Preparation and evaluation of fused silica capillary ... · separation etc. would not advance as...
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Preparation and evaluation of fused silica capillary columns forgas chromatography and micellar electrokineticchromatographyCitation for published version (APA):Wu, Q. (1992). Preparation and evaluation of fused silica capillary columns for gas chromatography and micellarelectrokinetic chromatography. Technische Universiteit Eindhoven. https://doi.org/10.6100/IR371918
DOI:10.6100/IR371918
Document status and date:Published: 01/01/1992
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PREPARATION AND EVALUATION OF FUSED SILICA
CAPILLARY COLUMNS FOR GAS CHROMATOGRAPHY
AND MICELLAR ELECTROKINETIC CHROMATOGRAPHY
QUANJIWU
. " . · ~- :" . ."
PREPARATION AND EV ALUATION OF FUSED SILICA
CAPILLARY COLUMNS FOR GAS CHROMATOGRAPHY
AND MICELLAR ELECTROKINETIC CHROMATOGRAPHY
PREPARATION AND EV ALUATION OF FUSED SILICA
CAPILLARY COLUMNS FOR GAS CBROMATOGRAPHY
AND l\fiCELLAR ELECTROKINETIC CBROMATOGRAPHY
PROEFSClll:FT
ter verkrijging van de graad van doctor aan de
Technische Universiteit Eindhoven, op gezag van
de Rector Magnificus, Prof,dr. J.H. van Lint, voor
een commissie aangewezen door het College
van Dekanen in het openbaar te verdedigen op
dinsdag 14 april 1992 om 16.00 uur
door
QtrANJI wt1
Geboren te Liuzhou (P.R. China)
Druk: Boek en Offsetdrukkerij Le1ru, Helmond, 04920·37797
Dit proefschrift is goedgekeurd
door de promotoren:
prof.dr.ir. C.A. cramers
en
prof.dr. P.J.F. Sandra
copromotor:
dr.ir. J.A. Rijks
To Rongrong and Fanda
To our parents
CONTENTS
CHAPTER 1 GENERAL INTROOUCTION
1.1
1.2
1.3
CHAPTER 2 •
Introduction Scope of This Thesis References
DEACTIVATION OF FUSED SILICA CAPILLARY COLUMNS BY POLY-HYOROSILOXANES WITH DIFFERENT
1
4
4
4
6
9
10
FUNCTIONAL SUBSTITUENTS • • • . • • • • • • • 10
2.1
2.2
2.3
2.4
2.5
2.6
CHAPTER 3 •
Introduction Theory Experimental Results and Discussion Conclusions • •
References
A SOLID STATE 29si NMR STUDY OF THE DEACTIVATION OF SILICA SURFACE USING NON-POROUS SILICA
10
14
17
21
34
37
39
PARTICLES AS MODEL SUBSTRATE • • • • • • • • • • • 39
3.1 Introduction . . . . . . . 3.2 Theory . . . . 3.3 Experimental . . . . . . . 3.4 Results and Discussion . . . . 3.5 conclusions . . . . 3.6 References . . . .
CHAPTER 4 • PREPARATION OF NON-POLAR AND MEDIUM POLAR FUSED
. .
. . 39
42
46
50
57
58
60
SILICA CAPILLARY COLUMNS FOR GC • • • • • • • • • 60
4.l
4.2
4.3
Introduction Experimental Results and Discussion
-1-
60 68
72
4.3.l Evaluation of Deactivated Capillaries 72 4.3.2 Evaluation of Columns coated with
Non-polar Stationary Phases . • • 4.3.3 Evaluation of Columns Coated with
Medium Polar Stationary Phases
4.4 conclusions 4.5 References
CHAPTER 5 • • • • APPLICATIONS IN GAS CHROMATOGRAPHY
5.l 5.2
Introduction Experimental
5.3 Results and Discussions • 5.3.l High Speed Analysis on Narrow-Bore
Capillaries • • • • • • • • • • • • • 5.3.2 High Temperature Columns •••••• 5.3.3 Analysis of Drugs, Pollutants, Amino
Acids, and Underivatized Steroids 5.4
5.5
CHAPTER 6 •
Conclusions • References
73
79
82
84
90
90
90
93
95
95
99
105 110 111
114 MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY ---THE INFLUENCE OF COLUMN MODIFICATION ON ELECTROOSMOTIC FLOW • • • • • • • • . . • . • . • 114
6.1
6.2 Introduction Theory
6.2.l Electrical Double Layer 6.2.2 t-Potential and Electroosmotic
Flow (EOF) • • • • • • • • 6.2.3 Factors Influencing EOF 6.3 Experimental ••••• 6.4 Results and Discussion 6.4.l Repeatability of EOF Data
and Peak Area • • • 6.4.2 Influence of Column surface
modif ication on EOF • • • •
-2-
114 119
119
120 l24 127 130
130
13l
6.4.3 Discussion on the Influence of surface Treatment and Addition of SOS on EOF
6.4.4 Stability of the Coatings • • •••• 6.5 Conclusions • • ••••••• 6.6 References ••••••••••
CHAPTER 7 •
SEPARATION OF HERBICIDES BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY
Introduction Theory Exper ilnental
7.1 7.2
7.3 7.4 Results and Discussion 7.4.1 Influence of surfactants 7.4.2 Influence of Methanol . 7.5 7.6
Conclusions • References
SUMMARY •• SAMENVATTING CURRICULUM VITAE ACKNOWLEDGEMENT • PUBLICATIONS
-3-
133 138 140
141 145
145
145 147 153 156 156 162 168 169
172
177 182 184 185
CBAPTER 1
GENERAL INTRODUCTION
1.1 INTRODUCTIOR
Capillary columns known for their high efficiency, separation
speed, inertness and thermal stability, nowadays dominate the
field of separation science. Capillary chromatography
(including GC, LC and SFC) and capillary electrophoresis have
been developed into powerful tools for scientists and analysts
specialists werking in widely different areas. Applications
include process control, environmental control, chemical,
medicine and food industry. Also biological and biochemical
research such as the analysis of trace amounts of drugs and
metabolites in various body fluids, the separation of proteins
and synthetic oligonucleotides and DNA sequencing fragment
separation etc. would not advance as fast without open tubular
column technology.
The capillary column is the heart of a capillary
chromatography system. Different materials such as stainless
steel, nickel, teflon, glass and fused silica can be used to
make capillary columns with inner diameters between ca. 10 µm
and 1 mm. Several manipulations are involved in the
preparation of capillary columns, some of them are still not
fully understood due to lack of suitable physical or chemical
techniques for direct characterization of the inner surface
of the capillaries.
-4-
In GC, generally, a glass or fused silica capillary column
needs a preliminary hydrothermal treatment in order to obtain
a controlled density of silanol groups on the column wall (l-
5]. The deactivation of the capillary column completes the
column pretreatment and gives the column its necessary
inertness, thermostability and wettability for stationary
phases of different polarity. After coating of the deactivated
capillaries with the desired stationary phase, the coated
stationary phase layer is usually subjected to immobilization.
The introduction of flexible fused silica columns by Dandeneau
and Zerenner [ 6] in 1979 brought a breakthrough in the
application of capillary columns. The main advantages of fused
silica capillaries are their mechanica! strength and the
increased inertness in comparison with glass columns. The
inner surface of the column can be easily modif ied using a
wide range of different modification methods (7-12] to be
compatible with the desired stationary phases.
Recently, capillary electrochromatography (EC), a separation
method in which the electroosmotic flow (EOF) is the driving
force for the mobile phase grows rapidly. This technique,
which is based on the basic principles of chromatography, uses
similar equipment as electrophoresis and can achieve very high
efficiencies in a relatively short time [13).
In EC, specially in micellar electrokinetic chromatography
(MEKC), a predictable and reproducible EOF through the
capillary column is important. The capillary column is used
-5-
with or without pretreatment in EC techniques. The column
modification, however, can both provide reproducible EOF
values and eliminate the interactions between the solutes and
the column wall. This is particular important for the
separations of proteins and biomolecules. Both dynamic and
statie modification of the capillary inner wall can be
obtained by different methods.
Numerous parameters are involved in the modification and
coating procedures for fused silica capillaries, all of them
control the quality and performance of the final columns. In
general, maxima! interaction of the solutes with the
stationary phase and minimal interaction with the residual
active sites on the column inner wall surface is desired. The
repeatability of the preparation and the stability of the
modification layer and stationary phase are very important
factors to obtain reproducible analytica! results.
1.2 SCOPE OF THIS THESIS
As pointed out in the general introduction fused silica is the
material of choice for capillary separations. For their
application it is essential that the column properties are
determined by the bulk properties of the stationary phases and
not by the residual active sites of the column wall. constant
surface properties are also essential to obtain controllable
and reproducible electroosmotic flow in EC.
Therefore deactivation of the column wall and compatibility
-6-
of the modified surface with the stationary phases is
essential for reproducible analytica! results.
This thesis describes some recent developments in methods of
deactivation and crosslinking of the stationary phases for
fused silica capillary columns. The experimental study of the
deactivation process is supported by solid state 29si NMR
measurements on model substrates like fumed silica.
Columns prepared following optimized procedures are evaluated
by applications both in capillary GC and in EC.
In Chapter 2 the deacti vation of fused silica capillary
columns by polyhydrosiloxanes with different functional
substituents is described. A series of vinyl modified
polyhydrosiloxane deactivation reagents containing different
functional groups is synthesized for this purpose. The
optimization of the deactivation reaction is investigated and
evaluated by GC with a dual column system. Also the
incorporation of different functional groups into the reagents
and their effect on the column performance is investigated.
Due to the small surface area of a capillary column, a model
substrate has to be used to characterize the deactivated
surfaces by solid state NMR. A study of the deactivation
process of silica surface using non-porous silica particles
as the model substrate by 29si NMR is described in Chapter 3.
The effect of the silylation temperatures and reaction times
on the deactivation process of silica surfaces is
investigated. The effect of the functional groups incorporated
-7-
in the reagents on the deactivation process is studied.
In Chapter 4 the preparation of non-polar and medium polar
capillary columns for GC is described, using the optimized
deactivation procedures outlined in the previous chapters.
crosslinking and/or chemical bonding of the stationary phases
by thermal treatment and radical initiation is investigated.
The effect of hydrogen and of vinyl groups, which exist in the
deactivation layer resulting from the deactivation reaction,
in the crosslinking process is studied.
The applicability of the prepared non-polar and medium polar
fused silica capillary columns in GC is illustrated in Chapter
5. Narrow-bore columns are used to demonstrate high speed
separations for selected samples, whereas normal bore columns
are used for the analysis of underi vatized steroids, drugs and
priority pollutants. The performance of the capillaries with
respect to inertness, thermostability and efficiency is
evaluated.
In Chapter 6, a systematic investigation is carried out on the
electroosmotic flow (EOF) in micellar electrokinetic capillary
chromatography (MECC). Fused silica capillaries are treated
with different reagents to investigate their effect on the
magnitude and reproducibility of the EOF. Experiments with and
without sodium dodecylsulphate (SDS) addition to the buffer
are carried out in order to elucidate the effect of this
surfactant on the EOF. A model is evaluated to explain the
mechanism of the influence of column wall modifications on the
-a-
electroosmotic flow.
MECC is applied for the separation of a number of
chlorophenoxy acid herbicides (Chapter 7). Sodium dodecyl
sulphate (SDS), Brij 35, cetyltrimethylammonium bromide (CTAB)
and methanol are added in various concentrations to the
buffering eluents to investigate their effects on the
separation of the herbicides. Besides that, the differences
in the electroosmotic flow (EOF), due to the different
composition of the various eluents, are correlated with the
viscosities of the mobile phases.
1.3 REFERENCES
1. K. Grob, G. Grob and K. Grob, Jr., Chromatographia, 10 (1977) 181.
2. K. Grob, J. High Resolut. chromatogr. & Chromatogr. Commun., 3 (1980) 493.
3. M. L. Lee and B.W. Wright, J. Chromatogr., 184 (1980) 235.
4. M.W. Ogden and H.M. McNair, J. High Resolut. Chromatogr. & Chromatogr. Commun., 8 (1985) 326.
5. B. Xu and N.P.E. Vermeulen, J. Chromatogr., 445 (1988) 1.
6. R.D. Dandeneau and E.H. Zerenner, J. High Resolut. Chromatogr. & Chromatogr. Commun., 2 (1979) 351.
7. K.K. Unger, Porous Silica, Elsevier, Amsterdam, 1978. 8. K. Grob, G. Grob and K. Grob, Jr., J. High Resolut.
Chromatogr. & Chromatogr. commun., 2 (1979) 31. 9. K.K. Unger, G. Schier and u. Beisel, Chromatographia, 6
(1973) 456. 10. R.C.M. de Nijs, J.J. Franken, R.P.M. Dooper, J.A. Rijks,
H.J.J.M. de Ruwe and F.L. Schulting, J. Chromatogr., 167 (1978) 231. .
ll. C.L. Woolley, R.C. Kong, B.E. Richter and M.L. Lee, J. High Resolut. Chromatogr. & Chromatogr. Commun., 7 (1984) 329.
12. C.L. Woolley, K.E. Markides and M.L. Lee, J. Chromatogr., 367 (1986) 23.
13. W.D. Preffer and E.S. Yeung, Anal. Chem., 62 (l.990) 2178.
-9-
CHAPTER 2
DEACTIV ATION OF FUSED SILICA CAPILLARY COLUMNS BY POLYHYDROSILOXANES WITH DIFFERENT FUNCTIONAL SUBSTITUENTS
A series of vinyl modified polyhydrosiloxane deactivation reagents containing different functional groups is synthesized. The optimization of the deactivation reaction is investigated and evaluated by GC with a dual column system. Due to the high activity of silylhydride groups in the polyhydrosiloxanes, relatively low temperatures and short reaction times can be used for the silylation. The incorporation of different functional groups into the reagents is emphasized. Some vinyl groups are intentionally left to facilitate crosslinking and/or chemical bonding of the stationary phases. The contribution of the functional groups of the deactivation layer to the polarity of the prepared column is discussed.
2.1 INTRODUCTION
It is generally accepted that inertness is one of the most
important parameters of capillary columns in gas
chromatography. This aspects is more significant in high-speed
GC and trace analysis where columns with reduced inner
diameters are used. To prepare highly efficient, inert, heat
and solvent resistant columns, they have to be subjected to
pretreatments before coating [ 1-3] • The inner surf ace of glass
and fused silica is, without adequate treatments, not
satisfactorily wettable, at least not by stationary phases
having high surface tensions. The films coated on such
surfaces are usually irregular and unstable, and tend to form
droplets which results in deteriorated column performance.
The weakly acidic silanol groups on the fused silica column
wall interact with and adsorb compounds with high peripheral
-10-
electron densities. The strained siloxane bridges on the
column surface can act as proton accepters in hydrogen bonding
interaetions, and cause adsorption of proton donating
components.
on a glass surface, in addition to the problems mentioned
above, alkali and alkaline earth metal ions cause even more
severe problems of adsorptive effects and catalytic activity.
The latter problem may also cause the stationary phases to
decompose at higher temperatures resulting in increased
bleeding of the stationary phases.
The main purpose of deactivation is to create a stable layer
which should shield all the active sites of the column
surface, resulting in perfectly neutra! columns. The other
goal of the deactivation is to obtain good wettability for the
stationary phases to be coated. This can be achieved by
applying similar materials as the stationary phases in the
deactivation procedure.
Tremendous amounts of research have been done in the
development of column technology and many methods and
techniques have been proposed and evaluated by different
authors. Aue et al. (4) introduced and ethers successfully
developed [S-9) the method of Carbowax 20M deactivation.
However, some investigators have shown (9, 10) that Carbowax
20M deactivation layers are not stable for extended use above
250°C. Grob and Grob [11] claimed that polar polyethylene
glycol deacti vated surf aces are 11poorly wettable by apolar
phases 11 •
-11-
currently, many deactivation techniques are based on
silylation reactions. Silylation reactions are flexible and
the regents are widely available. lt has been stated in
literature (12-18] that only the free surface hydroxyl groups
are involved in silylation reactions, whereas hydrogen-bonded
hydroxyl groups show virtually no reaction with the silylation
reagent.
..:L 'si-0-H /
>3.1 A ..........
T /Si-0-H
Free •U rfaee hydroxyl 11roup•
....i... 'si-O-H . I \/
2.4-2.8 A 0 " \ I \
T -Si-0-H /
Hydrogen·bonded .urface hydroxyl groupa
Trimethylchlorosilane (TMCS) and hexamethyldisilazane (HMDS)
were frequently used to silylate the column surface at lower
temperature (<250°C) in the early years of column technology
(19-21]. Welsch et al. (22] were the first to attempt high
temperature silylation {HTS) of glass capillary columns and
showed the benefits of high reaction temperatures in the
deactivation process. Their most favourable surface silylation
was obtained with pure HMDS at 300°C for 20 h. Based on
Welsch's work, different HTS methods ware suggested using
various reagents (23-42]. Schomburg et al. (43] described a
deactivation procedure known as the PSD {polysiloxane
degradation) in which a non-extractable layer of polysiloxane
phase is produced on the column surface (44-47]. OH-terminated
polysiloxanes were also applied to deactivate capillaries [4S,
49].
-12-
Quite high temperatures (normally between 370-450°C} and long
reaction times (4-20 hours) are required with above mentioned
methods in order to achieve a neutra! and inert surface prior
to coating. This might damage the polyimide outer coating of
the capillaries resulting in fragile columns.
Recently, methyl-, phenyl- ànd cyanopropyl-polysiloxanes
containing silylhydride groups are applied to deactivate fused
silica capillary columns at moderate temperatures (50-55]. The
deactivation reaction involves dehydrocondensation and yields
neutra! and highly inert capillaries.
It is essential that the coating and im.mobilization of the
stationary phase after deactivation should not deteriorate the
inertness of the deactivated surface. Deterioration is
observed when common free radical initiation techniques with
peroxides and azo compounds are used for the immobilization.
The peroxide residues can be bonded to the polymerie
stationary phase and result in an increased adsorptivity and
decreased thermal stability (56-58]. A second popular
deactivation reagent is azo-tert-butane (ATB). ATB only
slightly contributes to the polarity of the immobilized phases
(33). The performance of ATB, however has been found to
deteriorate with time [59, 60]. Moreover, columns treated with
aged ATB showed high activity levels, solute retention shifts,
high bleed and erratic immobilization efficiencies [59, 60).
Based upon these observations and considerations, an ideal
process consists of low temperature deactivation and
-13-
immobilization of the stationary phases without the use of any
additional reagents. Polyhydrosiloxanes can be an attractive
choice due to their relatively low reaction temperatures and
short deactivation times.
In this chapter, the applicability of polyhydrosiloxane
deactivation reagents is investigated. A series of vinyl
modified polyhydrosiloxane based deactivation reagents
containing different functional groups were synthesized f or
this purpose (61). Vinyl groups were intentionally
incorporated into the polymer chain for facilitating later
immobilization of the stationary phases. The reaction
conditions for the deactivation reaction are optimized. All
GC experiments were performed on a dual column system. Gas
chromatographic retention indices were measured for critical
solutes of different polarity in the test mixture. The peak
shapes of the recorded chromatograms were compared in order
to evaluate the degree of deactivation obtained with these
reagents. The influence of surface modification on the
polarity of the stationary coating is also discussed. The
effect of hydrogen and vinyl groups in the crosslinking
process of the stationary phases will be discussed in Chapters
3 and 4.
2.2 'l'HEORY
Fairly high temperatures are required for deactivation with
octamethylcyclotetrasiloxane and polydimethylsiloxanes, due
to the acidity of the silanols on the fused silica column wall
-14-
[50]. on the contrary, with polyhydrosiloxanes, covalent bonds
are formed from the dehydrocondensation between hydride
silicon bonds and silanols on the column wall [50, 62, 63]:
' 1 1 1 -si-OH + H-Si-Me ----> -si-o-si-Me + H2 t 1 1 1 1
(2 .1)
This reaction is favoured by acidic surface conditions and can
reach 100% conversion under medium temperature conditions.
Basic column surfaces cause deprotonation of silanols thereby
hindering the above mentioned reaction (64].
Hydrolysis of Si-H bonds according to equation (2. 2), is
essential for intra- and intermolecular cross-linking of PMHS.
1 1 Me-si-H + H20 ----> Me-si-OH + H2 t
1 1 (2.2)
The physisorbed water on the column surface induces initial
hydrolysis at the PMHS-water interface. The hydrolysis product
can react further with Si-H as shown in the equation (2.3).
1 1 1 1 Me-Si-OH + H-Si-Me ----> Me-si-o-si-Me + H2 t
1 1 1 1 (2.3}
The covalent bonds between the polymer molecules are formed
by eq 2. 3. The reactions shown in eq 2. 1 and 2. 3 are
irreversible at temperatures above 200°C. Therefore, under the
optimum conditions for deactivation, a thin and densely
crosslinked and unextractable layer was formed on the column
wall.
The hydrolysis products can react also according to:
-15-
1 1 Me-si-OH + HO-Si-Me ----> Me-Si-O-Si-Me + H20
1 1 1 1 (2.4)
This reaction is reversible in the presence of water formed
in the reaction. Therefore, the deactivation process will be
deteriorated by the presence of an excess of water on the
column wall [65].
Substi tut ion of other functional groups instead of methyl
groups will decrease the reactivity of the Si-H bonds because
of both the steric hindrance effect and the electron
withdrawing effect of the other functional groups [51, 52).
Additionally, the irregular distribution of the silanols and
siloxane bridges on the column surface should be noticed. The
increased molar ratio of other substituted functional groups
make it more difficult to obtain a complete deactivation
compared with PMHS.
(2.5)
The critical surface tension of a column deactivated with the
reagents shown above depends on the ratio of m, n, and p in
formula 2.5. In order to minimize the effect of polar sites
in the deactivation layèr on the retention of the solute in
coated columns, an optimized amount of the different
functional groups in the deactivation reagents should be used.
-16-
2.3 EXPERIMENT.AL
2.3.l Materials and Reaqents
1,3,5,7-tetramethylcyclotetrasiloxane (D4H), 1,3,5,7-tetra
vinylmethylcyclotetrasiloxane (D4Vi), bis-(cyanopropyl)di
chlorosilane, diphenyldimethoxysilane, polymethylhydrosiloxane
(PS 122, 85 es, fluid) were all purchased fro:m Petrarch
systems (Bristol, PA, USA). These reagents were used without
further purification.
Fused silica capillary columns with inner diameters of 0.22
mm and 0.32 mm were obtained fro:m Chrompack International BV
(Middelburg, NL), the 50 µ.m columns from Scientific Glass
Engineering PTY LTD. (Ringwood, Australia). All solvents were
PA grade (Merck AG, Oarmstadt, FRG), deionized water was used
throughout this study (Millipore Syste:m, Millipore Corp.,
Bedford, USA).
2.3.2 Synthesis of Polyvinylmethylhydrosiloxane (PVMHS)
D4Vi (2 .1 g) and o4H (13. 2 g) were mixed together (molar
ratio, 10:90) with 3 :ml dichloromethane. A few drops of
concentrated sulfuric acid were added to catalyze the
polymerization. The mixture was stirred at room temperature
for 48 h. Then 5 drops of hexamethyldisilazane were added to
the mixture and stirring was continued for another 16 h to
enhance the endcapping. This endcaps the ter:minated hydroxyl
groups of the linear polymers. The mixture was then dissolved
in 10 ml of CH2cl2 and washed 8 times with an equal volume of
-17-
water. The solvent was removed by distillation. Finally, the
fluid was dried at 60°C under vacuum for 6 hours.
2.3.3 synthesis of Polydiphenylvinylmethylhydrosiloxane (POPMHS)
Diphenyldimethoxysilane (4.88 g) and D4Vi (0.172 g) was mixed
in 2 ml CH2c12 and stirred after addition of 5 drops of
concentrated sulfuric acid at room temperature. Then D4H (1.08
g) dissolved in 2 ml CH2Cl2 was dropped into the mixture
(molar ratio, 50:5:45) during 30 minutes in an Argon
atmosphere. The mixture was stirred for 60 h. For the
endcapping, the same procedure as used for the synthesis of
PVMHS was applied.
2.3.4 synthesis of Polybis-(oyanopropyl)vinylmethylhydrosiloxane (PBCPVMHS)
The monomer, bis-(cyanopropyl)dimethoxysilane, was achieved
according to the following conversion reaction:
Cl 1
R-Si-R + 1
OCH3 1
R-Si-R + 1
(2.6)
Cl OCH3
The dichlorosilane was added in drops to the stirred excess
trimethylorthoformate to reach full conversion. Thereafter the
polymerization was performed according to the procedure for
the synthesis of PDPVMHS.
-18-
2.3.5 Pretreatment of the :rused Silica Capillaries
The capillary columns were hydrothermally treated by filling
the column with a 10% (w/w) solution of HCl to 90% of their
volume and then heated to 150°C for 1 h. Next, the column was
rinsed with a 2% HCl solution and with deionized water until
neutral. Finally the columns were washed with 5 ml of methanol
and dried at 250°C for l h under a flow of Helium.
2.3.6 Deactivation and ~esting Procedure
Each column was dynamically coated with a 5% or 10% (v/v)
solution of the deactivation reagents in pentane or
dichloromethane. The speed at which the coating solution was
pumped through the column was kept at about 25 to 30 cm/min.
After flushing the columns with Helium for 30 min and flame
sealing, the columns were heated to different temperatures
during different reaction times. After deactivation the
capillary columns were carefully rinsed with 10 ml of
dichloromethane and dried at 250°C for l h under a flow of
helium. The deactivated fused silica capillaries were tested
by GC with a dual column system consisting of a coated
precolumn and the column to be tested. A test mixture (cf.
Table 2 .1) which contains components with different
adsorptivity was used for evaluation of the deactivated
c.olumns ( 65) •
The test method is essentially as described by Schomburg et
al. [10]. It allows the examination of peak shapes and peak
areas for a quantitative comparison of the column activity for
the components in the test mixture. The column polarity is
-19-
measured by means of Kováts retention indices for the
deactivated column as well as for the coated capillary. Zero
dead volume connectors (Unimetrics Corp., CA, USA or Gerstel
GmbH, Mülheim a/d Ruhr, FRG) were used to connect the
deactivated column to a high quality coated column, (home
made, PMHS deactivated OV-1 precolumn, L = 10 m, i.d. = 0.25
mm, film thickness df = 0.25 µm). An average linear velocity
of 30-40 cm/sec (Helium) was used.
Tabla 2.1 Composition of the test mixture. Peak No. is the elution order on the precolumn.
Peak Components conc. Representation for No. (mg/l)
1 decane 119.0 ---2 1-octanol 112.4 silanol, hydrogen bond
3 2,6-dimethylphenol 118.3 basic sites
4 undecane 116.6 ---5 2,6-dimethylaniline 114.7 acid sites, silanol
6 dodecane 114.4 ---7 decylamine 116.4 acid sites, silanol,
exposed siloxane bridge
8 tridecane 107.8 ---9 nicotine 121.0 acid sites, silanol
10 tetradecane 128.4 ---The amount of each component injected onto the column system
is about 1 ng. The measurements are performed on a Packard 430
gas chromatograph (Packard, Delft, NL) equipped with a split
injector and an FID. All experiments were carried out
isothermally at ioo 0 c.
-20-
2.4 RESULTS AND DISCUSSION
2.4.l Deactivation with Pol1Jllethylhydrosiloxane (PMRS)
The deactivation conditions used for the deactivation of the
fused silica capillaries with PMHS are given in Table 2.2.
Table 2.2 survey of the deactivation conditions of fused silica capillaries.
Column Reaction conditions No.
ID (µm) L (m) T (OC) Time (h} Reagent
1 220 5 --- --- untreated
2 220 10 240 4 PMHS
3 220 10 280 4 PMHS
4 220 10 300 4 PMHS
5 1) 220 10 --- --- ---6 2) 220 10 280 4 PMHS
7 320 4 240 4 PVMHS
8 320 4 280 4 PVMHS
9 320 4 300 4 PVMHS
10 320 4 320 2 PVMHS
11 320 4 350 0.5 PVMHS
12 3) 320 4 --- --- ---l) After reconditioning of column No. 3 at 300°C for 30 h
with Helium flushing. 2) Flushed with water saturated Helium for l hour prior to
deactivation. 3) After rinsing and reconditioning column No. 10 at 300°C
for 30 h with Helium flushing.
-21-
l
4
2
6
7
0 2 4 6 B
4
5
6
8
9
0 2 4 6 8
Fiqure 2.1
a 1
4
3 6
s
10
10 min
1
c
!~ s
10
0 2
6
7
4
8
8 9
6
b
d
10
B 10 min
Representative chromatograms of the test mixture. (a) precolum.n; (b) precolumn plus untreated column No. 1; (c) precolumn plus PMHS deactivated column No. 2; (d) precolumn plus PMHS deactivated column No. 3. For test conditions see text and peak identification, see Table 2.1.
-22-
The normalized peak areas (NA), corrected for the weight
difference in the test mixture but not for FID response
factors and the corresponding Kováts retention indices
measured at 100 °c for both the precolumn and the evaluated
capillaries are given in Table 2.3.
Table 2.3 Normalized peak areas (NA) and Kováts retention indices (RI) at 100°c of the test components of the PMHS deactivated columns.
Col. Test components No.
1-octanol 12, 6·-DMA Decylamine Nicotine 2,6-DMP
NA RI NA RI NA RI NA RI NA RI
O* 86 1051.3 96 1081. 0 101 1136.5 86 1233.3 92 1309.1
1 11 1087.3 70 1093.8 61 1159. 0 0 -- 0 --2 73 1052.5 94 1093.7 99 1138.l 11 1251. 5 53 1313.8
3 86 1052.5 95 1081. 4 103 1136.l 81 1234.4 90 1310.8
4 85 1052.4 94 1081. 6 98 1137.0 75 1235.5 85 1311.1
5 86 1052.6 95 1081. 7 102 1136.8 80 1235.1 89 1311. 4
6 47 1053.0 80 1082.2 87 1137.9 21 1244.3 64 1312.6
* The precolumn.
Considering the representative chromatograms for the precolumn
and the differently deactivated columns given in Fig. 2.1 and
the data presented in Table 2.3, it is obvious that excellent
deactivation is obtained after heating the column to 280°c for
4 hours (#3) or at a higher temperature during a shorter time
{#4). The deactivated column #3 was heated at 300°C for 30 h
and rinsed with pentane and dichloromethane respectively. No
significant changes in the normalized peak areas and the
retention indices of the components were observed. This high
thermal stability and resistance to solvent rinsing was
explained by Woolley et al [55]. They assume that a very thin
-23-
(several monolayers) but densely crosslinked film was formed
by dehydrocondensation (eqns. 2.1-2.3) and that the surface
is covered by anchored Si-Me groups with methyl protruding
upwards from the surface. This is in agreement with the
observation that the Koväts retention indices of a well
deacti vated column are in acceptable agreement to those of the
precolumn.
As shown in equation (2.2), physically adsorbed water on the
surface plays an important role in the formation of a densely
cross-linked network. An excess of water, however, will
deteriorate the deactivation layer due to the presence of
unreacted Si-OH groups (cf. eq 2.4). This is illustrated by
the strong adsorption of 1-octanol and aminodecane on column
No. 6 (cf. Table 2.2 and 2.3).
2.4.2 Deactivation with Polyvinylmethylhydrosiloxane (PVMHS)
The deactivation conditions (e.g. temperatures and times) for
a number of short fused silica columns (L = 4 m) with PVMHS
are summarized in Table 2.2.
Representative chromatograms of the PVMHS deactivated columns
(at 320°C during 2 hours) before and after solvent rinsing are
gi ven in Fig. 2. 2. The corresponding normalized peak areas and
retention indices with this copolymer are presented in Table
2.4.
For reasons of comparison, the deactivated capillaries were
tested before and after solvent rinsing. Comparing the columns
#3 (Table 2. 3) and #9 (Table 2. 4) , it can be seen that a
-24-
slightly higher temperature is needed for complete
deactivation with PVMHS as compared to the PMHS deactivation.
This can be explained by steric effects so that a higher
deactivation temperature is required to form a densely cross
linked layer and to cover the residual active sites in this
case.
:5 5
6 a 6 b
8
7 8 7
9 9
10 1C
' ...... -.... __ _,..,, _____ --- L -·-~ .... .__ ... ..,, __ ~ ----0 2 4 6 8 min 0 2 4 6 8 min
Fiqure 2.2 Representative chromatograms of the test mixture on the precolumn plus PVMHS deactivated column No. 10 (a} before solvent rinsing; (b} after solvent rinsing. For test conditions see text and peak identification, see Table 2.1.
Comparing the Kováts indices given in Table 2.4 for one of the
most optimal columns (e.g. #9) and the precolumn an increase
of the retention indices can be observed. This indicates that
the incorporation of vinyl groups into the cross-linked
deactivation layer can be considered successful. Further
-25-
studies with solid state 29si NMR on model substrates, like
cab-O-Sil [66] were required to confirm these results and will
be described in Chapter 3.
'l'able 2.4 Normalized peak areas (NA) and Kováts retention indices (RI) at 100°c of the test components of the PVMHS deactivated columns.
Col. Test components No.
1-octanol 2,6-DMP 2,6-DMA Oecylamine Nicotine
NA RI NA RI NA RI NA RI !NA RI
0 86 1051.3 96 1081. 0 101 1136.5 86 1233.3 92 1309.1
Before rinsing wi:th solvent
7 80 1054.6 - - - - - ~~ •• ""0 ---8 85 1054.6 92
~1319.7 1086.5 99 1142.4 27 1241.8 1314.5
9 86 1054.8 93 ~1238,4 88 1315.5
10 87 1055.8 94 1238.8 88 1316.5
11 86 1055.7 95 1238.0 90 1316.4
After rinsing with solvent
7 80 1054.6 90 1086.l 99 1142.l 0 1--- 36 1319.0
8 86 1054.2 90 1086.6 100 1141. 5 30 1241.1 87 1313.6
9 86 1055.2 91 1085.6 100 1142.6 75 1238.l 89 1314.7
10 86 1055.7 93 1087.1 100 1143.5 78 1238.1 89 1315.5
11 85 1055.1 92 1087.4 100 1145.0 83 1238.8 90 1316.7
12 85 1053.0 96 1090.1 100 1147.2 80 1240.5 87 1318.5
2.4.3 Deactivation with PDPVMHS
The deactivation conditions (e.g. reaction times and tempera
tures) for the deactivation with PDPVMHS are summarized in
Table 2.5.
A survey of the corresponding normalized peak areas and Kováts
retention indices with POPVMHS before and after solvent
-26-
rinsing is given in Table 2.6. Corresponding representative
chromatograms of the test mixture (cf. Table 2.1) are shown
in Fig. 2.3.
Table 2.s Survey of the silylation conditions of fused silica capillaries, all columns are 0.32 mm i.d. * 4 m.
Column No. Reaction conditions
T (OC) Time (h) Reagent
13 260 4 PDPVMHS
14 280 4 PDPVMHS
15 300 4 PDPVMHS
16 320 2 PDPVMHS
17 350 2 PDPVMHS
18 1) --- --- ---19 260 4 PBCPVMHS
20 280 4 PBCPVMHS
21 300 2 PBCPVMHS
22 320 1 PBCPVMHS
23 350 0.5 PBCPVMHS
24 2) --- --- ---1) After rinsing and reconditioning column No. 17 at 350°C
for 40 h with Helium flushing. 2) After rinsing and reconditioning column No. 23 at 280°C
for 1 h with Helium flushing.
-27-
1 4
1
4
a b
2
1!3 '"'3
5 6 5
6
a a
7 9 10 10
[_ 9 l LJ. l
_...._ - ';
0 2 4 6 a min 0 2 4 6 a min
14
1,..3
5 d c
6 3
56
7 a
7 a
9 10
9 10
l~ ..._ -- ...............
0 2 4 6 a min 0 2 4 6 a min
Figure 2.3 Representati ve chromatograms of the test mixture on the precolumn plus PDPVMHS deactivated column No. 14 (a) before solvent rinsing; (b) after solvent rinsing; and precolumn plus PDPVMHS deactivated column No. 17 (c) before solvent rinsing; (d) after solvent rinsing. For test conditions see text and peak identification, see Table 2.1.
-28-
Table 2.6 Normalized peak areas (NA) and Kováts retention indices (RI) at 100°c of the test components of the pDPVMHS deactivated columns.
Col. Test components No.
1-0ctanol 2,6-DMP 2,6-DMA Decylamine Nicotine
NA RI NA RI NA RI NA RI 1
I
0 86 1051.3 96 1081. 0 101 1136.5 86 1233.3 92 1309.l
Before ~insing ~ith solvent
13 82 1056.7 88 1088.6 100 1147~ 1252.1 71 1344.5
14 83 1056.2 89 1092.4 99 1151 1243.5 81 1328.5
15 83 1058.6 93 1099.0 100 1157.5 53 1243.3 85 1337.6
16 85 1065.6 91 1113.7 100 1178.6 59 1254.0 86 1350.2
17 84 1067.3 91 1118.2 99 1183.8 77 1245.9 88 1351. 5
~ After dn1ing with solvent
1057. 2 87 1090. 3 100 1146. 2 33 1249.5 76 1335.6
1055.8 901089.6~ 1147.444 1241.7 70 1317.5
~055. 7 ~1091.8 99 1150.5158 1240.6 86 1325.l
057.0 921090.81001150.761 1242.7 86 1322. 5
17 84 1058.1 91 1093.7 100 1152.2 82 1240.5 90 1323.7
18 84 1057.9 92 1092.8 100 1152.0 59 1239.4 87 1322.7
Similar steric eff ects and shifts of Kováts retention indices
were observed with PDPVMHS as with PVMHS deactivation.
Obviously, a deactivation layer which incorporates polar
functional groups will show a shift of Kováts indices to a
higher value. It can be seen that there are larger retention
index shifts of the test components in the mixture for the
freshly deactivated columns compared to the rinsed columns
(#16 and #17). From that it can be concluded that some
unreacted or decomposed short chain materials are physically
attached or just left on the surface before solvent rinsing.
The residual contribution to the retention behaviour of the
-29-
test components after solvent rinsing, even if the
deactivation layer is very thin, indicates the incorporation
of the characteristic functional groups in the deactivation
layer.
After heating the column. No. 17 at 350°C for 40 hours, no
significant changes in the column activity were noted, which
indicates the good thermal stability of this deactivation
layer. In previous studies [23-25, 66], the loss of the phenyl
groups from the silica surface at temperature above 250°C was
observed when 1,3-diphenyltetramethyldisilazane {DPTMDS) was
used as the deactivation reagent [43, 67]. The two phenyl
groups are attached to different silicon atoms, bath together
with two methyl groups. Blum and Eglinton [68] pointed out
that asymmetrical substitution leads to a dipole moment at the
respective silicon atoms, which makes it more susceptible for
nucleophilic attacks by silanol groups or traces of water.
Improved thermal stability of the polymer will be obtained by
symmetrical substitution. This is in agreement with the
re sul ts shown above. Moreover, the deacti vation layer is
solvent resistant, neither the normalized peak areas nor the
retention indices were changed by rinsing the column with
10 ml each of n-pentane, acetone and dichloromethane.
2.4.4 Deactivation with PBCPVMHS
Deactivation conditions {e.g. reaction temperatures and times)
for this reagent are presented in Table 2.5.
-30-
A survey of the corresponding normalized peak areas and Kováts
retention indices is presented in Table 2. 7. Corresponding
representative chromatograms of the test mixture are shown in
Fig. 2. 4.
1 4
2
~5 6
8
9
7
a
10
12 4
5 6 3
b
8
9 10
L" t L _,_,_.........,.__1u1 __ _J _
0 J
2 4 6 8 min 0 2 4 6 8 min
Figure 2.4 Representati ve chromatograms of the test :mixture on the precolwnn plus PBCPVMHS deactivated column No. 21 {a) before solvent rinsing; (b) after solvent rinsing. For test conditions see text and peak identification, see Table 2.1.
-31-
Table 2.7 Normalized peak areas (NA) and Kováts retention indices (RI) at 100°c of the test components of the PBCPVMHS deactivated columns.
Col. Test components No.
1-0ctanol 2,6-DMP 2,6-DMA Decylamine !Nicotine
NA RI NA RI NA RI INA RI INA RI
0 86 1051.3 96 1081. 0 101 1136.5 86 1233.3 92 1309.1
Before rinsing with solvent
19 78 1060.5 89 1118. 6 99 1164.9 19 1236.5 93 1344.5
20 83 1063.1 90 1130.2 99 1173.2 25 1236.8 92 1328.5
21 83 1064.9 89 1139.6 100 1181.1 34 1237.5 96 1337.6
22 85 1065.9 91 1143.2 100 1182.9 45 1237.5 95 1350.2
23 83 1066.5 93 1143.4 100 1183.2 66 1237.8 97 1351. 5
After rinsing with solvent
19 81 1058.5 90 1107.0 99 1156.2 0 --- 87 1335.6
20 83 1061.9 90 1126.1 98 1169.5 0 --- 89 1317.5
21 84 1062.0 90 1125.5 99 1169.8 0 --- 92 1325.1
22 85 1063.3 93 1134.0 98 1174.5 0 --- 90 1322.5
23 84 1063.9 91 1134.5 100 1174.5 0 --- 91 1323.7
24 83 1065.6 91 1138.3 99 1178.4 21 1238.6 92 1322.7
As is shown in Table 2.7 the normalized peak areas and the
retention indices of the polar components in the mixture for
PBCPVMHS deactivated columns increase with silylation
temperature. Most probably this again can be explained from
the steric hindrance effect. In the case of higher
temperatures, the steric effect is decreased and a densely
cross-linked network is formed at the column wall.
From Fig. 2.4, it can be seen that the decylamine peak totally
vanished after solvent rinsing. This demonstrates that some
-32-
residual silanols still existed on the column surface after
the physically attached polymer layer was washed away.
Consequently this caused the adsorption of aminodecane, one
of the most critical components in the test mixture, After
shortly reconditioning the column (No. 23, cf. Table 2.5 and
2. 7) by heating, the decylamine peak elutes again, however not
quantitatively. Reheating the rinsed column resulted in a
further reaction between the silylhydride and the residual
silanols on the column wall. Apparently, higher temperatures
and/or longer reaction times are required to complete the
deactivation of fused silica capillaries with PBCPVMHS.
The wettability of the capillary columns by polar stationary
phases depends on the critical surface tension (CST) of the
column wall. The (CST) can be improved by increasing the
content of polar functional groups in the deactivation layer.
On the other hand, the polarity of the deactivation layer
should not be increased too much. A high content of polar
functional groups in the deactivation layer, which enhances
the wettability by the stationary phases, may result in an
increase of the overall column polarity expressed in the
Kováts retention indices. This effect is illustrated in a
chromatogram of the Grob test mixture given in Fig. 2.5 for
a column which has been deactivated with a 50% cyanopropyl
molar ratio of PBCPVMHS. The column was coated with OV-1701
(df • 0.3 µm), which contains only 7% cyanopropyl groups in
the polymer. The inverted elution order of 2,3-butanediol and
n-decane compared to the chromatograms in ref. [69)
illustrates that the deactivation layer significantly
-33-
contr ibutes to polarity of the column. Matching of the
polarities of the deactivated column wall and the stationary
phases should be carefully optimized in order to achieve not
only a high column efficiency but also a defined column
polarity.
4
3 s
6 7 s 9
LJ11ll •• ______ • ._ .......... ._~.____......_ __ _
0 10 20 min
Figure 2.s Representative chromatogram of Grob mixture #2 on a column (320 µm I.C. * 4 m) coated with OV-1701, df= 0.3 µm (see text for the details). GC condition: He, 25 cm/sec., oven temperature from 40°C to 200°c at 4°C/min; peak identification: 1 = decane, 2 = butanediol, 3 = octanol + dodecane, 4 = 2,6-dimethylphenol, 5 = 2,6-dimethylaniline, 6, s, 9 =methyl caprate, undecanoate and laurate respectively, 7 = dicyclohexylamine.
2.5 CORCLUSZOHS
The synthesized vinyl modified polymethylhydrosiloxanes with
different functional groups incorporated in the polymer chain
are very useful for silylation of silanol groups on the column
wall of fused silica capillaries. Excellent deactivation of
the fused silica surface was obtained with PMHS and PVMHS.
-34-
Relatively high temperature and/or long reaction times are
needed for diphenyl and bis-(cyanopropyl) substituted reagents
compared to the methyl substituted deactivation reagent. This
is due to steric hindrance effects and an electron withdrawing
effect of the functional groups in the polymer chain. Complete
silylation of the silanol groups on the column wall appears
most difficult for the bis-{cyanopropyl) substituted polymers.
Physisorbed water on the inner surface plays an important role
to achieve a thin and densely crosslinked layer on the column
wall. Too high a water content, however, will deteriorate the
deactivation layer due to the presence of unreacted Si-OH
groups caused by the hydrolysis of Si-H groups (see eq. 2.2).
Compared to methyl deactivated columns an increase of the
Kováts retention indices of polar test compounds indicates a
successful incorporation of functional groups in the
deactivation layer. If different functional groups are present
in the deactivation layer, the individual contribution of each
of these groups to the solute retention cannot be
distinguished. A careful optimization of the content of
functional groups in the deactivation layer and the stationary
phase is required in order to achieve efficient columns with
a reproducible polarity.
Confirmation of these conclusion with solid state 29si NMR on
a model substrate, Cab-0-Sil, and a more detailed
interpretation of the deactivation process will be presented
in Chapter 3.
-35-
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-37-
57. A. Aerts, J. Rijks. A. Bemgard and L. Blomberg, J. High Resolut. Chromatogr. Chromatogr. Commun., 9 (1986) 49.
58. L. Blomberg, J. Microcol. Sep., 2 (1990) 62. 59. B.A. Jones, T.J. Shaw and E.S. Francis, Pittsburgh
Conference, 1989, Abstract No. 1319. 60. T.J. Shaw and B.A. Jones, llth International Symp. on
Capillary Chromatogr., May 14-17, 1990, Monterey, CA, USA.
61. Q. Wu, c. Cramers and J. Rijks, lOth International Symp. on capillary Chromatogr., May 22-25, 1989, Riva del Garda, Italy.
62. M.G. Voronkov and N.V. Kalugin, J. Appl. Chem. USSR, 32 (1959) 1612.
63. I.A. Shikhiev, R.Yu. Gasanova and B.M. Guseinzade, J. Gen. Chem. USSR, 38 (1968) 188.
64. E. Wiberg and E. Amerger, "Hydrides of the Elements of Maingroups I-IV", Elsevier, New York, 1971, Chap. 7.
65. M. Hetem, G. Rutten, J. Rijks, L. van de Ven, J. de Haan and c. Cramers, J. Chromatogr., 477 (1989) 3.
66. G. Rutten, J. de Haan, L. van de Ven, H. van Cruchten and J. Rijks, J. High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 664.
67. T. Welsch and H. Frank, J. High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 709.
68. W. Blum and G. Eglinton, J. High Resolut. Chromatogr. Chromatogr. Commun., 12 (1989) 290.
69. K. Greb and G. Grob, J. High Resolut. Chromatogr. Chromatogr. Commun., 6 (1983) 153.
-38-
CHAP'l'ER 3
A SOLID STATE 29s1 NMR STUDY OF THE DEACTIV ATION OF SILICASURFACESUSING NON-POROUSSILICAPARTICLES AS THE MODEL SUBSTRATE
The effect of the silylation temperatures and reaction times on the deactivation process of silica sur.taces is investigated with solid state 29si NMR spectroscopy. These model experiments using fumed silica as a model substrate for fused silica can reveal the structure and the chemical properties of the deactivation layer on the surface of the particles. Interpretation of the results yields valuable information on the chemical details of the deactivation reaction. The advantages and disadvantages of solid state 29si NMR as a characterization method f or the surf ace properties are discussed.
3.1 INTRODUCTION
Tremendous improvements in the deactivation of capillary
surfaces have been achieved by high temperature silylation
and, more recently, by low temperature dehydrocondensation [ 1-
6]. The development of column technology clearly benefited
from the increasing understanding of the reaction mechanisms
involved in the deactivation process.
The characterization of deactivated surfaces has proved useful
for the description of the modif ied surface and is performed
mostly by chromatography. Other techniques such as cross
polarization magie angle spinning {CP-MAS) NMR and lH NMR
spectroscopy, normal and total reflectance IR spectroscopy,
GC-MS, scanning electron microscopy, secondary-ion mass
spectrometry, electron spectroscopy for chemical analysis and
capillary rise measurements have also been used to fulfil this
purpose. The surface area of the inner column wall, however,
-39-
is far too small to fit the requirement of most of these
characterization techniques. The combination of solid state
29si NMR spectroscopy and experiments with a fumed silica
material like Cab-o-sil as a model for the column wall may
give the researchers · some representative pictures of what
occurred inside the column during the deactivation process.
In fact, it has been proved to be a powerful tool for the
characterization of such species [7-14].
In the framework of a long term systematic study on column
technology, Chapter 2 presented the results of a study on the
deactivation of fused silica capillary columns with polyvinyl
methylhydrosiloxane (PVMHS) and polydiphenyl vinylmethylhydro
siloxane (PDPVMHS). Excellent deactivations were obtained at
a temperature of 300°C for PVMHS and 350°C for PDPVMHS,
respectively. According to Lee and coworkers [3-6] this type
of reagents serves two purposes in the process of
deactivation. The first one is to get rid of the active sites
of the column wal! by the reaction between the silylhydride
and surface silanols present at the column wall. The second
important characteristic of the deactivation layer is that it
increases the critical surface tension of the column inner
surface. Due to this increase in the surface tension of the
column wall wettability is obtained for phenyl containing
stationary phases without loss of the deactivation of the
column wall.
It has been stated in literature that with PMHS deactivation,
all silylhydride groups react with silanol groups to form a
-40-
layer of a densely crosslinked polymer with its methyl groups
protruding upwards from the surface [6]. It was found,
however, by 29si NMR studies [ 14] that the reaction of
silylhydride groups with the surface silanols is not complete
due to the intrinsically low reactivity of the silylhydride
groups and steric hindrance.
In previous studies [11, 15-19], the loss of phenyl groups
from the silica surface was observed at temperatures above
250°C when 1,3-diphenyltetramethyldisilazane (DPTMDS) was used
as the deacti vat ion reagent [ 11, 18, 19] • The two phenyl
groups are attached to different silicon atoms, both together
with two methyl groups. Blum and Eglinton [20) pointed out
that asymmetrical substi tution leads to a dipole moment at the
respective silicon atoms, which makes it easier accessible for
nucleophilic attacks by silanol groups or traces of water. An
improved stability of the polymer can be obtained with the
symmetrically substituted reagent. This was the third reason
to select PDPVMHS as the deactivation reagent in this study.
In this chapter, a parallel investigation of the deactivation
with PVMHS and PDPVMHS was carried out by solid state 29si NMR
spectroscopy. A fumed vitreous silica, Cab-o-Sil MS, was used
as the model substrate for detailed studies on the nature and
the chemical structure of the deactivation layer and for
confirmation of the intended chemical reactions at the column
wall. Estimation of the relative amounts of the functional
groups as determined by 29si cross-polarization magie angle
spinning (CP-MAS) NMR combined with elemental analysis can
elucidate the reaction mechanisms of the deactivation
-41-
reactions.
The effect of the vinyl groups, which were incorporated in the
synthesized copolymer on the deactivation process are
described. The advantages and disadvantages of 29si NMR as a
characterization method of the surface properties are also
discussed.
3.2 THBORY
The deactivationprocess which occurs on the surface of silica
particles can be studied by 29si CP-MAS NMR. The specific
chemical structure of the functional groups are determined by
NMR signals of deactivated silica samples. This gives
information on the properties and relative amounts of the
siliceous groups incorporated in the deactivation layer.
Typical capillary columns with inner diameters between 50-
530 µm have surface areas far too small to allow the
application of most spectroscopie analysis techniques for
surface characterization. Therefore, model materials such as
glass sheets (21, 22], silica gels (7-10] or fumed silica (11-
14, 18] have to be used for model studies on surface
deactivation and modification utilizing these techniques for
surface characterization.
A non-poreus fumed silica, Cab-O-Sil MS, was chosen because
of its compatibility with 29si CP-MAS NMR (high surface area
of about 200 m2 /g) and its close similarity to the fused
silica column wall (no pores, hence the reaction is not
limited by diffusion processes).
-42-
Below a brief overview of the principles of 29si CP-MAS NMR
is given.
If a nucleus is placed in an external magnetic field it will
interact with this external field. Moreover, it will interact
with the magnetic field generated by spinning neighbouring
nuclei. The spin states are determined by the spin quantum
number. The corresponding energy levels are governed by
Planck's law:
where E: the energy level, h: the Planck constant, vo: Larmor frequency.
( 3 .1)
v0 is related to Bo (magnetic strength of the external field)
according to:
\10
where
y *Bo 2 * 'lt
(3. 2)
-y: the gyromagnetic ratio, a physical property of the nucleus.
The sensitivity of NMR measurements is determined by the
natural abundance of a nucleus and its 'Y value. The shielding
of the nuclei by surrounding electrons causes the resonance
frequency to shift. The extend, to which the resonance
frequency shifts depends on the chemica! structure of the
molecules and provides qualitative information on the
structure.
NMR line in NMR spectra of stationary, solid samples are
-43-
broadened by a number of factors. Two of the more important
interactions are chemical shift anisotropy (C.S.A.) and
dipolar interactions between nuclei.
C.S.A. arises because the shielding of nuclei in a sample by
the bonding electrons depend on the direction of the sample
{e.g. the main axes of a crystal) with respect to the external
magnetic field. In fact, the shielding should be described by
a tensor, 3 . In liquids, rapid molecular tumbling reduces
this to the trace of the tensor: ae = (axx + ayy + Uzz)/3. The
line broadening, resulting from C.S.A. can be described a
Hamiltonian of the general form:
Hc.s.A. = Y * h * Ï * ä * B where .... I spin quantum number, = a shielding tensor, .... B external magnetic field.
(3.3)
In a sample consisting of small crystallites (powder) this can
be written as:
(3. 4) where c1 : a constant, r: the distance between the nuclei, 8: the angle between the magnetic field direction and the
internuclear axis.
When the sample is spun around an angle with the magnetic
field for which (3 * cos28 - 1) = 0 1 the C.S.A. broadening is
greatly reduced. This technique is called Magie Angle Spinning
(MAS) and 8 = 54°44' is called the magie angle. Moreover,
antifacts ("spinning side bands") remain in the spectrum of
-44-
the sample. They disappear gradually when the spinning speed
is enhanced.
Dipolar interactions between nuclei arise by coupling through
space. The appropriate Hamiltonian for these interactions is:
2 2 - '::t -HD = y * h * I * D * I (3.5)
where ::$
D : the dipolar coupling tensor.
In contrast with C.S.A., the dipolar interactions in Hertz
does not depend on the external field strength. Equation 3.5
can be simplified to:
where C2: a constant.
3 * cos26 - 1 r3
(3. 6)
The broadening effect of dipolar interactions will only
disappear when the MAS spinning frequency is larger than the
dipolar interaction in frequency units. In practice, this
means that small interactions (like e.g. in 29si-o-1H over
larger distances can be removed by MAS. In 13c solid state
NMR, however, one bas to cope with very large dipolar
interactions between 13c and lH nuclei (40 kHz for stationary,
solid samples). These interactions are removed by dipolar
decoupling (irradiation of the protons, while exciting and
observing e.g. 13c NMR signals).
Unfortunately, however, the sensitivity of 29si MAS-NMR is
quite low because of the low natural abundance of 29si (4.7%).
-45-
As the sensitivity is proportional to the square root of the
measuring time, a significant gain in sensitivity can only be
obtained at the expense of very long measuring time. On the
other hand, measuring time also depends on the relaxation time
T1 , the time necessary for a nuclear spin to return from
energy level E2 to E1 to restore equilibrium. Only when
equilibrium is restored can excitation occur again.
cross-Polarization (CP) techniques can shorten the relaxation
time. In this technique a large magnetization of e.g. protons
polarize the 29si nuclei and this is possible only between
closely spaced atoms. By adjusting the frequencies of the
proton and the 29si to meet the Hartmann-Hahn condition, a
link between the proton and the 29si magnetization is formed
(24). This results effectively in a reduction in the
relaxation time of 29si nuclei.
Only 29si nuclei near a spatially fixed proton which are
obviously present solely at the surf ace of the silica
particles contribute to the NMR signal in 29si CP-MAS NMR.
This makes 29si CP-MAS NMR a surface analysis technique suited
for silica materials.
3 • 3 EXPERJ:MEHTAL
3.3.1 Materials
1,3,5,7-tetramethylcyclotetrasiloxane (D4H), 1,3,5,7-tetra
vinylmethylcyclotetrasiloxane (D4Vi),diphenyldimethoxysilane
and polymethylhydrosiloxane (PMHS, 85 es, fluid) were all
purchased from Petrarch Systems (Bristol, PA, USA). These
-46-
reagents were used without further purification.
PVMHS was synthesized from o4H and o4vi (molar ratio= 90:10)
according to the procedure developed in our laboratory [25,
26). Similarly, PDPVMHS was synthesized from diphenyldi
methoxysilane, o4vi and D4H in a molar ratio of 50:7:43.
The Cab-O-Sil M5 (Cabot Corp., Tuscola, Ill., USA) was a gift
from Heybroek & Co. Handelmij. NV (Amsterdam, NL). The
specific area of the sample is 202 m:i /g determined by the saET
method after the heat and rehydration treatment. This value
will be used in this chapter. All solvents used were P.A.
grade from E. Merck (Darmstadt, FRG). The deionized water was
obtained from a Milli-Q System (Millipore, Bedford, MA, USA).
3.3.2 Silylation of Cab-O-Sil and Solid state 29si NMR Measurements
The Cab-o-sil was heated to 720-750°C for 48 h to remove
"water inclusions" [ 27). After cooling, the Cab-O-Sil was
rehydrated by refluxing it with pure water for 5 hours, then
the water was removed by a rotary evaporator. Afterwards, the
rehydrated sample was dried in a vacuum oven at 110°c
overnight and stored over P205 in a vacuum desiccator for
several weeks.
This dried Cab-O-Sil was coated with the deactivation
reagents. Approximately l g of Cab-O-Sil was added to
solutions of 10% of PVMHS and PDPVMHS in n-pentane
respectively and mixed by sonification. Aftar slowly
-47-
evaporating the solvent at room temperature with a rotary
evaporator, a polymer film with a film thickness of
approximately 5 nm was deposited on the silica surface.
The coated Cab-O-Sil samples were then dried in an oven at
100°C at reduced pressure for 1 h. A quartz ampoule which was
filled with about O. 3 g of coated Cab-O-Sil sample then
evacuated was repeatedly filled with Helium up to atmospheric
pressure. After sealing the tube to a volume of about 8 ml,
the ampoule was wrapped in an aluminum foil and heated to the
desired silylation temperature for 16 hours. After silylation
the ampoule was opened and the content was rinsed twice with
dichloromethane and subsequently dried overnight at 110°C
under reduced pressure.
Solid. state 29si NMR spectra of the samples listed in Table
3.1, were obtained with a Bruker CXP300 spectrometer at 59.63
MHz, in a similar way as discussed extensively by Hetero et al.
[ 14].
The carbon contents of the silylated Cab-O-Sil samples were
determined with a Perkin Elmer 240 Element Analyzer (Perkin
Elmer, Norwalk, CT, USA). The IR spectra of the synthesized
copolymers were measured with a Perkin Elmer 237 Grating
Infrared Spectrophotometer.
-48-
Tabla 3.1 Survey of the silylated Cab-O-Sil samples.
sample No.
1
2
3
4
5
6
7
8
(A}
-30 -32
Silylation conditions
Temperature ( oc) Reagents
240
280
320
360
280
300
320
360
-34 -36 -38 ppm
-32
PVMHS
PVMHS
PVMHS
PVMHS
PDPVMHS
s
PDPVMHS
PDPVMHS
(B)
-36 -40 -44 ppm
-48
:riqure 3.1 High resolution liquid 29si NMR spectra of the synthesized reagents, pulse interval time 10 s, acquisition time 0.1 s. (a) PVMHS, molar ratio= 90:10 (Si-H:Si-Vi), NS (numbers of free induced decays) = 666; (b) POPVMHS: molar ratio = 50:7:43 (Ph:H:Vi), NS= 5300.
-49-
3.4 RESULTS ABD DISCUSSION
3.4.1 Characterization of the Synthesized Reagents.
A representative high resolution liquid 29si NMR spectrum of
the synthesized PVMHS and PDPVMHS deactivation agents is given
in Figure 3.1. The relative amounts of the functional groups
determined by the intensity ratios of the 29si NMR signals are
comparable with the molar ratios as given in the experimental
section.
The structure and functionality notation codes of the most
relevant chemical shifts for the liquid reagent as well as the
silylated Cab-O-Sil samples are presented in Figure 3. 2.
Obviously all the required functional groups are present in
the synthesized deactivation reagents.
code: T, a,
CH, CH, 0 CH•CH,H H OH CH, OH O-
-o-l-cH-CH -l-~o-~-o-l1-o-~-o-L-o-~-o-L-o-:-~-È:
rl • ·ri ~I r' r' r' r' r' r.' r.1
t 0- 0- 0 CH CH CH CH 0 - 0- 0-
·20 -20 -46 -~ .3: .3: .5; -66 -101 ·110 ppm:
Figure 3.2 Structures and functionality notation codes of the most relevant siliceous surf ace moieties and their corresponding chemical shifts.
The IR spectra showed in Figure 3. 3 also confirmed the
presence of all the functional groups in the reagents.
-50-
100
1.1111 3000
Si-H 2000 1600
WÁVENUHBER ( ctf I )
î C•C
Si-C113 f 1000
Si-0-Si
\00
·-~ITT· , ! !J 1 ·1 ~ • " ,
0 /iOOO
, 1· _,.1, I' 1, ' ·1 • • . "
. : -~~ l· ·' .. • • .
t ". j
·il ~ J i i
Si-Ph ~ ~- ;, ; Si-H . ·jl·L · • ! ; .•• 1 • ; d1 11. " . ; 1.ILE~: l tl.
3000 2000 1609 WAVENlJMBER cci-r )
Fiqure 3.3
tiooo Si-0-Si
Infrared spectra of the synthesized copolymers. Upper: PVMHS, lower: PDPVMHS.
3.4.2 Evaluation of PVMKS Silylated Cab-o-sil Samples with Solid State 29si NKR. Spectroscopy
Solid state 29si CP-MAS NMR spectra of the PVMHS silylated
Cab-O-Sil samples (Table 3.1: #1-4) are given in Fig. 3.4. The
corresponding relative ratios of the 29si CP-MAS NMR signals
of bonded siliceous moieties of these samples are presented
in Table 3.2.
-51-
Table 3.2 Relative ratios of the 29si NMR signals of bonded siliceous moieties of the PVMHS silylated Cab-O-Sil samples.
Sample Relative ratio of the 29si NMR siqnals No.
D2ED2 D2H D'2H D2Vi T2 T3
1 4 61 29 2 -- 4
2 4 49 41 -- -- 6
3 5 39 45 -- -- 11
4 7 31 43 -- 2 17
Figure 3.4 clearly illustrates that different results were
obtained with different reaction conditions. rt is evident
that excellent deactivation was achieved by this reagent (note
the disappearance of the geminal and vicinal silanols which
usually give signals at -90 and -101 ppm respectively). The
main silylation products, having chemical shifts in the -35
ppm region show that methylhydrosiloxanes remained after the
silylation. A flexible polymer chain is formed on the surface
as is indicated by the appearance of narrow peaks at chemical
shifts around -35 ppm and -36 ppm in the spectra (Figure 3.4).
The presence of intra and intermolecular crosslinking in the
surf ace polymer layer of the Cab-O-Sil samples is indicated
by the signal of methyltrisiloxysilane (T3) (see eq. 2.1 in
Chapter 2). The intensity of the signal increased with
increasing temperatures. The T3 groups were formed mainly by
the reaction between silylhydrides and surface silanols. The
number of T3 groups increases at higher temperature due to the
degradation of the polymer chains.
The 29si NMR signal of D2ED2 , which is a non-desirable
crosslinking product of the silylation, indicates the reaction
of silicon hydride and vinyl groups incorporated in the
. -52-
polymer. The presence of vinyl qroups is not evident after the
silylation reaction. This is due to the relatively low amount
of vinyl groups in the polymer (even lower after the
silylation reaction) and the lack of sensitivity of the 29si
NMR technique.
4-'
3
2
----"'"\,.__
-20 -40 -60 -80 -100 -120 ppm
Figure 3.4 Solid state 29si NMR spectra of PVMHS silylated Cab-O-Sil samples (Table 3.1: #1-4). Q4 is the signal from the silica surf ace.
The amount of vinyl groups in the copolymer prior to the
deactivation is 5% on a molar basis. Because of the higher
density of silanol qroups on the cab-O-Sil particles as
compared to fused silica capillary columns, the silylation
-53-
reaction may not be exactly the same as that on the capillary
column surface. Higher surface concentration of silanol groups
may cause more pronounced crosslinking and chemical bonding
of the deactivation agent, which can also result in a higher
conversion of the vinyl groups. It can be seen from Table 3.2
that the amount of D2ED2 increases with increasing
temperature, this is in agreement with the above explanation.
The carbon content of the PVMHS silylated Cab-O-Sil samples
is 10.8%. This matches very well with the expected carbon
content of the samples and shows a complete crosslinking and
chemical bonding of the polymer layer to the particle surf ace.
3.4.3 Evaluation of PDPVMRS Silylated Cab-o-sil samples with Solid state 29si mm. Spectroscopy
Figure 3.5 presents the solid state 29si CP-MAS NMR spectra
of the PDPVMHS silylated Cab-O-Sil samples (Table 3.1: #5-8).
The corresponding relative ratios of the 29si NMR signals of
the bonded siliceous moieties of these samples are given in
Table 3.3.
Ta:ble 3.3 Relativa ratios of the 29si NMR signals of bonded siliceous moieties of the PDPVMHS silylated Cab-O-Sil sam les.
Sample No.
5
6
7
8
D ED
7
6
7
11
D H + D' H
26
17
18
14
-54-
D T
40 10 17
39 9 18
39 36
39 36
-20 ~60 ppm
-100
Figure 3.5 Solid state 29si CP-MAS NMR spectra of PDPVMHS silylated Cabo-sil samples. (Table 3.1: #5-8).
Analogous to the situation in the PVMHS experiments the intra
and intermolecular crosslinking in th.e surface polymer layer
of the Cab-O-Sil samples is observed. The amount of T3 groups
increases with increasing temperature again indicating the
formation of a more rigid and densely crosslinked layer at the
Cab-O-Sil surface.
Again, o2Eo2 (-20 ppm) was produced by the reaction of silicon
hydride and vinyl qroups incorporated in the polymer as a non-
desirable crosslinking product. Referring to a recently
-55-
published study on silylation reactions with PMHS reagent
(14], it can be estimated from the 29si CP-MAS NMR spectra of
the silylated Cab-O-Sil samples, that a relatively thin layer
was formed (l-2 nm) instead of 5 nm calculated from the amount
of the reaqent. It should be emphasized, however, that the
measurements with CP excitation are semiquantitative.
Experimentally, an averaqe carbon content of 17. 6% was
obtained by element analysis of the rinsed silylated Cab-O-Sil
samples. Compared to the expected carbon content (about 30%)
in the coated Cab-O-Sil, a considerable amount of the
deactivation reaqent is washed away by solvent flushing of the
deactivated Cab-0-Sil samples after silylation. Most probably,
further crosslinking of the polymer is obstructed by the large
ster ic hindrance effect of diphenyl groups. The thermal
degradation of the polymers should also be taken account in
this respect (26). Although, a significant part of the reagent
is lost by solvent rinsing, owing to inferior crosslinking,
the final result is a rigid, thin and stable network
consisting of short polymer chains bonded to the silica
surf ace. This is indicated by the appearance of broadened
peaks in the spectra (Figure 3.5).
Onder the silylation conditions applied in this study, the
29si CP-MAS NMR resonances showing thermal decomposition of
diphenyldisiloxysilane groups were not observed. Bath phenyl
groups remained chemically bonded to the siloxysilane
copolymer at silylation temperatures up to at least 360°C.
Loss of phenyl groups would have been accompanied by
-56-
transforxnation into other moieties, which have not been
observed in the spectra. With 1,3-diphenyltetramethyl
disilazane, which has been aften used as a silylation reagent,
phenyl losses were observed already at temperatures above
250°C [11]. This leads to a decreased wettability of the
capillaries for phenyl containing stationary phases.
3.S CONCLUSIONS
PVMHS and PDPVMHS are efficient deactivation reagents for
fused silica capillary columns. The main reaction between
silylhydride groups and the surface silanols produces a
densely crosslinked and chemically bonded layer which covers
the particle surface and blocks the interaction with active
sites on the surface.
29si NMR provides valuable information on the structures and
properties of organo-siliceous groups at a non-porous silica
surface after silylation. The reaction between Si-H and si-Vi
groups by thermal treatment is confirxned by solid state 29si
NMR experiments. The quantity of the reaction product, D2ED2
increases with increasing temperature. The existence of vinyl
groups on the silylated Cab-O-Sil samples, however, is not
evident, due to the low amount of vinyl groups in the
copolymer and the lack of sensitivity of the 29si NMR
technique.
Semi-quantitative results of the thickness of the polymer
layer can be obtained from the 29si NMR spectra and elemental
analysis. Thick layers (> 3 nm) give narrow and sharp peaks
while thin films (< 2.5 nm) show broadened signals. From this
-57-
point of view, the thickness of the modification layer of
PDPVMHS silylated Cab-O-Sil samples can be estimated to be
about 1-2 nm.
The spectra obtained show that the deacti vation layer is
stable up to at least 360°C. No loss of phenyl groups from the
modification layer was observed. The thermal stability of the
silylation layer is enhanced using the reagent with two phenyl
groups connected to one silicon atom. This is expected because
of the electron donating properties of the geminal phenyl
groups and their chemical stability.
3.6 REFERENCES
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3. K.E. Markides, B.J. Tarbet, C.M. Schregenberger, J.S. Bradshaw and M.L. Lee, J. High Resolut. Chromatogr. Chromatogr. commun., 8 (1985) 741.
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13. L. van de Ven, G. Rutten, J. Rijks and J. de Haan, J. Resolut. Chromatogr. & Chromatogr. Commun., 9 (1986) 741.
14. M. Hetem, G. Rutten, B. Vermeer, J. Rijks, L. van de
-!!>S-
ven, J. de Haan, and c. Cramers, J. Chromatogr., 477 {1989) 3.
15. K. Grob, G. Grob, and K. Grob, Jr., J. High Resolut. Chromatogr. & Chromatogr. Commun., 2 {1979) 31.
16. K. Grob, G. Grob, and K. Grob, Jr., J. High Resolut. Chromatogr. & Chromatogr. Commun., 2 (1979) 677.
17. K. Grob and G. Grob, J. High Resolut. Chromatogr. & Chromatogr. commun., 3 (1980) 197.
18. T. welsch and H. Frank, J. High Resolut. Chromatogr. & Chromatogr. Commun., 8 (1985) 709.
19. T. Reiher, J. High Resolut. Chromatogr. & Chromatogr. commun., 10 (1987) 158.
20. w. Blum and G. Eglinton, J. High Resolut. Chromatogr. & Chromatogr. commun., 12 (1989) 290.
21. M.L. Lee, D.L. Vassilaros, L.V. Phillips, O.M. Hercules H. Azumaya, J. W. Jorgenson, M. P.. Maskar inec and M. Novotny, Anal. Letters, 12 (1979) 191.
22. B.W. Wright, M.L. Lee, s.w. Graham, L.V. Phillips and O.M. Hercules, J. Chromatogr., 199 (1980) 355.
23. c.s. Yannoni, Acc. Chem. Res., 15 (1982) 201. 24. S.R. Hartmann and E.L. Hahn, Phys. Rev., 128 (1962)
2042. 25. Q. wu, C.A. cramers and J.A. Rijks, in P. Sandra (Ed.),
Proc. of the lOth International Symposium on Capillary Chromatography, Riva del Garda, Italy, May 1989, Hüthig, 1989, p. 188.
26. Q. wu, M. Hetero, C.A. Cramers and J.A. Rijks, J. High Resolut. Chromatogr., 13 (1990) 811.
27. J.A.G. Taylor and J.A. Hockey, J. Phys. Chem., 70 (1966) 2169.
28. N. Grassie and I.G. Mcfarlane, Europ. Polymer Journal, 14 (1978} 875.
-59-
CHAPTER 4
PREPARATION OF NON-POLAR AND :MEDIUM POLAR FUSED SILICA CAPILLARY COLUMNS FOR GC
Non-polar and medium polar capillary columns are prepared based on the deactivation desaribed in previous ahapters. Crosslinking and/or chemical bonding of the stationary phases by thermal treatment and the azo-tert-butane (ATB) initiation is investigated. The effect of hydrogen and of vinyl groups, whiah exist in the deactivation layer resulting :trom the deactivation reaation, on the crosslinking process are discussed. Evaluation of the prepared columns is carried out on the aspects o:t efficiency, inertness, thermal stability and resistance to commonly used solvents. Narrow bore capillary columns down to 23 µm i.d. with a non-polar stationary phase were successfully prepared by a similar procedure.
4.1 INTRODUCTION
Although efficient sample introduction systems, sensitive
detectors and other auxiliary devices are essential components
in modern high resolution gas chromatographic systems, the
column remains the haart of the analytical instrument. The
rapid growth in the application of capillary columns in
chromatography promotes the development of column technology
[1-3].
Tremendous eff orts have been invested in the pretreatment of
the inner surface of glass and fused silica capillaries in
order to improve the quality of the resulting columns. The aim
of the pretreatments is to eliminate and/or to shield the
active sites on the inner wall of the columns, while
simultaneously improving the wettability of the column wall
by the stationary phases. This has been extensively discussed
-60-
in Chapters 2 and 3.
For obtaining high separation efficiencies in any type of
capillary column it is essential that the stationary phase is
deposited as a smooth, thin homogeneous film. This film must
also maintain its integrity without forming droplets when the
column temperature is varied. The wettability of the column
is related both to the nature of the stationary phase and the
column surface to be coated. A number of studies (4-13] on the
wettability of column surfaces has been reported. capillary
rise method (1, 11) was used to determine the critica! surface
tension (CST, 70 ). From the Zisman plots [14, 15], in which
the eosine of the advanced contact angle (9) is plotted
against the surface tension of the test liquid (11) 1 can be
obtained (see Fig. 4.1 and 4.2). These graphs can be used to
characterize the column surface.
Fiqure 4.1. Diagram f or the contact angle
At equilibrium, the relation of three interfacial tensions can
be described by Young's equation:
COS 8 = Y sg - Y sl
Y1g ( 4 .1)
where îsg• îsl and îlg are the surface tension between solid
-61-
and gas (vapour of the wetting liquid) , solid and liquid,
liquid and its vapour respectively.
The significance of the critical surface tension ('Yc) is that,
in general, liquids with n :S 'Yc will completely wet the
surface while liquids with 'Yl > îc tend to form droplets.
0.80
o.so
0.40
0.20
0.00 ,__ _ ___._ __ _,__ _ ___,..__ _ _._ _ ____.
10 20 30 40 50 60 v1 (N/m)
Fiqure 4.2. Zisman plot of a silylated fused silica capillary column, data from ref. ( 11].
Wettability of the column can be enhanced by roughening the
surface and by chemica! modification of the surface.
Unfortunately, the first method is not applicable to fuse
silica columns as has been shown by Lee et al. (1, 16].
Replacement of surface silanols with silyl ether groups
(silylation} or other inert reagents containing functional
-62-
groups similar to those in the stationary phase is the most
successful way for both deactivation and enhancement of the
wettability of the surface (see Chapter 2).
Coating of the capillaries can be accomplished by either
dynamic or statie methods. The dynamic method described by
Dijkstra and De Goey [17] is a simple coating technique but
often results in non-uniform films [l-3, 18]. Many solutions
have been proposed to improve this method, such as controlling
the coating speed and temperature [19-22], controlling the
solvent evaporation rate (23] and the volatility o.f the
solvent [18], concentration of the coating solution [24J, and
using the "mercury plug" method [25, 26]. Different approaches
were suggested to calculate the film thickness {df) in dynami
cally coated capillaries (19, 23, 27-35). The following
equation was found to be the most useful (33], except for low
viscosity solutions.
where r: the column radius (m), u: the coating velocity (m/sec), c: the concentration of the solution (% V/V), ~= the viscosity of the solution (kgm-lsec-1), ~= the surface tension of the solution (N/m).
(4.2)
In contrast to dynamic coating, in statie coating the column
is filled with a solution of the stationary phase in a
volatile solvent. After sealing one end of the column the
solvent is evaporated and the stationary phase deposited as
a film on the column wall. This technique comm.only provides
-63-
more efficient columns [36-55]. The film thickness of the
stationary phase can be easily calculated from the equation:
(4.3)
where P is the phase ratio e.g. ~ = Vm/V6 , Vm and V6 are the
volumes of the mobile and stationary phase respectively. 1/~
is equal to the concentration (% V/V) of the coating solution.
An important advantage of statie method is that the p is
known, therefore, the df can be accurately determined. The
density of the stationary phase must be known for this
calculation. For a number of frequently used stationary phases
density values are tabulated in ref. [40]. In practica, it is
important that the coating solution is dust free and degassed
to eliminate bumping during the solvent evaporation step.
Furthermore it is important that no air or vapour bubbles
exist in the column, especially at the sealed end [36, 40, 43-
55].
The main drawback of the statie method is its time consuming
nature. Higher coating temperatures [56] and more volatile
solvents [57 / 58] were applied to accelerate the coating
procedure [59, 60). A high temperature-high pressure coating
method {61) and the free-release statie coating procedure (62-
65] were also proposed to speed up the coating procedure.
These methods are especially suitable f or coating narrow bore
capillary columns as the typical statie technique with
dichloromethane or n-pentane as the solvent is extremely time
consuming for this type of columns.
-64-
A precipitation coating method was introduced by Dluzneski and
Jorgenson ( 66 J to coat s µ;m. and 10 µ.m columns for open-tubular
LC. This method involves filling of a heated capillary column
with a hot stationary phase solution and then cooling the
column rapidly so that the stationary phase precipitates from
the solution onto the column wall.
Immobilization of the stationary phase by in situ crosslinking
and/or chemical bondinq on the column surface after coating
is one of the significant advances in column technology. The
physical and chemical stability of the stationary phase and
its ability to resist the strain caused by mechanica!
vibration, high temperatures and the injection of large volume
of polar solvents is enhanced (16, 67-69].
Early efforts on im:mobilization of the stationary phases
involved ionic reaction mechanism in the crosslinking process,
which results in Si-o-si linkages [70-81). High column
activities and low column efficiencies were obtained by these
approaches due to the large number of residual silanols and
alkoxy qroups. In addition, sample molecules generally become
less soluble in the stationary phase as a result of the high
degree of cros slinking. A more recent development ( 83] is
crosslinking by using OH- or methoxy terminated silicones with
different functional groups [84-98]. Excellent therm.al
stability was obtained by this method owing to the stability
of the Si-O-Si bonds formed by the condensation between the
surface silanols and the terminal silanol groups in the
stationary phase (16, 99].
-65-
currently, free radical initiation, first introduced by Grob
[100] is the most frequently used method to immobilize
stationary phases. Peroxides [67, 100-116] such as dicumyl
peroxide (DCP), and azo compounds (e.g. azo-tert-butane, ATB)
[16, 59, 103, 115-124] are normally applied for this purpose.
Radiation by high energy sources [108, 125-131] has also been
used as the means of initiation of stationary phase
immobilization. However, the necessity to have special
facilities severely hinders the application of this technique.
Also ozone has been used for the crosslinking of various
stationary phases [70., 106, 109, 132], even at room
temperature [70]. It has been found, however, that the
presence of tolyl groups in the stationary phases is a
prerequisite for this method. In the absence of tolyl groups
the immobilization of the stationary phases was not possible.
An excellent review on the immobilization of stationary phases
has recently been published [133].
The main drawback of immobilization initiated by peroxides is
that peroxide residues and by-products can be incorporated
into the phases (133-135]. This may result in an increased
adsorptive activity, decreased thermal stability and solute
retention indices shifts (133]. The oxidation of tolyl groups
in the stationary phases has also been observed when high
percentages of peroxide were used to cure the column [115,
116].
''
Immobilization of silanol-terminated stationary phases may
require fairly high temperatures or the addition of tri- or
tetra- functional crosslinking agents to improve the
-66-
immobilization yield. It has been stated that additional
silanol groups must be present throughout the polymer chain
in order to immobilize the stationary phases via thermally
induced condensation [136). In some case, a compromise had to
be made between the degree of immobilization and maximum
selectivity of hydroxyl terminated cyanopropylsilicones (137].
Azo-tert-butane (ATB) introduced by Wright et al. (103)
contributes only slightly to the polarity of the immobilized
stationary phases because its by-product is an alkane.
Unfortunately, however, a small portion of the radical
generator is inevitably incorporated in the phase matrix
[ 133 J. Additionally, the performance of ATB has. been found to
deteriorate with time (138, 139]. The columns treated with
aged ATB showed high activity levels, high bleed rates,
erratic immobilization eff iciencies and solute retention
shifts. The regeneration or clean-up of ATB is required to
maintain or increase its activity for the immobilization
process [133, 139].
Another type of immobilization was developed by Schomburg and
coworkers (70, 140, 141). In the crosslinking reactions,
stationary phases containing 1-alkene groups were
hydrosilylated by polymethylhydrosiloxane in the presence of
a chloroplatinic .acid catalyst, as shown in the following
equation:
j H2PtCl6 j 1
-ri-H + H2C=CH-ri- ---------> -ri-CH2-CH2-ri- (4.4)
In this chapter, the preparation of non-polar and medium po lar
-67-
capillary columns was investigated for columns deactivated
with the deactivation reagents described in Chapter 2. Vinyl
modified deactivation reagents were synthesized in order to
facilitate the inunobilization of the stationary phases. In
contrast to the platinum catalyzed method, the immobilization
was performed by thermal treatment to simplify the preparation
procedure and to avoid possible undesired products originating
from the catalyst. The radical ini tiation method was also used
to compare the performance of vinyl and non-vinyl containing
stationary phases and to elucidate the effect of hydrogen and
vinyl groups in the crosslinking process. Polysiloxanes with
varying methyl and phenyl contents were coated on the
deactivated fused silica capillary columns. The performance
of the prepared columns was evaluated with respect to
inertness, efficiency, thermal stability and crosslinking
efficiency.
4.2 EXPERIMENTAL
4.2.1 Materials and Apparatus
The materials, solvents and columns are described in the
experimental section of Chapter 2.
A Packard Becker GC (Model 430, Packard Becker, Delft, NL) and
a Varian GC (Model 3400, Varian Instruments, Walnut Creek, CA,
USA) were used to evaluate the deactivated and coated
capillaries. Evaluation of the narrow bore capillaries was
carried out on a carlo-Erba GC (Model 2900, Carlo-Erba
Instruments, Milan, Italy). All GC experiments were performed
with hot split injection and flame ionization detector (FID).
-68-
4.2.2 synthesis of the Deactivation Rea9ents
The synthesis of PVMHS and PDPVMHS is described in Chapter 2.
In order to obtain deactivation layer with different phenyl
9rOUp contents, a fixed volume of PDPVMHS copolymer was mixed
with various volumes of PMHS [142, 143] (see Table 4.1).
'l'a:ble 4 .1 Survey of the deactivation reagents and the stationary phases used in this study.
Stationary phases* Deactivation reagents Ratio of Ph:Vi:H silane
OV-73 (5.5% diPh) 6:1:93
OV-7 (20% Ph-Me) 21:3:76
OV-61 (33% diPh) 35:5:60
ov-11 (35% Ph-Me) 135:5:60
OV-17 (50% Ph-Me) 50:7:43
* The remaining substituant of the polymer chain is dimethyl.
4.2.3. Column Preparation
Fused silica capillary columns were pretreated and deactivated
under different deactivation conditions as described in
Chapter 2. The deactivated columns were statically coated with
an appropriate concentration of the stationary phase solution.
Then, i:mmobilization of the stationary phases was achieved
either by thermal treatment or radical initiation with ATB.
The following procedure was used for the thermal method: the
coated column was heated from 40°C to 250°C overnight (> 16 h)
under a slow flow of helium (5-10 cm/sec). The column was then
-69-
rinsed with 10 ml dichloromethane and conditioned by
progranuning the temperature from 40°C to 280°C at 4°C/min
under a normal flow of helium (25-30 cm/sec) and kept at 280°C
for 10 hours. For ATB cured columns, a procedure similar to
that described in ref. [103] was used for the immobilization
of the stationary phases. The regeneration of ATB was carried
out according to the procedure described in ref. [133]. Aged
ATB was used for the immobilization of one column to show its
effect on the quality of the crosslinked column.
4.2.4 Evaluation of the Prepared capillaries
The inertness of the columns was tested both aftar
deactivation and aftar coating using the test mixture listed
in Table 2.1. The split ratio was adjusted so that
approximately 1 ng of each component was injected onto the
column.
For the deactivated capillaries, a dual column system as
described by Schomburg et al. [144] was used, which consists
of a high quality home made PMHS deactivated ov-1 precolumn
(10 m * 250 µm i.d., film thickness df = 0.25 µm) coupled in
series to the column to be tested. Isothermal chromatograms
of the test mixture were recorded for the precolumn alone and
the precolumn connected to the deactivated column. The test
chromatograms were evaluated with respect to the peak shape,
peak retention ( expressed as kováts retention indices) and the
normalized peak area of the critical components. Additionally
for the coated capillaries, Grob 1 s test mixture [122, 145] was
used for the sake of comparison.
-10-
Column efficiency is characterized by calculating the coating
efficiency (C.E.). C.E. is defined as the ratio of the
theoretica! plate height to the experimental plate height,
Hexpi under optimal conditions [16, 36, 142):
C.E. = ( · Htbeor ) H•xr; min
(4. 5)
The minimum theoretica! plate height, Htheor1 is represented
by the simplified Golay-Giddings plate height [ 146, 14 7]
equation:
H =( r )*(ll tbeor {J3'(l+k)}
1
* k 2 + 6 * k + 1)2 (4.6)
where r is the column radius and k the capacity factor.
This simplification can only be used for thin film columns
under conditions of low pressure drop.
The Kováts retention index [148, 149] of a solute is
calculated by using the following equation:
( ( log t
1 log t
1 ) l I(x) = 100 * Z + 100 * / z,x - r,;
( log tr,(z+l) - log tr,x )
(4.7}
where I (x) is the retention index of component x,
t'r,z1 t'r,z+l are the adjusted retention times of component
x and of the alkanes just before and after the peak of
component x, respectively.
Thermostability of the deactivated and coated columns was
determined by platting the baseline drift against temperature.
The crosslinking efficiency, CLE, was obtained by determining
the capacity factor of tetradecane before and after rinsing
the crosslinked columns with dichloromethane. The crosslinking
efficiency is given by:
-71-
(4.8}
where ki and k2 are the capacity factors before and after
rinsing the column with the solvent respectively.
4.3 RESULTS AND DISCUSSIONS
4.3.1 Evaluation of the Deactivated capillaries
Representative chromatograms of the precolumn and the
precolumn connected to one of the deactivated columns are
shown in Fig. 4.3 (for more detailed discussion see Chapter
2). These chromatograms show no significant differences in
peak shape, which indicates the excellent deactivation
obtained by the reagents and the method used in this study.
1 34 ·z:
s b
' a
4
23 ~
1 5 7
1 6
8 7
9 8 10
9 1
i j ( l \ '--0 5 10 b 16 min
Figure 4.3 Representative. chromatograms of the test mixture. (a) precolumn; (b) precolumn plus a deactivated column; temperature 100°C isothermal, peak identification: l=decane, 2=octanol, 3=DMP, 4=undecane, S=DMA, 6=dodecane, 7=decylamine, S=tridecane, 9=nicotine, lO=tetradecane.
-72-
The thermostabili ty of these deacti vation layers is
illustrated in Fiq. 4.4. It can be seen that the deactivated
columns have a qood thermal stability due to the thin and
densely crosslinked polymer layer formed on the column wall.
In addition, the inertness of the deactivated columns was
tested before and after the bleeding test. In these
experiments no significant changes in peak shape, normalized
peak areas and retention indices were observed (see Chapter
2) •
10
8
< .s. El s 1 I! "" c '6 " 4 " ii5
2
2
0 100 200 300 400 500
Temperature (°C)
Fiqure 4.4 Bleeding test of the deactivated columns, 1-PMHS, 2-PVMHS, 3-PDPVMHS.
4.3.2 Evaluation of the Columns coate4 with Non-polar Stationary Phases
A number of columns with different non-polar stationary phases
has been prepared as described in the experimental section.
A survey of the experimental conditions and the characteristic
properties of these columns are outlined in Table 4.2.
-73-
Table 4.2 survey of the characteristics.
experimental
Col. Stat. Reagent I.D. df No. Phase (µm) (µm)
1 OV-1 c PMHS 50 0.23
2 OV-1 PMHS 250 0.25
3 OV-1 PMHS
4 OV-1 c PMHS • 25
5 ov-1 c PMHS 220 0.58
6 OV-101 PMHS 250 0.25
7 SE-54 PMHS 250 0.25
8 SE-54 PMHS 250 0.25
9 ov-1 c PVMHS 250 0.25
10 ov-1 PVMHS 250 0.25
11 ov-1 PVMHS 250 0.25
12 SE-30 PVMHS 250 0.25
13 SE-54 PVMHS 250 0.25
14 OV-101 PVMHS 250 0.25
15 OV-73 PVMHS 250 0.14
16 OV-73 PVMHS 250 0.14
17 OV-1 c PVMHS 23 0.12
conditions and column
curing C.E. CLE Method
a b
therm. 93 93 97
therm. 89 47 10
ATB 89 89 27
therm • 83 82 95
ATB d 95 91 96
therm. 88 70 15
therm. 90 90 94
ATB 86 86 95
therm. 93 93 96
therm. 86 71 21
ATB 85 89 83
94 78 24
99 99 95
85 85 85
88 87 17
88 88
therm. 89 89 95
C.E. = coating efficiency, CLE = crosslinking efficiency. a) coating efficiency before crosslinking treatment. b} coating efficiency aftar crosslinking treatment. c} vinyl modified. d) aged ATB reagent.
Some typical representative chromatograms of the coated
columns are shown in Figure 4.5. It can be seen from the data
in Tabla 4.2 and Figure 4.5 that highly efficient and very
inert columns can be prepared by the method described in this
study. In addition, high crosslinking efficiencies were
obtained for the columns coated with vinyl modified stationary
phases and crosslinked by thermal treatment without the
-74-
addition of a catalyst. The column efficiency and the column
activity were not significantly affected by this crosslinking
procedure. This indicates that the reaction shown in equation
( 4. 4) reaches a high conversion without the addi tion of a
catalyst.
p b 5
234
.5 a
6 7 8
7 9 JO
L 1 .__
a.~ .1-LLL ~ r l
0 10 20 min o !O min
Fiqure 4.5 Representative chromatograllls of the test mixture on the coated columns, (a) column No. 4, FID, f.s.d. 2•10-12 A; (b) column No. 7, FID, f.s.d. 16•10-12 A (cf. Table 4.2); temperature 100°c isothermal, peak identification see Fiqure 4.3.
In the case of PMHS deactivated columns, the hydrogen groups
left on the surface after deactivation play an important rele
in chemically binding the stationary phase to the column wall.
This role however, fainted for non-vinyl containing stationary
phases. Even if ATB was used as a radical initiator these
columns show very poer crosslinking efficiencies (cf. column
No. 2, 3 and 6 in Table 4.2). For PVMHS deactivated columns,
both hydrogen and vinyl groups play a role in the crosslinking
-75-
and chemica! bonding process. Thermal treatment of the non
vinyl containing phases did not yield a sufficiently high
crosslinking efficiency (cf. column No. 10, 12 and 15). By
using ATB or dicwnyl peroxide as the initiator, the cross
linking efficiency was improved. In this case the main
function of the vinyl groups in the deactivation layer is to
facilitate crosslinking and chemical bonding of the stationary
phases.
The advantage of immobilization of the stationary phases by
thermal treatment is that the introduction of any kind of
reagents needed to initiate the process is avoided. The
introduction of additional reagents is a potential source of
additional activity and thermal instability of the resulting
columns. This is illustrated in the chromatograms shown in
Figure 4.6, in which the column No. 5 was evaluated with the
test mixture before crosslinking and after crosslinking with
aged ATB. The activity of the column is increased, resulting
in the disappearance of the aminodecane peak. Moreover, the
bleeding of the column is also increased at elevated
temperatures.
Also narrow-bore capillary columns which are suitable for fast
GC analysis and for SFC, can be prepared using the procedures
described earlier in this chapter (cf. Figure 4. Sa). Inertness
of the low inner diameter columns is very important because
of the limited sample capacity of the column.
-76- .
a b
1 J
4
23 5
5
6
7 8 8 9
1 • ..,._,_._.I ......___.__. __ ___,[__[_ 1 1 L ~::::::::::::;:::=:::::::::==::;=:::::'.::=--
0 20 40 60 0 20 40 60 min
l!'igure 4.6 Representative chromatograms of the test mixture on column No. 5, (a) before crosslinking, and (b) after crosslinking with aged ATB (cf. Table 4.2); temperature 100°c isothermal, peak identification see Figure 4.3.
Another example is presented in Figure 4.7. This figure shows
the separation of the test mixture on a 23 µm i.d. column. The
injected amount was approximately 7 picogram per component.
As can be seen in the figure, the column has a very high
degree of inertness illustrating the potential of the methods
described here for the preparation of narrow-bore columns.
The thermostability of the coated columns is shown in Figure
4. S. From this figure it can be concluded that thermally
stable columns can be obtainedwith low bleeding levels in the
temperature ranges of interest.
-77-
0
Fiqure 4.7
4
3 5 6 2 7
5 min
Representative chromatogram of the test mixture on a narrow bore capillary column (23µm i.d. * 1 m), column.temperature 100°C isothermal, about 7 pg of each component onto the column, FID, f.s.d. l*lo-12 A, peak identification see Figure 4.3.
9
8
7
< 6
,!?,
" 5 ! "' c:
4 'ti " " iii
3
2
Figure 4.8
0 - 100 200 300
Temperature (°C)
1 2
3
400 500
Bleeding test for the coated columns, 1 = SE-54, 2 = ov-1, 3 = OV-17, 4 m OV-61.
-78-
4.3.3 Evaluation of the Columns coated with Medium Polar stationary Phases
A number of medium polar columns with different phenyl
contente (Table 4.1) in the stationary phases was prepared.
Corresponding experimental conditions and the performances of
these columns are outlined in Table 4.3.
'l'able 4.3 Survey of the experimental conditions and column character.
1
1
Col. Stat. Reagent df Curing C.E. CLE No. Phase (µ.m) Method
a b
18 OV-73 PDPVMHS 0.11 therm. 86 76 16
19 OV-73 PDPVMHS 0.11 ATB 85 85 87
20 OV-73 PDPVMHS c 0.11 ATB 86 85 86
21 OV-7 PDPVMHS 0.18 therm. 78 61 13
22 OV-7 PDPVMHS 0.18 ATB 77 79 83
23 OV-61 PDPVMHS 0.11 therm. 77 70 9
24 OV-61 PDPVMHS 0.11 ATB 77 80 79
25 OV-11 PDPVMHS 0.19 therm. 81 68 9
26 OV-11 PDPVMHS 0.19 ATB 80 80 78
27 OV-17 PDPVMHS 0.26 therm. 83 48 9
28 OV-17 PDPVMHS 0.26 ATB 83 82 70
C.E. = coating efficiency, CLE = crosslinking efficiency. a} coating efficiency before crosslinking treatment. b) coating efficiency after crosslinking treatment. c} with Ph:Vi:H ratio= 21:3:76.
Representati ve chromatograms of some of these columns are
presented in Figures 4.9-4.11. In agreement with the results
reported in Chapter 2, it fellows from Fig. 4.9, that the
effect of the retention behaviour of the deactivation layer
on the overall performance of the final column cannot be
neglected. The relativa positions of 2,6-dimethylaniline (OMA)
and n-dodecane and nicotine compared to n-tridecane have
-79-
initiator, such as ATB or OCP, is needed to obtain a
reasonable crosslinking efficiency.It can be seen that the
crosslinking efficiency decreased with an increasing phenyl
content in the stationary phases. This is most likely due to
the ster ie effect of the phenyl groups which tend to hinder
the crosslinking process [103, 151].
14
Fiqure 4.10 Representative chromatograms of different test mixtures on column No. 26, (a) the test mixture of Table 2.1, and (b) the Grob test mixture (cf. reL [145]), C10 = decane, C11 = undecane, 01 = octanol, al = nonanal, EH = 2-ethylhexanoic acid, P = 2,6-0MP, A = 2,6-0MA, E10' E11' E12 = methyl esters of decanoic acid, undecanoic acid, and dodecanoic acid respectively.
Oue to the electron donating properties of the geminal phenyl
groups and its chemical stability the diphenyl substituted
polysiloxanes showed an increased thermal stability as was
expected. This is illustrated in Chapter 5.
-81-
L o
Figure 4.11
1 4 6 2
3
8
10
5
9
L 6 12 min
Representative chromatogram of the test mixture on column No. 28, temperature 90°C isothermal, peak identification see Figure 4.3.
4.4 CONCLUSIONS
Using the methods described in this chapter highly efficient
and inert fused silica capillary columns with non-polar and
medium polar stationary phases were obtained. C.E. > 80% are
routinely obtained during this study. In addition these
columns have a high thermal stability and solvent resistance.
The highest thermostability was achieved by crosslinking
diphenyl substituted polysiloxane phases.
For vinyl modified non-polar stationary phases, immobilization
can be achieved by thermal treatment. This gi ves minimal
-82-
effects on column characteristics, such as the activity and
the retention behaviour. For non-vinyl containing stationary
phases, the immobilization of the PMHS deactivated columns is
less satisfactory both for thermal treatment and ATB
initiation. For these stationary phases, immobilization of the
PVMHS and POPVMHS deactivated columns is obtainable by the ATB
initiation method.
The crosslinking efficiency of the phenyl containing phases
decreases with an increasing phenyl content of the stationary
phase. This is caused by the steric hindrance effect of the
phenyl groups in the stationary phase.
Non-polar narrow bore capillary columns can be prepared using
the same method. Again highly inert and efficient columns were
obtained.
Aged ATB was observed to yield deteriorated performance of the
im:mobilized columns. Careful regeneration of the ATB can avo id
this problem.
Optimization of the deactivation procedure is required to
obtain proper wettability of column surface by the stationary
phases. The contribution of the deacti vation layer to the
retention mechanism of the coated column should be minimal to
avo id the dual solute retention mechanism. Otherwise the
selectivity of the stationary phase may be altered.
-83-
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89. S.R. Lipsky and M.L. Duffy, J. High Resolut. Chromatogr. Chromatogr. Conunun., 9 (1986) 376.
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151. W. Noll, Chemistry and Technology of Silicones, Academie Press, New York, 1968. ·
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CBAP'l'ER S
APPLICATIONS IN GAS CHROMATOGRAPHY
The applicability of the prepared non-polar and medium polar fused silica capillary columns was illustrated by different examples. High speed GC separations were performed on narrowbore capillary columns with inner diameters down to 23 µm. Columns with a high thermal stability were prepared for the analysis of less volatile and high molecular weight compounds. The separation of some underivatized steroids, drugs, priority pollutants and methyl esters of amino acids is also shown. The performance of the capillaries with respect to inertness, thermostability and efficiency in these applications is evaluated.
S.1 INTRODUC'l'IO:N
The knowledge of column technology has grown dramatically [1,
2). Nowadays, high resolution GC is one of the most widely
employed separation techniques in analytica! organic
chemistry.
capillary columns allow the complete separation and specific
analysis of many complex samples which were difficult to
analyze using packed columns. capillary GC has been applied
to a wide variety of analytica! problems because of its
repeatability, reliability in quantitative and qualitative
information, and shortened analysis time for a given plate
number.
The main application areas of high resolution gas
chromatography include chemical, petrochemical (3-5] t
pharmaceutical industries, life science (6-10), food (11) and
consumer products, and environmental analysis [12-15). The
-90-
demands of'these applications with regard to analysis time,
trace analysis and the ability to separate less volatile
compounds promoted column technology. Narrow bore capillary
columns for high speed analysis, high temperature columns for
high boiling point components and very well deactivated
columns for trace analysis were the results of these demands.
Theory [16-20] shows that the reduction of the inner diameter
of the capillary column is one of the most practical and
convenient ways to reduce the analysis time. High plate
numbers per unit of length ( inversely proportional to the
column diameter, see equation 5.1, [17, 18]), or per unit of
time (inversely proportional to the column diameter squared,
see equation 5. 3, [ 20 J) were obtained wi th small diameter
columns. For thin film columns the contribution of the
stationary phase for the plate height can be neglected and
follows:
where
2
F(k) = ( 1 + 6 * k + 11 * k 2 )
112
3 * { 1 + k 2 )
(5 .1)
(5.2)
and d0 is the column inner diameter, k is the capacity factor
of the component.
( dN) 48 * Dif dt U,O.Pt = --d-~--- *
( 1 + k )2 (5.3)
where DG 0 is the diffusion coefficient of the solute in the
mobile phase at the column outlet pressure.
In case of large values of P, P being the inlet to outlet
-91-
column pressure ratio, usually encountered in narrow-bore
columns, (dN/dt)u,opt is no longer proportional to 1/d20 but
to 1/dc (19].
Apart from high pressure drops other problems, such as sample
capacity and speed of sample introduction and detector
compatibility may be encountered in the use of narrow bore
capillary columns. Promising results, however, were obtained
by different authors [16-29].
The advantage of high temperature columns is that less
volatile or high bolling compounds can be analyzed on this
type of columns. High molecular weight samples such as the
oligomeric series of polyethylene glycol, polywax and triton
X-100 and simulated distillation products which up to now are
normally analyzed by supercritical fluid chromatography (SFC)
are now also amenable to high temperature GC (30-32]. Compared
with the results obtained by SFC, high temperature GC offers
superior speed of analysis, increased column efficiency and
improved resolution of the component bands. (30-44].
On the basis of the knowledge of column technology described
in the previous Chapters, different fused silica capillary
columns were prepared. Their applicability to the selected
samples is illustrated in this Chapter. Examples of high speed
GC separations, high temperature GC analysis and the
separation of methyl esters of amino acids are given. The
performance of the capillaries with respect to inertness,
thermostability and efficiency is evaluated.
-92-
5. 2 EXPERJ:HENTAL
s.2.1 Apparatus
The high temperature separations were carried out on a Varian
3400 gas chromatography (Varian, Walnut creek, CA, USA), or
on a Fractovap 4160 gas chromatography (Carlo Erba, Milan,
Italy) equipped with a split injector and a flame ionization
detector (FID). The hot split injection technique was applied
throughout the experiments. The chromatograms were recorded
with an SP 4000 integration system (Spectra Physics, Santa
Clara, CA, USA), or with a strip chart recorder (Kipp & Zonen,
Delft, The Netherlands).
The experiments with narrow bore capillary columns were
carried out on a Fractovap 2900 GC (Carlo Erba, Milan, Italy),
equipped with a hot split injector and a FID. According to the
specifications provided by the manufacturer, the time constant
of the electrometer amplifier was 50 ms for the 1 V integrator
output and about 150 ms for the 10 mV recorder output. A
Tescom high pressure regulator (Model 44-1100, Tescom Ine.,
Minnesota, USA) proceeded by a dust filter was used to control
the helium flow rate. The split injector was modified to allow
a high split ratio for delivery of a small amount of sample
onto the column.
The other experiments were performed on a Packard 430 gas
chromatograph (Packard, Delft, The Netherlands) or on the
Varian 3400 GC equipped with a split injector and a FID.
-93-
s.2.2 Materials and Chemicals
All the reagents used for the deactivation of the fused silica
capillary columns were detailed in Chapter 2. The empty
columns used in this study were purchased from Siemens
(Siemens, Germany) and can be used isothermally at 400°C. The
procedures for the preparation of different inner diameter
capillaries were summarized in Chapter 4 of this thesis.
All the solvents were PA grade and purchased from E. Merck AG
(Darmstadt, Germany).
Below follows a short description of the samples used.
The chemica! structures of the polymer additives, a gift from
DSM (Geleen, The Netherlands), are given in Table 5.1.
Pólyvinylmethylhydrosiloxane (PVMHS) was dissolved in pentane
(10%, v/v).
The fatty acid methyl esters (FAME) mixture (No. 4-7080) and
the volatile organic compounds mixture (VOC mix #3, No. 4-
8779) were obtained from Supelco (Bellefonte, PA, USA).
The standards of the sedative hypnotic barbiturates were a
gift from Professor Zlatkis (Houston, Texas, USA).
The methyl or ethyl esters of some amino acids and fatty acids
were obtained by derivatization from the corresponding acids
according to the procedure described in ref. [45].
The polyethylene glycol sample (PEG 400) was purchased from
E. Merck AG (Darmstadt, Germany).
-94-
Ta:ble 5.1 Chemical structures of the polymer additives
Trade name Chemica! name or •tr11ct11re MW
Oleamlde )·< 0 271 H, (CHt7( OK NH1
Cyasorb 531 Ó!-Ooc,H"
326
Tlnuvln 770 H ·0.c-(C!l,.1-o H () v 0 Q 480
C(CH,)1
lrgafH 168 P·(OO·C(CH,>~. 647
ID•
lrganox 1076 .,,.,.,,olc•,.c•Q-o• 531 . .. ,~ .
Tlnuvln 144 ...
685 C,H, COO.Q·CH,).
0 0
OSTOP Il Il 683 H,,C.j>-CClt,ctl,SC~,11,,,
lrganox 1010 M·l. 1178
5.3 RESULTS AND DISCUSSIONS
5.3.1 High Speed Analysis on Narrow Bore Capillaries
For narrow bore capillary columns (de< 100 µm), high inlet
pressures are normally needed for obtaining the optimum
velocity of the carrier gas. Special injection techniques
(such as cold trap injection) are required for very fast
analysis [19, 28, 29) to avoid an excessive contribution of
the injection to the overall band width. Under optima!
conditions, hot split injection can be used for narrow bore
capillary columns with inner diameters around 10 µm. High
-95-
column efficiencies were obtained using the hot split
injection technique (cf. Chapter 4).
0
Figure s.1
"' ...
2 4 min
Representative GC chromatogram of volatile organic compounds (VOC mix #3, Supelco) on a narrow bare column, 2 m * 23 µm, df = 0.12 µm OV-1, T0 from 60°C to 200°C at 20°C/min, inlet pressure, Pi = 15 Bar; peaks in elution order: 1,1-dicploropropylene, 1,2-dichloroethane, trichloroethylene, 1,2-dichloropropane, cis-1,3-dichloropropylene, trans-1,3-dichloropropylene, 1,1,2-trichloro-ethane, 1,3-dichloropropane, 1,2-dibromoethane, 1,1,1,2-tetra-chloroethane, 1,1,2,2-tetrachloroethane, 1,2,3-trichloropropane, 1,2-dibromo-3-chloropropane, hexachlorobutadiene.
Figure 5.1 presente the chromatogram of a mixture of volatile
organic compounds which was separated on a 23 µm column. On
this column 14 components can be separated within 3.5 min,
while it takes 35 min on a 60 m * 0.75 mm i.d. column.
Moreover, the GC oven could not reach the desired temperature
programming rate of 20°C/min. Otherwise, the analysis time
would have been even shorter than 3 min.
A representative chromatogram of the separation of a fatty
acids methyl ester mixture on the same column is depicted in
Figure 5.2. The total analysis time is less than 7 min. The
same mixture with compounds ranging from 2-hydroxydecanoic
acid to eicosanoic acid and having a wide bolling point range
required a separation time of more than 30 min on a 30 m * 0.25 mm i.d. column.
-96-
1 2 " ",,; ::J ..... ;
4
3
•• ...
... n
"" 0)
" . 1() i:"·f
... ...
5
.. " ... M
0 7 min Fiqure 5.2 Typical GC chromatogram of FAME mixture on the same column as in Figure 5.1, T0 at 200°C isothermal, peaks identification: 1-methyl dodecanoate, 2-methyl tetradecanoate, 3-methyl 3-hydroxytetradecanoate, 4-methyl cis-9-hexadecanoate, 5-methyl trans-9-octadecanoate, 6-methyl nonadecanoate, 7-methyl eicosanoate. For ether conditions see Fig. 5.1.
Il 3 13
19
4 10 22 9
1.
'l 24
2 l l 0 10 min
Fiqure 5.3 Representative GC chromatogram of methyl esters mixture of 24 benzoic and related acids, column, 3 m * 50 µm, df = 0.25 µm ov-1, Tç from 100°c to 290°C at 20°C/min, Pi = s Bar, peaks identification: 1) benzoic acid, 2) p-OMe benzoic acid, 3) cinnamic acid, 4), 9), 10) o-, m-, p-OH phenyl acetic acid, respectively, 5), 7), 8) o-, m-, p-OH benzoic acid respectively, 6)mandelic acid, 11) 3-0Me-4-0H-phenyl acetic acid, 12) 3-0Me-4-0H-benzoic acid, 13) m-OH-cinnamic acid, 17) 2, 5-diOH-phenyl acetic acid, 19) 3-0Me-4-0H-cinnamic acid, 22) sinapic acid, 24) 3,4-diOH-mandelic acid.
-97-
Figure 5. 3 represents another example of the high speed
separation of a methyl ester mixture containing 24 benzoic and
related acids. Except the isomers of m- and p-hydroxy-benzoic
acids, all other isomers were baseline separated within 8 min.
The problem again was the heating rate of the oven, which
delayed the elution of the later eluting components.
Typical chromatograms of the separation of a super gasoline
and a natural gas condensate are shown in the Figures 5.4 and
5. 5, respecti vely. It can be seen that these narrow bore
capillary columns are quite suitable for this kind of volatile
and complex hydrocarbon samples due to their relatively small
phase ratio {J.
0 7 min
Fiqure 5.4 Typical GC chromatogram of super gasoline on the same column as in Figure 5 .1, Pi = 15 Bar, Te from 40°C to 220°c at 20°C/min, no peak identification was performed.
-98-
O 5 JO 15 min P'igure s.s Typical GC chromatogram of a natural gas condensate. Column, 8 m * 50 µm, df = 0.25 µm OV-1, T0 from 40°C to 100°C at 8°C/min, then to 220°c at 15°C/min, no peak identification was performed.
s.3.2 Hiqh ~emperature columns
Fdr the elution of less volatile or high boiling compounds,
the GC oven has to be programmed to very high temperatures.
This dose not cause problems when glass capillary columns are
used. The outside coating of fused silica capillary columns,
however, can be damaged at temperatures above 370°C.
Generally, the capillary column becomes brittle after
prolonged use at high temperatures. Specially manufactured
polyimide coatings [39], or aluminum clad capillary columns
po, 31] have to be used in high temperature applications.
Polymer additives (antioxidants, uv stabilizers), which are
used in food packing materials or in pipes for water
distribution systems can represent a source of contamination
through the migration of these substances into the food or
-99-
water. Some of them are known to be toxic agents (46, 47].
Normally these additives are analyzed by SFC or HPLC [48, 49].
Figure 5.6 shows a chromatogram of a polymer additives mixture
on an OV-61 column. The last peak, Irganox 1010, having a
molecular weight of 1178 daltons (cf. Table 5.1), eluted from
the column at 420°C. Similar separations were achieved by Blum
and Damasceno [34] using an OH-terminated diphenyl phase.
Representative chromatograms of the separation of a mixture
of phenyl substituted cyclosiloxanes and of a triglyceride
sample on the same column as used above are shown in Figures
5.7 and 5.8, respectively. In the first example, octa- and
decaphenyl substituted cyclosiloxane (with a molecular weight
of 792 and 990 daltons, respectively) was easily separated and
quantitative results could be obtained. Similarly, quanti
tative analysis of triglyceride samples can be performed on
a routine basis with this type of columns.
2 3 4 s
'
j 0 10
Fiqure 5.6
7 1 Oleamide 2 cyasorb 3 Tinuvin 770 4 Irqafos 168 S Irqanox 1076 6 Tinuvin 144 7 DSTDP S Irqanox 1010
8
20 m.l.n
Representative GC chromatogram of polymer additives, column, 5 m * 0.25 mm i.d., df = 0.07 µm OV-61, Te from 150°C to 300°C at 20°C/min, then to 420°C at 10°C/min, (cf. Table 5.1).
-100-
2
Fig. 5.7 Fig. 5.8
:So
O 5 J 0 min 0 10 20 min i'igure 5.7 Representati ve GC chromatogram of a phenyl cyclosiloxane mixture on the same column as in Figure 5.6, Te from 180°C to 420°C at 15°C/min, peaks identification: 1 = octaphenylcyclotetrasiloxane, 2 = decaphenylcyclotetrasiloxane.
Figure S.B Representative GC chromatogram of a triglycerides sample on the same column as in Figure 5.6, Te from 40°C to 240°C at 20°C/min, then to 380°C at 10°C/min. Tso represents a triglyceride with a total carbon number of 50.
Figure 5. 9 presents the GC chromatogram of a polyethylene
glycol sample (PEG 400) with an average molecular weight of
400 daltons on the same column as used above. PEG samples can
also be analyzed by SFC, but the solubility of glycols in pure
carbon dioxide is limited. The addition of polar modifiers
such as methanol to the mobile phase is needed in order to
improve the separation of PEG samples.
Some other exa:mples are shown in Figures 5. 10 and 5 .11,
respecti vely. Figure 5. 10 is the chromatogram of a plant
extract containing hydrocarbons up to c50 • The separation of
the same sample with SFC takes about 40 min (19).
-101-
S 6 Fig 5.9 Fig. 5.10 i
7 4
c,_2 c " I 46
8
0 1 0 20 minO 10 20 min
Figure 5.9 Representative GC chromatogram of the polyethylene glycol (PEG 400) sample on the same column as in Figure 5.6, Te from 100°c to 370°C at 15°C/min, the numbers above the peak indicate the repeat unit in the polymer chain.
Fiqure s.10 Representative Ge chromatogram of a plant extraction product on the same column as in Figure 5.6, Te from 150°C to 350°C at 20°C/min, hot split injection.
0 40 min
Fiqure s.11 Representative GC chromatogram of a paraffin sample on a 15 m * 0.25 mm i.d. column, df = 0.18 µm ov-1, Te from so 0 c to 380°C at 8°C/min, hot split injection.
-102-
Another example of the high separation power that can be
obtained in GC is shown in Figure s.11. This figure shows the
separation of a paraffin sample on a 15 m * 0.25 mm i.d.
column.
Capillary GC can also be applied to analyze the deactivation
reagents used for the preparation of GC columns. Both the
cyclic and the linear polysiloxane molecules show a high
degree of dispersion with oligomers starting from the tetramer
up to some 27 siloxane units. Figure 5.12 shows the separation
of Polyvinylmethylhydrosiloxane (PVMHS) copolymer on an ov-1
column.
Solid state 29si NMR spectroscopy studies of the deactivation
process with this reagent using fumed silica particles as the
model substrate are described in Chapter 3. Figure 5. 13
represents a chromatogram of the extraction product of the
PVMHS silylated Cab-O-Sil samples. Comparing Figures 5.12 and
5.13 it can be concluded that the short chain siloxanes can
be extracted from the surface of Cab-O-Sil particles after
silylation. The crosslinked and longer chains are covalently
bonded to the surf ace and can not be extracted by
dichloromethane. The solid state 29si NMR results of this
silylated Cab-O-Sil sample indicated that a crosslinked thin
layer is formed on the surface of the Cab-O-Sil particles.
(cf. Chapter 3).
-103-
0 10 20 30 min
Fiqure s.12 Representative GC chromatogram of polyvinylmethylhydrosiloxane on the same colwnn as in Figure 5.11, Te from 80°C to 380°C at 10°C/min, hot split injection.
0 12 24 min
Figure 5.13 Representative GC chromatogram of the extraction product of the silylated Cab-O-Sil sample on the same column as in Figure 5.11, Te from 40°C to 200°C at 20°C/min, then to 340°C at 10°C/min, hot split injection.
-104-
S.3.3 Analysis of Drugs, Pollutants, Amino Aci4s, an4 On4erivatized steroids.
High resolution GC plays an essential role in the analysis of
pharmaceutical products and drugs. It is used in quality
control, analysis of new products and in monitoring
metabolites in biological fluids [50].
An example of the analysis of sedative hypnotic barbiturates
mixture is shown in Figure 5.14. With a short, narrow bore
column, this mixture could be analyzed in our laboratory
within 40 sec [29]. The symmetrical shape of all components
indicates that the column was very well deactivated and
quantitative analysis can be achieved.
34567 8 9 10
2
0 8 16 min
Figure S.14 Representative GC chromatogram of sedative hypnotic barbiturates mixture. GC conditions: column, 20 m * o.25 mm i.d., df = 0.25 µm ov-1, Te from 120°C to 300°C at 8°C/min; peaks in elution order: barbital, aprobarbital, amobarbital, pentobarbital, secobarbital, hexobarital, mephobarbital, phenobarbital, butabarbital, butabital.
Phenols are widely regarded as important priority pollutants
and are known to be difficult to analyze. They exhibit high
affinities to the activa sites on the column surface. It is
even more difficult to analyze halogen and nitro-substituted
phenols. Total or partial loss of the components and badly
-105-
tailing peaks can often be observed. Figure 5.15 presents the
GC chromatogram of a chlorophenols mixture analyzed on a 40 m
* 0.25 mm i.d. ov-1 column. The 40 m column consisted of two
20 m columns connected by a glass press-fit connector
(Unimetrics Corp., CA, USA). Apparently the separation
efficiencies and peak shapes were not affected by the
connection.
0 10 20 30 min
Figure s.1s Representative chromatogram of a chlorophenols mixture, GC conditions: 40 m * 0.25 mm i.d. column, ov-1, df = 0.25 µm, Te from 80°C to 200°C at 4°C/min, peaks in elution order: 1-chloro-, dichloro-, trichloro-, tetrachloro-, pentachlorosubstituted phenols.
The separation of a chlorobenzene mixture is given in Figure
5.16. Again it shows perfect inertness to these components and
almost baseline resolution for all critica! pairs.
The high separation power of the capillary columns makes them
suitable for the analysis of ester mixtures of fatty acids and
amino acids. Figure 5.17 shows a chromatogram of the c7-c9
fatty acids derivatized with 2-chloroethyl chloroformate, the
-106-
derivatization reaction is not complete therefore the sample
still contains free fatty acids. !t is surprising to see that
even the free fatty acids elute from the column with quite
good shapes. Normally, leading peaks were observed for free
fatty acids on non-polar columns.
1 2 -Fig. 5. 1 6 Fig. 5. 1
4 3 -- 6
5 M 4 ,..
1
•l 3 s
z "..,. 6 in -·· 1 •l
" ~ ~-) .
))
"· .;,; M
L~ ". ~:i,.; ~ - -
7
0 s 10 :n1n 0 a min
Fiqure s.u Representative chromatogram of chlorobenzene mixture, column, 20 m * 0.25 mm i.d., df = 0.25 µm ov-1, Te from so 0 c to 200°c at 4°C/min, peaks in elution order: l=chloro-, 2=dichloro-, 3=trichloro-, 4=tetrachloro-, 5=pentachloro-, 6=hexachlorosubstituted benzene.
Fiqure 5.17 Representative chromatogram of chloroethylation reaction product of c7-c9 free fatty acids, column, 19 m * 0.25 mm i.d., df = 0.25 µm ov-1, Te from 70°C to 200°C at 10°C/min, peaks identification: 1-3 and 4-6, c7-c9 free fatty acids and corresponding chlqroethyl esters respectively.
\
The chromatogram of another methyl ester mixture of fatty
acids on an OV-17 column is presented in Figure 5. 18. The
isomers containing more than two double honds are separated
from the saturated and one double bond isomers.
-107-
J 0
Fiqure 5.18
2 3
6
4 5 7 9 12 1011 1314
8 16 min
Representative chromatogram of the methyl esters of fatty acids, column, 20 m * 0.32 mm i.d., df = 0.26 µm OV-17, Te from l00°C to 2so 0 c at 10°C/min, peaks in elution order: C12:01 C14:01 C15:01 C16:0 + C16:l• C11:01 C1s:o + C1s:11 C1s:21 C1s:J1 C19:01 C20:01 C21:01 C22:0• C23:Q1 C24:0·
Fig. 5.19
7
1 2 4
9
6
J 8
5
j\ t lO
,\_ '
1
2
\._ -
3 56
4
!
Fig. 5.20 7 l l
s 10 9
- ..._.._ ____ _ O 10 O 10 min
Figure 5.19 Representative chromatogram of the separation of the ethyl esters of 10 amino acids, column: 4 m * 0.32 mm i.d., df = 0.26 µm OV-17, Te from 80°C to 270°C at 20°C/min, peaks in order: alanine, leucine, serine, proline, cysteine, hydroxyproline, aspartic acid, hysine, arginine.
Figure 5.20 Representative chromatogram of the separation of underivatized steroids, column, 20 m * 0.25 mm i.d., df = 0.25 µm ov-1, Te at 260°C isothermal, peaks in elution order: C2~· C241 ethylestrenol, c26 , androsterone, methandriol, dianabol, pregnanediol, c28 , estriol, C30·
-108-
The ethyl esters of 10 amino acids were chromatographed on an
OV-17 column (see Figure 5.19).
Steroids are often derivatized prior to analysis in order to
increase their volatility. Figure 5.20 shows, however, the
separation of an underivatized steroids mixture. This
separation was performed isothermally at 260°C. It can be seen
from Figure 5.20 that the column has a high efficiency and
inertness.
The same column also shows little interaction with oxygen
containing compounds at low temperatures (see Figure 5.21).
l 2
3
0
Figure s.21
4 7
6 5 8
9
10 14
12
11 13
10 min
Typical GC chromatogram of a test mixture on the same column as in Figure 5.20, Te from 40°C to 160°C at 4°C/min, peaks in elution order: 1-butanol, toluene, c8 , chlorobenzene, 3-heptanone, c9 , p-chlorotoluene, 1-heptanol, trimethylbenzene, o-dichlorobenzene, 2-nonanone, c11, 1-nonanol, benzylaceton.
The chromatograms shown in Figure 5. 22 are from hexane
extracts of oranges and of opium poppy straw respectively. No
-109-
attempt was made to identify the components present in these
two samples. The components in the poppy straw extract are
most likely steroids and alkaloids.
1 i
1 a
l 1 1 i
b
i 1
1 1
_l
! " 1ll .11 L 11 1
1 t •. l.
0 10 20 min 0 5 min
Fiqure s.22 Typical GC chromatograms of the hexane extraction products of (a) oranges, column, 20 m * 0.25 mm i.d., df = 0.58 µm OV-1, Te from 40°C to 250°C at 10°C/min, splitless injection, 1µ1; (b) poppy straw, column, 5 m * 0.25 mm i.d., df = 0.14 µm ov-73, Te from l80°C to 300°C at l0°C/min, split injection.
5.4 CONCLUSIONS
The narrow-bore capillary columns prepared according to the
methods described in Chapter 4 are very well suited for high
speed analysis of different samples. Besides the inertness,
a high separation efficiency was obtained with the small bore
columns and the time required to obtain a given separation was
reduced compared with the analysis time required using normal
bore columns.
The normal bore columns prepared in this study have a high
-110-
thermal stability and inertness. Less volatile and high
boiling compounds, such as polymer additives, glycerides and
polyethylene glycol 400 can be analyzed using these columns.
Compared with the results obtained by SFC, shorter analysis
time, higher column efficiencies for the separation of this
type of compounds can be obtained by high temperature columns.
The successful separation of a number of selected samples,
such as sedative hypnotic barbiturates, underivatized steroids
and chlorophenols demonstrates the inertness of the columns
prepared here. They are also suitable for the analysis of
ester mixtures of free fatty acids, amine acids and different
extracts.
5.5 REJ!'EREHCES
1. M.L. Lee and B. W. Wright, J. Chromatogr., 184 (1980) 235.
2. B. Xu and N.P.E. Vermeulen, J. Chromatogr., 445 (1988) l.
3. H. Boer, P. van Arkelp and W.J. Boersma, Chromatographia, 17 (1980) 500.
4. E.G. Boeren, J. Chromatogr., 349 (1985} 377. 5. J.P. Durand, Y. Boscher, N. Petroff and M. Berthelin, J.
Chromatogr., 395 (1987) 229. 6. v.w. Watts and T.F. Simonick, J. Anal. Toxical., 10
(1986) 198. 7. B. Newton and R.F. Foery, J. Anal. Toxical., 8 (1984}
129. 8. B. Rambeck and J.W.A. Meijer, Ther. Drug Monitor, 2
(1980) 385. 9. J.T. Burke and J.P. Therot, J. Chromatogr., 340 {1985)
199. 10. J.W.A. Meijer, B. Rambeck and M. Riedmann, Ther. Drug
Monitor, 5 (1983) 39. ll. H.T. Badings, c. De Jong, R.P.M. Dooper and R.C.M. De
Nijs, Progress in Flavour, Elsevier Science Publisher, Amsterdam, 1984.
12. N. Fehringer, J. High Resolut. Chromatogr. Chromatogr. commun., s (1985) 98.
13. B. Lau, Chemosphere, 14 (1985) 799. 14. J.C. Duinker, D.E. Schulz and G. Petrick, Anal. Chem.,
60 (1988) 478.
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15. M.N. Hasan and P.C. Jurs, Anal. Chem., 60 (1988) 978. 16. C.A. Cramers, G.J. Scherpenzeel and P.A. Leclercq, J.
Chromatogr., 203 (1981) 207. 17. C.P.J. Schutjas, E.A. Vermeer, J.A. Rijks and C.A.
Cramers, J. Chromatogr., 253 (1982) 1. 18. C.P.J. Schutjes, Ph.D. Dissertation, Eindhoven
University of Technology, Eindhoven, The Netherlands, 1983.
19. A. van Es, Ph. D. Dissertation, Eindhoven university of Technology, Eindhoven, The Netherlands, 1990.
20. C.A. Cramers and P.A. Leclercq, CRC Critical Reviews in Anal. Chem., 20 (1988) 117.
21. R.L. Henry, G.A. Baney and C.L. Woolley, in P. Sandra (Ed.), Proc. of llth International Symposium on Capillary Chromatography, Monterey, CA, USA, May 1990, Hüthig, 1990, p. 39.
22. D.H. Desty, A. Goldup and W.T. Swanton, In N. Brenner et al. (Eds.), Gas Chromatography, Academie Press, New York, 1962, p. 105.
23. P. Sandra and M.V. Roelenbosch, Chromatographia, 14 {1981) 345.
24. G. Gaspar, c. Vidal-Madjar and G. Guichon, Chromatographia, 15 (1982) 125.
25. J. Phillips, L. Dershingline and Ru-Po Lee, J. Chromatogr. Sci., 24 {1986) 296.
2 6. M. Proot and P. Sandra, J. High Resolut. Chromatogr. Chromatogr. Commun., 9 (1986) 618.
27. Th. Noij, E. Weiss, T. Herps, H. van cruchten and J. Rijks, J. High Resolut. Chromatogr. Chromatogr. Conunun., 11 ( 1988) 181.
28. A. van Es, J. Janssen, c. Cramers and J. Rijks, J. High Resolut. Chromatogr. Chromatogr. Commun., 11 (1988) 852.
29. J.P.E.M. Rijks, H.M.J. Snijders, A.J. Bombeek, H.E.M. van Leuken and J.A. Rijks, in P. Sandra (Ed.), Proc. of 13th International Symposium on Capillary Chromatography, Riva del Garda, Italy, May 1991, Hüthig, 1991, p. 35.
30. S.R. Lipsky and M.L. Duffy, J. High Resolut. Chromatogr. Chromatogr. commun., 9 (1986) 376.
31. S.R. Lipsky and M.L. Duffy, J. High Resolut. Chromatogr. Chromatogr. commun., 9 (1986) 725.
32. P. Sandra and F. David, in P. Sandra (Ed.), Proc. of llth International Symposium on Capillary Chromatography, Monterey, CA, USA, 1990, Hüthig, 1990, p. 382.
33. s. Trestianu, G. Zilioli, A. Sironi, c. Saravalle, F. Munari, M. Galli, G. Gaspar, J .M. Colin and J .L. Jovelin, J. High Resolut. Chromatogr. Chromatogr. Conunun., 8 (1985) 771.
34. w. Blum and L. Damasceno, J. High Resolut. Chromatogr. Chromatogr. Commun., 10 (1987) 472.
35. R.L. Firor and R.J. Phillips, in P. Sandra (Ed.), Proc. of 9th International Symposium on Capillary Chromatography, Monterey, CA, USA, May 1988, HUthig, 1988.
36. w. Blum, W.J. Richter and G. Eglinton, J. High Resolut. Chromatogr. Chromatogr. Commun., ll (1988) 148.
37. Y. Takayama, T. Takeichi and s. Kawai, J. High Resolut.
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Chromatogr. Chromatogr. Commun., 11 (1988) 732. 38. J. Curvers and P. van den Engel, J. High Resolut.
Chromatogr. Chromatogr. Com:mun., 12 (1989) 16. 3 9. J. Bui j ten, Ph. Mussche and J. Peene, in P. Sandra
(Ed.), Proc. of lOth International Symp. on capillary Chromatography, Riva del Garda, Italy, May 1989, Hüthig, 1989, p. 962.
40. W. Blum and G. Eglinton, J. High Resolut. Chromatogr. Chromatogr. Commun., 12 (1989) 290.
41. J. Buijten, c. Duvekot, J. Peene, J. Lips and M. Mohnke, in P. Sandra (Ed.), Proc. of llth International Symp. on capillary Chromatography, Monterey, CA, USA, May 1990, Hüthig, 1990 1 p. 91.
42. J. Buijten, c. Duvekot, J. Peene and Ph. Mussche, International Chromatography Lab., 2 (1990) 5.
43. L. Damasceno, J. cardoso and R. Coelho, in P. Sandra (Ed.), Proc. of 13th International Symp. on Capillary Chromatography, Riva del Garda, Italy, May 1991, Hüthig, 1991, p. 155.
44. J. Buijten, c. Duvekot, w. van der Poel, Ph. Mussche and M. Mohnke, in P. Sandra (Ed.), Proc. of 13th International Symp. on Capillary Chromatography, Riva del Garda, Italy, May 1991, Hüthig, 1991, p. 165.
45. P. Husek, J.A. Rijks, P.A. Leclercq and C.A. cramers, J. High Resolut. Chromatogr., 13 (1990} 633.
46. P.A. Tice and J.D. McGuinness, Food Addit. Contam., 4 (1987) 267.
47. O.A. Teirlynck and M.T. Rosseel, J. Chromatogr., 342 (1985) 399.
48. M.W. Raynor, K.O. Bartle, I.L. Davies, A. Williams, A.A. Clifford, J.M. Chalmers and B. W. Cook, Anal.. Chem., 60 (1988) 427.
49. P.J. Arpino, O. Dilettato, K. Nguyen and A. Bruchet, J. High Resolut. Chromatogr., 13 (1990) 5.
50. H.P. Burchfield and E.E. Storrs, Biochemica! Applications of Gas Chromatography, Academie Press, New York, 1962.
-113-
CBAP'l'ER 6
l\fiCELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY - THE INFLUENCE OF DIFFERENT COLUMN MODIFICATIONS ON THE ELECTROOSMOTIC FLOW
A systematic investigation was carried out to study the electroosmotic flow (EOF) in micellar electrokinetic capillary chromatography (MECC). Fused silica capillaries were treated with different reagents to investigate the parameters which influence . the magnitude and reproducibility of the EOF. The resul ts from untreated and treated columns were compared under the same experimental conditions. Furthermore, experiments with and without sodium dodecylsulfate (SDS) addition to the buffer were carried out in order to elucidate the effect of this surfactant on the EOF. Finally, the mechanism behind the effects of these modifiaations on the EOF and the stability of the coatings on the column wall are discussed.
6.1 IN'l'RODUC'l'ION
The term 11 electrochromatography11 {EC) was first used by Berraz
in 1943 (l) referring to a primitive form of paper electro-
phoresis. The whole process was described even earlier by
stain [2], though, he did not use this nomenclature in his
research.
At present, i t can be considered as a group of separation
methods in which the flow of the mobile phase is driven
through the column by electroosmosis {or electroendosmosis).
Generally, an EC-separation is accomplished by the combination
of electro-phoretic and chromatographic selectivity
mechanisms. Adopting the classification suggested by Knox and
Grant [3], electrochromato-graphy can be divided into four
major groups: micel lar electro-kinetic capillary
chromatography (MECC) [ 4-11] , capillary electroosmotic
chromatography (CEC) [12-14], capillary gel electrophoresis
-114-
(CGE) [15-21), and càpillary zone electrophoresis (CZE) (13,
22-26].
After the pioneer's works [22, 23] of using narrow bore tubes
in zone electrophoresis, CZE became a powerful tool in the
separation of ionized solutes [23). Very high efficiencies,
usually exceeding several hundred thousands theoretical plates
can be obtained. Two major contributions to that may be
distinguished (3, 23]. First, due to the plug like flow
profile in the column the peak broadening process of the
solute zones is in principal only determined by axial
diffusion of the solutes in the buffer. This condition is met
if no solute-wall interactions occur. Second, the currently
applied small diameter columns dissipate very efficiently the
generated joule heat in the column, so preventing the
disturbance of solute bands.
Complementary to CZE , which can separate only charged and
water soluble solutes, MECC can be used for the separation of
uncharged compounds, even when they are hardly soluble in
water [27). MECC, introduced in its present form by Terabe et
al. [4, 5), develops rapidly and a lot of attention is paid
to its application.
It must be mentioned, however, that the potentials of micellar
agents in separation technology were earlier recognized in
liquid chromatography as micellar or soap chromatography [28-
30).
As mentioned above,
compounds which are
in contrast to CZE, also uncharged
almost insoluble in water, can be
-115-
separated by MECC. In fact the separation in MECC is achieved
by the partitioning of the compounds over two phases, the
electroosmotically driven hydro (organic) mobile phase and the
micellar phase. It is a true chromatographic method since the
separation of the solutes depends on the different partition
of the solutes between two phases which move at different
velocities [5]. MECC can be performed in similar apparatus as
usually applied in CZE.
In electrically driven liquid chromatography (CEC), packed and
open capillary columns can be used. The buffer is driven
through the column by electroosmosis rather than by pressure.
Partitioning of solutes between the mobile and stationary
phases occurs in the usual way and promising results have been
reported recently (3, 13, 31, 32].
A predictable and reproducible electroosmotic flow (EOF)
through the capillary column is essential for MECC and CEC
techniques. Therefore it is important to control the magnitude
and the reproducibility of the EOF in order to obtain reliable
resul ts. The EOF is the movement of a conducting liquid
relative to a stationary surf ace of an isolator, which results
when an electrical field is applied along this liquid/solid
system.. At the interface between the solid and the liquid
phase an electrical double layer exists (Figure 6.1). In the
double layer a specific zeta-potential can be distinguished
as one of the factors determining the EOF. The expression for
the potential distribution in the electrical double layer has
been described (33-37} and lts relation to the EOF was derived
-116-
by von Smoluchowski [38).
Factors influencing the EOF include, the physical and chemical
properties of the column wall, the composition and
concentration of the buffer, the pH value and percentage of
organic modifiers in the mobile phase and the eventual in-situ
coating effects of the column wall by additives to the buffer.
î 111.: Stern layer dlffuae layer
1 1 1 1 r 1 1 LE-- plane ofrshear 1 1 1
i 1 1 1 I+ 1 1 i 1 1 1 IE 1 1 A - + 1 li 1 1
."':.. 1 + 1 --- + r 1 + 1 1
1 1 1 1 - + 1 + 1 + 1
~-- -----i-1
1 1 1 - + 1 1 1 +
1 + l 1
- + 1 1 1 aolld 1 1 1 bulk
1 + 1 1 aolutlon - + 1 1 1
11ë;;;-; ------T--------1 l - + l 1
lmmoblle 1 moblle reg Ion reg Ion
---+ dlstance x
Fiqure 6.1 Schematic diagram of the electrical double layer at a solid/liquid interface.
A number of reports bas been published about the factors which
may influence the EOF. Organic solvents such as methanol, 2-
propanol and acetonitrile were investigated ~s additives to
the buffer solution to affect the EOF [39-48]. Both decreased
EOF [ll, 45, 48) and increased EOF (41] have been observed
after the addition of acetonitrile. Also silanization by
trimethylchlorosilane (TMCS) of the column surface was used
-117-
to reduce the EOF (10, 23]. Polymers and a number of other
coatings were applied by different authors [ 49-55] and a
decreased EOF was generally observed.
Terabe et al. [ 56) investigated the effects on the EOF of
hydrophobic and hydrophilic coatings. The EOF was increased
in MECC by coating with polymethylsiloxane. This was explained
in terms of the adsorption of sodium dodecylsulfate (SOS) to
this coating at the column wall. A decreased EOF was obtained
in a carbowax 20M modified column which could not be explained
by the adsorption of SOS. Lux et al. [57] also studied the
influence of the same types of coatings. Similar results were
achieved as described by Terabe [56], it was also found that
the effect of the coating on the EOF increased with increasing
film thickness until the film thickness is larger than 40 nm
and a constant EOF value is reached. The observed changes of
the electroosmotic flow by the column coatings were attributed
to the changes of the f-potential in the electrical double
layer at the column wall, without further explanation.
In some cases confusing results of the observed EOF values
were reported and explained mainly in terms of the changes of
the f-potential in the electrical double layer at the column
wall (Fig. 6.1). The question, however, why the f-potential
changed in a specific situation by the different modifications
of the columns surface remains unanswered in most cases.
The aim of this chapter is to study these essential questions
by a systematic investigation of the influence of different
coatings on the EOF and to correlate that to the
-118-
physical/chemical properties of the column wall. FUsed silica
capillaries were treated with a variety of reagents to
investigate their effects on the magnitude and reproducibility
of the EOF. The results of untreated and treated columns were
compared under the same experimental conditions. Furthermore,
experiments with and without SDS addition to the buffer were
carried out in order to elucidate the effect of this
surfactant on the EOF. Finally interaction mechanisms at the
liquid/solid interface of the column wall are discussed to
explain the influence of the different modifications.
6.2 THEORY
6.2.1 Electrical double layer
An electrical double layer occurs at the interface between a
solid and a liquid phase which is a feature of all surfaces
i:m:mersed in a liquid [37). The surface of silica, glass and
Teflon tubes carry negative charges. In the case of silica,
the hydrated surf ace consists of a. o. silanol groups which can
be ionized and are balanced by positive charges (hydrated
cations) from the buffer solution. Some of these positive
charges firmly held on the surface by electrostatic force form
a "compact layer" or Stern layer [33). Part of the remaining
counterions diffuse away from the compact layer because of the
thermal motion and form a loosely held layer indicated as the
"diffuse layer" [34). The concentration of the diffuse layer
approaches finally the bulk value, as the distance from the
wall goes to infinity [34, 37). The potential drops linearly
in the Stern layer and exponentially within the diffuse layer
-119-
as depicted in Figure 6.1.
An expression for the potential distribution in the diffuse
layer in the solution was given by Gouy [35] and Chapman [36]
for a binary electrolyte system with the valency z+ = z-:
and
where
X = ( 2 * e2 * no * z2)~ t 0 * tr * k * T
ir: the electrical potential, x: the distance from the column wall, x: the Debye-Hilckel parameter, k: the Boltzmann constant, T: the absolute temperature, e: the electronic charge,
( 6 .1)
(6.2)
n°: the electrolyte concentration in the bulk solution, eo: the permittivity of vacuum, er: the dielectrical constant of the buffer, z: the valency of the electrolyte.
A similar result is obtained by the Debye-Hilckel theory, in
which, l/x is called the "thickness of the electrical double
layern, ó: 1
x (6.3)
ó is inversely proportional to the square root of the ionic
strength of the electrolyte solution (see eq 6.2).
6.2.2 t-Potential and Electroosmotic Flow (EOF)
When an external electrical field is applied across a
capillary column filled with the electrolyte solution, the
excess counterions within the diffuse layer will move towards
the appropriately charged electrode, dragging the solvating
-120-
liquid along with them. This movement of solution is known as
electro(end)osmosis and its direction and magnitude depends
both on the properties of the capillary wall and the nature
and concentration of the buffer (14, 58). Buffer ions, which
are tightly adsorbed in the Stern layer will not move under
the influence of an electrical field.
A boundary between the Stern layer and the diffuse layer is
assUllled to exist if the bulk solution f lows tangentially to
the surf ace and is indicated as the "plane of shear". The
corresponding potential at this shear is called the
electrokinetic potential or zeta-potential, denoted by f. The
question of localizing the shear plane remains open because
of the absence of independent methods of determining the rpotential which can be determined only by experiments on the
basis of various electrokinetic measurements (59).
The Smoluchowski equation (eq 6.4) can be used to describe the
relation between the electroosmotic Velocity, Veoi and the f
potential (38]. This equation, however, is restricted to
capillary systems with inner diameters considerably higher
than the double layer thickness.
where E: the applied electrical field strength, ~= the viscosity of the buffer, µe0 : the coefficient of the electroosmotic flow.
The assumption is made here that Er and ~ have the same value
in the double layer as in the bulk solution.
-121-
It is clear that a charged sheath around a core of uncharged
liquid is formed within the electrical double layer. Opposite
to the parabolic flow profile in pressure driven separation
systems, the flow profile in electrically driven systems
within the core is flat by the applied electrical field.
Because there is no charge unbalance within the core.
Furthermore, since the sheath itself is so thin that the flow
profile within the column as a whole is a near perfect plug
flow (see Figure 6.2). This plug like flow profile reduces the
band broadening process that occurs as a solute travels along
the tube, so resulting in very high efficiencies.
-u
© 1 J-u 0
Fiqure 6.2 Schematic diagram of the flow profile in open tubes under pressure driven (upper) and electroosmotic driven (lower).
several methods have been applied to measure the EOF. A simple
way is measuring the migration time of an electrically neutral
marker substance to calculate the EOF [ 4, 5, 14] • This
substance is transported only by the liquid flow and should
not show any electrophoretic mobility or any adsorption
effect. A similar problem is encountered with this method as
in liquid chromatography to find a good zero retention time
(to) marker.
The magnitude of the streaming potential, generated by
applying a pressure gradient across the length of the
-122-
capillary, can also be used to estimate the EOF [37, 60, 61].
The following equation correlates the streaming potential,
Estr to the r-potential of the column surface and thus to the
EOF:
(6.5)
where áp: the pressure drop across the tube, X: the specific conductivity of the electrolyte solution.
This method is limited to situation where the surface
conductance is negligible compared to the specific conductance
of the electrolyte solution and a laminar flow profile is
built up within the capillary.
Altria and Simpson [58, 62) introduced a weighing method by
using a microbalance to measure the electroosmotic volumetrie
flow of the electrolyte solution. This technique may approach
most closely the real experimental conditions because of the
direct determination of the flow. However, only time-averaged
results can be obtained by this method [61).
Another method based on the recording of the current during
CZE operation was proposed by Huang et al. [ 63) . In this
method different concentrations of the same electrolyte
solution were used while monitoring the changes in the
electrical current during this CZE operation. The displacement
of one solution by another solution in the tube can be
established and from that the EOF can be calculated. The
difference between the two concentrations of the solutions may
not be too large, otherwise, the j"-potential is changed during
-123-
the operation. Current disturbances may result in inaccurate
EOF measurements using this technique.
Also, colloidal particles, of similar materials as used for
the manufacturing of the capillary, were applied to determine
the EOF [52]. However, in general it is not recommended to
compare or to predict the EOF behaviour in an open tubular
capillary with EOF value obtained from colloidal particles
because of the dif f erence between this method and the real
experimental conditions.
6.2.3 Factors Influencinq the Electroosmotic Velocity
Glass, fused silica and Teflon capillary columns, which are
predominantly used in chromatography and electrophoresis, have
negative charges on the inner surface when they are filled
with an aqueous solution [37]. When an electrical field is
applied to the tube, the counterions in the diffuse layer move
to the negative electrode dragging the liquid with them and
so accomplishing the electroosmotic flow (EOF). The charge
density of the surface can be influenced in different ways and
also the sign of the charge at the column wall can be
reversed.
The control of the EOF in electrochromatography can be
subdivided in two major groups:
I. dynamic modification of the column wall by addition of
specific reagents to the mobile phase, resulting in
general in a reversible coating of it.
-124-
II. chemical treatment of the column wall, resulting
principally in the irreversible modification of it.
I. As mentioned earlier, organic solvents such as methanol,
ethanol, 2-propanol and acetonitrile can be added to the
buffer solution to influence the EOF [10, 11, 39-48, 64, 65].
Contradictory results were observed with the addition of
acetonitrile [41, 45, 48). More generally, the mechanism of
the effects on the EOF caused by addition of organic solvents
is still not quite clear.
The addition of surfactants to the mobile phase is also used
to influence or to reverse the electroosmotic flow. Decreased
EOF values were obtained by addition of mono- or.diamines [25,
66-69] to the electrolyte solutions. Simultaneously the
adsorption of proteins to the column wall was suppressed.
Divalent amines were found to be more effective than
monoamines in this particular case {69). Polyethylene glycol
(70), Triton-x (71), and Brij 35 [72) were also used to
decrease or eliminate the EOF by increasing the viscosity of
the buffers.
By applying cationic surfactants, such as dodecyltrimethyl
ammonium chloride (DTAC), tetradecyltrimethylammonium bromide
(TTAB), cetyltrimethylammonium chloride (CTAC) and cetyltri
methylammonium bromide (CTAB), not only the magnitude of the
EOF was changed, also the direction of the EOF was reversed
[8-10, 25, 62, 73-76]. This is caused by the adsorption of the
cationic molecules to the column wall, which consequently
changes the polarity of the f-potential in the electrical
-125-
double layer. Electroosmosis can be completely eliminated by
the addition of s-benzylthiouronium chloride to the buffer
according to Altria and Simpson (58].
II. By chemical modification methods, generally a thin layer
of a coating is formed on the inner column surface. Two
different aims may be pursued here. One is, e.g. in the case
of the application of fused silica columns, to cover or shield
the surface silanols to control the polarity and the magnitude
of the EOF. Second also a specific modification can be applied
to suppress or eliminate the interaction between the solute
molecules and the column surface. This is special important
for the separation of biopolymers such as proteins and other
biomolecules.
Jorgenson and Lukacs treated columns with trimethylchloro
silane (TMCS} and observed a decreased EOF (23]. Similar
results (10] were obtained with acrylamide (77], polyacryl
amide [49, 51], octadecylsilane [40], and aminopropyltri
ethoxysilane [78] coated columns. Due to the different
hydrophobic and hydrophilic properties of the coatings, mutual
differences in the observed EOF values may be expected. An
increased EOF was obtained in MECC applying a column coated
with polydimethylsiloxane [56, 57]. Here, this was explained
by the adsorption of the surfactant to the column wall (56].
Moreover, also the EOF was found to depend on the film
thickness of the coating and to reach a constant value after
the film thickness is larger than 40 nm (57]. The hydrophilic
coatings resulted in decreased EOF values and in some cases
in the reversal of the EOF direction. Glycol [50), glycerol
-126-
[55], polyethylene glycol (PEG) [52, 79] and Carbowax 20M [56,
57] coated columns showed suppression effects on the EOF. On
polyethyleneimine coated capillaries [80], the EOF is reversed
because of the positive charge of the coating when it is in
contact with aqueous buffers.
Recently, a method was suggested to control the electroosmotic
flow during an EC separation [64]. Here the external applied
potential is vectorially coupled with the potential inside the
capillary. In this way, the r-potential can be controlled and
changed by a definite positive, or negative value. Therefore,
the r-potential can be changed at any time during the analysis
to achieve improved separation results. However the problem
is that the instrumentation f or this technique is complicated
and not yet commercially available.
The concentration [81] and the pH value of the buffer solution
in EC separations are the most important werking parameters
to influence the EOF [58, 82, 83]. This is especially true for
fused silica and/or glass capillary columns because the
dissociation of the surface silanols strongly depend on the
pH of the buffer solution (see Figure 6.3).
6.3 EXPERIMENTAL
6.3.1 Equipment and reagents:
A home-built instrument system consisted of a Perspex box with
a pressure system for rinsing the capillary; an UV detector
(Unicam Analytica! Systems, Cambridge, UK) for on-column
-127-
detection; an HCN 35-35000 High voltage power supply (FuG,
Elektronik GmbH, FRG). A Tulip AT compact 3 computer connected
to a home made interface Multilab-TS allowed control of
electromigrative sample introduction and adjustment of the
separation voltage as well. Acquisition and calculation of the
data were performed with the software program Caesar (B-Wise
Software, Geleen, The Netherlands).
14 c
12
10
I î 8
l ... • :l
1
2
0 .__~ _ _,__....__,___,'----'---'--"'-----'
2 3 4 6 fl 7 8 9 10
pH
Fiqure 6.3 Schematic diagram for the effect of pH on electroosmotic flow. Data for line 1 and line 2 are from ref. ( 82] and ( 58) respectively.
The separations were performed in fused silica capillaries
(350 µm o.d., 50 µm i.d., Siemens, FRG) of 67 cm total length,
with an optical window at 50 cm from the injection side for
on-column detection. Samples were introduced into the column
by the electromigration technique applying 5 kV for 2 sec. The
separations were carried out at 20 kV using methanol as the
-128-
dead time marker and benzyl alcohol for the measurement of
peak area.
Polymethylhydrosiloxane (PMHS) and polymethyloctadecylsiloxane
(PMODS) were purchased from Petrarch systems (Bristol, PA,
USA). Polydimethylsiloxane (PDMS) and Carbowax 20M were
obtained from Chrompack (Middelburg, NL). These reagents were
used without any further purification.
6.3.2 Procedures:
The capillary column was pretreated according to the procedure
developed in our laboratory. [84]. For PMHS deactivation, the
column was dynamically coated with 15% (v/v) PMHS solution in
pentane (according to Bartle (85), generating a film thickness
of 40 nm). After flushing the column with He for 30 min, it
was sealed by a micro torch and heated in a GC oven from 40°C
to 300°C at 8°C/min and kept at 300°C for 2 h. Then, the
column was rinsed with l ml dichloromethane and l ml methanol
respectively.
For ether modifications, the capillaries were dynamically
coated with 15% (v/v) PMODS and statically coated with 0.8%
(v/v) PDMS and 0.5% (v/v) carbowax 20M solution respectively.
0.5-3% (w/w to the stationary phases) dicumyl peroxide (DCP)
was added to the coating solution f or immobilization of the
coatings. curing was carried out by heating the sealed column
from 40°C to 1ao 0 c at 8°C/min and then 180°C for 80 min.
Afterwards, the modified capillaries were rinsed with l ml
dichloromethane and l ml methanol respectively.
-129-
Before use in the MECC experiments all the capillaries ware
washed with water (30 min) and subsequently with the same
buffer solutions to be used in the experiments (20 min) , prior
to the separations.
The buffer solutions used in this study consisted of:
(1) 0.02 M sodium phosphate aqueous solution, pH 7.
(2) as (1) with addition of SOS (0.05 M), pH= 7.
(3) as (1) adjusted to pH 1 with concentrated HCl
solution.
(4) as (1) adjusted to pH 9 with concentrated NaOH
solution.
6.4 RESULTS AND DISCUSSION
6.4.1 Reproducibilities of the EOF data and peak areas
The reproducibility of the EOF expressed in migration times
and peak areas measured for some coated and untreated columns
are presented in Tabla 6.1. A rinsing step which included
washing the column with water for 10 min was involved between
soma of the analyses in order to investigate the influence of
rinsing on the reproducibility.
From the data in Tabla 6.1, it can be concluded that improved
reproducibility of the EOF and peak areas was obtained by
rinsing the capillary column with water between each analysis.
-130-
Table 6.1 Reproducibility of migration times and peak areas, buffer (1), UV detection at 210 nm.
Col. 1 migration time (min) peak areas (V*S) No.
(a) (b) (a) {b)
s a a/s s a a/s s a a/s s a o/s
1 3.20 .04 1.2 3.35 .os 2.3 5.75 .29 5.0 6.16 .48 7.8
3 4.02 .11 2.7 4.32 .26 6.0 9.58 .44 4.5 10.7 .86 8.0
4 4.92 .11 2.1 5.37 .27 5.0 14.5 .42 2.9 15.6 .92 5.9
Column No.: 1 = PDMS coated column; 3 = untreated column; 4 = PMODS coated column; s = average value, a = standard deviation, a/s = relative deviation, %; number of experiments n = 3; (a) with rinsing step; (b) without rinsing step.
6.4.2 J:nfluence on the EOF of the surface Modification of the Columns
1. Without addition of SDS to the buffer:
The EOF data together with the standard deviations for
untreated and modified capillary columns without addition of
sos to the buffer are summarized in Figure 6.4 and Table 6.2.
Table 6.2 Comparison of EOF data with and without addition of SDS to the buffer solution, UV detection at 210 nm.
col. EOF (mm/sec) No.
buffer treatment
(1) buffer (2)
s a a/s s a a/s 1 2.60 0.03 1.15 2.78 0.04 1.44 PDMS
2 2.25 0.07 3.11 2.39 0.06 2.51 PMHS
3 2.07 0.06 2.90 1.82 0.06 3.30 untreated
4 1.69 0.04 2.37 1.54 0.05 3.25 PMODS
5 1.04 0.03 2.88 0.93 0.03 3.23 Carbowax 20M
-131-
From the data in Table 6.2 it may be clear that compared to
the untreated column:
{l) The EOF is increased by the surface modification of PMHS
and PDMS coatings, which is in agreement to the
observation in (56, 57].
(2) A decreased EOF is observed for the PMODS and Carbowax
20M coatings.
3 "' without SOS 1
û I " Cl
~ "' s 2 6
Cl u.
wit! SOS
"' 0 Cl w
0
o~~~~~""-~~~~-'-~~~~-'-~~~~-'
1 2
Figure 6.4
3 column No.
4 5
EOF values in untreated and differently modified capillary columns, column No. see Table 6.2.
2. With addition of SDS to the buffer:
The EOF data together with the standard deviations for
untreated and modified capillary columns with SOS in the
buffer solution are included in Tabla 6.2.
Several conclusions can be drawn from the data in Table 6.2:
(1) In contrast to the situation without SOS in the buffer,
the EOF in the untreated column is decreased.
(2) The EOF is increased for PMHS and PDMS treated columns
-132-
compared with the results without SOS in the buffer.
(3) A significantly decreased EOF is observed for PMODS and
Carbowax 20M modified columns after SOS was added to the
buffer.
These conclusions will be discussed in paragraph 6.4.3.
Theoretically, an increased EOF will shorten the analysis time
with the sacrifice of resolution. On the other hand, the
resolution can be enhanced by decreasing the EOF,
simul taneously, the analysis time is prolonged. This was
confirmed by ref. [57] and our experiments. A detailed
discussion on the relation of analysis time and resolution is
given in Chapter 7.
6.4.3 Disoussion on the Znfluenoe of the surfaoe Treatment and Addition of SDS on the EOP
When an untreated fused silica capillary column is filled with
the buffering electrolyte, the dissociation of the surface
silanols is the main generation source of the ,S-potential. The
rate of dissociation of surface silanols depends strongly on
the pH value of the buffer solution. In the case the colUEn
is coated with hydrophobic and/or hydrophilic coatings,
different effects on the EOF, compared to a bare unmodified
column, can be expected.
In general, a coating at the wall of a capillary column is
supposed to eliminate and/or shield the active sites
(silanols) at the column surface. The accessibility of the
silanols on the column wall in contact with the water
molecules of the electrolyte solution depends a.o. on the
-133-
efficiency of the coverage. Both the dissociation of the
residual silanols and the specific properties of the coating
may contribute to the r-potential in the electrical double
layer.
To explain the effect of the silica substrate of the column
in controlling the EOF, the hypothesis of Tanabe et al. [86-
88] can be applied. This model was developed to describe the
surface acidity which was caused by the charge distribution
in binary metal oxides. When an inorganic oxide like silica
is contaminated with metal impurities it can be considered as
a mixture of oxides on molecular scale. These impurities may
cause charge imbalances leading to different contribution of
the positive or negative charges across the silica surface of
the column wall. In turn this may influence the EOF in
untreated silica columns at least. Depending on the purity of
the silica substrate and the efficiency of an eventual
modification of the column wall, this effect will become
manifest.
In addition to this effect also the charge distribution that
is the polarity of the column coating plays an important role.
For PMHS, PDMS and PMODS modified columns, the column wal! is
covered with a layer of a polysiloxane backbone, of which the
Si-o-si bridge is a major component (Fig. 6.5). Considering
the electronegativity of the elements involved in the chemica!
structure of the column surface given in Table 6.3, it can be
assumed that the two positive charges of the upper Si atom are
distributed to four bands, ie, +(2/4) ö of a valence unit per
bond. While the two negati ve charges of the o atom are
distributed to two bonds, i.e. one negative charge per bónd.
-134-
The total charge imbalance is (+2/4-2/2) ó * 2 = -1 ó. This
may increase the local negative charge of the column wall.
When in contact with aqueous buffer solutions this results in
an influence on the EOF.
Me 1 (+2/4 - 2/2) ll R "H, Me, C1s
R-Si-0-
1 charge difference 0 (2/4·2/2) 5 • 2 = -1 ll _____ t __________________ _
-O-Si-0- column wall 1
0
1 Figure 6.5 Schematic representation of the model structure of polysiloxane modif ied surface.
A more general explanation for the influence of the wall
coating on the EOF lies in the structure shown in Figure 6.5.
By the partial ionic character of the Si-o bond [89, 90] and
the electron donating properties of the alkyl groups, each
repeating unit in the backbone will gain an excess of electron
density. This makes them behave as a negatively charged centre
(spot) in contact with the electrolyte solution. Therefore the
EOF is strongly influenced in this way.
Besides the electronegativity, also the steric effects have
to be taken into account to explain the differences between
these three coatings. The PMHS modification shows an increased
EOF compared with the bare silica column, but smaller than the
corresponding value in the PDMS modified column. This may be
attributed to the weakening effect of the H atom in the PMHS
-135-
coating to the charge distribution of the siloxane bridge.
Moreover, the film thickness of the PMHS coating is smaller
(40 nm) than that of the PDMS coating (100 nm), which also may
influence the EOF.
Table 6.3 Electronegativity of the related elements
atom H c 0 Si
electronegativity 2.2 2.55 3.44 1.90
For the PMODS coated column, the electron donating properties
of the methyl and c18 groups are about the same. The steric
effect of the c1s ligands, however, may be dominant in
shielding the charged centres at the column wall resulting in
a decreased EOF.
In general the siloxane backbone will show a ó+ / ó- charge
distribution due to the difference in the electronegativity
between the involved elements. In addition this charge
distribution will be influenced by electron donating or
withdrawing groups.
(CH2CH20t H 1 n
0 --------~----------------------
Ji'iqure 6.6
- 0-Si- 0- column wall 1
0 1
Schematic representation of the model structure of Carbowax 20M modified silica surface.
-136-
The Carbowax 20M coating by which the column is covered with
a layer of polyethylene glycol (see Fig. 6.6) shows a
different effect on the EOF. The surface silanol groups are
supposed to be shielded from interaction with the electrolyte.
Here the electronegativity difference between carbon and
oxygen atoms is less than that between silicon and oxygen
atoms (see Table 6.3). Therefore, the &+/0-charge distribution
is less pronounced. In addi tion, the c-oH groups are more
difficult dissociated than silanol groups, in other words, the
acidity of silanol groups is higher than that of carbinol
groups [91]. Therefore, a reduced EOF is observed using
carbowax 20M coatings.
In general, when the column coating is very thin (df < 40 nm),
the shielding of the silanols may be not complete which will
contribute to the electroosmotic flow [57].
The adsorption of SOS to the column surface will influence the
f-potential and normally an increased EOF can be expected. For
untreated columns, however, this adsorption may be hindered
by the electrical repulsion of the surface silanols. In the
case of the untreated column, a decreased EOF was observed
after the addition of SOS to the buffer. This may be
attributed to the increased viscosity of the electrolyte
solution in the presence of sos. Eof is inversely proportional
to the viscosity of the electrolyte solution ( eq 6.4). An
increase in viscosity by the addition of 0.05 M SDS to the
buffer was estimated to be about 10% using the Einstein
equation [92, 93]. This is in agreement with the decrease of
-137-
the EOF by about 10% of column 3 in Table 6.2.
Taking into account a 10% decrease in the EOF due to the
increase of the viscosity of the buffer, the data in Table 6.2
show that SOS may adsorb to the PMHS and POMS coated surface.
This will be due to the hydrophobic interactions between these
coatings and the SOS. The enhancement of the EOF by the
adsorption overrules the decreasing effect of the viscosity,
so a net increased EOF was observed. For the PMOOS coating no
net EOF increase or decrease was observed, indicating that
very little or no SOS was adsorbed. In contrast to the PMHS
and POMS coatings here probably the occurrence of a Oonnan
exclusion effect prevented SOS adsorption at the column
coating.
The adsorption of SOS to the Carbowax 20M modified column wall
may be negligible since this material is a hydrophilic
coating, showing less or no hydrophobic interaction with SOS.
6.4.4 stability of the coatings
The PDMS and Carbowax 20M coated columns were selected to
study the stability of the coatings towards the applied
electrolytes. Also the effects of these possible instability
of the coating on the EOF were measured. A sequential
procedure was set up to test the coating stability and
consisted of:
(1) EOF is measured with buffer (1)
experimental section), respectively,
columns at the start of the procedure;
and (2) (see
in each of the
(2) next the columns were rinsed with buffer (3) for 1 day;
-138-
(3) followed by a rinsing step with buffer (4) for 1 day;
(4) finally, the columns were rinsed with water and again
the EOF was measured as in step (1) under the same
conditions.
This procedure was repeated during 2 weeks. Also, the column
activity and the film thickness were determined by GC
measurements before and aftar this rinsing procedure.
The results of these measurements are summarized in Fig. 6.7.
It can be seen that the EOF was decreased by 15% of the
starting value with PDMS coated columns after a 2 weeks
rinsing procedure. The EOF of the carbowax 20M modified column
is decreased by 5% of the starting value after this rinsing
test. This latter coating shows a higher st~bility than the
PDMS coating under the applied experimental conditions.
3.00
2.10 1.20
wlth SOS
! 0
A
î ·"·r·o• A 4
D o
t wlth SOS
POMS modlfied
0 A D
A D A
Carbowax 20M modlfied
0 D 0
D
A
D
0.70 .__ _ __. __ ......_ __ ...._ __ ..._ __ .__ _ __,_ __ _,
0 2 4 a 8 10 12 14
days
:riqure 6.7 Stability test of PDMS modified. column and Carbowax 20M modified column, experimental conditions as in Fig. 6.4.
It also turned out that the film thickness of both coatings
was decreased. For the PDMS modified column the EOF value is
-139-
approaching the value of an untreated capillary column. GC
measurements by comparing the capacity factors of tetradecane,
under the same conditions, before and after this rinsing test
show that the film thickness of PDMS and Carbowax 20M coated
columns is decreased by 15% and 7% respectively. The activity
of both columns to the polar components in the test mixture
[84] is significantly increased.
6.5 CONCLUSIONS
From the collected data it is clear that rinsing of the fused
silica capillary column between analyses is important to
obtain reproducible results.
Taking precaution with the experimental conditions a
repeatable EOF value with a relative standard deviation of
about 3% can be obtained throughout the experiments with the
home built instrument.
Different effects of the several coatings on the electro
osmotic flow were observed and explained with respect to their
specific influences on the f-potential. Enhanced EOF were
obtained by PMHS and PDMS surface modification of the columns
compared to untreated columns. This is due to the increased
charge distribution across the polysiloxane backbone of the
coatings, which increases the f-potential in the electrical
double layer and hence the EOF.
The PMODS coated column suppresses the EOF because of the
steric effect of the c18 ligands which may be dominant in the
shielding of the charged centres at the column wall.
A lower electroosmotic flow was observed for Carbowax 20M
-140- '
coated columns. This might be due to the less negative charge
distribution of the oxygen atom from the c-o bond in the
polyethylene glycol coating. In addition, the C-OH groups are
more difficult dissociated than the surface silanol groups.
The adsorption of SDS to the wall of a bare silica column is
hindered by the electrical repulsion of the ionized silanols.
The observed decrease of the EOF in this case can be
attributed to the increase of the viscosity of the electrolyte
solution by the addition of SDS. The enhancement of the EOF
in PMHS and PDMS modified columns indicate the adsorption of
SDS to the wall. For the PMODS coated columns we assume that
by a Donnan exclusion effect the SOS is prevented to adsorb
at the coating. consequently no significant change in the EOF
by the addition of SDS was observed. Since the Carbowax 20M
coating shows little or no hydrophobic properties SOS will not
be adsorbed. Therefore, again no significant change in the EOF
could be observed.
Finally, the stability of the Carbowax 20M modification is
superior to that of the PDMS coating under the applied
experimental conditions.
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CHAPTER 7
THE SEPARATION OF HERBICIDES BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY
A method was developed tor the separation by micellar electrokinetic capillary chromatography (MECC) of a number ot herbicides consisting ot chlorophenoxy acids. Sodium dodecyl sulphate (SDS), Brij 35, cetyltrimethylammonium bromide (CTAB) and methanol were applied in the buffering buffer to 1nvestigate their eftects on the separation of the herbicides. SDS combined with Brij 35 as micellar agents were tound to provide the best overall separation of these components. Besides that, the di:fterences in the electroosmotic flow (EOF), due to the different compositions of the various buffers, were correlated with the viscosities thereot. The theoretical calculations and the experimental results of the viscosity measurements were in good agreement with the observed shifts in EOF.
7.1 INTRODUCTIOH
The widespread use of chlorinated phenoxy acid herbicides in
agriculture has led to an increased pollution of soil, water
and food by their residues and their phenolic metabolites.
Their high toxicity to human and aquatic orqanisms [l], makes
the development of sensitive methods for the analysis of this
class of compounds essential.
A number of methods, such as spectrophotometr ic [ 2, 3) ,
differential pulse polarographic [4) and gas chromatographic
methods [5-18), have been reported for the analysis of
herbicides. A streng disadvantage of GC methods is that
derivatization of the target compounds is necessary. This is
time consuming and may introduce errors in the analytical
procedure.
High performance liquid chromatography (HPLC) and HPLC
-145-
combined with mass spectrometry (LC-MS) have been used also
to determine these compounds (19-28]. In this case no
derivatization is needed prior to LC separation. The coupling
of LC with MS can provide both the speed of analysis and the
specificity for non-volatile compounds such as chlorophenoxy
acids herbicides.
Enrichment and clean-up procedures are nearly always necessary
for the HPLC analysis of the chlorophenoxy acids in surf ace
water, urine, soil or sediment. Liquid-liquid extraction [9,
29, 30], solid-phase extraction (11, 22, 25, 31-34) and the
enrichment on a precolumn (33, 25, 35-37) or a supported
liquid membrane (38-40] can be applied in the sample
pretreatment procedure. Recently, a sample pretreatment
technique using isotachophoresis as the sample clean-up and
enrichment step prior to the LC analysis was developed [41].
Promising results were obtained by this method.
Capillary zone electrophoresis (CZE) and micel lar
electrokinetic capillary chromatography (MECC) may be also
attractive techniques for the analysis of the chlorophenoxy
acids. Cue to their outstanding separation efficiency, high
speed and potential for automation, CZE and MECC have already
been used to separate a large variety of compounds.
Applications include amino acids [42, 43], peptides [44-46),
proteins (47, 48), priority pollutants (49], vitamins [50-52]
and drugs (53-55].
In this study, a method for the separation of a number of
chlorophenoxy acid herbicides by MECC was developed. The
effects of some surfactants and methanol, in various
concentrations added to the buffer were investigated. The
-146-
herbicides were detected by a multi-wavelength UV detector.
Processing of the detector signals allowed three-dimensional
plots of the data, peak confirmation and peak purity
evaluation via comparison of spectra. Finally also, the
correlation between the variations of electroosmotic flow
(EOF) and viscosity changes due to the different buffer
compositions will be discussed.
7.2 THEORY
CZE is a one phase process where the separation is based on
the differences in electrophoretic mobilities of the solutes
[56, 57). In MECC [58 1 59] 1 however, two phases are
distinguished, a hydro (organic) and a micellar phase or
pseudo-stationary phase. These two phases are prepared and or
formed in the buffer solution containing surfactants, which
are added above their critical micellar concentration (CMC).
In Figure 7. 1 a schematic presentation of a micelle and a
solute partitioning over the mobile and the pseudo-phase is
given. Micelles are dynamic structures and are characterized
a.o. by their aggregation number. Both neutra! and charged
compounds can be separated by MECC according to their
dif f erences in hydrophobicities and electrophoretic
mobilities.
The electroosmotic veloci ty, 1.1eo, which was discussed in
detail in Chapter 6, in a capillary column is essential for
MECC. It can be described by [60):
(7 .1)
-147-
where
eo: the permittivity of vacuum,
Er: the dielectric constant of the buffer,
~= the viscosity of the buffer,
r1: the zeta potential at the column wall,
E: the external applied electrical field strength.
The negative sign means that when t is negative, the liquid
flow is towards the cathode [60). Typical ueo values are in
the range of 1-3 mm/sec. The migration time of an unretained
solute, t 0, is given by:
(7.2)
where 1 is the column length from the injection point to the
detector cell.
Figure 7.1 Schematic illustration of micelles and the partition of the solutes
Besides the EOF in a specif ic MECC separation system also a
distinct electrophoretic mobility of, at least charged
micelles, can be distinguished, resulting in the migration of
the micelles. The electrophoretic velocity, Vep, of a charged
-148-
micelle then is given by:
\) = ep 2 3
where f 2 is the zeta potential at the micelle.
(7. 3)
Here, it is asswned that € and ~ have the same values in the
electrical double layer and in the bulk solution. The overall
migration velocity of a micelle, Vmci under fixed experimental
conditions then follows from:
(7 .4)
Substitution of eq 7.1 and eq 7.3 into eq 7.4 gives a more
detailed insight in the parameters which determine. the
velocity of a micelle.
(7. 5)
In order to obtain satisfying separation, the velocity vectors
of Veo and liep are preferably opposite and differing in their
absolute values. The migration time, tmc1 of a neutral
compound completely retained in the micelle phase follows
from: 1 t = (7. 6) l1IC
"-When a specific neutral compound is solubilized by the
micellar phase an expression for the capacity factor, k, can
be described: k=
(7. 7)
where
tR: the migration time of the compound.
-149-
From this a general expression for the solute velocity in MECC
can be given:
\) = __ k_ * 1.l + __ 1_ * 1.leo 8 1 + k me 1+ k
(7. 8)
where
v8 : the velocity of the compound, k: capacity factor of an electrically neutral compounds.
It should be noted here that superimposed on the above
mentioned migration processes, charged compounds also move
electrophoretically along the capillary tube. The
corresponding velocity of such charged species equals:
where
v8 (i): the velocity of the charged solute, Vep(i): the electrophoretic velocity of the charged solute in
the aqueous phase, k1: capacity factor of the charged solute.
Non-ionic solutes partition between the two phases and elute
in between the range of the migration time of t 0 and tmc· The
migration time of a neutral solute is described by [59]:
l+k tR = ----/.,----*-k..- * to
1 + (to tmc) (7.10)
As usual in elution chromatography the following resolution
equation for a pair of neutral solutes can be derived:
a - 1 * .....,...._k_a_ a ka + 1
(7 .11)
where
ki and k2: the capacity factors of two neighbouring peaks, N: the number of theoretical plates,
-150-
a: the separation factor (equal to k2/ki)·
If two peaks are migrating close together, i.e. k1 ~ k2 ~ k,
eq 7.11 can be approximated by:
m IX - 1 * k * 1 - tol tmc R "' i::. * --- ---s 4 a k + 1 1 + (t01tmc) * k
(7.12)
The product of the last two terms in eq 7.12 is defined as
f(k) and bas a strong effect on the resolution.
f(k} = _k_ * 1 - t 0 /tmc 1 + k 1 + ( t 01 t.mc) * k
(7.13)
Figure 7.2 shows the relationship between f(k) and k for some
different values of t 0 /tmc· In practical situations to/tmc
ranges usually between 0.15 and 0.5.
By assuming that N and a are independent of k [61), the
dependence of the resolution on kis restricted to f(k). The
maximmn resolution can be derived from eq 7 .12 by setting
d{f(k)}/dk = O and yields the optimum value of k:
kopc(Rs,max) = ( tt: r/2 (7 .14)
As can be seen in Figure 7.2, maximum resolution in practical
situations is usually obtained for capacity factors between
2 and 5.
This relation indicates that the best resolution of neutral
solutes can be achieved by adjusting the working parameters
to give an average capacity factor of Ctmc/t0 )l/2,
-151-
1.00
0.80
0.60
0.40
0.20
0.00 '--~~~......_~~_._._ ......... ,__~~-=Cl:lll
0.1 10 100
capacity factor. k
Figure 7.2 Dependance of f(k) on the capacity factor, k. The numbers on the plot are the t 0/tmc values.
By substituting the kopt(Rs,max> from eq 7.14 into eq 7.10,
the analysis time at maximum resolution can be obtained:
(7 .15)
Finally, the result for Rs,max under the condition of
operating at the optimum capacity factor kopt is:
R = ./N * ~ * tmc - to s,max 4 (X { ti/2 + t~P )2 (7.16)
In conventional chromatography, Rs,max is obtained when k-ooo:
R = :/J!. s,max 4 «. - 1 *---
IX (7. 17)
Comparing eq 7.16 with eq 7.17, it can be seen that Rs,max in
MECC is 0.17 to 0.74 times value of Rs1max in conventional
chromatography (assuming t 0/tmc is between 0.15 and 0.5). This
-152-
difference however can be compensated by the superior
efficiency of MECC which also contributes to resolution. In
the case of a normal standard 15 cm LC column, N is 10,000 and
'V"N is 100. For a MECC system, 100, 000-200, 000 theoretica!
plates are easily obtained, 'V"N is between 316 and 447. Taking
account the contribution of efficiency, Rs,max in MECC is 0.5
to 3 times that in conventional chromatography.
Under conditions optimized for efficiency, f(k) is the most
important parameter for resolution optimization. Two
possibilities can be considered, that is the optimization of
capacity factor and the optimization of the elution range
(window). Again, these two possibilities are reflected by eq
7.14 and eq 7.16 respectively.
When tmc/to is infinite, Rs,max is given by eq 7.17. However,
at the same time, tR...co also (see eq 7 .15). In practical
situations, tmc/to lies between 3 and 6, then for the maximum
resolution, tR is between 1.73 t 0 and 2.45 to.
7 • 3 EXPERI:MBN'l'AL
7.3.1 Chemicals
2-(2,4-dichlorophenoxy)-propionic acid (2,4-DP), 4-(2,4-di
chlorophenoxy) butyric acid (2,4-DB), 4-chloro-2-methyl
phenoxy-acetic acid (MCPA), 2-(4-chloro-2-methylphenoxy)
propionic acid (MCPP), 4-(4-chloro-2-methylphenoxy) butyric
acid (MCPB), 2,4,5-trichlorophenoxy acetic acid (2,4,5-TPA),
2-(2,4,5-trichlorophenoxy)-propionic acid (2,4,5-TPP), 3,6-
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dichloro-2-methoxy benzoic acid (dicatnba), 2,3,6-trichloro
benzoic acid (2, 3, 6-TB) and 3-isopropyl-2, l, 3-benz:othiadiazin-
4-on-2, 2-dioxide (bentazon) were purchased from Schmidt
(Amsterdam, The Netherlands).
SodiUlll dodecylsulphate (SOS) and cetyltrimethylammonium
bromide (CTAB) were obtained from Aldrich Chemica! Co.
(Milwaukee, WI USA). All the other reagents and solvents were
purchased from Merck (Merck AG, Darmstadt, FRG) • HPLC grade
water prepared by filtering deionized water through a Milli-Q
Water purification system (Millipore Corp., Bedford, USA) was
used for the preparation of the buffer solutions and samples.
The buffer solutions and a standard mixture of the herbicides
were prepared by dissolving relevant amounts of the reagents
in the deionized water. Buffers and samples as well were
filtered over 0.45 µm Millex-GS filters (Millipore) prior to
use.
7.3.2 Equipment and Procedures
The experiments were carried out on a Spectra Physics iooo™
capillary electrophoresis system equipped with a multi
wavelength scanning UV detector (Spectra-physics Ine., San
Jose, CA, USA).
Automated capillary rinsing, sample introduction and execution
of the electrophoretic runs were controlled by an IBM personal
system/2 computer (Model 70 386) with software version 1.3.
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Multi-wavelength spectral data were collected, stored and
processed on the same computer. The high speed polychrome mode
by scanning from 200 nm to 300 nm at 5 nm intervals was
employed in these experiments. This permitted three
dimensional platting of the electrochromatograms, peak
confirmation and peak purity evaluation via the comparison of
absorption spectra. sample introduction on this equipment
could be performed either by the electromigration or the
vacuum technique. Here vacuum injection was applied throughout
the experiments to avoid sample discrimination. This was
performed by applying the preset vacuum at the cathode side
during 1 sec (typically about 2 nl fora 50 µm i.d. column).
Fused silica capillary columns with 50 µm i.d., total length
44 cm, effective separation length 37 cm (Siemens, Germany)
were used for all the experiments. Prior to use new capillary
columns were rinsed with 0.1 N sodium hydroxide solution for
20 min and subsequently by water and the buffer solutions, to
be used in the experiments, for 60 min and 20 min,
respectively. Thereafter, conditioning for each experiment was
effected by rinsing the capillary with water for 5 min and
with the buffers for 10 min.
In all the experiments a constant voltage of 15 kV was applied
and the temperature of the separation system was kept at 25°C.
The EOF value was determined by the migration time of
methanol, which can be seen from the disturbance of the
baseline. The viscosity of some of the buffers were measured
on a Rheometrics Fluids Spectrometer (model RFS II,
Rheometrics Ine., NJ, USA) at 25°C.
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7.4 RESULTS AND DISCUSSION
7.4.1 The Effect of surfactants on the Separations
From the chemical structures of the herbicides under study
shown in Table 7.1 and some preliminary experiments it was
concluded that three pairs of components in the mixture
difficult to be separated. They are assigned as:
pair a, consisting of compound 8 and 10;
pair b, consisting of compound 1 and 2;
pair c, consisting Of compound 6 and 7.
Table 7.1 Chemical structures of the investigated herbicides.
1. R1
•H, R3
•CH3
, R2
•Cl: 2,4·DP
2. R1
•H, 112
•R3
•CH3
: MCPP
3. R1
•R2
•Il, R3
•Cll 3 : MCPA
4. R1
•R2
•Cl, R3
•H: 2,4,S·TPA
5. R1 •R2 •Cl, R 3
•CH3
: 2,4,$·TPP
Cl·o~ ·O·CH ·CH ·CH -c"'0
_ 2 2 2 'oH
R 8. Il • Cl: 2,4·08
7. R • CH3 : MCP8
/Cl
c•-o~ .c"'0
- 'oH 'R
8. R - OCH 3 : dlcamba
9. R • Cl : 2,3,6-TB
10. bentazon
were
At the selected pH value of the buffers used in this study,
all compounds are partially or totally dissociated. Therefore,
they carry a negative charge and their migration is governed
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by the electroosmotic flow, the electrophoretic mobility and
eventually by their interaction with the micellar phase in
case MECC is applied. Very high efficiencies {average plate
numbers about 120,000 for each compound) were obtained
throughout the experiments. In spite of that it was hardly
possible to achieve satisfactory separations by using
phosphate buffer solutions only. This due to the very similar
chemical structures of the target compounds. Therefore, a
separation based on MECC was developed.
0
'i>igure 7.3
0.05 concentration of SOS {M)
0.1
Plot of resolution versus concentration of SDS. The experiments were conducted with a voltage of 15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm, buffer: 0.02 M phosphate solution. T = 25°C. (a) bentazon and dicamba; {b) MCPP and 2,4-DP; (c) MCPB and 2,4-DB.
The solubilization of the compounds by specific micelles will
contribute to the migration behaviour and thus to a selective
separation. · The results obtained at different SDS
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concentrations for the three pairs of the components are
presented in Figure 7.3. The resolution of the three pairs
increases with the concentration of SOS. This is due to the
increased hydrophobicity of the components 8, 1 and 6 in the
pairs of a, band c respectively (Table 7.1). So an increased
interaction of these compounds corresponding to the
concentrations of micelles could be expected. A typical three
dimensional electrochromatogram of the 10 herbicides under
study is presented in Figure 7.4.
AU
O.OOEIO
0.0060
0.00S9
0 .0019
-0.0001 ' tS. 150
Figure 7.4
4
"'1"~1
l.O 7'" •• 90 e"''70
Migration time (min)
6
-> "'eso
wavelength (nm)
200 21.0
240
:öl?O :::eo
300
Three dimensional electrochromatograms of a standard mixture of ten chlorophenoxy acids. The experiments were conducted with a voltage of 15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm, buffer: 0.1 M SOS and 0.02 M phosphate solution. T = 25°C. Peak numbers see Table 7.1.
At a concentration of 0.1 M SOS, the resolution of 2,4-DB and
MCPB approaches unity. However, the two peaks are asymmetrie.
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A further increase in SDS concentration will increase the
electrical current to intolerable values. This will cause
additional peak broadening due to the temperature gradients
inside of the column.
It has been shown that the addition of Brij 35 (non-ionic
surfactant) to an anionic surfactant system may improve the
resolution in MECC [62). A distinct advantage of using Brij
35 is that a high concentration of Brij 35 can be used without
influencing the current in the electrophoretic system. Brij
35 was employed in this investigation in combination with SDS
to study its effect on the separation of the chlorinated
phenoxy acids. The results obtained with various concentration
of Brij 35 is shown in Figure 7.5. A slightly increased
resolution for pairs a and b was observed at · increasing
concentrations of Brij 35. This indicates that the interaction
between these solutes and Brij 35 is less effective. The
resolution of pair c, however, was almost proportionally
increased with the concentration of Brij 35. compared with the
other compounds, 2,4-DB and MCPB have relatively longer
molecular chains and Brij 35 is a linear molecule. This may
result in strenger interaction between these two solutes and
Brij 35,
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3.00
2.50
2.00 c c:
.Q ;; ö " f
1.50
a
t 1.00
b
0.50 0 2 3 4 5
conc. of Brij 35 {mM)
Figure 7.5 Plot of resolution versus concentration of Brij 35. The experiments were conducted with a voltage of 15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm, buffer: 0.1 M SOS in 0.02 M phosphate solution. T = 25°C. (a) bentazon and dicamba; (b) MCPP and 2,4-DP; (c) MCPB and 2,4-DB.
Further improvement in the resolution of the three pairs of
the solutes can be obtained by increasing the concentration
of Brij 35. However, the migration times change as well
resulting in the overlapping of compounds 3 and 5 and
deteriorate the overall separation performance.
CetyltrimethylaI!lll\onium (CTAB) was demonstrated to be effective
for the separation of a number of negatively charged compounds
[63]. It was also applied in this work to evaluate its effect
on the separation of the chlorophenoxy acids. An example of
the potential of CTAB to separate acids and related compounds
is presented in Figure 7.6.
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AU 3
1 2
1 1
l 1 1 1
1 1
~ 1 t
L~ i i 1 1 1
J 1
~ 0 2 3 4 5
· Migration time (mii:l.)
Figure 7.6 Electrokinetic chromatogram of a three components mixture .. The experiments were.conducted with a voltage of -15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm, buffer: o. 01 M CTAB in o. 02 M aqueous phosphate solution. T == 25°C. Peak identification: l==isonicotinic acid; 2=nicotinic acid; 3=nicotinamide.
The application of CTAB did not result in a sufficient
separation for the chlorophenoxy acids. The target compounds
are very alike and interact probably in a similar way with
CTAB.
Optimization of the resolution by the capacity factor was
discussed already in the theoretica! section. It treats the
separation conditions concerning one pair of closely migrating
compounds. It is more complicated to separate three pairs of
difficultly to be resolved solutes because of their similar
dependence on the experimental parameters. To achieve a
satisfactory separation of the target compounds a compromise
has to be made in the composition of the buffer. The best
overall separation of the herbicides under study was achieved
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by the application of 0.02 M phosphate buffer solution
containing 0.1 M.SDS and 3*10-3 M Brij 35 (Figure 7.7).
21 4 7 6
wavelength
0. O:l.00 "'~ ( 310
0.001!10
0.0060
0.0039
0. 00:!.9
7.':i.o 7 .'.:>o e .'7o Migration time {min)
i /
.,. .'tso
2:20 230
240
1 :öltlO
1 260 270
::eo 390
300
Fiqure 7.7 Three dimensional electrochromatograms of a standard mixture of ten chlorophenoxy acids. All experiments were carried out with a voltage of 15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm, buffer: 0.1 M SDS and 3*10-3 M Brij 35 in 0.02 M phosphate solution. T = 25°C. Peak numbers see Table 7.1.
7.4.2 The influence of Methanol on the Separation
It has been show that a small percentage of organic modifiers
can also improve the separation efficiency in MECC [64, 65].
The resolution of the three pairs of compounds with addition
of various portions of methanol to the buffer are depicted in
Figure 7.S.
An improvement in resolution for the pairs a and b was
observed with increasing amounts of methanol. A remarkable
enhancement in the resolution of the pair c was achieved by
increasing the methanol percentages.
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The improvement in the resolution can be explained by tl:Î.e
increase in the elution range (window) as a result of the
addition of the modifiers [64). In fact, a smaller value of
to/tmc is obtained by the addition of methanol. From eq. 7.13,
and Figure 7.2, it is evident that f(k) increases with
decreasing values of to/tmc· consequently, increased
resolutions can be expected in the case of decreasing values
of to/tmc (cf. eq 7.12).
3.00 c
( A)
2.50
i:: 0 .....
2.00 .... ::!
..-1 0 tl)
a.I 1.50 ...
1.00
o.so 0 5 10 15 20 0 s 10 15
percentage of methanol
Figure 7.8 Plot of resolution versus methanol percentage. (A) O. 02 M phosphate buffer with 0.1 M SDS; (B} 0.02 M phosphate buffer with 0.1 M SOS and 3*10-3 M Brij 35. All experiments were conducted with a voltage of 15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm. T = 25°C. (a) bentazon and dicamba; (b) MCPP and 2,4-DP; (c) MCPB and 2,4-DB.
The migration times of the ether four components (peaks 3 1 4,
5, and 9) in Table 7.1 are also influenced by the addition of
methanol to the buffer solution. As in the case of the
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addition of Brij 35 an increase in migration time is observed
for these compounds. This has a negative effect on the overall
separation of the sample, e.g. compound 3 starts to overlap
compound 5 {Figure 7.9).
AU
Figure 7.9
wavelength (run)
200
Three dimensional electrochromatograms of a standard mixture of ten chlorophenoxy acids. The experiments were carried out with a voltage of 15 kV across 44 cm * 50 µm i.d. column, effective length 37 cm, buffer: 0.1 M SOS and 3*10-3 M Brij 35 and 10% methanol in 0.02 M phosphate solution. T = 25"C. Peak numbers see Table 7.1.
With respect to the qualitative and quantitative analysis,
multi-wavelength detection allowed three-dimensional plotting
of the electrochromatograms (Figure 7.4 and 7.7) showing the
relationships of absorbance vs. migration times vs.
wavelength.
The absorption spectrum of each component can be extracted
from the collected data points indicated as time slices
(Figure 7 .10). This provides additional possibilities for the
identification of a compound.
-164~
AU
aJ,o wavalength (nm)
AU
wavelength ( lllll)
Figure 7.10 Absorption spectra obtained as a time slice from the data in Figure 7.7. (A) Pairs a and b; (B) pairs band c.
Figure 7.10 shows that in contradiction to compounds s and 10
(pair a), the spectra of compounds 1 and 2 (pair b) and
compounds 6 and 7 (pair c) are quite similar. This indicates
that the sufficient separation of these chlorophenoxy acids
is mandatory to achieve reliable quantitative and qualitative
analytica! results.
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As an example the results of migration times and peak areas
for 6 compounds (the three pairs} are presented in Table 7.2.
Table 7.2 Repeatability of migration times and peak areas.
Peak Migration times Peak areas No. s u /s (%) s u/s(%} (1 a
10 5.67 0.05 0.9 3613 44 1.2
8 5. 72 0.05 0.9 8232 199 2.4
2 5.81 0.05 0.9 13470 193 1.4
l 5.87 0.06 1.0 14217 311 2.2
7 8.11 0.09 1.1 14772 209 1.4
6 8.32 0.11 1.4 14148 247 l. 7
conditions: 0.02 M phosphate containing 0.1 M SDS and 3*10-3 M Brij 35 buffer solution. Sa111.ple concentration about o. 2 mg/ml for each compound, constant voltage 15 kV, column: 44 cm * 50 µm i.d. column and effective length 37 cm. T = 25°C. Peak numbers see Table 7. 1. s-average value, a-standard deviation, a/s (%)-relative standard deviation, number of experiments n = 3,
It was observed that the electroosmotic flow altered with the
various buffer solutions used in the experiments. This was
examined with respect to the contribution of the viscosity
changes of the buffer solutions to the EOF.
The viscosity of a dilute suspension of rigid particles in a
continuous medium can be approximated by the Einstein equation
(66, 67). The result, restricted to the case of small volume
fractions of spherical particles, is given by:
11 = 11 0 * ( 1 + 2. 5 * cf> )
where ~: the viscosity of the suspension, ~0 : the viscosity of the medium, ~: the volume fraction occupied by the particles.
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(7. 18)
This equation can also be applied for s'olutions containing
micelles which are relative large compared to the solvent
molecules [68]. Therefore it can be applied here to estimate
the viscosities of the micellar buffer solutions.
The ratio of ~/~o indicates the relative change in viscosity
of the solutions with and without micelles and can be
determined if t;/> is known. several methods can be used to
calculate the volume fraction, t;f>. One of the methods in based
on the following equation:
(7. 19)
where c is the concentration of the micelles in the buffer
solution and Vi is the average mole volume of the micelles.
The viscosity change of a 0.02 M phosphate buffer solution by
addition of 0.05 M SDS can be estimated as following. The
average radius of an SDS micelle is 2. 5 nm and the aggregation
number in this case is 62. The average mole volume of the SDS
then, is O. 64 lf:mol and from this t;/> can be calculated as
0.032. Therefore, ~/~o = 1 + 2.5 * t;/> = l.OS, or a 8% increase
in the viscosity of the SDS buffer solution compared to the
original phosphate buffer solution.
The corresponding experimental results
measurements of 0.02 M phosphate and
of the
0.02 M
viscosity
phosphate
containing O. 05 M SOS buffer solution show viscosi ties of
0.995 and 1.096 e.p. (25°C}, respectively. The ratio of these
two values (l.10) is in reasonable agreement with the value
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calculated from the Einstein equation.
Assum.ing that with the exception of " all the variables in eq
7.1 are constant, under defined experimental conditions, then
Cveo)ph/(ueo)sos = "sos/"ph· Where (veo>ph and (ueo)sos are
the electroosmotic velocities in phosphate and SOS buffers,
"SOS and ?jph are the viscosity of the corresponding buffers
respectively. The experimentally observed (1.1eo>ph/(11e0 )sos
value is about 1.09, which is in fair agreement to the value
of the measured ?!sos/77ph value.
7.5 CONCLUSIONS
The selectivity of the MECC technique is demonstrated in this
study for compounds which have similar chemica! structures.
Tuned selectivity in the separation was obtained by optimizing
parameters such as the concentration of SOS and Brij 35 and
the percentage of methanol in the buffers.
SDS combined with Brij· 35 as the micellar agents provided
satisfactory separations and relatively short analysis time.
Also, high efficiencies (more than 100,000 theoretica! plates
were obtained for the separation of these chlorinated phenoxy
acids herbicides under MECC conditions.
Relative standard deviations for migration times are about 1%
and the repeatabilities for peak areas are approximately 2%
for all the investigated compounds.
The multi-wavelength detection provided useful additional
information. The characterization of the sample zones by their
migration times and adsorption spectra proved to be a powerful
-168-
tool for solute identification.
Experimentally measured viscosity data were in good agreement
with the calculated results from the Einstein equation. The
differences in the viscosities match well with the observed
changes in the EOF.
MECC offers great selectivity and flexibility. Therefore the
possibilities of this technique in other application areas
should be further investigated.
7.6 REFERENCES
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-171-
SUMMARY
Chromatography is of extraordinary practical importance in
both analytica! applications and preparative separations. No
ether method of instrumental analysis can match its
efficiency, speed, reproducibility, and versatility.
Chromatographic methods are often the only way to analyze
target compounds in different matrices. Biochemistry is
another important area that benef its from the application of
chromatography and the complementary technique of capillary
electrophoresis.
In gas chromatography and electrophoresis the best performance
is obtained on capillary columns.
It is widely accepted that fused silica is the material of
choice for capillary separations. For their application in
chromatography it is essential that the column properties are
determined by the bulk properties of the stationary phase and
not by contributions of residual active sites on the column
wall. Deactivation of the column wall and compatibility of the
modified surface. with the stationary phase to be coated is
essential for obtaining reproducible analytica! results.
Constant surf ace properties are also essential in obtaining
a controllable and reproducible electroosmotic flow in
electrochromatography (EC).
This thesis describes some recent developments in the
preparation of capillary columns for gas chromatography (GC)
and EC. The emphasis is on deactivation of the column wall and
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crosslinking of the stationary phase. The experimental study
of the deactivation process is supported by solid state 29si
NMR measurements on fumed silica (Cab-O-Sil) as model
substrate.
Columns prepared according to the optimized procedures are
evaluated both in capillary GC and in EC.
chapter 2 describes the synthesis of a series of vinyl
modified polymethylhydrosiloxane deactivation reagents
containing different functional groups. After deactivation
some vinyl groups are left to facilitate crosslinking and/or
chemical bonding of the stationary phase.
The optimization of the deactivation reaction is investigated
and evaluated by GC with a dual column system. Excellent
deactivation of the fused silica surface is obtained with the
reagents studied. Relatively high temperatures and/or long
reaction times are needed for diphenyl and bis-(cyanopropyl)
substituted reagents compared to methyl substituted
deactivation reagents.
Physisorbed water on the inner surface plays an important role
in achieving a thin and densely crosslinked layer on the
column wall.
compared to methyl deacti vated columns an increase of the
Kováts retention indices of polar test compounds is observed.
This indicates a successful incorporation of polar functional
groups in the deactivation layer.
The effect of the silylation temperature and the reaction time
on the deactivation of silica surfaces is investigated using
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solid state 29si NMR spectroscopy (Chapter 3). The reaction
between Si-H and Si-vinyl groups by thermal treatment is
confirmed by NMR experiments using fumed silica as model
substrate. The existence of vinyl groups on the silylated cab
o-sil samples, however, is not evident, due to the low amount
of vinyl groups in the copolymer and the lack of sensitivity
of the 29si NMR technique. The spectra obtained show that the
deactivation layer is stable up to at least 360°C.
The preparation of non-polar and medium polar capillary
columns based on deactivation reactions described in previous
chapters is presented in Chapter 4. The crosslinking and/or
chemica! bonding of various stationary phases by thermal
treatment or azo-tert-butane (ATB) initiation is investigated.
The immobilization of vinyl modified non-polar stationary
phases can be achieved by thermal treatment. This results in
very inert columns and has minimal ef f ects on the retention
behaviour. To obtain a sufficiently high degree of
immobilization of non-vinyl containing stationary phases, a
combination of ATB initiation and a vinyl modified
deactivation layer is required.
Non-polar narrow bore capillary columns down to approximately
20 µm i.d. could be prepared using the procedure described in
this chapter. Highly inert and efficient columns were
obtained.
In chapter 5, the applicability of the prepared non-polar and
medium polar fused silica capillary columns is illustrated by
different examples. High speed GC separations are performed
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on 'narrow-bore capillary columns with inner diameters of
20 µ:m. Columns with high thermal stability were successfully
prepared for the analysis of less volatile compounds, such as
poly:mer additives, glycerides and polyethylene glycol 400. The
successful separation of some underivatized steroids, drugs,
priority pollutants and derivatized amino acids de:monstrates
the high degree of inertness of the columns prepared according
to the methods described here.
In chapter 6, a systematic investigation is carried out to
study the electroosmotic flow (EOF) in micellar electrokinetic
capillary chro:matography (MECC). The chemical nature of the
modification layer is shown to have a strong influence on the
:magnitude of the EOF. This can be explained by studying in
detail the specific influence of the modification layer on the
r-potential. The results from untreated and treated columns
were co:mpared under the sa:me experimental conditions.
Further:more, experiments with and without sodiu:m
dodecylsulfate (SDS) addition to the buffer were carried out
in order to elucidate the effect of this surfactant on the
EOF.
Under the proper experimental conditions reproducible EOF
values with a relativa standard deviation of about 3% can be
obtained using a home-built instrument.
In chapter 7, the selectivity of MECC for the separation of
herbicides consisting of chlorophenoxy acids is de:monstrated.
Tuned selectivity was obtained by optimizing parameters such
as the concentration of SDS and Brij 35 and the percentage of
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methanol in the buffer. SOS combined with Brij 35 as micellar
agents were found to provide the best overall separation of
these components.
The analysis of the herbicides was carried out using multi
wavelength detection. The characterization of the sample zones
by their absorption spectra and migration times proved to be
a powerful tool for solute identification. In this particular
MECC separation system, high efficiencies (more than 100,000
theoretical plates) were obtained. Relative standard
deviations for migration times of the components were about
1% and for peak areas approximately 2%.
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SAMENVATTING
Chromatografie is van uitzonderlijk belang in het analytisch
laboratorium, met name in combinatie met spectroscopische
identificatietechnieken, in het bijzonder massaspectrometrie.
Preparatieve chromatografie wordt meer en meer toegepast voor
de scheiding van produkten met hoge toegevoegde waarde in de
farmaceutische en biotechnologische industrie.
Geen enkele andere analysetechniek benadert het scheidend
vermogen, snelheid van analyse en de nauwkeurigheid van
kwantitatieve resultaten. De verscheidenheid aan toepassings
mogelijkheden is ongeëvenaard.
Vaak zijn chromatografische methoden de enige mogelijkheid om
"doelcomponententt te bepalen in ingewikkelde matrices, b.v.
urine, bloed, water en bodemmonsters.
Milieuresearch en monitoring is meestal alleen mogelijk door
chromatografische spor·enanalyse uitgevoerd met zeer hoge
gevoeligheid en scheidend vermogen. Biochemie en biotech
nologie zijn twee andere belangrijke gebieden waarvan de
vooruitgang mede afhangt van de toepassing van chromatogra
fische en de verwante.en aanvullende techniek, elektroforese.
Zowel voor gaschromatografie als elektroforese geldt dat met
capillaire kolommen de beste resultaten worden verkregen.
Sinds 1979 worden voornamelijk de door Dandeneau
geïntroduceerde 'fused silica' kolommen gebruikt.
Het is van wezenlijk belang dat de chromatografische
eigenschappen van een kolom worden bepaald door de toegepaste
stationaire fase en niet door aktieve plaatsen op de
kolomwand. Daartoe wordt voor het opbrengen van de stationaire
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fase de kolomwand gedeactiveerd. Voor de spreiding van de
stationaire fase op het gedeaktiveerde kolomoppervlak is het
van groot belang dat de polariteit van de wand is aangepast
aan die van de op te brengen laag.
Ook bij de toepassing van 'fused silica' kolommen in de
electrochrOlllatografie (EC) zijn konstante oppervlakte-
eigenschappen essentieel, zij bepalen namelijk de grootte en
richting van de elektro-osmotische stroming.
Dit proefschrift beschrijft nieuwe methoden voor de bereiding
van 'fused silica' capillaire kolommen voor zowel gaschromato
grafie als micellaire elektrokinetische capillaire
chromatografie (MECC).
In Hoofdstuk 2 van dit proefschrift wordt de synthese van een
aantal vinylgemodif iceerde polymethylhydrosiloxaan deacti
veringsreagentia besproken. De bij deactivering met dit
reagens in de deactiveringslaag ingebouwde vinyl-groepen
vereenvoudigen de crosslinking en het chemisch verankeren van
de stationaire fase. De kinetiek van de deactiveringsreacties
wordt bestudeerd en geoptimaliseerd met twee-dimensionale
capillaire GC. Het blijkt dat deactivering met diphenyl en
bis-{cyanopropyl) gesubstitueerde reagentia hogere deacti
veringstemperaturen en/of langere reactietijden vereist dan
reacties met methyl gesubstitueerde reagentia. Verder blijkt
gephysisorbeerd water op het kolomoppervlak een essentiële rol
te spelen in het deactiveringsproces. Met de bestudeerde
reagentia kan een hoge mate van inertheid van het fused silica
oppervlak bereikt worden.
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In Hoofdstuk 3 worden vaste stof 29si NMR technieken toegepast
voor de bestudering van de invloed van de silylerings
temperatuur en de reactietijd op de deactivering van fused
silica oppervlakken. Met NMR experimenten aan het model
substraat 11 fumed" silica (Cab-O-Sil) kan de reactie tussen
si-H en Si-vinylgroepen bij verhoogde temperaturen gevolgd
worden. De aanwezigheid van vinylgroepen op de gesilyleerde
Cab-O-Sil monsters kan met NMR door het percentage
vinylgroepen in het uitgangsmateriaal en de relatief lage
gevoeligheid de 29si NMR, niet onomstotelijk bewezen worden.
Wel kan worden aangetoond dat de verkregen deactiveringslaag
stabiel is tot een temperatuur van tenminste 360°C.
Het onderwerp van Hoofdstuk 4 is de bereiding van niet-polaire
en zwak-polaire capillaire kolommen gebruikmakend van de
deactiveringsreagentia bestudeerd in de voorgaande hoofd
stukken. Een belangrijk onderwerp beschreven in dit hoofdstuk
is de vergelijking van thermisch geïnitieerde en azo-tert
butane geïnitieerde crosslinking en chemische verankering van
diverse stationaire fasen. Thermisch geïnitieerde crosslinking
van vinyl-gemodificeerde stationaire fasen geeft zeer stabiele
en inerte kolommen en heeft een verwaarloosbare invloed op de
retentiegeigenschappen van de stationaire fase. Voor het
verkrijgen van een vergelijkbare deactivering op een vinyl
vrije stationaire fase is een combinatie van ATB initiatie met
een vinyl gemodificeerde deactiveringslaag vereist. De in dit
hoofdstuk beschreven procedures bleken bij uitstek geschikt
voor de bereiding van narrow-bore capillaire kolommen met
diameters tot ongeveer 20 µm.
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In Hoofdstuk 5 wordt de toepasbaarheid van de kolommen, bereid
via de procedures beschreven in de voorafgaande hoofdstukken,
aan de hand van een aantal applicaties geïllustreerd. In dit
hoofdstuk worden enkele zeer snelle scheidingen uitgevoerd met
20 µm kolommen. Verder worden de bereide kolommen succesvol
toegepast voor de hoge temperatuurscheiding van zeer hoog
kokende componenten zoals polymeer additieven, glyceriden en
polyethyleen glycol 400. De goede resultaten verkregen bij de
scheiding van ongederivatiseerde steroïden, alkaloïden en de
zogenaamde 'priority pollutants' is een bewijs voor de hoge
mate van inertheid van de kolommen.
In hoofdstuk 6 is het onderzoek beschreven naar de effecten
van verschillende coatings van capillaire kolommen op het
elektroosmotisch debiet in micellaire elecktrokinetische
capillaire chromatografie (MECC).
Gebleken is dat het physisch-chemisch karakter van de
kolomwand een sterke invloed heeft op de grootte van de
electroosmotische snelheid (EOF) in de kolom. Een verklaring
hiervoor kan worden gegeven door de polariteit van de
verschillende niet-gecoate en gecoate kolommen in beschouwing
te nemen. Daarnaast is ook het effect bestudeerd van de
toevoeging van het surfactant natriumdodecylsulfaat (SOS) aan
de buffer op de EOF.
In dit deel van het onderzoek werd aangetoond dat onder de
toegepaste experimentele kondities reproduceerbare EOF-waarden
met een relatieve standaardafwijking van 3% te realiseren
zijn.
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In hoofdstuk 7 wordt, aan de hand van de scheiding van
herbiciden, de toepassing van verschillende surfactanten in
de buffer en de invloed hiervan op de selektiviteit in MECC
behandeld. Daarnaast is ook de rol van de toevoeging van
methanol aan de buffer op de selektiviteit onderzocht.
De effecten van verschillende concentraties van de
surfactanten SDS en Brij-35 en de organische modifier methanol
in de buffer werden bestudeerd.
Gebleken is dat combinaties van de verschillende typen en
concentraties van deze additieven aan de buffer de selec
tiviteit in MECC sterk kunnen verbeteren.
Met betrekking tot de kwalitatieve en kwantitatieve analyse
bleken de relatieve standaardafwijkingen van migratietijden
en piekoppervlakken bij de scheiding van deze herbiciden,
respectievelijk 1 en 2%.
samenvattend, blijkt MECC een potentieel krachtige scheidings
methode door de mogelijkheden tot beïnvloeding van de
selektiviteit in combinatie met de hoge efficiency, die met
deze techniek te realiseren is.
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ACDOWLEDGEMENT
At the moment of releasing of this thesis, I feel obliged to
acknowledge all of thosepeople, without whose contributions,
direct or indirect, I would have been unable to finish this
work.
First I would like to express my gratitude to Prof.Or. Carel
cramers, who gave me the opportuni ty to carry out my doctorate
research at the Eindhoven University of Technology. It is his
liberal way of guidance and his encouragement throughout the
investigations that led to the completion of this thesis.
Special thanks are going to Jacques Rijks sr. and Henk
Claessens for their permanent support. I additionally thank
Jacques Rijks sr. for his help in introducing me to the "world
of chromatography" • His help in personal matters is kindly
appreciated.
I thank Hans-Gerd Janssen, Martin He tem, Robert Houben, Gerard
Rutten, Jacek Staniewski, Andrew van Es, Dana Conron, Hans
Mol, Andrea Houben, Pieter Hendriks, Marion van straten, Janet
Eleveld, Hans Verhoeven, Pavel Coufal, Pim Muijslaar, Marc
Bergman, Door Platje, and Gerlie Heemels for the many
stimulating discussions on separation sciences. Jacques Rijks,
Hans-Gerd Janssen, Henk Claessens, Jan de Haan are further
acknowledged for their help in revising the first concept of
this thesis and the published papers.
I am obliged to Denise Tjallema, Hans van Rijsewijk, Jack
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Rijks jr., Henri snijders, Anja Huygen, Anton Bombeeck, Piet
Leclercq, Harry Maathuis and Dick Kroonenberg for their
kindness and help. Special thanks to Huub van Leuken for the
construction of the electrophoresis system.
I cordially thank Rongrong, my wife, and Fanda, my daughter
for their patience and their unreserved support for my work.
Finally, I acknowledge all members and students of the
Laboratory of Instrumental Analysis of Eindhoven University
of Technology, who in one way or another contributed to my
work and my stay in The Netherlands.
Quanji Wu
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~ 1 CURRICULUM VJ:Tll
3 $"
The author was born in Liuzhou, Guangxi Province, China, on
December 2, 1956. He finished his middle school education in
1973 and entered the Guangxi University in 1978, after 4 years
study, he obtained his Bachelor degree. He continued his study
in 1978 at the Lanzhou Institute of Chemica! Physics,
belonging to the Chinese Academy Science. There he obtained
his Master degree in ~ In 1987 he came to the Eindhoven
University of Technology in The Netherlands, where he started
his Ph.D. research work which led to this thesis.
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PtJBLICATIOHS
1. Q.J. wu and W.L. Yu, Analytical Instruments (Chinese), 1987, (1) 35 (Ch.). "Characterization of Hall Electrolytic conductivity detector for GC 11 •
2. Quanji Wu, Q.Y. Ou and W.L. Yu, Fenxi Huaxue (Chinese), 1987, 15(5) 390 (Ch.). "The selectivity and the response factor of Hall electrolytic conductivity detector for GC11 •
3. M. Hetem, Q. Wu, J. Rijks, L. van de Ven, G. Rutten, J. de Haan and C. Cramers, Proc. of 17th International Symposium on Chromatography, Vienna, Austria, september 1988, I, P32. "Deactivation of silica surface with polyhydrosiloxanes: a quantitative evaluation by chromatography and 29si NMR".
4. Q. wu, C.A. Cramers and J .A. Rijks, Proc. ·of lOth International Symposium on capillary Chromatography, Riva del Garda, Italy, May 1989, P. 188. "Preparation of capillary columns with vinyl modified polyhydrosiloxane deactivation reagents-characterization and optimization of the deactivationtt,
5. Q. Wu, M. Hetem, C.A. Cramers and J.A. Rijks, J. High Resolut. Chromatogr., 13 (1990) 811. 11 Preparation of thermally stable phenylpolysiloxane fused silica capillary columns-optimization and evaluation of the deactivation by capillary GC and solid state 29si NMR".
6. Q. Wu, C.A. Cramers and J.A. Rijks, Proc. of 13th International Symposium on Capillary Chromatography, Riva del Garda, Italy, May 1991, P. 176. "Some aspects of crosslinking and chemical bonding of stationary phases in the preparation of non-polar and medium polar GC capillary columns".
7. G.A.F. Rutten, Q. Wu, J.A. Rijks and C.A. Cramers, Proc. of 13th International symposium on Capillary Chromatography, Riva del Garda, Italy, May 1991, P. 143. "Deactivation of fused silica capillaries with polydiphenylvinylmethyl-hydrosiloxane copolymer. Optimization of reaction conditions using a new modification of intermediate test".
8. Q. Wu, H.A. Claessens and C.A. cramers, "The influence of surface treatment on electroosmotic flow in micellar electrokinetic capillary chromatography". Chromatographia, in press.
9. Q. Wu, H.A. Claessens, C.A. cramers, "The separation of Herbicides by micellar.electrokinetic chromatography". Submitted for publication.
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STELLINGEN
1. The contribution of the deactivation layer to the retention behaviour of solutes cannot be neqlected.
-This thesis, chapter 2.
2. The ratio of the mobility and the diffusion coefficient of a solute in the equation for plate num.ber N = µE / 2D is not an intrinsic property of the solute, independent of buffer properties.
-J.W. Jorqenson and R.D. Lukacs, Anal. chem., 53 (1981) 1298. -H.'1'. Rasmussen and H.M. McNair, J. chromatoqr., 516 (1990) 223. -H.J. Issaq, I.Z. Atamna, G. M. Muschik and G.M. Janini, chromatographia, 32 (1991) 155.
3. The statement that there are no exposed Si-H bonds on the column surface after deactivatinq fused silica capillary columns with polymethylhydrosiloxane is incorrect.
-C.L. woolley, R.E. Markides and M.L. Lee, J. Chromatoqr., 367 (1986) 23. -M. He tem, G. Rutten, B, Vermeer, J. Rijks, L. van de Ven, J. de Haan and c. cramers, J. chromatoqr., 477 (1989) 3.
4. The conclusion that in micel lar electrokinetic chromatography the effect of a temperature gradient on plate height is negligible is based on a series of incorrectly derived equations.
-s. Teral:>e, R. otsuka and T. Ando, Anal. Chem., 61 (1989) 251. -s. Teral:>e, K. Otsuka and T. Ando, Anal. Chem., 57 (1985) 834.
5. Capillary columns coated with polydimethylsiloxane, a non-polar stationary phase, result in an increased electroosmotic flow in micel lar electrokinetic chromatography. This is not expected because silanol
groups on the column surface are covered by the nonpolar coating.
-This thesis, chapter 6.
6. Hanai's observation that the migration rate of fructose increases with the concentration of sodium chloride is in contradiction with theory.
-T. Hanai, H. Hatano, N. Nimura and T. Kinoshita, J. High Resolut. Chromatoqr., 14 (1991) 481.
7. Quantitative 29si CP-MAS NMR measurements of derivatized silica surfaces require more precautions than published by Köhler c.s. 1). In fact, the cross-polarization (CP) characteristics should be measured and accounted for explicitly.
-J. Köhler, D.B. Chase, R.D. Furlee, A.J. Vega and J.J. Rirkland, J. Chromatoqr., 352 (1986) 275. -B. Pfleiderer, K. Albert, E. Bayer, L. van de Ven, J. de Haan and c. cra.mers, J. Phys. Chem., 94 (1990) 4189.
8. The contribution of the term describing resistance to mass transfer in the liquid phase cannot be neglected in vacuwn outlet capillary gas chromatography.
-N. Bel.lier and G. Guiochon, J. Chromatoqr. sci., 8 (1970) 147.
9. A linear behaviour of the logarithm of the capacity ratio of a solute vs. the percentage modifier is not observed in supercritical chromatography even if the action of modifiers is solely due to interactions between the modifier and the stationary phase.
-L.J. Mul.cahey and L.T. Taylor, J. Hiqh Résolut. Chromatoqr., 13 (1990) 393.
10. Modification is an important factor in science and technology but also in society. This is clearly reflected by the food served in most Chinese restaurants in The Netherlands, which is quite different frorn the original.