-1105 phase was
promising, but here again a phase combining the selective properties
of the silicone nitrile fluids and the thermel stability of silicone
polymers was indicated.
After preliminary investigations with different silicone
elastermers, silicone nitrile polymers and various polyester phases,
both singly and in mixtures, four stationary phases which differed in
degrees of polarity and selectivity were chosen: the methyl substituted
silicone gum SK-30 (non-selective, heat stable), the silicone nitrile
polymer XB-60 (similar to silicone nitrile fluids but more stable at
elevated temperatures) and neopentylglyeol sebacate and Hi-Bff 8B
(heat stable, polar) polyester phases.
The effect of varying the chemical composition of the stationary
phase for the separation of a wide range of steroids had been reported
(82-86). Lipsky and Landowne (83) had reported the thermal stability
and polar properties of neopentylglyeol sebacate* SB-30 silicone
polymer had found wide use as stationary phase since the early appli¬
cations of the gas-liquid chromatographic methods to steroid analyses.
60

XE-60 and Hi-Eff 8B were two new polymers developed specifically for
high temperature uses.
- 61 -

Comparative Exper traen te
All of the androatañes and the derivatives listed under
Materials and Methods were utilised In this portion of the study. The
concentration of the solutions were 2 jug/pi and 0.2 to 1 ;ul aliquots
were Injected. The detector and vaporiser temperatures were the same
as those given In the preliminary experiments. The gas chromatograph
equipped with the p-ioniration detector (operated at 1500 volts) was
employed unless Indicated otherwise.
Following columns were used:
SB-30. A column of 3X SE-30 coated on 40-50 mesh Anakrom ABS
In a 5-foot 3/16" (o.d.) copper tube was prepared. The chromatograph
equipped with the flame ionisation detector was employed. Coluan
conditions : T • 260°, P ■ 10 psl* Retention time for cholestane •
12-14 minutes.
XK-60. A column of 3X XE-60 coated on 80-100 mesh Dlatoport S
in a 6-foot 1/8" (o.d.) stainless steel tube was prepared. The chro¬
matograph equipped with the flame ionisation detector was employed.
Column conditions: T * 260-262°, P • 14 psl. Retention time of
cholestane â–  6-9 minutes.
62

Hl-Bff 8B. A column of 3X Hi-Eff 8B coatad on 40-50 mash
Anakrom ABS in a 5-foot 3/16" (o.d.) copper tuba was prepared.
Column conditions: T ■ 238°, P • 15 psi and flow rata ■ 39 ml.
Retention time for cholaatana * 6-8 minutes.
A column of IX Hi-Eff 8B coated on 80-100 mesh Diataport S
in a 6-foot 1/8" (o.d.) stainless steel tube was prepared. Column
conditions: T ■ 242°, P • 33 psi and flow rate * 24 ml/min.
Retention time for cholestane - 5-7 minutes.
t
NGSeb. A column of 3X NGSeb on 30-60 mesh Chromosorb V in a
6-foot 1/8" (o.d.) stainless steel tube «ms prepared. Coltaan
conditions: T • 232°, P » 25 psi and flow rate ■ 74 ml/min.
Retention time for cholestane • 8-9 minutes for the measurements
of the free steroids and the steroid acetates and 11 minutes for all
other compounds.
A column of IX NGSeb coated on 80-90 mesh Anakrom ABS in a
6-foot 1/8" (o.d.) stainless steel tube was prepared. Column conditions:
T ■ 231°, P • 30 pal and flow rate « 24 ml/min. Retention time for
cholestane â–  10 minutes.
The relative retention time values with respect to cholestane
observed on the six columns under the conditions described above are
susmarlzed in Table 11.
63 -

TABLE 11 - SE-30 3%*
A Comparison of the Relative Retention Times of Substituted Androstanes
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.21
-
-
-
5(3-Androstane
0.20
m
m
--
5a-A-17p-ol
0.32
0.39
0.33
-
5p-A-17p-ol
0.41
0.52
0.49
-
5a-A-17-one
0.32
-
-
-
5p-A-17-one
0.42
-
-
--
5a-A-3p-ol
0.31
0.39
0.34
0.24
5p-A-3p-ol
0.29
0.35
0.31
0.22
5a-A-3-one
0.33
-
-
5(3-A-3-one
0.32
-
-
-
5a-A-3a,17p-diol
0.54
0.89
0.58
0.28
5(3-A-3a,17p-diol
0.51
0.87
0.61
0.32
5a-A-3p,17p-diol
0.59
0.98
0.69
0.36
5p-A-3p,17(3-diol
0.49
%
0.86
0.56
-
5a-A-3a-ol,17-one
0.51
0.58
0.47
-
5p-A-3a-ol,17-one
0.47
0.56
0.46
0.28
5a-A-3p-ol,17-one
0.55
0.66
0.55
0.30
5p-A-3p-ol,17-one
•»
-
-
-
5a-A-3-one,17p-ol
0.59
0.76
0.64
0.35
5p-A-3-one,17f3-ol
0.54
0.69
0.59
0.33
5a-A-3-one,17a-ol
0.56
0.75
0.60
-
5(3-A-3-one,17a-ol
0.53
0.61
0.47
0.33
5a-A-3,17-dione
0.58
-
-
-
5p-A-3,17-dione
0.50
â– B
-
-
A-5-en-3p-ol,17-one
0.61
0.64
0.55
0.33

TABLE 11 - XE-60 3%b
(cont.)
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.27
-
-
-
5(3-Andró stane
0.25
-
-
-
5a-A-17p-ol
0.60
0.71
0.38
-
5(3-A-17(3-ol
0.89
1.04
0.52
5a-A-17-one
0.65
-
-
5{3-A-17-one
0.95
-
-
-
5a-A-3p-ol
0.65
0.70
0.39
-
5p-A-3p-ol
0.53
0.59
0.32
-
5a-A-3-one
0.77
-
am
-
5(3-A-3-one
0.68
-
-
-
5a-A-3a, 17(3-diol
1.67
2.05
0.49
-
5(3-A-3a,17p-diol
1.68
1.95
0.50
-
5a-A-3(3,17(3-diol
1.89
2.21
0.66
-
5(3-A-3(3,17(3-diol
1.56
1.90
0.51
-
5a-A-3a-ol,17-one
2.00
2.14
0.87
-
5p-A-3a-ol,17-one
1.90
2.15
0.94
-
5a-A-3(3-ol,17-one
2.10
2.44
1.16
-
5(3-A-3(3-ol,17-one
1.98
2.98
0.86
-
5a-A-3-one,17(3-ol
2.58
2.81
1.32
-
5(3-A-3-one,17(3-ol
%
2.44
2.53
1.18
--
5a-A-3-one,17a-ol
2.03
2.32
1.16
5p-A-3-one,17a-ol
2.14
2.14
0.91
-
5a-A-3,17-dione
2.64
-
m
-
5(3-A-3,17 -dione
2.48
-
m
-
A-5-en-3(3-ol,17-one
2.01
- 65 -
2.38
1.10
*

TABLE 11 - Hi-Eff 3%C
(cont.)
Steroids Free Acetate TMSi.XFA
5a-Androstane
0.13
-
-
-
5(3-Androstane
0.12
-
m
-
5a-A-17(3“ol
0.71
0.58
0.22
0.16
5p-A-17p-ol
1.48
1.27
-
-
5a-A-17-one
0.61
-
-
-
5(3-A-17-one
1.15
-
-
-
5a-A-3p-ol
0.75
0.71
0.24
0.20
5p-A-3p-ol
0.61
0.57
-
0.15
5a-A-3-one
0.73
m
-
-
5(3-A-3-one
0.67
-
-
-
5a-A-3a,17(3-diol
3.94
2.48
0.28
0.27
5p-A-3a,17(3-diol
3.85
2.82
0.37
0.30
5a-A-3p,17(3-diol
4.19
3.18
0.42
0.24
5(3-A-3(3,17(3-diol
3.50
2.42
0.28
0.23
5a-A-3a-ol,17-one
3.73
2.62
0.74
0.80
5p-A-3a-ol,17-one
3.58
2.86
1.03
0.61
5a-A-3p-ol,17-one
3.97
3.44
1.20
0.67
5p-A-3p-ol,17-one
-
-
-
-
5a-A-3-one,17p-ol
4.61
3.88
1.27
1.24
5p-A-3-one,17p-ol
4.18
3.44
1.13
1.14
5a-A-3-one,17a-ol
4.36
3.31
1.09
-
5p-A-3-one,17a-ol
3.97
2.91
0.81
0.78
5a-A-3,17-dione
4.00
-
-
-
5p-A-3,17-dione
3.64
-
-
-
A-5-en-3p-ol,17-one
4.00
- 66 -
3.38
1.16
1.02

TABLE 11 - Hi-Eff l%d
(cont.)
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.27
-
-
-
5(3-Andros tane
0.26
-
a*
-
5a-A-17(3-ol
0.74
0.65
0.29
0.16
5(3-A-17(3-ol
1.32
1.04
0.41
-
5a-A-17-one
0.63
-
-
-
5(3-A-17-one
1.14
-
-
-
5a-A-3(3-ol
0.75
0.67
0.32
0.20
5(3-A-3(3-ol
0.63
0.57
0.25
0.15
5a-A-3-one
0.78
-
-
-
5(3-A-3-one
0.70
-
-
-
5a-A“3a,17(3-diol
3.23
3.27
0.88
0.27
5(3-A-3a, 17(3-diol
3.20
2.52
1.07
0.30
5a-A-3p,17(3-diol
3.60
2.86
1.18
0.24
5(3-A-3(3,17(3-diol
2.93
2.23
0.86
0.23
5a-A-3a-ol,17-one
2.88
2.43
0.78
0.80
5f3-A-3a-ol,17-one
2.92
2.61
0.99
0.61
5a-A-3(3-ol, 17-one
3.24
2.95
1.15
0.67
5(3-A-3(3-ol,17-one
-
-
-
-
5a-A-3-one, 17(3-ol
3.84
3.28
1.24
1.24
5p-A-3-one,17p-ol
3.49
3.04
1.13
1.14
5a-A-3-one,17a-ol
3.18
2.92
1.11
-
5(3-A-3-one, 17a-ol
3.26
2.58
0.85
0.78
5a-A-3,17-dione
3.63
-
-
-
5p-A-3,17-dione
3.32
-
-
-
A-5-en-3(3-ol, 17-one
3.32
- 67 -
2.92
1.14
.1.02

TABLE 11 - NGSeb 37,®
(cont.)
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane
0.09
-
-
-
5f3-Androstane
0.08
-
-
-
5a-A-17(3-ol
0.47
0.44
0.20
0.16
5p-A-17p-ol
0.84
0.80
-
0.32
5a-A-17-one
0.39
-
-
-
5(3-A-17-one
0.71
-
-
-
5a-A-3(3-ol
0.47
0.47
0.21
0.18
5p-A-3(3-ol
0.38
0.37
-
0.13
5a-A-3-one
0.49
-
-
5p-A-3-one
0.42
-
-
-
5a-A-3a,17(3-diol
2.27
1.69
0.31
0.29
5(3-A-3a,17p-diol
2.08
1.89
0.35
0.34
5a-A-3p,17(3-diol
2.44
2.20
0.44
0.41
5p-A-3p,17p-diol
1.93
1.66
0.28
0.28
5a-A-3a-ol,17-one
1.83
1.61
0.58
0.67
5f3-A-3a-ol,17-one
1.80
1.76
0.72
0.73
5a-A-3p-ol,17-one
2.10
2.00
0.91
0.94
5(3-A-3f3-ol,17-one
1.88
-
-
-
5a-A-3-one, 17|3-ol
2.61
2.38
1.07
1.18
5p-A-3-one,17p-ol
2.20
1.98
0.91
0.97
5a-A-3-one,17a-ol
2.22
2.12
0.94
-
5(3-A-3-one, 17a-ol
2.05
1.76
0.62
0.50
5a-A-3,17-dione
2.27
-
-
-
5p-A-3,17-dione
1.98
P»
-
-
A-5-en-3p-ol,17-one
2.09
1.99
0.90
0.84
- 68 -

f
TABLE 11 - NGSeb 1%
(cont.)
Steroids
Free
Acetate
TMSi TFA
5a-Androstane
0.14
-
-
5|3-Androstane
0.12
-
-
5a-A-17p-ol
0.46
0.45
0.24
5f3-A-17f3-ol
0.80
0.80
0.39
5a-A-17-one
0.39
-
-
5(3-A-17-one
0.68
-
-
5a-A-3(3-ol
0.48
0.49
0.26
5(3-A-3(3-ol
0.39
0.41
0.40
5a-A-3-one
0.48
-
-
5(3-A-3-one
0.42
-
-
5 a-A-3a,17(3-diol
1.88
0.73
0.36
5(3-A-3a,17(3-diol
1.82
0.84
0.40
5a-A-3(3,17p-diol
2.14
0.82
0.50
5p-A-3p,17(3-diol
1.70
0.65
0.35
5a-A-3a-ol,17-one
1.64
1.50
0.60
5p-A-3a-ol,17-one
1.59
1.57
0.73
5a-A-3(3-ol,17-one
1.85
1.88
0.88
5(3-A-3(3-ol,17-one
1.53
1.48
0.74
5a-A-3-one,17(3-ol
2.24
2.10
0.96
5p-A-3-one,17(3-ol
1.96
1.91
0.87
5a-A-3-one, 17a-ol
-
-
-
5(3-A-3-one,17a-ol
1.82
1.61
0.63
5a-A-3,17-dione
2.00
-
-
5(3-A-3,17-dione
1.78
-
-
A*5-en-3(3-ol, 17-one
1.86
1.78
0.84
Retention time of Cholestane:
a b
12-14, 6-9,
c e
6-8, 9-10,
f
10 minutes.
6?

Elution pattern of the leomerlc free steroids on the six
columns ere given In Table 12.
The calculated group retention factors for the hydroxyl,
ketone, acetyl and trlmethylsilyl groups are given In Table 13. A
comparison of observed and calculated retention time values based
on average log k values are reported in Table 14.
The changes In retention time upon derivative formation are
calculated according to the equation.
^ 'derivative ^ 'free steroid^ * 100 * r* *
The results are summarised In Table 15.
The average change In retention time consequent to derivative
formation and the dependence of this change upon the number of
substituent groups are shown In Table 16.
Table 17 gives the values of the T term calculated according
to the following equation
t* t'
T - XE-60 - SB-30
SB-30
A comparison of the ketone selective properties of the four
column phases Is given In Table 18.
70

TABLE 12 - SE-30 3%
Comparison of Retention Data of
Isomeric Pairs: Substituent Effects
Functional Group
5a
. 5P
No Substitution
0.21
0.20
170-ol
0.32
0.41
30-ol
0.31
0.29
3a,170-diol
0.54
0.51
30,170-diol
0.59
0.49
17-one
0.32
0.42
3-one
0.33
0.32
3,17-dione
0.58
0.50
3a-ol,17-one
0.51
0.47
30-ol,17-one
0.55
-
3-one,170-ol
0.59
0.54
3-one,17a-ol
0.56
0.53
71

TABLE 12 - XE-60 3%
( cont. )
Functional Group
5a
5P
No Substitution
0.27
0.25
170-ol
0.60
0.89
30-ol ..
0.65
0.53
3a,170-diol
1.67
1.68
30,170-diol
1.89
1.56
17-one
0.65
0.95
3-one
0.77
0.68
J â– 
3,17-dione
2.64
2.48
3a-ol,17-one
2.00
1.90
30-ol,17-one
2.10
1.98
3-one,170-ol
2.58
2.44
3-one,17a-ol
2.09
2.14

TABLE 12 - Hi-Eff 3%
( cont. )
Functional Group
5a
5P
No Substitution
0.13
0.12
17p-ol
0.71
1.48
3£-ol
0.75
0.61
3a,17^-diol
3.94
3.85
3£,17p-diol
4.19
3.18
17-one
0.61
1.15
3-one
0.73
0.67
3,17-dione
4.00
3.64
3a-ol,17-one
3.73
3.58
3£-ol,17-one
3.97
-
3-one,17¡3-ol
4.61
4.18
3-one, 17a-ol-
4.36
3.97
73

TABLE 12 - Hi-Eff 1%
( -cont. )
Functional Group
5a
No Substitution
170-ol
3f}-ol
3a,17p-diol
3p,17^-diol
17-one
3-one
3,17-dione
3a-ol,17-one
3p-ol,17-one
3-one,17¡3-ol
3-one,17a-ol
0.27
0.74
0. /5
3.23
3.60
0.63
0.78
3.63
2.88
3.24
3.84
3.18
0.26
1.32
0.63
3.20
2.93
1.14
0.70
3.32
2.92
3.49
3.26

TABLE 12 - NGSeb 37»
( cont. )
Functional Group
5a
53
No Substitution
0.09
0.08
170-ol
0.47
0.84
30-ol
0.47
0.38
3a,170-diol
2.27
2.08
30,170-diol
2.44
1.93
17-one
0.39
0.71
3-one
0.49
0.42
3,17-dione
2.27
1.98
3a-ol,17-one
1.83
1.80
30-ol,17-one
2.10
1.88
3-one,170-ol
2.61
2.20
3-one,17a-ol
2.22
2.05
75

TABLE 12 - NGSeb 1%
( cont. )
Fuñe tiona1 Group
5a
5P
No Substitution
0.13
0.12
170-01
0.46
0.80
30-ol
0.48
0.39
3a,170-diol
1.88
1.82
30,170-diol
2.14
1.70
17-one
0.39
0.68
3-one
0.48
0.42
3,17-dione
2.00
1.78
3a-ol,17-one
1.64
1.59
30-ol,17-one
1.85
1.53
3-one,170-ol
2.25
1.96
3-one,17a-ol
-
1.82

TABLE 13 - SE-30 37.
Calculated Group Retention Factors
(log r = log rn + log ka + log kb)
Functional 5a 5 P>
Group Free Acet TMSi Free Acet ' TMSi
17ft-ol
17£-ol / Androstane
0.19
0.28
0.20
0.31
0.41
3p,17£-diol / 3p-ol
0.27
0.40
0.31
0.23
0.39
3-one,17£-ol / 3-one
0.25
0.36
0.29
0.23
0.34
17a-ol
3-one,17a-ol / 3-one
0.23
0.36
0.26
0.22
0.28
3B-o1
3<3-o1 / Androstane
0.18
0.28
0.22
0.16
0.25
3¡3,17(3-diol / 17p-ol
0.26
0.40
0.32
0.08
0.22
3¡3-ol, 17-one / 17-one
0.24
0.31
0.23
-
-
3a-ol
3a,170-diol / 17p-ol •'
0.22
0.35
0.25
0.10
0.23
3a-ol,17-one / 17-one
0.21
0.26
0.17
0.05
0.13
17-one
17-one / Androstane
0.19
-
-
0.32
0.32
3¡3-ol, 17-one / 3p-ol
0.25
0.22
0.20
•
_
3,17-dione / 3-one
0.24
-
-
0.20
-
3-one
3-one / Androstane
0.20
-
-
0.20
—
3-one, 17f3-ol / 17¡3-ol
0.26
0.29
0.29
0.12
0.13
3,17-dione / 17-one
0.26
-
-
0.08
-
77- -

TABLE 13
(_cont.
- XE-60 3%
5 a
56
Grouo
Free
Acet
TMSi
Free
Acet
TMSi
176-ol
17p-ol / Androstane
0.35
0.42
0.14
0.55
0.62
0.32
36,176-diol / 3g-ol
0.41
0.50
0.23
0.47'
0.51
0.20
3-one, 17f}-ol / 3-one
0.51
0.63
0.23
0.55
0.57
0.24
17a-ol
3-one, 17a-ol / 3-one .
0.42
0.48
0.18
0.50
0.50
0.13
3p-ol
3fJ-ol / Androstane
0.38
0.41
0.16
0.33
0.37
0.11
3p, 17p-diol / 176-ol
0.45
0.49
0.25
0.24
0.26
-0.01
36-ol,17-one / 17-one
0.51
0.57
0.25
0.32
0.50
-0.04
3a-ol
3a,176-diol / 176~ol
0.44
0.46
0.12
0.28
0.27
-0.02
3a-ol,17-one / 17-one
0.49
0.47
0.07
0.30
0.35
0.00
17-one
17-one / Androstane
0.38
.
0.58
36~ol,17-one / 36-ol
0.51
0.54
0.47
0.57
0.70
0.43
3,17-dione / 3-one
0.53
-
-
0.56
-
-
3-one
3-one / Androstane
0.45
-
-
0.43
-
-
3-one,176-ol / 176_ol
0.62
0.60
0.55
0.44
0.38
0.36
3,17-dione / 17-one
0.61
-
-
0.42
-
-
p
-
78 -

TABLE 13 - Hi-Eff 37.
( cont. )
5a '5p
ÍU11Ü LJLUIlcUL
Group
Free
Acet
TMSi
Free
Acet .
TMSi
176-oi
170-ol / Androstane
0.74
0.66
0.23
1.09
1.02
30,170-dio1 / 30-ol
0.75
0.65
0.26
0.76
0.63
-
3-one,170-ol / 3-one
0.80
0.73
0.24
0.79
0.71
0.23
17a-ol
3-one,17a-ol / 3-one
0.78
0.66
0.18
0.77
0.64
0.08
36-ol
30-ol / Andróscene
0.77
0.74
0.27
0.70
0.67
30,170-dio 1 / 170-ol
0.77
0.74
0.28
0.37
0.28
-
30-o1,17-one / 17-one
0.81
0.75
-0.70
-
-
-
3a-ol
3a, 170-diol / 170-ol â– 
0.74
0.63
0.10
0.41
0.35
-
3a-ol,17-one / 17-one
0.79
0.63
0.08
0.49
0.40
-0.05
17-one
17-one / Androstane
0.68
0.98
30-ol,17-one / 30-ol
0.72
0.68
-0.30
-
-
-
3,17-dione / 3-one
0.74
-
-
0.73
-
3-one
3-one / Androstane
0.76
-
-
0.74
-
-
3-one,170-ol / 170-ol
0-81
0.82
0.76
0.45
0.43
-
3,17-dione / 17-one
0.82
-
-
0.50
-
-
79

TABLE 13 - Hi-Eff 1%
( cont. )
Functional
Group
Uft-ol
179-ol / Androstane
30,17^-diol / 3p-ol
3-one, U9-0I / 3-one
17Q-ol
3-one,17a-ol / 3-one .
39-Ql
3p-ol / Androstane
39,179-diol / 17p-ol
39-01,17-one / 17-one
3a-ol
3a, 179-diol / 179-0I
3a-ol,17-one / 17-one
17-one
17-one / Androstane
39-0I,17-one / 39-0I
3,17-dione / 3-one
3-one
3-one / Androstane
3-one,I79-0I / I79-0I
3,17-dione / 17-one
5a 59
Free
Acet
TMSi
^
Free
Acet
TMSi
0.44
0.38
0.03
0.71
0.61
0.20
0.68
0.63
0.57
0.67
0.59
0.54
0.69
0.62
0.20
0.70
0.64
0.21
0.61
0.57
0.15
0.67
0.57
JL.08
0.45
0.40
0.08
0.39
0.35
-0.01
0.69
0.64
0.61
0.35
0.33
0.45
0.71
0.67
0.26
0.48
0.50
-0.18
0.64
0.54
0.48
0.38
0.38
0.42
0.66
0.59
0.09
0.40
0.36
-0.06
0.37
.
0.65
.
.
0.64
0.64
0.56
0.74
0.80
0.48
0.67
-
—
0.67
“
—
0.46
0.43
0.71
0.70
0.63
0.42
0.47
0.44
0.76
-
-
0.46
-
-
- 80 -

TABLE 13-NGSeb 3%
(,'cont. )
5a 53
r LLuua jl
Group
Free
AceC
TMSi
Free
AceC
TMSi
• /
176-01
17¡3-ol / Andros cane
0.72
0.69
0.35
1.02
1.00
33,173-diol / 3p-ol
0.71
0.67
0.32
0.71
0.65
3-one, U3-0I / 3-one
0.73
0.69
0.34
0.72
0.67
0.34
l7a-ol
3-one,17a-ol / 3-one
0.66
0.64
0.28
0.69
0.62
0.17
3p-ol
33-ol / AndrosCane
0.72
0.72
0.37
0.68
0.66
--
33,173-dio 1 / 173-01
0.71
0.70
0.34
0.36
0.32
--
33~ol,17-one / 17-one
0.73
0.71
0.37
0.42
--
--
3a-ol
3a,173-dio 1 / 173-ol •
0.68
0.58
0.19
0.39
0.37
--
3a-ol,17-one / 17-one
0.67
0.62
0.17
0.40
0.39
0
17-one
17-one / Androscane
0.64
--
—
0.94
—
—
33-ol,17-one / 33-ol
0.65
0.63
0.64
0.69
—
—
3,17-dione / 3-one
0.67
—
—
0.67
—
- -
3-one
3-one / Androscane
0.73
—
—
0.72
--
—
3-one,I73-0I / I73-0I
0.74
0.73
0.73
0.42
0.39
—
3,17-dione / 17-one
0.76
—
--
0.44
--
81

TABLE 13 - NGSeb 1%
( cont. )
5a 5p
Functional ,
Group Free Acet TMSi Free Acet TMSi
175-Ql
17p-ol / Androstane
0.53
0.52
0.25
0.82
0.82
0.51
3g,17B-diol / 3p-ol
0.65
0.22
0.28
0.64
--
-.06
3-one, 17f}-ol / 3-one
0.67
0.64
0.30
0.67
0.66
0.32
17a-ol
3-one,17a-ol / 3-one •
-
-
-
.64
.58
.18
3b-o1
33-0I / Androstane
0*55
0.56
0.28
0.51
0.53
0.52
3^,17p-diol / 17B-ol
0.67
0.26
0.32
0.33
-
-.05
3p-ol,17-one /17-one
0.68
0.68
0.35
0.35
0.34
0.03
3a-ol
3a, 17f3-diol / 17£-ol
0.61
0.21
0.18
0.36
0.02
0.01
3a-ol,17-one / 17-one
0.62
0.58
0.19
0.37
0.36
0.03
17-one
17-one / Androstane
0.46
--
--
0.75
—
—
33-01,17-one / 3j3-ol
0.58,
0.58
0.53
0.59
0.5,6
0.27
3,17-dione / 3-one
0.61
- “
0.63
3-one
3-one / Androstane
0.55
. --
0.54
“ —
” “
3-one,17p-ol / 17g-ol
0.69
0.67
0.60
0.39
0.38
0.35
3,17-dione / 17-one
0.71
“ -
- “
- 0.42
“ “
82

TABLE 14
a
Comparison of .Calculated and Observed Retention Time Values
Relative to Cholestane
Steroids
3% SE-
-30
37,
XE-60
37» Hi
-Eff 8B
37o
NGSeb
FREE
5CC-A-3,17-dione
Caled.
0.60
Obsd.
0.58
Caled,
2.88
, Obsd.
2.64
Caled.
4.17
Obsd.
4.00
Caled.
2.19
Obsd.
2.27
5p-A-3,17-dione
0.50
0.50
2.52
2.48
3.17
3.64
1.59
1.98
5a-A-3-one, 17(3-ol
0.63
0.59
2.58
2.58
4.68
4.61
2.57
2.61
5(3-A-3-one, 17(3-ol
0.50
0.54
2.24
2.44
3.32
4.18
1.74
2.20
ACETATES
5a-A-3p,17p-diol
1.00
0.98
2.75
2.21
3.39
3.18
2.19 2.20
5p-A-3p,17p-diol
0.81
0.86
2.24
1.90
_
2.42
1.66
TMSi Ether
5a-A-3(3,17p-diol
0.69
0.69
0.71
0.66
-
0.42
0.40
0.44
5p-A-3(3,17p-diol
-
-
0.47
0.51
0.28
0.28
Based on average log k values

TABLE
15 - SE-30 370a
Ratio of Relative
Retention
Times of Derivatives
to Free
Steroids
Steroids
Acetate
TMSi
TFA
5a-Androstane
-
-
-
5p-Andróstane
m
-
-
5a-A-17(3-oi
1.22
1.02
-
5p-A-17p-ol
1.27
1.19
-
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3p-ol
1.25
1.09
0.75
5p-A-3p-ol
1.22
1.09
0.75
5a-A-3-one
-
-
-
5p-A-3-one
-
-
--
5a-A-3a, 17f3-diol
1.65
1.09
0.52
5p-A-3a,17(3-diol
1.69
1.09
0.63
5a-A-3(3,17f3-diol
1.68
1.17
0.62
5f3-A-3(3,17(3-diol
1.75
1.14
-
5a-A-3a-ol,17-one
1.13
0.91
-
5(3-A-3a-ol, 17-one
1.20
0.98
0.60
5a-A-3p-ol,17-one
1.89
0.99
0.54
5p-A-3p-ol,17-one
-
-
-
5a-A-3-one,17p-ol
1.30
1.09
0.60
5(3-A-3-one, 17f3-ol
1.28
1.09
0.61
5a-A-3-one,17a-ol
1.32
1.07
-
5p-A-3-one,17a-ol
1.14
0.90
0.62
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
84

TABLE 15 - XE-60 3%b
(Cont.)
Steroids
Acetate
TMSi
5a-Androstane
5(3-Androstane
-
«.
5a-A-17p-ol
1.18
0.63
5p-A-17p-ol
1.17
0.58
5a-A-17-one
-
5p-A-17-one
-
-
5a-A-3p-ol
1.08
0.59
5p-A-3p-ol
1.11
0.60
5a-A-3-one
-
-
5p-A-3-one
-
-
C
5a-A-3a,17p-diol
1.23
0.29
5p-A-3a,17p-diol
1.16
0.30
5a-A-3p,17p-diol
1.17
0.30
5p-A-3p,17p-diol
1.22
0.33
5a-A-3a-ol,17-one
1.07
0.44
5p-A-3a-ol,17-one
1.13
0.50
5a-A-3p-ol,17-one
1.16
0.55
5(3-A-3(3-ol,17-one
1.51
0.43
5a-A-3-one,17(3-ol
1.09
0.51
5(3-A-3-one,17p-ol
1.04
0.48
5a-A-3-one,17a-ol
1.14
0.57
50-A-3-one,17a-ol
1.00
0.43
5a-A-3,17-dione
-
-
5p-A-3,17-dione
•»
- 85

I
TABLE 15
(Cont.)
- Hi-Eff 3%c
Steroids
Acetate
TMSi
TFA
5a-Androstane
-
-
-
5^-Androstane
-
• -
5a-A-17p-ol
0.82
0.31
0.23
5p-A-17p-ol
0.86
-
-
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3p-ol
0.95
0.32
0.27
5(3-A-3j3-ol
0.93
-
0.25
5a-A-3-one
-
-
-
5(3-A-3-one
-
-
-
5a-A-3a,17p-diol
0.63
0.07
0.07
5p-A-3a,17f3-diol
0.73
0.10
0.08
5a-A-3p,17p-diol
0.76
0.10
0.06
5p-A-3p,17p-diol
0.69
0.08
0.07
5a-A-3a-ol,17-one
0.70
0.20
0.21
5(3-A-3a-ol,17-one
0.80
0.29
0.17
5a-A-3p-ol,17-one
0.87
0.30
0.17
5f}-A-3p-ol, 17-one
-
-
-
5a-A-3-one,17p-ol
0.84
0.28
0.27
5p-A-3-one, 17f3-ol
0.82
0.27
0.27
5a-A-3-one,17a-ol
0.76
0.25
-
5p-A-3-one,17a-ol
0.73
0.20
0.20
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
_
mm
86

TABLE 15
(Cont.)
- Hi-Eff 17od
Steroid
Acetate
TMSi
TFA
5a-Androstane
-
-
•
5(3-Androstane
-
-
-
5a-A-17p-ol
0.88
0.39
0.23
5f3-A-17f3-ol
0.79
0.31
-
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3j3-ol
0.89
0.43
0.27
5f3-A-3g-ol
0.90
0.40
0.25
5a-A-3-one
-
-
-
5p-A-3-one
-
-
-
5a-A-3a, 17p-diol
0.70
0.27
0.07
5p-A-3a,17p-diol
0.79
0.33
0.08
5a-A-3p,17p-diol
0.79
0.33
0.05
5(3-A-3f3, 17(3-diol
0.76
0.29
0.06
5a-A-3a-ol,17-one
0.84
0.27
0.21
5p-A-3a-ol,17-one
0.89
0.34
0.17
5a-A-3p-ol,17-one
0.91
0.35
0.17
5f3-A-3(3-ol,17-one
-
-
-
5a-A-3-one, 17(3-ol
0.85
0.32
0.27
5p-A-3-one,17f3-ol
0.87
0.32
0.27
5a-A-3-one,17a-ol
0.92
0.35
-
5(3-A-3-one, 17a-ol
0.79
0.26
0.20
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
_
87

TABLE 15 - NGSeb 3%e
(Cont.)
Steroids
Acetate
TMSi
TFA
5a-Androstane
-
-
-
5|3-Androstane
-
-
-
5a-A-17(3-ol
0.94
0.43
0.34
5p-A-17p-ol
0.95
-
0.38
5a-A-17-one
-
-
-
5p-A-17-one
-
-
-
5a-A-3p-ol
1.00
0.45
0.38
5p-A-3p-ol
0.97
-
0.34
5a-A-3-one
-
-
-
5p-A-3-one
-
-
-
5a-A-3a,17p-diol
0.74
0.14
0.13
5 (3-A-3a, 17f3-diol
0.91
0.17
0.16
5a-A-3p, 17p-diol
0.91
0.18
0.17
5f3-A-3¡3,17p-diol
0.86
0.15
0.15
5a-A-3a-ol,17-one
0.88
0.32
0.37
5p-A-3a-ol,17-one
0.98
0.40
0.41
5a-A-3j3-ol,17-one
0.95
0.43
0.45
5f3-A-3j3-ol,17-one
-
-
-
5a-A-3-one,17p-ol
0.91
0.41
0.45
5p-A-3-one,17p-ol
0.90
0.41
0.44
5a-A-3-one,17a-ol
0.95
0.42
-
5p-A-3-one,17a-ol
0.86
0.30
0.24
5a-A-3,17-dione
-
-
-
5p-A-3,17-dione
m
88

TABLE 15 - NGSeb 17of
Steroids Acetate TMSi
5a-Androstane
5(3-Androstane
5a-A-17£-ol
5p-A-17p-ol
5a-A-17-one
5¡3-A-17-one
5a-A-3p-ol
5p-A-3p-ol
5a-A-3-one
5f3-A-3-one
5a-A-3a,17p-diol
5f3-A-3a,17p-diol
5a-A-3p,17p-diol
5f3-A-3(3,17(3-diol
5a-A-3a-ol,17-one
5p-A-3a-ol,17-one
5a-A-3(3-ol,17-one
5f3-A-3(3-ol,17-one
5a-A-3-one,17p-ol
5(3-A-3-one, 17(3-ol
5a-A-3-one,17a-ol
5p-A-3-one,17a-ol
5a-A-3,17-dione
5p-A-3,17-dione
Retention time of Cholestane: a12-14
0.98
0.52
0.10
0.49
1.02
0.54
1.05
0.39
0.19
0.46
0.22
0.38
0.23
-
0.21
0.92
0.37
0.99
0.46
1.02
0.48
0.97
0.48
0.93
0.43
0.97
0.44
0.89
0.35
b6-9, C6-8, d5-7, e9-ll, f10 minutes
89

TABLE -16
The Average Change in Retention Time Occurring in Derivative Formation
( rderiv/ rfree x 100 = r'>4 )
Column
Acetate
TMSi
TFA
Mono
Di
Mono
Di
Mono
Di
SE-30 3%
123
169
102
115
69
59
XE-60 37o
110
120
53
30
—
—
Hi-Eff 8B 37o
83
70
27
9
23
7
NGSeb 37o
93
85
41
16
40
15
- 90

TABLE 17
Values of T at 260° Based on Measurements Obtained with
3% SE-30 and 3% XE-60 Columns
( T
t' t'
XE-60 - SE-30
i
t SE-30
)
Functional Group
5a
17p-ol
0.88
1.14
17-one
1.03
1.26
3(3-ol
1.09
0.83
3-one
1.33
1.13
3a,17(3-diol
2.09
3.08
3(3, 17(3-diol
2.21
2.19
3a-ol,17-one
2.92
3.04
3p-ol,17-one
2.82
-
3-one,17(3-ol
3.38
3.52
3-one,17a-ol
2.63
3.04
3,17-dione
3.55
3.98
- 91 -

TABLE 18
Comparison of Selective Retention of the Four Phases
for Ketone and Hydroxyl Groups
Relative Retention Times
Steroids
XE-60a SE-30b NGSebc Hi-Effd
5a-Androstan-3(3-ol
0.40 0.32 0.47 0.75
5(3-Andros tan-3(3-ol
0.35 0.26 0.39 0.63
5a-Androstan-3-one
0.98 0.36 0.48 0.78
5(3-Andros tan-3-one
0.88 0.31 0.42 0.70
a
Cholestane 9 minutes. 37« XE-60 on 80-100 mesh Diatoport S.
b Cholestane 13.6 minutes.
. 1% SE-30 on 80-90 mesh Anakrom ABS.
c
Cholestane 10 minutes.
17o Neopentyl glycol sebacate on 80-90 mesh
d Cholestane 6.3 minutes.
17o Hi-Eff 8B on 80-100 mesh Diatoport S.
92 -

The A log r parameters for tha oxidation of a hydroxyl group
to a ketone are given in Table 19.
Separation factors of androstane Isomers calculated according
to the equation
r 5a / r 5p " separation factor
are given in Table 20.
Discussion of Comparative Experiments
This study has confirmed the observations of Lipsky and Lendovne
(83) with respect to the properties of the neopentylglycol sebacate
phase. In concentrations as low as one per cent (w/w) reproducible
results were obtained with the neopentylglycol sebacate over a period
of two to three months. Furthermore, after the initial curing process,
this column could be used any time thereafter with remarkable reproduci¬
bility of initial retention data, a fact which may be indicative of the
absence of any further loss of the phase during repeated use.
The Hi-Eff 8B phase, a polymer of dlmethylcyclohexylsucclnate,
exhibited about the same degree of polarity and thermal stability as did
neopentylglycol sebacate. It is of Interest that while there was no
observable difference in the performance and characteristics of the one
and three per cent neopentylglycol sebacate columns, scam variation in
the retention pattern between the one and three per cent Hi-Eff 8B
- 93

TABLE 19 - SE-30 3%
The Change in log r Contribution from Hydroxyl to Ketone Transitions
( Log Ar OH -» one)
.
Functional Groups
5a
50 .
17(3-ol / 17-one
0-
0.01
3(3-ol / 3-one
0.02
0.04
3o-o1-17-one / 3,17-dione
0.05
0.03
3(3-ol-17-one / 3,17-dione
0.02
-
3-one-17(3-ol / 3,17-dione
0.01
0.03
3-one-17a-ol / 3,17-dione
0.01
0.02
3a,170-diol / 3,17-dione
0.03
0.01
3a,170-diol / 3a-ol,17-one
0.02
0.04
3a, 17{3-diol / 3-one,170-ol
0.04
0.02
3(3,17(3.-diol / 3,17-dione
0.01
0.01
3p, 17(3-diol / 30-ol-17-one
0.02
30,17(3-diol / 3-one-17p-ol
0-
0.04
- 94

I
TABLE 19 - XE-60 3%
( cont. )
Functional Groups
5a
56
17f}-ol / 17-one
0.50
0.20
3f3-ol / 3-one
0.40
0.40
3a-ol-17-one / 3,17-dione
0.19
0.19
33~ol-17-one / 3,17-dione
0.17
0.17
3-one-17p-ol / 3,17-dione
0.08
0.08
3-one-17a-ol / 3,17-dione
0.18
0.14-
3a, 17(3-diol / 3,17-dione
0.27
0.24
3a,17p-diol / 3a-ol,17-one
0.08
0.06
3a,17p-diol / 3-one,17p-ol
0.19
0.17
3g,17p-diol / 3,17-dione
0.23
0.27
3¡3,17p-diol / 3p-ol-17-one
0.07
0.10
3p,17p-diol / 3-one-17p-ol
0.16
0.19
95

TABLE 19 -Hi-Eff 3%
( cone.)
Functional Groups
170-ol / 17-one
3f3-ol / 3-one
3a-ol-17-one / 3,17-dione
30-ol-17-one / 3,17-dione
3-one-170-ol / 3,17-dione
3-one-17a-ol / 3,17-dione
3a, 170-diol / 3,17-dione
3a, 170-diol / 3a-ol, 17-one
3a, 170-diol / 3-one,17p-ol
30,170-diol / 3,17-dione
30,-170-diol / 30-ol-17-one
30,170-diol / 3-one-170-ol
5a
50
0.06
0.11
0.01
0.04
0.03
0.06
0
-
0.06
0.06
0.03
0.03
0.01
0.02
0.02
0.03
0.07
0.04
0.02
0.02
0.02
-
0.04
0.08
96 -

TABLE 19 -Hi-Eff 17.
c cont. )
Functional Groups
5a
56
17{3-ol / 17-one
0.07
0.06
3(3-ol / 3-one
0.02
0.05
3a-ol-17-one / 3,17-dione
0.02
0.06
3(3-ol- 17-one / 3,17-dione
0.05
-
3-one-17(3-ol / 3,17-dione
0.02
0.02
3-one-17a-ol / 3,17-dione
0.06
0.01
3a,17(3-diol / 3,17-dione
0.05
0.02
3a,17p-diol / 3a-ol,17-one
0.05
0.04
3a, 17p-diol / 3-one,17p-ol
0.07
0.04
3^,17p-diol / 3,17-dione
0
0.05
3^,17p-diol / 3£}-ol-17-one
0.05
-
3p,17^-diol / 3-one-17f3-ol
0.03
0.08
- 97 -

TABLE 19 - NGSeb 37.
( cont. )
Functional Groups
5a
56
17(3-ol / 17-one
0.08
0.07
3(3-ol / 3-one
0.02
0.04
3a-o1-17-one / 3,17-dione
0.09
0.04
3|3-ol-17-one / 3,17-dione
0.03
0.02
3-one-17(3-ol / 3,17-dione
0.06
0.04
3-one-17a-ol / 3,17-dione
0.01
0.02
3a, 17f3-diol / 3,17-dione
0
0.02
3a,17p-diol / 3a-ol,17-one
0.09
0.06
3a,17(3-diol / 3-one, 17(3-ol
0.06
0.02
3p,17p-diol / 3,17-dione
0.03
0.01
3p,-17p-diol / 3(3-ol-17-one
0.06
0.01
3p,17p-diol / 3-one-17p-ol
0.03
0.06
•- 98 -

TABLE 19 - NGSeb 1%
( cont. )
Functional Groups
5a
56
17f3-ol / 17-one
0.07
0.07
3p-ol / 3-one
o-
0.03
3a-ol-17-one /'3,17-dione
0.09
0.05
3p-ol-17-one / 3,17-dione
0.03
0.07
3-one-17f3-ol / 3,17-dione
0.06
0.04
3-one-17a-ol / 3,17-dione
-
0.01
3a, 17¡3-diol / 3,17-dione
0.03
0.01
3a, 17p-diol / 3a-ol,17-one
0.06
0.06
3a, 17p-diol / 3-one, 17j3-ol
0.08
0.03
3g,17p-diol / 3,17-dione
0.03
0.02
3(3,-17{3-diol / 3p-ol-17-one
0.06
0.05
3g,17p-diol / 3-one-17p-ol
0.02
0.06
99

TABLE 20 - SE-30 3%
Isomeric Separation Factors
( 5a / 5p ).
Steroids
Free
Acetate
TMSi
TFA
5a-Androstane /5(3-Andró stane
1.04
-
-
-
5a-A-17p-ol/5p-A-17p-ol
0.80
0.76
-
-
5a-A-17-one/5¡3-A-17-one
0.76
-
-
-
5a-A-3p-ol/5g-A-3p-ol
1.09
1.11
-
1.08
5a-A-3-one/5^-A-3-one
1.03
-
-
-
5a-A-3a, 17^-diol/5p-A-3a, 17p-diol
1.04
1.02 .
0.95
0.87
5a-A-3p, 17fJ-diol/5f3-A-3p, 17p-diol
1.19
1.14
1.22
-
5a-A-3a-ol, 17-one/5p-A-3a-ol, 17-one
1.10
1.03
1.02
-
5a-A-3£-ol,17-one/5p-A-3p-ol,17-one
-
-
-
-
5a-A-3a-ol,17-one/A-5-ene-3p-ol,17-one
0.85
0.90
0.86
-
5p-A-3a-ol,17-one/A-5-ene-3(3-ol,17-one
0.77
0.87
0.84
0.85
5a-A-3p-ol,17-one/A-5-ene-3p-ol,17-one
0.91
1.02
1.00
0.91
5¡3-A-3(3-ol, 17-one/A-5-ene-3¡3-ol, 17-one
-
-
-
-
5a-A-3-one, 17f3-ol/5£-A-3-one, 17£-ol
1.08
1.10
1.08
1.06
5G¡-A-3-one, 17a-ol/5{3-A-3-one, 17a-ol
1.06
1.23
1.26
-
5a-A-3,17-dione/5f}-A-3,17-dione
«
100

TABLE 20 - XE-60 3%
( cont. )
Steroids
Free
Acetate
TMSi
TFA
5a-Andros tane/5£J-Andros tane
1.08
-
-
-
5a-A-17£-ol/5(3-A-17p-ol
0.70
0.68
0.73
-
5a-A-17-one/5p-A-17-one
0.68
-
-
-
5a-A-3p-ol/50-A-3p-ol
1.23
1.17
1.19
-
5a-A-3-one/5(3-A-3-one
1.13
-
-
-
5a-A-3a, 17p-diol/5p-A-3a, 17(3-diol
0.99
1.05
0.98
-
5a-A-3p, 17p-diol/5p-A-3p, 17p-dio 1
1.21
1.16
1.29
-
5a-A-3a-ol,17-one/5f3-A-3a-ol,17-one
1.05
1.00
0.93
-
5a-A-3j3-o1,17-one/5p-A-3p-o1,17-one
1.06
0.82
1.35
-
5a-A-3a-ol,17-one/A-5-ene-3p-ol,17-one
1.00
0.90
0.79
-
5p-A-3a-ol,17-one/A-5-ene-3p-ol,17-one
0.95
0.90
0.85
' -
5a-A-3f}-ol, 17-one/A-5-ene-3p-ol, 17-one
1.04
1.03
1.05
-
5p-A-3p-ol,17-one/A-5-ene-3p-ol,17-one
0.99
1.25
0.78
-
5a-A-3-one,17¡3-ol/5¡3-A-3-one,17p-ol
1.06
1.11
1.12
-
5a-A-3-one,17a-ol/5£-A-3-one,17a-ol
0.98
1.08
1.28
-
5a-A-3,17-dione/5(3-A-3,17-dione
-
-
-
-
101

TABLE 20 - Hi-Eff 8B 3%
( cont. )
Steroids
Free
Acetate
TMSi
TFA
5a-Andros tane/5j3-Andros tane
1.06
-
-
-
5a-A-17^-0l/5g-A-17f3-ol
0.48
0.46
-
-
5a-A-17-one/5g-A-17-one
0.53
-
-
-
5a-A-3p-ol/5£-A-3p-ol
1.23
1.25
-
1.33
5a-A-3-one/5[3-A-3-one
1.09
-
-
-
5a-A-3a, 17p-diol/5p-A-3a,17p-diol
1.02
0.88
0.76
0.90
5a-A-3p,17p-diol/5p-A-3p,17p-diol
1.20
1.33
1.50
1.03
5a-A-3a-ol,17-one/5(3-A-3a-ol,17-one
1.04
0.92
0.72
1.31
5a-A-3p-ol,17-one/5p-A-3(3-ol,17-one
-
-
-
-
5a-A-3a-ol, 17-one/A-5-ene-3(3-ol, 17-one
. 0.93
0.78
0.64
0.78
5p-A-3a-ol,17-one/A-5-ene-3f3-ol,17-one
0.90
0.85
0.89
0.60
5a-A-3p-ol,17-one/A-5-ene-3p-ol,17-one
0.99
1.02
1.03
0.66
5p-A-3g-ol,17-one/A-5-ene-3j3-ol,17-one
-
-
-
-
5a-A-3-one,17p-ol/5p-A-3-one, 17(3-ol
1.10
1.13
1.12
1.09
5a-A-3-one,17a-o 1/5(3-A-3-one,17a-ol
1.10
1.14
1.35
-
5a-A-3,17-dione/5^-A-3,17-dione
1.10
-
-
-
102

TABLE 20 - Hi-Eff 8B 1%
C cont. )
Steroids
5a-Andros tane/5¡3-Andros tane
5a-A-17p-ol/5£-A-17p-ol
5a-A-17-one/5(3-A-17-one
5a-A-3£-o1/5p-A-3p-o1
5a-A-3-one/5f3-A-3-one
5a-A-3a,17p-diol/5p-A-3a, 170-diol
5a-A-3p,17p-diol/5p-A-3p,17p-diol
5a-A-3a-ol,17-one/5£-A-3a-ol,17-one
5a-A-3g-ol,17-one/5f3-A-3f3-ol,17-one
5a-A-3a-ol,17-one/A-5-ene-3p-ol,17-one
5f3-A-3a-ol, 17-one/A-5-ene-3g-ol, 17-one
50A-3(3-ol, 17-one/A-5-ene-3(3-ol, 17-one
5p-A-3p-ol,17-one/A-5-ene-3p-ol,17-one
5a-A-3-one,17f3-ol/5f3-A-3-one, 17f3-ol
5a-A-3-one, 17a-o 1/5(3-A-3-one, 17Cü-ol
5a-A-3,17-dione/5(3-A-3,17-dione
Free
Acetate
TMSi
TFA
1.04
-
-
-
0.56
0.62
0.71
-â– 
0.55
-
-
-
1.19
1.18
1.28
1.33
1.11
-
-
-
1.01
0.90
0.82
0.90
1.23
1.28
1.37
1.04
0.99
0.93
0.79
1.31
0.94
0.82
1.54
0.87
0.83
0.68
0.78
0.88
0.89
0.87
0.60
0.98
1.01
1.00
-
1.04
- 1.23
0.66 -
0.65
1.10
1.18
1.10
1.09
0.98
1.13
1.31
-
1.09
_
103

TABLE 20 - NGSeb 37.
( cont. )
Stfc.; oids
Free
Acetate
TMSi
TFA
5a-Andros tane/5(3-Andros tane
1.13
-
-
-
5a-A-17p-ol/5p-A-17(3-ol
0.56
0.55
-
0.50
5a-A-17-one/5f3-A-17-one
0.55
-
-
-
5a-A-3p-o 1/5(3-A-3(3-ol
1.24
1.27
-
1.38
5a-A-3-one/5(3-A-3-one
1.17
-
-
-
5a-A-3a, 17f}-diol/5(3-A-3a, 17p-dlol
1.09
0.89
0.89
0.85
5a-A-3p, 17p-diol/5p-A-3p, 17p-diol
1.26
1.33
1.57
1.46
5a-A-3a-ol, 17-one/5(3-A-3a-ol, 17-one
1.02
0.91
0.81
0.92
5a-A-3(3-ol,17-one/5(3-A-3(3-ol, 17-one
1.12
-
-
-
5a-A-3a-ol,17-one/A-5-ene-3(3-ol,17-one
0.88
- 0.81 -
0.64-
'0.80
5p-A-3a-ol,17-one/A-5-ene-3(3-ol,17-one
0.86
0.88
0.80
0.87
5a-A-3(3-ol, 17-one/A-5-ene-3(3-ol, 17-one
1.00
1.01
1.00
1.12
5|3-A-3[3-ol, 17-one/A-5-ene-3(3-ol, 17-one
0.90
-
-
-
5a-A-3-one,17(3-ol/5(3-A-3-one, 17(3-ol
1.19
1.20
1.18
1.22
5a-A-3-one, 17a-ol/5f3-A-3-one, 17a-ol
1.08
1.20
1.52
-
5a-A-3,17-dione/5(3-A-3,17-dione
1.15
-
-
-
104 -

TABLE 20 - NGSeb 1%
( cont. )
Steroids
5a-Androstane/5|3-Andros tane
5a-A-17p-ol/5p-A-17p-ol
5a-A-17-one/5(3-A-17-one
5a-A-3p-ol/5(3-A-3p-ol
5a-A-3-one/5f3-A-3-one
5a-A-3a, 17p-diol/5p-A-3a,170-diol
5a-A-3p, 17p-diol/5f3-A-3p, 17p-dio 1
5a-A-3a-ol, 17-one /5p-A-3a-ol, 17-one
5a-A-3p-ol,17-one/5p-A-3p-ol,17-one
5a-A-3a-0l,17-one/A-5-ene-3p-ol,17-one
5(3-A-3a-ol, 17-one/A-5-ene-3f3-ol, 17-one
5a-A-3p-ol, 17-one/A-5-ene-3j3-ol, 17-one
5£-A-3(3-ol, 17-one/A-5-ene-3p-ol, 17-one
5ct-A-3-one, 17p-ol/5p-A-3-one, 17p-ol
5a-A-3-one, 17a-ol/5p-A-3-one, 17a-ol
5a-A-3,17-dione/5j3-A-3,17-dione
Free
Acetate
TMSi
TFA
1.13
-
-
-
0.58
0.56
0.61
-
0.57
-
-
-
1.23
1.20
0.65
-
1.14
-
-
1.03
0.87
0.90
-
1.26
-
1.43
-
1.03
0.96
0.82
-
1.21
1.27
1.19
-
0.88
0.84
0.71
0.80
0.86
0.88
0.87
0.72
1.00
1.06
1.02
-
0.82
0.83
0.88
0.76
1.15
1.10
1.10
-
-
-
-
-
1.12

colimns was observed. One explanation for the change In the
retention pattern of steroids on the one per cent Hl-Eff 8B coluna
night be the presence of uncoated support surfaces. Either the
viscosity of the polyester or possibly the porosity of the Dlataport S
support nay result In Incomplete covering of the solid surface by the
liquid polymer.
XE-60, a recently developed silicone co-polymer consisting of
equal amounts of dimathylslloxane and cyanoethylmethylslloxane,
exhibited selective retention of compounds containing ketone substi¬
tuents. Even though the experience of the author with this stationary
phase has been most satisfactory, a word of caution scans necessary.
Changes In the degree of selectivity which were observed after a few
weeks of constant use at 260° might be Indicative of loss of stationary
phase or loss of a volatile component which was responsible for se¬
lective ketone retention. However, excellent stability of this phase
at 350° was reported (87).
The least polar and the least selective phase studied was the
methyl silicone gum SE-30. This elastomer was characterised by high
thermal stability over a long period of time and exhibited retention
which was directly proportional to the molecular weight of the solute.
Wotlz (88), using an SE-30 column, has reported the separation of several
major estrogen derivatives, which differ In the number of acetoxy groups.
It has been reported that the particular non-selectlve property of the
SB-30 elastomer with respect to various functional groups might make
106

this an ideal stationary phase In the determination of a parameter
called steroid number (89). During the comparative study each liquid
phase was studied Individually and mixed phases were not employed.
It was felt that with a mixed stationary phase the contribution of one
group might be masked while that of others might dominate. The
possibility of employing mixed phases to effect separation Is, however,
a very attractive one In gas chromatography and might be the key to
successful applications. Son» Investigators have reported the use of
mixed packing but their results appear inconclusive (90).
Concentration of the Stationary Phase and Mash Sise of Support
The second consideration, after the choice of suitable stationary
phase for the column, was the concentration of this phase. The de¬
pendence of the height equivalent to a theoretical plate to the amount
of stationary phase Is given In the van Deemter equation (22).
£ * 2 Ad + -h.üíM + 811 á.. U
T “ TT* 2 Dllq
Experiments designed to test the Interrelationship of the various terms
of the above equation have been reported.
Bohemsn and Purnell have reported that the eddy diffusion
term (or the multiple peth effect), which was considered to be Inde¬
pendent of the velocity of the carrier gas, may actually be Inversely
proportional to it (24). Improved column efficiencies were achieved
by a decrease In mesh sise and an increase in uniformity of support.
107

Similarly, increased column efficiency consequent to reduced retie
of liquid phase to solid support vas observed (91) and led to the
use of low load columns of one to five per emit instead of the ten
to thirty per cent range employed during the early stages of appli¬
cations of gas-liquid chromatography. Recently Hishta and his
co-workers have reported the reduction of the time required for
analysis by the simultaneous reduction of temperature and concentra¬
tion of stationary phase on column support (92).
A word of caution is necessary on low-load columns. Incomplete
and non-uniform distribution within the support as a result of
insufficient amount of liquid phase may lead to exposed sites on the
column support. The result would be a system of not two but three
components* This point was recently discussed by Keller and Stewart
(93). According to these authors low load columns are usually of
such a composition that neither the two-phase system of liquid
partition nor that of a solid adsorption predominates and the description
and treatment of these systems as two phase systems is incorrect.
Thus, while liquid-liquid distribution certainly dominates most
successful solvent systems designed as partition systems, complications
due to adsorption and other factors are quite probable and should not be
minimised. Martin (94) has suggested that the law Rp values of
basic amino acids in chromatograms based on starch or cellulose, which
ve a smaller than the Rp values expected \on the basis of partition
108

coefficients In the pure solvents of a given system, might be due
to ion-exchange with carboxyl groups or to association with the
partially dissolved polysaccharide molecules of the stationary
phase.
While evidence for the thermal stability of steroids, which
allows their vapor phase separations Is abundant (95-97) occasional
Instances of alteration during gas chromatography of the more sensi¬
tive type of molecules such as the corticosteroids have been re¬
ported (98). The reason for this decomposition Is not clear.
Recently Wotiz (99) has reported incomplete coating of the column
as a possible cause of the decomposition of steroid molecules.
Wotiz studied the behavior of several types of steroids on column
supports coated with stationary phases of various concentrations.
Decomposition was observed on very low-load columns ( 0.25 - 0.50
w/w per cent ) even for very stable steroids. A possible expla¬
nation suggested by Wotiz Is that during gas chromatography the
solute molecule which was activated In the vapor phase my be de¬
activated In the liquid phase by dissipating Its energy of activation
to the solvent molecules. If there Is Insufficient amount of solvent
to absorb the activated molecule, thermal degradation might occur.
109

The interrelationship* of the terms In the van Deeater
equation and the effect of each tens of the equation on 5 may be
summarised as follows:
The smaller and more uniform the particle sise of
the solid support, the smaller Is H. The contri*
butlon of mesh sise Is negligible at low flow rates
but becosaes a major contributing factor to 5 at
high rates of flow.
The smaller the concentration of the liquid coating
on the column, the smaller Is H. When the range
of flow Is wide, a larger concentration of the liquid
coating results In Increased column efficiency* When
the range of flow Is narrow, better performance Is
achieved with low*load columns.
The smaller the quantity of the sample, the higher Is
m ' *
the number of theoretical plates and the smaller Is H*
A clarification of the last point Is necessary* The sise of
the sample Is directly dependent on the concentration of the liquid
phase. Heavily coated columns can tolerate larger amounts samples
than can low*load coltsons* Therefore, a discussion of the Influence
of sample sise is meaningful only as expressed simultaneously with
the percentage of stationary phase.
* 110

The theoretical dependence of H upon experimental parameters
in terms of the extended van Deemter equation and the experimental
evaluation of rate equation constants are discussed in a compre¬
hensive manner by Purnell (25).
On the basis of preliminary results obtained, liquid concen¬
trations of three per cent and mesh range of 40-50 were chosen for
the comparative study. These conditions could be altered in either
direction if necessary. For the comparative study on structure of
steroids and their chromatographic behavior the above conditions
vare found to be satisfactory for all columns. These conditions
allowed a sufficient amount of phase for interactions in a partition
system and a vide range for the variation of flow.
On the XB-60 columns the peaks were sharp and symmetrical*
On the remaining three columns, free steroids and their acetylated
derivatives tended to give wide peaks, especially for the dioxygenated
compounds, but the trimsthylsilyl and trlfluoroacetate derivatives
were eluted rapidly with sharp peaks.
The separation of the 17-ketosterolds and of the two pregnane
isomers could be effected only on the XK-60 column and not on the
other columns in spite of efforts to effect separations by variations
of temperature and flow rate. Columns with one per cent liquid
phase on finer mesh supports were constructed for the S8-30,
neopentylglycol sebacate and Hl-Kff 8B phases.
Ill

As predicted by the van Deenter equation, the combined
effect of the finer and more uniform particle size of the support
and the reduced concentration of the stationary phase «as to in¬
crease column resolution. It must be emphasized that column ef¬
ficiency Is not the sole factor of importance In gas-liquid
chromatography. Some excellent separations can be achieved on
columns not operating at maximum efficiency. Selective retention
of the liquid plays a role here. Furthermore, column efficiency
Is not an absolute quantity but varies for different compounds.
The van Deemter equation makes available to the Investigator a
tool for improving and revising column conditions and efficiency
when desired.
112

Separation of Urinary 17-Ketosterolds(100)
Known Mixture»
A known mixture of the trliMthylsllyl ethers of the principal
17-ketosterolds and two pregnane Isomers were chromatographed In the
usual manner on four columns.
A column of IX SB-30 coated on 80-90 mesh Anakrom ABS In a
6-foot 1/8" (o.d.) stainless steel tube was prepared. The chromato¬
graph equipped with the flame Ionization detector was employed. Column
conditions: T • 251°, P • 14 psl, Retention time for cholestane »
13.6 minutes.
Other columns used In the separation study were IX Hi-Bff 8B,
IX NGSeb and 3X XE-60 under the same conditions as described above.
Results are presented In Table 21. The five major urinary 17-ketosterolds
were resolved on the XE-60, Hl-Eff 8B and NGSeb columns. Only partial
separations were obtained on the SB*30 column.
Chromatograms showing the separation of the urinary keto-
sterolds and the progesterone metabolites are shown In Figures 9-11.
The seperatlon of the two pregnane isomers simultaneously with the 17-keto¬
sterolds was achieved only on the Hl-Eff 8B and XE-60 columns.
- 113 -

TABLE 21
Separation of Principal 17-Ketosteroids and Progesterone Metabolites
as Trimethylsilyl Ethers
Steroids
XE-60a
SE-30b
NGSebc
Hi-Effd
•. I
3a-Hydroxy-5a-androstan-17-one
1.02
0.47
0.60
0.78
3o:-Hydroxy-5f3-androstan-17-one
1.18
0.50
0.73
0.98
3p-Hydroxyandrost-5-en-17-one
1.36
0.56
0.84
1.14
3a-Hydroxy-5p-androstane-ll,17-dione
2.40
-
1.36
2.03
3a-llp-Dihydroxy-5Q:-androstan-17-one
2.57
-
1.87
2.59
5p-Pregnane-3a,20a-diol
0.78
-
0.70
0.68
3a-Hydroxy-5p-pregnan-20-one
1.44
-
1.08
1.38
a Cholestane 9 minutes. 37« XE-60 on 80-100 Diatoport S.
b Cholestane 13.6 minutes. 1% SE-30 on 80-90 mesh Anakrom ABS.
0
Cholestane 10 minutes. 1% Neopentyl glycol sebacate on 80-90 mesh.
Cholestane 6.3 minutes. 1% Hi-Eff 8B on 80-100 mesh Diatoport S.
114

115
MINUTES
FIGURE 9
Gas-liquid chromatogram of a mixture of known steroid trimethylsilyl ethers on 37» XE-60 column.
Conditions as described in text. (A) 5p-Pregnane-3a,20a-diol, (B) 3a-hydroxy-5á-androstan-l7-one,
(C) 3a-hydroxy-5f3-androstan-l7-one, (D) 3p-hydroxyandrost-5-en-l7-one, (E) 3cc-hydroxy-5p-pregnan-
20-one, (F) 3a-hydroxy-5B-androstane-ll,17-dione, (C) 3a,lip-dihydroxy-5a-androstan-l7-one.
height units

FIGURE 10 . . .
Gas-liquid chromatogram of a mixture of known steroid trimethylsilyl ethers on
1% Hi-Eff 8B column. Conditions as described in text. (A) 5p-Pregnane-3a,20a-
diol. (B) 3a-hydroxy-5a-andros tan-17-one, (C) 3a-hydroxy-5£-androstan-17-one,
(D) 3(3-hydroxyandrost-5-en-17-one, (E) 3a-hydroxy-5p-pregnan-20-one, (F) 3a-
hydroxy-5p-androstane-ll,17-dione, (G) 3a,llp-dihydroxy-5a-androstan-17-one.
- 116 -
A.

FIGURE 11
Gas-liquid chromatogram of a mixture of known steroid trimethylsilyl ethers
on 1% NGSeb column. Conditions as described in text. (A) 3a-Hydroxy-5a-
androstan-17-one, (B) 3a-hydroxy-5p-androstan-17-one, (C) 3p-hydroxyandrost-
5-en-17-one, (D) 3a-hydroxy-5p-pregnan-20-one, (E) 3a-hydroxy-5p-androstane-
11,17-dione, (F) 3a,ll£-dihydroxy-5a-androstan-17-one.
- 117

Preparation of Urine Sample
A portion (24 ml) of a 24 hour specimen was treated
with 2000 units/«I of helix pomatia extract ( Gluaulase, Endo
Laboratories) at pH 4.8 and 37° for 24 hours followed by extraction
with dlchloroisethane. The extract was washed with 0.1 N NaOH and
water and the extract was carefully evaporated in vacuo to complete
dryness. The residue was then treated with hexamethyldlsllazane
and trlaethylchloro8ilane as described under Materials and Methods.
The chromatogram of a 1/2040 aliquot of the total urine is shown in
Figure 12.
Discussion of the Separation of Urinary 17-Ketosterolda
All measurements were made on the trlmethylsllyl deri¬
vatives. The five major urinary 17-ketosterolds and the two preg¬
nane metabolites were separated on the Hl-Eff 8B column when the
column conditions were adjusted to give maximum resolution for the
17-ketosteroids exclusively. The importance of low-load columns
in achieving higher column resolution was thus evident. On the one
per cent neopentylglycol sebacate column, separations could be ac¬
complished for the five principal urinary ketosteroids, however,
3a-hydroxy-5p-androstan-17-one could not be separated from 5p-
pregnane-3a,20a-dlol. All seven steroids were resolved on the three
per cent XE-60 column. Only partially resolved peaks could be
effected on the one per cent SE-30 column. This result was not
surprising since the non-selectivity properties of the SE-30 polymer
118

I I "T
20 10 0
MINUTES
FIGURE 12
Gas-liquid chromatogram of urinary components as trimethylsilyl ethers on
3% XE-60 column. Conditions as described in text. (A) 3a-hydroxy-5a-
androstan-17-one, (B) 3a-hydroxy-5p-androstan-17-one, (C) 3p-hydroxy-
androst-5-en-17-one, (D) 3a-hydroxy-5p-androstane?ll, 17-dione, (E) 3a-
llp-dihydroxy-5a-androstan-17-one.
119 -
HEIGHT UNITS

were evident In the preliminary experiments. The purpose for In¬
cluding the SE-30 phase In the present investigation ves primarily
as a reference for comparison of the more selective phases.
120

Study of the Dependence of Relative Retention Tine on
Temperature and on the Nature of the Reference Standard
In order to aaaeas the variation of relative retention time
with changes in temperature the retention times of three androstane
trlasthylallyl ethers and of choiestane were determined on the one per
cent SK-30 column (described above) at four different temperatures.
Relative retention times were then calculated first as related to
choiestane and then as related to the trlmethylsllyl ether of 3a-
hydroxy-Sa-androstan-17-one. The results are given In Table 22.
Discussion of Factors Affecting Retention Tine Values
Temperature is a major factor which affects the elution time
of a solute in a gas-liquid chromatographic column. The rate of flow
is also a contributing factor to the retention time values* However,
the rate of flow varies inversely with temperature, hence its effect is
not always obvious.
While changes in absolute retention times with changes in
temperature are normally expected, the variation of relative retention
values with temperature poses a problem to the investigator. The
changes in relative retention time over a twenty degree temperature
range were recently reported (89) and the recommendation was made
121 -

122
TABLE 22
The Effect of the Reference Standard on the Variation of Relative Retention Time with Temperature
on 1% SE-30 Column
Steroids
Temp.,
°C
251
240
230
221
a
b
c
a
b
c
a
b
c
a
b
c
(1)
5q:-A-3q:-o1, 17-one
6.3
0.46
1.00
8.8
0.43
1.00
12.3
0.40
1.00
15.5
0.38
1.00
(2)
5p-A-3a-ol,17-one
6.8
0.50
1.08
9.4
0.46
1.07
13.0
0.42
1.06
16.5
0.40
1.07
(3)
A-5-en-3p-ol,17-one
7.6
0.56
1.21
10.8
0.53
1.23
15.2
0.50
1.24
19.3
0.47
1.24
(4)
Cholestane
13.6
-
-
20.4
30.6
_
•
41.0
a - Observed absolute retention time in minutes. b.- Retention
relative to cholestane. c - Retention relative to (1).

that all measurements on all columns should ba made at the same
temperature. While such a precautionary measure might be desirable
in comparative work, it may not always be feasible. For solutes
of low mobility it is more advantageous to employ higher tempera¬
tures than higher flow rates, because retention time varies ex¬
ponentially with temperature and linearly with the velocity of the
carrier gas. Furthermore, high-load columns require higher
temperatures than low-load columns.
An alternative to the constant temperature conditions as
suggested by Vandenheuvel and Horning (89) is the use of a reference
standard other than cholestane. If a standard structurally similar
to the group of compounds under investigation is employed, then,
under the same conditions, variations with temperature in the re¬
tention time of such a standard would be expected to be in approxi¬
mately the same ratio as the variations in the retention time of the
samples. Hie experiment designed to test this assumption supported
the above argument (Table 22). When cholestane is used as the
reference standard, the mean variation of the relative retention
time over the 30° range is 3.5 per cent. When one of the androatane
isomers is employed as the reference standard, the mean variation is
0.5 per cent.
The dependency of retention times on the quantity and volume
of the sample was also reported by the same authors (89). Small
123 -

volumes and concentrations of samples preferrably In the same
solvent were suggested. In the course of the present Investi¬
gation 0.2 to 1 pi aliquots of 2 pg/pl concentrated solutions
were chromatographed with good reproducibility of retention times.
However, in the Isolated Instances where larger volumes were used,
reproducibility of the data was not compromised. large quantities
of samples which adversely affect column efficiency should be
avoided, expeclally in low-load columns. Variation in retention
times was not observed when different solvents were used. Never¬
theless, it is desirable to employ the same solvent especially in
comparative studies provided all of the solutes are soluble in one
solvent

DISCUSSION
Relationship of Chromatographic Behavior to Chemical Structure
Retention Patterns
*
Certain deductions concerning the polarities of the four
stationary phases nay be drawn from the relative retention times of
the free steroids and the three derivatives (Table 11). The
polarltlea of these phases are approximated as Hl-Bff 8B â–  NGSeb }
XE-60 ) SB-30. The general pattern of relative retention time
values is: free steroid) acetate ) trimethyls llyl > trlfluoroacetate
for the two polyester columns; acetate ) free steroid > trlmethyl-
sllyl for the XE-60 polymer; acetate ) trimethylsllyl - free
steroid ) trlfluoroacetate for the non-polar SB-30 elastosMr.
Derivatives have been employed since the early days of
chromatography (101-106). The use of derivatives in steroid analysis
was advocated by Wotlz (86,105) and VandenHeuvel at al..(73.75.106).
There are several advantages to the use of derivatives in preference
to the free steroids. Derivatives are more stable to heat.
Polarity can be varied by the formation of a derivative. Molecular
125

weight difference* «ay result in better separation* where differences
in the number of substituent groups exist. Greater sensitivity may
be obtained with derivatives when p-ionisation detectors are employed
(79).
The retention time of a solute in the chromatographic column
is the sum of its residence time in each phase of the column and la
dependent on the degree of interaction of the solute and the solvent.
Hence, a change in the polarity of a molecule consequent to derivative,
formation is reflected in its retention time. A substituent such as
the acetyl group will decrease the polarity due to the hydroxyl group
and contribute to an increased retention time value of the steroid
acetate on non-polar columns and to a decreased value on polar columns.
The results obtained in the present investigation were in agreement
with the qualitative predictions.
In the case of trlmethylsilyl ethers the great reduction in
retention times on polar columns is due to the fact that the tri-
methyls ilyl group behaves like a non-polar functional group. The
group is sufficiently bulky to affect molecular weight and shape,
but does not show selective functional-group retention effects. The
high volatility of these ethers is indicated by the lower than ex¬
pected retention times on the non-polar columns and by the unexpec¬
tedly low retention times on the polar coluasts.
126

The changes brought about in the steroid nolecule by the
substitution of the trifluoroacetyl group are ambiguous. In
general, trifluoroacetyl derivatives have been found to be less
satisfactory than the trismthylsilyl derivatives (107). The ease
of decomposition of the trifluoroacetyl derivatives on chromato¬
graphic columns Introduce some unreliability in their data.
Research was not pursued on these derivatives.
One further observation emerges from the results summarised
in Table 11. Changes in retention time values of tbs steroids con¬
sequent to derivative formation notwithstanding, the derivatives
follow the retention pattern of the isomeric free steroids in all
but a few Instances. However, the retention time values of the
isomeric derivatives do not always rataln tha same ratio to each
other as do the corresponding free compounds. A similar observa¬
tion was made by Lipsky and Landowne (83), who reported that the
observed decreases in retention times of steroid acetates on polar
columns were not the same in all casas. The reversal of the order
of elution upon derivative formation was seen in the case of the
lsomtirlc 3a,17£-dlol and 3a-ol, 17-one pairs with almost all of the
columns studied. However, no explanation was evident for this
anomaly.
- 127

Effect of Structure on Retention Pata
On «11 columns studied the retention time values of the
5a-endrostene nucleus Is consistently greeter then that of the 5p-
lsomer as seen in Table 12. Although this difference In the values
of the 5a- and the 5p-androstane nuclei la small, It la nevertheless
constant and reproducible on all columns and Is reflected In the
retention time values of the oxygenated nuclei as well. A basic
structural difference or a unique chemical property must account
for the greater mobility of the 5p-androstane molecule. A possible
explanation may lie In the planarity of the 5a-androatane nucleus.
The vide area of the planar molécula enhances the Interactions of
the steroid with the liquid phase of the column, hence decreases
the mobility of the 5a-compound.
The pattern established by the parent nuclei Is followed
by all the C-3 oxygen and C-3,17 dioxygen substituted Isomers. The
length of time by which the substituted 5p«steroids precede the 5a-
lsomers varies from column to column and the variation Is not In tha
same ratio as those of the parent nuclei.
The Isomeric pairs of androstan-17p-ol and androstan-17-one
not only do not follow the pattern of their respective nuclei but
reverse it markedly. Thus 17-oxygenated Sp-androstanes exhibit
retention values nearly double those of the 5a-Isomers. This reversal
is most pronouncad In the case of the two polar columns and less
128

pronounced for the XE-60 and SE-30 columns. It is evident that
the presence of an oxygen function In the C-17 of the steroid
nucleus strongly Influences the elution ratlc of the 5a/5p Isomers
and that this Influence Is dominant regardless of the nature of the
liquid solvent. To our knowledge this Is the first observation
of the anomalous effect of the 17-oxygen function In androstanes
or in any other class of steroids. The consistent observation of
the effect of this group In all the solvents under study Is In
agreement with the general observation In other types of chromato¬
graphic studies. Tt has been reported (3) that changes In the
values brought about by the secondary hydroxyl groups In different
positions on the steroid nucleus were usually the same for a variety
of solvent systems.
The two Isomeric pairs of 3,17-dioxygenated androstanes
maintain the characteristic elution order of the hydrocarbon nuclei,
a fact Indicating an opposing effect of the oxygen function on carbon
atom 3. However, a marked difference between the effect of the 3a-
and 3p-hydroxyl function Is evident from the results In Table 12. In
all cases studied, the presence of the 3p-hydroxyl group appears to
enhance the normal difference between the 5a- and 5p-Isomers while
the presence of the 3a-hydroxy group appears to counteract the
Influence of the 17-oxygen only to the point where the Isomeric 3a,-
17p-diol and 3a-ol,17-one androstanes exhibit nearly Identical re¬
tention times. These observations were made with each column, In¬
cluding the SE-30 column In which case the Influence of the 17-oxygen
129

function was found to ba the least evident. Thus, the oxidation of
the C-3 hydroxy function to the corresponding ketone brings about a
significant change in retention time values only when coopered to the
3a,17£-dlol compound. The contribution of the 3p-hydroxyl group in
increasing the retention value of the 5a-androstane isomers can also
be deduced from the comparative data given in a recent paper for the
purpose of illustrating certain other factors (107).
The influence of the 3p-hydroxyl group may be considered in
terms of the conformations of the steroid molecules, that is, in
terms of the orientation of the C-3 hydroxyl group within the 5a -
and the 5£-serles. A further correlation between chromatographic
behavior, structure and conformation then emerges. In the 5p-isomers
the 3p-hydroxyl Isomers (axial conformation) exhibit greater mobility
than the corresponding 3a-hydroxyl compounds (equatorial conformation)
In the 5a-serles, on the other hand, this pattern is reversed and
compounds with the hydroxyl group in the 3a-orlentatlon ( axial confer
station) exhibit greater mobility than their 3^-lsoswrs (equatorial
conformation). It is significant that in both series the orientation
which is els between the C-3 and C-5 results In greater mobility. It
is possible that intramolecular interaction may exist between the C-5
hydrogen and the C-3 hydroxyl group. These observations are in agree¬
ment with those of Savard on the paper partition chromatography of
C¿9 and C21 steroids (65) and with the general concept defined by
Barton (31,66).
130

Theoretical Aspects of Partition Chromatography; Relationship of
Structure to Chromatographic Mobility
Martin (94) has developed a theory of partition coefficients
based on the assumption that the chemical potential of a substance is
an additive function of the constituent parts of its molecule. If pA
is the chemical potential of the substance A and A pA is equal to the
free energy required to transport one mole of A from one phase to
another, then as a first approximation A juA may be regarded as being
equal to the sum of the potential differences (¿jua,A.pt>) of the various
groups which make up molecule A.
A pA - A pa + A ^ + A fie
To a first approximation, the free energy required to trans¬
port a given group X from one solvent to another is independent of the
rest of the molecule. The addition of a group X to a molecule will
change the partition coefficient, and hence the retention time value,
of a substance by a definite amount in a particular solvent system.
This change will be dependent on the nature of group X and independent
of the structural features of the rest of the molecule (94). Intro¬
duction of more than one group into a molecule will cause a change in
the partition coefficient directly proportional to the number of groups
of X.
131

Martin, in pointing out the limitations of the application
of his theory, suggested that stereochemical factors night be a
major cause of deviation from theory in the relationship of structure
to partition coefficient. Owing to steric factors, the solvent
nolacules nay be unable to approach the solute molecules and the
energy of association between the solute and the solvent will be
lower than expected from the considerations of the sun of various
groups of the solute molecule. Bate-Snlth and Westell (108) have
reported excellent agreement with the theory in their study of a wide
range of polyphenolic compounds with substituents where little or no
steric hinderanee was involved.
It has been observed that deviations from theory begin to
appear when the solute contains fused ring systems. Since most
steroid molecules contain at least four fused alicyclic rings, it is
not surprising that in this class of compounds steric factors should
be more pronounced and that more of the substituent groups should
show deviations from theory. However, the steric limitations in
the steroid molecule might introduce an advantage to the applicability
of the theory to this group of compounds. The rigidly joined ring
system of the steroid nucleus has little possibility of conformational
inversion. Consequently the conformations of any one steroid are
limited in number. Furthermore, substituents on the large extended
steroid molecule might be expected to be sufficiently far apart to
exert little or no influence on each other. This is an ideal
132

requirement for any application of the theory. (Note, however, the
dependence of the influence of the 17-oxygen function on subatitutlon
at 03.) Bush has made the practical suggestion that in order to
make the theory workable over a wide range of steroids, the word
"group" be defined to mean not only the chemical nature of the group,
as suggested by Martin, but its position and orientation on the
nucleus as well (109).
Martin's concept of the additive contribution to chroma¬
tographic mobility of the components of a molecule was recently applied
to the behavior of steroid molecules during gas-liquid chromatography
by Clayton (110-112) and Knights and Thomas (113). Clayton expressed
the mobility of a substituted molecule in tens of the sK>bllltes
attributable to the components of that molecule.
r â–  rn x ka * ^
where rn is the retention time of the unsubstituted nucleus, and
ka, kjj etc. are group retention time factors of non-interacting
groups at positions a, b on the nucleus. The contribution of the
relatively non-polar methoxy group at C-3 was found to be constant
«dille that of the polar So-hydroxyl group was not. Clayton suggested
that the observed variation in the contribution of the 5a-hydroxyl
group may be in part due to steric hlnderance which restricts the
Interaction of the polar liquid phase with a double bond In the
molecule.
133

Knights and Thomas (113) have used the above equation in
its logarithmic form
log r - log rQ + log ka + log
These investigators have demonstrated that the contribution to
retention time of the C-10 methyl group for several steroids vas
constant. Other observations of these authors Include the constancy
in the A log r contributions for 03 equatorial hydroxyl to 03
ketone transitions on a stationary phase selective for ketones. The
vicinal group effects observed in the variation in the log r para¬
meters of ll£>-hydroxyl and 11-ketone groups were in keeping with the
theory. K negative contribution of the 016 methyl group was attri¬
buted to the change in the contribution of the 20-ketone in the 16a-
end 16^-methyl pregn-4-ene-3,20-dione, as a result of sterlc hlnderance.
It must be emphasized, however, that not all discrepancies with respect
to theory are explicable in terms of steric factor. The data of
Knights and Thomas are too limited to permit such a generalization.
Calculated group retention factors (Table 13) obtained on
the two polar phases exhibit similarities in the over-all pattern as
well as in the individual values. Similarly, the values obtained on
the two non-polar phases are alike, though some of the extremely low
and high values obtained on the SE-30 column are moderated on the XE-60
column.
134

The Magnitude of log k4 Is determined by the extent of
the Interaction of group (a) with the liquid phase. Since functional
group effects are accentuated on polar columns and minimized on non¬
polar ones, log k values of functional groups should be larger for
polar solvents than for non-polar phases. The results suggest a
greater polarity of the Hi-Eff 8B polyester as compared to the
neopentylglycol sebacate phase.
A comparison of the group retention time factors of the
acetyl versus the trlmethylsllyl groups show better agreement among
the log k . values, which is In agreement with other observations
made on the acetate group(Table 16).
Retention time values were calculated using the average log k
values obtained from Table 13 and compared with the observed retention
time values (Table 14). In general, the agreement between the observed
and the calculated figures Is quite good. It Is Interesting that
deviations occur when the Influence of the C-17 oxygen function Is
most pronounced, that Is, on the two polar columns for the 5p-
androstanes»
The changes in retention time upon derivative formation are
expressed as the ratio of the relative retention times of the deri¬
vatives to those of the free steroids (Table 15). Interactions of
the non-polar acetate group with the non-polar SE-30 and to a lesser
extent with the XE-60 phases are Indicated In the Increased retention
135 -

times of the steroid acetates on these coloans. The decreased
Interactions of the acetates with the polar phases are reflected
In the faster elution tlae of the steroid acetates as coopered to
t
the free steroid on the Hi-lff 8B and NGSeb coluans.
The average change brought about In the retention of the
parent compound upon derivative formation is given in Table 16. It
is evident that of the three derivatives, the contribution to
retention tlae values is additive only in the case of the acetate
group. The absence of a direct relationship between contribution
to retention tine and nunber of substituent groups in the case of
trlmethylsllyl ethers and the trlfluoroacetates is indicative of the
presence of factors such as volatility and starlc hinderanca. The
use of the acetyl group in preference to the trlmethylsllyl or tri-
fluoroacetate derivatives for structural studies is suggested by
these results*
VandenHeuvel and others (89,90) have suggested two new terms,
steroid number (SN) and T, as parameters for the elucidation of the
structural features of a steroid molecule. Both of these terms are
directly derived from the work of Woodford and van Gent (114) in the
gas-liquid chromatography of fatty acids. Steroid number is
analogous to carbon number of fatty acids, which is related to retention
time r by
log r â–  k z no of C in fatty acid molecule
136

The manner of determining the carbon number la to construct
a reference graph according to the above equation based on the
carbon number of known fatty acids. The carbon number of an un¬
known acid can then be read off at a point corresponding to the
retention time of the acid. The concept of carbon number may be
useful because It Is Independent of slight changes in temperature
and eliminates the need for an arbitrary reference standard to
which retention times must be related.
Steroid number Is expressed as a summation of terms
dependent on the nature of the carbon skeleton and the functional
groups of the steroid molecule*
SN - S + rm + rb
where SN Is the steroid number, S Is the carbon content of the
steroid skeleton and Fa, Fb are values characteristic of the functional
groups of the steroids.
In the opinion of this Investigator there are at least
three drawbacks to the use of SN term for the elucidation of structure.
1. Steroid numbers are determined from relative retention
values, so that the advantage cited by Woodford and van
Gent In eliminating the reference standard no longer
exists. Furthermore the temperature dependency of the
relative retention time value will be present In the
steroid number term.
137

2. Woodford and van Gant «aployad a calibration curva
basad on five fatty acids of known chain length.
VandenHeuvel and Horning draw their standard curve
between only two points. The reliability of any
information derived from such a calibration graph
is questionable.
3» The steroid number term neither reflects the actual
• i,.
carbon content of the steroid molecule nor indicates
substituent groups on the nucleus with any accuracy.
For example, the value of SN is 29.4 for choleatanol,
29*2 for cholestenyl methyl ether and 28.8 for
cholestanyl trlfluoroacetate. The term is therefore
misleading.
The second parameter derived from the early work of Woodford
and van Gent is called the T term which is a function of the select*
ivity of the stationary phase. This term is defined as
where t' is the relative retention time observed with a selective
stationary phase and t„ is the relative retention time observed
with a non-aelectivo stationary phase. Woodford and van Gent had
employed polar and non-polar columns to study the behavior of satu¬
rated fatty acids.
138 -

T values of androstane Isomers (Table 17) calculated from
measurements obtained on the XE-60 and the SE-30 phases Indicate
the dominating Influence of the C-17 oxygen function in the 5p-lsoaers
when the C-3 configuration is of the alpha type. Thus T values
might be useful in reflecting stereochemical relationships of
functional groups and might provide clues for intramolecular inter¬
actions.
The ketone selective properties of the four phases were
compared and changes in retention time accompanying the oxidation of
a hydroxyl group to the corresponding ketone were calculated (Tables 18
and 19). It is seen that the value of A log r is fairly constant and
very smell for Si-30, NGSeb and Hl-Eff 8B columns. Furthermore, there
» i • • i •
was no difference in the A log r value for ol _*one and dlol _* dlone
transitions. The preferential selectivity of the XE-60 phase for
ketones is evident in the relatively large values of log r0]_pne
in the greater retention time value on this phase of the 3-ketosterolds
as compared to the 3-hydroxyl IsoaMrs (Table 18). The unexpectedly
small values of A log *dioL-4ione *** * direct consequence of the
anomalous effect of C-17 substitution. However, it is also possible
that boom loss of the XE-60 phase may be the cause of the unexpectedly
small value of these dlol-dlone transformations.
In Table 20, where separation factors for the 5a- end 5p-
isamers ere given, it can be seen that these separations are in general
better for the trlmethylsllyl derivatives, especially within the 17-
ketosteroid group.
139

From the comparative data presented It le evident that
the gas-liquid chromatographic separation of almost any isomeric
pair can be achieved by the judicious choice of the stationary
¡díase or of the derivative of the steroids under study. It is
worthy of note that the isomeric 3,17-dione androstanes which
have eluded separation by paper chromatography methods, can be
resolved on gas-liquid chromatography columns coated with polar
liquids (Table 11).
- 140 -

The Application of Gas-liquid Chromatography to the Isolation and
Measurement of Urinary 17-Ketosterolds
A Method for the Simultaneous Separation of CioOg and CioCH
17-Ketosterolds and Progasterona Metabolites (100)
The term "urinary 17-ketosterold" has been used for a group
of substituted androstanes excreted by man. During the last twenty
years a large body of data has become available on the excretion of
17-ketosterolds. Bioassay methods, although necessary, have been
limited in usefulness because the steroids are excreted In a less
active form or In the Inactive form of their hormonal precursors.
The chemical method used most widely for the determination of 17-keto-
steroids is the assay based on the Zlmmermann reaction (57,58). Since
the urinary 17-ketosterolds are end products of the metabolism of
hormones secreted principally by the adrenal cortices (115-118) and
the gonads (119,120) any method which can measure the amounts of the
17-ketosterolds may also Indirectly help to assess the gonadal and
adrenocortical functions In man*
The excretion of 17-ketosterolds In urine Is Influenced by
the basal metabolic status of the Individual as well as by conditions
of stress. The former Includes age and sex of the Individual while
141

the latter ten «ay involve chronic stress resulting from a disease
or a shorter ten emotional or physical stimulus. The 17-ketosteroid
excretion at any one time reflects the combined contributions of all
of these factors.
The array of excreted C19 steroids, the urinary 17-ketosterolds,
mas first disclosed by the chromatographic studies of Dlngenanse et al.
(121,122). Subsequently moderately successful separations of 17-kato-
steroids by adsorption and partition systems were accomplished (65,123,
124). While many conventional chromatographic techniques for the
separation and measurement of Individual 17-ketosterolds have been
developed and are in actual use today, all suffer from the disadvantages
of tediousness and Inefficiency* The classical column chromatographic
method (121,122) required the collection of multiple discreet samples
of eluent each of which then had to be analysed colorimetrically
using the Zlmmermsna reaction (57,58)* This reaction Is at best a
non-specific chromogenlc reaction subject to Interference by non¬
steroidal Impurities. The paper partition method of Savard (65) has
represented an improvement In efficiency In that the actual number of
samples to be finally aluted and measured as Zinmermann chromogen are
limited to those being resolved and studied. However, running times
are long In these systems. By virtue of very short running times,
thin layer partition chromatography vl11 obviate this disadvantage.
All of these modalities have a common limitation In resolving power
attributable to practical limits to which the chromatographic column,
142

pinte or paper strip can be lengthened. Specifically one encounters
difficulty In trying to separate sore than five or six components at
a time by any of these liquid-liquid methods.
Following the demonstration of the feasibility of gas-liquid
chromatographic analysis of steroids (83,86,112) concerted efforts
have been directed to the development of methods for the analysis of
the urinary CI9 steroids. The first attempts Involved the simultane¬
ous use of two columns (125,126). The disadvantages were found to
be those Inaccuracies and Inconveniences Inherent In such a complex
system. Partial separation of urinary 17-ketosterolds on SK-30
column have been reported (127). More recently separation of three
17-ketosterolds was reported cm a cyanomethylslllcone column (107).
However, a comprehensive method of analysis should separate and
measure at least seven urinary end products, which are: 3a-hydroxy-
5a-endrosten-17-one, 3a-hydroxy-5f-andros ten-17-one, 3p-hydroxyandrost-
5-en-17-one, 3a-lip-dihydroxy-5a-*ndrostan-17-one, 3c-hydroxy-5p-
androstan-11,17-dione, 5p-pregnane-3a,20a-diol and 5p-pregnan-3a-o1,
20-one.
A review of the results obtained In the first phase of the
study Indicated that the trlmsthylsllyl ethers were the most efficacious
derivatives for biological application. This vas attributed to their
high volatility and fast elution time with subsequent sharp syasaetrlcal
peaks. Separation factors for the trlmsthylsllyl ethers of the
143 -

steroids were also more favorable than for the free steroids or for
the other derivatives tested. In addition, column efficiencies were
generally higher for these derivatives. When mixtures of trimethyl»
sllyl ethers of the C19 steroids and of two progesterone metabolites
were chromatographed on the column phases described earlier, adequate
separations were obtained in the case of the three per cent XE-60 and
the one per cent Hi-Eff 8B columns; NGSeb column provided only margin¬
al resolution of the 3a-hydroxy-5p-androsten-17-one and 5f-pregnane-
3a,2Qof-diol. The results are shown in Table 21 and are graphically
depicted in Figures 9-11. Thus the method developed analyzed not
only the five major C19 steroids but in addition two progesterone
metabolites of great interest in human metabolism.
In an actual application of this basic technique to the study
of human urine it was found that the initial objectives of the investi¬
gation eould be realized. Chromatography on a three per cent XE-60
column of the trlmethylsllyl derivatives of a glusulase hydrolyzed
and solvent extracted 24 hour urine sample from a normal male subject
gave the results shown in Figure 12. By comparison of retention
times for major peaks with those obtained for pure crysalllne standards
it was possible to quantitatively detect and identify the urinary
components. Although no attempt at measurement was made, the distri¬
bution and the relative proportion to each other of these compounds,
represented as peak height responses should be noted. Since the
chromatogram was obtained using a 1/2040 aliquot of the urine sample,
the indications are that the method will permit the requisite
144

sensitivity for determinations at both the physiological and
pathophysiological levels. Indeed it is conceivable that plasma
levels of these steroids may be determined by this technique.
Advantages of the present method include the factor of
speed, efficiency and a one-step process of separation and measure¬
ment. The complete analysis of a prepared extract can be accomplished
in 25 minutes as compared to the seventy-two hours necessary for the
development of a paper chromatogram.
The success of the biological application supported the
original premise that access to a useful analytical technique may
come from a thorough knowledge of the compounds to be studied and
from the generalisations gained in the investigation of their proper¬
ties which in the present research was their chromatographic behavior.
- 145 -

Summary
The relative polarities of the four stationary phases
•■ployed were established as Hi-Eff 8B ) NGSeb > XE-60 ) SB-30.
The general pattern of retention tines of the free steroids and
their derivatives were: acetate > trlmethylsilyl ether > free
steroid > trlfluoroacetate on the SE-30 column; acetate >
free steroid > trlmethylsilyl ether on the XE-60 column; free
steroid > acetate ) trlmethylsilyl ether > trlfluoroacetate on
the Hi-Eff 8B and NGSeb columns.
On all four columns 3a-androstañes showed a greater re¬
tention time than the corresponding 5$-isomers. This was attri¬
buted to the planarity of the 5a-androstane nucleus and enhanced
Interaction with the stationary phase. In agreesient with earlier
observations on paper partition systems, the mobility of the axial
Isomer was greater than Its equatorial Isomer. In the 5^-androstane
series, the steroids with an oxygen function at C-17 exhibited
anomalous behavior In that they had greater retention times than
the corresponding 5a-isomers. The absence of the anomalous be¬
havior In the 5p -androstanes containing oxygen substituents In both
the C-3 and C-17 positions Indicated the opposing Influence of the
C-3 oxygen function.
- 146 -

Among the three derivatives the acetates were found to
be the most satisfactory in correlation of chrosmtogrephic be¬
havior to structure. The best resolutions were effected with
the trimethylsllyl ethers of the steroids.
Separation of the principal urinary 17-ketosterolds was
achieved on one per cent NGSeb, one per cent Hi-Kff 8B and
three per cent XE-60 columns. The two pregnane leasers studied
were resolved simultaneously with the 17-ketosterolds on the
latter two colons.
The gas-liquid chromatographic method developed was
successfully applied to the separation of the major 17-ketosterolds
on a crude urine extract.
147

BIBLIOGRAPHY
1. tel Mogare, 8. aad Juvet, ft. 8., Jr., "Gas-Liquid Chromatography,"
Interscienc* Publishers, New York, 1982, ft. A.
2. Tswett, M.. Bar. deut. lot». 6m., 24, 316, 384 (1906).
3. teeh, X. I., "The Cm Chromatography of Steroids," Parganon Presa,
Mew York, 1961, chapters 1 aad 2.
A. Raichstain, T. and Shoppaa, C. M., Discussions Faraday Sec., 7,
303 (1949).
5. Dobriner, ft., Llaberaan, 8. and Rhoads, 0. P., J. Biol. Chew.,
172. 2A1 (1948).
6. Callow, N. H., Blochea. J., 33, 559 (1939).
7. Callow, M. H., and Callow, ft. ft., Blochea. J., 33, 931 (1939).
8. Pincus, 6. and Roaanoff, L. P., Federation Proc., 9, 101 (1950).
9. Butt, N. ft., Norris, P. and Morris, C. J. 0. ft., Abst. Int.
Coagr. Blochea., Caabrldge, 405 (1949).
10. Butt, W. ft., Morris, P., Morris, C. J. 0. ft. and Williams, D. C.,
Blochea. J., 49, 434 (1951).
11. Saffaronl, A., Burton, ft. B. and Keutaean, B. I., Science, 111.
6(1950). W
12. Heftasan, ft., Scioace, 111, 571 (1950).
13. Bush, X. ft.. Mature, 166, 445 (1950).
14. Bush, X. I., J. Physiol., ¿l¿, 12P (1951).
15. Savard, ft., Federation Proc., Jl, 281 (1952).
16. Martin, A. J. P. sad Synge, ft. X.. M., Blochea. J., 35, 1358 (1941).
17. Jasas, A. T. aad Martin, A. J. P., Blochea. J., ¿0, 679 (1952).
18. Tlsellus, A., Arkiv Real, Mineral Gaol., 14B, Mo. 22 (1940).
19. ClaoMoa, 8., Arkiv Real, Mineral Gaol., 23A, Mo. 1 (1946).
- 148 -

20* Ambrose, D. and Ambrose, B. A., "Gas Chromatography," D, Van
Mostrand Company, Inc,, Princeton, Nov Jersey, 1962, p. 9.
21. Reference 1, p. 23.
22. van Deem ter, J. #., Zulderveg, P. J. and Kllnkanberg, A., Cham.
Eng. Sci., 5, 271 (1956).
23. Patten, H. W. in "Principles sad Practice of Gas Chromatography,"
R. L. Pecsok, editor, John Wiley and Sons, Inc., 1959, p. 16.
24. Bohemen, J. and Purnell, J. H. In *H5as Chromatography 1958,"
D. H. Desty, editor, Academic Press Inc., Publishers, Nov York,
1958, p. 8-16.
25. Purnell, H., "Gas Chromatography," John Wiley and Sons, Inc., Nov
York, 1962, chapters 8 and 9.
26. James, A. T., Martin, A. J. P. and Smith, G. H., Blochem. J., 52,
238 (1932). *
27. Greene, 8. A. In "Principles and Practise of Gas Chromatography,"
R. L. Pecsok, editor, Jehu Wiley and Sons, Inc., Nsv York,
1959, p. 47.
28. Klyne, W., "The Chemistry of Steroids," John Wiley and Sons, Inc.,
Nev York, 1957, p. 31.
29. flaser, L. F. and Fleser, M., "Steroids," Relnhold Publishing Corp.,
Nev York, 1959, p. 1-14.
30. Wilson, I. B., Jr., Proc. Natl. Acad. Sci., 0. 8., 43. 816 (1957).
31. Barton, D. B. R., Experientla, 6, 316 (1950).
32. Barton, D. B. R., J. Cham. Soc., 1027 (1953).
33. Barton, D. B. R. and Cooks on, R. C., Quart. Rev. Chen. Soc., 10,
. 44 (1956).
34. Klyne, W. la "Progress Is Stereochemistry," Butteruorths Scientific
Publications, London, 1954, p. 36-89.
35. Reference 28, p. 34.
36. Pésard, A., Compt. rend., ¿§3, 1027 (1911).
37. Gallagher, X. F. and Koch, F. C., J. Biol. Cham., 84, 495 (1929).
38. Butenaadt, A., Angev Cham., 44, 905 (1931).
39. Butenaadt, A. and Tschernlng, K., Z. physiol, chan., 229, 167, 185
(1934). “
- 149 -

40. Rnclcka, L., Goldberg. M. W. end Brüngger, H., Helv. Chin, Acta»
17, 1389 (1934).
41. Ruaicka, L., Goldberg, K. V.» Meyer, J., Brttngger, H. and
Klchenberger, I.» Helv. Chin. Acte, ¿2, 1395 (1934).
42. Kttslcke, L., BrUngger, 1.» Klchenberger» K. end Meyer» J., Helv.
Chian. Acte. ¿2. 1407 (1934).
43. Xnaicka, L. end Klchenberger, X.» Helv. Chin. Acte, 18, 430 (1935).
44. Cello», H. H. and Cello», K. X.» Blochca. J., 34, 276 (1940).
45. Peerlnen, If. H., J. Biol. Chea., 1J6, 807 (1940).
46. Meeon, H. L., J. Biol. Chen., 158. 719 (1943).
47. Meeon, X. L. ead Kepler, K. J., J. Biol. Chou, 161, 235 (1945).
48. Meeon, H. L., J. Biol. Chen., 162» 745 (1945).
49. Miller, A. M., Dorfnen, &. Z. end Sevrlnghoaee, E. I»., Endocrinology,
38, 19 (1946).
50. Bnteadeadt, A. end Daaaenbaua, H., Z. pbyelol. Chen., 229. 192 (1934).
51. Bnteadeadt, A., Daanenbaua, H., Healeeh, G. and Kodaene, H., Z.
pbyelol. Chen., ¿32, 37 (1935).
52. Lleberaaa, 8.» Dobrlner, K., Hill, B. K., Fleeer, L. f. end Ihoade,
C. P., J. Biol. Chen., ¿72, 263 (1948).
53. Cardwell, H. M. K., Cornforth, J. «., Duff, 8. K., Holtemenn, H.
and Xoblaeon, R., J. Chen. Soc., 361 (1953).
54. Voodward, t. B., Sondhelner, f., Taub, D., Henaler, K. and McLaaore,
H. M., J. An. Chen. Soc., Jfc, 4223 (1952).
S3. Johaaon, W. 8., Bennie ter, B.» Bloon, B. M., Reap, A. D., Pappo,
R., Rogler, B. R. and Ssnueekovlcs, J., J. An. Chea. Soc., 75,
2275 (1933).
56. 8aratt, L. H., Arth, 6. X., Lukee, R. M., Beyler, R. E., Pooe, G. I.,
Johna, V. P. «ad Cone ten tin, J. M., J. An. Chea. 8oc., 74,
4975 (1952).
57. Zianeraaaa, W., Z. pbyelol. Chen., 233 . 257 (1935).
58. Zlanemean, ¥., Z. pbyelol. Chen., 300, 141 (1955).
59. Dorfnen, L.. Chen. Rave., 53, 47 (1953).
- 150 -

60. Jonu, R. H. «nd Sandorfy, C., "Techniques of Organic Chemistry,"
A. Velssberger, editor, Interscience Publishers, Row York, 1956,
p. 462-498.
61. Djerasal, C., Bull. soc. chin. Franco, 741 (1957).
62. Djerasal, C., Halpern, 0., Hal pom, V. and Rinlkar, 1., J. An.
Chon. Soc., 80, 4001 (1958).
63. Parsons, J., Beher, W. T. and Baker, G. D., Anal. Chon., 28, 1514
(1956)* 29, 762 a»37).
64. Burton, A. B., Zaffaronl, A. and Kautaaan, K. H., J. Biol. Chon.,
188. 763 (1951).
65. Bauard, K., J. Biol. Chan., 202, 457 (1953).
66. Barton, D. H. R. and Xoaanfoldar, W. J., J. Chen. Soc., 1048 (1951).
67. Llabemaa, S., Bari ton, L. B., Humphries, p., Rhoads, C. P. and
Detainer. K., J. Biol. Chon., ¿6 , 793 (1952).
68. Grundy, H. M., Simpson, 8. A. and Tait, J. F., Roturo, 169, 795
(1952).
69. do Courcy, C., Gray, C. H. and Lunnon, J. B., Nature, 170, 494
(1952).
70. Zaffaronl, A. and Burton, 1. B., J. Biol. Chon., 193, 749 (1951).
71. 3. Chon. Soc., 3526 (1951).
72. Coopt, rend., 240, 190 (1955).
73. VandenHeuvel, V. J. A., SjÓOall, J. and Horning, B. C., Bloehin.
at Blophya. Acta, 4B, 596 (1961).
74. Longer, 8. H., Frledel, 1. A., Wander, Z. and Sharkey, A. G., Jr.,
Anal. Chon., 30, 1353 (1958).
75. Luukkainer, T., VandenHeuvel, W. J. A., Haahtl, 1. 0. A. and
Horning, I. C., Bloehin. at Blophya. Acta, 52, 599 (1961).
76. Homing, I. C., Hoscatelll, E. A. and Sweeley, C. C., Chon, and
Znd., London, 751 (1959).
77. Lovelock, J. I., Roturo, JJ2, 1663 (1958).
78. McWllllan, X. G. and Dewar, R. A., Nature, 1&, 760 (1958).
79. Sweeley, C. C. and Ta-Chuang, L. C., Anal. Chon., 33, 1860 (1961).
80. Tenney, H. M. and Harria, H. J., Anal. Chen., 29, 317 (1957).
- 151 -

81. London, F., J. Phys. Chon., 46, 305 (1946).
• f . *. 1 • • v • • - • i i ■ ’ ■ k- * • . i » ., ^ ■ ; . ; / • *
82. VandenHouvol, H. J. A., Sweeley, C. C. and Homing, I. C., J. An.
Chon. Soo., 82, 3481 (1960).
83. Lipsky, 8. E. and Landovne, E. A., Anal. Chon. 33, 818 (1961).
84* Haahei, E. 0. A., VandenHeuvel, W. J. A. and Homing, I. C., J.
Org. Chon., ¿6, 626 (1961).
' , . '. ... • " • -
85. VandenHeuvol, W. J. A., Haahti, E. 0. A. and Homing, E. C., J.
An. Chon. Soe., 83, 15U (1961).
86. Hotia, H. H. and Martin, H. »., J. Biol. Chon., 23£, 1312 (1961).
87. Facts and Methods, 3, 11 (1962).
88. Votis, H. H., Biochin. ot Biophya. Acta, in prana.
89. VaadonHeuvol, H. J. A. and Horning, E. C., Biochin. ot Biophya.
Acta, 64, 416 (1962).
90. Haahti, E. 0. A., VandenHeuvel, H. J. A. and Homing, E. C., Anal.
Biochon., 2, 344 (1961).
91. Sawyer, D. T, and Barr, Jf. K., Anal. Chon., 34, 1052 (1962).
92. Biahta, C., Measerly, J. F., Reachko, E. F.» Fredericks, 0. H. and
Cooke, V. 0., Anal. Chon., 32, 880 (1960).
93. Keller, E. A. and Stewart, 6. H., Anal. Chon., 34, 1834 (1962).
94. Martin, A. J. P., Biochon. Soo. Symposia, 3 , 4 (1949),
95. Boorthuia, E. K. and Recourt, J. H., Nature, 1868 372 (I960).
96. VaadonHeuvol, W. J. A., Sweeley, C. C. and Homing, E. C,, Biochon.
Biophya. Research Consuno., 3, 33 (I960).
97. Hotia, H. H. and Martin, H. F., Abstracta 138th Mooting An. Chon.
Soc., 58C, 1960.
98. VandenHouvol, V. J. A. and Horning, B. C., Biochen. Biophya.
Research Consuno., 3, 356 (I960).
99. Votis, H. H., Biochin. ot Biophya. Acta, 63, 180 (1962).
100. Hartnan, Z. 8. and Hotia, H. H., Steroids, 1, 33 (1963).
101. Janos, A. T. and Martin, A. J. F., Biochon. J., 63, 144 (1956).
102. Lipaky, 8. E. and Landovne, E. A., Biochin. ot Biophya. Acta, 27,
666 (1958).
152 -

*502 * *WMD *l®w T *’0
*(5561) 629
«•rsbtj pm •% 1 'xnmjxoQ **i •* ‘n^qn^
*(5561) 895 *51 ••loaTaoopnj ««no *f
“0 *cmoirj¿ pm *i *i •mmgsoa *•% ‘samaqumo* ••j *f •ujqnjg
*921
*521
* (2561) 99 *21 * •lootiDopoi «uno
T *’l *$ pm *0 •! *pm *,«* »ft»H •’! * ••mantra •*«
*(9961) 555 *? * *lo«|Jto«p««
*«110 *f *H ** *a*»l *P F» *9 *1 «Pm *,«* »T"H «’* 'MmmtBTa •jzx
*(¿561) 596 ‘iff **jmq»s »•* t *W|nj *021
*(0261) ¿25 *9 *X8ot«xi*ioopaa **v «pxe*aj *$tt
_ *(9561) 90t *?? **P*fl
*t®1* ’136*51 ’0®8 *0®*6 ,#0 *«notrf¿ pm *i •« ‘mmjxoq *qooxf *ft1
*(1961) ¿68 *W ‘na? ’*T«D ‘*I«H '** *n?®3«|aT®* pm 'A *f *A»a •¿n
*(8561) ¿611 *12 *»3aV **T«10 ’Am «*a *f ««ng pm *a «nrmttpm *911
*(9561) 522 *62 *57 **3»Y ’*1*10 ’Am * \L‘**W*«jai®H *511
. *(0961)
8«I *x 'qomwi pidn *r *«k *0 'amo m pm *| *1 «paojpooA
*(2961) 9901 *9? ***«10 *1«Y *’H *8 ‘waoqi pm *y •§ ’«aqfjux
*(2961) ¿55 *7 *** *(1961) 925 *757 *•»«»* •’« H *mal9t0
*(1961) U01 *061 *»*na»|i *•* •* *w»al»io
*(0961) 1 *8t *»1*oAb¿3 ‘oog U*qa©Tf "j *j *q*ng
. . ■' *(0561) ¿29 *?
«1®? ’«¿q6oig }• **iqooi8 «*0 *l *n*3«*A 9*» *0 *1 *q3T*S-»3*8
. ___ *(2961) 161 *9 **w*|aoi8
•imp * ’D *1 ‘fn-ptxoH pm *0 *t ‘«jamao **Y T ’ft *imn»HmpmA
*(1961) 829 *57 ****«10 *830 *f
**H '«wpjl pm *i *«a*8 ‘*3 *1 ‘8W1BJWH **Y *f ’A *l*An»HU»po»A
*(1961) 699 *5? *•»»? •«Al6ei*
3® '«rjqooTa *‘*f *’I *H **»0 pw ’I *omi«npjmH ‘*H T| **T30ft
*(1961) 665 *¥ **ioi—bo «jaammH
'■Xqdoxs *mqaoif • *0 ’I *fttjuxoH pm *Y *f ’A * t»An»Hmpu»A
*(8561) 692 *3? **»•* **®«I0 **Y *f *’I *f '»n»0 pm *h *0 '«0
•911
*511
*211
*111
*011
*60t
*801
•¿01
*901
•501
*901
*501

125* Huhti( Ei O* A«i VanduHtwoli W. J• A. snd Horning, E< C., Anal,
Blochen., 2, 182 (1961).
V :V> ! • «i V :■ . ">• >• ,f ri'J ■ ■ - í, !•>' :*/
126. Cooper, J. A. and Creech, B. 6., Anal. Blochen., 2, 502 (1961).
127. Spar agane, M., Mason, V. B. and Rsutaann, X. H., Anal. Ches.,
|4, 1157 (1962).
S
;n
%•
154

BIOGRAPHICAL SKETCH
Iclal Sirel Hartman vas born December 22, 1930 at Blaslg,
Turkey. She attended Preparatory School In Iatanbul, Turkey and
received the degree of Bachelor of Arts In June 1950 from Mount
\ . . . â– . . . ..
Holyoke College. In June, 1951 she received the degree of Master
of Arts from Mount Holyoke College. From 1952 to 1955 she vas
employed by B. R. Squibb and Sons, first as a trainee and later
as the Head of tha Quality Control Department In Squibb Istanbul.
In 1955, she enrolled in the Graduate School of the University of
Florida. She Interrupted her graduate study to teach. She
joined the faculty of Simmons College in 1959 vfaere she Is at
present teaching Chemistry. She resumed her graduate study In 1961
and since that date has pursued her graduate vork toward the degree
of Doctor of Philosophy simultaneously with her teaching.
Iclal Sirel Hartman Is married to Standlsh Chard Hartman,Jr.
- 155

This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to the
Dean of the College of Arts and Sciences and to the Graduate Council,
and was approved as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
April 20, 1963
Dean, College of Arts and Sciences
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08553 7842



UNIVERSITY OF FLORIDA
3 1262 08553 7842



PAGE 1

$ 678'< 2) 7+( 5(/$7,216+,36 %(7:((1 &+520$72*5$3+,& %(+$9,25 $1' 6758&785( 2) 67(52,' +25021(6 %\ ,&/$/ 6,5(/ +$570$1 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( &281&,/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$ $SULO

PAGE 2

7KLV GLVVHUWDWLRQ LV GHGLFDWHG WR D\ KXVEDQG

PAGE 3

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

PAGE 4

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

PAGE 5

3DJH 3UHSDUDWLRQ RI 'HULYDWLYHV 6WXGLHV RQ WKH )UHH 6WHURLGV DQG WKHLU 'HULYDWLYHV 5HIHUHQFH 6WDQGDUG 6ROYHQWV DQG 5HDJHQWV &ROXPQ 6XSSRUWV 6WDWLRQDU\ 3KDVH 3UHSDUDWLRQ RI &ROXPQV (TXLSPHQW 3UHOLPLQDU\ ([SHULPHQWV 6( 0HWK\O 6LOLFRQH 3RO\PHU 6(f 0L[HG 3KDVHV &RQWDLQLQJ 6( 6( 0HWK\O 3KHQ\O 6LOLFRQH 3RO\PHU 6(f 'RZ &RUQLQJ +LJK 9DFXXP 6LOLFRQH *UHDVH (WK\OHQHJO\FRO 6XFFLQDWH 3RO\HVWHU (*6f 1HRSHQW\OJO\FRO $GLSDWH 3RO\HVWHU 1*$Gf 1HRSHQW\OJO\FRO 6HEDFDWH 3RO\HVWHU 1*6HEf 6LOLFRQH 1LWULOH )OXLGV ;) DQG ;) 4) )OXRURDOIF\O 3RO\PHU 4)f 'LVFXVVLRQ RI 3UHOLPLQDU\ ([SHULPHQWV &RPSDUDWLYH ([SHULPHQWV 'LVFXVVLRQ RI &RPSDUDWLYH ([SHULPHQWV &RQFHQWUDWLRQ RI WKH 6WDWLRQDU\ 3KDVH DQG 0HVK 6LVH RI 6XSSRUW Y

PAGE 6

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`J DQG &LJ2M .HWRVWHUROGV DQG 3URJHVWHURQH 0HWDEROLWHV 6XPPDU\ %,%/,2*5$3+< %,2*5$3+,&$/ 6.(7&+ 9L

PAGE 7

/,67 2) 7$%/(6 7DEOD 9DULDWLRQ RI 5HWHQWLRQ 7LQD ZLWK &KDQJHV ,Q 7HPSHUDWXUH DQG 3UHVVXUH RQ = 6% &ROXPQ 9DULDWLRQ RI 6HSDUDWLRQ )DFWRU ZLWK 7HPSHUDWXUH DQG 3UHVVXUH RQ = 6% &ROXPQ 5HWHQWLRQ 7LPHV RQ = 6% &ROXPQ 5HWHQWLRQ 7LPHV DQG 6HSDUDWLRQ )DFWRUV RQ = 6% &ROXPQ &RPSDULVRQ RI 6HSDUDWLRQ )DFWRUV RQ 6% DQG (WK\OHQHn JO\FRO ,HRSKWDODWH (*,3f &ROXPQV &RPSDULVRQ RI 5HWHQWLRQ 7LPHV RQ 6% DQG %LVPSKDQR[\ SKDQ\OfHWKHU &ROXPQV 5HWHQWLRQ 7LPHV RQ ; 6% &ROXP ,VRPHULF 5HWHQWLRQ 7LPHV RQ = 1HRSHQW\OJO\FRO $GOSHWH &ROXPQ 5HWHQWLRQ 7LPHV RI 5HWRVWHUROG $FHWDWHV RQ 1HRSHQW\OJO\FRO $GLSDWH &ROXPQ 5HWHQWLRQ 7LPHV DQG 6HSDUDWLRQ )DFWRUV RQ b ;) &ROXPQ $ &RPSDULVRQ RI WKH 5HODWLYH 5HWHQWLRQ 7LPHV RI 6XEVWLWXWHG $QGURVWHQHV &RPSDULVRQ RI 5HWHQWLRQ 'DWH RI ,VRPHULF 3DLUV 6XEVWLWXHQW (IIHFWV &DOFXODWHG *URXS 5HWHQWLRQ )DFWRUV ORJ U f ORJ UQ ORJ N ORJ IFMf &RPSDULVRQ RI &DOFXODWHG DQG 2EVHUYHG 5HWHQWLRQ 7LPH 9DOXHV 5HODWLYH WR &KROHVWDQH 5DWLR RI 5HODWLYH 5HWHQWLRQ 7LPHV RI 'HULYDWLYHV WR )UHH 6WHURLGV YLO

PAGE 8

F 7rE8 3DJH 7KH $YHUDJH &KDQJH ,Q 5HWHQWLRQ 7LQH 2FFXUULQJ LQ 'HULYDWLYH )RUPDWLRQ UAAUAr [ U?=f 9DOXHV RI 7 DW r %DVHG RQ 0HDVXUHDHQWV 2EWDLQHG ZLWK b 6( DQG ; ;( &ROXPQV 7 Wf[% WnJJ WnJJf &RPSDULVRQ RI 6HOHFWLYH 5HWHQWLRQ RI WKH )RXU 3KDVHV IRU .HWRQH DQG +\GUR[\O *URXSV 7KH &KDQJH LQ ORJ U &RQWULEXWLRQ IURP +\GUR[\O WR .HWRQH 7UDQVLWLRQV /RJ$U 2+RQHf ,VRPHULF 6HSDUDWLRQ )DFWRUV D If 6HSDUDWLRQ RI 3ULQFLSDO .HWRVWHUROGV DQG 3URJHVWHURQH 0HWDEROLWHV DV 7UOPHWK\OVOO\O (WKHUV 7KH (IIHFW RI WKH 5HIHUHQFH 6WDQGDUG RQ WKH 9DULDWLRQ RI 5HODWLYH 5HWHQWLRQ 7LPH ZLWK 7HPSHUDWXUH RQ ,; 6( &ROXPQ YLLL

PAGE 9

/,67 2) ),*85(6 )LJXUH 3DJr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

PAGE 10

,1752'8&7,21 6WDWHPHQW RI 3XUSRVH 7KLV VWXG\ KDG WZR PDLQ SXUSRVHV WWLH LQYHVWLJDWLRQ RI WKH JDVOLTXLG FKURPDWRJUDSKLF EHKDYLRU RI D VHULHV RI LVRPHULF DQGURVWDHV ZLWK WKH DOD RI FRUUHODWLQJ FKHPLn FDO VWUXFWXUH WR FKURPDWRJUDSKLF PRELOLW\ 7KURXJK WKH NQRZOHGJH DQG LQVLJKW JDLQHG LQ WKH FRXUVH RI WKH DERYH VWXG\ WR DSSO\ WKH JDVOLTXLG FKURPDWRJUDSKLF PHWKRG DV DQ DQDO\WLFDO WHFKQLTXH LQ WKH ,VRODWLRQ DQG PHDVXUHPHQW RI &A VWHURLGV RI LPSRUWDQFH LQ PDQ 7KH XOWLPDWH REMHFWLYH ZDV WKH DFWXDO PHDVXUHn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

PAGE 11

&KURPDWRJUDSK\ +LVWRULFDO %DFNJURXQG 7VZHWW OD JHQHUDOO\ JLYHQ FUHGLW IRU WKH ,QYHQWLRQ RI WKH PHWKRG RI FKURPDWRJUDSK\ DOWKRXJK ,Q D *HUPDQ G\H FKHPLVW 3 ) %XQJH KDG GHVFULEHG D VHSDUDWLRQ SURFHVV ZKLFK PLJKW EH FODVVLILHG DV SDSHU FKURPDWRJUDSK\ f ,Q 7VYHWW f VHSDn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n DOV 7KH SDUWLFXODUO\ GLVWLQJXLVKLQJ IHDWXUH RI FKURPDWRJUDSK\ ,V WKH DPSOLILFDWLRQ RI WKH GLVWULEXWLRQ SURFHVV 7KLV DPSOLILFDWLRQ ,V DFFRPSOLVKHG E\ WKH ,QWLPDWH FRQWDFW RI HDFK HOHPHQW RI WKH PRYLQJ SKDVH ZLWK HDFK HOHPHQW RI WKH YHU\ ILQHO\ GLYLGHG IL[HG SKDVH 7KH

PAGE 12

SURFHVV RI FRQWLQXRXV DQG VXFFHVVLYH FRQWDFWV UHVXOWV LQ WKH GLVWULn EXWLRQ RI WKH VROXWH EHWZHHQ WKH HOHPHQWV RI WKH WZR SKDVHV DQG WKH DWWDLQPHQW RI HTXLOLEULXP LQ D YHU\ VPDOO OHQJWK RI WLPH 7KH WZR EDVLF W\SHV RI FKURPDWRJUDSK\ DUH DGVRUSWLRQ DQG SDUWLWLRQ DQG DUH GLVWLQJXLVKHG E\ WKH QDWXUH RI WKH SURFHVV RI GLVWULn EXWLRQ RI WKH VROXWH EHWZHHQ WKH SKDVHV 6HH )LJXUH f ,Q DGn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f DUH OLQHDU IRU D ZLGH UDQJH RI FRQFHQWUDWLRQ RI WKH VROXWH DV ZHOO DV IRU PRGHUDWH FRQFHQWUDWLRQV RI RWKHU VXEVWDQFHV LQ WKH V\VWHP 7KLV LV WKH PDMRU GLVWLQJXLVKLQJ IHDWXUH RI SDUWLWLRQ V\VWHP DQG DFFRXQWV IRU WKHLU WUHn PHQGRXV VXFFHVV f :LWK WKH H[FHSWLRQ RI WKH ZRUN RI %XQJH HDUO\ SKDVHV RI WKH GHYHORSPHQW RI FKURPDWRJUDSK\ ZHUH OLPLWHG WR FROXPQ FKURPDWRJUDSK\ ,Q WKH VWHURLG ILHOG ERWK DGVRUSWLRQ DQG SDUWLWLRQ PHWKRGV IRXQG ZLGH DSSOLFDWLRQ

PAGE 13

$ % ),*85( 'LVWULEXWLRQ LVRWKHUPV $ $GVRUSWLRQ LVRWKHUP % 3DUWLWLRQ LVRWKHUP

PAGE 14

7ZR W\SHV RI DGVRUSWLRQ PHWKRGV ZHUH HPSOR\HG E\ WKH HDUO\ LQYHVWLJDWRUV 7KH ILUVW W\SH ,QYROYHG WKH XVH RI ODUJH FROXPQV IRU ODUJH VDPSOHV DQG ZHUH SULPDULO\ SUHSDUDWLYH LQ SXUSRVH f 7KH VHFRQG W\SH ZDV IRU VHSDUDWLRQ DQG HVWLPDWLRQ RI VPDOO TXDQWLWLHV RI VDVLSOHV RQ VPDOO FROXPQV 7KLV PHWKRG IRXQG DSSOLFDWLRQ LQ WKH VHSDUDWLRQ DQG HVWLPDWLRQ RI XULQDU\ NHWRVWHURLGV f DQG RI DGUHQDO VWHURLGV DQG PHWDEROLWHV f $OXPLQD PDJQHVLD DQG VLOLFD JHO ZHUH XVHG VLRVW IUHTXHQWO\ IRU FROXPQ SDFNLQJ 7KH IOUDW VXFFHVVIXO SDUWLWLRQ PHWKRGV ZHUH GHYHORSHG E\ %XWW 0RUULV DQG 0RUULV f ZKR XWLOL]HG &HOOWH FROXPQV 6KRUWO\ DIWHU WKH SXEOLFDWLRQ RI WKHVH PHWKRGV LQWHQVLYH UHVHDUFK ZDV GLn UHFWHG WRZDUGV SDUWLWLRQ FKURPDWRJUDSK\ WHFKQLTXHV +LH SUREOHP RI WKH ORZ VROXELOLW\ RI VWHURLGV LQ V\VWHPV EDVHG RQ ZDWHU DV WKH VWDWLRQDU\ SKDVH ZDV RYHUFRPH E\ WKH GHYHORSPHQW RI RUJDQLF V\VWHPV DQG D ZKROH QHZ ILHOG RI SDUWLWLRQ FKURPDWRJUDSK\ RI VWHURLGV EHFDPH DYDLODEOH $PRQJ WKH SLRQHHUV LQ WKLV ILHOG ZHUH 0RUULV DQG KLV JURXS f =DIIDURQO DQG KLV DVVRFLDWHV f +HIWPDQQf %XVK f DQG 6DYDUG f WR QDPH D IHZ 7KH V\VWHPV RI SDUWLWLRQ FKURPDWRJUDSK\ FRQVLVWHG RI WKUHH FRPSRQHQWV D PDMRU FRPSRQHQW RI WKH PRELOH SKDVH ZKLFK ZDV UHODWLYHO\ QRQSRODU D FRPSRQHQW RI WKH VWDWLRQDU\ SKDVH PRUH RU OHVV SRODU LQ FKDUDFWHU WR HQKDQFH WKH VROXELOLW\ RI WKH VWHURLG DQG D WKLUG FRPn SRQHQW RI D VPDOO DPRXQW RI ZDWHU 7KH ZDWHU ZDV ODWHU RPLWWHG IURP V\VWHPV XVLQJ LPSUHJQDWHG SDSHU DQG IURP FHUWDLQ FROXPQ PHWKRGV ,Q

PAGE 15

UHYHUVHGSKDVH SDUWLWLRQ FKURPDWRJUDSK\ V\VWHPV} WKH PRELOH SKDVH ZDV PRUH SRODU WKDQ WKH VWDWLRQDU\ SKDVH DQG WKH VXSSRUW YDV K\GURSKRELF 7KH SULQFLSOHV RI OLTXLGOLTXLG DQG JDVOLTXLG SDUWLWLRQ FKURPDn WRJUDSK\ ZHUH GHVFULEHG E\ 0DUWLQ DQG 6\QJH LQ f EXW WKH FRQFHSW RI D JDVHRXV PRYLQJ SKDVH ZDV QRW DSSOLHG XQWLO ZKHQ -DPHV DQG 0DUWLQ SXEOLVKHG WKHLU QRZ FODVVLFDO SDSHU f 6LQFH WKDW WLPH} H[WHQVLYH UHVHDUFK RQ JDV FKURPDWRJUDSK\ KDV EHHQ FDUULHG RXW LQ ODERn UDWRULHV WKURXJKRXW WKH ZRUOG IRU PDQ\ W\SHV RI SUREOHPV 7KH JUHDW ,QWHUHVW LQ YDULRXV IRUPV RI JDV FKURPDWRJUDSK\ LV GXH WR LWV ZLGH UDQJH RI DSSOLFDWLRQ} VSHHG RI DQDO\VLV DQG WKH VHQVLWLYLW\ RI GHn WHFWLRQ IRU PLFURTXDQWLWLHV RI VDPSOH *DV &KURPDWRJUDSKLF 0HWKRGV *DV FKURPDWRJUDSK\ LV WKH SURFHVV E\ ZKLFK D PL[WXUH LV VHSDn UDWHG LQWR LWV LQGLYLGXDO FRPSRQHQWV E\ D JDVHRXV PRELOH SKDVH ,I WKH VWDWLRQDU\ SKDVH LV OLTXLG} WKH PHWKRG LV FDOOHG JDVOLTXLG FKURPDWRJUDSK\ DQG WKH VHSDUDWLRQ LV GXH WR WKH GLIIHUHQWLDO VROXELOLW\ RI WKH PDWHULDOV EHWZHHQ WKH OLTXLG DQG JDV ,I WKH VWDWLRQDU\ SKDVH LV D VROLG} WKH PHWKRG LV FDOOHG JDVVROLG FKURPDWRJUDSK\ DQG WKH VHSDUDWLRQ LV GXH WR WKH DGVRUSWLRQ RI WKH VDPSOH FRPSRQHQWV RQ WKH VROLG VXUIDFH 7KH WZR W\SHV RI JDV FKURPDWRJUDSKLF PHWKRGV DUH IXUWKHU GLVn WLQJXLVKHG E\ WKH PDQQHU LQ ZKLFK WKH VDPSOH PRYHV WKURXJK WKH FROXPQ

PAGE 16

(OXWLRQ GLVSODFHPHQW DQG IURQWDO DQDO\VHV DUH WKUHH WHFKQLTXHV ZKLFK QD\ EH XVHG ,Q JDVVROLG FKURPDWRJUDSK\ 7KH HOXWLRQ WHFKQLTXH ,V XVHG DOPRVW H[FOXVLYHO\ ,Q JDVOLTXLG FKURPDWRJUDSK\ )URQWDO DQDO\VLV ,V WKH SDVVDJH RI D FRQWLQXRXV IORZ RI FDUULHU JDV FRQWDLQLQJ WKH VDPSOH PL[WXUH WKURXJK D FROXPQ RI DGVRUEHQW 7KH ,GHD RI XVLQJ D FRQWLQXRXV VWUHDP RI WKH VDPSOH ,WVHOI DV D GLVSODFHU ZKHUH HDFK VROXWH DWWDLQV ,WV RZQ FRPSHWLWLYH DGVRUSWLRQ HTXLOLEULXP ZDV WKH FRQWULEXWLRQ RI 7OVHOOXD f 6HSDn UDWLRQ ,V DFKLHYHG RQO\ IRU WKDW FRPSRQHQW ZKLFK HPHUJHV ILUVW IURP WKH FROXPQ FRQVHTXHQWO\ IURQWDO DQDO\VLV OD RI OLPLWHG XVH DV D PHWKRG RI VHSDUDWLRQ 'LVSODFHPHQW DQDO\VLV ZKLFK ZDV HODERUDWHG E\ &ODHVVRQ f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

PAGE 17

GRZQ WKH FROXPQ &RPSRQHQWV RI WKH VDPSOH GLVWULEXWH WKHPVHOYHV EHWZHHQ WKH PRELOH JDVHRXV SKDVH DQG WKH VWDWLRQDU\ SKDVH (DFK FRPSRQHQW HYHQWXDOO\ HPHUJHV IURP WKH FROXPQ DIWHU WKH SDVVDJH RI D FKDUDFWHULVWLF YROXPH RI FDUULHU JDV 7KH XQLTXH IHDWXUH RI HOXWLRQ WHFKQLTXH DV FRPSDUHG WR IURQWDO DQG GOVSODF mVHQW DQDO\VLV ,V WKH PHUJHQFH RI WKH VROXWH DV D SHDN 7KH UHWHQWLRQ WLPH Wt GHILQHG DV WKH OHQJWK RI WLPH EHWZHHQ WKH ,QWURGXFWLRQ RI WKH VDPSOH ,QWR WKH FKURPDWRJUDSKLF V\VWHP DQG WKH HPHUJHQFH RI WKH SHDN PD[Ln PXP LV D PHDQV RI TXDOLWDWLYH ,GHQWLILFDWLRQ RI WKH FRPSRXQG 7KH SHDN DUHD FDQ XVXDOO\ EH UHODWHG GLUHFWO\ WR WKH FRQFHQWUDWLRQ RI WKH 3ULQFLSOHV RI JDV &KURPDWRJUDSK\ $ GLVWLQJXLVKLQJ IHDWXUH RI JDVFKURPDWRJUDSK\ OD WKH XWLOLVDWLRQ RI KLJK WHPSHUDWXUHV ,Q RUGHU WR PDLQWDLQ WKH VROXWHV ,Q WKH YDSRU VWDWH 7KH YLGH VHOHFWLRQ RI OLTXLG SKDVHV HQKDQFHV WKH DSSOLFDELOLW\ RI JDV OLTXLG FKURPDWRJUDSK\ PHWKRGV WR D YLGH UDQJH RI DQDO\VHV 7KH WHFKn QLTXH RI JDVOLTXLG FKURPDWRJUDSK\ ,V VLPLODU WR OLTXLGOLTXLG SDUWLWLRQ DQG WKH JDVOOTXOG FROXPQ OD DQDORJRXV WR D GLVWLOODWLRQ FROXPQ +RZHYHU WKHUH LV RQH PDMRU GLIIHUHQFH EHWZHHQ WKH WZR V\VWHPV ZKLFK ,V RI JUHDW SUDFWLFDO ,PSRUWDQFH ,Q V\VWHPV RI JDVOOTXOG FKURPDn WRJUDSK\ WKHUH ,V QR ,QWHUIHUHQFH ZLWK VHSDUDWLRQ GXH WR D]HRWURS\ $W WKH ,QOHW RI D JDVOOTXOG FKURPDWRJUDSK\ FROXPQ WKHUH PLJKW RFFXU DQ ,QWHUDFWLRQ EHWZHHQ WKH FRPSRQHQWV RI WKH VDPSOH EXW RYHU WKH ZKROH OHQJWK RI WKH FROXPQ WKH ,QWHUDFWLRQ RI WKHVH FRPSRQHQWV ZLWK WKH

PAGE 18

OLTXLG SKDVH IDU RXWZHLJK DQ\ PXWXDO HIIHFWV f 7KXV HDFK FRPSRQHQW LV FDUULHG WKURXJK WKH FROXPQ LQGHSHQGHQWO\ RI DQ\ RWKHU FRPSRQHQW $ WKHRUHWLFDO SODWH WUHDWPHQW LV DSSOLFDEOH WR ERWK OLTXLGr OLTXLG DQG JDVrOLTXLG FROXPQV $ WKHRUHWLFDO SODWH LV GHILQHG DV D VHFWLRQ RI WKH FROXPQ LQ ZKLFK WKH YDSRU OHDYLQJ WKH VHFWLRQ KDV WKH FRPSRVLWLRQ WKDW ZRXOG EH LQ HTXLOLEULXP ZLWK WKH DYHUDJH FRQn FHQWUDWLRQ RI OLTXLG VROXWLRQ ZLWKLQ WKDW VHFWLRQ 7KH PRVW LPn SRUWDQW DVVXPSWLRQ XQGHUO\LQJ WKH WKHRUHWLFDO GHVFULSWLRQ RI WKH FKURPDWRJUDSKLF SURFHVV LV WKDW HTXLOLEULXP EHWZHHQ WKH WZR SKDVHV LV REWDLQHG DIWHU HDFK PLQXWH PRYHPHQW RI WKH PRELOH SKDVH 7KH VHSDUDWLRQ SHU SODWH VHSDUDWLRQ IDFWRUf DQG WKH WRWDO QXPEHU RI SODWHV 1f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f 7KH QXPEHU RI WKHRUHWLFDO SODWHV H[KLELWHG RQ D FROXPQ IRU D VSHFLILF OLTXLG SKDVH

PAGE 19

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f 7KH XQLWV PD\ EH WLPH RU OHQJWK SURYLGHG ERWK W5 DQG W DUH LQ WKH VDPH XQLWV 7KLV HTXDWLRQ IRU 1 DVVXPHV WKDW WKH FRPSRQHQW EHLQJ VWXGLHG LV LQ HTXLOLEULXP ZLWK WKH FROXPQ 1 FDOFXODWHG LQ WKLV PDQQHU LV VLJQLILn FDQW RQO\ IRU V\PPHWULFDO SHDNV VLQFH WKH DERYH HTXDWLRQ LV EDVHG RQ SUREDELOLW\ WKHRU\ f 7KH KHLJKW HTXLYDOHQW WR D WKHRUHWLFDO SODWH LV JLYHQ E\ WKH HTXDWLRQ + / 1 ZKHUH / LV WKH OHQJWK RI WKH FROXPQ XVXDOO\ LQ PLOOLPHWHUV 7KH YDOXH RI + LV JHQHUDOO\ ORZ IRU FROXPQV RSHUDWLQJ DW KLJK HIILFLHQF\ DQG GHFUHDVHV ZLWK ,QFUHDVHV LQ UHWHQWLRQ WLPH 7KH SODWH WKHRU\ SURYLGHV D XVHIXO PHDQV RI HYDOXDWLQJ FROXPQ HIILFLHQF\ 7KH

PAGE 20

5HVSRQVH ),*85( $ PHWKRG IRU FDOFXODWLQJ QXPEHU RI WKHRUHWLFDO SODWHV

PAGE 21

UHODWLRQVKLS RI YDULRXV WHUPV ZKLFK GHWHUPLQH WKH YDOXH RI + DUH JLYHQ LQ WKH YDQ 'HHDWHU HTXDWLRQ f 6 nM8 7rDm} f VN 9G Nnf '8T ZKHUH ? LV D TXDQWLW\ FKDUDFWHULVWLF RI WKH SDFNLQJ GS ,V WKH DYHUDJH SDUWLFOH GLDPHWHU \ ,V D FRUUHFWLRQ IDFWRU IRU WKH WRUWXRVLW\ RI WKH SDWK RI WKH JDV ,Q WKH LQWHUSDUWLFOH VSDFH ,V WKH GLIIXVLRQ FRHIILFLHQW RI WKH VROXWH ,Q WKH JDVHRXV SKDVH X ,V WKH OLQHDU JDV YHORFLW\ N LV WKH UDWLR RI WKH IUDFWLRQ RI VDPSOH LQ WKH OLTXLG SKDVH WR WKH IUDFWLRQ ,Q WKH YDSRU SKDVH G LV WKH WKLFNQHVV RI WKH OLTXLG ILOP DQG 'MA LV WKH GLIIXVLRQ FRn HIILFLHQW RI WKH VROXWH LQ WKH OLTXLG SKDVH ,Q WKH VLPSOLILHG YDQ 'HHPWHU HTXDWLRQ + $ %X &X $ UHSUHVHQWV WKH HGG\ GLIIXVLRQ WHUP DOVR FDOOHG WKH PXOWLSOH SDWK HIIHFW %X WHUP LV WKH PROHFXODU GLIIXVLRQ DQG &X ,V WKH WHUP WKDW GHVFULEHV WKH UHVLVWDQFH WR PDVV WUDQVIHU 7KH HGG\ GLIIXVLRQ ,V WKH UHVXOW RI WKH ,UUHJXODU SDWK ZKLFK WKH JDV IROORZV WKURXJK WKH SHHNHG FROXPQ 7KH LUUHJXODULWLHV ,Q WKLV SDWK FRQWULEXWH WR D EURDGHQn LQJ RI WKH FKURPDWRJUDSKLF EDQG $ UHGXFWLRQ LQ SDUWLFOH VL]H GSf XQLIRUP SDFNLQJ DQG XQLIRUP SDUWLFOH VL]H WHQG WR GHFUHDVH WKH FRQWULn EXWLRQ WR + RI WKLV WHUP 7KH VHFRQG IDFWRU ,V WKH GLIIXVLRQ RI WKH VDPSOH PROHFXOHV LQ WKH FDUULHU JDV ZKLFK UHVXOWV LQ EDQG EURDGHQLQJ 7KH ORZHU WKH PROHFXODU ZHLJKW RI WKH FDUULHU JDV WKH JUHDWHU ,V WKLV

PAGE 22

IDFWRU 'LIIXVLRQ LQFUHDVHV ZLWK ,QFUHDVH LQ WHPSHUDWXUH $Q LQFUHDVH LQ SUHVVXUH RQ WKH RWKHU KDQG E\ LQFUHDVLQJ WKH GHQVLW\ RI WKH JDV WHQGV WR GHFUHDVH WKH GLIIXVLRQ )RU WKLV UHDVRQ KLJK WHPSHUDWXUHV DUH HPSOR\HG WRJHWKHU ZLWK KLJK SUHVVXUHV RI WKH FDUULHU JDV 7KH WHUP &X UHVLVWDQFH WR PDVV WUDQVIHU ,QFOXGHV VHYHUDO SDUDPHWHUV 7KH YDOXH RI Nn LV GHSHQGHQW RQ WKH SDUWLWLRQ FRHIn ILFLHQW DV ZHOO DV WKH UHODWLYH YROXPHV RI WKH OLTXLG DQG WKH JDVHRXV SKDVH LQ WKH FROXPQ Nn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r LI WKH DPRXQW RI OLTXLG SKDVH LV UHGXFHG 7KH OLTXLG GLIIXVLRQ FRHIILFLHQW 'LLTf LV GHSHQGHQW RQ WKH YLVFRVLW\ RI WKH OLTXLG SKDVH $Q ,QFUHDVH LQ WHPSHUDWXUH PLJKW EH H[SHFWHG WR UHGXFH WKH YLVFRVLW\ EXW VLQFH ,QFUHDVHG WHPSHUDWXUHV DIIHFW Nn DQG 'JDD DGYHUVHO\ WKH QHW UHVXOW LV GLIILFXOW WR DVVHVV 7KH ODVW WHUP LQ WKH YDQ 'HHPWHU HTXDWLRQ LV WKH UDWH RI IORZ RI WKH FDUULHU JDV 7KHRUHWLFDOO\ HGG\ GLIIXVLRQ LV ,QGHSHQGHQW RI WKH UDWH RI IORZ 7KH FRQWULEXWLRQ WR + RI PROHFXODU GLIIXVLRQ LV LQ LQYHUVH UDWLR WR WKH UDWH RI IORZ 5HVLVWDQFH WR PDVV WUDQVIHU

PAGE 23

KRZHYHU YDULHV GLUHFWO\ ZLWK WKH YHORFLW\ RI WKH JDV ,I DOO RWKHU YDULDEOHV DUH NHSW FRQVWDQW WKHUH ZLOO H[LVW DQ RSWLPD UDWH RI IORZ IRU WKH PRVW HIILFLHQW RSHUDWLRQ RI D FROPDQ DV ,QGLFDWHG ,Q )LJXUH $W IORZ UDWHV OHVV WKDQ WKH RSWOVXP YDOXH WKH FRQWULn EXWLRQ RI QR ORFXODU GLIIXVLRQ WR + ZLOO EH DSSUHFLDEOH $ERYH RSWLPD IORZ UDWH UHVLVWDQFH WR QHVV WUDQVIHU EHFRPHV ,PSRUWDQW +RZHYHU WKLV SDUW RI WKH FXUYH ,V UHODWLYHO\ IODW DQG RSHUDWLRQV DERYH RSWLPXP YHORFLW\ ZLOO QRW KDYH WRR GHOHWHULRXV DQ HIIHFW RQ + 7KH PROHFXODU GLIIXVLRQ WHUP OD ODUJH IRU FDUULHU JDVHV RI KLJK GOIIXVOEOOOW\r 5HVLVWDQFH WR PDVV WUDQVIHU ZLOO EH GRPLQDQW IRU OLTXLGV RI KLJK YLVFRVLW\ DQG IRU KLJIU FRQFHQWUDWLRQV RI VWDWLRQDU\ SKDVH 4XDOLWDWLYH DQG TXDQWLWDWLYH SUHGLFWLRQV RI WKH HIIHFW RI + RI FKDQJHV ,Q WKH SDUDPHWHUV RI WKH YDQ 'HHPWHU HTXDWLRQ DUH VXPPDULVHG E\ 3DWWRQ f %RKHPHQ DQG 3XUQHOO f DQG 3XUQHOO f )RU DQ\ SDFNHG FROXPQ WKHUH H[LVWV D SUHVVXUH GURS EHWZHHQ WKH ,QOHW DQG WKH RXWOHW HQGV RI WKH FROXPQ $Q ,QFUHDVH ,Q WKH DEVROXWH SUHVVXUH DW HLWKHU HQG RI WKH FROXPQ RU WKH XVH RI D WDSHUHG FROXPQ ZLWK D ZLGHQLQJ GLDPHWHU WRZDUGV WKH RXWOHW HQG DUH SRVVLEOH VROXWLRQV WR PLQLPLVLQJ WKH SUHVVXUH GURS $ FRUUHFWLRQ IDFWRU IRU WKH SUHVVXUH GURS DFURVV WKH FROXPQ KDV EHHQ GHULYHG E\ -DPV DQG 0DUWLQ f 7KH UHWHQWLRQ YROXPH GHILQHG DV 95 )Fr5 ZKHUH )H ,V WKH YROXPHWULF IORZ UDWH RI JDV FDQ KH FRUUHFWHG WR JLYH WKH OLPLWLQJ UHWHQWLRQ YROXPH ZKLFK ,V ,QGHSHQGHQW RI SUHVVXUH f

PAGE 24

+ 0ROFXOD GLIIXVLRQ 5HVLVWDQFH WR PDVV WUDQVIHU (GG\ GLIIXVLRQ *DV YHORFLW\ FPVHF ),*85( ,QIOXHQFH RI FDUULHU JDV YHORFLW\ RQ FROXPQ HIILFLHQF\

PAGE 25

3R 3 ZKHUH 95 ,V WKH 8QLWLQJ UHWHQWLRQ YROXPH HW ]HUR SUHVVXUH GURS VFURVV WKH FROXPQ 95 LV WKH UHWHQWLRQ YROXPH S4 ,V WKH FRLWLRQ SUHVVXUH HW WKH RXWOHW HQG DQG I ,V WKH HYHUHJH FROXPQ SUHVVXUH 9DULRXV H[SHULPHQWV KDYH EHHQ GHYLVHG WR WHVW WKH DSSOLHVr EOOOW\ RI WKH YDQ 'HHPWHU WKHRU\ DQG ZLOO EH FRQVLGHUHG LQ WKH 'LVFXVVLRQ VHFWLRQ 7KH WKHRU\ KDV EHHQ XVHIXO ,Q DWWDLQLQJ KLJK FROXPQ HIILFLHQFLHV %DVLF *DV &KURPDWRJUDSKLF $SSDUDWXV 7KH DSSDUDWXV IRU DQ\ JDV FKURPDWRJUDSKLF VHSDUDWLRQ FRQVLVWV RI D FDUULHU JDV VXSSO\ D IORZ UHJXODWRU D SUHVVXUH JDXJH D UHFRUGHU VDPSOH SRUW m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n VLGHUDWLRQ ,Q JDV FKURPDWRJUDSK\ 7KH PLQLPXP GHWHFWRU WHPSHUDWXUH

PAGE 26

VKRXOG EH WKVW RI WKH FROXPQ WR SUHYHQW FRQGHQVDWLRQ RI VROXWHV ZKLFK ZRXOG UHVXOW LQ FKDQJH LQ FRQFHQWUDWLRQ 7KH VDPSOH SRUW WHVSHUDWXUHV VKRXOG EH DGMXVWDEOH WR SHUPLW WKH GHYHORSPHQW RI WHPSHUDWXUHV VXIILFLHQWO\ KLJK IRU WKH UDSLG YDSRUL]DWLRQ RI VROXWHV

PAGE 27

$QGURVWRQHV $QGURVWDHV EHORQJ WR WKH EURRG JURXS RI ELRORJOFROO\ LPSRUWDQW RUJDQLF FRPSRXQGV FROOHG WKH VWHURLGV ZKLFK SRVVHVV WKH F\FORSHQWDQRSHUK\GURSKHQDQWKUHQH QXFOHXV FRQWHOQLQJ FRUERQ DWRPV 7KLV QRPHQFODWXUH ,QFOXGHV PHPEHUV SUHYLRXVO\ GHVLJQDWHG DV HWLR FKRODQHVf 7KH QXFOHXV FRQWDLQV FRUERQ DWRPV ZKLFK PRNH XS WKH FRQGHQVHG V\VWHP RI IRXU ULQJV )LJXUH f 7KH ULQJV DUH GHVLJQDWHG RV $ % & DQG DQG WKH FRUERQ DWRPV RUH QXPEHUHG RV ,QGLFDWHG LQ WKH DERYH VWUXFWXUH 7KH WZR DQJXODU PHWK\O JURXSV GHVLJQDWHG RV FDUERQ DWRPV DQG f SURMHFW WR WKH IURQW RI WKH VWHURLG VZOHFXOH 7KLV SURMHFWLRQ LV GHVFULEHG DV ARULHQWHG HQG LV UHSUHVHQWHG E\ D VROLG OLQH 6LPLODUO\ RQ\ SURMHFWLRQ WR WKH UHDU RI WKH ULQJ V\VWHP LV GHVLJQDWHG RV DRULHQWHG DQG LV UHSUHVHQWHG E\ R GRWWHG OLQHr &RQIRUPDWLRQV RI $QGURVWRQH 5LQJ 6\VWHP 7KH WHUP FRQIRUPDWLRQ GHVFULEHV WKH GLIIHUHQW VSDWLDO DUUDQJHn PHQWV RI WKH DWRPV LQ D VLQJOH FODVVLFDO VWUXFWXUH RU FRQILJXUDWLRQ 7UDQVIRUPDWLRQV IURP RQH DUUDQJHPHQW WR DQRWKHU WDNH SODFH E\ WKH VLPSOH URWDWLRQ RI D VLQJOH ERQG 7KH EHVW NQRZQ H[DPSOHV DUH WKH FKDLU DQG ERDW IRUPV RI WKH F\FORKH[DQH PRGHO )LJXUH f 9LH GLIIHUn HQFHV LQ HQHUJ\ ZKLFK DUH SULPDULO\ GXH WR GLIIHUHQFHV LQ WKH

PAGE 28

),*85( &RQIRUPDWLRQV RI F\FORKH[DQH $ FKDLU % ERDW ),*85( $ f (TXDWRULDO ERQGV % $[LDO ERQGV

PAGE 29

UHSXOVLRQr EHWZHHQ FORVHO\ DSSURDFKLQJ DWRPV DPRQJ YDULRXV FRQIRUn PDWLRQV GHWHUPLQH WKH VWDELOLW\ RI WKH SDUWLFXODU FRQIRUPDWLRQ ,Q JHQHUDO LW PD\ EH VWDWHG WKDW HYHQ WKRXJK UHDFWLRQV RIWHQ SURFHHG WKURXJK XQVWDEOH IRUPV WKH XQH[FLWHG PROHFXOHV H[LVW LQ WKH FRQIRUn PDWLRQ RI ORZHVW HQHUJ\ DQG WKHUHIRUH RI KLJKHVW VWDELOLW\ f 7KH ERQGV RI WKH FDUERQ DWRPV LQ D F\FORKH[DQH PROHFXOH DUH RI WZR W\SHV )LJXUH f 7KRVH PRUH RU OHVV ,Q WKH SODQH RI WKH ULQJ DUH FDOOHG HTXDWRULDO 7KRVH SHUSHQGLFXODU WR WKH SODQH RI WKH ULQJ DUH FDOOHG D[LDO 7KHVH ZHUH IRUPHUO\ GHVLJQDWHG DV SRODU ERQGV 7KH VWDELOLW\ UHODWLRQVKLS FDQ EH LQIHUUHG E\ FRQVLGHUDWLRQ RI QRQERQGHG ++ LQWHUDFWLRQV YDQ GHU :DDOV RYHUODS fr 6LQFH WKHVH LQWHUDFWLRQV FRQWULEXWH WR LQVWDELOLW\ WR DQ H[WDQW ZKLFK ,QFUHDVHV ZLWK GHFUHDVLQJ LQWHUDWRPLF GLVWDQFH WKLV IDFWRU FDQ EH XVHG WR HVWLPDWH WKH UHODWLYH VWDELOLWLHV RI GLIIHUHQW FRQIRUPDWLRQV f ,Q WKH ERDW IRUP WKH DQG JURXSV DUH FORVHU WR HDFK RWKHU WKDQ LQ WKH FKDLU IRUP 7KHUHIRUH WKH FKDLU LV WKH PRUH VWDEOH FRQIRUPDWLRQ ,Q DGGLWLRQ WR YDQ GHU :DDOV RYHUODS HOHFWURQ GHQVLW\ LQ RU QHDU D[LDO ERQGV KDV EHHQ UHSRUWHG WR EH D IDFWRU LQ VWHUOF LQWHUDFWLRQV f 7KH LQWHUDWRPLF GLVWDQFHV EHWZHHQ DQ\ SDLU RI D[LDO JURXSV DUH VPDOOHU WKDQ WKRVH EHWZHHQ HTXDWRULDO JURXSV WKHUHIRUH D[LDO JURXSV H[KLELW JUHDWn HU UHSXOVLRQ WRZDUGV HDFK RWKHU 7KXV D FRPSRXQG FDUU\LQJ D[LDO

PAGE 30

VXEVWLWXHQWV LV OHVV VWDEOH WKDQ WKH FRUUHVSRQGLQJ FRPSRXQG ZLWK HTXDWRULDO VXEVWLWXHQWV ZKHUH QRQERQGHG UHSXOVLRQV DUH DW D PLQLPXP 7KH JHQHUDOL]DWLRQ WKDW FDQ EH PDGH IRU FRQGHQVHG ULQJ V\VWHPV PDGH XS RI VL[ PHPEHUHG ULQJV VXFK DV WKH VWHURLGV LV WKDW WKH VWDEOH VWUXFWXUHV DUH FKDLU FRQIRUPDWLRQV ZLWK DV PDQ\ DV SRVVLEOH RI WKH VXEVWLWXHQWV LQ WKH HTXDWRULDO SRVLWLRQV $V D UHSUHVHQWDWLYH VWUXFWXUH RI WKH VWHURLG QXFOHXV WKH FKROHVWDQRO PROHFXOH )LJXUH f PD\ EH H[DPLQHG ,Q WKLV PROHFXOH ULQJV % DQG & DUH ORFNHG LQ WKH FKDLU FRQIRUPDWLRQ E\ WUDQVIXVLRQ WR ULQJV $ DQG 5LQJ $ LV IUHH WR DVVXPH WKH ERDW RU FKDLU IRUPV %XW WKH LQVWDELOLW\ DVVRFLDWHG ZLWK WKH ERDW IRUP RI WKH F\FORKH[DQH PROHFXOH LV DXJPHQWHG E\ WKH LQWHUDFWLRQ EHWZHHQ WKH PHWK\O JURXS RQ & DQG WKH K\GUR[\O JURXS RQ &r 6LPLODUO\ LQ FKROHVWDQH WKH SK\GURJHQ DQG WKH & PHWK\O JURXS RSSRVH WKH ERDW IRUP f ,Q WKH DQGURVWDQH PROHFXOH )LJXUH f WKH ULQJ V\VWHP RI FKROHVWDQH LV SUHVHQW EXW WKH VLGH FKDLQ DW LV ODFNLQJ 7KH DQGURVWDQH PROHFXOH LV GHVFULEHG DV KDYLQJ WKUHH VL[PHPEHUHG FKDLU ULQJV DQG RQH ILYHPHPEHUHG ULQJ 7KH D[LDO K\GURJHQV DW SRVLWLRQ DQG DUH LQ D OLQH SDUDOOHO WR RQH DQRWKHU 7KH FDUERQ SDLUV DQG OLH LQ RQH SODQH DQG FDUERQ DWRPV DQG OLH LQ D VHFRQG SODQH SDUDOOHO WR WKH ILUVW f )RUn PDWLRQ RI ULQJ SURGXFHV D VOLJKWO\ SXFNHUHG ULQJ GXH WR WKH EHQGLQJ RI WKH ERQGV 7KH K\GURJHQV DWWDFKHG WR & DQG & DUH HFOLSVHG

PAGE 32

DQG WKH HTXDWRULDO DQG D[LDO FRQFHSW LV QRW DSSOLFDEOH 7KH ERQGV DW DQG KDYH TXDVLD[LDO Dnf DQG TXDVLHTXDWRUODO Vnf UHODWLRQVKLSV ZLWK UHVSHFW WR ULQJ & 8VHIXO JHQHUDOLVDWLRQV UHJDUGLQJ VWDELOLW\ DQG UHDFWLYLW\ RI HTXDWRULDO DQG D[LDO VXEVWLWXHQWV KDYH EHHQ GHYHORSHG f DQG D IHZ RI WKH SHUWLQHQW RQHV VD\ EH VXPPDULVHG DV IROORZV $ VXEVWLWXHQW LV JHQHUDOO\ PRUH VWDEOH LQ WKH HTXDWRULDO WKDQ LQ WKH D[LDO RULHQWDWLRQ 7KXV LI D VXEVWLWXHQW FDQ EH HTXLOLEUDWHG LQ GLIIHUHQW RULHQWDWLRQV WKH HTXDWRULDO ZLOO SUHGRPLQDWH LQ HTXLOLEULXP PL[WXUHV $Q HTXDWRULDO K\GUR[\O JURXS LV PRUH DFFHVVLEOH WKDQ WKH D[LDO DQG WKHUHIRUH UHDFWV PRUH UDSLGO\ WKDQ WKH D[LDO JURXS LQ DVWDUOIOHHWLRQ RU K\GURO\VLV W\SH UHDFWLRQV ,Q FKURPDWRJUDSK\ RQ SDSHU RU RQ DOXPLQD DQ HTXDn WRULDO DOFRKRO LV PRUH VWURQJO\ DGVRUEHG WKDQ LWV D[LDO ,VRPHU 7KH HTXDWRULDO RU D[LDO RULHQWDWLRQ RI D VXEVWLWXHQW LV RIWHQ UHIOHFWHG LQ ,WV ,QIUDUHG DEVRUSWLRQ VSHFWUXP

PAGE 33

,DRUQD ULVD ,Q $QGURVWDH} 2ULHQWDWLRQ RI VXEVWLWXHQWV DW VSHFLILF SRVLWLRQV DUH VXDQDUL]HG EHORZ f 3RVLWLRQ *6HUOHV 4FRQILD D H % FRQILD H D D$%f D D} DV f$%f D DQG % f6HULHV H D D r D D H D D H D D P Hn D Dr H S6HULHV TFRQILD EFRQILD $fH%f $fr%f

PAGE 34

7KH VSDWLDO RULHQWDWLRQ RI D VXEVWLWXHQW JURXS ,V GHVLJn QDWHG ZLWK UHIHUHQFH WR WKH DQJXODU PHWK\O JURXSV ZKLFK DUH ARULHQWHG 7KXV D JURXS SURMHFWLQJ LQ IURQW RI WKH QXFOHXV LV UHIHUUHG WR DV EHLQJ HOV WR WKH PHWK\O JURXSV DQG D JURXS SURMHFWn LQJ WR WKH UHDU ,V WUDQV WR WKHP 7KH WZR PDMRU FODVVDV RI ,VRPHULF DQGURVWDQHV DUH GHVLJn QDWHG DV D DQG I"DQGUVWDQH DQG GLIIHU LQ WKH VSDWLDO FRQILJXn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

PAGE 35

RI WKH KRUPRQH WR SURPRWH FRPE JURZWK ,Q FHSRQV f %XWHQDQGW f XVLQJ XULQH DV WKH VRXUFH RI WKH KRUPRQH ZDV WKH ILUVW WR LVRODWH DQG LGHQWLI\ RQH RI WKH SULQFLSDO PHWDEROLWHV RI WKH PDOD KRUPRQH 7KH ,VRODWHG VXEVWDQFH H[KLELWHG ELRORJLFDO DFWLYLW\ &KHPLFDO FKDUDFWHUL]DWLRQ LQGLFDWHG LW WR EH D VWHUROOLNH NHWRQH DQG %XWHQDQGW QDPHG WKH KRUPRQH DQGURVWHURQH 7KH VWUXFWXUH RI DQGURVWHURQH ZDV IXUWKHU HOXFLGDWHG E\ SDUWLDO V\QWKHVLV IURP FKROHVWHURO E\ 5X]OFND DQG KLV DVVRFLDWHV f 7KH V\QWKHWLF FRPSRXQG ZDV LGHQWLFDO WR WKDW ,VRODWHG IURP XULQH E\ %XWHQDQGW ERWK FKHPLFDOO\ DQG SK\VLRORJLFDOO\ DQG LWV VWUXFWXUH ZDV VKRZQ WR EH RK\GUR[\DDQGURVWDQ-RQH 7ZR RWKHU ELRORJLFDOO\ DFWLYH LVRPHUV RI DQGURVWDQ ZHUH V\QWKHVL]HG DQG LGHQWLILHG VKRUWO\ WKHUHn DIWHU 7KHVH ZHUH S K\GUR[\SDQGURVWDQRQH DQG SK\GUR[\ 6DDQGURVWDQRQH f ERWK RI ZKLFK KDYH VLQFH EHHQ ,VRODWHG IURP KXPDQ XULQH f $PRQJ WKH &A2M DQGU RV WDHV WKH ILUVW WR EH ,VRODWHG ZDV D OSGOK\GUR[\DDQGURVWDQRQH ZKLFK ZDV IROORZHG E\ WKH LVRODWLRQ RI DK\GUR[\DDQGURVWDQHOOGORQH DK\GUR[\SDQGURVWDQH GLRQH DQG DOOeGOK\GUD[\SDQGURVWDQRQH f $QGURJHQLF DFWLYLW\ ZDV GHPRQVWUDWHG IRU DSGLK\GUR[\DDQGURVWDQ RQH f +XPDQ XULQH DOVR FRQWDLQV VWHURLGV KDYLQJ D GRXEOH ERQG EHn WZHHQ FDUERQV DQG %XWHQGDQGW DQG KLV FRZRUNHUV f ,VRODWHG DQG LGHQWLILHG SK\GUR[\DQGURVWHQRQH

PAGE 36

/LHEHUPDQ DQG KLW JURXS f UHSRUWHG WKH LVRODWLRQ RI D DQGURVWDQHGLRQH DQG IDQGURWDQHGLRQH IURP QRUPDO PDOH DQG IHPDOH XULQHV 'XULQJ WKH ODVW GHFDGH WKH FRPSOHWH V\QWKHVLV RI WKH VWHURLG QXFOHXV DQG RI DOPRVW DOO RI WKH QDWXUDOO\ RFFXUULQJ DQGURJHQV KDYH EHHQ DFFRPSOLVKHG f 0HWKRGV RI $VVD\ IRU $QGURJHQV DQG .HWRVWDUROGD $QGURJHQV H[WUDFWHG IURP XULQH RU WLVVXHV ZHUH ILUVW VWXGLHG f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f 7UHDWPHQW RI D NHWRQH ZLWK PGLQLWUREHQ]HQH LQ WKH SUHVHQFH RI DONDOL JLYHV D WUDQVLHQW UHDFWLRQ SURGXFW RI YLROHW FRORU EXW RI XQn NQRZQ VWUXFWXUH 7KH FRORUHG FRPSRXQG KDV DQ DEVRUSWLRQ PD[LPXP DW PIL DQG LV VXIILFLHQWO\ VWDEOH WR SHUPLW UHSURGXFLEOH UHDGLQJV +RZHYHU WKH NHWRQH JURXS RQ FDUERQ FDQ ,QWHUIHUH ZLWK WKH UHVXOWV 0RGLILFDWLRQV RI WKH DERYH SURFHGXUH KDYH EHHQ UHYLHZHG E\ =OPQHUPDQQf

PAGE 37

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nV DQG EHFDPH H[WUHPHO\ SRSXODU DV D WRRO IRU WKH GHWHFWLRQ DQG GHWHUPLQDWLRQ RI WUDFH DPRXQWV RI FRPSRXQGV ZLWK

PAGE 38

FKURPRSKRUOF JURXSV :LWK WKH DFFXPXODWLRQ RI GDWD FRUUHODWLRQ RI VWUXFWXUH DQG XOWUDYLROHW DEVRUSWLRQ DIIRUGHG D SRZHUIXO QHZ PHWKRG IRU WKH HOXFLGDWLRQ RI WKH VWUXFWXUH RI D ODUJH YDULHW\ RI VWHURLGV 7KH ILHOG KDV EHHQ UHYLHZHG E\ 'RUIPDQ f 0RUH UHFHQWO\ -RQHV DQG KLV FRZRUNHUV f ,QYHVWLJDWHG WKH LQIUDUHG DEVRUSWLRQ FKDUDFn WHULVWLFV RI VWHURLGV 7KH HOXFLGDWLRQ RI D[LDO DQG HTXDWRULDO RULHQWDWLRQ ,V ,Q SDUW GXH WR ,QIUDUHG VSHFWUDO DQDO\VLV 7KH YDULn DWLRQ RI RSWLFDO DFWLYLW\ ZLWK WKH ZDYH OHQJWK RI WKH OLJKW ZDV NQRZQ DQG XVHG E\ HDUO\ ,QYHVWLJDWRUV XQWLO ZKDQ %XQVHQ GHYHORSHG KLV VRGLXP ODPS DQG D PRQRFKURPDWLF OLJKW VRXUFH EHFDPH DYDLODEOH 7KH DSSOLFDWLRQ RI URWDWRU\ GLVSHUVLRQ PHWKRGV WR WKH VWHURLG ILHOG ZDV SLRQHHUHG E\ 'MHUDVVO DQG KLV DVVRFLDWHV f 7KH DEVRUSWLRQ EDQGV ,Q URWDWRU\ GLVSHUVLRQ PHDVXUHPHQWV DUH VHQVLWLYH WR FRQIRUn PDWLRQDO FKDQJHV DQG KDYH EHHQ XVHIXO LQ HVWDEOLVKLQJ WKH YDOLGLW\ RI FHUWDLQ VWUXFWXUDO UHODWLRQVKLSV ;UD\ GLIIUDFWLRQ SDWWHUQ RI VWHURLGV KDYH EHHQ XVHIXO ,Q VXSSOHPHQWLQJ ,QIUDUHG DQDO\VLV DQG PHOWn LQJ SRLQW GHWHUPLQDWLRQV f :LWK WKH DGYHQW RI FKURPDWRJUDSKLF WHFKQLTXHV D YDOXDEOH WRRO ZDV DGGHG WR VWHURLG DQDO\VLV $GVRUSWLRQ FKURPDWRJUDSK\ ZDV DSSOLHG WR WKH SXULILFDWLRQ DQG VHSDUDWLRQ RI VWHURLGV f 6ROYHQW V\VWHPV RI GLIIHUHQW SRODULWLHV ZHUH GHVLJQHG IRU VXFFHVVIXO VHSDUDWLRQV E\ SDUWLWLRQ FKURPDWRJUDSK\ f 6DYDUG f LQYHVWLJDWHG WKH EHKDYLRU RI D ZLGH UDQJH RI & DQG & NHWRVWHURLGV DQG HVWDEOLVKHG WKH DSSOLFDELOLW\ RI WKH OOJUROQSURS\OHQH JO\FRO V\VWHP WR WKH UHVROXWLRQ RI WKHVH NHWRVWHURLGV &HUWDLQ FRUUHODWLRQV EHWZHHQ FKURPDWRJUDSKLF PRELOLW\ DQG VWUXFWXUH EHJDQ WR HPHUJH 7KH PRELOLW\

PAGE 39

RI WKH VWHURLG PROHFXOH ZDV REVHUYHG WR GHFUHDVH ZLWK ,QFUHDVLQJ QXPEHU RI R[\JHQ IXQFWLRQV 7KH OHVV WKDQ H[SHFWHG UHWDUGDWLRQ LQ WKH PRELOLW\ RI D VWHURLG ZLWK D & K\GUR[\O VXEVWLWXHQW ZDV LQWHUSUHWHG DV DQ ,QGLFDWLRQ RI ,QWUDPROHFXODU K\GURJHQ ERQGLQJ 7KH JHQHUDO SDWWHUQ RI PRELOLWLHV REVHUYHG E\ 6DYDUG ZDV FRQVLVWHQW ZLWK WKH FRQFHSW GHYHORSHG E\ %DUWRQ WKDW VWHURLGV SRVVHVVLQJ HTXDWRULDO K\GUR[\O JURXSV H[KLELW ORZHU PRELOLWLHV WKDQ WKH FRUUHVSRQGLQJ ,VRPHUV ZLWK DQ D[LDO K\GUR[\O JURXSV f )XUWKHUPRUH WKH FRUUHODWLRQV EHWZHHQ PRELOLW\ QXPEHU DQG QDWXUH RI R[\JHQ IXQFWLRQ SRVLWLRQ DQG RULHQWDWLRQ RI K\GUR[\O JURXSV WRJHWKHU ZLWK WKH UHODWLRQr VKLS RI PRELOLWLHV EHWZHHQ D DQG SLVRPHUV SURYLGHG JHQHUDOL]DWLRQV ZKLFK ZHUH ,Q DJUHHPHQW ZLWK WKH REVHUYDWLRQV RI /OHEHUPDQ DQG KLV JURXS f )URP WKH UHODWLRQVKLS RI FKURPDWRJUDSKLF HOXWLRQ WLPH WR VWUXFWXUH FHUWDLQ FOXHV WR WKH QDWXUH RI XQNQRZQ FRPSRXQGV ZHUH REWDLQHG f &KURPDWRJUDSK\ EHIRUH DQG DIWHU DFHW\ODWLRQ HQDEOHG WKH ,QYHVWLJDWRU WR DVVHVV WKH QXPEHU RI DFHW\ODEOH K\GUR[\O JURXSV f 1RPHQFODWXUH 6WHURLG QRPHQFODWXUH OD EDVHG RQ WKH UXOHV GHILQHG DW WKH &OED )RXQGDWLRQ &RQIHUHQFH ,Q /RQGRQ f 7KHVH UXOHV KDYH EHHQ SURYLVLRQDOO\ DSSURYHG E\ WKH ,QWHUQDWLRQDO 2QLRQ RI 3XUH DQG $SSOLHG &KHPLVWU\ 6LQFH WKH QRPHQFODWXUH V\VWHP ,V RI UHFHQW RULJLQ DQG VLQFH PDQ\ ,QYHVWLJDWRUV VWLOO HPSOR\ FRPPRQ QDPHV D EULHI VXPPDU\ RI WKH DSSURYHG QRPHQFODWXUH ZLOO EH JLYHQ

PAGE 40

$FFRUGLQJ WR WKH QHZ V\VWHP WKH QHPH RI WKH VWHURLG ,V EDVHG RQ ,WV K\GURFDUERQ QXFOHXV WR ZKLFK SUHIL[HV DQG VXIIL[HV DUH DGGHG WR ,QGLFDWH WKH QDWXUH RI WKH VXEVWLWXHQW 7KH SRVLWLRQ RI WKHVH VXEVWLWXHQWV DUH LQGLFDWHG E\ WKH QXPEHU RI WKH FDUERQ DWRP WR ZKLFK WKH\ DUH DWWDFKHG 7KH SURMHFWLRQV RI WKH VXEVWLn WXHQWV WR WKH IURQW DQG WR WKH UHDU RI WKH K\GURFDUERQ VNHOHWRQ DUH GHVLJQDWHG DV S DQG D UHVSHFWLYHO\ 7KH SDUHQW FRPSRXQGV DUH DQGURVWDQH SUHJQDQH FKROHVWDQH HWF DQG ZLWK HDFK QDPH WKH FRQILJXUDWLRQ DW ,V ,QGLFDWHG E\ WKH SUHIL[ D RU ^ 8QVDWXUDWLRQ ,V ,QGLFDWHG E\ WKH VXIIL[ nHQH HJ HQH 7KH UXOHV IRU WKH VXEVWLWXHQWV VWDWH WKDW RQO\ RQH NLQG RI VXEVWLWXHQW LQ HDFK FRPSRXQG ,V ,QGLFDWHG E\ D VXIIL[ WKH UHPDLQLQJ VXEVWLWXHQWV DUH LQGLFDWHG E\ SUHIL[HV 7KH VXEVWLWXHQW WR EH GHVLJQDWHG E\ D VXIIL[ ,V FKRVHQ DFFRUGLQJ WR WKH IROORZLQJ RUGHU RI SULRULW\ FDUER[\OLF DFLG RU GHULYDWLYH ,QFOXGLQJ HVWHUV FDUERQ\O DOFRKRO DPLQH HWKHU KDORJHQ

PAGE 41

(;3(5,0(17$/ 0DWHULDOV DQG 0HWKRGV &RPSRXQGV VWXGLHG 6WHURLGV ,QYHVWLJDWHG ,Q WKH FRXUVH RI WKLV VWXG\ ZHUH FKRVHQ IRU WKHLU SDUWLFXODU VWUXFWXUDO IHDWXUHV DQG ELRORJLFDO LPSRUWDQFH $OO H[FHSW WZR ZHUH DQGURVWDQH LVRPHUV WKH UHPDLQLQJ WZR ZHUH SURJHVWHURQH PHWDEROLWHV 7KH LVRPHULF DQGURVWDHV HPSOR\HG ZHUH 6D6HULDV DDQGURV WDQH DDQGURVWDQSRO D HQGURV WDQI }RO DDQGURVWDQRQH DDQGURVWDQRQH DDQGURVWDQHDSGORO DDQGURVWDQHSSGLRO DK\GUR[\ DDQGURVWDQRQH DK\GUR[\DDQGURVWDQRQH IK\GUR[\ DDQGURVWDQRQH DK\GUR[\DDQGURVWDQRQH DDQGURVWDQH GLRQH D OSGLK\GUR[\DDQGURV WDQRQH DK\GUR[\D HQGURVWHQHOOGORQH

PAGE 42

m6HUOH} 7KH LVRXHUV RI HOO WKH FRPSRXQG} OLVWHG DERYH ZHUH VWXGLHG VLPXOWDQHRXVO\ ZLWK WKH DFRZSRXQGm 7KH QRPHQFODWXUH ,V H[DFWO\ WKH VDPH DV VWDWHG DERYH H[FHSW WKDW WKH G HV,JQDW,RQ ZLOO EH VXEVWLWXWHG IRU D ,Q HDFK ,QVWDQFH 7KH RQO\ XQVDWXUDWHG DQGURVWDQH LVRPHU LQYHVWLJDWHG ZDV K\GUR[\DQGURVWHQORQH ZKLFK ZDV LQFOXGHG EHFDXVH RI LWV ELRORJLFDO LPSRUWDQFH 7KH WZR SUHJQDQH LVRPHUV ZHUH MSUHJQDQHDFDGLRO HQG DK\GUR[\I SUHJQHQRQH ,Q WKH VHFWLRQ RI WKLV ZRUN LQYROYLQJ WKH VHSDUDWLRQ RI NHWRVWHURLGV UHIHUHQFH ZLOO EH PDGH WR FRPSRXQGV IRU ZKLFK FRPPRQ QDPHV DUH VWLOO ZLGHO\ XVHG LQ WKH OLWHUDWXUH} HVSHFLDOO\ LQ WKH DUHD RI ELRORJLFDO DSSOLFDWLRQV 7KH FRVQRQ DQG WKH DSSURYHG QDPHV RI WKHVH FRPSRXQGV DUH VXPPDULVHG EHORZ $QGURVWHURQH .WORFKRODQRORQH 'HK\GURHSODQGURVWHURQH .HWRHWORFKRODRORQH +\GUR[\DQGURVWHURQH 3UHJQDQHGORO 3UHJQHQRORQH D+\GUR[\DDQGURVWDQRQH D+\GUR[\DQGURVWDQRQH +\GUR[\DQGURVWHQRQH D+\GUR[\DQGURHWDQHOO GORQH D OO'LK\GUR[\DHQGURVWDQRQH 3UHJQDQHDIODGORO D+\GUR[\I SUHJQDQRQH

PAGE 43

,Q WKH WDEOHV VXDPDUO]OQJ WKH UHVXOWH REWDLQHG ,Q WKLV VWXG\ DQ DEEUHYLDWHG IRUP RI QDPLQJ LV XVHG DQG VLQFH RQH SXUSRVH RI WKLV LQYHVWLJDWLRQ ZDV WKH FRPSDULVRQ RI DS LVRPHULF SDLUV WKH RULHQWDWLRQ DW ,V ZULWWHQ DV WKH SUHIL[ LQ WKHVH DEEUHYLDWHG IRUPV )RU H[DPSOH DK\GUR[\DDQGURVWDQRQH ,V DEEUHYLDWHG DV D$DRORQH DQG SK\GUR[\SDQGURVWDQRQV ,V DEEUHYLn DWHG HV S$SRORQH 7KH VWHURLGV ZHUH REWDLQHG IURP FRPPHUFLDO VRXUFHV DQG ZHUH RI VXIILFLHQWO\ KLJK SXULW\ IRU GLUHFW XVH 6RPH KRZHYHU VKRZHG PLQXWH DPRXQWV RI ,PSXULWLHV ZKHQ H[DPLQHG E\ JDV FKURPDWRJUDSK\ 7KUHH RI WKH NDWRVWHUROGV DK\GUR[\DDQGURVWDQRQH D K\GUR[\SDQGURVWDQRQH DQG SK\GUR[\DQGURVWHQRQHf ZKHQ DQDO\VHG E\ SDSHU FKURPDWRJUDSK\ H[KLELWHG QR ,PSXULWLHV 3UHSDUDWLRQ RI 3HU,YHW,YHV 7KH GHULYDWLYHV RI WKH VWHURLGV ZHUH SUHSDUHG E\ WKH IROORZLQJ SURFHGXUHV $FHWDWHV 7KH VWHURLG PJf ZDV DOORZHG WR UHDFW ZLWK DFHWLF DQK\GULGH POf DQG S\ULGLQH POf DW r ,Q D GU\ EDWK IRU WZR KRXUV 7KH VROYHQWV ZHUH HYDSRUDWHG XQGHU QLWURJHQ DQG WKH VWHURLG DFHWDWH ZDV GLVVROYHG ,Q DFHWRQH

PAGE 44

7UOIOXRURDFHWDWHV 7)$f 7KH VWHURLG PJf YDV DOORZHG WR UHDFW ZLWK WUOIOXRURDHHWOF DQK\GULGH POf DQG S\ULGLQH mf 7KH UHDFWLRQ ZDV FRRSOHWV LQ D IHZ PLQXWHV DW URRP WHPSHUDWXUH f 7KH VROYHQWV ZHUH HYDSRUDWHG XQGHU QLWURJHQ DQG WKH GHULYDWLYH ZDV GLVVROYHG ,Q DFHWRQH 7UOPHWK\OVOO\O HWKHUV 706f 7KH VWHURLG PJf ZDV DOORZHG WR UHDFW ZLWK KH[DPHWK\OGOVOOD]DQH POf DQG D IHZ GURSV RI WULDHWK\OFKORURVOODQH DV FDWDO\VW IRU KRXUV DW URRP WHPSHUDWXUH f 7KH UHDFWLRQ PL[WXUH ZDV FHQWULIXJHG DQG WKH VXSHUQDWDQW VROYHQWV ZHUH HYDSRUDWHG XQGHU QLWURJHQ 7KH UHVLGXH ZDV WULWXUDWHG ZLWK KH[DQH DQG UHFHQWULIXJHG 7KH VXSHUn QDWDQW ZDV DJDLQ HYDSRUDWHG XQGHU QLWURJHQ DQG WKH WUOPHWK\OVOO\O HWKHU RI WKH VWHURLG ZDV GLVVROYHG ,Q WHWUDK\GURIXUDQ f 6WXGLHV RQ WKH )UHH 6WHURLGV DQG WKHLU 'HULYDWLYHV 7KH IUHH VWHURLGV DFHWDWHV DQG WUOIOXRURDFHWDWHV ZHUH FKURn PDWRJUDSKHG IURP DFHWRQH VROXWLRQV WKH WUOPHWK\OVOO\O HWKHUV ZHUH FKURPDWRJUDSKHG IURP WHWUDK\GURIXUDQ VROXWLRQV $OO FRQFHQWUDWLRQV ZHUH SJSO 9ROXPHV RI WR XO ZHUH XVHG IRU ,QMHFWLRQV $OO PHDVXUHPHQWV ZHUH PHGH LQ GXSOLFDWH

PAGE 45

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f DQG UHDJHQW JUDGH WHWUDK\GURIXUDQ (DVWPDQ 2UJDQLF &KHPLFDOVf FKORURIRUP S\ULGLQH KH[DQH DQG DFHWLF DQK\GULGH DOO IURP )LVKHU 6FLHQWLILF &Rf ZHUH HPSOR\HG 3\ULGLQH DQG DFHWLF DQK\GULGH ZHUH IUHVKO\ GLVWLOOHG EHIRUH XVH +H[DPHWK\OGOVLOD]DQH WULPHWK\OFKORURVOLDQD DQG WUOIOXRURDFHWOF DQK\GULGH ZHUH REWDLQHG . /DERUDWRULHV DQG ZHUH XVHG ZLWKRXW IXUWKHU SXULILFDWLRQ

PAGE 46

&ROXPQ 6XSSRUWV &DXVDUHWDOO\ REWDLQHG DFLGZDVKHG DQG LQ WKH FDVH RI WKH ODVW WZR VLOLFRQL]HGf GLDWRPDFHRXV HDUWKV ZHUH XVHG DV FROXDQ VXSSRUWV 7KHVH ZHUH $SSOLHG 6FLHQFH /DERUDWRULHV6WDWH &ROOHJH3Df %XUUHOO &RUS 3LWWVEXUJ 3Df $QDODEV +DPGHQ &RQQHFWLFXWf ) 0 6FLHQWLILF &RUS $YRQGDOH3Df &KURPRVRUE : .URPDW &( $QDNURP $%6 'LDWDSRUW 6 'LIIHUHQW PHVK VL]HV HPSOR\HG DUH LQGLFDWHG XQGHU LQGLYLGXDO FROXDQ SUHSDUDWLRQV 6WDWLRQDU\ 3KDVH 7KH IROORZLQJ FRPPHUFLDOO\ REWDLQHG SRO\PHUV ZHUH XWLOLVHG DV WKH VWDWLRQDU\ SKDVHV 6LOLFRQH 5XEEHU *XPV 6( PHWK\O VLOLFRQH SRO\PHU 6( PHWK\O SKHQ\O VLOLFRQH SRO\PHU 6LOLFRQH )OXLGV ;) QLWULOH VLOLFRQH SRO\PHU PROH SHU FHQW F\DQRHWK\Of ;) QLWULOH VLOLFRQH SRO\PHU PROH SHU FHQW F\DQRHWK\Of 7KH DERYH SRO\PHUV ZDUH REWDLQHG IURP *HQHUDO (OHFWULF &RPSDQ\ :DWHUIRUG 1HZ
PAGE 47

6LOLFRQr &RSRO\PHU ;( VLOLFRQH SRO\PHU PDGH XS RI GOPHWK\OVOOR[DQH HQG 2; F\DQRHWK\OPHWK\O VLOR[DQH 7KLV ZDV REWDLQHG IURP ) + 6FLHQWLILF &RUS $YRQGDOH 3HQQV O\YDQLD )OXRUOQDWHG 6LOLFRQH 3RO\PHU 4) IOXRURDON\O VLOLFRQH SRO\PHU 6LOLFRQH &UHDVH 'R} &RUQLQJ KLJK YDFXXP VLOLFRQH JUHDVH '&VLOLHRQHf HWK\O HFHWDWH H[WUDFWf 7KH DERYH WZR PHUH REWDLQHG IURP 'RZ &RUQLQJ &RUS 0LGODQG 0LFKLJDQ 3RO\HVWHUV (WK\OHQHJO\FRO ,VRSKW£OHWH (&;3f (WK\OHQHJO\FRO VXFFLQDWH (6f 1HRSHQW\OJO\FRO HGOSDWH 1*$Gf 1HRSHQW\OJO\FR VHEDFDWH 1*6HEf 7KHVH ZHUH REWDLQHG IURP $QDODEV +DPGHQ &RQQHFWLFXW +Lr}(II % f 'OPHWK\OF\F ORKH[\OVXFFLQDWH 7KLV ZDV REWDLQHG IURP $SSOLHG 6FLHQFH /DERUDWRULHV 6WDWH &ROOHJH 3HQQV\OYDQLD

PAGE 48

%OD IHSKHQR[\SKHQ\fHWKHU &+&+f 7KLV FRPSRXQG ZDV REWDLQHG IURP (DVWPDQ 2UJDQLF &KHPLFDOV 3UHSDUDWLRQ RI &ROXPQV 7KH GHVLUHG DPRXQW RI OLTXLG SKDVH ZDV ZHLJKHG DQG GLVVROYHG ,Q D VPDOO DPRXQW RI FKORURIRUP IRU VLOLFRQH SRO\PHUVf RU DFHWRQH IRU DOO RWKHU SKDVHVf 7KH VROXWLRQ ZDV WUDQVIHUUHG ZLWK ULQVLQJV WR D EHDNHU FRQWDLQLQJ WKH ZHLJKHG VXSSRUW VXVSHQGHG LQ WKH VDPH VROYHQW DV XVHG IRU GLVVROYLQJ WKH OLTXLG SRO\PHU 7KH PL[WXUH ZDV KHDWHG JHQWO\ ZLWK FRQVWDQW VWLUULQJ XQWLO WKH VROYHQW KDG HYDSRn UDWHG FRPSOHWHO\ DQG D IUHH IORZLQJ XQLIRUPO\ FRDWHG VXSSRUW ZDV REWDLQHG 7KLV PHWKRG RI GHSRVLWLRQ RI WKH OLTXLG SKDVH RQWR WKH VXSSRUW ZDV UHOLDEOH DQG UHSURGXFLEOH DQG ZDV SUHIHUDEOH WR WKH ILOWUDWLRQ PHWKRG fr ,Q WKH ODWWHU PHWKRG D VOXUU\ RI OLTXLG SKDVH VROYHQW DQG VXSSRUW ZDV ILOWHUHG DQG WKHQ GULHG ZLWK VWLUULQJr 7KH FRQFHQn WUDWLRQ RI WKH OLTXLG SKDVH GHSRVLWHG RQ WKH VXSSRUW ZDV WKXV GHn SHQGHQW RQ WKH PHVK VL]H RI WKH VXSSRUW DV ZHOO DV RQ WKH YROXPH RI VROYHQW XVHG

PAGE 49

7KH FRDWHG VXSSRUW ZDV SDFNHG ZLWK JHQWOH WDSSLQJ ,QWR D FRSSHU RU VWDLQOHVV VWHHO FROXPQ RQH HQG RI ZKLFK ZDV SOXJJHG ZLWK JODVV ZRRO 7KH RSHQ HQG RI WKH FROXPQ ZDV IODUHG IRU HDVH RI SDFNLQJ $ YLEUDWRU DV DQ DLG ,Q SDFNLQJ ZDV IRXQG WR EH XQn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n ODWLRQ RI YRODWLOH VXEVWDQFHV ,Q WKH GHWHFWRU $IWHU WKH FROXPQ ZDV FXUHG FRQQHFWLRQV WR WKH GHWHFWRU ZHUH PDGH DQG WKH FROXPQ ZDV VDWXUDWHG ZLWK D SJ TXDQWLW\ RI WKH UHIHUHQFH VWDQGDUG FKROHVWDQD 7KH VR FDOOHG nSULPLQJn RU VDWXUDWLRQ SURFHVV ZDV QHFHVVDU\ RQ WKRVH FROXPQV ZKHUH D FHUWDLQ DPRXQW RI VDPSOH EVV DWWULEXWDEOH WR LUUHn YHUVLEOH DGVRUSWLRQ ZDV REVHUYHG 7KLV SKHQRPHQRQ YDULHG IURP FROXPQ WR FROXPQ DQG ZDV GHSHQGHQW RQ WKH QDWXUH RI WKH VDPSOH )RU H[DPSOH RQ WKH ;% FROXPQ WKHUH ZDV QR REVHUYDEOH ORVV RI DQGURVWDQHV ZKLOH HVWURJHQV ZHUH DGVRUEHG LUUHYHUVLEO\ (TXLSPHQW 0HDVXUHPHQWV ZHUH PDGH RQ WZR JDV FKURPDWRJUDSKLF LQVWUXPHQWV 0RGHO IURP 5HVHDUFK 6SHFLDOWLHV &RPSDQ\ 5LFKPRQG &DOLIRUQLD RQH HTXLSSHG ZLWK D A,RQL]DWLRQ GHWHFWRU f DQG WKH RWKHU ZLWK D IODPH

PAGE 50

LRQL]DWLRQ GHWHFWRU f 6OLJKWO\ KLJKHU VHQVLWLYLWLHV ZHUH REVHUYHG ZLWK WKH IODPH LRQL]DWLRQ XQLW ,W KDV EHHQ UHSRUWHG f WKDW PXOWLSOH K\GUR[\O JURXSV WHQG WR GHSUHVV WKH VHQVLWLYLn W\ RI WKH SLRQL]DWLRQ GHWHFWRU %RWK LQVWUXPHQWV ZHUH HTXLSSHG ZLWK DQ LQMHFWLRQ V\VWHP GHVLJQHG IRU 7HQQH\ DQG +DUULV W\SH PLFURGLSSHU LQMHFWRUV f ,Q WKH DXWKRUnV H[SHULHQFH WKLV W\SH RI LQMHFWRU ZDV PRUH FRQYHQLHQW WR XVH DQG JDYH PRUH UHSURGXFLEOH UHVXOWV WKDQ V\ULQJH W\SH LQMHFWRUV 7KH FROXPQ WHPSHUDWXUH ZDV FRQWUROOHG E\ D SURSRUWLRQDO FRQWUROOHU $ IDQ LQVLGH WKH KHDWLQJ FDELQHW ZDV HPSOR\HG WR LQVXUH XQLIRUP GLVWULEXWLRQ RI KHDW WKURXJKRXW WKH FROXPQ 7KH FDUULHU JDVHV XVHG ZHUH DUJRQ IRU WKH SLRQL]DWLRQ GHWHFWRU XQLW DQG QLWURJHQ IRU WKH IODPH XQLW ,Q WKH ODWWHU XQLW FRPSUHVVHG DLU DQG K\GURJHQ ZHUH HPSOR\HG IRU WKH IODPH

PAGE 51

3UHOLPLQDU\ ([SHULPHQWV 5HVXOWV RI SUHOLPLQDU\ H[SHULPHQWV ZLOO EH UHSRUWHG EULHIO\ DQG RQO\ IRU D IHZ UHSUHVHQWDWLYH FDVHV $OO PHDVXUHPHQWV ZHUH PDGH RQ WKH FKURPDWRJUDSK HTXLSSHG ZLWK WKH SORQL]DWORQ GHWHFWRU XQOHVV LQGLFDWHG RWKHUZLVH &ROXPQV ZHUH SDFNHG DQG FXUHG DV GHn VFULEHG 5HWHQWLRQ WLPHV W5f DUH UHSRUWHG LQ PLQXWHV $OO LQMHFWn LRQV ZHUH PDGH ZLWK D SL DOLTXRWV RI SJ SL VROXWLRQV 7KH SORQL]DWLRQ GHWHFWRU ZDV RSHUDWHG DW YROWV 'HWHFWRU WHPSHUn DWXUH } r 9DSRUL]HU WHPSHUDWXUH f r 6( 0HWK\O 6LOLFRQH 3RO\PHU 6(f 7KUHH FROXPQV GLIIHULQJ LQ FRQFHQWUDWLRQ RI 6% PHVK VL]H RI VXSSRUW RU WKH OHQJWK RU GLDPHWHU RI WKH FROXPQ ZHUH HPSOR\HG r $ FROXPQ RI 6( FRDWHG RQ PHVK &KURPRVRUE : LQ D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG 9DULDWLRQV RI UHWHQWLRQ WLPH ZLWK WHPSHUDWXUH RI WKH FROXPQ DQG SUHVVXUH RI WKH FDUULHU JDV DUH JLYHQ LQ 7DEOH $ FROXPQ RI 6( FRDWHG RQ PHVK &KURPRVRUE : LQ D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG

PAGE 52

7$%/( 9DULDWLRQ RI 5HWHQWLRQ 7LPH ZLWK &KDQJHV LQ 7HPSHUDWXUH DQG 3UHVVXUH RQ 6( &ROXPQ &RQGLWLRQV 6WHURLGV 7HPSr& 3UHV LQ SVL D$QGURVWDQf c %RORQH DRORQH RQH _fRO GLRQH _$DRORQH M If f ,Q PLQXWHV

PAGE 53

6HSDUDWLRQ IDFWRUV IRU DQGURDWDQH LVPHUD XQGHU GLIIHUHQW FRQGLWLRQV RI WHPSHUDWXUH DQG SUHVVXUH ZHUH PHDVXUHG 7DEOH f $FHWDWH GHULYDWLYHV RI WKH VWHURLGV ZHUH SUHSDUHG ZLWK WKH DLP RI LPSURYn LQJ VHSDUDWLRQ IDFWRUV &RPSDUDWLYH UHWHQWLRQ WLPH YDOXHV RI IUHH VWHURLGV DQG VWHURLG DFHWDWHV DUH JLYHQ ,Q 7DEOH &ROXPQ FRQGLWLRQV 7 r r 3 SVO $ FROXPQ RI b 6% FRDWHG RQ PHVK .URPDW &( ,Q D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG 5HWHQWLRQ YDOXHV DQG VHSDUDWLRQ IDFWRUV RI WKUHH NHWRWHUROGV DQG WKH UHVSHFWLYH DFHWDWHV DUH JLYHQ ,Q 7DEOH &ROXPQ FRQGLWLRQV 7 f r 3 SVO ,QGLYLGXDO FRPSRQHQWV FRXOG QRW EH UHVROYHG ZKHQ D PL[WXUH FRQWDLQLQJ WKH WKUHH SULQFLSDO XULQDU\ NHWRVWHUROGV ZDV FKURPDWRn JUDSKHG $FHW\ODWLRQ RI WKH VWHURLGV GLG QRW UHVXOW LQ LPSURYHG VHSDUDWLRQ 6HH VHFWLRQV DQG DERYHf 0L[HG 3KDVHV &RQWDLQLQJ 6( 6( DQG '&VLFRQH $ FROXPQ FRDWHG ZLWK b 6% DQG b '&VLFRQH ZDV IRXQG XQVDWLVIDFWRU\ 5HVXOWV ZHUH QRW UHSURGXFLEOH 7KH FROXPQ ZDV GLVFDUGHG

PAGE 54

7$%/( 9DULDWLRQ RI 6HSDUDWLRQ )DFWRU ZLWK 7HPSHUDWXUH DQG 3UHVVXUH RQ b 6( &ROXPQ 6WHURLGV &RQGLWLRQV r SVL r SVL r SVL S$DRORQH D$DRO RQH D$_RO RQH D$RQHSRO D$GLRQH

PAGE 55

7$%/( D 5HWHQWLRQ 7LPHV RQ b 6( &ROXPQ 6WHURLGV )UHH $FHWDWH S$DRORQH D$DRORQH D$RQHRO I$RQH_RO D$SRORQH &$ GLRQH D$DGLR ,Q PLQXWHV ?

PAGE 56

7$%/( D 5HWHQWLRQ 7LPHV DQG 6HSDUDWLRQ )DFWRUV RQ R 6( &ROXPQ 6WHURLGV )UHH $FHWDWH W5 6HSIDF &5 6HSIDF S$DRORQH D$DRORQH $HQRO RQH ,Q PLQXWHV

PAGE 57

6( DQG (WK\OHQHJO\FRO ,VRSWL W£OHWH (*,3f 7KUHH FROXDQD YHUD SUHSDUHG D ; 6( ; (*,3 E ; (*,3 H ; 6( &RPSDUDWLYH UHVXOWV REWDLQHG RQ WKH WKUHH FROXPQV DUH JLYHQ ,Q 7DEOH ZKHUH WKH ,QGLYLGXDO FRQWULn EXWLRQV RI HDFK SKDVH OD YLVLEOH HJ D YV E 6( DQG %LVDSKHQR[\SKHQ\fHWKHU 0L[HG 3KDVH 7KUHH FROXPQV ZHUH SUHSDUHG D ; %LVPSKHQR[\SKDQ\fHWKHU $EEUHYLDWHG DV SKHQR[\ ,Q 7DEOH f E ; %LVPSKHQR[\SKHQ\fHWKHU ; 6( UDWLRf Fr ; %LVPSKHQR[\SKHQ\fHWKHU ; 6( UDWLRf &RPSDULVRQ RI WKH UHWHQWLRQ WLPHV DUH JLYHQ ,Q 7DEOH r &ROXPQ FRQGLWLRQV 7 ‘ r 3 m SVLm 7KH GHSHQGHQFH RI WKH FKURPDWRJUDSKLF SDWWHUQ RI WKH VWHURLGV RQ WKH FRQFHQWUDWLRQ RI HDFK FRPSRQHQW RI WKH VWDWLRQDU\ SKDVH ZDV QRWHZRUWK\ E DQG F DERYHf 6HSDUDWLRQV FRXOG QRW EH DIIHFWHG GXH WR ODUJH SHDN DUHDV DQG VRPH VNHZLQJ RI WKH SHDNV $IWHU D VKRUW SHULRG RI XVH ,UUHJXODU EL]DUUH SDWWHUQV ,QGLFDn WLYH RI SKDVH GHFRPSRVLWLRQ ZHUH REVHUYHG RQ WKLV FROXPQ DQG IXUWKHU ZRUN ZDV QRW FRQWLQXHG

PAGE 58

7$%/( &RPSDULVRQ RI 6HSDUDWLRQ )DFWRUV RQ 6( DQG (WK\OHQHJO\FRO ,VRSKWDODWH (*,3f &ROXPQV b 6( )5(( f (*,3 b (*,3 b 6( $DRO RQH D$DRORQHD S$RQHIRO D$RQH IRO D$SRRQH $HQSRORQH D$GLRQH D$DSGLRO $&(7$7(6 I$DRO RQH D D$DRO RQH S$RQHRO D$RQHSR $HQRORQH D$DSGLRO D 7KH YDOXHV RI WKHVH FRPSRXQGV ZHUH IRU WKH FDOFXODWLRQ RI VHSDUDWLRQ WKH DFHWDWHV UHVSHFWLYHO\ XVHG DV WKH SRLQW IDFWRUV RI WKH IUHH RI UHIHUHQFH VWHURLGV DQG

PAGE 59

7$%/( D &RPSDULVRQ RI 5HWHQWLRQ 7LPHV RQ 6( DQG %LVPSKHQR[\SKHQ\OfHWKHU &ROXPQV 6WHURLG $FHWDWHV R 3KHQR[\ f 6( DQG R 3KHQR[\ UDWLRf UDWLRf 5 6HSIDF &5 6HSIDF 6HSIDF D$DRORQH $DRO RQH $HQRO RQH &KROHVWDQH D ,Q PLQXWHV 6 m

PAGE 60

6( 0HWK\O 3KHQ\O 6LOLFRQH 3RO\PHU 6%f $ FROXPQ RI b 6( FRDWHG RQ PHVK $QDNURP $%6 ,Q D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG 5HWHQWLRQ WLPH YDOXHV DUH JLYHQ ,Q 7DEOH &ROXPQ FRQGLWLRQV 7 ‘ r S f SVO DQG IORZ UDWH ‘ POPOQ r 7KHUH ZDV QR REVHUYDEOH DGYDQWDJH WR EH JDLQHG E\ WKH XVH RI 6( SRO\PHU DV FRPSDUHG WR 66 SKDVH 7KH ILQH PHVK VXSSRUW FDXVHG D FRQVLGHUDEOH GHFUHDVH ,Q IORZ UDWH DQG ,W ZDV GHFLGHG WR SHUn IRUP VXEVHTXHQW PHDVXUHPHQWV RQ FRDUVHU WKDQ PHVK VXSSRUWVr Lff L L L }f 'RZ &RUQLQJ +LJK 9DFXXP 6LOLFRQH JUHDVH f fn r ? f n n r f nr nr $ FROXPQ RI b '&VLOLHRQH FRDWHG RQ PHVK $QDNURP $%6 ,Q D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG &ROXPQ ZDV WRR UHWHQWLYH HYHQ DIWHU ,W ZDV FXW GRZQ WR D OHQJWK RI IHHW %WK\OHQHJO\FRO 6XFFLQDWH 3RO\HVWHU (*6f $ FROXPQ RI b (*6 FRDWHG RQ PHVK &KURPRVRUE : ,Q IRRW RGf FRSSHU FROXPQ ZDV SUHSDUHG &ROXPQ ZDV YHU\ UHWHQWLYH QR SHDNV ZHUH REVHUYHG 1HRSHQW\OJO\FRO $GLSDWH 3RO\HVWHU 1*$Gf $ FROXPQ RI b 1*$G FRDWHG RQ PHVK &KURPRVRUE : LQ D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG +LH FROXPQ UHTXLUHG

PAGE 61

7$%/( 5HWHQWLRQ 7LPHV RQ b 6( &ROXPQ 6WHURLG IF5 5HO W &KROHVWDQH _$RQH S$RQHSRO $D GLRO D$D IGLRO D$S SGLRO I$GLRQH ,Q PLQXWHV

PAGE 62

GDLO\ SULPLQJ ZLWK HKROHDWDDH ODRQHUOF UWWZWLRY WLQHD DUF JOZDD Wr 7DEOD &ROPD FRQGLWLRQr 7 f r 3 } -2 SFL IORZ UDWD PODLD ,KD UFWHQWLRD RI DROXWHV FP WKH IRRW %20 FRORQ ZDV ZDU\ WDZ 7KHUHIRUH D FRORQ RI IW :%$G FRDWHG DP r PHDK &KUHQRDRUE 9 OD D }IRRW RGf FRSSHU WXED ZDD SUHSDUHG 7KH UHWHQWLRQ GDWD RQ WKUHH NDWRHWDURLG DFHWDWHV DUH UHSRUWHG OD 7DEOH &RODQD FRQGLWLRQV 7 f r W f SDO IOHZ UDWH r QODOD $ FROHDQ RI IW 726HK FRDWHG RQ r PDVK &KURDRDRUE : LQ D IRRW 8 RGf FRSSHU WXEH ZDD SUHSDUHG LKD UHPLWD DSSHDUHG DDWODIDFWRU\ EHW WKH FROXP ZDD VXEMHFWHG WH KLJK WHPSHUDWXUFD RYHUQLJKW E\ H EUHDNGRZQ OD WKH WUDSHUDWXUH FRQWURO XQLW ,W XDH GLVFDUGHG $ FROXP RI ;) FRDWHG RQ PXFK 3URPW &O LQ m IRRW f RGf FRSSHU WXED ZDV SUHSDUHG 7KH IORZ UDWH ZHD H[WUHPHO\ FORZ HHG OHFD HI SKDVH ZHH GHWHFWHG 7KH HHOXP ZHH GLVFDUGHG

PAGE 63

7$%/( D ,VRPHULF 5HWHQWLRQ 7LPHV RQ b 1HRSHQW\OJO\FRO $GLSDWH O &ROXPQ 6WHURLG W7" 5HOW MAS$ GLRQH YD$GLRQH AR$S SGLRO O &&$4 SGLRO D$DGLRO & S$DSGLRO SD$DRO RQH A‘S$DRO RQH n D$DRO RQH A &&$RO RQH U$HQSRORQH YD$SRORQH US$GLRQH S$RQH IRO nS$RQHRO AS$DROSGLRO AD$RO ? 9&KROHVWDQH ,Q PLQXWHV

PAGE 64

7$%/( 5HWHQWLRQ 7LPHV RI .HWRVWHURLG $FHWDWHV RQ b 1HRSHQW\OJO\FRO $GLSDWH &ROXPQ 6WHURLG $FHWDWHVA5HOWS D$DRO RQH $DRORQH r $HQRO RQH &KROHVWDQH D ,Q PLQXWHV

PAGE 65

$ FROXPQ RI ;) FRDWHG RQ PHVK .URPDW &( LQ D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG 5HWHQWLRQ GDWD DQG VHSDUDWLRQ IDFWRUV DUH JLYHQ LQ 7DEOH &ROXPQ FRQGLWLRQV 7 ‘ r 3 ‘ SVL IORZ UDWH ‘ POPLQ 7KH IORZ UDWH ZDV PD[LPDO (YHQ WKRXJK WKH VHSDUDWLRQ IDFWRUV LQGLFDWHG WKHRUHWLFDO VHSDUDWLRQV DFWXDO UHVROXWLRQV RI WKH FRPSRQHQWV RI WKH PL[WXUH ZHUH QRW DFFRPSOLVKHG 7KH ZLGH SHDN DUHD SUHVHQFH RI VRPH VNHZLQJ DQG WKH KLJK UHWHQWLYOW\ RI WKH FROXPQ LQ VSLWH RI WKH KLJK WHPSHUDWXUH RI RSHUDWLRQ ZHUH PDMRU GUDZEDFNV 2) )OXRURDNO\O 3RO\PHU 2)f $ FROXPQ RI b 4) FRDWHG RQ PHVK $QDNURP LQ D IRRW RGf VWDLQOHVV VWHHO WXEH ZDV SUHSDUHG 0HDVXUHPHQWV ZHUH PDGH RQ WKH FKURPDWRJUDSK HTXLSSHG ZLWK WKH IODPH LRQLVDWLRQ GHWHFWRU &ROXPQ FRQGLWLRQV 7 ‘ r 3 ‘ SVL ,W ZDV KRSHG WKDW VHOHFWLYH UHWHQWLRQ RI WKLV FROXPQ IRU NHWRQHV FRXOG EH XVHG WR DGYDQWDJH LQ HIIHFWLQJ VHSDUDWLRQV /RVV RI SKDVH DQG ,UUHJXODU FKURPDWRJUDSKLF SDWWHUQV ZHUH REVHUYHG DQG LQ VSLWH RI QXPHURXV HIIRUWV QR UHOLDEOH GDWD FRXOG EH REWDLQHG 2QH LQWHUSUHn WDWLRQ RI WKH GDWD ZDV WKDW WKH SKDVH KDG UHDFWHG ZLWK WKH JODVV ZRRO SOXJ

PAGE 66

7$%/( D 5HWHQWLRQ 7LPHV DQG 6HSDUDWLRQ )DFWRUV RQ b ;) &ROXPQ 6WHURLG IF5 5HO 6HSIDF I$DRO RQH D$DRORQH $HQSRORQH _$DRO GLRQH D$DeGLRRQH &KROHVWDQH D ,Q PLQXWHV

PAGE 67

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f 7KH LQWHUDFWLRQV EHWZHHQ WKH SDUWV RI WKH VROXWH HQG WKH VROYHQW PROHFXOHV FRQSHWH ZLWK ,QWHUDFWLRQV EHWZHHQ WKH VROYHQW PROHn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a

PAGE 68

,Q KLJKO\ XQVDWXUDWHG PROHFXOHV VWURQJHU ,QWHUDFWLRQV QD\ RFFXU GXH WR GHORFDOLVDWLRQ RI HOHFWURQV DQG UHVXOW ,Q DQ ,QFUHDVH ,Q WKH GLVSHUVLRQ IRUFHV f 7KH WKHRUHWLFDO EDVLV RI VXFK ,QWHUn DFWLRQV ,V VWLOO ,QFRPSOHWD EXW HPSLULFDOO\ ,W ,V NQRZQ WKDW XQn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r DV HPSOR\HG LQ WKLV VWXG\ WKH FKRLFH RI SKDVHV ZDV OLPLWHG 6DWLVIDFWRU\ SKDVHV DW WKHVH KLJKHU WHPSHUDWXUHV ,QFOXGHG VLOLFRQH JXPV DQG SRO\ W ‘ f HVWHUV RI ORZ YRODWLOLW\ 6LQFH R[\JHQ VXEVWLWXWHG VWHURLGV RI UHODWLYHO\ KLJK SRODULW\ ZHUH WR EH VWXGLHG VLPXOWDQHRXVO\ ZLWK WKHLU GHULYDWLYHV RI KLJKHU RU ORZHU SRODULW\ WZR EDVLF W\SHV RI OLTXLG SKDVHV ZHUH ,QGLFDWHG $ SRODU VWDWLRQDU\ SKDVH ZRXOG HQKDQFH WKH ,QWHUDFWLRQ RI WKH SRODU JURXSV RI WKH VWHURLG PROHFXOH ZLWK WKH SKDVH DQG D QRQSRODU SKDVH ZRXOG EH H[SHFWHG SUHIHUUHQWODOO\ WR DWWUDFW WKH QRQSRODU IXQFWLRQV

PAGE 69

6% SRO\PHU ZDV IRXQG WR EH D VDWLVIDFWRU\ QRQVHOHFWLYD SKDVH %LVPSKHQR[\SKHQ\OfHWKHU ZDV LQWHUHVWLQJ LQ WKH XQLTXH UHWHQWLRQ SDWWHUQ RI WKH IFHWRVWHURLG DFHWDWHV RQ WKLV VWDWLRQDU\ SKDVH7DEOH f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f WKH VLOLFRQH QLWULOH SRO\PHU ;% VLPLODU WR VLOLFRQH QLWULOH IOXLGV EXW PRUH VWDEOH DW HOHYDWHG WHPSHUDWXUHVf DQG QHRSHQW\OJO\HRO VHEDFDWH DQG +LJII % KHDW VWDEOH SRODUf SRO\HVWHU SKDVHV 7KH HIIHFW RI YDU\LQJ WKH FKHPLFDO FRPSRVLWLRQ RI WKH VWDWLRQDU\ SKDVH IRU WKH VHSDUDWLRQ RI D ZLGH UDQJH RI VWHURLGV KDG EHHQ UHSRUWHG f /LSVN\ DQG /DQGRZQH f KDG UHSRUWHG WKH WKHUPDO VWDELOLW\ DQG SRODU SURSHUWLHV RI QHRSHQW\OJO\HRO VHEDFDWHr 6% VLOLFRQH SRO\PHU KDG IRXQG ZLGH XVH DV VWDWLRQDU\ SKDVH VLQFH WKH HDUO\ DSSOLn FDWLRQV RI WKH JDVOLTXLG FKURPDWRJUDSKLF PHWKRGV WR VWHURLG DQDO\VHV

PAGE 70

;( DQG +L(II % ZHUH WZR QHZ SRO\PHUr GHYHORSHG VSHFLILFDOO\ IRU KLJK WDPSHUDWXUH XVHV

PAGE 71

&RPSDUDWLYH ([SHU WUDHQ WH $OO RI WKH DQGURDWDHV DQG WKH GHULYDWLYHV OLVWHG XQGHU 0DWHULDOV DQG 0HWKRGV ZHUH XWLOLVHG ,Q WKLV SRUWLRQ RI WKH VWXG\ 7KH FRQFHQWUDWLRQ RI WKH VROXWLRQV ZHUH MXJSL DQG WR MXO DOLTXRWV ZHUH ,QMHFWHG 7KH GHWHFWRU DQG YDSRULVHU WHPSHUDWXUHV ZHUH WKH VDPH DV WKRVH JLYHQ ,Q WKH SUHOLPLQDU\ H[SHULPHQWV 7KH JDV FKURPDWRJUDSK HTXLSSHG ZLWK WKH SLRQOUDWLRQ GHWHFWRU RSHUDWHG DW YROWVf ZDV HPSOR\HG XQOHVV ,QGLFDWHG RWKHUZLVH )ROORZLQJ FROXPQV ZHUH XVHG 6% $ FROXPQ RI ; 6( FRDWHG RQ PHVK $QDNURP $%6 ,Q D IRRW RGf FRSSHU WXEH ZDV SUHSDUHG 7KH FKURPDWRJUDSK HTXLSSHG ZLWK WKH IODPH ,RQLVDWLRQ GHWHFWRU ZDV HPSOR\HG &ROXPQ FRQGLWLRQV 7 f r ) r SVOr 5HWHQWLRQ WLPH IRU FKROHVWDQH r PLQXWHV ;. $ FROXPQ RI ; ;( FRDWHG RQ PHVK 'ODWRSRUW 6 ,Q D IRRW RGf VWDLQOHVV VWHHO WXEH ZDV SUHSDUHG 7KH FKURn PDWRJUDSK HTXLSSHG ZLWK WKH IODPH ,RQLVDWLRQ GHWHFWRU ZDV HPSOR\HG &ROXPQ FRQGLWLRQV 7 r r 3 P SVO 5HWHQWLRQ WLPH RI FKROHVWDQH ‘ PLQXWHV

PAGE 72

+O%II % $ FROXPQ RI ; +L(II % FRDWDG RQ PDVK $QDNURP $%6 LQ D IRRW RGf FRSSHU WXED ZDV SUHSDUHG &ROXPQ FRQGLWLRQV 7 ‘ r 3 f SVL DQG IORZ UDWD ‘ PO 5HWHQWLRQ WLPH IRU FKROHDWDQH r PLQXWHV $ FROXPQ RI ,; +L(II % FRDWHG RQ PHVK 'LDWDSRUW 6 LQ D IRRW RGf VWDLQOHVV VWHHO WXEH ZDV SUHSDUHG &ROXPQ FRQGLWLRQV 7 ‘ r 3 f SVL DQG IORZ UDWH } POPLQ 5HWHQWLRQ WLPH IRU FEROHVWDQH PLQXWHV 1*6HE $ FROXPQ RI ; 1*6HE RQ PHVK &KURPRVRUE 9 LQ D IRRW RGf VWDLQOHVV VWHHO WXEH mPV SUHSDUHG &ROWDVQ FRQGLWLRQV 7 f r 3 } SVL DQG IORZ UDWH ‘ POPOQ 5HWHQWLRQ WLPH IRU FKROHDWDQH r PLQXWHV IRU WKH PHDVXUHPHQWV RI WKH IUHH VWHURLGV DQG WKH VWHURLG DFHWDWHV DQG PLQXWHV IRU DOO RWKHU FRPSRXQGV $ FROXPQ RI ,; 1*6HE FRDWHG RQ PHVK $QDNURP $%6 LQ D IRRW RGf VWDLQOHVV VWHHO WXEH ZDV SUHSDUHG &ROXPQ FRQGLWLRQV 7 ‘ r 3 f SDO DQG IORZ UDWH m POPOQ 5HWHQWLRQ WLPH IRU FKROHVWDQH ‘ PLQXWHV 7KH UHODWLYH UHWHQWLRQ WLPH YDOXHV ZLWK UHVSHFW WR FKROHVWDQH REVHUYHG RQ WKH VL[ FROXPQV XQGHU WKH FRQGLWLRQV GHVFULEHG DERYH DUH VXVPDUO]HG LQ 7DEOH

PAGE 73

7$%/( 6( br $ &RPSDULVRQ RI WKH 5HODWLYH 5HWHQWLRQ 7LPHV RI 6XEVWLWXWHG $QGURVWDQHV 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURVWDQH _$QGURVWDQH PP P D$SRO S$SRO D$RQH S$RQH D$SRO S$SRO D$RQH $RQH D$D_GLRO $DSGLRO D$SSGLRO S$SGLRO b D$DRORQH S$DRORQH D$SRORQH S$SRORQH f} D$RQHSRO S$RQHIRO D$RQHDRO $RQHDRO D$GLRQH S$GLRQH D $HQSRORQH

PAGE 74

7$%/( ;( bE FRQWf 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURVWDQH $QGU VWDQH D$SRO $RO D$RQH ^$RQH D$SRO S$SRO D$RQH DP c$RQH D$D GLRO $DSGLRO D$GLRO $GLRO D$DRORQH S$DRORQH D$RORQH $RORQH D$RQHRO $RQHRO b D$RQHDRO S$RQHDRO D$GLRQH ff $ GLRQH P PP $HQRORQH r

PAGE 75

7$%/( +L(II b& FRQWf 6WHURLGV )UHH $FHWDWH 706L;)$ D$QGURVWDQH $QGURVWDQH P D$fRO S$SRO D$RQH $RQH D$SRO S$SRO D$RQH P $RQH D$DGLRO S$DGLRO D$_GLRO $ GLRO D$DRORQH $DRORQH D$SRORQH S$SRORQH D$RQHSRO S$RQHSRO D$RQHDRO S$RQHDRO D$GLRQH $GLRQH $HQSRORQH

PAGE 76

7$%/( +L(II ObG FRQWf 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURVWDQH $QGURV WDQH Dr D$RO $RO D$RQH $RQH D$RO $RO D$RQH $RQH D$fDGLRO $D GLRO D$GLRO $GLRO D$DRORQH $DRRQH D$RO RQH $RORQH D$RQHSRO S$RQHSRO D$RQHDRO S$RQHDRO D$GLRQH S$GLRQH $HQRO RQH

PAGE 77

7$%/( 1*6HE p FRQWf 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURVWDQH I$QGURVWDQH D$RO S$SRO D$RQH $RQH D$RO S$RO D$RQH S$RQH D$D`GLRO $DSGLRO D$SGLRO S$SSGLRO D$DRORQH I$DRORQH D$SRORQH $IRORQH D$RQH _RO S$RQHSRO D$RQHDRO $RQH DRO D$GLRQH S$GLRQH P $HQSRORQH

PAGE 78

I 7$%/( 1*6HE } FRQWf 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURVWDQH _$QGURVWDQH D$SRO I$IRO D$RQH $RQH D$RO $RO D$RQH $RQH D$DGLRO $DGLRO D$SGLRO $GLRO D$DRORQH S$DRORQH D$RORQH $RORQH D$RQHRO S$RQHRO D$RQH DRO $RQHDRO D$GLRQH $GLRQH $WLHQRO RQH D E F H I 5HWHQWLRQ WLPH RI &KROHVWDQH PLQXWHV "

PAGE 79

(OXWLRQ SDWWHUQ RI WKH OHRPHUOF IUHH VWHURLGV RQ WKH VL[ FROXPQV HUH JLYHQ ,Q 7DEOH 7KH FDOFXODWHG JURXS UHWHQWLRQ IDFWRUV IRU WKH K\GUR[\O NHWRQH DFHW\O DQG WUOPHWK\OVOO\O JURXSV DUH JLYHQ ,Q 7DEOH $ FRPSDULVRQ RI REVHUYHG DQG FDOFXODWHG UHWHQWLRQ WLPH YDOXHV EDVHG RQ DYHUDJH ORJ N YDOXHV DUH UHSRUWHG ,Q 7DEOH 7KH FKDQJHV ,Q UHWHQWLRQ WLPH XSRQ GHULYDWLYH IRUPDWLRQ DUH FDOFXODWHG DFFRUGLQJ WR WKH HTXDWLRQ A nGHULYDWLYH A nIUHH VWHURLGA r r Ur r 7KH UHVXOWV DUH VXPPDULVHG ,Q 7DEOH 7KH DYHUDJH FKDQJH ,Q UHWHQWLRQ WLPH FRQVHTXHQW WR GHULYDWLYH IRUPDWLRQ DQG WKH GHSHQGHQFH RI WKLV FKDQJH XSRQ WKH QXPEHU RI VXEVWLWXHQW JURXSV DUH VKRZQ ,Q 7DEOH 7DEOH JLYHV WKH YDOXHV RI WKH 7 WHUP FDOFXODWHG DFFRUGLQJ WR WKH IROORZLQJ HTXDWLRQ Wr Wn 7 ;( 6% 6% $ FRPSDULVRQ RI WKH NHWRQH VHOHFWLYH SURSHUWLHV RI WKH IRXU FROXPQ SKHVHV ,V JLYHQ ,Q 7DEOH

PAGE 80

7$%/( 6( b &RPSDULVRQ RI 5HWHQWLRQ 'DWD RI ,VRPHULF 3DLUV 6XEVWLWXHQW (IIHFWV )XQFWLRQDO *URXS D H 1R 6XEVWLWXWLRQ RO RO DGLRO GLRO RQH RQH GLRQH DRORQH RORQH RQHRO RQHDRO

PAGE 81

7$%/( ;( b FRQW f )XQFWLRQDO *URXS D 1R 6XEVWLWXWLRQ RO ARL DGLRO GLRO RQH RQH GLRQH DRORQH RORQH RQHRO RQHDRO ‘

PAGE 82

7$%/( +L(II b FRQW f )XQFWLRQDO *URXS D 3 1R 6XEVWLWXWLRQ SRO eRO D AGLRO eSGLRO RQH RQH GLRQH DRORQH eRORQH RQHcRO RQH DRO

PAGE 83

7$%/( +L(II b FRQW f )XQFWLRQDO *URXS D 1R 6XEVWLWXWLRQ RO SRO DSGLRO ? SeGLRO RQH RQH GLRQH DRORQH r SRORQH RQHIRO RQHDRO }

PAGE 84

7$%/( 1*6HE } FRQW f )XQFWLRQDO *URXS D 1R 6XEVWLWXWLRQ RO RO DGLRO GLRO RQH RQH GLRQH DRORQH RORQH RQHRO RQHDRO

PAGE 85

7$%/( 1*6HE b FRQW f )XH WLRQD *URXS D 3 1R 6XEVWLWXWLRQ SRO SRO DAGLRO SJGLRO RQH RQH GLRQH DRORQH MRO RQH RQHSRO RQHDRO

PAGE 86

7$%/( 6( b &DOFXODWHG *URXS 5HWHQWLRQ )DFWRUV ORJ U ORJ UQ ORJ ND ORJ NEf )XQFWLRQDO D S *URXS )UHH $FHW 706L )UHH $FHW n 706L RO RO $QGURVWDQH GLRO SRO RQHSRO RQH DRO RQHDRO RQH RO RO $QGURVWDQH GLRO RO RO RQH RQH DRO DSGLRO SRO fn DRORQH RQH RQH RQH $QGURVWDQH SRO RQH RO f B GLRQH RQH RQH RQH $QGURVWDQH B RQH RO RO GLRQH RQH

PAGE 87

7$%/( & FRQW ;( D *URXWf )UHH $FHW 706L )UHH $FHW 706L RO SRO $QGURVWDQH GLRO JRO n RQH I`RO RQH DRO RQD DRO RQH SRO JRO $QGURVWDQH SAGLRO RO RORQH RQH DRO DGLRO aRO DRORQH RQH RQH RQH $QGURVWDQH aRORQH RO GLRQH RQH RQH RQH $QGURVWDQH RQHBRO BRO GLRQH RQH S

PAGE 88

7$%/( +L(II b FRQW f D nS UXQF/LXQDL *URXR )UHH $FHW 706L )UHH $FHW 706L 4O 8, $QGURVWDQH SGLR RO RQH 8, RQH DRO RQHDRO RQH IR RO $QGURVWHQH GLRO RO RQH RQH DRO D GLRO ‘ DRORQH RQH RQH RQH $QGURVWDQH ,RQH GLRQH RQH RQH RQH $QGURVWDQH RQH,, ,, GLRQH RQH ?

PAGE 89

7$%/( +L(II b FRQW f D J )XQFWLRQDO f§ Af§ *URXS )UHH $FHW 706L)UHH $FHW 706L RO RO $QGURVWDQH eeGLRO SRO RQHSRO RQH DRO RQHDRO RQH -K VRO RO $QGURVWDQH eSGLRO RO RORQH RQH DRO D GLRO RO DRORQH RQH RQH RQH $QGURVWDQH SRORQH SRO GLRQH RQH RQH RQH $QGURVWDQH RQHSRO SRO GLRQH RQH

PAGE 90

7$%/( 1*6HE b nFRQW f D U /LXXFL MO *URXS )UHH $FH& 706L )UHH $FH& 706L f cRO $QGURV FDQH GLRO SRO RQHSRO RQH DRO RQHDRO RQH SRO RO $QGURV&DQH GLR aRORQH RQH DRO DGLR RO f DRORQH RQH RQH RQH $QGURVFDQH f§ f§ f§ RORQH f§ f§ GLRQH RQH f§ f§ f§ RQH RQH $QGURVFDQH f§ f§ f§ RQH,, ,, f§ GLRQH RQH f§

PAGE 91

7$%/( 1*6HE b FRQW f D S )XQFWLRQDO *URXS )UHH $FHW 706L )UHH $FHW 706L 4O SRO $QGURVWDQH J%GLRO SRO f§ RQH I`RO RQH DRO RQHDRO RQH f ER $QGURVWDQH r A IGLRO %RO IRO RQH RQH DRO D IGLRO eRO DRORQH RQH RQH RQH $QGURVWDQH f§ f§ RQH MRO GLRQH RQH f RQH RQH $QGURVWDQH f§ f f RQHSRO JRO GLRQH RQH f f f f

PAGE 92

7$%/( D &RPSDULVRQ RI &DOFXODWHG DQG 2EVHUYHG 5HWHQWLRQ 7LPH 9DOXHV 5HODWLYH WR &KROHVWDQH 6WHURLGV b 6( } ;( } +L (II % R 1*6HE )5(( &&$GLRQH &DOHG 2EVG &DOHG 2EVG &DOHG 2EVG &DOHG 2EVG S$GLRQH D$RQH RO $RQH RO $&(7$7(6 D$SSGLRO S$SSGLRO B 706L (WKHU D$SSGLRO S$SGLRO %DVHG RQ DYHUDJH ORJ N YDOXHV

PAGE 93

7$%/( 6( b 5DWLR RI 5HODWLYH 5HWHQWLRQ 7LPHV RI 'HULYDWLYHV WR )UHH 6WHURLGV 6WHURLGV $FHWDWH 706L 7)$ D$QGURVWDQH S$QGUVWDQH ‘} D$RL S$SRO D$RQH S$RQH D$SRO S$SRO D$RQH S$RQH ff§ D$DcGLRO S$DSGLRO D$ScGLRO S$SGLRO D$DRORQH S$DRORQH D$SRORQH S$SRORQH D$RQHSRO S$RQHSRO D$RQHDRO S$RQHDRO D$GLRQH S$GLRQH

PAGE 94

7$%/( ;( bE &RQWf 6WHURLGV $FHWDWH 706L D$QGURVWDQH $QGURVWDQH D$SRO S$SRO D$RQH f S$RQH D$SRO S$SRO D$RQH S$RQH U D$DSGLRO S$DSGLRO D$SSGLRO S$SSGLRO D$DRORQH S$DRORQH D$SRORQH $RORQH D$RQHRO $RQHSRO D$RQHDRO $RQHDRO D$GLRQH S$GLRQH f}

PAGE 95

, 7$%/( &RQWf +L(II bF 6WHURLGV $FHWDWH 706L 7)$ D$QGURVWDQH A$QGURVWDQH f D$SRO S$SRO D$RQH S$RQH D$SRO $MRO D$RQH $RQH D$DSGLRO S$DIGLRO D$SSGLRO S$SSGLRO D$DRORQH $DRORQH D$SRORQH I`$SRO RQH D$RQHSRO S$RQH IRO D$RQHDRO S$RQHDRO D$GLRQH S$GLRQH B PP

PAGE 96

7$%/( &RQWf +L(II RG 6WHURLG $FHWDWH 706L 7)$ D$QGURVWDQH f $QGURVWDQH D$SRO I$IRO D$RQH S$RQH D$MRO S$SRO D$RQH S$RQH D$D SGLRO S$DSGLRO D$SSGLRO $I cGLRO D$DRORQH S$DRORQH D$SRORQH I$RORQH D$RQH RO S$RQHIRO D$RQHDRO $RQH DRO D$GLRQH S$GLRQH B

PAGE 97

7$%/( 1*6HE bH &RQWf 6WHURLGV $FHWDWH 706L 7)$ D$QGURVWDQH $QGURVWDQH D$RO S$I`RO D$RQH S$RQH D$SRO S$SRO D$RQH S$RQH D$DSGLRO $DIGLRO D$SSGLRO I$cSGLRO D$DRORQH S$DRORQH D$MRORQH I$MRORQH D$RQHSRO S$RQHSRO D$RQHDRO S$RQHDRO D$GLRQH S$GLRQH P

PAGE 98

F 7$%/( 1*6HE I 6WHURLGV $FHWDWH 706L D$QGURV WDQH $QGURVWDQH D$eRO S$SRO D$RQH S$RQH D$SRO S$SRO D$RQH S$RQH D$DSGLRO I$DGLRO D$SSGLRO I$SGLRO D$DRORQH _$DRO RQH D$RO RQH _$RORQH D$RQHSRO $RQH RO D$RQHDRO S$RQHDRO D$GLRQH S$GLRQH 5HWHQWLRQ WLPH RI &KROHVWDQH D E & G HOO I PLQXWHV

PAGE 99

7$%/( 7KH $YHUDJH &KDQJH LQ 5HWHQWLRQ 7LPH 2FFXUULQJ LQ 'HULYDWLYH )RUPDWLRQ UGHULY UIUHH [ Un! f &ROXPQ $FHWDWH 706L 7)$ 0RQR 'L 0RQR 'L 0RQR 'L 6( b ;( R f§ f§ +L(II % R 1*6HE R

PAGE 100

7$%/( 9DOXHV RI 7 DW r %DVHG RQ 0HDVXUHPHQWV 2EWDLQHG ZLWK b 6( DQG b ;( &ROXPQV 7 Wn Wn ;( a 6( L W 6( f )XQFWLRQDO *URXS D SRO RQH SRO RQH DGLRO SGLRO DRORQH SRORQH RQHRO RQHDRO GLRQH

PAGE 101

7$%/( &RPSDULVRQ RI 6HOHFWLYH 5HWHQWLRQ RI WKH )RXU 3KDVHV IRU .HWRQH DQG +\GUR[\O *URXSV 5HODWLYH 5HWHQWLRQ 7LPHV 6WHURLGV ;( 6(E 1*6HEF +L(IIn D$QGURVWDQSRO $QGURV WDQRO D$QGURVWDQRQH $QGURV WDQRQH D &KROHVWDQH PLQXWHV } ;( RQ PHVK 'LDWRSRUW 6 E &KROHVWDQH PLQXWHV b 6( RQ PHVK $QDNURP $%6 F &KROHVWDQH PLQXWHV R 1HRSHQW\O JO\FRO VHEDFDWH RQ PHVK A &KROHVWDQH PLQXWHV } +L(II % RQ PHVK 'LDWRSRUW 6

PAGE 102

7KH $ ORJ U SDUDPHWHUV IRU WKH R[LGDWLRQ RI D K\GUR[\O JURXS WR D NHWRQH DUH JLYHQ LQ 7DEOH 6HSDUDWLRQ IDFWRUV RI DQGURVWDQH LVRPHUV FDOFXODWHG DFFRUGLQJ WR WKH HTXDWLRQ U D U A VHSDUDWLRQ IDFWRU DUH JLYHQ LQ 7DEOH 'LVFXVVLRQ RI &RPSDUDWLYH ([SHULPHQWD 7KLV VWXG\ KDV FRQILUPHG WKH REVHUYDWLRQV RI /LSVN\ DQG /HQGRYQH f ZLWK UHVSHFW WR WKH SURSHUWLHV RI WKH QHRSHQW\OJO\FRO VHEDFDWH SKDVH ,Q FRQFHQWUDWLRQV DV ORZ DV RQH SHU FHQW ZZf UHSURGXFLEOH UHVXOWV ZHUH REWDLQHG ZLWK WKH QHRSHQW\OJO\FRO VHEDFDWH RYHU D SHULRG RI WZR WR WKUHH PRQWKV )XUWKHUPRUH DIWHU WKH LQLWLDO FXULQJ SURFHVV WKLV FROXPQ FRXOG EH XVHG DQ\ WLPH WKHUHDIWHU ZLWK UHPDUNDEOH UHSURGXFLn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

PAGE 103

7$%/( 6( b 7KH &KDQJH LQ ORJ U &RQWULEXWLRQ IURP +\GUR[\O WR .HWRQH 7UDQVLWLRQV /RJ $U 2+ } RQHf )XQFWLRQDO *URXSV D IRO RQH IRO RQH RRRQH GLRQH >RORQH GLRQH RQHRO GLRQH RQHDRO GLRQH D GLRO GLRQH DGLRO DRORQH D ^GLRO RQHSRO GLRO GLRQH S GLRO RORQH GLRO RQHRO

PAGE 104

, 7$%/( ;( b FRQW f )XQFWLRQDO *URXSV D I`RO RQH IRO RQH DRORQH GLRQH aRORQH GLRQH RQHSRO GLRQH RQHDRO GLRQH D GLRO GLRQH DSGLRO DRORQH DSGLRO RQH RO cIGLRO GLRQH cSGLRO SRORQH SSGLRO RQHSRO

PAGE 105

7$%/( +L(II b FRQHf )XQFWLRQDO *URXSV RO RQH IRO RQH DRORQH GLRQH RORQH GLRQH RQHRO GLRQH RQHDRO GLRQH D GLRO GLRQH D GLRO DRO RQH D GLRO RQHSRO GLRO GLRQH GLRO RORQH GLRO RQHRO D

PAGE 106

7$%/( +L(II F FRQW f )XQFWLRQDO *URXSV D ^RO RQH RO RQH DRORQH GLRQH SRORQH GLRQH RQHJRO GLRQH RQHDRO GLRQH DGLRO GLRQH DSGLRO DRORQH D cGLRO RQH SRO eGLRO GLRQH SSGLRO I`RORQH SAGLRO RQHSRO

PAGE 107

7$%/( 1*6HE FRQW f )XQFWLRQDO *URXSV D RO RQH RO RQH DRRQH GLRQH _RORQH GLRQH RQHRO GLRQH RQHDRO GLRQH DJGLRO GLRQH DSGLRO DRORQH DSGLRO RQH RO SSGLRO GLRQH IGLRO SRORQH SSGLRO RQHSRO f

PAGE 108

7$%/( 1*6HE b FRQW f )XQFWLRQDO *URXSV D IRO RQH SRO RQH R DRORQH nGLRQH SRORQH GLRQH RQHIRO GLRQH RQHDRO GLRQH D cGLRO GLRQH D SGLRO DRORQH D SGLRO RQHSRO SSGLRO GLRQH IGLRO SRORQH SSGLRO RQHSRO

PAGE 109

7$%/( 6( b ,VRPHULF 6HSDUDWLRQ )DFWRUV D S f 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURVWDQH $QGU VWDQH D$SROS$SRO D$RQHc$RQH D$SROJ$SRO D$RQHA$RQH D$D AGLROS$D SGLRO D$S SGLROS$S SGLRO D$DRO RQHS$DRO RQH D$eRORQHM$RORQH D$DRORQH$HQHSRORQH S$DRORQH$HQHRORQH D$SRORQH$HQHSRORQH S$SRORQH$HQHRORQH D$RQHIROI`$RQH eRO F$RQH DRO^$RQH DRO D$GLRQHI`$GLRQH

PAGE 110

7$%/( ;( b FRQW f 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURV WDQHe-$QGURV WDQH f D$eRO$SRO D$RQHS$RQH D$SRO$SRO D$RQH$RQH D$D SGLROS$D GLRO D$S SGLROS$S SGLR D$DRORQHI$DRORQH D$MRRQHS$SRRQH D$DRORQH$HQHSRORQH S$DRORQH$HQHSRORQH n D$I`RO RQH$HQHSRO RQH S$SRORQH$HQHSRORQH D$RQHROM$RQHSRO D$RQHDROS$RQHDRO D$GLRQHI$GLRQH

PAGE 111

7$%/( QL(II % b FRQW f 6WHURLGV )UHH $FHWDWH 706L 7)$ D$QGURV WDQH_$QGURV WDQH D$AROJ$IRO D$RQHJ$RQH D$SROe$SRO D$RQH>$RQH D$DSGLROS$DSGLRO D$SSGLROS$SSGLRO D$DRORQH$DRORQH 6D$6SRORQHS$RORQH D$DRORQH$HQHRORQH S$DRORQH$HQHIRORQH D$SRORQH$HQHMRORQH e$IRO RQH$HQHMRO RQH D$RQH SROM$RQH cRO BD$RQH DR $RQH DRO &$GLRQHM$GLRQH

PAGE 112

7$%/( +L(II % b & FRQW f 6WHURLGV D$QGURV WDQHc$QGURV WDQH D$SROe$SRO D$RQH$RQH D$eRS$SR D$RQHI$RQH D$DSGLROS$D GLRO D$A SGLR $ GLR D$DRO RQH$DRO RQH D$JRORQH$SRORQH D$DRORQH$HQHSRORQH $DRO RQH$HQHRO RQH $RO RQH$HQHRO RQH $RORQH$HQHRORQH D$RQHRO$RQHRO D$RQH DRO$RQH &RO D$GLRQH$GLRQH )UHH $FHWDWH 706L 7) f B

PAGE 113

7$%/( 1*6HE FRQW f 6WIF RLGV )UHH $FHWDWH 706L 7)$ D$QGURV WDQH$QGURV WDQH D$SROS$RO D$RQHI$RQH D$SR $RO D$RQH$RQH D$D I`GLRO$D SGORO D$e SGLROS$S SGLRO D$DRO RQH$DRO RQH D$RORQH$RO RQH D$DRORQH$HQHRORQH n S$DRORQH$HQHRORQH D$RO RQH$HQHRO RQH _$>RO RQH$HQHRO RQH D$RQHIRO$RQH RO D$RQH DROI$RQH DRO D$GLRQH$GLRQH

PAGE 114

7$%/( 1*6HE b FRQW f 6WHURLGV D$QGURVWDQH$QGURV WDQH D$SRO$SRO D$RQH$RQH D$RO$IRO D$RQH$RQH D$D SGLROS$DGLRO D$S GLR $ SGLR D$DRO RQH I$DRO RQH D$RO RQH$RO RQH D$DORQH$HQHRORQH $DRO RQH$HQHRO RQH D$RO RQH$HQHRO RQH $RO RQH$HQHRO RQH FW$RQH SROS$RQH SRO D$RQH DROS$RQH DRO D$GLRQH$GLRQH )UHH $FHWDWH 706L 7)$

PAGE 115

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n WXHQWV (YHQ WKRXJK WKH H[SHULHQFH RI WKH DXWKRU ZLWK WKLV VWDWLRQDU\ SKDVH KDV EHHQ PRVW VDWLVIDFWRU\ D ZRUG RI FDXWLRQ VHHPV QHFHVVDU\ &KDQJHV ,Q WKH GHJUHH RI VHOHFWLYLW\ ZKLFK ZHUH REVHUYHG DIWHU D IHZ ZHHNV RI FRQVWDQW XVH DW r PLJKW EH ,QGLFDWLYH RI ORVV RI VWDWLRQDU\ SKDVH RU ORVV RI D YRODWLOH FRPSRQHQW ZKLFK ZDV UHVSRQVLEOH IRU VHn OHFWLYH NHWRQH UHWHQWLRQ +RZHYHU H[FHOOHQW VWDELOLW\ RI WKLV SKDVH DW r ZDV UHSRUWHG f 7KH OHDVW SRODU DQG WKH OHDVW VHOHFWLYH SKDVH VWXGLHG ZDV WKH PHWK\O VLOLFRQH JXP 6( 7KLV HODVWRPHU ZDV FKDUDFWHULVHG E\ KLJK WKHUPDO VWDELOLW\ RYHU D ORQJ SHULRG RI WLPH DQG H[KLELWHG UHWHQWLRQ ZKLFK ZDV GLUHFWO\ SURSRUWLRQDO WR WKH PROHFXODU ZHLJKW RI WKH VROXWH :RWO] f XVLQJ DQ 6( FROXPQ KDV UHSRUWHG WKH VHSDUDWLRQ RI VHYHUDO PDMRU HVWURJHQ GHULYDWLYHV ZKLFK GLIIHU ,Q WKH QXPEHU RI DFHWR[\ JURXSV ,W KDV EHHQ UHSRUWHG WKDW WKH SDUWLFXODU QRQVHOHFWOYH SURSHUW\ RI WKH 6% HODVWRPHU ZLWK UHVSHFW WR YDULRXV IXQFWLRQDO JURXSV PLJKW PDNH

PAGE 116

WKLV DQ LGHDO VWDWLRQDU\ SKDVH ,Q WKH GHWHUPLQDWLRQ RI D SDUDPHWHU FDOOHG VWHURLG QXPEHU f 'XULQJ WKH FRPSDUDWLYH VWXG\ HDFK OLTXLG SKDVH ZDV VWXGLHG ,QGLYLGXDOO\ DQG PL[HG SKDVHV ZHUH QRW HPSOR\HG ,W ZDV IHOW WKDW ZLWK D PL[HG VWDWLRQDU\ SKDVH WKH FRQWULEXWLRQ RI RQH JURXS PLJKW EH PDVNHG ZKLOH WKDW RI RWKHUV PLJKW GRPLQDWH 7KH SRVVLELOLW\ RI HPSOR\LQJ PL[HG SKDVHV WR HIIHFW VHSDUDWLRQ ,V KRZHYHU D YHU\ DWWUDFWLYH RQH ,Q JDV FKURPDWRJUDSK\ DQG PLJKW EH WKH NH\ WR VXFFHVVIXO DSSOLFDWLRQV 6RQ} ,QYHVWLJDWRUV KDYH UHSRUWHG WKH XVH RI PL[HG SDFNLQJ EXW WKHLU UHVXOWV DSSHDU LQFRQFOXVLYH f &RQFHQWUDWLRQ RI WKH 6WDWLRQDU\ 3KDVH DQG 0DVK 6LVH RI 6XSSRUW 7KH VHFRQG FRQVLGHUDWLRQ DIWHU WKH FKRLFH RI VXLWDEOH VWDWLRQDU\ SKDVH IRU WKH FROXPQ ZDV WKH FRQFHQWUDWLRQ RI WKLV SKDVH 7KH GHn SHQGHQFH RI WKH KHLJKW HTXLYDOHQW WR D WKHRUHWLFDO SODWH WR WKH DPRXQW RI VWDWLRQDU\ SKDVH ,V JLYHQ ,Q WKH YDQ 'HHPWHU HTXDWLRQ f e r $G K0 LW X 7 f 77r IFNnf 'OOT ([SHULPHQWV GHVLJQHG WR WHVW WKH ,QWHUUHODWLRQVKLS RI WKH YDULRXV WHUPV RI WKH DERYH HTXDWLRQ KDYH EHHQ UHSRUWHG %RKHPVQ DQG 3XUQHOO KDYH UHSRUWHG WKDW WKH HGG\ GLIIXVLRQ WHUP RU WKH PXOWLSOH SHWK HIIHFWf ZKLFK ZDV FRQVLGHUHG WR EH ,QGHn SHQGHQW RI WKH YHORFLW\ RI WKH FDUULHU JDV PD\ DFWXDOO\ EH ,QYHUVHO\ SURSRUWLRQDO WR LW f ,PSURYHG FROXPQ HIILFLHQFLHV ZHUH DFKLHYHG E\ D GHFUHDVH ,Q PHVK VLVH DQG DQ LQFUHDVH LQ XQLIRUPLW\ RI VXSSRUW

PAGE 117

6LPLODUO\ LQFUHDVHG FROXPQ HIILFLHQF\ FRQVHTXHQW WR UHGXFHG UHWLH RI OLTXLG ^GLHVH WR VROLG VXSSRUW YHV REVHUYHG f HQG OHG WR WKH XVH RI ORZ ORDG FROXPQV RI RQH WR ILYH SHU HPLW LQVWHDG RI WKH WHQ WR WKLUW\ SHU FHQW UDQJH HPSOR\HG GXULQJ WKH HDUO\ VWDJHV RI DSSOLn FDWLRQV RI JDVOLTXLG FKURPDWRJUDSK\ 5HFHQWO\ +LVKWD DQG KLV FRZRUNHUV KDYH UHSRUWHG WKH UHGXFWLRQ RI WKH WLPH UHTXLUHG IRU DQDO\VLV E\ WKH VLPXOWDQHRXV UHGXFWLRQ RI WHPSHUDWXUH DQG FRQFHQWUDn WLRQ RI VWDWLRQDU\ SKDVH RQ FROXPQ VXSSRUW f $ ZRUG RI FDXWLRQ LV QHFHVVDU\ RQ ORZORDG FROXPQV ,QFRPSOHWH DQG QRQXQLIRUP GLVWULEXWLRQ ZLWKLQ WKH VXSSRUW DV D UHVXOW RI LQVXIILFLHQW DPRXQW RI OLTXLG SKDVH PD\ OHDG WR H[SRVHG VLWHV RQ WKH FROXPQ VXSSRUW 7KH UHVXOW ZRXOG EH D V\VWHP RI QRW WZR EXW WKUHH FRPSRQHQWV 7KLV SRLQW ZDV UHFHQWO\ GLVFXVVHG E\ .HOOHU DQG 6WHZDUW f $FFRUGLQJ WR WKHVH DXWKRUV ORZ ORDG FROXPQV DUH XVXDOO\ RI VXFK D FRPSRVLWLRQ WKDW QHLWKHU WKH WZRSKDVH V\VWHP RI OLTXLG SDUWLWLRQ QRU WKDW RI D VROLG DGVRUSWLRQ SUHGRPLQDWHV DQG WKH GHVFULSWLRQ DQG WUHDWPHQW RI WKHVH V\VWHPV DV WZR SKDVH V\VWHPV LV LQFRUUHFW 7KXV ZKLOH OLTXLGOLTXLG GLVWULEXWLRQ FHUWDLQO\ GRPLQDWHV PRVW VXFFHVVIXO VROYHQW V\VWHPV GHVLJQHG DV SDUWLWLRQ V\VWHPV FRPSOLFDWLRQV GXH WR DGVRUSWLRQ DQG RWKHU IDFWRUV DUH TXLWH SUREDEOH DQG VKRXOG QRW EH PLQLPLVHG 0DUWLQ f KDV VXJJHVWHG WKDW WKH ODZ 5S YDOXHV RI EDVLF DPLQR DFLGV LQ FKURPDWRJUDPV EDVHG RQ VWDUFK RU FHOOXORVH ZKLFK YH D VPDOOHU WKDQ WKH 5S YDOXHV H[SHFWHG ?RQ WKH EDVLV RI SDUWLWLRQ

PAGE 118

FRHIILFLHQWV ,Q WKH SXUH VROYHQWV RI D JLYHQ V\VWHP PLJKW EH GXH WR LRQH[FKDQJH ZLWK FDUER[\O JURXSV RU WR DVVRFLDWLRQ ZLWK WKH SDUWLDOO\ GLVVROYHG SRO\VDFFKDULGH PROHFXOHV RI WKH VWDWLRQDU\ SKDVH :KLOH HYLGHQFH IRU WKH WKHUPDO VWDELOLW\ RI VWHURLGV ZKLFK DOORZV WKHLU YDSRU SKDVH VHSDUDWLRQV ,V DEXQGDQW f RFFDVLRQDO ,QVWDQFHV RI DOWHUDWLRQ GXULQJ JDV FKURPDWRJUDSK\ RI WKH PRUH VHQVLn WLYH W\SH RI PROHFXOHV VXFK DV WKH FRUWLFRVWHURLGV KDYH EHHQ UHn SRUWHG f 7KH UHDVRQ IRU WKLV GHFRPSRVLWLRQ ,V QRW FOHDU 5HFHQWO\ :RWL] f KDV UHSRUWHG LQFRPSOHWH FRDWLQJ RI WKH FROXPQ DV D SRVVLEOH FDXVH RI WKH GHFRPSRVLWLRQ RI VWHURLG PROHFXOHV :RWL] VWXGLHG WKH EHKDYLRU RI VHYHUDO W\SHV RI VWHURLGV RQ FROXPQ VXSSRUWV FRDWHG ZLWK VWDWLRQDU\ SKDVHV RI YDULRXV FRQFHQWUDWLRQV 'HFRPSRVLWLRQ ZDV REVHUYHG RQ YHU\ ORZORDG FROXPQV ZZ SHU FHQW f HYHQ IRU YHU\ VWDEOH VWHURLGV $ SRVVLEOH H[SODn QDWLRQ VXJJHVWHG E\ :RWL] ,V WKDW GXULQJ JDV FKURPDWRJUDSK\ WKH VROXWH PROHFXOH ZKLFK ZDV DFWLYDWHG ,Q WKH YDSRU SKDVH P\ EH GHn DFWLYDWHG ,Q WKH OLTXLG SKDVH E\ GLVVLSDWLQJ ,WV HQHUJ\ RI DFWLYDWLRQ WR WKH VROYHQW PROHFXOHV ,I WKHUH ,V ,QVXIILFLHQW DPRXQW RI VROYHQW WR DEVRUE WKH DFWLYDWHG PROHFXOH WKHUPDO GHJUDGDWLRQ PLJKW RFFXU

PAGE 119

7KH LQWHUUHODWLRQVKLSr RI WKH WHUPV ,Q WKH YDQ 'HHDWHU HTXDWLRQ DQG WKH HIIHFW RI HDFK WHUP RI WKH HTXDWLRQ RQ PD\ EH VXPPDULVHG DV IROORZV 7KH VPDOOHU DQG PRUH XQLIRUP WKH SDUWLFOH VLVH RI WKH VROLG VXSSRUW WKH VPDOOHU ,V + 7KH FRQWULr EXWORQ RI PHVK VLVH ,V QHJOLJLEOH DW ORZ IORZ UDWHV EXW EHFRPHV D PDMRU FRQWULEXWLQJ IDFWRU WR + DW KLJK UDWHV RI IORZ 7KH VPDOOHU WKH FRQFHQWUDWLRQ RI WKH OLTXLG FRDWLQJ RQ WKH FROXPQ WKH VPDOOHU ,V + :KHQ WKH UDQJH RI IORZ ,V ZLGH D ODUJHU FRQFHQWUDWLRQ RI WKH OLTXLG FRDWLQJ UHVXOWV ,Q ,QFUHDVHG FROXPQ HIILFLHQF\r :KHQ WKH UDQJH RI IORZ ,V QDUURZ EHWWHU SHUIRUPDQFH ,V DFKLHYHG ZLWK ORZrORDG FROXPQV 7KH VPDOOHU WKH TXDQWLW\ RI WKH VDPSOH WKH KLJKHU ,V P n r WKH QXPEHU RI WKHRUHWLFDO SODWHV DQG WKH VPDOOHU ,V +r $ FODULILFDWLRQ RI WKH ODVW SRLQW ,V QHFHVVDU\r 7KH VLVH RI WKH VDPSOH ,V GLUHFWO\ GHSHQGHQW RQ WKH FRQFHQWUDWLRQ RI WKH OLTXLG SKDVH +HDYLO\ FRDWHG FROXPQV FDQ WROHUDWH ODUJHU DPRXQWV VDPSOHV WKDQ FDQ ORZrORDG FROWVRQV 7KHUHIRUH D GLVFXVVLRQ RI WKH ,QIOXHQFH RI VDPSOH VLVH ,V PHDQLQJIXO RQO\ DV H[SUHVVHG VLPXOWDQHRXVO\ ZLWK WKH SHUFHQWDJH RI VWDWLRQDU\ SKDVH r

PAGE 120

7KH WKHRUHWLFDO GHSHQGHQFH RI + XSRQ H[SHULPHQWDO SDUDPHWHUV LQ WHUPV RI WKH H[WHQGHG YDQ 'HHPWHU HTXDWLRQ DQG WKH H[SHULPHQWDO HYDOXDWLRQ RI UDWH HTXDWLRQ FRQVWDQWV DUH GLVFXVVHG LQ D FRPSUHn KHQVLYH PDQQHU E\ 3XUQHOO f 2Q WKH EDVLV RI SUHOLPLQDU\ UHVXOWV REWDLQHG OLTXLG FRQFHQn WUDWLRQV RI WKUHH SHU FHQW DQG PHVK UDQJH RI ZHUH FKRVHQ IRU WKH FRPSDUDWLYH VWXG\ 7KHVH FRQGLWLRQV FRXOG EH DOWHUHG LQ HLWKHU GLUHFWLRQ LI QHFHVVDU\ )RU WKH FRPSDUDWLYH VWXG\ RQ VWUXFWXUH RI VWHURLGV DQG WKHLU FKURPDWRJUDSKLF EHKDYLRU WKH DERYH FRQGLWLRQV YDUH IRXQG WR EH VDWLVIDFWRU\ IRU DOO FROXPQV 7KHVH FRQGLWLRQV DOORZHG D VXIILFLHQW DPRXQW RI SKDVH IRU LQWHUDFWLRQV LQ D SDUWLWLRQ V\VWHP DQG D YLGH UDQJH IRU WKH YDULDWLRQ RI IORZ 2Q WKH ;% FROXPQV WKH SHDNV ZHUH VKDUS DQG V\PPHWULFDOr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

PAGE 121

$V SUHGLFWHG E\ WKH YDQ 'HHQWHU HTXDWLRQ WKH FRPELQHG HIIHFW RI WKH ILQHU DQG PRUH XQLIRUP SDUWLFOH VL]H RI WKH VXSSRUW DQG WKH UHGXFHG FRQFHQWUDWLRQ RI WKH VWDWLRQDU\ SKDVH mDV WR LQn FUHDVH FROXPQ UHVROXWLRQ ,W PXVW EH HPSKDVL]HG WKDW FROXPQ HIn ILFLHQF\ ,V QRW WKH VROH IDFWRU RI ,PSRUWDQFH ,Q JDVOLTXLG FKURPDWRJUDSK\ 6RPH H[FHOOHQW VHSDUDWLRQV FDQ EH DFKLHYHG RQ FROXPQV QRW RSHUDWLQJ DW PD[LPXP HIILFLHQF\ 6HOHFWLYH UHWHQWLRQ RI WKH OLTXLG SOD\V D UROH KHUH )XUWKHUPRUH FROXPQ HIILFLHQF\ ,V QRW DQ DEVROXWH TXDQWLW\ EXW YDULHV IRU GLIIHUHQW FRPSRXQGV WKH YDQ 'HHPWHU HTXDWLRQ PDNHV DYDLODEOH WR WKH ,QYHVWLJDWRU D WRRO IRU ,PSURYLQJ DQG UHYLVLQJ FROXPQ FRQGLWLRQV DQG HIILFLHQF\ mKHQ GHVLUHG

PAGE 122

6HSDUDWLRQ RI 8ULQDU\ .HWRVWHUROGVf .QRZQ 0L[WXUH} $ NQRZQ PL[WXUH RI WKH WUOL0WK\OVOO\O HWKHUV RI WKH SULQFLSDO NHWRVWHUROGV DQG WZR SUHJQDQH ,VRPHUV ZHUH FKURPDWRJUDSKHG ,Q WKH XVXDO PDQQHU RQ IRXU FROXPQV $ FROXPQ RI ,; 6% FRDWHG RQ PHVK $QDNURP $%6 ,Q D IRRW RGf VWDLQOHVV VWHHO WXEH ZDV SUHSDUHG 7KH FKURPDWRn JUDSK HTXLSSHG ZLWK WKH IODPH ,RQL]DWLRQ GHWHFWRU ZDV HPSOR\HG &ROXPQ FRQGLWLRQV 7 ‘ r 3 f SDO 5HWHQWLRQ WLPH IRU FKROHVWDQH } PLQXWHV 2WKHU FROXPQV XVHG ,Q WKH VHSDUDWLRQ VWXG\ ZHUH ,; +L%II % ,; 1*6HE DQG ; ;% XQGHU WKH VDPH FRQGLWLRQV DV GHVFULEHG DERYH 5HVXOWV DUH SUHVHQWHG ,Q 7DEOH 7KH ILYH PDMRU XULQDU\ NHWRVWHUROGV ZHUH UHVROYHG RQ WKH ;% +O%II % DQG 1*6HE FROXPQV 2QO\ SDUWLDO VHSDUDWLRQV ZHUH REWDLQHG RQ WKH 6%r FROXPQ &KURPDWRJUDPV VKRZLQJ WKH VHSDUDWLRQ RI WKH XULQDU\ NHWR VWHUROGV DQG WKH SURJHVWHURQH PHWDEROLWHV DUH VKRZQ ,Q )LJXUHV 7KH VHSDUDWLRQ RI WKH WZR SUHJQDQH LVRPHUV VLPXOWDQHRXVO\ ZLWK WKH NHWRn VWHUROGV ZDV DFKLHYHG RQO\ RQ WKH +O(II % DQG ;% FROXPQV

PAGE 123

7$%/( 6HSDUDWLRQ RI 3ULQFLSDO .HWRVWHURLGV DQG 3URJHVWHURQH 0HWDEROLWHV DV 7ULPHWK\OVLO\O (WKHUV 6WHURLGV ;(D 6(E 1*6HEF +L(IIG ‘ D+\GUR[\DDQGURVWDQRQH D+\GUR[\^DQGURVWDQRQH +\ GU R[\ DQGU R V W HQ RQH FW+\GUR[\DQGURVWDQHOO GLRQH DOOS'LK\GUR[\4DQGURVWDQRQH S3UHJQDQHDDGLRO D+\GUR[\SUHJQDQRQH D &KROHVWDQH PLQXWHV m ;( RQ 'LDWRSRUW 6 E &KROHVWDQH PLQXWHV b 6( RQ PHVK $QDNURP $%6 &KROHVWDQH PLQXWHV b 1HRSHQW\O JO\FRO VHEDFDWH RQ PHVK &KROHVWDQH PLQXWHV b +L(II % RQ PHVK 'LDWRSRUW 6

PAGE 124

+(,*+7 XQLWV

PAGE 125

),*85( *DVOLTXLG FKURPDWRJUDP RI D PL[WXUH RI NQRZQ VWHURLG WULPHWK\OVLO\O HWKHUV RQ b +L(II % FROXPQ &RQGLWLRQV DV GHVFULEHG LQ WH[W $f S3UHJQDQHDD GLRO %f DK\GUR[\DDQGURV WDQRQH &f DK\GUR[\eDQGURVWDQRQH 'f K\GUR[\DQGURVWHQRQH (f DK\GUR[\SSUHJQDQRQH )f D K\GUR[\SDQGURVWDQHOOGLRQH *f DOOGLK\GUR[\DDQGURVWDQRQH

PAGE 126

) m 0,187(6 ),*85( *DVOLTXLG FKURPDWRJUDP RI D PL[WXUH RI NQRZQ VWHURLG WULPHWK\OVLO\O HWKHUV RQ b 1*6HE FROXPQ &RQGLWLRQV DV GHVFULEHG LQ WH[W $f D+\GUR[\D DQGURVWDQRQH %f DK\GUR[\SDQGURVWDQRQH &f SK\GUR[\DQGURVW HQRQH 'f DK\GUR[\SSUHJQDQRQH (f DK\GUR[\SDQGURVWDQH GLRQH )f DOOeGLK\GUR[\DDQGURVWDQRQH

PAGE 127

3UHSDUDWLRQ RI 8ULQH 6DPSOH $ SRUWLRQ POf RI D KRXU VSHFLPHQ ZDV WUHDWHG ZLWK XQLWVm, RI KHOL[ SRPDWLD H[WUDFW *OXVXODVH (QGR /DERUDWRULHVf DW S+ DQG r IRU KRXUV IROORZHG E\ H[WUDFWLRQ ZLWK GOFKORURQHWKDQH 7KH H[WUDFW ZDV ZDVKHG ZLWK 1 1D2+ DQG ZDWHU DQG WKH H[WUDFW ZDV FDUHIXOO\ HYDSRUDWHG LQ YDFXR WR FRPSOHWH GU\QHVV 7KH UHVLGXH ZDV WKHQ WUHDWHG ZLWK KH[DPHWK\OGOVOOD]DQH DQG WUODHWK\OFKORURLODQH DV GHVFULEHG XQGHU 0DWHULDOV DQG 0HWKRGV 7KH FKURPDWRJUDP RI D DOLTXRW RI WKH WRWDO XULQH LV VKRZQ LQ )LJXUH -LVFXRVLRQ RI WKH 6HSDUDWLRQ RI 8ULQDU\ .DWRVWHUROGV $OO PHDVXUHPHQWV ZHUH PDGH RQ WKH WUOPHWK\OVOO\O GHULn YDWLYHV 7KH ILYH PDMRU XULQDU\ NHWRVWHUROGV DQG WKH WZR SUHJn QDQH PHWDEROLWHV ZHUH VHSDUDWHG RQ WKH +O(II % FROXPQ ZKHQ WKH FROXPQ FRQGLWLRQV ZHUH DGMXVWHG WR JLYH PD[LPXP UHVROXWLRQ IRU WKH NHWRVWHURLGV H[FOXVLYHO\ 7KH LPSRUWDQFH RI ORZORDG FROXPQV LQ DFKLHYLQJ KLJKHU FROXPQ UHVROXWLRQ ZDV WKXV HYLGHQW 2Q WKH RQH SHU FHQW QHRSHQW\OJO\FRO VHEDFDWH FROXPQ VHSDUDWLRQV FRXOG EH DFn FRPSOLVKHG IRU WKH ILYH SULQFLSDO XULQDU\ NHWRVWHUROGV KRZHYHU DK\GUR[\SDQGURVWDQRQH FRXOG QRW EH VHSDUDWHG IURP S SUHJQDQHDDGORO $OO VHYHQ VWHURLGV ZHUH UHVROYHG RQ WKH WKUHH SHU FHQW ;( FROXPQ 2QO\ SDUWLDOO\ UHVROYHG SHDNV FRXOG EH HIIHFWHG RQ WKH RQH SHU FHQW 6( FROXPQ 7KLV UHVXOW ZDV QRW VXUSULVLQJ VLQFH WKH QRQVHOHFWLYLW\ SURSHUWLHV RI WKH 6( SRO\PHU

PAGE 128

0,187(6 ),*85( *DVOLTXLG FKURPDWRJUDP RI XULQDU\ FRPSRQHQWV DV WULPHWK\OVLO\O HWKHUV RQ b ;( FROXPQ &RQGLWLRQV DV GHVFULEHG LQ WH[W $f DK\GUR[\D DQGURVWDQRQH %f DK\GUR[\SDQGURVWDQRQH &f SK\GUR[\ DQGURVWHQRQH 'f DK\GUR[\SDQGURVWDQH"OOGLRQH (f D OOeGLK\GUR[\DDQGURVWDQRQH +(,*+7 81,76

PAGE 129

ZHUH HYLGHQW ,Q WKH SUHOLPLQDU\ H[SHULPHQWV 7KH SXUSRVH IRU ,Qn FOXGLQJ WKH 6( SKDVH ,Q WKH SUHVHQW LQYHVWLJDWLRQ ZDV SULPDULO\ DV D UHIHUHQFH IRU FRPSDULVRQ RI WKH PRUH VHOHFWLYH SKDVHV

PAGE 130

6WXG\ RI WKH 'HSHQGHQFH RI 5HODWLYH 5HWHQWLRQ 7LQH RQ 7HPSHUDWXUH DQG RQ WKH 1DWXUH RI WKH 5HIHUHQFH 6WDQGDUG ,Q RUGHU WR DDDHDD WKH YDULDWLRQ RI UHODWLYH UHWHQWLRQ WLPH ZLWK FKDQJHV LQ WHPSHUDWXUH WKH UHWHQWLRQ WLHVD RI WKUHH DQGURVWDQH WUOPHWK\ODOO\O HWKHUV DQG RI FKROHDWDQH ZHUH GDWHUDLQHG RQ WKH RQH SHU FHQW 6( FROXPQ GHVFULEHG DERYHf DW IRXU GLIIHUHQW WHPSHUDWXUHV 5HODWLYH UHWHQWLRQ WLPHV ZHUH WKHQ FDOFXODWHG ILUVW DV UHODWHG WR FKROHDWDQH DQG WKHQ DV UHODWHG WR WKH WULQHWK\OVOO\O HWKHU RI D K\GUR[\6DDQGURVWDQRQH 7KH UHVXOWV DUH JLYHQ ,Q 7DEOH 'LVFXVVLRQ RI )DFWRUV $IIHFWLQJ 5HWHQWLRQ 7LQH 9DOXHV 7HPSHUDWXUH OD D PDMRU IDFWRU ZKLFK DIIHFWV WKH HOXWLRQ WLPH RI D VROXWH ,Q D JDVOLTXLG FKURPDWRJUDSKLF FROXPQ 7KH UDWH RI IORZ LV DOVR D FRQWULEXWLQJ IDFWRU WR WKH UHWHQWLRQ WLPH YDOXHVr +RZHYHU WKH UDWH RI IORZ YDULHV ,QYHUVHO\ ZLWK WHPSHUDWXUH KHQFH ,WV HIIHFW LV QRW DOZD\V REYLRXV :KLOH FKDQJHV ,Q DEVROXWH UHWHQWLRQ WLPHV ZLWK FKDQJHV ,Q WHPSHUDWXUH DUH QRUPDOO\ H[SHFWHG WKH YDULDWLRQ RI UHODWLYH UHWHQWLRQ YDOXHV ZLWK WHPSHUDWXUH SRVHD D SUREOHP WR WKH LQYHVWLJDWRU 7KH FKDQJHV ,Q UHODWLYH UHWHQWLRQ WLPH RYHU D WZHQW\ GHJUHH WHPSHUDWXUH UDQJH ZHUH UHFHQWO\ UHSRUWHG f DQG WKH UHFRPPHQGDWLRQ ZDV PDGH

PAGE 131

7$%/( 7KH (IIHFW RI WKH 5HIHUHQFH 6WDQGDUG RQ WKH 9DULDWLRQ RI 5HODWLYH 5HWHQWLRQ 7LPH ZLWK 7HPSHUDWXUH RQ b 6( &ROXPQ 6WHURLGV 7HPS r& D E F D E F D E F D E F f T$TR RQH f S$DRORQH f $HQSRORQH f &KROHVWDQH B f D 2EVHUYHG DEVROXWH UHWHQWLRQ WLPH LQ PLQXWHV E 5HWHQWLRQ UHODWLYH WR FKROHVWDQH F 5HWHQWLRQ UHODWLYH WR f

PAGE 132

WKDW DOO PHDVXUHPHQWV RQ DOO FROXPQV VKRXOG ED PDGH DW WKD VDPH WHPSHUDWXUH :KLOH VXFK D SUHFDXWLRQDU\ PHDVXUH PLJKW ED GHVLUDEOH LQ FRPSDUDWLYH ZRUN LW PD\ QRW DOZD\V EH IHDVLEOH )RU VROXWHV RI ORZ PRELOLW\ LW LV PRUH DGYDQWDJHRXV WR HPSOR\ KLJKHU WHPSHUDn WXUHV WKDQ KLJKHU IORZ UDWHV EHFDXVH UHWHQWLRQ WLPH YDULHV H[n SRQHQWLDOO\ ZLWK WHPSHUDWXUH DQG OLQHDUO\ ZLWK WKH YHORFLW\ RI WKH FDUULHU JDV )XUWKHUPRUH KOJKORDG FROXPQV UHTXLUH KLJKHU WHPSHUDWXUHV WKDQ ORZORDG FROXPQV $Q DOWHUQDWLYH WR WKH FRQVWDQW WHPSHUDWXUH FRQGLWLRQV DV VXJJHVWHG E\ 9DQGHQKHXYHO DQG +RUQLQJ f LV WKH XVH RI D UHIHUHQFH VWDQGDUG RWKHU WKDQ FKROHVWDQH ,I D VWDQGDUG VWUXFWXUDOO\ VLPLODU WR WKH JURXS RI FRPSRXQGV XQGHU LQYHVWLJDWLRQ LV HPSOR\HG WKHQ XQGHU WKH VDPH FRQGLWLRQV YDULDWLRQV ZLWK WHPSHUDWXUH LQ WKH UHn WHQWLRQ WLPH RI VXFK D VWDQGDUG ZRXOG EH H[SHFWHG WR EH LQ DSSUR[Ln PDWHO\ WKH VDPH UDWLR DV WKH YDULDWLRQV LQ WKH UHWHQWLRQ WLPH RI WKH VDPSOHV +LH H[SHULPHQW GHVLJQHG WR WHVW WKLV DVVXPSWLRQ VXSSRUWHG WKH DERYH DUJXPHQW 7DEOH f :KHQ FKROHVWDQH LV XVHG DV WKH UHIHUHQFH VWDQGDUG WKH PHDQ YDULDWLRQ RI WKH UHODWLYH UHWHQWLRQ WLPH RYHU WKH r UDQJH LV SHU FHQW :KHQ RQH RI WKH DQGURDWDQH LVRPHUV LV HPSOR\HG DV WKH UHIHUHQFH VWDQGDUG WKH PHDQ YDULDWLRQ LV SHU FHQW 7KH GHSHQGHQF\ RI UHWHQWLRQ WLPHV RQ WKH TXDQWLW\ DQG YROXPH RI WKH VDPSOH ZDV DOVR UHSRUWHG E\ WKH VDPH DXWKRUV f 6PDOO

PAGE 133

YROXPHV DQG FRQFHQWUDWLRQV RI VDPSOHV SUHIHUUDEO\ ,Q WKH VDPH VROYHQW ZHUH VXJJHVWHG ,Q WKH FRXUVH RI WKH SUHVHQW ,QYHVWLn JDWLRQ WR SL DOLTXRWV RI SJSO FRQFHQWUDWHG VROXWLRQV ZHUH FKURPDWRJUDSKHG ZLWK JRRG UHSURGXFLELOLW\ RI UHWHQWLRQ WLPHV +RZHYHU ,Q WKH ,VRODWHG ,QVWDQFHV ZKHUH ODUJHU YROXPHV ZHUH XVHG UHSURGXFLELOLW\ RI WKH GDWD ZDV QRW FRPSURPLVHG ODUJH TXDQWLWLHV RI VDPSOHV ZKLFK DGYHUVHO\ DIIHFW FROXQQ HIILFLHQF\ VKRXOG EH DYRLGHG H[SHFODOO\ LQ ORZORDG FROXPQV 9DULDWLRQ LQ UHWHQWLRQ WLPHV ZDV QRW REVHUYHG ZKHQ GLIIHUHQW VROYHQWV ZHUH XVHG 1HYHUn WKHOHVV LW LV GHVLUDEOH WR HPSOR\ WKH VDPH VROYHQW HVSHFLDOO\ LQ FRPSDUDWLYH VWXGLHV SURYLGHG DOO RI WKH VROXWHV DUH VROXEOH LQ RQH VROYHQW

PAGE 134

',6&866,21 5HODWLRQVKLS RI &KURPDWRJUDSKLF %HKDYLRU WR &KHPLFDO 6WUXFWXUH 5HWHQWLRQ 3DWWHUQV &HUWDLQ GHGXFWLRQV FRQFHUQLQJ WKH SRODULWLHV RI WKH IRXU VWDWLRQDU\ SKDVHV PD\ EH GUDZQ IURP WKH UHODWLYH UHWHQWLRQ WLPHV RI WKH IUHH VWHURLGV DQG WKH WKUHH GHULYDWLYHV 7DEOH f 7KH SRODUOWOHD RI WKHVH SKDVHV DUH DSSUR[LPDWHG DV +O%II % f 1*6HE ;( f 6% 7KH JHQHUDO SDWWHUQ RI UHODWLYH UHWHQWLRQ WLPH YDOXHV LV IUHH VWHURLGf DFHWDWH f WULPHWK\OV OO\O WUOIOXRURDFHWDWH IRU WKH WZR SRO\HVWHU FROXPQV DFHWDWH f IUHH VWHURLG WUOPHWK\O VOO\O IRU WKH ;( SRO\PHU DFHWDWH f WULPHWK\OVOO\O IUHH VWHURLG f WUOIOXRURDFHWDWH IRU WKH QRQSRODU 6% HODVWRV0U 'HULYDWLYHV KDYH EHHQ HPSOR\HG VLQFH WKH HDUO\ GD\V RI FKURPDWRJUDSK\ f 7KH XVH RI GHULYDWLYHV LQ VWHURLG DQDO\VLV ZDV DGYRFDWHG E\ :RWO] f DQG 9DQGHQ+HXYHO DW DOf 7KHUH DUH VHYHUDO DGYDQWDJHV WR WKH XVH RI GHULYDWLYHV LQ SUHIHUHQFH WR WKH IUHH VWHURLGV 'HULYDWLYHV DUH PRUH VWDEOH WR KHDW 3RODULW\ FDQ EH YDULHG E\ WKH IRUPDWLRQ RI D GHULYDWLYH 0ROHFXODU

PAGE 135

ZHLJKW GLIIHUHQFHV mD\ UHVXOW LQ EHWWHU VHSDUDWLRQV ZKHUH GLIIHUHQFHV LQ WKH QXPEHU RI VXEVWLWXHQW JURXSV H[LVW *UHDWHU VHQVLWLYLW\ PD\ EH REWDLQHG ZLWK GHULYDWLYHV ZKHQ SLRQLVDWLRQ GHWHFWRUV DUH HPSOR\HG f 7KH UHWHQWLRQ WLPH RI D VROXWH LQ WKH FKURPDWRJUDSKLF FROXPQ LV WKH VXP RI LWV UHVLGHQFH WLPH LQ HDFK SKDVH RI WKH FROXPQ DQG OD GHSHQGHQW RQ WKH GHJUHH RI LQWHUDFWLRQ RI WKH VROXWH DQG WKH VROYHQW +HQFH D FKDQJH LQ WKH SRODULW\ RI D PROHFXOH FRQVHTXHQW WR GHULYDWLYH IRUPDWLRQ LV UHIOHFWHG LQ LWV UHWHQWLRQ WLPH $ VXEVWLWXHQW VXFK DV WKH DFHW\O JURXS ZLOO GHFUHDVH WKH SRODULW\ GXH WR WKH K\GUR[\O JURXS DQG FRQWULEXWH WR DQ LQFUHDVHG UHWHQWLRQ WLPH YDOXH RI WKH VWHURLG DFHWDWH RQ QRQSRODU FROXPQV DQG WR D GHFUHDVHG YDOXH RQ SRODU FROXPQV 7KH UHVXOWV REWDLQHG LQ WKH SUHVHQW LQYHVWLJDWLRQ ZHUH LQ DJUHHPHQW ZLWK WKH TXDOLWDWLYH SUHGLFWLRQV ,Q WKH FDVH RI WUOPHWK\OVLO\O HWKHUV WKH JUHDW UHGXFWLRQ LQ UHWHQWLRQ WLPHV RQ SRODU FROXPQV LV GXH WR WKH IDFW WKDW WKH WUL PHWK\OV LO\O JURXS EHKDYHV OLNH D QRQSRODU IXQFWLRQDO JURXS 7KH JURXS LV VXIILFLHQWO\ EXON\ WR DIIHFW PROHFXODU ZHLJKW DQG VKDSH EXW GRHV QRW VKRZ VHOHFWLYH IXQFWLRQDOJURXS UHWHQWLRQ HIIHFWV 7KH KLJK YRODWLOLW\ RI WKHVH HWKHUV LV LQGLFDWHG E\ WKH ORZHU WKDQ H[n SHFWHG UHWHQWLRQ WLPHV RQ WKH QRQSRODU FROXPQV DQG E\ WKH XQH[SHFn WHGO\ ORZ UHWHQWLRQ WLPHV RQ WKH SRODU FROXDVWV

PAGE 136

7KH FKDQJHV EURXJKW DERXW ,Q WKH VWHURLG VROHHXOH E\ WKH VXEVWLWXWLRQ RI WKH WULIOXRURDFHW\O JURXS DUH DPELJXRXV ,Q JHQHUDO WULIOXRURDFHW\O GHULYDWLYHV KDYH EHHQ IRXQG WR EH OHVV VDWLVIDFWRU\ WKDQ WKH WULVZWK\OVLO\O GHULYDWLYHV f 7KH HDVH RI GHFRPSRVLWLRQ RI WKH WULIOXRURDFHW\O GHULYDWLYHV RQ FKURUQDWR} JUDSKLF FROXPQV ,QWURGXFH VRPH XQUHOLDELOLW\ LQ WKHLU GDWD 5HVHDUFK ZDV QRW SXUVXHG RQ WKHVH GHULYDWLYHV 2QH IXUWKHU REVHUYDWLRQ HPHUJHV IURP WKH UHVXOWV VXPPDULVHG LQ 7DEOH &KDQJHV LQ UHWHQWLRQ WLPH YDOXHV RI WEV VWHURLGV FRQn VHTXHQW WR GHULYDWLYH IRUPDWLRQ QRWZLWKVWDQGLQJ WKH GHULYDWLYHV IROORZ WKH UHWHQWLRQ SDWWHUQ RI WKH LVRLDHULF IUHH VWHURLGV LQ DOO EXW D IHZ ,QVWDQFHV +RZHYHU WKH UHWHQWLRQ WLPH YDOXHV RI WKH LVRPHULF GHULYDWLYHV GR QRW DOZD\V UHWDLQ WKH VDPH UDWLR WR HDFK RWKHU DV GR WKH FRUUHVSRQGLQJ IUHH FRPSRXQGVr $ VLPLODU REVHUYDn WLRQ ZDV PDGH E\ /LSVN\ DQG /DQGRZQH f ZKR UHSRUWHG WKDW WKH REVHUYHG GHFUHDVHV LQ UHWHQWLRQ WLPHW RI VWHURLG DFHWDWHV RQ SRODU FROXPQV ZHUH QRW WKH VDPH LQ DOO FDVHV 7KH UHYHUVDO RI WKH RUGHU RI HOXWLRQ XSRQ GHULYDWLYH IRUPDWLRQ ZDV VHHQ LQ WKH FDVH RI WKH OVRPQUOF DeGORO HQG DRORQH SDLUV ZLWK DOPRVW DOO RI WKH FROXPQV VWXGLHG +RZHYHU QR H[SODQDWLRQ ZDV HYLGHQW IRU WKLV DQRPDO\

PAGE 137

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mVWHURLGV SUHFHGH WKH D OVRPHUV YDULHV IURP FROXPQ WR FROXPQ DQG WKH YDULDWLRQ ,V QRW ,Q WKD VDPH UDWLR DV WKRVH RI WKH SDUHQW QXFOHL 7KH ,VRPHULF SDLUV RI DQGURVWDQSRO DQG DQGURDWDQRQH QRW RQO\ GR QRW IROORZ WKH SDWWHUQ RI WKHLU UHVSHFWLYH QXFOHL EXW UHYHUVH LW PDUNHGO\ 7KXV R[\JHQDWHG 6SDQGURVWDQHV H[KLELW UHWHQWLRQ YDOXHV QHDUO\ GRXEOH WKRVH RI WKH DLVRPHUV 7KLV UHYHUVDO LV PRVW SURQRXQFHG ,Q WKH FDVH RI WKH WZR SRODU FROXPQV DQG OHVV

PAGE 138

SURQRXQFHG IRU WKH ;( DQG 6( FROXPQV ,W LV HYLGHQW WKDW WKH SUHVHQFH RI DQ R[\JHQ IXQFWLRQ ,Q WKH RI WKH VWHURLG QXFOHXV VWURQJO\ ,QIOXHQFHV WKH HOXWLRQ UDWOF RI WKH De ,VRPHUV DQG WKDW WKLV ,QIOXHQFH ,V GRPLQDQW UHJDUGOHVV RI WKH QDWXUH RI WKH OLTXLG VROYHQW 7R RXU NQRZOHGJH WKLV ,V WKH ILUVW REVHUYDWLRQ RI WKH DQRPDORXV HIIHFW RI WKH R[\JHQ IXQFWLRQ ,Q DQGURVWDQHV RU ,Q DQ\ RWKHU FODVV RI VWHURLGV 7KH FRQVLVWHQW REVHUYDWLRQ RI WKH HIIHFW RI WKLV JURXS ,Q DOO WKH VROYHQWV XQGHU VWXG\ ,V ,Q DJUHHPHQW ZLWK WKH JHQHUDO REVHUYDWLRQ ,Q RWKHU W\SHV RI FKURPDWRn JUDSKLF VWXGLHV 7W KDV EHHQ UHSRUWHG f WKDW FKDQJHV ,Q WKH YDOXHV EURXJKW DERXW E\ WKH VHFRQGDU\ K\GUR[\O JURXSV ,Q GLIIHUHQW SRVLWLRQV RQ WKH VWHURLG QXFOHXV ZHUH XVXDOO\ WKH VDPH IRU D YDULHW\ RI VROYHQW V\VWHPV 7KH WZR ,VRPHULF SDLUV RI GLR[\JHQDWHG DQGURVWDQHV PDLQWDLQ WKH FKDUDFWHULVWLF HOXWLRQ RUGHU RI WKH K\GURFDUERQ QXFOHL D IDFW ,QGLFDWLQJ DQ RSSRVLQJ HIIHFW RI WKH R[\JHQ IXQFWLRQ RQ FDUERQ DWRP +RZHYHU D PDUNHG GLIIHUHQFH EHWZHHQ WKH HIIHFW RI WKH D DQG SK\GUR[\O IXQFWLRQ ,V HYLGHQW IURP WKH UHVXOWV LQ 7DEOH ,Q DOO FDVHV VWXGLHG WKH SUHVHQFH RI WKH SK\GUR[\O JURXS DSSHDUV WR HQKDQFH WKH QRUPDO GLIIHUHQFH EHWZHHQ WKH D DQG S,VRPHUV ZKLOH WKH SUHVHQFH RI WKH DK\GUR[\ JURXS DSSHDUV WR FRXQWHUDFW WKH ,QIOXHQFH RI WKH R[\JHQ RQO\ WR WKH SRLQW ZKHUH WKH ,VRPHULF D SGLRO DQG DRORQH DQGURVWDQHV H[KLELW QHDUO\ ,GHQWLFDO UHn WHQWLRQ WLPHV 7KHVH REVHUYDWLRQV ZHUH PDGH ZLWK HDFK FROXPQ ,Qn FOXGLQJ WKH 6( FROXPQ ,Q ZKLFK FDVH WKH ,QIOXHQFH RI WKH R[\JHQ

PAGE 139

IXQFWLRQ ZDV IRXQG WR ED WKH OHDVW HYLGHQW 7KXV WKH R[LGDWLRQ RI WKH K\GUR[\ IXQFWLRQ WR WKH FRUUHVSRQGLQJ NHWRQH EULQJV DERXW D VLJQLILFDQW FKDQJH LQ UHWHQWLRQ WLPH YDOXHV RQO\ ZKHQ FRRSHUHG WR WKH DSGLRO FRPSRXQG 7KH FRQWULEXWLRQ RI WKH SK\GUR[\O JURXS LQ LQFUHDVLQJ WKH UHWHQWLRQ YDOXH RI WKH DDQGURVWDQH LVRPHUV FDQ DOVR EH GHGXFHG IURP WKH FRPSDUDWLYH GDWD JLYHQ LQ D UHFHQW SDSHU IRU WKH SXUSRVH RI LOOXVWUDWLQJ FHUWDLQ RWKHU IDFWRUV f 7KH LQIOXHQFH RI WKH SK\GUR[\O JURXS PD\ EH FRQVLGHUHG LQ WHUPV RI WKH FRQIRUPDWLRQV RI WKH VWHURLG PROHFXOHV WKDW LV LQ WHUPV RI WKH RULHQWDWLRQ RI WKH K\GUR[\O JURXS ZLWKLQ WKH Dr DQG WKH AVHUOHV D IXUWKHU FRUUHODWLRQ EHWZHHQ FKURPDWRJUDSKLF EHKDYLRU VWUXFWXUH DQG FRQIRUPDWLRQ WKHQ HPHUJHV ,Q WKH e,VRPHUV WKH SK\GUR[\O ,VRPHUV D[LDO FRQIRUPDWLRQf H[KLELW JUHDWHU PRELOLW\ WKDQ WKH FRUUHVSRQGLQJ DK\GUR[\O FRPSRXQGV HTXDWRULDO FRQIRUPDWLRQf ,Q WKH DVHUOHV RQ WKH RWKHU KDQG WKLV SDWWHUQ LV UHYHUVHG DQG FRPSRXQGV ZLWK WKH K\GUR[\O JURXS LQ WKH DRUOHQWDWORQ D[LDO FRQIHU PDWLRQf H[KLELW JUHDWHU PRELOLW\ WKDQ WKHLU AOVRVZUV HTXDWRULDO FRQIRUPDWLRQf ,W LV VLJQLILFDQW WKDW LQ ERWK VHULHV WKH RULHQWDWLRQ ZKLFK LV HOV EHWZHHQ WKH & DQG UHVXOWV LQ JUHDWHU PRELOLW\ ,W LV SRVVLEOH WKDW LQWUDPROHFXODU LQWHUDFWLRQ PD\ H[LVW EHWZHHQ WKH K\GURJHQ DQG WKH & K\GUR[\O JURXS 7KHVH REVHUYDWLRQV DUH LQ DJUHHn PHQW ZLWK WKRVH RI 6DYDUG RQ WKH SDSHU SDUWLWLRQ FKURPDWRJUDSK\ RI & DQG & VWHURLGV f DQG ZLWK WKH JHQHUDO FRQFHSW GHILQHG E\ %DUWRQ f

PAGE 140

7KHRUHWLFDO $VSHFWV RI 3DUWLWLRQ &KURPDWRJUDSK\ 5HODWLRQVKLS RI 6WUXFWXUH WR &KURPDWRJUDSKLF 0RELOLW\ 0DUWLQ f KDV GHYHORSHG D WKHRU\ RI SDUWLWLRQ FRHIILFLHQWV EDVHG RQ WKH DVVXPSWLRQ WKDW WKH FKHPLFDO SRWHQWLDO RI D VXEVWDQFH LV DQ DGGLWLYH IXQFWLRQ RI WKH FRQVWLWXHQW SDUWV RI LWV PROHFXOH ,I S$ LV WKH FKHPLFDO SRWHQWLDO RI WKH VXEVWDQFH $ DQG $ S$ LV HTXDO WR WKH IUHH HQHUJ\ UHTXLUHG WR WUDQVSRUW RQH PROH RI $ IURP RQH SKDVH WR DQRWKHU WKHQ DV D ILUVW DSSUR[LPDWLRQ $ S$ PD\ EH UHJDUGHG DV EHLQJ HTXDO WR WKH VXP RI WKH SRWHQWLDO GLIIHUHQFHV MXD$3Ef RI WKH YDULRXV JURXSV ZKLFK PDNH XS PROHFXOH $ $ S$ $IL $ A $ ILF 7R D ILUVW DSSUR[LPDWLRQ WKH IUHH HQHUJ\ UHTXLUHG WR WUDQVn SRUW D JLYHQ JURXS ; IURP RQH VROYHQW WR DQRWKHU LV LQGHSHQGHQW RI WKH UHVW RI WKH PROHFXOH 7KH DGGLWLRQ RI D JURXS ; WR D PROHFXOH ZLOO FKDQJH WKH SDUWLWLRQ FRHIILFLHQW DQG KHQFH WKH UHWHQWLRQ WLPH YDOXH RI D VXEVWDQFH E\ D GHILQLWH DPRXQW LQ D SDUWLFXODU VROYHQW V\VWHP 7KLV FKDQJH ZLOO EH GHSHQGHQW RQ WKH QDWXUH RI JURXS ; DQG LQGHSHQGHQW RI WKH VWUXFWXUDO IHDWXUHV RI WKH UHVW RI WKH PROHFXOH f LQWURn GXFWLRQ RI PRUH WKDQ RQH JURXS LQWR D PROHFXOH ZLOO FDXVH D FKDQJH LQ WKH SDUWLWLRQ FRHIILFLHQW GLUHFWO\ SURSRUWLRQDO WR WKH QXPEHU RI JURXSV RI ;

PAGE 141

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f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

PAGE 142

UHTXLUHPHQW IRU DQ\ DSSOLFDWLRQ RI WKH WKHRU\ 1RWH KRZHYHU WKH GHSHQGHQFH RI WKH LQIOXHQFH RI WKH R[\JHQ IXQFWLRQ RQ VXEVWLWXWLRQ DW f %XVK KDV PDGH WKH SUDFWLFDO VXJJHVWLRQ WKDW LQ RUGHU WR ‘VKH WKH WKHRU\ ZRUNDEOH RYHU D ZLGH UDQJH RI VWHURLGV WKH ZRUG JURXS EH GHILQHG WR PHDQ QRW RQO\ WKH FKHPLFDO QDWXUH RI WKH JURXS DV VXJJHVWHG E\ 0DUWLQ EXW LWV SRVLWLRQ DQG RULHQWDWLRQ RQ WKH QXFOHXV DV ZHOO f 0DUWLQnV FRQFHSW RI WKH DGGLWLYH FRQWULEXWLRQ WR FKURPDn WRJUDSKLF PRELOLW\ RI WKH FRPSRQHQWV RI D PROHFXOH ZDV UHFHQWO\ DSSOLHG WR WKH EHKDYLRU RI VWHURLG PROHFXOHV GXULQJ JDVOLTXLG FKURPDWRJUDSK\ E\ &OD\WRQ f DQG .QLJKWV DQG 7KRPDV f &OD\WRQ H[SUHVVHG WKH PRELOLW\ RI D VXEVWLWXWHG PROHFXOH LQ WHQV RI WKH V.!EOOOWHV DWWULEXWDEOH WR WKH FRPSRQHQWV RI WKDW PROHFXOH U ‘ UQ [ ND r r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

PAGE 143

.QLJKWV DQG 7KRPDV f KDYH XVHG WKH DERYH HTXDWLRQ LQ LWV ORJDULWKPLF IRUP ORJ U ‘ ORJ U4 ORJ ND ORJ NA 7KHVH LQYHVWLJDWRUV KDYH GHPRQVWUDWHG WKDW WKH FRQWULEXWLRQ WR UHWHQWLRQ WLPH RI WKH & PHWK\O JURXS IRU VHYHUDO VWHURLGV ZDV FRQVWDQW 2WKHU REVHUYDWLRQV RI WKHVH DXWKRUV ,QFOXGH WKH FRQVWDQF\ LQ WKH $ ORJ U FRQWULEXWLRQV IRU HTXDWRULDO K\GUR[\O WR NHWRQH WUDQVLWLRQV RQ D VWDWLRQDU\ SKDVH VHOHFWLYH IRU NHWRQHV 7KH YLFLQDO JURXS HIIHFWV REVHUYHG LQ WKH YDULDWLRQ LQ WKH ORJ U SDUDn PHWHUV RI OOeK\GUR[\O DQG NHWRQH JURXSV ZHUH LQ NHHSLQJ ZLWK WKH WKHRU\ $ QHJDWLYH FRQWULEXWLRQ RI WKH PHWK\O JURXS ZDV DWWULn EXWHG WR WKH FKDQJH LQ WKH FRQWULEXWLRQ RI WKH NHWRQH LQ WKH D HQG APHWK\O SUHJQHQHGLRQH DV D UHVXOW RI VWHUOF KOQGHUDQHH ,W PXVW EH HPSKDVL]HG KRZHYHU WKDW QRW DOO GLVFUHSDQFLHV ZLWK UHVSHFW WR WKHRU\ DUH H[SOLFDEOH LQ WHUPV RI VWHULF IDFWRU 7KH GDWD RI .QLJKWV DQG 7KRPDV DUH WRR OLPLWHG WR SHUPLW VXFK D JHQHUDOL]DWLRQ &DOFXODWHG JURXS UHWHQWLRQ IDFWRUV 7DEOH f REWDLQHG RQ WKH WZR SRODU SKDVHV H[KLELW VLPLODULWLHV LQ WKH RYHUDOO SDWWHUQ DV ZHOO DV LQ WKH LQGLYLGXDO YDOXHV 6LPLODUO\ WKH YDOXHV REWDLQHG RQ WKH WZR QRQSRODU SKDVHV DUH DOLNH WKRXJK VRPH RI WKH H[WUHPHO\ ORZ DQG KLJK YDOXHV REWDLQHG RQ WKH 6( FROXPQ DUH PRGHUDWHG RQ WKH ;( FROXPQ

PAGE 144

7KH 0DJQLWXGH RI ORJ OH GHWHUPLQHG E\ WKH H[WHQW RI WKH ,QWHUDFWLRQ RI JURXS Df ZLWK WKH OLTXLG SKDVH 6LQFH IXQFWLRQDO JURXS HIIHFWV DUH DFFHQWXDWHG RQ SRODU FROXPQD DQG PLQLPL]HG RQ QRQn SRODU RQHV ORJ N YDOXHV RI IXQFWLRQDO JURXSV VKRXOG EH ODUJHU IRU SRODU VROYHQWV WKDQ IRU QRQSRODU SKDVHV 7KH UHVXOWV VXJJHVW D JUHDWHU SRODULW\ RI WKH +O(II % SRO\HVWHU DV FRPSDUHG WR WKH QHRSHQW\OJO\FRO VHEDFDWH SKDVH $ FRPSDULVRQ RI WKH JURXS UHWHQWLRQ WLPH IDFWRUV RI WKH DFHW\O YHUVXV WKH WUOPHWK\OVOO\O JURXSV VKRZ EHWWHU DJUHHPHQW DPRQJ WKH ORJ N YDOXHV ZKLFK ,V ,Q DJUHHPHQW ZLWK RWKHU REVHUYDWLRQV PDGH RQ WKH DFHWDWH JURXS7DEOH f 5HWHQWLRQ WLPH YDOXHV ZHUH FDOFXODWHG XVLQJ WKH DYHUDJH ORJ N YDOXHV REWDLQHG IURP 7DEOH DQG FRPSDUHG ZLWK WKH REVHUYHG UHWHQWLRQ WLPH YDOXHV 7DEOH f ,Q JHQHUDO WKH DJUHHPHQW EHWZHHQ WKH REVHUYHG DQG WKH FDOFXODWHG ILJXUHV ,V TXLWH JRRG ,W ,V ,QWHUHVWLQJ WKDW GHYLDWLRQV RFFXU ZKHQ WKH LQIOXHQFH RI WKH & R[\JHQ IXQFWLRQ ,V PRVW SURQRXQFHG WKDW ,V RQ WKH WZR SRODU FROXPQV IRU WKH S DQGURVWDQHV} 7KH FKDQJHV LQ UHWHQWLRQ WLPH XSRQ GHULYDWLYH IRUPDWLRQ DUH H[SUHVVHG DV WKH UDWLR RI WKH UHODWLYH UHWHQWLRQ WLPHV RI WKH GHULn YDWLYHV WR WKRVH RI WKH IUHH VWHURLGV 7DEOH f ,QWHUDFWLRQV RI WKH QRQSRODU DFHWDWH JURXS ZLWK WKH QRQSRODU 6% DQG WR D OHVVHU H[WHQW ZLWK WKH ;% SKDVHV DUH ,QGLFDWHG ,Q WKH ,QFUHDVHG UHWHQWLRQ

PAGE 145

WLPHV RI WKH VWHURLG HFHWHWHV RQ WKHVH FROXPQV 7KH GHFUHVVHG LQWHUDFWLRQV RI WKH DFHWDWHV ZLWK WKH SRODU SKDVHV DUH UHIOHFWHG LQ WKH IDVWHU HOXWLRQ WLPH RI WKH VWHURLG DFHWDWHV DV FRRSHUHG WR W WKH IUHH VWHURLG RQ WKH +LOII % DQG 1*6HE FROXPQV 7KH DYHUDJH FKDQJH EURXJKW DERXW LQ WKH UHWHQWLRQ RI WKH SDUHQW FRPSRXQG XSRQ GHULYDWLYH IRUPDWLRQ LV JLYHQ LQ 7DEOH ,W LV HYLGHQW WKDW RI WKH WKUHH GHULYDWLYHV WKH FRQWULEXWLRQ WR UHWHQWLRQ WLPH YDOXHV LV DGGLWLYH RQO\ LQ WKH FDVH RI WKH HFHWDWH JURXS 7KH DEVHQFH RI D GLUHFW UHODWLRQVKLS EHWZHHQ FRQWULEXWLRQ WR UHWHQWLRQ WLPH DQG QXPEHU RI VXEVWLWXHQW JURXSV LQ WKH FDVH RI WULPHWK\OVLO\O HWKHUV DQG WKH WULIOXRURDFHWDWHV LV LQGLFDWLYH RI WKH SUHVHQFH RI IDFWRUV VXFK DV YRODWLOLW\ DQG VWHULH KLQGHUDQHH 7KH XVH RI WKH DFHW\O JURXS LQ SUHIHUHQFH WR WKH WULPHWK\OVLO\O RU WUL IOXRURDFHWDWH GHULYDWLYHV IRU VWUXFWXUDO VWXGLHV LV VXJJHVWHG E\ WKHVH UHVXOWV 9DQGHQ+HXYHO DQG RWKHUV f KDYH VXJJHVWHG WZR QHZ WHUPV VWHURLG QXPEHU 61f DQG 7 DV SDUDPHWHUV IRU WKH HOXFLGDWLRQ RI WKH VWUXFWXUDO IHDWXUHV RI D VWHURLG PROHFXOH %RWK RI WKHVH WHUPV DUH GLUHFWO\ GHULYHG IURP WKH ZRUN RI :RRGIRUG DQG YDQ *HQW f LQ WKH JDVOLTXLG FKURPDWRJUDSK\ RI IDWW\ DFLGV 6WHURLG QXPEHU LV DQDORJRXV WR FDUERQ QXPEHU RI IDWW\ DFLGV ZKLFK LV UHODWHG WR UHWHQWLRQ WLPH U E\ ORJ U ‘ N ] QR RI & LQ IDWW\ DFLG PROHFXOH

PAGE 146

7KH PDQQHU RI GHWHUPLQLQJ WKH FDUERQ QXPEHU OD WR FRQVWUXFW D UHIHUHQFH JUDSK DFFRUGLQJ WR WKH DERYH HTXDWLRQ EDVHG RQ WKH FDUERQ QXPEHU RI NQRZQ IDWW\ DFLGV 7KH FDUERQ QXPEHU RI DQ XQn NQRZQ DFLG FDQ WKHQ EH UHDG RII DW D SRLQW FRUUHVSRQGLQJ WR WKH UHWHQWLRQ WLPH RI WKH DFLG 7KH FRQFHSW RI FDUERQ QXPEHU PD\ EH XVHIXO EHFDXVH ,W ,V ,QGHSHQGHQW RI VOLJKW FKDQJHV LQ WHPSHUDWXUH DQG HOLPLQDWHV WKH QHHG IRU DQ DUELWUDU\ UHIHUHQFH VWDQGDUG WR ZKLFK UHWHQWLRQ WLPHV PXVW EH UHODWHG 6WHURLG QXPEHU ,V H[SUHVVHG DV D VXPPDWLRQ RI WHUPV GHSHQGHQW RQ WKH QDWXUH RI WKH FDUERQ VNHOHWRQ DQG WKH IXQFWLRQDO JURXSV RI WKH VWHURLG PROHFXOHr VK V UP )E ZKHUH 61 ,V WKH VWHURLG QXPEHU 6 ,V WKH FDUERQ FRQWHQW RI WKH VWHURLG VNHOHWRQ DQG )D )E DUH YDOXHV FKDUDFWHULVWLF RI WKH IXQFWLRQDO JURXSV RI WKH VWHURLGV ,Q WKH RSLQLRQ RI WKLV ,QYHVWLJDWRU WKHUH DUH DW OHDVW WKUHH GUDZEDFNV WR WKH XVH RI 61 WHUP IRU WKH HOXFLGDWLRQ RI VWUXFWXUH 6WHURLG QXPEHUV DUH GHWHUPLQHG IURP UHODWLYH UHWHQWLRQ YDOXHV VR WKDW WKH DGYDQWDJH FLWHG E\ :RRGIRUG DQG YDQ *HQW ,Q HOLPLQDWLQJ WKH UHIHUHQFH VWDQGDUG QR ORQJHU H[LVWV )XUWKHUPRUH WKH WHPSHUDWXUH GHSHQGHQF\ RI WKH UHODWLYH UHWHQWLRQ WLPH YDOXH ZLOO EH SUHVHQW ,Q WKH VWHURLG QXPEHU WHUQ

PAGE 147

:RRGIRUG DQG YDQ *DQW HPSOR\HG D FDOLEUDWLRQ FXUYH EDVHG RQ ILYH IDWW\ DFLGV RI NQRZQ FKDLQ OHQJWK 9DQGHQ+HXYHO DQG +RUQLQJ GUDZ WKHLU VWDQGDUG FXUYH EHWZHHQ RQO\ WZR SRLQWV 7KH UHOLDELOLW\ RI DQ\ LQIRUPDWLRQ GHULYHG IURP VXFK D FDOLEUDWLRQ JUDSK LV TXHVWLRQDEOH } 7KH VWHURLG QXPEHU WHUP QHLWKHU UHIOHFWV WKH DFWXDO L FDUERQ FRQWHQW RI WKH VWHURLG PROHFXOH QRU LQGLFDWHV VXEVWLWXHQW JURXSV RQ WKH QXFOHXV ZLWK DQ\ DFFXUDF\ )RU H[DPSOH WKH YDOXH RI 61 LV IRU FKROHDWDQRO r IRU FKROHVWHQ\O PHWK\O HWKHU DQG IRU FKROHVWDQ\O WUOIOXRURDFVWDWH 7KH WHUP LV WKHUHIRUH PLVOHDGLQJ 7KH VHFRQG SDUDPHWHU GHULYHG IURP WKH HDUO\ ZRUN RI :RRGIRUG DQG YDQ *HQW LV FDOOHG WKH 7 WHUP ZKLFK LV D IXQFWLRQ RI WKH VHOHFWr LYLW\ RI WKH VWDWLRQDU\ SKDVH 7KLV WHUP OD GHILQHG DV ZKHUH Wn LV WKH UHODWLYH UHWHQWLRQ WLPH REVHUYHG ZLWK D VHOHFWLYH VWDWLRQDU\ SKDVH DQG Wf LV WKH UHODWLYH UHWHQWLRQ WLPH REVHUYHG ZLWK D QRQrVHOHFWOYH VWDWLRQDU\ SKDVH :RRGIRUG DQG YDQ *HQW KDG HPSOR\HG SRODU DQG QRQSRODU FROXPQV WR VWXG\ WKH EHKDYLRU RI VDWXn UDWHG IDWW\ DFLGV

PAGE 148

7 YDOXHV RI DQGURVWDQH ,VRPHUV 7DEOH f FDOFXODWHG IURP PHDVXUHPHQWV REWDLQHG RQ WKH ;( DQG WKH 6% SKDVHV ,QGLFDWH WKH GRPLQDWLQJ ,QIOXHQFH RI WKH & R[\JHQ IXQFWLRQ LQ WKH SOVRPHUV ZKHQ WKH & FRQILJXUDWLRQ LV RI WKH DOSKD W\SH 7KXV 7 YDOXHV PLJKW EH XVHIXO LQ UHIOHFWLQJ VWHUHRFKHPLFDO UHODWLRQVKLSV RI IXQFWLRQDO JURXSV DQG PLJKW SURYLGH FOXHV IRU LQWUDPROHFXODU LQWHUn DFWLRQV 7KH NHWRQH VHOHFWLYH SURSHUWLHV RI WKH IRXU SKDVHV ZHUH FRPSDUHG DQG FKDQJHV LQ UHWHQWLRQ WLPH DFFRPSDQ\LQJ WKH R[LGDWLRQ RI D K\GUR[\O JURXS WR WKH FRUUHVSRQGLQJ NHWRQH ZHUH FDOFXODWHG 7DEOHV DQG f ,W LV VHHQ WKDW WKH YDOXH RI $ ORJ U LV IDLUO\ FRQVWDQW DQG YHU\ VPHOO IRU 6L 1*6HE DQG +O%II % FROXPQV )XUWKHUPRUH WKHUH ZDV QR GLIIHUHQFH LQ WKH $ ORJ U YDOXH IRU RO BrRQH DQG GORO Br GORQH WUDQVLWLRQV 7KH SUHIHUHQWLDO VHOHFWLYLW\ RI WKH ;( SKDVH IRU NHWRQHV LV HYLGHQW LQ WKH UHODWLYHO\ ODUJH YDOXHV RI ORJ UMBSQH mQG LQ WKH JUHDWHU UHWHQWLRQ WLPH YDOXH RQ WKLV SKDVH RI WKH NHWRVWHUROGV DV FRPSDUHG WR WKH K\GUR[\O LVRPHUV 7DEOH f 7KH XQH[SHFWHGO\ VPDOO YDOXHV RI $ ORJ rGLR/LRQH ZN EH f GLUHFW FRQVHTXHQFH RI WKH DQRPDORXV HIIHFW RI & VXEVWLWXWLRQ +RZHYHU LW LV DOVR SRVVLEOH WKDW ERRP ORVV RI WKH ;; fSKDVH PD\ EH WKH FDXVH RI WKH XQH[SHFWHGO\ VVPOO YDOXH RI WKHVH GOROGORQH WUDQVIRUPDWLRQV ,Q 7DEOH ZKHUH VHSDUDWLRQ IDFWRUV IRU WKH D HQG LVDPHUV HUH JLYHQ LW FDQ EH VHHQ WKDW WKHVH VHSDUDWLRQV DUH LQ JHQHUDO EHWWHU IRU WKH WUOPHWK\OVOO\O GHULYDWLYHV HVSHFLDOO\ ZLWKLQ WKH NHWRVWHURLG JURXS

PAGE 149

)URP WKH FRPSDUDWLYH GDWD SUHVHQWHG ,W ,V HYLGHQW WKDW WKH JDVOLTXLG FKURPDWRJUDSKLF VHSDUDWLRQ RI DOPRVW DQ\ ,VRPHULF SDLU FDQ EH DFKLHYHG E\ WKH MXGLFLRXV FKRLFH RI WKH VWDWLRQDU\ cGDVH RU RI WKH GHULYDWLYH RI WKH VWHURLGV XQGHU VWXG\ ,W ,V ZRUWK\ RI QRWH WKDW WKH ,VRPHULF GORQH DQGURVWDQHV ZKLFK KDYH HOXGHG VHSDUDWLRQ E\ SDSHU FKURPDWRJUDSK\ PHWKRGV FDQ EH UHVROYHG RQ JDVOOTXOG FKURPDWRJUDSK\ FROXPQV FRDWHG ZLWK SRODU OLTXLGV 7DEOH f

PAGE 150

7KH $SSOLFDWLRQ RI *DVOLTXLG &KURPDWRJUDSK\ WR WKD ,VRODWLRQ DQG 0HDDXUDPHQW RI 8ULQDU\ .HWRWWHUROGV $ 0HWKRG IRU WKD 6LPXOWDQHRXV 6HSDUDWLRQ RI &LR2J DQG &LR2A .HWRVWDUROGV DQG 3URJHVWHURQH 0HWDEROLWHV f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f 6LQFH WKH XULQDU\ NHWRVWDUROGV DUH HQG SURGXFWV RI WKH PHWDEROLVP RI KRUPRQHV VHFUHWHG SULQFLSDOO\ E\ WKH DGUHQDO FRUWLFHV f DQG WKH JRQDGV f DQ\ PHWKRG ZKLFK FDQ PHDVXUH WKH DPRXQWV RI WKH NHWRVWHUROGV PD\ DOVR ,QGLUHFWO\ KHOS WR DVVHVV WKH JRQDGDO DQG DGUHQRFRUWLFDO IXQFWLRQV ,Q PDQ 7KH H[FUHWLRQ RI NHWRVWHUROGV ,Q XULQH ,V ,QIOXHQFHG E\ WKH EDVDO PHWDEROLF VWDWXV RI WKH LQGLYLGXDO DV ZHOO DV E\ FRQGLWLRQV RI VWUHVV 7KH IRUPHU ,QFOXGHV DJH DQG VH[ RI WKH ,QGLYLGXDO ZKLOH

PAGE 151

WKH OHWWHU WHQ mD\ LQYROYH FKURQLF VWUHVV UHVXOWLQJ IURP H GLVHDVH RU D VKRUWHU WHQ HPRWLRQDO RU SK\VLFDO VWLPXOXV 7KH NHWRVWHUROG H[FUHWLRQ DW DQ\ RQH WLPH UHIOHFWV WKH FRPELQHG FRQWULEXWLRQV RI DOO RI WKHVH IDFWRUV 7KH DUUD\ RI H[FUHWHG & VWHURLGV WKH XULQDU\ NHWRVWHURLGV PDV ILUVW GLVFORVHG E\ WKH FKURPDWRJUDSKLF VWXGLHV RI 'LQJHQDQVH DW DO f 6XEVHTXHQWO\ PRGHUDWHO\ VXFFHVVIXO VHSDUDWLRQV RI NDWR VWHURLGV E\ DGVRUSWLRQ DQG SDUWLWLRQ V\VWHPV ZHUH DFFRPSOLVKHG f :KLOH PDQ\ FRQYHQWLRQDO FKURPDWRJUDSKLF WHFKQLTXHV IRU WKH VHSDUDWLRQ DQG PHDVXUHPHQW RI LQGLYLGXDO NHWRVWHURLGV KDYH EHHQ GHYHORSHG DQG DUH LQ DFWXDO XVH WRGD\ DOO VXIIHU IURP WKH GLVDGYDQWDJHV RI WHGLRXVQHVV DQG LQHIILFLHQF\} 7KH FODVVLFDO FROXPQ FKURPDWRJUDSKLF PHWKRG f UHTXLUHG WKH FROOHFWLRQ RI PXOWLSOH GLVFUHHW VDPSOHV RI HOXHQW HDFK RI ZKLFK WKHQ KDG WR EH DQDO\VHG FRORULPHWULFDOO\ XVLQJ WKH =LPPDUPDQQ UHDFWLRQ f} +LOD UHDFWLRQ LV DW EHVW D QRQVSHFLILF FKURPRJHQOF UHDFWLRQ VXEMHFW WR LQWHUIHUHQFH E\ QRQn VWHURLGDO LPSXULWLHV 7KH SDSHU SDUWLWLRQ PHWKRG RI 6DYDUG f KDV UHSUHVHQWHG DQ LPSURYHPHQW LQ HIILFLHQF\ LQ WKDW WKH DFWXDO QXPEHU RI VDPSOHV WR EH ILQDOO\ HOXWHG DQG PHDVXUHG DV =LPPHUDDQQ FKURPRJHQ DUH OLPLWHG WR WKRVH EHLQJ UHVROYHG DQG VWXGLHG +RZHYHU UXQQLQJ WLPHV DUH ORQJ LQ WKHVH V\VWHPV %\ YLUWXH RI YHU\ VKRUW UXQQLQJ WLPHV WKLQ OD\HU SDUWLWLRQ FKURPDWRJUDSK\ YOOO REYLDWH WKLV GLVDGYDQWDJH $OO RI WKHVH PRGDOLWLHV KDYH D FRPPRQ OLPLWDWLRQ LQ UHVROYLQJ SRZHU DWWULEXWDEOH WR SUDFWLFDO OLPLWV WR ZKLFK WKH FKURPDWRJUDSKLF FROXPQ

PAGE 152

SLQWH RU SDSHU VWULS FDQ EH OHQJWKHQHG 6SHFLILFDOO\ RQH HQFRXQWHUV GLIILFXOW\ ,Q WU\LQJ WR VHSDUDWH VRUH WKDQ ILYH RU VL[ FRPSRQHQWV DW D WLQH E\ DQ\ RI WKHVH OLTXLGOLTXLG PHWKRGV )ROORZLQJ WKH GHPRQVWUDWLRQ RI WKH IHDVLELOLW\ RI JDVOLTXLG FKURPDWRJUDSKLF DQDO\VLV RI VWHURLGV f FRQFHUWHG HIIRUWV KDYH EHHQ GLUHFWHG WR WKH GHYHORSPHQW RI PHWKRGV IRU WKH DQDO\VLV RI WKH XULQDU\ &, VWHURLGV 7KH ILUVW DWWHPSWV ,QYROYHG WKH VLPXOWDQHn RXV XVH RI WZR FROXPQV f 7KH GLVDGYDQWDJHV ZHUH IRXQG WR EH WKRVH ,QDFFXUDFLHV DQG ,QFRQYHQLHQFHV ,QKHUHQW ,Q VXFK D FRPSOH[ V\VWHP 3DUWLDO VHSDUDWLRQ RI XULQDU\ NHWRVWHUROGV RQ 6( FROXPQ KDYH EHHQ UHSRUWHG f 0RUH UHFHQWO\ VHSDUDWLRQ RI WKUHH NHWRVWHUROGV ZDV UHSRUWHG FP D F\DQRPHWK\OVOOOFRQH FROXPQ f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

PAGE 153

VWHURLGV ZHUH DOVR PRUH IDYRUDEOH WKDQ IRU WKH IUHH VWHURLGV RU IRU WKH RWKHU GHULYDWLYHV WHVWHG ,Q DGGLWLRQ FROXPQ HIILFLHQFLHV ZHUH JHQHUDOO\ KLJKHU IRU WKHVH GHULYDWLYHV :KHQ PL[WXUHV RI WULPHWK\O} VOO\O HWKHUV RI WKH & VWHURLGV DQG RI WZR SURJHVWHURQH PHWDEROLWHV ZHUH FKURPDWRJUDSKHG RQ WKH FROXPQ SKDVHV GHVFULEHG HDUOLHU DGHTXDWH VHSDUDWLRQV ZHUH REWDLQHG LQ WKH FDVH RI WKH WKUHH SHU FHQW ;( DQG WKH RQH SHU FHQW 5O(II % FROXPQV 1*6HE FROXPQ SURYLGHG RQO\ PDUJLQ} DO UHVROXWLRQ RI WKH DK\GUR[\SDQGURVWHQRQH DQG ISUHJQDQH D4IGLRO 7KH UHVXOWV DUH VKRZQ LQ 7DEOH DQG DUH JUDSKLFDOO\ GHSLFWHG LQ )LJXUHV } 7KXV WKH PHWKRG GHYHORSHG DQDO\]HG QRW RQO\ WKH ILYH PDMRU & VWHURLGV EXW LQ DGGLWLRQ WZR SURJHVWHURQH PHWDEROLWHV RI JUHDW LQWHUHVW LQ KXPDQ PHWDEROLVP ,Q DQ DFWXDO DSSOLFDWLRQ RI WKLV EDVLF WHFKQLTXH WR WKH VWXG\ RI KXPDQ XULQH LW ZDV IRXQG WKDW WKH LQLWLDO REMHFWLYHV RI WKH LQYHVWLn JDWLRQ HRXOG EH UHDOL]HG &KURPDWRJUDSK\ RQ D WKUHH SHU FHQW ;( FROXPQ RI WKH WUOPHWK\OVOO\O GHULYDWLYHV RI D JOXVXODVH K\GURO\]HG DQG VROYHQW H[WUDFWHG KRXU XULQH VDPSOH IURP D QRUPDO PDOH VXEMHFW JDYH WKH UHVXOWV VKRZQ LQ )LJXUH %\ FRPSDULVRQ RI UHWHQWLRQ WLPHV IRU PDMRU SHDNV ZLWK WKRVH REWDLQHG IRU SXUH FU\VDOOOQH VWDQGDUGV LW ZDV SRVVLEOH WR TXDQWLWDWLYHO\ GHWHFW DQG LGHQWLI\ WKH XULQDU\ FRPSRQHQWV $OWKRXJK QR DWWHPSW DW PHDVXUHPHQW ZDV PDGH WKH GLVWULn EXWLRQ DQG WKH UHODWLYH SURSRUWLRQ WR HDFK RWKHU RI WKHVH FRPSRXQGV UHSUHVHQWHG DV SHDN KHLJKW UHVSRQVHV VKRXOG EH QRWHG 6LQFH WKH FKURPDWRJUDP ZDV REWDLQHG XVLQJ D DOLTXRW RI WKH XULQH VDPSOH WKH LQGLFDWLRQV DUH WKDW WKH PHWKRG ZLOO SHUPLW WKH UHTXLVLWH

PAGE 154

VHQVLWLYLW\ IRU GHWHUPLQDWLRQV DW ERWK WKH SK\VLRORJLFDO DQG SDWKRSK\VLRORJLFDO OHYHOV ,QGHHG ,W ,V FRQFHLYDEOH WKDW SODVPD OHYHOV RI WKHVH VWHURLGV PD\ EH GHWHUPLQHG E\ WKLV WHFKQLTXH $GYDQWDJHV RI WKH SUHVHQW PHWKRG ,QFOXGH WKH IDFWRU RI VSHHG HIILFLHQF\ DQG D RQHVWHS SURFHVV RI VHSDUDWLRQ DQG PHDVXUHn PHQW 7KH FRPSOHWH DQDO\VLV RI D SUHSDUHG H[WUDFW FDQ EH DFFRPSOLVKHG ,Q PLQXWHV DV FRPSDUHG WR WKH VHYHQW\WZR KRXUV QHFHVVDU\ IRU WKH GHYHORSPHQW RI D SDSHU FKURPDWRJUDP 7KH VXFFHVV RI WKH ELRORJLFDO DSSOLFDWLRQ VXSSRUWHG WKH RULJLQDO SUHPLVH WKDW DFFHVV WR D XVHIXO DQDO\WLFDO WHFKQLTXH PD\ FRPH IURP D WKRURXJK NQRZOHGJH RI WKH FRPSRXQGV WR EH VWXGLHG DQG IURP WKH JHQHUDOLVDWLRQV JDLQHG ,Q WKH ,QYHVWLJDWLRQ RI WKHLU SURSHUn WLHV ZKLFK ,Q WKH SUHVHQW UHVHDUFK ZDV WKHLU FKURPDWRJUDSKLF EHKDYLRU

PAGE 155

6XPPDU\ 7KH UHODWLYH SRODULWLHV RI WKH IRXU VWDWLRQDU\ SKDVHV f‘SOR\HG ZHUH HVWDEOLVKHG DV +L(II % f 1*6HE ;( \ 6% 7KH JHQHUDO SDWWHUQ RI UHWHQWLRQ WLQHV RI WKH IUHH VWHURLGV DQG WKHLU GHULYDWLYHV ZHUH DFHWDWH WUODHWK\OVOO\O HWKHU IUHH VWHURLG WUOIOXRURDFHWDWH RQ WKH 6( FROXPQ DFHWDWH IUHH VWHURLG WUODHWK\OVOO\O HWKHU RQ WKH ;( FROXDQ IUHH VWHURLG DFHWDWH \ WUODHWK\OVOO\O HWKHU WUOIOXRURDFHWDWH RQ WKH +L(II % DQG 1*6HE FROXPQV 2Q DOO IRXU HROXDQV 6DDQGURVWDQHV VKRZHG D JUHDWHU UHn WHQWLRQ WLPH WKDQ WKH FRUUHVSRQGLQJ SLVRPHUV 7KLV ZDV DWWULn EXWHG WR WKH SODQDULW\ RI WKH DDQGURVWDQH QXFOHXV DQG HQKDQFHG ,QWHUDFWLRQ ZLWK WKH VWDWLRQDU\ SKDVH ,Q DJUHHDLHQW ZLWK HDUOLHU REVHUYDWLRQV RQ SDSHU SDUWLWLRQ V\VWHPV WKH DREOOOW\ RI WKH D[LDO ,VRPHU ZDV JUHDWHU WKDQ ,WV HTXDWRULDO OVRQHU ,Q WKH 6SDQGURVWDQH VHULHV WKH VWHURLGV ZLWK DQ R[\JHQ IXQFWLRQ DW & H[KLELWHG DQRPDORXV EHKDYLRU ,Q WKDW WKH\ KDG JUHDWHU UHWHQWLRQ WLPHV WKDQ WKH FRUUHVSRQGLQJ DLVRQHUV 7KH DEVHQFH RI WKH DQRPDORXV EHn KDYLRU ,Q WKH S DQGURVWDQHV FRQWDLQLQJ R[\JHQ VXEVWLWXHQWV ,Q ERWK WKH & DQG & SRVLWLRQV LQGLFDWHG WKH RSSRVLQJ ,QIOXHQFH RI WKH & R[\JHQ IXQFWLRQ

PAGE 156

$PRQJ WKH WKUHH GHULYDWLYHV WKH DFHWDWHV ZHUH IRXQG WR EH WKH PRVW VDWLVIDFWRU\ LQ FRUUHODWLRQ RI FKURVPWRJUDSKOF EHn KDYLRU WR VWUXFWXUH 7KH EHVW UHVROXWLRQV ZHUH DIIHFWHG ZLWK WKH WULDDWK\OVLO\O HWKHUV RI WKH VWHURLGV 6HSDUDWLRQ RI WKH SULQFLSDO XULQDU\ NHWRVWHUROGV ZDV DFKLHYHG RQ RQH SHU FHQW 1*6HE RQH SHU FHQW +L.II % DQG WKUHH SHU FHQW ;( FROXPQV 7KH WZR SUHJQDQH ,VRPHUV VWXGLHG ZHUH UHVROYHG VLPXOWDQHRXVO\ ZLWK WKH NHWRVWHUROGV RQ WKH ODWWHU WZR FROXPQV 7KH JDVOLTXLG FKURPDWRJUDSKLF PHWKRG GHYHORSHG ZDV VXFFHVVIXOO\ DSSOLHG WR WKH VHSDUDWLRQ RI WKH PDMRU NHWRVWHUROGV RQ D FUXGH XULQH H[WUDFW

PAGE 157

%,%/,2*5$3+< WHO %RJDUH mDG -XYHW 5 -U *DV/LTXLG &KURQDWRJUDSK\ ,Q WHUVH OHQFD 3XEOLVKHUV +DZ
PAGE 158

r $PEURVH DQG $PEURVH % $ *DV &KURPDWRJUDSK\ 9DQ +RHWUDQG &RPSDQ\ ,QF 3ULQFHWRQ 1RY -HUVH\ S ,QIHUHQFH O S YDQ 'HHPWDU =XOGHUZHJ 3 DQG .OLQNHQEHUJ $ &KDP (QJ 6FL f 3DWWHQ + ,, LQ 3ULQFLSLDV DQG 3UDFWLFH RI *DV &KURPDWRJUDSK\ t / 3HFVRN HGLWRU -RKQ :LOH\ DQG 6RQV ,QF S %RKDQHQ DQG 3XUQROO + LQ r+DD &KURPDWRJUDSK\ + 'HVW\ HGLWRU $FDGHPLF 3UHVV ,QF 3XEOLVKHUV +HZ
PAGE 159

5QVOFND / *ROGEHUJ 0 WI HQG %UQJJHU + +HOY &KLQ $FWD} f 5XVLFNH / *ROGEHUJ 0 0H\HU %UWWQJJHU + VDG ;OFKHQEHUJHU +HOY &KLQ $FWH  f 5XVLFNH / %UWWQJJHU + (OFKHQEHUJHU ; HQG 0H\HU +HOY &KLQ $FWH  f ;HHLFND / HQG ;OFKHQEHUJHU ; +HOY &KLQ $FWH f &HOOR} + ; DQG &HOOR} ; 0RFKHQ f 3HHUOQHH + + %LRO &KHQ f 0HVRQ + / %LRO &KHQ f 0DVRQ ; / HDG .HSOHU ; %LRO &KRX f 0HHRD + / %LRO &KHQ f 0LOOHU $ 0 'RUIDDQ % = HDG 6HYUOQJKRHHH ; / (QGRFULQRORJ\ f %QWHQGHQGW $ HQG 'DDDHQEDXQ + = SK\VLRO &KHQ f %XWHQGHQGW $ 'HDQHQEHXQ + +HQOVFK DQG .QGHHQH % = SK\VLRO &KHQ  f /OHEHUDDD 'REUOQHU +LOO % )OHVHU W I HQG ;KRHGV & 3 %LRO &KHQ  f &DUGZHOO + 0 ; &RPIRUWK 'XII ; +ROWHUQHQQ + DQG 5RELQVRQ 5 &KHQ 6HF f :RRGZDUG 5 } 6RQGKHOQHU I 7HXE +HXVOHU DQG 0F/DQRUH : 0 $Q &KHQ 6RF f -RKQVRQ + %DQQLVWHU %} %LVHQ % 0 5HDS $ 3HSSR 5 5RJOHU ; 5 HQG 6VQXVVNRYOFV $Q &KHQ 6RF f DUVWW / + $UWK ; /DNHV 5 0 %H\OHU 5 ; 3RRV -RKQV 9 ) HQG &RQVWDQWLQ 0 $Q &KHQ RF f =LDDHUQHQD : = SK\VLRO &KHQ f =ODDHPDDD 9f = SK\VLRO &KHQ f 'RUIQHQ O &KHQ 5HYV f

PAGE 160

-DQHD 5 % DQG 6DQGRUI\ & 7HFKQLTXHV RI 2UJDQLF &KHPLVWU\ $ :HOVVEHUJHU HGLWRU ,QWHUDFODQFH 3XEOLVKHUV 5RZ
PAGE 161

/RQGRQ ) 3K\D &KRQ f W I f ‘ r ‘ f f Y f V L ? rn Y ‘ L f cL N r f L } R f ‘ 9RQGHQ+HXYHO : $ 6ZHFOH\ & & DQG +RPLQJ & $Q &KRQ 6RR f /LSVN\ ( DQG /DQGRYQH ( $ $QDO &KRQ f f ? f f ? n 8 f f 9 n f f ‘n n f f f f L n b ‘ } +DDKWL ( $ 9RQGHQ+HXYHO : $ DQG +RPLQJ & 2UJ &KRQ  f n f f 9RQGHQ+HXYHO : $ +DDKWL $ DQG +RPLQJ & $Q &KRQ 6RH 8 f +RWLQ % + DQG 0DUWLQ + 9 %LRO &KRQ  f )DFWR DQG 0HWKRGV f 9RWLV + + %LRFKLQ RW %LRSK\D $FWD LQ SURQD 9DDGRQ+HXYRO : $ DQG +RUQLQJ ( & %LRFKLQ DW %LRSK\D $FWD f +DDKWL ( $ 9RQGHQ+HXYHO : $ DQG +RPLQJ ( & $QDO %LRFKRQ f 6DZ\HU 7 DQG %DUU $QDO &KRQ f +LDKWD & 0HDDRUO\ )m 5HDFKNR ( )} )UHGHUOFND + DQG &RRNH : $QDO &KRQ f .HOOHU ( $ DQG 6WHZDUW + $QDO &KRQ f 0DUWLQ $ 3 %LRFKHP 6RR 6\QSRDLD f %RRUWKXLD ( DQG 5RFRXUW + 1DWXUH ,f 9DDGRQ+HXYRO : $ 6ZHHOH\ & & DQG +RPLQJ ( & %LRFKHP %LRSK\D %RDHDUFK &RQPLQD ,f :RWLD + + DQG 0DUWLQ + ) $KD WUDFWR WK 0RRWLQJ $Q &KRQ 6RF & F n !! U n f 9RQGHQ+HXYHO 9 $ DQG +RUQLQJ % & %LRFKHP %LRSK\D %HDDDUFK &RPPXQH ,f 9RWOD + + %LRFKLQ RW %LRSK\D $FWD f +DUWQDD = DQG :RWLD + + 6WRUROGD f -DQHD $ 7 DQG 0DUWLQ $ )m %LRFKRQ f /LSHN\ ( DQG /DQGRYQH ( $ %LRFKLQ RW %LRSK\D $FWD f

PAGE 162

2UU & + mDG &DOOHQ ( $EX &KHQ 6RF f 9DDGHD+HXYHO : $ mDG +RUQLQJ 5 F %ORFKHQ %ORSK\V 5HVHDUFK &RZX f :RW LV % + 1DXNNDUOQHQ DDG &DUU % 6 -U %ORFKLQ HW %ORSK\V $FWH f 9DQGHQ+HLUUHO : $ +RUQLQJ F 6DWH DDG ;NHNDZH 1 2UJ &KH} f 9DQGHQ+HXYHO : $ &UHHFK % DDG +RUQLQJ & $QDO %ORFKHQ f %DWH6DO WK ( & DDG :HV WDOO 5 %ORFKLQ HW %ORSK\V $FWD f A %XVK ; % %ORFKHQ 6RF 6\DSRDOD f &OD\WRQ % 0DWXUH f &OD\WRQ 5 % 0DWDUH r f &OD\WRQ 5 %} %ORFKHQ  f ;DOJKWH % $ mDG 7KRQHD + $QDO &KHQ f :RRGIRUG U ) DDG YDQ *HQW & 0r /LSLG 5HVHDUFK f 5HOFKDWHOQr 7 +HOY &KLQ $FWD  f 5HOFKDWHOQ 7 DQG %XU %HOY &KLQ $FWD f (LDU Y DQG 5HOFKDWHOQ 7 +HOY &KLQ $FWD f %ORFK % 'RUIDDQ 5 ; DQG 3ODFDV )UHH 6RF ([SWO %LRO 0HG f 3HDDUG $ (QGRFULQRORJ\ f 3DUNDV $ 6 0DWXUH t f 'OQJHQDDVH % +XOD LQnW 9HOG / DDG GH /DVW % 0 &OLQ (QGRFULQRO f 'ODJHQHDDH % +XOD ,QnW 9HOG / mDG +DUWRJK.DW] 6 / &OLQ (QGRFULQRO f 5XELQ % / 5RVHDNUDDWV + 'RUIQDD 5 ; mLG 3,QFXV &OLQ (QGRFULQRO f 5XELD % / 'RUIDDQ 5 DQG 3LQFXH %LRO &KHQ f

PAGE 163

+DDKWL ( $ 9DQGHQ+HXYDO : $ DQG +RPLQJ ( & $QDO %ORFKHQ f f rYn nf f ‘ f r f f r Y\ f r f f r r} &RRSDU $ DQG &UHHFK % $QDO %LRFKDD f 6SDUDJDQD 0 0DVRQ 9 % DQG.HXWDDQQ ( + $QDO &KHP f

PAGE 164

%,2*5$3+,&$/ 6.(7&+ ,FODO 6LUHO +DUWPDQ YDV ERUQ 'DFHPEHU DW %ODVOJ 7XUNH\ 6KH DWWHQGHG 3UHSDUDWRU\ 6FKRRO ,Q ,VWDQEXO 7XUNH\ DQG UHFHLYHG WKH GHJUHH RI %DFKHORU RI $UWV ,Q -XQH IURP 0RXQW ? ‘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

PAGE 165

7KLV GLVVHUWDWLRQ ZDV SUHSDUHG XQGHU WKH GLUHFWLRQ RI WKH FKDLUPDQ RI WKH FDQGLGDWHnV VXSHUYLVRU\ FRPPLWWHH DQG KDV EHHQ DSSURYHG E\ DOO PHPEHUV RI WKDW FRPPLWWHH ,W ZDV VXEPLWWHG WR WKH 'HDQ RI WKH &ROOHJH RI $UWV DQG 6FLHQFHV DQG WR WKH *UDGXDWH &RXQFLO DQG ZDV DSSURYHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ $SULO 'HDQ &ROOHJH RI $UWV DQG 6FLHQFHV 'HDQ *UDGXDWH 6FKRRO 6XSHUYLVRU\ &RPPLWWHH

PAGE 166

81,9(56,7< 2) )/25,'$