,..i,u 'i! - open...
TRANSCRIPT
Factors affecting selectivity of covalent chromatography.
Item Type text; Dissertation-Reproduction (electronic)
Authors Crampton, Mary Catherine.
Publisher The University of Arizona.
Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.
Download date 16/07/2018 13:11:50
Link to Item http://hdl.handle.net/10150/184356
INFORMATION TO USERS
The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the original text directly from the copy submitted. Thus, some dissertation copies are in typewriter face, while others may be from a computer printer.
In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyrighted material had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each oversize page is available as one exposure on a standard 35 mm slide or as a 17" x 23" black and white photographic print for an additional charge.
Photographs included in the original manuscript have been reproduced xerographically in this copy. 35 mm slides or 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order.
,."""".I,U 'I! ,
I" I'li II I, ail I , "1 lii~L I
300 North Zeeb Road, Ann Arbor, M148106· 1346 USA
Order Number 8814226
Factors affecting selectivity of covalent chromatography
Crampton, Mary Catherine, Ph.D.
The University of Arizona, 1988
V·M·I 300 N. Zccb Rd. Ann Arbor, MI 48106
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark_./_.
1. Glossy photographs or pages __
2. Colored illustrations, paper or print __ _
3. Photographs with dark background __
4. Illustrations are poor copy __ _
5. Pages with black marks, not original copy V 6. Print shows through as there is t~xt on both sides of page __ _
7. Indistinct, broken or small print on several pages __ _
8. Print exceeds margin requirements __
9. Tightly bound copy with print lost in spine __ _
10. Computer printout pages with indistinct print __ _
11. Page(s) lacking when material received, and not available from school or author.
12. Page(s) seem to be missing in numbering only as text follows.
13. Two pages numbered . Text follows.
14. Curling and wrinkled pages __
15. Dissertation contains pages with print at a slant, filmed as received /
16. Other _________________________________ __
U·M-I
FACTORS AFFECTING SELECTIVITY OF COVALENT CHROMATOGRAPHY
by
Mary Catherine Crampton
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF CHEMISTRY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
198 8
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read
the dissertation prepared by Mary Catherine Crampton
entitled Factors Affecting Selectivit~ of Covalent Chromatography
and recommend that it be accepted as fulfilling the dissertation requirement
for the Degree of Doctor of Philosophy
August 20, 1987 Date
August 20, 1987 Date
August 20, 1987 Date
August 20, 1987
Date
Dr. Quintus Fernando ;;.f_
VJ :L~/JilI/k1-< Dr. W. Ronald Salzman
August 20, 1987
Date Dr.~:ft.~d~~ Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
:if" /", !T'/5:" ~ August 20, 1987 Dissertation Director Date
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
Dedication:
To my parents.
iii
ACKNOWLEDGMENTS
The author would like to thank Analytichem International for
their support and all the people there who impacted on this work,
especially
Dr. Mark Stolowi·tz.
The author would like to thank Dr. M. F. Burke for his
support,
encouragement and guidance throughout the entire course of study.
The author would like to thank all my committee members,
group members and the rest of the members of the staff, faculty and
students at the University of Arizona who offered encouragement and
guidance.
iv
TABLE OF CONTENTS
LIST OF TABLES . . . .
LIST OF ILLUSTRATIONS.
ABSTRACT ..
1. INTRODUCTION
Principles of Chromatography General Elution Chromatography. Solid Phase Extraction. Types of Interactions
Bonded Functiona1ities . Pheny1boronic Acid. Thio1 Phases.
The Solid Support.
2. BACKGROUND AND PROCEDURES.
Chemically Modified Surfaces Supports ....... .
Organic Supports . Inorganic Supports
Immobilized Functiona1ities Modification. . . . .
Procedures . . . . . . . . . . . Modification Methods ....
Reactions with bare silica Aminopropy1 based bonded silicas Activation of mercaptopropy1 bonded silica Propy1urea based bonded silicas.
Characterization of Bonded Silicas.
Summary.
Elemental Analysis . . . . Solid Phase Extraction . . Diagnostic Chromatography.
3. EFFECTS OF MODIFICATION.
Control of Non-specific Interactions Mobile Phase .. . Stationary Phase ....... .
v
Page
viii
ix
xiv
1
5 5 8 9
11 11 13 17
19
19 20 20 21 22 24 30 30 30 31 31 37 40 40 45 46 49
51
51 51 52
TABLE OF CONTENTS--Continued
Experimental . . . . . . . . Results and Discussion . . .
Dispersive Interactions Lipophilicity of the Bonded Phases Effects of Solvent Strength.
Electrostatic Interactions. Anion Exchange Sites . . . Cation Exchange Sites ... Effects of Ionic Strength.
Hydrogen Bonding. . . . . . . . Conclusions. . . . . . . . . . . . .
Controlling Liophilic Interactions. Controlling Electronic Interactions
4. PHENYLBORONIC ACID
Introduction . Acid base chemistry Compounds Expected to Interact with PBA
Diols ..... Polyhydroxyls. Hydroxyacids . Hydroxyamines. Other Functiona1ities.
Isolation of Compounds. Experimental . . . . . . . . .
Solid Phase Extraction. . Diagnostic Chromatography
Results and Discussion . . . . Retention Dependence on pH.
Diol ..... . Hydroxyamines .. . Mercaptoa1cohols . Salicylic Acid and Related Compounds Diketone . . . . . . . . . Keto-carboxylic Acid . . .
Effects of Modification on Retention. Secondary Interactions . . . Shift in pKa of PBA. . . . .
Demonstration of the Isolation of Compounds Diols .... Hydroxyacids
Conclusion. . . .
vi
Page
53 55 55 55 57 57 57 61 64 68 70 70 71
73
73 73 75 77 78 78 78 79 79 80 80 80 82 82 82 89 92 92
.100
.100
.104
.104
.104
.105
.106
.106
.106
TABLE OF CONTENTS--Continued
5. COVALENT CHROMATOGRAPHY OF THIOL CONTAINING COMPOUNDS.
Traditional Thiol Chemistry .. Separation of Thiol Containing Solid Phase Thiol Chemistry.
Mixed disulfide . . Phenylmercurial . . Phenylarsonous acid
Experimental . . . . Results & Discussion
Compounds
Disulfide Phase Organomercurial Phenylarsonous Acid Extraction of Reducing Agent.
Conclusions ......... .
6. CONCLUSIONS & REFERENCE WORKS.
REFERENCES . . . . . . . . . .
vii
Page
.111
.111
.114
.115
.115
.117
.121
.124
.125
.125
.133
.138
.144
.146
.148
.154
LIST OF TABLES
Table Page
2.1 Elemental analysis of modified silicas ... .43
2.2 Capacity factors of several probe molecules on propy1aminoPBA with methanol as the organic modifier .. 47
4.1 Molecules that interact with PBA. . .74
4.2 Buffers for solid phase extraction. .81
5.1 Dissociation Constants for Complexes with Methy1mercuric Acid. . . . . .................... 120
viii
Figure
1.1
1.2
1.3
1.4
LIST OF ILLUSTRATIONS
Page
A. Ionization of phenylboronic acid to the tetrahedral form
B. Interaction with diols. . . . . . . 12
Retention, detection and elution of thiols using a mixed disulfide phase. 14
Retention and elution of thiols on an immobilized organomercurial. 15
A. B.
Hydration of immobilized arsine oxide to arsonous acid Interaction of immobilized arsonous acid with dithio1
C. compounds to form a ring structure Interaction with monothio1s .16
2.1 Polymeric C18 bonded silica. . . .25
2.2 Propylaminophenylboronic acid andthe second interactions occurring on this phase. .... ..... .27
2.3 Propylureaphenylboronic acid immobilized on silica .29
2.4 Reaction of triethoxyaminopropyl silane with silica. .32
2.5 Reaction of triethoxychloropropyl silane with silica .33
2.6 Reaction of triethoxymercaptopropyl silane with silica .34
2.7 Reaction of chloropropyl bonded silica with m-aminophenylboronic acid bonded silica . . . . . 35
2.8 Reaction of 2,2-dithiodipyridine with mercaptopropyl bonded silica to form the mixed disulfide phase ......... 36
2.9 Reaction of propylamino bonded silica with 1,1'carbodiimidizole followed by m-aminophenylboronic acid to form propylureaphenylboronic acid bonded silica.. . .38
2.10 Reaction of a CDI activated aminopropyl phase with p-aminophenyl-mercurial hydroxide . . . . . . 39
ix
LIST OF ILLUSTRATIONS--Continued
Figure Page
2.11 Reaction of a COl activated aminopropy1 phase with p-aminopheny1-arseneoxide . . . . . . . . . . . . . . 41
2.12 Reaction of a COl activated aminopropy1 phase with ammonia to form a propy1urea phase . . . . . . . . . .. . ... 42
3.1 Comparison of ionization of the two modification methods. 54
3.2 Retention of benzene and toluene on three modified silicas as a function of pH . . . . . . . . . . . . . . . . 56
3.3 Effects of acetonitrile concentration on the retention of benzene, aniline, phenol, and nitrate on propy1aminopheny1boronic acid bonded silica.
3.4 Retention of benzoic acid and phenylacetic acid on three
.58
bonded silicas as a function of pH . . . . . . . . .59
3.5 Effects of pH on the retention of benzene sulfonic acid and pheny1trimethy1ammonium on three modified silicas . 60
3.6 Retention of aniline and benzy1amine on three modified silicas as a function of pH . . . . . . . . . . . . 62
3.7 Effects of ionic strength on theretention of benzy1amine and benzoic acid on propy1urea (a), propy1ureapheny1boronic acid (b), propy1aminopheny1boronic acid (c) and aminopropyl bonded silica (d). . . . .. .............65
3.8 Effects of ionic strength on the retention of phenylacetic acid and benzenesulfonic acid on propylurea (a), propylureaphenylboronic acid (b), propylaminophenylboronic acid (c) and aminopropyl bonded silica (d) ...... 66
3.9 Ionic strength dependence on the retention of phenyltrimethylammonium on propylureaphenylboronic acid (a) and propylaminophenylboronic acid (b) . . . . . . . 67
3.10 Retention of phenol and benzyl alcohol on three bonded silicas as a function of pH ................ 69
4.1 Equilibration of PBA to its tetrahedral form. Reaction with diols and thereverse reaction when the pH is dropped ... 76
x
LIST OF ILLUSTRATIONS--Continued
Figure Page
4.2 Two PBA modified silica phases, a 1iophi1ic phase (a) and a hydrophilic phase (b). . . . . . . . . . .. .. 83
4.3 Retention of catechol on three modified silicas as a function of pH . . . . . . . . . . .. .. 84
4.4 Retention of 2-hydroxybenzy1 alcohol as a function of pH. . . . . . . . . . . . . ......... 86
4.5 Retention of aminothio1pheno1 (a), 2-hydroxybenzy1 alcohol (b) and 2-aminobenzy1 alcohol (c) on commercially available PBA columns by solid phase extraction. 88
4.6 Retention of aminopheno1 as a function of pH 90
4.7 Retention of 2-aminobenzy1 alcohol as a function of pH 91
4.8 Retention of 2-mercaptobenzy1 alcohol as a function of pH. 93
4.9 Retention of salicylic acid as a function of pH. 94
4.10 Salicylic acid (a) and salicylamide (b) retained on PBA as a function of pH . . . . . . . . .96
4.11 Retention of salicylamide as a function of pH on pheny1boronic acid . . . . . 98
4.12 Retention of thiosa1icy1ic acid as a function of pH on pheny1boronic acid ., .......... 99
4.13 Retention of benzi1 as a function of pH on phenyl~oronic acid. . . . . . .. ..... . . . .. . 101
4.14 Retention of benzi1 (a) and benzillic acid (b) on PBA as a function of pH .102
4.15 Retention of benzi11ic acid as a function of pH on pheny1boronic acid .103
4.16 Demonstration of the isolation of 1,3-dio1s on PBA .107
4.17 Demonstration of the retention of hydroxyacids on PBA. .108
xi
LIST OF ILLUSTRATIONS--Continued
Figure Page
5.1 Reduction of disulfide bonds by dithiothreitol followed by reaction with 2,2-dithiodipyridine . . . . . .. 113
5.2 Retention, detection and elution of thiols using a mixed disulfide phase. .. ........ . . .116
5.3 Retention and elution of thiols on an immobilized
5.4
organomercurial ..... . .. 118
A. B.
c.
Hydration of arsine oxide to arsonous acid Interaction of immobilized arsonous acid with dithiol compounds to form a ring structure Interaction with monothiols . . . . .. 122
5.5 Use of te mixed disulfide phase for the quantitation of cystiene (a) and dithithrietol (b). 126
5.6 Secondary interactions on the mixed disulfide phase as a function of % methanol (a) and -log ionic strength (b). 128
5.7 Retention of aniline (a) and benzoic acid (b) as a function of % methanol on the mixed disulfide phase at pH 4 and 6 . . . . . .................... 130
5.8 Reaction of 2-mercaptoetthanol with themixed disulfide phase .. ............ . . . . . 131
5.9 Retention as a function of % methanol (a) and -log ionic strength (b) on an exhausted mixed disulfide phase ... 132
5.10 Non-specific interaction of organomercurial bonded silica using 5% acetonitrile and 95% ammonium acetate buffer with an ionic strength of 0.1 M ................ 134
5.11 Oxytocin retained and eluted on an immobilized organomercurial . ..... . . . . . . . . . .137
5.12 Automated system for the retention of thiols on organomercurial. 139
5.13 Non-specific interactions on phenylarsonous acid bonded silica 140
xii
LIST OF ILLUSTRATIONS--Continued
Figure Page
5.14 Retention of cystiene on immobilized arsonous acid as a function of solvent strength. . . . . . . . . . . .. .142
5.15 Use of arsonous acid bonded silica for the separation of dithiols (DTT) and monothiols (cystiene) detected by the mixed disulfide phase. A. Without arsonous acid column. B. With arsonous acid collunn .............. 143
xiii
ABSTRACT
Of the interactions utilized in the separation of chemical
species, the reversible covalent bond is the strongest and most
selective. In order to exploit the selectivity of this interaction,
an understanding of the effects of several factors on the formation of
the covalent bond have been studied.
Covalent chromatography is most useful in solid phase
extraction. The strength of the covalent bond will allow for high
distribution coefficients needed to quantitatively retain chemical
species. The selectivity of the covalent interaction allows for the
retention of a specific compound or class of compounds. Under
conditions where the covalent bond is no longer formed or will break,
a low distribution coefficient is possible, and the compounds may be
eluted from the sorbent in a small volume allowing for
preconcentration.
The sorbent consists of three parts, the active functionality
capable of forming reversible covalent bonds, the solid support and
the spacer arm that tethers the active functionality to the support.
Silica supports demonstrate several advantages over organic supports.
However, silica supports have been limited by the activity of the
residual silanols and the use of hydrophobic spacer arms. This
research describes the preparation and characterization of a
modification method for silica with a hydrophilic spacer arm that
restricts the sample from the residual silanols.
xiv
Immobilized phenylboronic acid (PBA) interacts with compounds
containing polar functionalities in the correct configuration, but
this interaction is dependent on the local environment. Controlling
the local environment allows for the control of the interaction
provided the effects are understood. Diagnostic chromatography was
used to determine the effects of the solvent strength, ionic strength,
pH and composition of the mobile phase and the effects of the spacer
arm on the interactions of PBA with several compounds.
Three phases selective for thiols were also characterized.
Thiopyridone attached to mercaptopropyl bonded silica through a
disulfide linkage, is used for isolation and detection. Immobilized
phenylmercury was utilized for the extraction of thiol containing
species while immobilized phenylarsonous acid selectively extracts
dithiol compounds. A unique and powerful means of separation of
monothiol from dithiol compounds has been demonstrated.
xv
CHAPTER 1
INTRODUCTION
Solid phase biochemistry can be defined as any biochemical
process where one component is immobilized on a solid support(l).
This approach is popular due to the ease of sample handling and ease
of automation, as well as the fact that the immobilization of a
chemical species often allows for more efficient and more selective
interactions or reactions. This approach to chemistry is not limited
to biochemistry and can be separated into three types: synthesis,
sensors or detection systems, and separation systems. Solid phase
synthesis has been shown to be both simpler and more efficient than
solution synthesis especially for large molecules such as DNA or
proteins (1,2,3,4). Immobilized enzymes (5) as well as immobilized
redox reagents (6) are often used as catalysts in organic synthesis.
Immobilized enzymes are not only useful in synthesis, but are also
used as chemical sensors such as immobilized enzyme electrodes (7)
which provide selective and sensitive methods for quantitative analy
sis. In addition, solid phase reactors coupled to traditional UV
liquid chromatography detectors have been employed in liquid chromato
graphy as sensitive, selective and versatile detection systems (8).
Separation methods in solid phase chemistry have traditionally relied
on bioselective adsorption (affinity chromatography) or chemiselective
adsorption (e.g. covalent chromatography)(l).
1
2
In each of these types of chemistry, it is important to
understand the factors that affect the interactions of chemical
species with the surface. In general, the unique selectivity, which
is the goal of any solid phase chemistry, relies on the formation of a
reversible linkage between the species of interest and the solid
phase. This linkage usually consists of at least one covalent bond,
although other high energy interactions such as ion exchange or charge
transfer interactions, have also been used. The exploitation of solid
phase chemistry to its full potential relies on understanding the
conditions which allow one to form and break this linkage in a rapid
and efficient manner.
The covalent bond is not the only interaction possible between
the surface and the spec~es of interest. In some cases, the molecule
adsorbs to the surface through non-specific interactions. Understand
ing these non-specific or secondary interactions and the factors which
affect them allows for the utilization of these interactions to
enhance the formation of the selective interaction or the minimization
of these interactions when adsorption is undesirable.
Chromatography has not only proven itself as a separations
technique, but also as a method of studying solid surfaces, (9,lO) to
gain insight into the types of interaction and the relative strengths
of the interactions available at a solid surface. Diagnostic chro
matography is the use of the principles of chromatography to determine
a wide variety of information about a solid surface.
3
While covalent chromatography has proven itself for many
separations, its use has been limited by a lack of understanding of
the factors which affect the separations. Several factors affect the
formation of the covalent bond including pH, solvent strength, ionic
strength, solvent composition of the mobile phase and environment of
the solvated stationary phase. The optimum conditions for the cova
lent reaction depend on the analyte and the matrix, understanding the
effects will allow for the determination of mobile and stationary
phases for an efficient separation.
In addition to the covalent interaction several other types of
interaction may be occurring on these phases. These secondary
interactions include electrostatic, H-bonding and dispersive inter
actions. These interactions allow for the compound of interest to be
solvated by the stationary phase. The sample must spend some time in
the stationary phase to allow for the covalent reaction to occur.
These secondary interactions are also responsible for non-specific
retention. Molecules that are unable to form a covalent bond may be
retained on the surface through non-specific interactions. Recog
nizing these interactions allow for the removal of contaminants that
might otherwise be left on the surface or eluted with the compound of
interest.
The physical stability and the efficiency of silica are not
matched by any of the soft gels commonly used as supports in covalent
chromatography. The limitation of silica as a solid support has been
due to a lack of creativity in the modification of silica, in that
4
traditional modification results in a lipophilic surface. Since a
hydrophilic support is desired for work with biomolecules(22 ,23) , a
hydrophilicly modified silica support has been developed and charac
terized. This modification method allows for a broader range of
mobile phases due to a decrease in dispersive and anionic interactions
and will expand the use of silica as a support in all areas of solid
phase chemistry.
Four different functionalities have been immobilized on silica
using two different modification methods. These have been character
ized through elemental analysis, solid phase extraction and diagnostic
chromatography. The effects of pH, ionic strength, solvent strength
and composition on the retention of several probe molecules have been
determined. The probe molecules were chosen because of their ability
to probe different interactions occurring on the phases. Thus the
effects of the changes in mobile phase and stationary phase on spe
cific interactions are determined.
Once the factors affecting the selectivity of these phases is
understood the versatility can be increased by coupling several
columns together. The selectivity of the covalent bond allows for the
isolation of several different classes of compounds, each on a differ
ent column-. Compounds that interfere with the immobilization of the
compound of interest may be removed prior to the immobilization
provided the interferants contain a distinctive functional group.
However choosing optimum conditions for this type of separation will
depend on an understanding of the selectivity of each column.
Principles of Chromatography
General Elution Chromatography
5
In liquid chromatography, the solute molecules are swept by
the flowing stream of the mobile phase past a second phase, the
stationary phase, which is most commonly a chemically modified solid
substrate. The general elution equation for chromatographic processes
can account for a partition mechanism, an adsorption mechanism and an
exclusion mechanism as shown in equation 1.1. (11)
Eq 1.1
where
Vr1 - the retention volume of the solute
Vm the volume of the mobile phase in the column
K the thermodynamic equilibrium constant (Eq l. 3)
Vs the volume of the stationary phase
K' the adsorption thermodynamic equilibrium constant
As the surface area of the support
K" the exclusion thermodynamic equilibrium constant
Vi the interstitial volume
The retention of a probe to partitioning is defined as KVs . This
mechanism "involves the intercalation of a molecule into the stationary
phase, so as to change the solvation of the ana1yte. The adsorption
mechanism defined as K'As involves the adsorption of a molecule on the
surface of the solid support. Retention due to exclusion is defined
as K"Vi' Smaller molecules are able to diffuse into small pores while
large molecules are excluded, and therefore, K" represents the frac
tion of the pore volume available to a given analyte. Since the
volume of the stationary phase available to smaller molecules is
larger, the retention volume is also larger. In some cases, all of
these mechanisms may be occurring at the same time; however, the
separation is more easily controlled when one mechanism predominates.
6
If partitioning is the predominate mechanism for retention the
elution equation can be given as equation 1.2.
Eq 1.2
The thermodynamic equilibrium constant is defined by:
K [Als / [Alm Eq 1.3
where [Als is the concentration of the analyte in the stationary phase
and [Alm is the concentration in the mobile phase. If the analyte has
a strong interaction with the stationary phase, the distribution
coefficient will be large and the molecule will spend a significant
amount of time associated with the stationary phase. The molecules
which interact strongly with the surface will be retained longer than
those that interact weakly with the surface. Thus, molecules are
separated because of the different strengths of interaction with the
surface.
The distribution coefficient is difficult to determine
experimentally, because the volume of the stationary phase is unknown
and depends on the amount of mobile phase imbibed in the stationary
phase. The capacity factor (k') is defined as:
7
Eq. 1.4
where ns is the number of analyte molecules in the stationary phase,
nm is the number of analyte molecules in the mobile phase, K, Vs and
Vm are defined in equation 1.1 and Vm/Vs is the phase ratio. The
capacity factor is measured as
Eq. 1.5
where Vr is the retention volume.
In differential migration chromatography, a k' of less than 2
does not allow for the resolution of compounds. Above a k' of 20 the
increase in resolution is minimal and the increase in analysis time
limits the number of analyses possible. For this reason, a k' between
2 and 20 is considered optimal (12). Assuming a phase ratio between
.1 and 10 -means optimal distribution coefficients are between 0.2 and
200. In order to control the retention of a molecule the distribution
coefficient may be altered through changes in the mobile or stationary
phases or the altering the phase ratio.
8
Solid Phase Extraction
The general elution equation (equation 1.1) is incomplete, in
that it does not take into account the capability of a phase to form a
covalent or an ionic bond with an analyte. Including this type of
retention, the equation is expanded to
Eq 1.6
where K'IIN accounts for retention due to bonds occurring with specific
sites. N is defined as the number of sites and K'II is the bonding
coefficient.
The strong interactions that are described by the last term in
the general elution equation are most useful in solid phase extraction
(SPE). This extraction involves the same principles as the more
traditional differential elution separations. In SPE an analyte is
quantitatively retained and isolated on a solid sorbent. In order to
quantitatively retain the analyte while eluting any contaminants a
distribution coefficient of 1000 or greater is needed. The strength
of the covalent bond allows for this high distribution coefficient
while the selectivity allows for the retention of only compounds with
specific functional groups. Once the analyte is isolated on the
sorbent, the conditions are changed to allow for a distribution
coefficient to be 0.001 or less. The analyte is eluted in the small
est volume possible to allow for preconcentration. This has also been
called digital chromatography, since in one set of conditions, the
solute is retained on the column and a second elutes the solute off
the column. SPE usually isolates a class of compounds rather than
single components. This type of separation is useful to clean up a
sample prior to HPLC or GC separations and for analyses that do not
require a pure compound but are affected by a complex matrix.
Types of Interactions
9
The most commonly used interaction in liquid chromatography
today is the dispersive or induced dipole- induced dipole interactions
of reverse phase chromatography. The dispersion interactions, as
with all of the Van der Waals forces, are relatively weak and involve
about 2 kcal/mol. These interactions result from the instantaneous
dipoles formed in molecules due to the movement of electrons and the
formation of corresponding dipoles in nearby molecules. While the
universal nature of these interactions make them useful for many
separations, it also limits selectivity.
Normal phase chromatography is older than reverse phase, but a
lack of reproducibility due to experimental parameters limits it's
use. Normal phase chromatography relies on hydrogen bonding inter
actions. These interactions are stronger than dispersion forces and
average about 10 kcal/mol.
The electrostatic interactions of ion chromatography, at about
100 kcal/mol, are stronger than those of normal or reverse phase
chromatography. They are also more selective, occurring only with
ionized solutes. The strength of electrostatic interactions allows
10
for quantitative retention of compounds and early uses of electro
static interactions for separations employed in this ion exchange
chromatography. More recently with the work of H. Small electrostatic
interactions have found wide use in elution chromatography (13).
The strongest and most selective interaction available in
chromatography today is the covalent bond. Reversible covalent bonds
that form between functional groups bound to a solid support and
selective solutes have been used in both general elution chromato
graphy and SPE. One type of chromatography that has utilized covalent
bonds for elution chromatography has been ligand exchange chromato
graphy. In this type of chromatography the mobile phase contains a
compound which is capable of competing with the species of interest
for the active site. The real advantages of this interaction are
realized in SPE. The strength of the covalent bond from 50-200
kcal/mol allows for large distribution coefficients under conditions
where the solute reacts. These large K values needed for quantitative
retention in SPE are achieved. Under conditions where the solute does
not react or the bond will break, very small distribution coefficients
exist allowing for a quantitative elution in small volumes. Changes
in the mobile phase allow for the retention or elution of the solute.
The chemical nature of the functionality on the surface dictates the
factors that control the interaction. The physical nature of the
solid support and the spacer arm that links the support and the
functionality will also affect the selectivity of a functionality.
11
Bonded Functionalities
The ideal functionality in covalent chromatography would be
able to form reversible stable bonds with specific functional groups.
For efficient separations, the bond formation must be more rapid than
the diffusion which controls band spreading on the column. In addi
tion, the bond would be easily and controllably reversed in order
to elute the molecule from the packed bed. Once again the reverse
reaction must be fast to allow for elution in the smallest possible
volume.
Several different functionalities have been bound to a silica
surface through two bonding methods. The effects of bonding methods
as well as solvent strength and composition, ionic strength and pH on
the retention of molecules on these phases has been studied.
Phenylboronic Acid
Phenylboronic acid (PBA) (figure 1.1) reacts readily with a
wide variety of polar functionalities if they exist with the appro
priate geometric configuration. The most commonly studied molecules
are the 1,2- and l,3-diols (14). However, other combinations of
amines, mercaptans, carboxylic acids, ketones or hydroxy groups will
also react with PBA (24).
PBA is a Lewis acid with a pKa near 8.3. It ionizes through
acceptance of a hydroxy group at high pH. It is the acid-base chem
istry that controls the formation of covalent complexes. At high pH,
it is tetrahedral and ionized, at low pH it is trigonal planar and
A
+2H,O~
Figure 1.1 A. Ionization of phenylboronic acid to the tetrahedral form. B. Interaction with diols.
12
neutral. These characteristics can be used to explain the pH depen
dence of retention for several functiona1ities.
Thio1 Phases
13
The use of a mixed disulfide to covalently link thiol contain
ing compounds to surfaces has been used for the immobilization of
compounds (15,16,17,18). A thiol bonded surface activated by
reaction with 2,2'-dithiodipyridine (figure 1.2) makes it possible to
separate thiol containing peptides from non-thiol containing peptides
or to separate enzymes with active thiol sites from denatured enzymes
or to protect or store enzymes with active thiol sites. In addition
the thiopyridone is displaced when a solute interacts with the sur
face. The chromophore released may allow for an increase in the
sensitivity of detection for thiol containing compounds (15). The
ability to use this phase for isolation and quantification makes this
a very useful phase. Unfortunately, this phase has a few limitations
due to the ease of oxidation of the free thio1s, which will be dis
cussed in Chapter 5.
A second phase selective for thio1 containing compounds is
shown in figure 1.3. This phase is an organomercurial bonded phase.
The mercu~y is very stable and forms strong interactions with thio1s
(19,20) under a wide range of conditions. This phase is useful for
the isolation of thio1s, but the displacement of a chromophore has not
been demonstrated.
Immobilized phenylarsonous acid (figure 1.4) also interacts
with free thio1s. This phase is able to interact at two sites. When
z
}- S-S-R 0 }-S-S-o + i: III. R-SH ". 11& 0
'" Q c
S=O ,N
H z
}-S-S-R }-S-S-R' 0
-= + It'-Slot '\ ::) -.. R-SH
Figure 1.2 Retention, detection and elution of thiols using a mixed disulfide phase.
14
Z i }-H9-OH + R-SH c(
0_ Z}-~ Hg-S-R + R'-SH "\ • }-H9-S-R'
R-SH
15
Figure 1.3 Retention and elution of thio1s on an immobilized organomercurial.
A
JO _all I O-AS"'OH HS-C-
B + I
HS-C-HN I
~
JO A ,all C 0- s'OH
+ 2 R-SH HN
~
~ ~
rA-r .... OH M AS'OII
HN
~
I
,..5-<;-)O-AS'S-T-
HN
~
JOT ,S-R . a AS"' S_ R ~ ~ HN
~
+2 1120
+2 H2O
Figure 1.4 A. Hydration of immobilized arsine oxide to arsonous acid. B. Interaction of immobilized arsonous acid with dithiol compounds to form a ring structure. C. Interaction with monothiols.
16
interacting with a dithiol, a ring structure is formed. This ring
structure is more stable than the structure formed with monothiols,
and one can immobilize dithiol compounds without retaining the mono
thiol containing compounds (21).
17
These phases used separately or in series will allow for easy
methods of separating and detecting thiol containing compounds.
The Solid Support
All areas of solid phase chemistry, including chromatography,
rely on properly modified solid supports. While no support to date is
considered ideal, several different types have been used. The ideal
support would be physically stable. It would be rigid so as to
withstand high pressure drops necessary to facilitate fast flow rates.
In addition, it would have a large surface area to allow for a large
number of active sites in a small volume. The active sites need to be
accessible to the solute requiring a pore size to be of a known
uniform size. The support must be insoluble under a wide range of
conditions. It must not be easily degraded through microbial attack.
In addition to the physical characteristics of the support, it is also
important to covalently bind the active functionality to the support.
Once chemically modified, the ideal support would be com
pletely reacted to contain the highest concentration of active sites
possible. The modified support would not diminish the ability of the
functionality to interact and would not interact directly with the
solute.
18
Of the solid supports available, silica gels are physically
strong, easily reacted and available in specific and consistent sizes
and pore volumes. Silica gels have found limited use in covalent
chromatography due to the surface activity of silica and the modifi
cation methods used. To expand the use of silica gel in covalent
chromatography and all areas of solid phase chemistry, the silica must
be properly modified to interact with chemical species in a controlled
manner. Proper modification relies on understanding the silica
surface and the types of active groups present.
CHAPTER 2
BACKGROUND AND PROCEDURES
Chemically modified silica surfaces are routinely used for
separations, however, the mechanism of most separations are not well
understood. The general elution equation (Eq. 1.6) defines retention
of molecules as due to several different mechanisms. In addition,
each mechanism may rely on several different parameters; for example,
partition chromatography may rely on more than one interaction for the
retention of molecules. Usually in cases with multiple interactions,
one interaction is considered to predominate and experimental condi
tions are set to assure this. However, the separation depends on all
the interactions occurring at the surface, and the predominant or
primary interaction is affected by the secondary or nonspecific
interactions. If a molecule is excluded from the surface due to ionic
interactions, it will not form a covalent bond even when the condi
tions are such that the covalent bond should form. In addition, if a
molecule is well solvated by the stationary phase, a covalent bond
will be stabilized. Understanding all the interactions occurring at
the surface allows for better control of the separation.
Chemically Modified Surfaces
A chemically modified surface consists of three parts, the
solid support, the immobilized functionality and the spacer arm that
attaches the functionality to the surface.
19
20 supports
There are many different types of supports available today. A
complete review of these supports can be found elsewhere (12,22,23).
The important properties of solid supports are physical and chemical
stability, porosity, particle shape and particle size. Solid supports
may be classified as inorganic or organic, with further classification
depending on chemical or physical properties.
Organic Supports. Organic supports include natural macro
molecules, polymers and synthetic polymers. The organic supports may
be hydrophilic or lipophilic. The lipophilic supports have not been
widely used in biochemical applications due to the aqueous nature of
the samples. Of these supports, the most commonly used are the
hydrophilic natural polysaccharides.
Polysaccharides include cellulose, dextran, agarose and
starch. These supports are hydrophilic, and the surface hydroxyl
groups are easily reacted to bind surface moieties. Dextran and
agarose gels are the most widely used of the polysaccharides and have
been utilized as macroporous supports in several areas of chromato
graphy. Starch has found little use because it is degraded easily
through microbial attack. Cellulose has found limited use, because
until recently it was not available in rigid macroporous particles.
Of dextran and agarose supports, the agarose gels are mechanically
more stable and more resistant to microbial attack; however, they
suffer from several disadvantages. They cannot be heat sterilized;
they disintegrate in alkaline solutions and organic solvents; they
shrink irreversibly on drying and are more expensive than other
21
supports. The main disadvantage of both dextran and agarose is that
they are soft gels compared to silica. They lack pressure stability
and are not useful in high performance systems. Even in medium
pressure systems, one must be concerned with the compressibility of
these gels.
Commonly used synthetic polymers include polystyrene, poly
acrylate types, polyacrylamides, and many others. A wide range of
synthetic polymers are easily prepared with different porosities due
to the various polymers and crosslinkers available. Unfortunately
many of these supports shrink and swell with changes in ionic or
solvent strength. This shrinking and swelling will cause concern in
many types of separations, especially in SPE where the change in
mobile phases is extreme.
Inorganic Supports. The inorganic supports have high mechan
ical strength, thermal stability, resistance to organic solvents and
microbial attack, easy handling, long shelf lives and do not change
structure over wide ranges of pH, pressure, or temperature. Highly
porous inorganic supports with high surface areas are available and
allow for a high concentration of binding sites in a small volume.
The mechanical stability of these supports allows for small particle
sizes, since they are able to withstand the pressure caused by small
particles. Inorganic supports offer more efficient separations than
any of the organic supports.
Porous inorganic supports inclUde several natural products;
however, these have a wide range of pore sizes. Controlled pore glass
has been used for some applications, but is expensive and has proven
to be less stable than silica or alumina.
Silica is available in a wide range of sizes and porosities
to fit any application. Furthermore, the chemical modification of
silica has been widely studied, and a wide range of alkylsilanes is
commercially available.
22
The availability of silica in small uniform particles offers
an advantage in reducing band broadening. Decreasing band broadening
will allow for more efficient separations, and in SPE, eluting the
compound of interest in a smaller volume. Band broadening in chro
matography is commonly expressed as the theoretical plate height. The
five major contributors to band broadening are eddy diffusion, mobile
phase transfer, longitudinal diffusion, stagnant mobile phase transfer
and stationary phase mass transfer, but longitudinal diffusion is
negligible in liquid chromatography. Since smaller particles decrease
eddy diffusion, mobile phase transfer and stagnant mobile phase
transfer, smaller particles will have a significant effect on the
reduction of band broadening. If all other parameters are optimal,
the minimum plate height is proportional to the particle size.
Immobilized Functionalities
In covalent chromatography, the efficiency of the separation
also depends on the kinetics of the covalent interaction. Retention
of molecules depends on two steps,much like the kinetics of ion
chromatography (11). The first step is the partitioning of the
molecule into the stationary phase. The second step is the reaction
23
of the molecule with the immobilized functionality. The first step is
governed by the non-specific interactions, and an understanding and
optimization of these interactions are necessary prior to the the
study of the covalent interaction. Exclusion of the compound of
interest will inhibit the formation of the covalent bond, and reten
tion through non-specific interactions will stabilize the covalent
interaction. At the same time it is important that non-specific
interactions are not so strong that contaminants are not removed from
the phase.
The amount of time an unretained molecule spends in contact
with the packed bed is dependent on flow rate and void volume of the
column as well as the nonspecific retention. A small packed bed with
a void volume of 50 u1 using a flow rate of 2 m1/minute has a resi
dence time of 1.5 seconds for an unretained molecule. Fo~ a k' of 1,
the compound spends 1.5 seconds in the stationary phase. For a k' of
5 there is a residence time of 7.5 seconds. Using the theoretical
plate hypothesis (10) and assuming 1000 plates means that each encoun
ter with the stationary phase lasts, on the average, 7.5 milliseconds
and the reaction will occur in this time. If the molecule does not
react in this time, the flow rate may be slowed or stopped to allow
more time for the reaction. Alternatively, the time of reaction could
be decreased by raising the temperature.
In order to assure that an adsorbent is not overloaded, the
amount of the sample should be about 1% of the stationary phase. In
these cases, the reaction kinetics are pseudo first order. For 99.9%
24
of the compound of interest to react in 7.5 milliseconds, a first
order rate constant of 1000 seconds- 1 is needed. Reactions with rate
constants of this magnitude or higher can easily be utilized in
covalent chromatography.
Modification
The most commonly used surface for chromatography is an
octadecy1si1ane modified si1ica(C18). Extensive research into an
understanding of the nature of this surface has been carried out
(9,10). Figure 2.1 is a polymerically bound octadecy1si1ane surface
equilibrated with a methanol/water mobile phase. On the molecular
level this surface is a heterogeneous phase. Associated with the
residual si1ano1s on the surface is about two layers of water mole
cules. Moving away from the surface, the hydrocarbon chain solvated
with the organic component of the mobile phase forms a nonpolar
region. Moving to the end of the chain, the polarity will gradually
increase to the polarity of the mobile phase. Thus, the surface shows
a polarity gradient (9) with different molecules finding their most
favorable interactions at different areas of the phase, and therefore
intercalating to the area of its most favorable interaction. Separa
tions on C18 phases rely on several types of interactions. The
residual si1ano1s of the surface ionize above a pH of 6. Thus, at
neutral to basic pH, the si1ano1s are ionized and are capable of
electrostatic interactions. Unionized si1ano1s and the associated
water are capable of hydrogen bonding. The C18 chain is capable of
dispersive interactions. Incorporated solvent molecules will also
H H H H If HCH HCH HCH HCH HCH I \ \',
HCH HCH HCH HCH HCH \ / / I I HCH HCH HCH HCH I1CH I \ \ \ \
HCH HCH HCH HCH HCH ,I 1// HCH HCH HCH HCH HCH
HC' JcH ~H ~H ~H \ / / / / HCH HCH HCH HCH HCH I \ \ \ \
HCH HCH HCH HCH HCH \ / I 1 / HCH HCH HCH HCH HCH I \ \' \
HCH HCH HCH HCH HCH \. / I / / HCH HCH HCH HCH HCH I \ \' \ HC~;eH 1H ;CH H~
HCH HCH HCH HCH HCH / \ \ \ /
HCH HCH HCH HCH HCH \ 1 I 1 I HCH HCH HCH HCH HCH I \ \' \
HCH HCH HCH HCH HCH \ I / I I HCH HCH HCH HCH HCH I '\ \ \ \
HCH HCH HCH HCH HCH \ I '\ I /
-51-Q-5i-O-H H H-O- 5i-0-5i-0-5i-0-H I '\ , 1 \ '\
o 0 0 0 0 '\ " \ , \
51 5i 51 51 51 51 Si 51 Si /\/,/\.1'1'1"'1\1
o 0 0 0 0 0 0 0 0
Figure 2.1 Polymeric C18 bonded silica.
25
26
interact with the sample. There is a great difference in the energet
ics of these interactions; however, all of these interactions are
important in the retention.
While alkyl bonded silicas have proven themselves for reverse
phase chromatography, the hydrocarbon nature of these phases has been
a limitation in covalent chromatography. Covalent chromatography is
most applicable to biochemical separations. Most of these samples are
aqueous and often the compounds of interest are denatured in organic
solutions. If a lipophilic phase is used, organic solutions are
needed to assure that non-specific adsorption does not occur and that
sufficient solvation of the sorbent is occurring.
The residual silanols which may be important in the separa
tions of reverse phase chromatography (9) result in irreversible
retention of biopolyrners. If a silica support is used in covalent
chromatography, care must be taken to assure that the residual sila
nols are unable to interact with the sample.
Silica based stationary phases used in covalent chromatography
have traditionally relied on alkyl silane backbones. N-phenylboronic
acid-N-propylsilylamino silica shown in figure 2.2 is a commercially
available phenyl boronic acid phase. Applications of this phase are
discussed in more detail in chapter 4. The non-specific or secondary
interactions available to the sample are demonstrated in figure 2.2b.
Molecules may form electrostatic interactions at the ionized sites.
They may interact through hydrogen bonding or dispersive forces.
These non-specific interactions may stabilize a covalent bond, but
they may also cause non-specific retention of unwanted molecules.
c;BCQ0
2 d CQf)2 ~
Cl HH Cl lIf Cl / I 1/1
HCH HCH HCH HCH HCH \ / I / I HCH HCH HCH HCH HCH / \ \ \ \
HCH HCH HCH HCH HCH \ / \ / / Si-O-Si-O-H H H-O- 5i-0-Si-0-5i-0-H I, \ /'
0, 0, 0 0 0 \ \ 1
Si 51 5i 5i Si 5i 5i 5i 5i
t \/\/ \1 \/\1\1\/\/ o 000 0 000
HVOROPHOBIC. w""
+ rQI .ARGE'TRANSFER
....... N~B,.OH H I ~ HVOROGEN + OH'" BONOING
. ,1~ ..... OH
IONIC -0- e~""OH OM
Figure 2.2 Prc:.pylaminophenylboronic acid and the secondary interactions occurring on this phase.
27
28
Non-specific retention may be eliminated by changing the
mobile phase to reduce the strength of the non-specific interactions.
Increasing the solvent strength decreases retention due to dispersion
forces but may increase retention of ionic compounds. Increasing
ionic strength will decrease retention due to electrostatic interac
tions but may increase retention due to dispersion forces. Changes in
the pH will affect the ionization of bQ~h the stationary phase and the
sample, thus affecting the electrostatic interactions. Non-specific
retention of molecules on residual silanols may be reduced by the
addition of an amine to the mobile phase. The amines will interact
strongly with the silanols, competing with the sample for this inter
action. Since the mobile phase additive is in a much higher concen
tration than the sample, it will effectively eliminate retention due
to silanol interaction. The silanols are often reacted with tri
methylchlorosilane. This endcapping reaction removes most of the
available residual silanols but increases the lipophilicity of the
surface.
N-phenylboronic acid-N'-propylsilylurea silica is shown in
figure 2.3. The difference in this phase is the hydrophilicity of the
surface. On this surface, the urea forms a hydrogen bonding barrier
that restricts molecules from intercalating to the silica surface. In
addition to the hydrogen bonding of the urea group the residual
silanols are also capable of hydrogen bonding. There are two to three
layers of water molecules associated with the surface silanols (10).
The water associated with the surface silanols is highly structured
29
g-(OO) 2
U. HO,PH NIl 0 N /!\,H \ II I HO C=O ----- HN- C ,O=C
/ H \, \ HNH HN NH NH / / \ \
HCH HCH HCH HCH \ \ / / HCH HCH HCH HCH / / / \ \
HCH HCH HCH HCH HCH \ / '\ / /
HO-Si-O-Si-OH H0-5i-O-Si- O-Si-OH I \ H / \ ,
o a '0 0 0
\ " " '\ '\ Si Si Si Si Si Si Si Si Si / \/ \/ \/ \1 \/\/ \/ \/ 000000 000
Figure 2.3 Propylureaphenylboronic acii immobilized on silica.
30
through hydrogen bonding, and this structure will extend past the
propyl linkage to merge with the water that is hydrogen bonded to the
urea group. In order to interact with the surface silanols, the
molecule would have to break the hydrogen bonds and disrupt the
structure of the water molecule. In addition, the residual amine
groups that remain unreacted are capable of interaction with the
silanols and will compete with the sample for the interaction.
Procedures
Several silica based adsorbents were prepared using several
active functionalities and two different types of modification
methods. Elemental analysis, diagnostic chromatography and solid
phase extraction were employed to characterize the phases.
Modification Methods
Modification of the silica was done in two steps. The silica
was first modified with a reactive organic moiety, either an amino
propyl, thiopropyl or a chloropropyl silane, followed by reaction with
the covalently reactive species, the disulfide, phenylmercurial,
phenylarsonous or phenyl boronic acid group.
Reactions With Bare Silica. To prepare aminopropy1 bonded
silica, forty micron irregular silica gel with 60 A average porosity
was acid washed, rinsed with deionized water and dried overnight at
80°C. The silica gel was suspended in dried 80% carbon tetrachloride
20% methanol. For each gram of silica, 1.2 mi11iequiva1ents of
triethoxyaminopropy1 silane (Petrach) was diluted by a factor of five
31
with dried carbon tetrachloride. This was added slowly to the silica
gel suspension. The reaction mixture was covered and left on the
shaker table for 6 hours. The reaction mixture was filtered and the
silica was washed thoroughly with SO% carbon tetrachloride 20% meth
anol with a final rinse with methanol. The silica was dried with
suction, then placed in an SO°C oven overnight. This reaction is
shown in figure 2.4.
Mercaptopropyl and chloropropyl bonded silicas were prepared
in the same manner as the aminopropyl bonded silica using triethoxy
mercaptopropyl silane and triethoxychloropropyl silane (Petrach) as
the silating reagents. These reactions are shown in figure 2.5 and
2.6.
Aminopropyl based bonded silicas. To prepare N-phenylboronic
acid-N-propylsilylamine silica (figure 2.7), chloropropyl silica was
reacted with m-aminopheny1boronic acid. For each gram of silica, 1
milliequivalent of m-aminophenylboronic acid was used. The reactants
are slurried in an aqueous solution with triethylamine as an acid
scavenger and left on a shaker table for 6 hours.
Activation of the mercaptopropy1 bonded silica. Mercapto
propyl bonded silica oxidizes easily, so care was taken to insure this
phase is kept in a non-oxidizing atmosphere. This phase was reacted
with 2,2'-dithiodipyridine (Aldrich), figure 2.S. 2,2-dithiodipyri
dine was dissolved in deaerated methanol. Thiopropyl modified silica
was added to the solution, and the reaction mixture was stirred
overnight. The phase was washed with methanol and dried overnight at
SO°C.
+ H H H H H
I I 1// 0, \ 0\ 0\ 0,
Si Si Si Si Si Si Si Si Si /\/\/\/\/\1\/\/\/ 00000 0 000
HNH HNH HNH HNH HNH I / I / /
HCH HCH HCH HCH HCH \ / / I / HCH HCH HCH Ht:H HCH / \ \ \ \
HCH HCH HCH HCH HCH
_ \Si-O-S:-'O-H H H-O - 'Si.-o-Sr- 0 -Si:O-H I , \ / \ 00000 \ \ \ , \
5i 5i 5i 5i 51 5i 51 5i 5i / \/ ,/\1 \/ \/ \1 \1\ /
o 0 0 0 0 0 0 0 0
32
Figure 2.4 Reaction of triethoxyaminopropyl silane with silica.
(CH3CH20)351CH2CH2CH2CI
+ H H H H H
I I I I / 0 0 0 0 0
\ '\ '\ \ '\ 5i 51 51 51 51 51 5i 5i 51
1\/ \./,\/\/ \ / \ / \ I \/ 0 0 0 0 0 0 0 0 0
~ Cl Cl CI Cl CI
/ / / / / HCH HCH HCH HCH HCH
\ / I / I HCH HCH HCH HCH HCH / \ \ \ \
HCH HCH HCH HCH HCH \ / \ / / 5i-O-Sl-0-H H H-O-51-0-Si-O-5i-O-H
I '\ \ 1 '\ o 0 0 0 0 \ \ \ \ /
51 51 51 5i 51 51 5i 51 51 / \/\/ \/\/\/\/\1\/ 600000000
33
Figure 2.5 Reaction of triethoxychloropropyl silane with silica.
H H I /
0 0
(CH3CH20)3SiC~CH2CH2SH
+
H H / /
0 0
H /
0
'SI , \ \ \
5i Si Si SI Si Si 51 5i I \ / \ / \ / ,I \ I \ I " / \ /
0 0 0 0 0 0 0 0 . 0
SH)H 5H 5H 5H I / / I
HCH HCH HCH HCH HCH \ I / I / j'H HC~ HC~ HC~ H~
HeH HCH HCH HCH HCH \ / \ / /
Si 0 SI-0-H H H-O-Si-O-Si-O-Si-OooH / \ \ I \
o 0 0, O. 0 \ \ \ \
5i 51 Si 51 51 51 5i SI 51 . / \ / \ / \. / '\ / ,/ \ /., / \ I 000000000
Figure 2.6 Reaction of triethoxymercaptopropyl silane with silica.
34
CI CI CI CI CI / / / / /
HeH HCH JH HeH HCH \ 1 1 / HeH HeH HCH HCH HeH / \ \ \ \
~ ItCH HeH HCH HeH HCH \ / , / /
iJ(oH)') SI-O-SI- O-H H H- 0- SI-O-SI-O-SI-O-H / \ \ 1 ,
0 0 0 0 , 0, \ , /
SI SI 51 51 SI 51 51 51 51 ('/\/\/ \1\1\1\1\/
000 0 0 0 0 0
d(af)2 Cl HH Cl NH CI
/ / / / / HCH HCH HCH HCH HCH
\ 1 / / / HeH HCH HeH HCH HCH / \ \ \ \.
HCH HCH HeH HeH HCH \ / ,/ / SI-O-SI-O-H H H-0-SI-0-51-0-SI-0-H
/ \ \ 1 \ o 0 0 0 .J , \ \ , /
51 51 51 SI 51 51 51 SI SI / ,/\/ \/\/\/\/\1\/ o 0 000 0 0 0 0
Figure 2.7 Reaction of chloropropyl bonded silica with maminophenylboronic acid to form propylaminophenylboronic acid bonded silica.
35
36
SH JH SH SH SH I / / /
HOt HCH HCH HeH HeH \ / / / /
@-s~~ ItCH HCH HeH HCH H~ I \ \ \ HeH HCH HCH HeH HCH
) \ / , / / - 51-0-51-0- H H H-0-Sl-0-51-0 -51-0-tf I \ \ I \ "-
0 0 0, 0 0 \ \ \ \
51 51 51 51 51 51 51 51 51 \ / \ / \ 1 \/ \ /, / ,/, /' I "-
o 0 000 0 0 0 0
5H S/S@ S/S-@ 5H SH
I / / / I HeH HCH . HCH HeH HeH \ / / / I
/HCH HCH HeH HeH HCH
\ \ \. \ HeH HeH HeH HeH HCH
\ / '/ / -51-0-51-0-H H H-0-51-0-SI-0-SI-Oif I \ , I \ , o 0 0, 0 0 \ \ \ \
51 51 51 51 51 51 51 51 51 \/\/\1\/\1'\./\/\/\ I' o 0 000 0 0 0 0
Figure 2.8 Reaction of 2,2-dithiodipyrirline with mercaptopropyl bonded silica to form the mixed disulfide phase.
37
Propylurea based bonded silicas. To prepare N-Phenylboronic
acid-N'-propylsilylurea silica (figure 2.9), aminopropyl bonded silica
was dried for 3 hours at sooe and cooled to room temperature in a
desiccator. The aminopropyl bonded silica was activated by reaction
with N,N-carbonyldiimidizole in methylene chloride. Triethylamine was
added as an acid scavenger. This reaction is left for 3 hours at room
temperature. The.activated silica is removed from the solution by
filtration and washed with methylene chloride and dimethylsulfoxide
(DMSO). The silica is added to a solution of m-aminophenylboronic
acid hemisulfate in DMSO. This reaction is heated to 40 0 e and left
for 24 hours. The silica is filtered and then placed in a solution of
ammonia saturated DMSO and reacted for three hours. The ammonia will
react with any residual carbo imide sites. The final product is
filtered and washed with 50% DMSO, water, 0.02 M hydrochloric acid, 1
M sodium chloride and again with water. The product is dried at room
temperature.
To prepare N-phenylmercury-N'-propylsilylurea silica, (figure
2.10), aminopropyl silica is activated in the same manner as for
N-phenylboronic acid-N'-propylsilylurea silica (figure 2.9). The
activated silica gel is added to a solution of p-aminophenylmercuric
acetate in DMSO. The reaction is heated to 40 0 e for 24 hours. The
silica is filtered and placed in a solution of ammonia saturated DMSO
and stirred for 3 hours at room temperature. The finished product is
rinsed and dried just as the previous reaction.
l1li HIIH HIIH-
/ / I ltC' ~HeH ? ,H /
HeH HeH HCH / \ \ \ ()
HCH HeH HCH HeH ~O \ / ,I'
_ Si-0.51-G-H H H-O- SI-0-51-0' ,'N
/ \ \ / \ 00000
~,;
\ \ \ \ \ SI SI SI 51 51 51 51 51 SI 1\/\/\1\1'\/\/\/\1 000 0 0 0 0 0 0
Q 0 QQ ~~
Nt. HIIIt "" HHH 1 / I /
HCH' HeH HCH ItCH ,/ I / HCH HCH HCH HCH
1 \ " ItCH HCH HCH HCH \ / \ /
-SI-0.51-0-H" H-O- 51-0·SI-0-/ \ , /"
o 0 0 0 0 , '\. '\. \ \ ~ ~ ~ g ~ ~ ~ ~ ~ /\ /,/\1,1 \/ \1 \/ \/
d. 0 0 0 0 0 0 0 0
d(QIl'1. 4~ ~ 1:.'/ e.o ~'Q /
HH HIlI HH HHH / / / 1
HCH HCH HCH He"
HCH ~ ~ ~ / \ \ \
HCN HCH HCH HCH \ / \ / I
-51-0.51-0-H" H-O- 51-0·SI- 0-51· / \ \ / \ I
o 0 0 0 0 " " \ , " 51 SI 51 51 51 51 51 51 SI 1\/\/\/\/\/\1'\/\1 000000000
>
>
Figure 2.9 Reaction of propylamino bonded silica with 1,1'carbodiimidizole followed by m-aminophenylboronic acid to form propylureaphenylboronic acid bonded silica.
38
Q () I ro "0 I
NH HHH NH HNH / / / /
HeH HCH HCH HeH \ / / I HCH HCH HCH HeH / \ \ \
HeH HeH HCH HeH \ / \ / I
H2N-@-~ -5i-0-51-0- H H H- 0 - 5i· 0-5i- O-SI-/ '\ \ / \ ,
0 0 0 0 0 \ \ \ \ \
Si Si 5i Si Si Si 5i Si Si /\/\/\/'\/'/'\1'\1\1
o 0 0 0 0 0 I) a 0
Q '-0 I '-0 I NH HUH NH HNH
/ / / / HeH HeH HCH HeH
\ / 1 / HeH HCH HeH HeH / \ \ \
HeH HeH . HCH HeH \ / \ / J
-5i-0-5i-0- H H H- 0 - 5i· 0-5i- 0-5i-/, \ / \ I
o 0 0 0 0 \ \ \ \ \
5i 5i 5i 5i Si 5i 5i Si 5i 1\/\/\/,\/,/,\/,/\/ 000000000
~
Figure 2.10 Reaction of a cor activated aminopropy1 phase with p-aminopheny1mercurial hydroxide.
39
40
To prepare N-phenylarseneoxide-N'propylsilylurea silica
(Figure 2.11), aminopropyl bonded silica is activated with N,N'
carbonyldiimidazole as in the previous reaction. The activated
silica is then reacted with p-aminophenylarsine oxide in DMSO for
three hours at room temperature. The silica is filtered and placed in
a solution of ammonia saturated DMSO and stirred for 3 hours at room
temperature. The finished product is rinsed and drie~ just as the
previous reactions.
A ureidopropyl phase (figure 2.12) was prepared by activating
aminopropyl bonded silica with N,N'-carbonyldiimidazole as in the
previous reaction. The silica is filtered and placed in a solution of
ammonia saturated DMSO and stirred for 3 hours at room temperature.
The finished product is rinsed and dried as described previously.
In order to assure that these phases are consistent, the same
batch of activated aminopropyl silica was used in all three reactions.
Characterization of Bonded Silicas
Elemental Analysis. The surfaces were characterized at the
through elemental analysis both at the University of Arizona and at
Analytichem (Harbor City, CA). The results are found in table 2.1.
This information is complemented with the ICP emission data for boron,
mercury and arsenic. These data allow for the determination of the
the different types of groups bound to the surface.
In the preparation of the propylaminoPBA phase (figure 2.7),
it is possible for it to contain 2 bonded moieties, the chloropropyl
group and the propylaminoPBA moiety. From the elemental analysis
41
Q [) Coro c·o I I
NH HNH NH HNH I / / /
IICH HCH HeH HCH \ / I / HCH HeH HeH HCH
'7~""IIIO / \ \ ,
HCH HeH HeH HCH \ / \ / I )
-Si-O-Si-O-H It H-O- Si· O-Si- O-Si-I " \ / \ I
0 0 0 0 0 \ \ \ \ " Si Si' Si Si Si Si Si Si Si /\1\1\1,,1'1'1'1"1
0 0 0 a 0 0 0 0 0
Q ~. C·O CIO I I
NH HNH NH HNH I / I /
HOt HCH HeH HCH \ / I / HCH HeH HCH HCH / \ \'
HCH HCH HeH HeH \ / \ / I
-Si-O-Si-O-H It H-O- Si· O-Si- O-Si-/ \ \ / \ I
o 0 0 0 a \ \ \ \ \
Si Si Si Si Si Si Si Si Si 1\/\1\1,,1'1'1'1,,1 00 a 000 000
Figure 2.11 Reaction of a CDr activated aminopropy1 phase with p-aminopheny1arseneoxide.
Q f) C-o C·o I I
NH HHH NH HNH / / / I
HCH HCH HCH HCH \ / / / HCH HCH HCH HCH / \ \ \
HCH HCH HCH HeH \ / I \ /
-SI-O-SI-O- H H H- 0 - SI· O-SI- O-SI-/ '\ \ / \ I
0 0 0 0 0 \ \ \ \ \
SI SI SI SI SI Si SI Si Si /\/\/\/\/,/'\/'\/\/
0 0 0 0 0 0 0 0 0
KV" H~H e-o e.o I I
NH HNH NH HNH / / / I
HCH HeH HCH HCH \ / / / HCH HCH HCH HCH / \ \ \
HCH HCH HCM HCM \ / \ / I
-SI-O-SI-O-H H H-O- SI· O-Si- O-SI-/ '\ \ / \ I
o 0 0 0 0 \ \ \ \ \
51 SI Si SI SI 51 Si SI 5i /\/\/\/,\/'/'\/'\/\/ 000000000
42
NH3
;,.
Figure 2.12 Reaction of a CDr activated aminopropyl phase with ammonia to form a propylurea phase.
43
Table 2.1 Elemental analysis of modified silicas.
Bonded Silica %C %H %N %M
Aminopropyl 5.44 1. 52 1.77
Mercaptopropyl 3.16 0.74 0.00
Mixed Disulfide 6.80 0.66 0.13
PropylaminoPBA 5.95 0.97 0.47
PropylureaPBA 9.26 1.71 2.40 0.53!!
PropylureaHgOH 7.50 1. 52 2.19 6.43h
PropylureaAsO 7.76 1.52 2.37 1.22£
Propy1urea 6.73 1.59 2.99
a %Boron, b %Mercury, c %Arsenic
44
(table 2.1), the concentration of both can be determined. The chloro
propyl phase initially bound with 0.98 millimoles/gram. Approximately
35% reacted with m-aminophenylboronic acid to give 0.34 millimoles/
gram PBA and 0.64 millimoles/gram unreacted chloropropyl. The cova
lent interactions in this phase are occurring on a lipophilic hydro
carbon backbone.
The preparation of the hydrophilic propylureaPBA phase is
more complicated, and there are four possibilities of surface
moieties: the PBA phase, an unactivated aminopropyl, an activated
aminopropyl and the urea propyl phase. Assuming that the ammonia in
DMSO was able to react with all the activated sites, we can assume
that no activated aminopropyl sites remain. The aminopropyl bonded
silica contains 1.42 millimoles/gram. During the reaction of this
phase to the ureapropyl phenylboronic acid phase (figure 2.9), it is
unlikely that any of the bonded groups were removed, so the total
number of bonded moieties is the same for both phases. From the boron
analysis, the concentration of the PBA functionality is 0.49
millimoles/gram, or 35% of the bonded functionalities. Using the
carbon data and solving for the remaining variables indicates that
0.32 millimoles/gram or 23% remain unreacted as the aminopropyl and
0.61 millimoles/gram or 43% are ureapropyl. The unreacted aminopropyl
groups are capable of ionizing, and the hydrophilic urea groups will
provide a completely different environment than in the hydrocarbon
phase.
The ureapropylsilane silica shown in figure 2.12 may have
three functionalities on the surface: the unreacted aminopropyl, the
45
activated aminopropyl and the ureapropyl functionality. Once again,
the activated aminopropyl has been fully reacted with the ammonia in
DMSO. Using the carbon data and the assumption that 1.42 millimoles/
gram is the total number of functionalities on the surface, it can be
determined that 0.34 millimoles/gram or 24% remain as the aminopropyl
and 1.08 millimoles/gram or 76% are reacted to the ureapropyl.
Using the same assumptions, the data can be used to determine
the concentrations of the different functionalities on the propyl
ureaorganometallic phases. For the phenylmercurial phase, there are
0.32 milliequivalents/gram or 20% of the propylureamercurial function
ality, 0.48 milliequivalents/gram or 34% of the ureidopropyl function
ality and 0.62 milliequivalents/gram or 46% aminopropyl groups. The
arsonous acid phase has 0.16 milliequivalents/gram or 10.6% propyl
ureaphenylarsonous acid, 0.79 milliequivalents/gram or 52%
uriedopropyl groups and 0.56 milliequivalents/gram or 37% aminopropyl
groups.
Solid Phase Extraction. The capacity of the phases was
determined using solid phase extraction. Plastic syringe barrels were
filled with 100 milligrams of aminopropylPBA bonded silica. The
silica was washed first with methanol or acetonitrile, then with water
and finally the mobile phase. Approximately two micromoles of sample
dissolved in the mobile phase was applied to the column. The effluent
from the column was captured in a 5 ml volumetric flask and the
concentration of sample was determined through HPLC with a UV detec
tor. HPLC was used to separate the compound of interest from the
solvent. During the aspiration of the sample into the volumetric
flask, some of the organic solvent would evaporate. This change in
the solvent made simple UV experiments to determine concentration
difficult. UV data was used to confirm the identification of eluted
components.
46
Solid phase extraction experiments were used to determine the
capacity of the phases and the changes in capacity when the mobile
phase was changed.
Diagnostic Chromatography. The chromatographic results
indicate relative strengths of interactions and the trends in inter
actions as the ionic strength, solvent strength, solvent composition
and pH are varied. In all cases, only one variable is changed while
the rest are held constant.
Careful consideration of mobile phases is needed to assure
that the mobile phase is not competing with the sample for the active
sites on the surface. Alcohols are expected to bind to PBA (14),
therefore acetonitrile was chosen as the organic modifier. Data in
table 2.2 confirms that as the methanol concentration increases, the
retention of benzene does not decrease. This indicates that the
lipophilic nature of the stationary phase is increasing with the
increase in methanol concentration. The increase in lipophilicity of
the phase is caused by the covalent interaction between the methanol
and the phenylboronic acid. The interaction of PBA with monofunc
tional compounds is weak and is only noticed at high concentrations
(greater than 10%).
Table 2.2 Capacity factors of several probe molecules on
propylaminoPBA with methanol as the organic modifier
Solvent
Concentration
5
10
15
5
10
25
5
10
25
Probe
Benzene
Benzene
Benzene
Phenol
Phenol
Phenol
Nitrate
Nitrate
Nitrate
Capacity
Factor
27.6
40.5
4l.5
7.3
6.3
6.8
0.0
0.0
0.0
47
48
The choice of buffer composition is based on the buffer
capacity, range of pH and a limited interaction with the stationary
phase. Phosphate buffers bind with PBA and are not acceptable buffers
(14). Ammonium acetate is a commonly used buffer for liquid chromato
graphy. While both ammonia and carboxylic acids show some interaction
with PBA (chapter 4), these interactions are weaker than that of
phosphate since each contains only a single interaction site while
phosphate has four. The ammonium acetate also demonstrates inter
action with the mercury phase (20). However a great number of mole
cules will act as ligands to mercury but the interaction with thiols
is so strong it easily displaces the buffer even with the difference
in concentrations.
The modified silica was packed into a 2 cm long 2 mm inner
diameter column. A second column containing octadecylsilyl silica was
placed in system just prior to the injection valve. In the case of
high pH mobile phases, the CIS column would dissolve into the mobile
phase so that the mobile phase would be equilibrated with silanes and
would not dissolve the phase of interest.
Mobile phases were prepared using doubly distilled filtered
water and filtered reagent grade acetonitrile or methanol. All
mobile phases are a volume/volume percent and were measured using
graduated cylinders. Stock solutions of the ammonium acetate and KCl
were prepared by weighing the salts and dissolving them in doubly
distilled filtered water. These solutions were then added to mobile
phases to assure that the buffer concentration and the the ionic
strength were consistent.
49
Chromatographic experiments were done using an IBM 9533
chromatograph, with a 9522 detector. This is a 254nm fixed wavelength
ultraviolet detector. The data collection was an IBM 9000 lab com
puter or a strip chart recorder.
Retention of the molecules is reported as capacity factor
(eq. 1.5). The void volume was measured as the volume needed to
elute nitrate from the column. This volume was assumed to be the
same for all pH ranges but was measured at a pH of 9.
Several probe molecules were chosen to determine the relative
strengths of interactions occurring at the surface. These probe
molecules include compounds expected to interact with the covalent
functionality as well as molecules which should not interact. The
retention of these molecules on different phases and when using
different mobile phases will allow for determination of the factors
which affect selectivity of the surface and which factors must be
controlled when using or preparing the silica surface.
Summary
Diagnostic chromatography has been used to characterize two
different classes of modified silica surfaces. Careful choice of
probe molecules allows for the determination of the relative
strengths of interactions and the effects of different mobile phases
and modifications on the versatility of silica adsorbents.
Of the commonly used solid sorbents, silica is physically
strong and easily modified. The reactivity of silica may be a
50
limitation. Unless the surface is completely reacted the unreacted
sites of the silica can interact with many compounds causing irrever
sible adsorption. Controlling the modification of the surface is
important when using silica.
CHAPTER 3
EFFECTS OF MODIFICATION
While the covalent interaction is the primary interaction and
the control of this interaction will be most important in covalent
separations, the control of the non-specific or secondary interactions
will also be important. Secondary interactions are most commonly
dispersive, hydrogen bonding or electrostatic interactions that may
occur between the compound of interest and the sorbent, the matrix and
the sorbent or between the compound of interest and the matrix. Non
specific interactions may be necessary for the formation of the
covalent bond, or they may cause retention of contaminants. Optimiz
ing non-specific interactions is necessary for efficient separations.
Control of the Non-specific Interactions
Mobile Phase
Traditionally, non-specific interactions are controlled by
altering the mobile phase. Dispersive interactions can be reduced by
increasing the concentration of the organic modifier. Ionic inter
actions can be reduced by increasing the ionic strength. Electro
static repulsion is minimized by an increase in ionic strength.
Changing the pH to suppress ionization of the sample or the stationary
phase may also eliminate electrostatic interactions. Interactions
51
52
with residual silanols has been reduced by the addition of an amine to
compete with the sample for these sites.
In some cases, altering the mobile phase may not be possible.
In many biochemical applications, increasing the organic modifier may
not be an option. The compound of interest is often denatured ir
reversibly at concentrations of 20% acetonitrile or less. Increasing
the ionic strength above 0.1 molar is difficult when working with
liquid chromatographic pumping systems because of the salt deposits
that block lines or ruin seals. In automated systems the ionic
strength must be kept to a minimum to increase the lifetime of the
pumps.
Stationary Phase
In addition to altering the mobile phase, the stationary phase
may also be altered. Residual silanols have been reacted with tri
methylchlorosilane. This molecule is smaller than other silanes and
is believed to react with silanols that are sterically not available
to the larger molecules. Even the most carefully reacted silica
surfaces will have residual silanols. These silanols may irreversibly
adsorb samples so that any residual silanols must be sterically unable
to interact with the compound of interest.
A more versatile modification of silica is needed in order to
overcome the present limitations while still allowing for the effi
ciency of the small rigid particles. A new modification method with a
hydrophilic surface that restricts samples from the residual silanols
has been developed. This propylurea phase (figure 2.3) is described
in chapter two.
Experimental
Diagnostic chromatography as described in chapter two was
employed to compare the differences in a traditionally modified
53
silica and the new modification. The effects of solvent strength,
ionic strength and pH on dispersive, hydrogen bonding and electro
static interactions were determined using molecules that probe each of
these interactions.
The modified silicas characterized in this work all contain
some ionizable species. These are shown in figure 3.1. In addition.
each contains residual silanols that ionize at pH 6 and above. PBA
will ionize through the acceptance of a hydroxyl group with a pKa of
9.2 (24). The meta amino functionality which binds the PBA to the
surface in the lipophilic phase will accept a proton with a pKa
similar to that of N-ethylaniline which is 5.12 (25). The urea or
hydrophilic phases have two types of nitrogen atoms, the aminopropyl,
and the urea nitrogens. The pKa of propyl amine is 10.7 (25) and the
pKa of urea is 0.10. Due to the low pKa of urea, the urea type
nitrogens of the phase are not ionizing. Conversely, due to the high
pKa of propylamine, the amine groups on the surface are ionized
throughout the experiments.
As the pH of the solution is altered, the different bonded
moieties will ionize. A study of retention vs pH will allow for the
54
S(al)3"
Q "hydrophilic" "lipophilic" d(01I) 3 NH ~ f ~ I C-O C-O
;m; I , NH NH Cl NH Cl I I I I I
~ fIlz ~Z ~ 1HZ 9Hz ~ ~ ~ ~ C~ 9~ - - - , -f2 0
~ 0 C~ ~
0 CHZ 0 C~ I I I I I I I
Q«(JI)2 Basic pH
d(01I) 2 NH ~ f ~ I C-O C-O
~ I , .
NH NH Cl NH Cl I I I I I
~ fH2 ~Z ~ ~ 9~ ~ - F2 - ~ ~ ~ <fiz - -~ 0
~ 0
~ ~ 0 f2 0
~ I I I I QB«(JI) 2 Neutral pH
~ J 0(011)2 NH F2 f ~ I C-O Cm()
~ I I NH NH Cl NHz Cl r , I
~ I
~ ~. ~ fHz 7HZ
~ H ~ ~ ~z ~2 H 9H2 ~ ~z CHZ 0 f2 0
~ ~ 6
~ 0 CHZ I I I I I I
Acidic pH Figure 3.1 ~o\Uparison of ionization of the two modification
methods.
determination of the types surface charges on the phase. The pH of
the solution was varied between 3.4 and 9.2, and the retention of
several molecules able to probe different interactions was measured.
For these data, the ionic strength was held at 0.1 molar.
55
In order to assure that the bonded silica is solvated, it is
necessary that the mobile phase contain some organic modifier. In
this work 5% acetonitrile was chosen. Below 5%, it is difficult to
keep the organic strength constant throughout the experiments. Above
5% acetonitrile, the capability of the mobile phase to dissolve salts
decreases, and the compatibility of the mobile phase with biopo1ymers
is diminished. Therefore, 5% was chosen as a good trade off between
reproducibility, compatibility and practicality.
Results and Discussion
Dispersive Interactions
Lipophi1icity of the Bonded Phases. The difference in
lipophilicity in the two phases is demonstrated by the retention of
benzene and toluene (figure 3.2). Toluene has a capacity factor of 10
on aminopropylPBA and 2 on propylureaPBA. Benzene has a capacity
factor of 5 on aminopropylPBA and 1 on propy1ureaPBA. Both molecules
show a lower capacity factor on propylureaPBA with a difference of a
factor of five. AminopropylPBA is more lipophilic and the dispersive
interactions are much stronger on this phase causing the higher
capacity factor. The variations in capacity factor with a change in
pH may be due to slight differences in the concentration of the
BENZENE 7
.,
5
4
:.. 3
2
: ; :~ 0
3 5 7 9
Cl proPvNrea ~ar prop~A
TOLUENE 13
IZ
n
~ 10
9
IS
:.. 7
., 5
4
3 ==---: 2 ... -CJ Ei EI -r--
3 5 7 9
Cl propylur .. propylonaPar 0 propytP8A
Figure 3.2 Retention of benzene and toluene on three modified silicas as a function of pH.
56
57
organic modifier in the mobile phase which would cause noticeable
differences in k', or due to the difference in surface charge as the
pH changes. A highly charged surface will exclude hydrocarbon
samples and decrease retention. Electrostatic interactions are a
better measure of charged surface sites than lipophilic interactions.
Effects of Solvent Strength. The effect of increasing the
concentration of acetonitrile on the lipophilic surface is shown in
figure 3.3. Above 10%, the addition of more organic modifier results
in only small decreases in retention for these small probe molecules.
The effects of increasing ionic strength would be greater for larger
molecules that are well retained on the phase at 5%.
Electrostatic Interactions
Anion Exchange Sites. As the pH of the mobile phase changes,
the charge on the surface will change. Ionizable sites on the
surface or the immobilized functionality will be charged or neutral
due to the pH of the surrounding solvent. While the pH at the surface
will not always be the same as the mobile phase, an increase in mobile
phase pH will increase the pH at the surface. At low pH, amine
functionalities on the surface will be positively charged. As the pH
of the surface increases, the amines may neutralize or the the ioniza
tion of surface silanols and the PBA functionality will produce a
negatively charged surface. Retention of anions is expected to be
high at low pH and decrease as the surface becomes negative. Benzoic
acid (figure 3.4), phenylacetic acid (figure 3.4), and benzenesulfonic
acid (figure 3.5) probe the anion exchange sites on the surface.
SOL VENT STRENGTH DEPENDENCE
6,---------------------------------------------------~
5
4
:.. 3
2
5 7 9 II 15 f7 19 21 23 2S
" ACETOHITRLE Q BENZENE o ~E A NITRATE
Figure 3.3 Effects of acetonitrile concentration on the retention of benzene, aniline, phenol, and nitrate on propylaminophenylboronic acid bonded silica.
58
BENZOIC ACID
7,----------------------------------------------------, 6
5
4
2
0 ~ 5 7 9
0 PfO\>yIon. ptOfJ."....,el' prupytP8A
PHENYLACETIC ACID 7
6
5
4
:.. ~
2
" "
O+-------r------,-------r------.-------~----~~----~ 5 7 9
Figure 3.4 Retention of benzoic acid and phenylacetic a:id on three bonded silicas as a function of pH.
59
60
BENZENESULFONIC ACID
8~--------~-----------------------------r
.~
..
2
0 ---i :J 5 7 9
0 propytura. _yturaJ _yIP9A
PHENYL TRIMETHYLAMMONIUM 4S
40
:IS
:JO
2S
:.. 20
IS
10
S
0 :J 5 7 9
0 propyka'aa _ytura""ett 0 propylP9A
Figure 3.5 Effects of pH on the retention of benzenesulfonic acid and phenyltrimethylammonium on three modified silicas.
61
Benzenesulfonic acid retention shows a continuous decrease in reten
tion as the pH is increased. Higher retention of this probe on the
hydrophilic surface at low pH indicates that this phase has more
positively charged sites than the lipophilic surface up to a pH of 7.
The increase in anion exchange capacity is due to the residual amino
propyl groups on the silica. The decrease in retention at
neutral pH is not due to the suppressed ionization of these sites but
to the ionization of the PBA to its anionic form and the ionization of
the surface silanols. These sites compete with the sample for inter
action with the cations. Above a pH of 7, all the phases are nega
tively charged and the anions are not retained on the surface.
Benzoic acid and phenylacetic acid show the same trend as benzene
sulfonic acid. The difference is that the carboxylic acids with pKa
near 4.0 are not ionized at the low pH, so they are not retained
through electrostatic interactions. As the pH increases to the pKa of
these probes, the retention increases.
Cation Exchange Sites. Phenyltrimethylammonium (figure 3.5),
aniline (figure 3.6) and benzyl amine (figure 3.6) will probe the
cation exchange characteristics of the surface. The cation exchange
sites will occur with the ionization of residual silanols or the
immobilized PBA. Elemental analysis shows that the hydrophilic phase
has 0.49 millimoles/gram of PBA and the lipophilic phase contains 0.34
millimoles/gram. If the PBA is the only important site, we would
expect more cationic exchange sites on the lipophilic phase. The
amines on the surface are capable of interaction with the PBA to
:..
0
:..
=
ANILINE 2..8
2.4
2.2
2
L8
L8
L4
1.2
\ 0.0
0.0
0.4
Q.2
\ 0
3 S 7 9
PIOCIvlllrea prc,PVtVINfi'9r 0 propvtP9A
BENZYLAMINE 7
8
S ~~ ..
2
G-- -----.. 9 9
~ 3 S 7 9
propykn_ PIOCIvtureePal" 0 PIOCIyiPtlA
Figure 3.6 Retention of aniline and benzylamine on three modified silicas as a function of pH ..
62
63
decrease the pKa (26) or to neutralize the the charge. The only
amines on the lipophilic phase is the meta amine which does not
interact with the PBA groups and is not protonated at the pH where
the PBA ionizes. The hydrophilic phase contains a large number of
amine functionalities that are protonated at high pH and are able to
interact with the PBA. The affects of this amine will be to neutral
ize the surface.
The quaternary amine, phenyltrimethylammonium, shows little
retention on any of the phases at low pH. As the pH increases, the
retention increases, but the retention on the lipophilic phase is much
higher. Comparing these data to the data of catechol (figure 4.3),
the catechol is retained much better on the hydrophilic phase.
Catechol interacts only with the ionized PBA indicating that the
hydrophilic phase contains more ionized PBA functionality. The
residual silanols and the propyl amine functionalities are important
sites for electrostatic interactions. The residual silanols are
available to the sample on the lipophilic phase, but the organized
water of the hydrophilic phase block these sites.
Aniline with a pka of 4.6 is not retained at low pH (less than
5), since the surface and the probe are positively charged. At
neutral pH (between 5 and 8), aniline interacts with the silica
surface on the lipophilic phase or with PBA on either phase. At high
pH, the hydroxyl groups compete for the PBA site and the retention
drops.
Benzylamine with a pKa of 9.33 (25) remains charged throughout
the pH region studied and there is no change in retention on the
64
lipophilic phase. The drop in retention at pH 9 for the hydrophilic
phase is due to competition for the boronic acid from hydroxyl groups.
This interaction will be discussed later.
Effects of Ionic Strength. Changing the ionic strength of
the mobile phase will allow further probing of the electrostatic
interactions. The retention of benzene, toluene, phenol, benzyl
alcohol and aniline is not changed as the ionic strength is increased.
These molecules are not charged and electrostatic interactions are not
expected. Benzylamine (figure 3.7) is a cation at pH of 7. There is
little change in the retention as the ionic strength is increased.
Benzyl amine is not retained through electrostatic interactions. The
anions, benzoic acid (figure 3.7) phenylacetic acid (figure 3.8) and
benzenesulfonic acid (figure 3.8), demonstrate ionic strength depend
ence on the urea phases. These molecules are retained on the amino
propyl groups of the urea phases. This interaction is weak, however,
because of the competition for these sites from the residual silanols
and the PBA functionalities. At pH 7, the lipophilic phase has little
cationic character and the anions show little retention and no ionic
strength dependence.
The retention of phenyltrimethylammonium (figure 3.9) on the
lipophilic phase decreases as the ionic strength increases, indicating
that the cation is retained through electrostatic interactions. On
the urea phase, there is no ionic strength dependence indicating that
there is no electrostatic interaction. The negative charges on the
surface are not likely due to ionized PBA groups because these
Figure 3.8 Effects of ionic strength on the retention of phenylacetic acid and benzenesulfonic acid on propylurea (a), propylureaphenylboronic acid (b), propylaminophenylboronic acid (c) and aminopropyl bonded silica (d).
1% pH 7
111
a
k 6
4 a e e
&= z
a 12.' 31.5 63
ion ie stzoongth
pH 9
18
38
~ 28
18
8--------~--------------~--------------~ 8 68
ionic strength 115
67
Fi~ure 3.9 Ionic strength dependence on the retention of phenyltrimethylammonium on propylureaphenylboronic acid (a) and propylaminophenylboronic acid (b).
68
groups, would not be significantly charged at this pH. This retention
is therefore due to the availability of ionized residual si1ano1s on
the surface.
If the pH is increased to nine, the retention of the cation
shows ionic strength dependence for both surfaces (figure 3.9). At
this pH, the PBA functionality is charged on both phases, but the k'
for the cation is higher on the hydrophobic phase indicating an
increase in ion exchange sites. The increase in the number of cation
exchange sites is due to the cationic aminopropy1 groups on the
hydrophilic surface which interact with the anions on the surface, and
to the availability of the si1ano1s to the sample on the lipophilic
surface. The residual si1ano1s are not available to the sample on the
lipophilic surface due to the hydrogen bonding of the urea groups and
the organization of water which forms a barrier to the molecule.
Hydrogen Bonding
The hydrogen bonding capability of the surface was probed with
phenol (figure 3.10) and benzyl alcohol (figure 3.10). In 95% water
the hydrogen bonding characteristics are expected to be insignificant.
The retention of the molecules is similar to retention of benzylamine
(figure 3.6). On the lipophilic phase the retention is not changed as
the pH changes. On this surface these molecules are retained at the
silica surface. The polar end of the molecule interacts with the
surface silanols or with the associated water while the lipophilic
phenyl ring interacts with the hydrocarbon chains (9). This inter
action is sufficiently removed from the PBA that the ionization of the
69
PHENOL 2.1 2
1.9
1.8 L7
US
L5 L4
L3
:.. L2 LI
o.s o..a 0.7 o..a o.s 0.4
Q.!I :) 5 7 9
0 propyluru PIOf'y/AIrotIi'att 0 propyfP8A
BENZYL ALCOHOL 2.:)
2.2 2.1 2
L9 1.8 L7 US L5 L4 :.. L3 L2 LI I
0.9 0 o..a 0.7 o..a o.s 0.4
:) 5 7 9
0 propykn- prop.,....J 0 PIOf'yIPfIA
Fig~re 3.10 Retention of phenol and benzyl alcohol on three bonded silicas as a function of pH.
70
PBA does not affect the retention of these molecules. On the hydro
philic phase, a drop in the retention at pH 9.2 for both the amines
and alcohols indicates that these molecules are retained at the PBA
and the ionization of this site has removed the site of interaction
for these probes. These molecules are retained through interaction
with the boron. The k' values are smaller for the hydrophilic than
the lipophilic phase indicating that the interaction with the PBA
molecule is weaker than the interaction at the surface. On the hydro
philic phase the molecules are unable to intercalate past the urea
functionalities to the silica surface.
Conclusions
The retention of these probe molecules containing only one
functionality can be used to understand the complex interactions of
larger molecules with more than one functional group.
Controlling Lipophilic Interactions
Lipophilic interactions were much stronger on the propyl
aminoPBA phase. These interactions are due to the availability of the
propyl linkage for interaction with the sample. For biochemical
interactions, increasing the organic modifier is not an acceptable
way to eliminate these interactions. While the addition of a small
amount of organic modifier was able to minimize these interactions for
the small molecule, the much larger biopolymers may have stronger
interactions with the surface. The advantage of the more hydrophilic
71
propylureaPBA phase is the minimization of these interactions without
excluding lipophilic molecules from the surface.
Controlling Electrostatic Interactions
The residual aminopropyl groups on the propylureaPBA surface
help control the anionic nature of the surface. The cationic amine
sites are capable of neutralizing the surface most likely with direct
interaction with the residual silanols or with the ionized PBA sites.
The ability of the residual amines on the lipophilic surface
also helps to assure that anions are not excluded from the surface.
While it is important that the retention of molecules be minimized to
restrict irreversible adsorption or nonspecific retention of mole
cules, molecules excluded from the surface will not be able to
covalently react with the functionality. The presence of the amines
keeps exclusion to a minimum.
Interaction of the molecule with the residual silanols is
always unwanted. The interactions with these sites is often very
strong and often irreversible. Since it is not possible to complete
ly react all of the residual silanols, and since most endcapping
reagents increase the lipophilicity of the resulting surface, develop
ing a surface that will restrict the molecule from the surface is
important. The hydrogen bonding of the urea group as well as the
highly structured water molecules imbibed in the lipophilic phase
restrict sample from the residual silanols and the lipophilic inter
actions of the propyl functionality.
72
Controlling the retention of molecules on the surface depends
on the ability to reproduce a surface exactly. Since there are fewer
steps and variables in the preparation of the propylaminoPBA, the
preparation of this phase is more consistent from batch to batch. In
addition, since the unreacted amine functionalities on the propylurea
phases interact with other functionalities on the phase, an increase
in the unreacted sites will have an even greater effect on the inter
actions, both primary and secondary.
CHAPTER 4
PHENYLBORONIC ACID
Introduction
Boronic acid and phenylboronic acid (PBA) readily and reversi
bly react with molecules containing polar functionalities. The
interaction of borate with sugars was first reported by Biot in 1842
(14). While a boric acid with four reactive sites is able to interact
with two sugar molecules, organoboronic acids, with three sites, can
only interact with a single sugar molecule (27). The organoboronic
acids are often used because of the simplicity of the one to one
interaction.
While diols and polyols are the most common ligands to PBA,
there are several types of polar functionalities capable of interac
tion with the boronates. 1,2-hydroxyacids (28,29) and 1,2-hydroxy
amines (30) as well as several other functionalities are expected to
bind to PBA. Examples of substances expected to react with PBA are
found in table 4.1.
Acid Base Chemistry
The interaction of the PBA with polar functionalities is
intrinsically dependent on the acid base chemistry of the PBA as well
as the acid base characteristics of the sample and its matrix.
73
Table 4.1 Molecules that interact with PBA.
Compound Class
Polyhydroxy
1 ,2 dihydroxy
1,3 hydroxy
Hydroxy acids
Diketo
Hydroxyamines
Mercaptoalcohols
Examples
Mannitol, Carbohydrates
Catechol, Tannis
Tris Pyridoxine
Lactate, Benzillic, Salicylic acids
Benzil
Aminophenol
2-mercapto 'benzylalcohol
74
75
Therefore, the pH of the solution can be used to control the selectiv
ity of the PBA, provided the effects of pH on the formation of the
covalent complex are understood.
Phenylboronic acid is a Lewis acid, accepting a hydroxyl group
as shown in figure 4.1 with a pKa of 8.86 (27,31) in an aqueous
solution. The pKa is alte-ed by substituent groups on the ring thus
the pKa of immobilized PBA will depend upon the immobilization proce
dure. In general, PBA is bound through an amine functionality in the
meta position. Maestas et al (24) report the pKa of PBA bound in this
way as 9.2. The increase in pKa is attributed to the meta amine
functionality. The addition of a nitro group on the benzene ring in
the ortho position will decrease the pKa of the boronate substantial
ly. Transfer RNA has been immobilized on ortho-nitro substituted
meta-aminophenylboronic acid at neutral pH (32). Residual amine
groups on a surface reduce the apparent pKa of immobilized PBA through
a surface buffering effect or direct interaction with a PBA (26).
Compounds Expected to React with PBA
Monofunctionalities such as amines and alcohols are reported
to interact with PBA(l). However, monofunctional interactions are
weaker than those that involve di- or trifunctionalities, because the
di or tri functionality will interact with two or three sites to
stabilize the interaction.
Difunctionalities, such as diols, must have the correct
configuration. They must be cis, coaxial or able to rotate into the
same plane in order to physically reach the PBA. In addition the
EQUIL1UIVU lON
~B .... OH+2H'O I
~3:'OH+ • "'OH
OH OH
REACTION
lQl R,@ O H '""H.,.... 0 e OH + +-- e 0 B:: H~"'H .B.# 'l._ R i "OH R HO\\~ A .t<L
. OM 0 \""1' I'
RH ELUTION
76
R
+ HO-f"", H H01""H
R
Figure 4.1 Equilibration of PBA to its tetrahedral form. Reaction with diols and the reverse reaction when the pH is dropped.
77
geometry of the PBA will determine the ability to bind different
molecules. At low pH, the PBA is in the trigonal planar neutral form
and will form charge transfer interactions. At high pH, the PBA is a
tetrahedral anion, and it is capable of undergoing condensation
reactions with dio1s and po1yols. Since the pH governs the conforma
tion and the conformation governs the reactivity, the pH of the mobile
phase will control the reactions occurring on an immobilized PBA.
The charge on the compound is also important. Molecules that
bind to PBA in the anion form may be excluded from PBA sorbents if the
compound of interest is also an anion. Cationic functionalities on
the compound of interest may stabilize the covalent interactions with
the anionic form through electrostatic interactions.
Dio1s. A great deal of attention has been paid to the
interaction of PBA with cis or l,2-dio1s, especially catecholamines,
nuc1eotides and t-RNA. These compounds form a covalent complex with
PBA under basic conditions, when the PBA is in the ionized or tetrahe
dral form. Under acidic conditions, the l,2-diols do not interact
with the PBA. Under these conditions, the PBA is in the trigonal form
with a 120 0 bond angle and the ring strain is believed to be too great
to form the complex (14).
l,3-dio1s interact with PBA in the same way as the l,2-diols;
however, the l,3-diol has more freedom and spends less time with its
hydroxy groups in the same plane. The decrease in the number of
molecules with the correct configuration weakens the interaction.
78
Po1yhydroxy1s. Po1yhydroxyl containing compounds interact
with PBA similarly to vicinal diols except that the possibility of a
tridentate interaction is possible for compounds with the correct
configuration. (14,27) This extra interaction further stabilizes the
complex. This type of interaction explains the high formation con
stants reported for molecules capable of tridentate interactions (14).
Molecules able to form tridentate complexes have higher formation
complexes than the bidentate compounds.
Hydroxyacids. The interactions of alpha-hydroxy aliphatic
acids or ortho-hydroxy aromatic acids with PBA have been studied by
several researchers (28,29). These interact at low pH to form a
complex but are excluded at high pH due to the electrostatic repul
sions. Recognition of these electrostatic repulsions is important in
the primary interactions of PBA. Lactic acid has also been shown to
interact with PBA in solutions of high ionic strength and pH of 2 and
6 (28).
Hydroxyamines. Amines are able to donate an electron pair to
the boron atom in its trigonal state (30). The bond formed between
nitrogen and boron is weaker than the bond between oxygen and boron.
The interaction of the nitrogen with boronic acid is further stabi
lized by the interaction of the hydroxy group with the PBA. At low
pH, the protons compete with the boron for the nitrogen, and at high
pH, the hydroxide competes with the nitrogen for the boron, causing
the strongest interactions at neutral pH. The interaction of amines
with boron is further stabilized by a vicinal hydroxy group that will
react with the boron in the same fashion as the diols.
79
Other Functiona1ities. The interaction of PBA with dike to
and triketo compounds with PBA has been reported (24). Understanding
the effects of pH on the retention of these molecules and other
related compounds will allow for a better understanding of the effects
of pH on biologically significant molecules.
Isolation of Compounds
The reaction of PBA with compounds containing polar function
a1ities has been studied using pH titration (28) and pH depression
(33) techniques, as well as, infrared (29) and Raman (34) spectro
scopic methods in solution. Complexation with cell walls has been
determined using fluorescent boronic acids (35). Pheny1boronic acid
solid supports have been used to isolate catecho1amines (36, 37,38),
nuc1eosides (39,40,41), tRNA (32,36,42,43), carbohydrates (41), and
enzymes (44). Automated systems using PBA have been developed (36,
40,45) for the isolation of compounds. A great deal of work on PBA
has been done to demonstrate its ability to isolate compounds, but few
studies to determine the factors controlling the interactions have
been carried out.
The association constants for PBA and several compounds have
been measured by spectroscopic methods in unbuffered solutions. As
mentioned, the pH dependence of complexation with PBA is important in
controlling the isolation and subsequent elution of compounds. A
study using UV spectroscopy to determine the effects of pH on the
complexation of PBA with different dihydroxy substituted naphthalenes
80
(46), shows that the retention of these molecules will reach a maximum
at a pH of 9-10 and that this maximum depends on the molecule.
While spectroscopic data on PBA in solution can yield a
great deal of information, the best method for determining conditions
for efficient separations is the use of diagnostic chromatography.
Determining the factors which affect retention of molecules on immobi
lized PBA using solvents suitable for chromatography will be more
informative for developing chromatographic methods.
Experimental
The strength of interactions of immobilized PBA was investi
gated in two different experiments. Solid phase extraction experi
ments and diagnostic chromatography were used to determine the rela
tive strengths of interactions of several probe molecules from a pH of
3 to a pH of 9.
Solid Phase Extraction
SPE experiments as described in chapter two were carried out
using application buffers covering a wide range of pH. These buffers
are listed in table 4.2. The amount of sample eluted in 3 milliliters
(k'-30) was measured using HPLC. The HPLC analysis separated the
sample from the application buffer allowing for better reproducibility
and accuracy.
Diagnostic Chromatography
The retention of several probe molecules over a range of pH
was measured by liquid chromatography as described in chapter two.
Table 4.2
pH
2.0
4.1
6.1
9.0
Buffers for solid phase extraction
Components
30 mM Dich1oroacetic Acid, 17 mM NaOH
8.7 mM Acetic Acid, 1.8 mM Sodium Acetate
4.35 mM Acetic Acid, 94.4 mM Sodium Acetate
10 mM Ammonium Acetate, 9.9 mM NaOH
81
82
The different modification methods for PBA immobilized on silica
(figure 4.2) described in chapter two were used, and the effects of
modification as well as the effects of pH on the covalent interaction
were determined.
Results and Discussion
Retention Dependence on pH
Diol. A comparison of the retention of catechol (figure 4.3)
on a surface containing no PBA with surfaces with immobilized PBA
demonstrates the retention of catechol through covalent interactions.
At pH above 7, the retention of catechol is much less on the phase
containing no PBA, indicating that a covalent bond between catechol
and PBA is occurring on the PBA phases. The difference in the reten
tion is too large to be explained by retention through non-specific
interactions. The increase in retention as the pH increases is
consistent with the spectroscopic data (4.10). The increase in pH
increases number of PBA functionalities in the ionized or tetrahedral
form; therefore, the catechol is interacting with the ionized PBA, but
not with the trigonal planer molecule. The lack of interaction with
the trigonal planar form is attributed to the ring strain from the
120 0 bond angle of this form.
The interaction of PBA with catechol depends on the modifica
tion methods. Catechol interaction is stronger on the hydrophilic
urea phase (figure 4.2b) than on the lipophilic propyl phase (figure
4.2a) in the pH range from 4.4 to 7. Equation 1.5 demonstrates that
the retention of molecules due to covalent bonds is proportional to
the number of reactive sites. The number of pheny1boronic sites on
;==<B«(Jd) 2 cfat
)2 '-(
f r ,H ~ HCH HCH tiCH HCH
\ \ I / HCH HCH HCH HCH / / / \ \
HCH HCH HCH HCH HCH \ / \ I /
HO-5i-0-5i-Cii HQ-Si-0-5i- Q-SI-OH I \ H / \
o '0 0 0 '\ " " \ " 5i 5i 5i 5i 5i 5i 5i 5i 5i /\/\/\/\/\/\1\/\/
o 0 0 0 0 0 000
,-((Jd) 2
\..(NH 0 H ':JL,.,pH \ 1/ r Hoe"oH C=O ----- HN- C .O.C
1 H \ \ HNH HIl NH ~IH
HC/H / \ \ HCH HCH. HOI
\ \ I / HCH HCH HCH HCH / / \ \ /
HCH HCH HCH HCH HCH \ 1 \ I /
HO-5i-0-5i-(- HQ-Si-0-51- Q-5i-OH / \ '\. / \ . ~ 0 I.i 0 0
\ " " " " 5i /51 5i 5i 51 5i 5i 5i ~l /\ \/\/\/\/\1\/\/-COOO 00 Q 00
Figure 4.2 Two PBA modified silica phases, a lipophilic phase (a) and a hydrophilic phase (b).
83
CATECHOL 100
90
eo
70
60
:.. 50
40
30
20
10
0 3 5 7 9
a propyl .... ll""PylunaPBlH
o propylPBA
-~-fc5\ . HO-fc5\
~ "OH ._)::::Y "B HO
HO
-N-\Q/ H 0JQJ / Be
HO/ '0 0
Figure 4.3 Retention of catechol on three modified silicas as a function of pH.
84
85
the surface is determined from the elemental analysis (chapter two);
the lipophilic phase contains 0.34 milliequivalents/gram and the
hydrophilic phase contains 0.48 milliequivalents/gram of PBA. The
capacity of the hydrophilic phase is 1.4 times that of the lipophilic
phase, but the capacity factor of the hydrophilic phase at pH 7 is 3
times the capacity factor of the lipophilic phase. The difference in
retention is not solely due to the difference in the number of PBA
groups bound to the surface.
Catechol interacts only with the PBA that is ionized. There
fore, the number of active sites is not the number of PBA function
alities on the surface, but the number of these sites that are ion
ized. The acid base equilibrium of any acid depends on its environ
ment and the non-specific interactions (chapter three) have shown that
the there is a significant difference in the environment of the PBA on
the differently modified surfaces. In addition, the presence of amine
functionalities on the surface have been shown to cause a decrease in
the pKa of PBA through direct interaction or through a surface buffer
ing effect (26). A significant number of amines are left unreacted on
the hydrophilic phase, while there are no amines in the lipophilic
phase that are capable of interaction with the PBA. The residual
amines on the surface are causing a decrease in the pKa of the PBA so
that more of the PBA on the hydrophilic surface are ionized at a
given mobile phase pH.
The retention of the 1,3 diol, 2-hydroxybenzyl alcohol (figure
4.4) shows the same trends as the retention of catechol. This mole
cule interacts with the tetrahedral form of PBA, and the
100
90
80
70
00
:.. 50
40
30
20
10
0 3
Cl propyl ....
-~-«S\ . ~,OH
8 , OH
2-HYDROXYBENZYL ALCOHOL
5
PfOPyl ... aPBr
HO-@ HOCH z
7 9
o propylPBA
-N-!c3\ ~ H ~I /0 0 Be
/ "-HO OCHz
Figure 4.4 Retention of 2-hydroxybenzyl alcohol as a function of pH.
86
difference in the ionization of PBA due to the modification of the
silica is apparent.
87
Comparing the capacity factor for catechol, a cis diol, with
the 1,3-diol it is apparent that the catechol interaction is occurs at
lower pH. At pH of 7 on the hydrophilic phase, the capacity factor of
the catechol is 3.7 times the capacity factor of 2-hydroxybenzyl
alcohol. The capacity factor of the 1,3-diol at pH of 8 is the same
as the capacity factor for catechol at a pH of 7. For the 1,3-diol to
have the same retention volume as the 1,2-diol, the pH, and therefore,
the number of ionized PBA sites is increased. Thus, the reaction
constant, K (equation 1.5), for the 1,3-diol is weaker under these
conditions. This weakness is due to the ability of the 1,3-diol to
rotate the hydroxy groups into more than one configuration. The cis
diol retains its hydroxyl groups in the correct geometry for reaction
with the PBA. The hydroxyl groups of the 1,3 diol are capable of
rotating into the correct geometry, but not all of the 1,3 diols will
have the correct configuration at anyone time.
The trends in the chromatographic data fit with the trends in
the solid phase extraction data for 2-hydroxybenzyl alcohol (figure
4.5), except that at a pH of 9, the diol should have been strongly
retained. The difference may be in the equilibration of the station
ary phase. In order to assure that the phase was equilibrated, it was
washed with 2 ml of O.02M NaOH. The continuous flow of the chromato
graphic system allows for easy equilibration of the phase, but with
SPE, care must be taken to assure that the phase is equilibrated.
Figure 4.5
i ~
~ ~
SdLWPHASEEXTRAcnON roo
00
00
70
60 b 50
40
30
W
~
0 2 4 6 8
~H
Figure 4.5 Retention of aminothiolphenol (a), 2-hydroxybenzyl alcohol (b) and 2-aminobenzyl alcohol (c) on commercially available PBA columns by solid phase extraction.
88
89
Hydroxyamines. Amines have been reported to interact with the
unfilled orbital on boronic acid in the trigonal planar form (figure
4.6 and 4.7). This interaction is stabilized by a hydroxy group that
is capable of interaction with the PBA to form a five or six membered
ring. Competition between protons and the PBA for the amine site and
competition of hydroxide ions and the amine for the boron cause this
interaction to be strongest at neutral pH. The retention data for
aminophenol (figure 4.6) and 2-aminobenzyl alcohol (figure 4.7)
demonstrate an increase in retention at neutral pH. However, this
retention is very low and these compounds could not be isolated by
covalent chromatography under these condition. In fact, the retention
of these molecules is lower than the retention of the monofunction
ali ties (phenol (figure 3.10) and aniline (figure 3.6». The SPE data
(figure 4.5) is consistent with the chromatography data, but the
retention of these probes at pH 6 is higher. This increase is due to
the buffer used in SPE at this pH which does not contain a competing
amine. The retention is still too low to be useful for covalent
chromatography.
The conditions chosen for the SPE and the chromatographic
experiments were not optimized for the retention of amines. The
ammonia in the buffer used for covalent chromatography is competing
with the amines for the boron interaction. Carboxylic acids also
complex with PBA. The ammonium acetate buffer may be competing for
the PBA sites.
The SPE retention of aminothiolphenol (figure 4.5) is much
greater than that of the hydroxyamines. This increase in retention is
90
AMINOPHENOl 1.8
L7
us L5
L4
L3
L2
U
:.. I
0.9
0..8
0.7
0,8
o.s 0.4
o.:l
Q.2
:J 5 7 9
c PfOIIvturea propwturoaPett Q 1W000vtPBA
+
-@,oH -@OH ©rN~ OH Sr B' ~ HO' 'OM
Figure 4.6 Retention of aminophenol as a function of pH.
91
2-AMINOBENZYL ALCOHOL. L9
La
L7
La
L5
L4
L3
:.. 1.2
L1
0.9
0.8
0.7
0.8
o.s 3 5 7 9
c:J propyluree o propyIPBA
Figure 4.7 Retention of 2-aminobenzyl alcohol as a function of pH.
92
due in part to the lipophilic nature of this molecule. This molecule
is retained through lipophilic interaction with the propyl chains.
Once this molecule is solvated by the propyl chains, the covalent
interaction occurs. The ability of this molecule to be well solvated
by the phase allows for the stabilization of the covalent bond. The
data demonstrate that this interaction predominates at neutral pH for
the same reasons as the aminophenol.
Mercaptoalcohols. The retention of 2-mercaptobenzyl alcohol
(figure 4.8) is different on all the phases so each phase will be
discussed separately. On the ureaPBA phase, the retention is similar
to the retention of l,3-diol at low pH (figure 4.4), but at a pH of
above 5.6, the diol is well retained and the mercaptoalcohol retention
decreases. This decrease is due to the ionization of the thiol and
the PBA to anions. The electrostatic repulsions cause the molecule to
be excluded from the stationary phase and no covalent bonds may form.
On the lipophilic phase, at this solvent strength the probe is
well retained through the non-specific interactions due to the
lipophilicity of the probe. The dispersion forces are not the only
interaction responsible for the retention on this phase, but these
interactions stabilize the formation of a complex as shown in figure
4.11. At high pH where the PBA is ionized as well as the thiol, the
probe is excluded from the phase due to electrostatic repulsion.
Salicyclic Acid and Related Compounds. The effect of pH on
the retention salicylic acid (figure 4.9) is the opposite of the
effect on diol compounds. For the salicylic acid, retention decreases
as the pH is increased in this case the covalent complex forms with
:..
100
go
00
70
00
50
40
:xJ
20
10
0 3
CJ propylur ••
2-MERCAPTOBENZYL ALCOHOL
5
propyUuPol"
7
-<9!~OH Be
Ha' 'OH
9
o propyIPBA
Figure 4.8 Retention of 2-mercaptobenzyl alcohol as a function of pH.
93
94
SALICYLIC ACID 100
90
eo
70
60
:.. 50
40
:JO
20
10
0 3 5 7 9
CJ propylun. PfOfJvUeel'afi 0 propylP9A
-~-©>/o~ -~-©> HO~ "OH
/B~ B e 0
0_
C HO O=C HO" 'OH II
I 0 OH
Figure 4.9 Retention of salicylic acid as a function of pH.
the trigonal planar form of PBA not with the ionized form. This is
consistent with the SPE data (figure 4.10) and with reported separa
tions of alpha-hydroxy aliphatic acids (28,29). Some salicylic acid
with a pKa of 3.0 is ionized throughout the entire pH range studied.
At low pH, the salicylic acid complexes with the PBA as shown in
figure 4.9. As the pH increases, the PBA ionizes and the this mole
cule is excluded from the surface due to electrostatic repulsion.
95
Comparing the retention of salicylic acid on the different
phases, a shift in the pKa is apparent. The PBA on the lipophilic
phase is ionizing at a higher pH than the hydrophilic phase. In
addition, the decrease in retention of salicylic acid occurs over a
wide pH range for the hydrophilic phase but is very sharp on the
lipophilic phase. The retention data for the hydrophilic phase
implies that the PBA ionizes over a wide range. This indicates that a
full range of PBA groups exist with the extremes being a PBA group
surrounded by arnines or interacting directly with an amine and a PBA
group completely removed from all amine functionalities and their
buffering effects. The decrease in retention on the lipophilic phase
is much sharper and occurs between 5.6 and 7. This pH is consistent
with the pH range for the ionization of silanols (47) which are shown
by the non-specific interactions (Chapter 3) to be available to the
sample. The ionization of the PBA occurs at a higher pH (24). The
electrostatic repulsions are occurring between the ionized surface
silanols and the salicylic acid and are excluding the probe from the
surface. Salicylic acid is unable to probe the ionization of the PBA
SOLID PHASE EXTRACTION
wo~-------------------------------------------------~
90
00 a 70
60
50
20
\0
o+-----.-----.-----~~~~-===~==:~~----~ 2 4 6 8
Cl SAUCYUC ACID pH
SAUCYLAMDE'
Figure 4.10 Salicylic acid (a) and salicylamide (b) retained on PBA as a function of pH.
96
on the hydrophobic phase, due to the exclusion of the probe from the
phase at the pH where PBA ionizes.
97
The interaction of salicylamide (figure 4.11) with PBA is much
weaker than the interaction of salicylic acid, but it follows the same
trend except that the amide is capable of interacting with the boron
through the oxygen or the nitrogen (48), as shown in figure 4.11. At
low pH, the interaction occurs through the keto group. As the pH
increases, the keto interaction decreases and the amine interaction
increases. The dip at pH 4.4 occurs because of the two types of
interactions. At high pH, hydroxide ions compete with both interac
tions and the molecule is not retained. The SPE retention demon
strates the same trends.
The pH dependence on the retention of thiosalicylic acid
(figure 4.12) shows different behavior on the different PBA phases.
The difference in retention is due to the differences in non-specific
interactions. Thiosalicylic interacts with the PBA as shown in figure
4.12. This interaction is weaker than the interaction of salicylic
acid. The propylPBA phase is lipophilic and the thiosalicylic acid
will intercalate into this phase at pH 3, above this the thiosalicylic
acid ionizes (49) and the lipophilic interactions decrease. At a pH of
5.6, the ionization of the silanols excludes the probe from the phase.
The hydrophilic phase excludes the lipophilic probe molecule
at low pH where the molecule is unionized. When the thiosalicylic
acid ionizes, the positively charged amine groups on the surface allow
for solvation and covalent interaction. As the pH increases, the PBA
ionizes and the thiosalicylic acid is no longer retained.
Cl
98
SALICYLAMIDE
7~-------------------------------------------
8
5
4
3
2
0 3 5 7 9
propyl .... propyl ... ePB1H
0 propylPBA
-~-©; o-@ -~-@/o-@ -~-@ S/ 0 ' "OH
/B", B0 / '" Hci 'OH
HO O::~ HO HzN-C
NHz 8
Figure 4.11 Retention of salicylamide as a function of pH on phenylboronic acid.
100
90
60
70
60
:.. 50
40
:lO
20
10
0 :l
0 propy" •••
THIOSALlCYLlC ACID
5 7 9
prOpy .... ePBIH
0 propylPBA
-~~/o-fc5\ /,\ );::Y
HO S-C
" o
:0-PJ s-c
" o
Figure 4.12 Retention of thiosa1icy1ic acid as a function of pH on pheny1boronic acid.
99
100
Diketone. Benzil interaction with PBA (Figure 4.13) is
dependent on the non-specific interactions. Benzil is a lipophilic
molecule, and the dispersive interactions available to this probe on
the lipophilic phase stabilize the interactions with the PBA (figure
4.13). At pH 9, the hydroxide ions compete for the site on the boron
and benzil is no longer retained. The lipophilic molecule is excluded
from the charged surface. On the hydrophilic phase at a very low pH
the phase has enough of a positive nature to exclude benzil. As the
pH increases the benzil is retained as shown in figure 4.20, but above
a pH of 5, the surface amines or hydroxide ions compete with the probe
for the Boronic acid and it is no longer retained.
The SPE retention of benzil (figure 4.14) is consistent with
the chromatography data. For these data, 20% acetonitrile was used
and the dispersion forces were weakened causing a decrease in the
retention.
Keto-carboxylic acid. The pH dependence of the retention of
benzillic acid on the lipophilic phase (figure 4.15) is consistent
with the SPE data and with salicylic acid. It is retained at low pH
but elutes when the residual silanols ionize due to electrostatic
repulsion. On the hydrophilic phase, the retention data are similar
to benzil. Benzillic acid is unretained at a low pH due to poor
solubility in the phase. At a pH of 4.4, the carboxylic acid is
ionized but the PBA is not. For this small pH range, the molecule is
well solvated by the surface and the covalent interaction occurs. At
higher pH, about 5.6, the PBA begins to ionize and the covalent
:..
Cl
100
90
80
70
80
50
40
:10
20
10
0 3 5
propyl .... +
BENZIL
7
propylunoaPBIH
0
-<Q(,OH Be
Ho' 'OH
9
Pl'opylPBA
Figure 4.13 Retention of benzi1 as a function of pH on pheny1boronic acid.
101
i ~ ~
SOLID PHASE EXTRACTION 100
90
80 a 70
80
50
40
:lO b 20
10
0 2 4 6 8
CJ BENlD. ~Zl.UCACID
Figure 4.14 Retention of benzil (a) and benzillic acid (b) on PBA as a function of pH.
102
:..
0
103
BENZILLIC ACID 100
90
60
70
60
50
40
30
20
10
0 3 5 7 9
propylur •• propylureaPoiH
0 propyiPBA
-~-<Qf./~-@ -Z-<Qf ~ ,OH Ho-~~ B0 / '\ 4'c H<1 'OH HO 0 bH C o~ be
Figure 4.15 Retention of benzi11ic acid as a function of pH on pheny1boronic acid.
interaction cannot occur. At higher pH, 7 and above, the electro
static repulsion excludes the molecule from the phase.
Effects of Modification on Retention
104
In comparison, the two PBA phases show a great deal of simi
larity in the capability of forming covalent interactions with
compounds containing polar functionalities. The differences in the
local environment do cause disparities in the retention values. These
differences are due to the effects of the non-specific interactions on
the covalent bond and the apparent shift in the pKa of PBA.
Secondary Interactions
The secondary interactions discussed in detail in chapter
three may support or resist the formation of the covalent bond. The
formation of the covalent bond requires the diffusion of the molecule
of interest to the surface and the solvation of the molecule of
interest into the stationary phase prior to the reaction. The solva
tion of a species of interest is more important for the reaction of
the ketones and thiol compounds than the other polar functionalities.
An understanding of the effects of the secondary interactions will
allow for the optimization of solvents for efficient separations.
Shift in pKa of PBA. The retention data for several probe
molecules indicate a shift in the pKa of PBA on the urea phase. While
the chromatographic data does not give an absolute pKa for either
phase, the shift in pKa is apparent. This shift is due to electronic
and solvation effects. The amine functionality in the meta position
of the PBA increase its pKa of the boronic acid from 8.6 to 9.2. The
105
urea functionality in the meta position would also increase the pKa of
PBA but not to the same extent as the amine functionality. The
solvation effects of the different modification methods will also
affect the pKa . It has been reported (26) that amine functionalities
on the surface will lower the pKa of immobilized PBA. These amines
either interact with the PBA directly or cause a surface buffering
effect. These data do not distinguish between the two models.
A related effect of amines on the PBA is the increase in the
heterogeneity of the PBA groups. While a decrease in the retention of
salicylic acid or the increase in retention of the diols is very sharp
on the lipophilic phase, it is not sharp on the hydrophilic phase.
This difference is due to the availability of the amines, to different
PBA groups. While some groups may be isolated from amines others are
very close to one or more causing a disparity in the interaction
energies of PBA.
Demonstration of Isolation of Compounds
Once the effects of pH and modification on retention are
understood, efficient methods for the isolation of specific compounds
or classes of compounds can be developed. The sample matrix and the
wash solvent should be optimized to assure strong interaction of the
compound of interest with the stationary phase, while removing contam
inants. The elution solvent should be chosen to assure that minimal
interaction is occurring.
To demonstrate the use of PBA sorbents, a 2 centimeter long
2mm id column was dry packed with 40 micron propylureaPBA bonded
106
silica and coupled to a 10 centimeter 10 micron octadecylsilyl modi
fied silica column in a traditional liquid chromatographic system.
The system was equilibrated with a retaining solvent and the sample
was injected. Fifty column volumes of the retaining solvent was
washed through the column. The PBA column was then removed from the
system. The system without the PBA column was equilibrated with an
elution solvent and the PBA column was replaced in the system.
Diols. Figure 4.16 shows the isolation of diols. Trace A is
is the 2-hydroxybenzyl alcohol injected through the system at pH 4.4.
The retention time is 1.5 minutes. Trace B is the same compound
injected at a pH of 9.2. At time 1, the PBA column is removed and
the system equilibrated to pH of 4.4. At time 2, the PBA is returned
to the system and the peak at 1.5 minutes was saved. The UV spectra
of this peak compared to a standard (figure 4.17) confirms that this
peak is 2-hydroxybenzyl alcohol.
Hydroxyacid. A similar experiment with salicylic acid (figure
4.17) demonstrates the retention using a pH of 4.4 and elution with a
pH of 9.2. The tailing in the elution peak demonstrates slow kinetics
of the equilibration of PBA to the ionized form.
Conclusions
While PBA has been used for the isolation of compounds, the
effects of pH on the retention and elution of compounds was not well
understood (50). Understanding the effects of pH on the retention of
molecules will allow for more efficient use of this phase for the
immobilization of compounds. Recognizing that many types of polar
107
e
Time (min)
Figure 4.16 Demonstration of the retention of 1,3-dio1s on PBA.
PBA coupled to a C18 column using a mobile phase of 5% acetonitrile and .1M ionic strength and a flow rate of 1 m1/min. A) Mobile phase pH 4.4 and a sample of 2-hydroxybenzy1 alcohol. B) Injection of 2-hydroxybenzy1 alcohol at pH 9.2, after 2.5 minutes the PBA column is removed from the system and the mobile phase pH is dropped to pH 4.4 at 5.5 minutes the PBA column is returned to the system.
Time (min)
Figure 4.17 Demonstration of the retention of hydroxyacids on PBA.
108
PBA coupled to C1S column with a mobile phase of 5% acetonitrile and an ionic strength 0.1 M. and a flow rate of 1 m1/min. A. Injection of salicylic acid using a mobile phase of pH 9.2. B. Injection of salicylic acid with a pH of 4.4. After 2.5 minutes the PBA column is removed and the system is equilibrated with pH 9.2 and the PBA column is returned to the system after 5.5 minutes.
109
functionalities are capable of binding to PBA will allow for the use
of this phase to isolate these molecules. This recognition will also
help one to realize what types of contaminants will be retained on the
surface.
tVhile the pH controls the retention of compounds on the
surface, the modification of the surface can alter the pH range where
different molecules are retained.
The compounds studied interacted with the immobilized PBA in a
manner consistent with the solution data. Diols interact through a
condensation reaction with two hydroxy groups on the boron. This
interaction only occurs when the hydroxy groups on the diol are cis or
coplanar and when the boronic acid is in a tetrahedral form because of
the bond angle of the hydroxy groups. Since the diols only interact
with this form, the interaction increases as the pH increases.
Amines interact with PBA as Lewis bases donating electrons to
the empty orbital of the boron. This interaction may be stabilized
through the reaction of a vicinal diol, but under the conditions used,
the interaction was weak. The amines interact only with the trigonal
planar form of the PBA and protons compete with the boron for the
amine causing this interaction to be strongest at neutral pH.
The interaction of 2-mercaptobenzyl alcohol with PBA on the
hydrophilic phase seemed to follow the interaction of alcohols. The
ionization of the thiol group, however, eliminated the interaction due
to the electrostatic repulsion. On the lipophilic phase, the lipo
philic compound was well solvated. The thiol interacts with the PBA
as a lewis base, and the alcohol can react with the hydroxy group
of the boron. This is a weak interaction, but it is stabilized
through the lipophilic interactions.
110
Salicylic acid interacts with the PBA through a carboxylic
acid and a hydroxy group. The carboxylic acid donates electrons to
the boronic acid in the trigonal planar form. The ionized PBA and the
carboxylic acid do not interact because of electrostatic repulsion.
As the pH increases and the PBA ionizes, the retention decreases.
Salicylamide interacts through a hydroxy group and the amide.
The amide has two possibilities of interaction, the keto group or the
amine group. The keto interaction predominates at pH 3.6, and the
amine interaction predominates at pH 5.6. At high pH, the hydroxide
ions compete with the amide for the boron site and the amide is not
retained.
Thiosalicylic acid, benzil and benzillic acid all interact
with PBA as Lewis bases, and none of these molecules contain a hydroxy
groups. The interaction of these molecules with PBA are dependent on
the modification method. Each of these compounds shows different
retention behavior for the two different modification methods. The
dispersive interactions are important in the solvation of these
molecules to stabilize the otherwise weak interactions with PBA.
CHAPTER 5
COVALENT CHROMATOGRAPHY OF THIOL CONTAINING COMPOUNDS
Sulfide and disulfide groups play an important role in the
structure and activity of biochemical compounds (51). The tertiary
structure of proteins depends in part on the formation of disulfide
bonds between cysteine residues, and the active sites in enzymes often
contain free thiols. In order to prepare synthetic biomolecules or to
understand their functions, an understanding of the thiol groups or
the disulfide linkage is needed (52). Several different reagents have
been developed to work with thiol chemistry (8,15,52,53,54,55). These
reagents all utilize the redox potentials of sulfide disulfide exc
hanges to study thiol chemistry. In general, there are three types of
reagents traditionally used with thiols in solution chemistry, the
reducing agent, assay compounds and blocking compounds. Each of
these types of solution chemistry could be simplified with the
development of separation methods.
Traditional Thiol Chemistry
Several disulfide compounds have been employed to assay thiols
by colorimetric or spectroscopic methods (15,52,53,56). The number of
thiol groups may be utilized to determine the concentration of a thiol
containing compound or the extent of denaturation of a biomolecule
III
112
which in turn gives insight into the structure of the molecule. Free
thiols in solution may react with the disulfide forming a mixed
disulfide and a molecule that contains a chromophore or electrophore.
For example, 2,2-dithiodipyridine may be reacted with a free thiol to
form a mixed disulfide and 2-thiopyridone as shown in figure 5.1 (15).
Thiopyridone is almost exclusively in the tautomeric thio form with
the hydrogen on the nitrogen. This causes a large difference in the
UV spectra of the thiopyridone and its corresponding disulfide,
allowing one to spectrometrically follow the reaction of dithiodipy
ridine with free thiols (18).
Much of the work involving thiols requires the use of a
reducing agent to reduce any disulfide bonds to thiols and to assure
that no reoxidation of the thiols occurs. The most commonly used
reducing agents are dithiothreitol (OTT), dithioerythritol (DTE) and
2-mercaptoethanol (ME) (57). The interaction of OTT with a disulfide
is shown in figure 5.1. The reducing agent is added in excess and
contains free thiols. The free thiols of the reducing agent will
interfere with the assay of the free thiols on the compound of inter
est unless they are removed or blocked. Arsenite has been used to
block the thiols of OTT and DTE in solution by binding to the dithiols
and not the monothiols (54). The rates of formation and breakdown of
the complexes are needed to determine the number of uncomplexed thiols
at equilibrium. This assay could be simplified by the use of immobi
lized arsonous acid to remove the dithiol from solution.
R-S-S-R • U - R-SH •
HO OH
J-\ . H R-SH
HO OH
113
D~-HO OH
@-S-S-@ 'ZR-SH - --'N~ 2 S"==/_" • R-S-S-R'
Figure 5.1 Reduction of disulfide bonds by dithiothreitol followed by reaction with 2,2-dithiodipyridine.
114
Due to the reactivity of thiols, these groups are often
blocked during reactions or storage. Reagents that reversibly react
with thiols are often used for this purpose.
Separations of Thiol Containing Compounds
It is often advantageous to separate the thiol containing
compounds from the non-thiol containing compounds. When working with
a mixture of peptides, the free thiols may need to be blocked. Prior
to the blocking reaction, it may be desirable to separate the thiol
containing peptides from the non-thiol containing peptides. Because
the amount of active enzyme is important, the separation of the active
from the denatured enzyme is of interest. When the active site of the
enzyme is a thiol, the active enzyme will contain an available and
active free thiol and the denatured enzyme will not. A separation of
thiol containing compounds from nonthiol containing compounds will
allow for the separation of the isolation of the active enzyme for
accurate measurements. When working with a reducing agent, some of
this reagent may have oxidized to the disulfide, separation of the
oxidized and reduced forms of DTT, DTE or ME will allow for accurate
measurements of the active reducing reagent (57). The use of a solid
adsorbent that reversibly reacts with the thiol containing compounds
to isolate these compounds on the surface will be demonstrated as a
quick, efficient method of separation.
In addition to these separations, the separation of a compound
of interest and the reducing reagent is often necessary or the excess
115
reducing agent will compete with the compound of interest for blocking
or assay reagents. When the reducing agent is extracted either by
solid phase chemistry or solution chemistry, care must be taken to
assure that the free thiol of the compound of interest is not
oxidized to the disulfide bond prior to the assay. Disulfides form
readily and the free thiols must be kept in a non-oxidizing
atmosphere. The environment of the sample is easily controlled when
using solid phase adsorbents by directly coupling the extraction
column to a second column or a reaction vessel.
Solid Phase Thiol Chemistry
The use of solid phase chemistry rather than solution chemis
try provides simpler sample handling, especially with smaller sample
size. In addition, covalent chromatography has been shown to be an
effective method for the separation of thiol containing compounds from
non-thiol containing compounds (58). Several types of phases have
been employed for the immobilization of thiol containing compounds
(17,57,58,59). Thiols can then be stored on these phases to prevent
oxidation or the thiol may be eluted for further analysis.
Mixed disulfide. The reversibility of sulfide/disulfide
reactions is utilized to reversibly immobilize compounds of interest
on a solid support (15,16,17,18,60). A mercaptopropyl derivatized
surface is activated by reaction with 2,2'-dithiodipyridine (figure
2.8). A thiol containing compound introduced to the phase will
displace the thiopyridone and be immobilized on the column (figure
5.2). A competing thiol may then be used to elute the compound. This
z 0
~5-S-o + }- S-S-R ~ ..
R-SH • '"~ 0
'" a c sD
,N H
z
~S-S-R }-S-S-R' 0 .: + R~SH ~ '\ .. ..
R-SH
Figure 5.2 Retention, detection and elution of thiols using a mixed disulfide phase.
116
phase is useful in the isolation of compounds through SPE or the
storage of compounds with active thiol groups.
117
While the isolation of compounds is an important area of
separations research, it is also important to develop selective and
inexpensive methods of detection. The displacement of the thiopy
ridone can also be used to improve the sensitivity of detection of
thiol containing compounds with a simple fixed wavelength ultraviolet
flow through detector. Extra column volume and the kinetics of the
displacement reaction cause wider peaks, but the trade off between
wider peaks and an increase in sensitivity is often acceptable.
Phenylmercurial. A second phase which is commonly used for
the separation of thiol and non-thiol containing compounds is a
phenylmercurial phase (figure 2.10). Mercurials are among the most
reactive and selective of the thiol reagents. Immobilized organomer
curials interact with thiol containing compounds, immobilizing the
thiol compound on the surface (figure 5.3) allowing for an effective
method to separate thiol containing compounds from non-thiol contain
ing compounds (61). A competing thiol or mercuric chloride can be
used to elute the compound of interest. An excellent review of
mercurial chemistry is available (20).
Organomercurials interact with thiols similar to mercuric
chloride (HgC12) or inorganic mercury through a bond with covalent and
ionic character. The interaction is generally somewhat weaker for the
organomercurials but may be stabilized through dispersive interac
tions. Inorganic mercury interacts with four ligands, but only two
are bound tightly. The other two are bound weakly due possibly to a
I }-H9-S-R + R'-SH '\' }-H9-S-R'
R-SH
ll8
Figure 5.3 Retention and elution of thiols on an immobilized organomercurial.
119
change in bond configuration (20). Organomercurials are capable of
binding to three ligands but only one is bound tightly. Under the
conditions of covalent chromatography were there is an excess of
mercury sites, only one thiol will bind with the surface. The ligands
bound to the immobilized organomercurial during its use will depend on
the pH, the buffer components, the organic modifier and any other
molecules present in the mobile phase and their concentration. The
association constants for methyl mercuric ion with several ions is
given in table 5.1. The thiol compound is capable of displacing any
ligand bound to the mercury so the buffer choice and composition will
not affect the retention of thiols but it will affect nonspecific
interactions.
Organomercurials interact with a thiol functionality and are
selective for this group, not for the compound; however, the chemical
nature of the compound as well as the immobilized reagent will affect
the optimal conditions for the interaction (20). At high pH, the
interaction of mercury with thiols is expected to improve due to the
electrostatic forces resulting from the ionization of the thiol. The
electrostatic attraction between the anionic thiol group and the
cationic mercury would stabilize the formation of a covalent bond.
This is not always the case because of competition from hydroxide ions
and the increase in pH will affect the conformation of a protein
making the thiols less accessible. The effects of pH will be differ
ent for every compound and the optimization of the phase for individ
ual compounds may be necessary. However, optimum interaction usually
occurs in the pH range between 4 and 8.
Table 5.1
Ligand
C1
OH
Acetate
Ammonia
Cysteine
Dissociation Constants for Complexes with Methy1mercuric Acid
pK
5.4
9.5
3.6
8.4
15.7
120
121
Immobilized organomercurials have been used in the study of DNA
replication through the separation of newly synthesized DNA and RNA
from the parental strands (19,6~,63,64,65) and separations of syn
thetic peptides (58) and proteins (66,67). It has also been used for
the isolation of compounds from natural products (68). The separation
of active enzymes from inactive enzymes (5.24) is possible on this
phase if the inactivity is due to an oxidized thiol from the active
site.
Pheny1arsonous Acid. Phenylarsonous acid (PAA) is the hydrat
ed form of phenylarseneoxide also called phenylarsine oxide or
arsenosobenzene (figure 5.4). In aqueous solutions, the hydrated form
is dominant (19). Organoarsonous acids are capable of interacting
with thiols to form an arsenic thiol complex and water. These mole
cules are capable of interaction with both mono and dithiol compounds.
Several studies have seen that the interaction of monothiols with PAA
are weaker than the interactions of dithiols (69). This is due to the
ring formed by the dithio1 complex. The ring structure stabilizes the
complex. The stability of the ring depends on the size of the ring,
with the most stable rings having 5 or 6 atoms and the least stable
having 7. With rings larger than 7, the interaction is stronger
because the disruption of the intramolecular interactions of the
dithiol with rings larger than 7, the disruption of the intramolecular
interactions decreases as the ring size increases (70). While the 7
membered ring may be the least stable under certain conditions, this
ring structure is still stronger than the interaction with monothiol
compounds.
A
)OJ A _all I 0- s .... OH HS-C-
B + I
HS-C-HN I
~
)OJ A -DH C 0- s"'OH + 2 R-SH
HN
~
~ ~
rAtA ,..OH N s,{)11
HN
~
I )OJ fl-C-o -AS,S_~_ HN
~
,0- ,5-" o AS'S_R f--~ HN
~
122
+2 li20
+2 H 0 2
Figure 5.4 A. Hydration of arsine oxide to arsonous acid. B. Interaction of immobilized arsonous acid with dithiol compounds to form a ring structure. C. Interaction with monothiols.
123
A study of arsonous acid immobilized on an agarose support (21)
demonstrated the selectivity of immobilized arsine oxide for dithiols
over monothiols. In this study, the monothiols, cysteine (cys) and
2-mercaptoethanol were eluted at neutral or acid pH but were retained
with O.OIM NaOH. Dihydrolipoic acid (DHLA) , a dithiol, was retained
using 0.01 M NaOH but eluted at 0.2 M NaOH. This study showed weaker
interactions of DTT and DTE than DHLA, because the DHLA forms a 5
membered ring while DTT and DTE form 7 membered rings. In order to
use this phase for the extraction of DTT and DTE the optimization of
conditions for retention will be important.
This phase will allow for the extraction of a dithiol from a
solution. With the appropriate conditions, this phase will extract
dithiol reducing reagents from a solution containing monothiols with
minimal retention of monothiols. When the reducing agent is extract
ed, either by solid phase chemistry or solution chemistry, care must
be taken to assure that the free thiol of the compound of interest is
not oxidized to the disulfide bond prior to the assay. Disulfides
form readily, and the free thiols must be kept in a non-oxidizing
atmosphere. The environment of the sample is easily controlled when
using solid phase adsorbents in a column that is directly coupled to a
second column which may contain a sorbent for the immobilization of
the compound through the free thiol. Coupling the immobilized
arsonous acid column to a mixed disulfide column allows for the
extraction of the dithiol reducing agent followed by the
124
quantification of free thiols in the sample through the detection of
the displaced thiopyridone. In addition, the thiol containing com
pounds may be isolated on this phase and eluted with a competing
thiol. This reduction in sample handling can increase the ease and
efficiency of the quantification of thiols.
The immobilization of thiol reagents will allow for the
isolation, detection or storage of thiol containing compounds with an
ease of sample handling, small sample volumes and speed. In order to
fully exploit this chemistry, the effects of immobilization on selec
tivity and reactivity must be understood. In addition, the efficiency
and selectivity of the immobilized reagents may be controlled through
the mobile phase. Diagnostic chromatography was employed to determine
the selectivity and the ability to control the selectivity of the
immobilized thiol reagents discussed in this chapter.
Experimental
The capacity of the phases as measured by elemental analysis
and described in chapter 2 is not necessarily the capacity available
to the sample, because it may be sterically restricted from some or
even all of the active sites. However, it is useful to compare the
number of sites bound to the surface and the number available to the
sample. The capacity of the mixed disulfide phase available to
cysteine was measured using frontal elution chromatography. A mobile
phase containing cysteine was used to completely wash the thiopyridone
from the phase. The effluent was captured in a volumetric flask and
the concentration of thiopyridone was measured using UV spectroscopy.
125
The capacity of the phenyl mercurial phase was measured using repeated
injections of cysteine. When the cysteine is eluted, the column was
considered overloaded and the capacity of the phase was determined
from the number of injections.
Diagnostic chromatography as described in chapter two was
performed to measure the retention of several probe molecules on the
column to determine the extent of non-specific interactions and their
contributions to the retention of thiol compounds.
The detection of the thiol compounds was either through direct
measurement of absorption at 254 nm or through the use of the mixed
disulfide phase as a reactor bed. The use of this phase allows for an
increase in sensitivity and therefore the use of lower concentrations.
Results & Discussion
Disulfide Phase
Studies of the mixed disulfide phase (figure 5.2) involving
the determination of peak height vs concentration show that the
amount of thiopyridone eluted from the surface is proportional to the
amount of cysteine or DTT immobilized over a small concentration range
(figure 5.5). This phase may be used to quantify the amount of thiols
injected into the column provided it has been carefully standardized.
The capacity of this phase for immobilizing cysteine was
measured by frontal elution chromatography. A solution of cysteine in
50% methanol/ 50% water was washed through a column which contained 71
milligrams of the mixed disulfide phase. UV spectroscopy was used to
126
CONCENTRATION OF CYSTIENE VERSUS PEAK AREA ON THE YXED 0ISUI.I'1De: PtfASE
28
ae a 24
22
20
• I •
M ~ 12 ~
10
8
a .. 2
J 0 0 2 3 ..
CONCENTRA 110H ImMI
CONCENTRA TION VERSUS PEAK HEIGHT AND AREA FOR on ON IIIXED DISlAJ'lOE: PtfASE
24
22 b
20
IG
IG 1/1 .... ; M ... ~ 12 e: I 10
/ /
: ~ / /
/ / .. /
/ 2
0 0 ..
Figure 5.5 Use of the mixed disulfide phase for the quantification of cysteine (a) and dithiothreitol (b).
127
determine the concentration of thiopyridone in the effluent. It was
determined that 38 micromoles were eluted from the system, which
corresponds to 0.5 milliequivalents per gram. The initial concentra
tion of propylmercaptan was 0.9 milliequivalents/gram. About
60% of the original mercaptopropyl groups were accessible to the
cysteine. This is consistent with the elemental analysis. Cysteine
is not expected to be excluded from the active sites, because it is
smaller than the activation molecule. The available capacity will be
more important as larger molecules are used. The larger molecules may
be sterically or otherwise restricted from the active site and there
fore unable to react. In those cases, the modification of the silica
would need to include a longer spacer arm that could reach to the free
thiol on the compound of interest.
Determination of the non-specific interactions on this phase
are needed in order to develop effective isolation and detection
procedures. The retention of benzene, phenol and benzoic acid at a pH
of 4 show typical reverse phase retention (figure 5.6). As the
methanol concentration is increased, the retention of these probes
decreases. These molecules are most likely interacting with the
propyl linkages and the mobile phase associated with these chains (9).
As the ionic strength changes (figure 5.6), retention for these
molecules is unaffected, indicating that these molecules are not
retained through electrostatic interactions. The retention of aniline
at a pH of 4 demonstrates electrostatic interactions. The retention
is unaffected by a change in methanol (figure 5.6) but decreases as
FORI·1A TE BUFF ER pH 4
A
2.5
1 • 5
. 5
5 25 4S
B
~ ANILINE
2.5 1!l8ENZENE
~PHENOL
2.0
..:.: 1.5
1.0
:z!s:
2." 3." Figu~e 5.6 Secondary interactions on the mixed disulfide phase
as a functio~ of % methanol (A) and -log ionic strength (B).
128
129
the ionic strength is increased (figure 5.6). At pH 6, aniline
retention decreases as the methanol concentration is increased (figure
5.7) which is consistent, because at pH 6, the aniline is not charged
and electrostatic interactions are not responsible for its retention.
At pH 4, benzoic acid is not charged and demonstrates typical reverse
phase interactions. When the pH is increased to pH 6, the benzoic
acid is charged and elutes near the void volume and the retention is
unaffected by a change in methanol. These data indicate a strong
cation exchange site on the surface. Since the charge exists at a pH
of 4, it is not likely residual silanols, since these ionize at pH 6
and above (47). The negative charge on this surface is attributed to
the oxidation of the free thiols to a sulfonic acid.
In order to demonstrate how the secondary interactions might
change as the surface is exhausted, the thiopyridone was washed from
the surface using 2-mercaptoethanol (figure 5.8). The lipophilic
interactions are not significantly affected as demonstrated by the
retention of benzene and phenol, but the retention of aniline increas
es (figure 5.9). The retention of aniline is dependent on ionic
strength but not on methanol concentration, demonstrating electrosta
tic interactions between aniline and negative surface charge. The
increase in retention is due to an increase in cation exchange capac
ity. 2-mercaptoethanol reduces the disulfide on the surface to leave
a free thiol as shown in figure 5.10. When the disulfide bond is
cleaved, the free thiols then oxidized to sulfonate groups, increasing
the cation excha.nge capability of the surface.
Figure 5.7
a
4.9
3.5
3.0 .:.::
2.5
2.0
5
3." b
2.5
2."
1.5 .:.::
1.9
.5
5
Retention of aniline
FORI·1A TE BUFF ER
ANILINE
<!> pH = 4
I!l = 6
15 25 35 45
BENZOIC ACID
<!> pH = 4
[!] p II 6
[!I
15 25 35 45
(a) and benzoic acid (b) as a function of % methanol on the mixed disulfide phase at pH 4 and 6.
130
-s-@ + 2 HOCH 2CH 2 SH
+
+
Figure 5.8 Reaction of 2-mercaptoethanol with the mixed disulfide phase.
131
FORMATE BUFFER pH = 4 MERCAPTOPYRIDIHE REMOVED FROM SURFACE
a
7.0
4.111
1.111
b
8.
5.5
3.0
.s
5 15
~ ANILINE
(!l8ENZENE
~PHENOL
25 35 45.13
,.W 2.5 3.0 3.5 4.111 Figure 5.9 Retention as a function of % methanol (a) and - log
ionic strength (b) on an exhausted mixed disulfide phase.
132
133
When using the mixed disulfide phase, care must be taken to
assure that all mobile phases are carefully deaerated to assure that
all oxygen is removed. Secondly, it is important to keep this phase
in a non oxidizing atmosphere during storage and preparation. The
oxidation of a thiol to a disulfide is reversible but the oxidation a
sulfonate is not. Once the thiols have oxidized to sulfonates,
regeneration of the phase is impossible.
Organomercurial
The secondary interactions of the organomercurial phase were
studied at pH 5 and 7 with a 4lmM ammonium acetate buffer and 5%
acetonitrile. The capacity factors of several probe molecules are
shown in figure 5.10. The ligand strongly attached to the mercury may
be the acetate or the ammonia from the buffer or hydroxide ions.
Assuming that the dissociation constants for methylmercury (table 5.1)
are similar to those for phenylmercury (20), the extent of complex
ation can be determined. Only the deprotonated form of the ammonia is
capable of complexation with the mercury, but the complexation is
strong, and even at pH 5 when only a negligible amount of ammonia is
deprotonated, the ammonia complex exists in the same concentration
range as the acetate complex. The concentration of unprotonated
ammonium is about 4 orders of magnitude lower than the acetate but the
dissociation constant is four orders of magnitude lower. When the pH
is increased to 7, the ammonia concentration increases by a factor of
100 and this is the dominant ligand complexed to mercury.
134
Mercurial
~ ~------------------------------------------------------~
9
o
7
:.. S
4
3
2
PAA BSA
IZZI pHS
Figure 5.10 Non-specific interactions of organomercurial bonded silica using 5% acntonitrile and 95 % ammonium acetate buffer with c:'.n ionic strength of 0.1 M.
135
The acetate complex with mercury is neutral. The negative
acetate neutralizes the positive mercury. The ammonia is a neutral
compound and unable to neutralize the cation. At pH 5, some of the
mercury sites are complexed with acetate, but as the pH is increased
to 7, the mercury complexes with ammonia and the acetate sites are
negligible. Therefore, the anion exchange capability of the surface
increases.
The increase in anion exchange capacity of the phase with an
increase in pH is demonstrated by the increase in retention of
benzene sulfonic acid at pH 7 (figure 5.10). One expects the anion
exchange capability of the surface to decrease as the pH increases,
and this is the case for the phases discussed in chapter 3. The
retention of the rest of the probes does not change as drastically;
however, the decrease in retention of benzene and toluene at pH 7 is
consistent with an increase in positive surface charges.
The retention of these probe molecules under these conditions
are, in general, within the optimum range for covalent chromatography.
The molecules are not excluded from the phase and they are not strong
ly retained. The exceptions are the ionic species. The cation,
phenyltrimethylammonium, is excluded from the column and the anion,
benzenesulfonic acid, at pH 7 is retained for almost 10 column vol
umes. An increase in the ionic strength will reduce the electrostatic
interactions and eliminate the excessive retention of the anion and
the exclusion of the cation.
136
The positive nature of the surface is an advantage in retain
ing thiol containing compounds. The positive charge on the surface
will stabilize the retention of ionized thiols. Cysteine was well
retained on the mercury phase in a wide variety of conditions.
Cysteine was retained at a k' of over 100 on this phase with aceto
nitrile concentrations up to 100% and with ionic strengths from O.lM
to lmM. A great advantage of this phase is the versatility of the
mobile phase, allowing for a wide range of sample matrixes without a
loss of efficiency.
The use of a phenylmercuria1 column for the retention, isola
tion and subsequent elution of several thiol containing compounds has
been demonstrated. The strong retention of cysteine on this column
demonstrates its possibility for this use. Oxytocin (figure 5.11) was
dissolved in acetonitrile with a ten fold excess of DTT (figure 5.1).
Using a octadecy1si1ane 10 centimeter 2 millimeter inner diameter
column in a typical chromatographic experiment the oxytocin was eluted
in 1 minute (figure 5.11, Trace A). When an organomercurial column is
placed in front of the column the octadecylsilane column (figure 5.11
Trace B), the oxytocin is not eluted after 5 minutes. At 5 minutes,
the mercurial column is removed and the system is equilibrated with a
mobile phase of 100% acetonitrile and 0.02 M cysteine. The mercurial
column was replaced in the system, and a peak is eluted 1.2 minutes.
When using a mobile phase with no cysteine, the oxytocin interacts
with the mercury forming a covalent bond with at least one of the free
thiols from the cysteine residues. When a mobile phase with cysteine
137
OXYTOCIN
H_Cys_Tyr_Ile_Gln_ASn_Cys-Pro-Leu-Gly-NH2
B
A ---
t t t 1 2 3
Figure 5.11 Oxytocin retained and eluted on an immobilized organomercurial.
A. Chromatogram of oxytocin on CIS with an acetonitrile mobile phase and a flow rate of 1 ml/min. B. Chromatogram with the phenylmercurial column and the same conditions. At 2 the mercurial column was removed and the system was equilibrated with .02 M cysteine in acetonitrile. At 3 the mercurial column is replaced in the system.
is used the cysteine displaces the oxytocin through mass action,
eluting it from the column.
138
The DTT used to reduce the disulfide bridges does not appear
in the chromatogram because the absorption of DTT at 254 nm is too low
to detect it. In most cases, it is necessary to remove the DTT prior
to isolation on the organomercurial column. Removal of the DTT would
assure that the reducing agent does not overload the mercurial column
A second demonstration of the usefulness of an immobilized
organomercurial column for the isolation of thio1 containing compounds
is shown in figure 5.12. The same experimental procedure was used
except the sample was 2,2-dithiodipyridine reduced with DTT and the
mobile phase was 5% acetonitrile and 95% 20 mM ammonium acetate
buffer. The elution mobile phase contains 0.02 M cysteine. Trace A
is an unreduced dithiodipyridine which is not retained on the mercury
column. This compound elutes in the void volume. Trace B is the
reduced sample which is retained on the column and then eluted by the
cysteine mobile phase.
Pheny1arsonous Acid
The non-specific interactions of the pheny1arsonous acid phase
were probed at pH 5 and 7 (figure 5.13) with a mobile phase of 41mM
ammonium acetate buffer and 5% acetonitrile. The effects of pH on
retention of the probe molecules is consistent with the PBA phases
(chapter 3). As the pH increases, the retention of the cations
increase and the retention of anions decrease, demonstrating fewer
A
1
B
10
2 3
20 Time (min)
Figure 5.12 Automated system to using organomercurial column coupled to a CIS column with a mobile phase of 5% acetonitrile 95% 20 roM ammonium acetate and a flow rate of lml/min.
139
At 1 the sample is injected. At 2 the mercurial column is removed and the system is equilibrated with the a .02 M solution of cysteine in the original mobile phase. At 3 the mercurial column is replaced in the system. Trace A is unreduced dithiodipyridine and trace B is dithiodipyridine in an excess of OTT.
3.2
3
2.8
2.8
2..4
2.2
2
La
1.6
L4
L2
o.a 0.8
0.4
a.z 0
11;(11
Figure 5.13 Nonspecific interactions on phenylarsono\ls acid bonded silica.
140
141
positive sites or an increase in negative sites on the surface as the
pH increases.
The non-specific interactions are within the optimum range for
covalent chromatography. However, an increase in ionic strength to
reduce the exclusion of the cation at pH 5 may be necessary to retain
cations.
The retention of cysteine as a function of acetonitrile
concentration was measured (figure 5.14). As the acetonitrile concen
tration increases, the zwitterionic cysteine is better solvated
by the hydrophilic urea phase and the retention increases. To mini
mize the interaction of cysteine with this phase, 5% acetonitrile was
chosen as the concentration of organic modifier.
In order to demonstrate the ability for this phase to separate
mono and dithiols, a sample of DTT and cysteine was used. The concen
tration of DTT was twice the concentration of cysteine. The results
of chromatographic separations using an arsonous acid column coupled
to a C1S column is shown in figure 5.15. Trace A is the sample using
a propylurea column coupled to an octadecylsilane column. The first
peak at 2.1 minutes is the cysteine the second peak at 4.5 minutes is
the DTT. In trace B, the urea column is replaced with a phenylar
sonous acid column. The cysteine peak is eluted at 3.5 minutes and
the DTT peak is not eluted after 20 minutes. The arsonous acid column
is effective at separating mono and dithiols.
The poor peak shape is partly due to the use of a mixed
disulfide column as a means of detection. A small packed bed was
j j c . -:
142
Retention versus Organic Modifier
4.8
4.8
4.4
4.2
4
3.8
3.8
3.4
3.2
3
2.8
2.8
2.4
2.2 5 10 15
" Acstonitil
Figure 5.14 Retention of cysteine on immobilized arsonous acid as a function of solvent strength.
OTT
ffi COLUMN I Ii CIS
A COLUMN I BLANK B COLUMN I PHENYLARSENEOX IDE
TimeCmin)
143
~ DETECTION I
Figure 5.15 Use of arsonous acid bonded silica for the separation of dithiols (OTT) and monothiols (cysteine) detected by the mixed disulfide phase. A. Without arsonous acid column. B. With arsonous acid column.
144
placed in front of the 254 fixed wavelength detector to allow for
greater sensitivity in detecting these molecules. This detection does
cause some band broadening. This column, the urea column and the
arsonous acid column are all dry packed beds of 40 micron particles
that also add to band broadening.
Extraction of the Reducing Agent
Phenylarsonous acid has demonstrated its ability to separate
dithiol containing compounds from monothiol containing compounds.
This is an effective way to isolate the compound of interest from the
reducing agent provided that one is a dithiol and the other is a
monothiol. In the case of DTT, the arsonous acid only interacts with
the reduced form. The cyclized DTT will not be retained. The cycliz
ed form will not interact with the mercuric or the mixed disulfide
phases, so in most cases the presence of this form of DTT will not
matter.
If the reducing agent has another functionality that is
capable of forming a reversible covalent bond, then the extraction of
the agent may be accomplished through this this functionality. DTT
contains a diol which is capable of reaction with PBA in either its
oxidized or reduced form. SPE studies to immobilize DTT on PBA
however proved difficult. A 100 milligram Bond Elut (Analytichem Int)
column with a capacity of 0.49 millieqivalents/gram was conditioned 2
milliliters of acetonitrile, 2 milliliters of water and 2 milliliters
of pH 12 buffer, followed by 2 milliliters of 20% acetonitrile 80% pH
145
9 ammonium acetate buffer (84mM). Two micromoles of DTT were washed
through the column with two milliliters of the final solution. The
effluent was saved and measured by liquid chromatography with a solid
phase detection using a mixed disulfide bed coupled to a 254 fixed
wavelength detector. Only 50% of the sample or less was retained on
the column. Only when the ionic strength of the wash buffer is raised
to 2 M NaCl is 97% of the sample retained.
The difficulty in retaining DTT on the PBA phase is due to the
exclusion of DTT from the surface. PBA only interacts with diols when
it is in a tetrahedral ionized form. DTT with pKa values of 7.8 and
8.4 for the two thiol groups, is ionized at the pH where it would form
a covalent complex with PBA. The electrostatic repulsion decreases
the ability of these molecules to react. At high ionic strength, the
DTT is no longer excluded from the column and the retention increases.
Unfortunately, 2 M NaCl is not an acceptable mobile phase for a
liquid chromatograph due to experimental limitations. In order to
automate a system for this extraction, it is important that the mobile
phase be compatible with pumping systems.
In comparison, the use of immobilized PBA to extract DTT will
not be as rugged nor as versatile as the use of immobilized arsonous
acid. The difficulty in using PBA is attributed to electrostatic
repulsion between the PBA and the DTT. The non-specific interactions
occurring on a phase are very important in the development of solid
phase extractions as well as any solid phase chemistry.
146 Conclusions
There thiol reagents were immobilized on a solid support with
no apparent change in the selectivity or reactivity from solution
chemistry. The advantage of solid phase chemistry is the ability to
immobilize these molecules on a solid surface for ease in separation
and sample handling and storage. The effects of the mobile phase
composition on the selectivity of the immobilized reagents were
determined for more efficient use of these phases.
The mixed disulfide phase may be used for the detection of
thiols as in solution chemistry (15) or for isolation or storage of
thiol containing compounds. One limitation of this phase is the care
needed to assure that the thiols on the surface do not oxidize to
sulfonate groups. Unlike the oxidation to disulfide bonds, the
oxidation to sulfonates is not reversible and the phase cannot be
regenerated. The non-specific retention of cations at the sulfonate
sites can be minimized through the use of high ionic strength mobile
phases. While the mobile phase controls the non-specific interaction,
the covalent interaction forms under a wide variety of conditions
from 50% methanol to 5% acetonitrile, and within the pH range of 4 to
9. This phase is compatible with a wide variety of sample matrixes
and will be useful for detection in a variety of applications.
The organomercurial phase is useful for the isolation and
storage of thiol containing compounds. This phase demonstrates
compatibility with a wide variety of mobile phases and limited
secondary interactions, making this a very versatile and easy to use
147
phase. The ability for mercury to complex with so many different
ligands will not affect is reaction with thio1s since these are
strongly retained but will affect the secondary interactions. Changes
in the mobile phase may cause unexpected changes in the non-specific
interactions.
The immobilized arsine oxide is useful for the extraction of
dithiol compounds from a solution of monothiol compounds. Tnis
extraction will simplify much of thiol chemistry since the extraction
may be performed just prior to the assay, immobilization, or storage
of the monothiol in controlled conditions. This simplifies sample
handling and eliminates the reoxidation of the thiols to a disulfide.
CHAPTER SIX
CONCLUSIONS AND FUTURE WORKS
Reversible covalent bonds between an immobilized reagent and
a compound of interest may be exploited for a wide variety of uses,
including the isolation of compounds through covalent or affinity
chromatography, solid phase synthesis or solid phase reactor beds. In
all areas of solid phase chemistry, the efficiency may be improved
through the use of rigid inert supports and an understanding of the
factors which affect the selectivity and reactivity of the immobilized
reagent.
The physical stability and the efficiency of chemically
modified silica adsorbents is not matched by any of the soft gels
commonly used as supports in covalent chromatography. One limitation
of silica has been the use of lipophilic modification methods which
are not compatible with the aqueous samples of biochemical applica
tions. In order to assure that the lipophilic phase is solvated, an
organic solvent must be added to the mobile phases. The amount of
solvent needed to solvate the traditionally bonded silicas was detri
mental to the stability of some biochemical compounds. A hydrophilic
phase is desirable for most biochemical applications.
A second limitation to silica as a solid support is due to
the reactivity of the support. Residual si1ano1s are capable of
148
149
strongly interacting with some compounds causing irreversible adsorp
tion. Traditionally, these sites were reacted with trimethylchloro
silane; however, this increases the lipophilicity of the phase.
In order to retain the advantages of silica, a new modifica
tion method that would exclude the sample from residual silanols and
provide a homoenerget~c hydrophilic surface was developed and charac
terized. This phase is a propylurea bonded silica. The urea groups
will hydrogen bond directly to other urea groups or thorough water
molecules to form a hydrogen bonded barrier that will restrict com
pounds from intercalating past the urea into the surface silanols.
Chemically modified silica surfaces have been shown to have 2-3 layers
of highly structured water at the surface. Between the hydrogen
bonding nature of the silica surface and the urea groups, this entire
phase is a highly structured hydrogen bonded hydrophilic surface.
Molecules do not intercalate to the surface because of the energy
required to break the hydrogen bonds and displace a water molecule.
As demonstrated by electrostatic and hydrogen bonding inter
actions, the residual silanols that are available to the sample on the
traditionally modified silica sorbents are not available to the sample
on the urea modified phases. The elimination of the non-specific
retention at these sites will expand the use of silica in covalent
chromatography, and all areas of chemistry utilizing immobilized
reagents by allowing for compatibility with a wider range of samples
and mobile phases.
150
Comparisons of the data of immobilized functionalities to the
solution work shows no apparent change on reactivity or selectivity.
Diagnostic chromatography and solid phase extraction were used to
determine the effects of pH on the reaction of several molecules with
PBA. The effects of pH on the reaction with immobilized PBA was
consistent with solution chemistry.
Diagnostic chromatography of several immobilized thiol rea
gents showed that immobilization did not affect the selectivity of
these phases. Thiopyridone immobilized on a support through a disul
fide bond will be displaced by thiol containing compounds. The
thiopyridine may be detected as it elutes from the system to quantify
the compound of interest or the compound of interest may be immobi
lized, isolated and eluted with a competing thiol. Mercurial com
pounds immobilized on solid supports will strongly interact with thiol
containing compounds under a wide variety of conditions. Arsonous
acid immobilized on a surface will interact with thiols but the
interaction with dithiols is much stronger than the interaction with
monothiols. The interactions of immobilized organomercurial and
arsonous acid are consistent with the interactions of these molecules
in solution.
The covalent reaction between the compound of interest and the
immobilized reagent occurs in two steps. The first step is the
diffusion of the molecule to the immobilized reagent and the second is
the formation of the covalent bond. The nonspecific interactions can
improve the first step by solvating the molecule in the stationary
151
phase. For example, electrostatic forces between opposite charges
would attract the molecule to the surface improving the first step,
while the repulsion of like charges would cause exclusion of the
molecule from the surface and the covalent bond will not form. While
non-specific repulsion may be minimized by changing the mobile phase,
a more effective method is to modify the surface so the local envi
ronment of the reagent is compatible with the sample.
The local environment may affect the formation of the covalent
bond. The pKa of PBA on the hydrophilic propylurea modified silica is
shifted to a lower value. The amine groups on this surface are
interacting directly with the PBA or causing a surface buffering
effect to decrease the pKa . The preparation of a lipophilic phase
with residual propyl amine functionalities will have the same affect
(27). The retention of molecules will be affected by the shift in
pKa . Catechol and other diols are retained at a lower pH on the
hydrophilic phase, because these molecules interact with the ionized
form of PBA. Compounds that interact with the neutral form of PBA
will require lower pH in order for quantitative retention.
Understanding the effects of the modification on the retention
of molecules is necessary in order to develop separation methods. In
addition, modification methods of silica may be altered to fit with
the requirements for a separation to improve selectivity. Once an
adsorbent has been designed, the mobile phase may be used to fine tune
the separation as long as the non-specific interactions are recognized
and their effects are understood.
152
The use of several phase coupled together would allow the
isolation of several classes of compounds from one sample or for the
removal of an interferant prior to the isolation of a compound of
interest. Successfully coupling columns together requires an under
standing of the interactions of all the columns and the use of samples
and mobile phases that will be compatible with all the sorbents. In
figure 1.4, an immobilized arsonous acid was coupled to immobilized
mercurial column. The arsonous acid column allowed for the extraction
of the dithio1 reducing agent so this would not compete with the
thiopyridone for the active sites in the organomercurial column. The
mixed disulfide adsorbent was used with the PBA, the mercurial and the
arsonous acid column as a method to detect low concentrations of thio1
containing compounds. The ability to couple the columns together to
isolate several classes of compounds will allow for high speed
separations of a number of compounds from a small sample.
Extending the reported studies using different buffers and
organic solvents to measure the effects of these on the selectivity
could be carried out, as well as the use of other techniques to
characterize the surface. Titration of the surface would allow for
the determination of the pKa of immobilized PBA under a wide variety
of mobile phases. This would complement the retention data of mole
cules that interact with only one form of PBA. Chromatographic
temperature studies and IR spectroscopy as described by Schunk (9)
could be used to determine the organization of the solvated stationary
phase and the retention mechanisms occurring on the phase. The
153
isolation of biologically significant molecules is simplified when the
retention of small probe molecules is understood. While the retention
of the small probes will allow for the estimation of the interactions
of larger and more complicated molecules, systematic investigations of
the complicated molecules is needed for the isolation of these com
pounds, and will allow for further understanding of the interactions
of the surface.
One complication of larger molecules is the inability of
reagents immobilized using short spacer arms to reach into the mole
cule to react with the active sites. The folding of proteins or
enzymes often causes active sites to exist within the folded chains.
Modification methods should be developed that would have spacer arms
long enough to reach into folded proteins, have a chemical nature
compatible with the protein and restrict interactions of the compound
of interest with residual silanols. Development of these spacer arms
requires an understanding of the effect of the spacer arm on retention
and the effects of the spacer arm on the compound of interest.
The isolation of compounds as well as the use of multidimen
sional chromatography would be significantly enhanced through the
automation of system with column switching capability and mobile phase
control. The preparation of an automated system would require the
understanding of the sample and the matrix to assure complete and
efficient separation.
REFERENCES
1. Scouten, William H. Solid Phase Chemistry Wiley, NY (1983) 1-16.
2. Stewart, John M. "Solid Phase Pepti1e and Protein Synthesis" Solid Phase Biochemistry Wiley, NY (1983) 507-534.
3. Wallace, R. B., Keiichi Itakura "Solid Phase Synthesis and Biological Applications of Po1ydeoxyribonuc1eotides." Solid Phase Biochemistry Wiley, NY (1983) 631-664.
4. Merrifield, R. B. "Solid Phase Synthesis I. Synthesis of a Tetrapeptide" J. Amer. Chern. Soc 85 (1963) 2419-2430.
5. Tramper, J. "Organic Synthesis Using Immobilized Enzymes" Solid Phase Biochemistry Wiley, NY (1983) 393-478.
6. Bergo1d, Alan, William Scouten "Borate Chemistry" Solid Phase Biochemistry Wiley, NY (1983) 149-187.
7. Guiban1t, G. G. "Immobilized Enzyme Electrode Probes" Solid Phase Biochemistry Wiley, NY (1983) 479-503.
8. Keen, James, William Jakoby "A Convenient Assay Method for Disulfide Interchange and Other Thio1 Transfer Reactions" Biochem 90 (1978) 136-140.
9. Schunk, T. C. "Chemical Interactions at the Solid-Liquid Interface: Investigations Employing Diagnostic Separations" Ph.D. Dissertation, University of Arizona (1985).
10. Gorse, J., "Characterization of the Solid-Liquid Interface on Chemically Modified Particulate Surfaces" Ph.D. Dissertation, University of Arizona (1985).
11. Karger, Barry L., Lloyd R. Snyder, Casaba Horvath An Introduction to Separation Science, Wiley, NY (1973).
12. Snyder, L. R., J. J. Kirkland Introduction to Modern Liquid Chromatography 2nd Edition, Wiley, NY (1979).
13. Small, Hamish, Timothy S. Stevens, William C. Bauman "Novel Ion Exchange Chromatographic Method Using Condeuctimetric Detection" Anal Chern 17 (1975) 11 1801-1809.
14. Lewis, C., W. Scouten "Immobilized Protein Modification Reagents" Solid Phase Biochemistry Wiley, NY (1983) 671-675.
154
155
15. Grassetti, D. R., J. F. Murry, .Tr. "Determination of Sulfhydryl Groups with 2,2/- or 4,4/-Dithiodipyridine" Arch. Biochem. Biophys. 119 (1967) 41-49.
16. Carlsson, Jan, Rolf Axen, Keith Brocklehurst, Eric M. Crook "Immobilization of Urease by Thiol Disulfide Interchange with Concommitant Purification" Eur. J Biochem. 44 (1974) 189-194.
17. Brocklehurst, Keith, Jan Carlsson, Marek P.J. Kierstan, Eric M. Crook "Covalent Chromatography" Biochem. J. 133 (1973) 573-584.
18. Carlsson, Jan, Anders Svenson "Preparation of Bovine Mercaptoalbumin by Means of Covalent Chromatography" FEBS Letters 42 (1974) 2 183-186.
19. Yoshida, Shonen, Toshiteru Morita, Mineo Saneyoshi "Isolation of the Newly Synthesized DNA of HeLa cells Containing p-2/-Deoxy-6-Thioguanylate on an Organomercurial Agarose Column" Biochem Biophis Acta 607 (1980) 527-529.
20. Webb, J. Leyden "Mercurials" Enzvrne and Metobolic Inhibitors Vol. II Academic Press, NY (1966) 729-907.
21. Hannes tad , Ulf, Per Lunquist, Bo Sorbo "An Agarose Derivative Containing an Arsenical for Affinity Chromatography of Thiol Compounds" Anal Biochem. 126 (1982) 200-204.
22. Kennedy, J. F., J.M. Cabral "Immobilized Enzymes" Solid Phase Biochemistry Wiley, NY (1983) 253-292.
23. Unger, K. K., R. Jansen "Packings and Stationary Phases in Preparative Column Liquid Chromatography" J Chromatogr. 373 (1986) 227-264.
24. Maestas, R. R., J. R. Preito, G. D. Juehn, J. H. Hageman "Polyacrylamide-Boronate Beads Saturated with Biomolecules: A New General Support for Affinity Chromatography" J. Chromatogr. 189 (1980) 225-231.
25. Dean, John A., editor Langes Handbook of Chemistry 11th ed., McGraw Hill, NY (1973).
26. Lochmuller, C. H., Walter B. Hill, P. M. Gross "Manipulation of Stationary Phase Acid-Base Properties by a Surface Buffering Effect: Boric Acid-Saccharide Complexation" Chromatography and Separations Chemistry, Advances and Developments (1986) 210-225.
27. Yabroff, David L., G. E.K. Branch, H. J. Almquist "The Interactions of the Boron Sextet with Adjacent Groups" J. Amer. Chern. Soc. 55 (1933) 2935-2941.
156
28. Freidman, Samuel, Benjamin Pace, Richard Pizer "Complexation of Phenylboronic Acid with Lactic Acid. Stability Constant and Reaction Kinetics" J. Amer. Chern Soc. 56 (1934) 5381-5384.
29. Larsson, Ragnar, Gennaro Nunziata "An Infrared Spectroscopic Investigation of the Complexes Formed Between Boric Acid and Lactic Acid in Aqueous Solutions" Acta Chern. Scand 24 (1970) 2156-2168.
30. Torssell, Kurt, John H. McClendon, G. Fred Somers "Chemistry of Arylboric Acids VIII. The Relationship Between Physico-Chemical Properties and Activity in Plants" Acta Chern. Scand 12 (1958) 7 1373-1385.
31. Branch, G.E.K., David L. Yabroff, Bernard Bettman "The Dissociation Constants of the Chlorophenyl and Phenethyl Boric Acids" J. Amer. Chern. Soc. 56 (1934) 937-941.
32. Johnson, Barbara J. B. "Synthesis of a Nitrobenzene Acid Substituted Polyacrylamide and Its Use in Purifying Isoaccepting Transfer Ribonucleic Acids" Biochemistry 20 (1981) 21 6103-6108.
33. Lorand, John P., John O. Edwards "Polyol Complexes and Structure of the Benceneboronate Ion" J Org Chern. 24 (1959) 769.-774.
34. Oertel, Richard P. "Raman Study of Aqueous Monoborate-Polyol Complexes. Equilibria in the Monoborate-l,2 Ethanediol System" Inorg Chern. 11 (1972) 3 544-549.
35. Burnett, Thomas J. Henry C. Peebles, James M. Hageman "Synthesis of a Fluorescent Boronic Acid Which Reversibly Binds to Cell Walls and a Diboronic Acid Which Agglutinates Erythorocytes" Biochem. Biophys. Res. Comm. 96 (1980) 1 157-162.
36. Kemper, K., E. Hagemier, D. Ahrens, K-S. Boos, E. Schlimme "Group Selective Prefractionation and Analysis of Urinary Catecholamines by On-Line HPLAC-HPLC Chromatography" Chromatographica 19 (1984) 288-291.
37. Kemper, K., E. Hagemier, K-S. Boos, E. Schlimme "Group Selective Prefractionation and Analysis of Nucleosicles and Catecholamines by High Performacne Liquid Chromatographic Techniques" Fresenius Z. Anal. Chern. 317 (1984) 680-681.
38. Higa, Sadayoshi, Tomokazu Suzuki, Akira Hayashi, Isao Tsuge, Yuichi Yamamura "Isolation of Catecholamines in Biological Fluids by Boric Acid Gels" Anal Biochem. 77 (1977) 18-42.
39. Gehrke, Charles W., Kenneth C. Kuo, George E. Davis, Robert D. Suits, T. Phillip Waalkes, Ernest J. Borek" Quantitative High Performance Liquid Chromatography of Nucleosides in Biological Materials" J. Chromatogr. 150 (1978) 455-476.
157
40. Hagemier, E., K-S. Boos, E. Schlimme, K. Lechtenborger, A. Kettrup "Synthesis and Application of a Boronic Acid-Substituted Silica for High-Performance Affinity Chromatography" J. Chromatogr. 208 (1983) 291-295.
41. Glad, Mangus, Sten Olsen, Lennart Hansson, Mats-olle Mansson, Klaus Mosbach "High Performance Liquid Affinity Chromatography of Nucleosides, Nucleotides and Carbohydrates with Boronic AcidSubstituted Microparticulate Silica" J Chromatogr. 200 (1980) 254-260.
42. Singhal, Ram P., Raui K. Baja, Charles M. Buess, David B. Smoll, Vikram N. Vakharia "Reversed Phase Boronate Chromatography for the Separation of O-Methylribose Nucleosides and AminoacyltRNA's" Anal. Biochem. 109 (1980) 1-11.
43. Hagemier, E., K. Kemper, K-S. Boos, E. Schlimme "Development of a Chromatographic Method for the Quantitative Determination of Minor Ribonucleosides in Physical Fluids" J. CUn. Chern. CUn. Biochem. 22 (1984) 175-184.
44. Bouriotis, V., I. J. Galpin, P.D.G. Dean "Applications of Immobilized Phenylboronic Acids as Supports for Group Selective Ligands in the Affinity Cl'\romatography of Enzymes" J. Chromatogr. 210 (1981) 267-278.
45. Hagemier, E., K. Kemper, K-S. Boos, E. Schlimme "On-Line High Performance Liquid Affinity Chromatography-High Performance Liquid Chromatography Analysis of Monomeric Ribonucleoside Compounds in Biological Fluid" J. Chromatogr. 208 (1983) 663-669.
46. Seinkiewicz, Paul A., David C. Roberts "Chemical Affinity Systems I. pH Dependence of Boronic Acid-Diol Affinity in Aqueous Solution" Inorg. Nucl Chern. 42 (1980) 1559-1575.
47. Iler, R. K. The Chemistry of Silica Wiley, NY (1979).
48. Homer, R. B., C. D. Johnson "Acid Base and Complexing Properties of Amides" The Chemistry of Amides Wiley, NY (1970).
49. Crampton, M. R. "Acidity and Hydrogen Bonding" The Chemistry of the Thiol Group, Wiley, NY (1974) 379-415.
50. Sotowitz, Mark L. "Covalent Chromatography: Immobilized Phenyl boronic Acid for Sample Preparation" Sample Preparation and Isolation Using Bonded Silicas Analytichem Int. Harbon City, CA (1985) 41-44.
51. Anfinsen, Christian B. "Principles That Govern the Folding of Protein Chains" Science 181 (1973) 4096 223-230.
52. Ellman, George L. "A Colorimetric Method for Determining Low Concentrations of Mercaptans" Arch Biochem. Biphys. 74 (1958) 443-450.
53. Ellman, George L. "Tissue Sulfhydryl Groups" Arch Biochem. Biophys. 82 (1959) 70-77.
158
54. Zahler, W. L., Cleland, W. W. "A Specific and Sensitive Assay for Disulfides" J. BioI. Chern. 243 (1968) 4 716-719.
55. Cleland, W.W. "Dithiothrieto1, A New Protective Reagent for SH Groups" Biochem 3 (1964) 480-482.
56. Anderson, W. L., D. B. Wet1aufer "A New Method for Disulfide Analysis of Peptides" Anal. Biochem. 67 (1975) 493-502.
57. Ingebretsen, Ole Christian, Mikae1 Farstad "Separation of the Oxidized and Reduced Forms of Dithiothrieto1 and 2-Mercaptoethanol by reversed-phase high-performance liquid chromatography. Application of the Method to Biological Extracts and the Determination of Disu1fides" J. Chromatogr. 210 (1981) 522-526.
58. Krieger, David E., Bruce W. Ericson, R. B. 11errifie1d "Affinity Purification of Synthetic Pepddes" Proc Nat1. Acad. Sci. 73 (1976) 9 3160-3164.
59. Yu, Mu-quing, Gui-qui Liu, Qinhan Jin "Determination of Trace Arsenic, Antimony, Selinium and Tellurium in Various Oxidation States in Water by Hydride Generation and Atomic Absorption Spectrophotometry After Enrichment and Separation with Thio1 Cotton" Ta1anta 30 (1983) 4 265-270.
60. Bishop, C.A., T. M. Kittson, D. R. K. Harding, W. S. Hancock "Investigation of the isolation of a mixed disulfide, diethy1thiocarbamoy1 2-pyridy1 disulfide by analytical and preparative high-performance liquid chromatography" :L. Chromatogr. 208 (1981) 141-147.
61. Liener, Irvin E. "Binding of Thio1s by a Water Insoluble Organo Mercurial Copolymer of Ethylene and Maleic Acid" Arch. Biochem. Biophys. 121 (1967) 67-72.
62. Ono, Masao, Masya Kawakami "Separation of Newly Synthesized RNA by Organomercurial Agarsoe Affinity Chromatography" J. Biochem. 81 (1977) 1247-1252.
63. Smith, Michael M. Anthony Reeve, Ru Chih C. Huang "Transcription of Bateriophase ADNA in Vitro Using Purine Nucleoside 5'-(~S]Triphosphates as Affinity Probes for RNA Chain Initiation" Biochem 17 (1978) 3 493-500.
159
64. Mar, Eng-Chun, Eng Shang Huang "Comparative Studies of Herpes Group Virus Induced DNA Polymeraces" Intervirology 12 (1979) 73-83.
65. Melvin, William T., Hamish M. Keir "Affinity Chromatography of Thiol Containing Purines and Ribo Nucleic Acid" Biochem J. 168 (1977) 595-597.
66. Sun, Irene Yi Chi, Edward M. Johnson, Vincent G. Allfrey "Affinity Purification of Newly Phosphorylated Protein Molecules" J Biochem. 255 (1980) 2 742-747.
67. Shami, Y. S. Ship, A. Rothstein "Rapid Quantitative Separation of the Major Glycoproteins (PAS 1, 2 and 3) From Other Red Cell Membrane Proteins in a Nondenaturing Medium by Affinity Chromatography" Anal. Biochem. 80 (1977) 438-445.
68. Carroll, Anthony R., Robert R. Wagner "Adenosin-5'-O- (3-thiotriphosphate) as an Affinity Probe for Studying Leader RNA's Transcribed by Vesicular Stomatitis Viris" J. Biol. Chern. 254 (1979) 19 9339-9341.
69. Webb, J. Leyden "Arsenicals" Enzvrne and Metobolic Inhibitors Vol. III Academic Press, NY (1966) 595-793.
70. Whittaker, V. P. "An Experimental Investigation of the Ring Hypothesis of Arsenical Toxicity" Biochem. J. 41 (1947) 56-61.