DOI:
10.1039/A908102J
(Communication)
Analyst, 2000,
125, 1-4
Electro-osmotic and pressure-driven flow properties of
frits for packed column capillary electrochromatography prepared from
functionalised and bare silica packings
Received 8th October 1999, Accepted 15th November 1999
First published on UnassignedUnassigned7th January 2000
Abstract
Changes in electro-osmotic flow (EOF) induced in an open fused
silica capillary by the introduction of a single packing retainer (referred
to as a ‘frit’ throughout the article) are investigated. Frits
located in a 34.5 cm long capillary close to the detection window, as used
commonly in packed column capillary electro-chromatography, were made by
sintering silica based packing materials having varying functionalities
(bare silica, ODS-silica, a strong cation exchange packing (SCX) and a
strong anion exchanger (SAX) at 430 °C for 15 s. The contributions of
such frits to the total EOF measured in the capillary are discussed.
Despite the high temperature of heating of the packing, sufficient residual
functional groups remained on the frit such that distinctively different
EOF behaviour was observed for each type of packing. Frits made from
materials providing negative surface charges, such as silica, ODS-silica
and SCX, increased the magnitude of the cathodic EOF compared to the open
capillary. On the other hand, a substantial anodic EOF of −1.88
× 10−8 m2 V−1
s−1 was produced by introduction of a single frit made
from the SAX material. An explanation of this behaviour is given, based on
the hypothesis that the EOF generated by the frit determines the overall
flow in the whole open capillary. The frit is considered to work as a pump
and overrides the EOF generated at the capillary wall.
Introduction
Capillary electrochromatography (CEC) using packed columns has been an
emerging technique over recent years and since suitable instrumentation for
CEC has become available commercially, increased interest in this new
separation method has resulted. This fact is demonstrated clearly by the
steadily growing number of publications within this field.1–8 The
success of CEC can be attributed to several potential advantages of this
approach over other liquid phase separation methods, such as
high-performance liquid chromatography (HPLC) or capillary zone
electrophoresis (CZE). First, the almost plug-like flow profile of the
electro-osmotic flow (EOF) helps to minimise the dispersion of analyte
zones which occurs in HPLC. Second, the velocity of the electroosmotically
generated flow is independent of the capillary diameter, the column length
and the particle size of the packing, and this allows the use of microbore
capillaries and microparticulate chromatographic supports without the
limitation of increased back pressure as occurs with pressure-driven
methods such as HPLC. Finally, CEC offers additional separation selectivity
because of its combined electrophoretic and chromatographic separation
mechanisms.Since the beginnings of CEC various methods for packing of columns have
been described, such as employing high pressure,9,10 EOF11 or even
centripetal forces.12 Recently, so-called
monolithic columns for CEC, prepared by in situ polymerisation of
a stationary phase inside the capillary, have been reported.13 In the latter case the chromatographic support
is immobilised by chemical bonding to the capillary walls, but with all
other packing methods a retainer is necessary to keep the particles inside
the column. This can be accomplished either by modification of the
capillary design using tapered capillaries as described recently14 or through the use of frits, which can be
inserted into the column mechanically,15 or
generated inside the capillary by various chemical or physical means. In
the literature published so far, the latter method is still the most widely
used and a number of techniques for the preparation of frits have been
described.16–18
There have been several reports on the effects of frits and packings on
EOF, but the results have been inconsistent and vary from reporting a
reduction of 44% in EOF when two frits were introduced into an open
capillary,19 to an increase in EOF for a
capillary packed with an ODS material compared to an equivalent open
capillary.20,21 As there were only
minor differences in the column packing procedures reported in these
studies, the observed discrepancies might be attributable to differences in
the materials and procedures used for making the frits, since these vary
considerably between the studies. Furthermore, it is well known that many
of the problems associated with CEC column operation (e.g., bubble
formation) can be due to the interface between packed and open zones within
the capillary column.21–23 This means that it is desirable that the
resistivity of the frits be as similar to either the packed or the open
zone as possible, thus avoiding further discontinuities in the column
structure.
Therefore, whilst the influence of the frits on the performance of the
entire packed column can be regarded as crucial, only a few detailed
reports investigating these important components of the CEC column
exist.19,21,24,25 In this
paper we investigate the effects on key parameters such as pressure
resistance and EOF caused by the introduction into a fused silica capillary
of a single frit made of bare or functionalised silica.
Experimental
Instrumentation
Experiments were performed using an HP3D CE system
(Hewlett-Packard, Waldbronn, Germany), equipped with a diode array detector
and connected to an HP 3D-CE Chemstation (Hewlett-Packard) for data
processing. The temperature of the column was maintained at 25 °C
during all separations. Samples were injected hydrodynamically by applying
a pressure of 20 mbar (open capillary) and 2 bar (capillary with frit) for
3 s.Materials and reagents
Fused silica capillaries (75 μm or 100 μm id × 360 μm od)
obtained from Polymicro Technologies Inc. (Phoenix, AZ, USA) were used
throughout this work. All capillaries were flushed with 0.1 M NaOH (at
least 20 capillary volumes) before use, and flushed with BGE (at least 5
capillary volumes) between runs.The following packing materials (all from XTec Consultants Ltd., Clwyd,
UK) were used: 3 μm silica (190 m2 g−1, 8 nm
pore size), 3 μm silica-based reversed phase material (OSD1, 6% carbon,
8 nm pore size) 3 μm silica-based strong anion exchange material (SAX,
capacity approx. 200 μequiv. g−1, 2.5% carbon, 8 nm
pore size) 3 μm silica-based strong cation exchange material (SCX,
capacity approx. 120 μequiv. g−1, 1% carbon, 8 nm pore
size). Additionally, frits were prepared from Hypersil ODS 3 μm silica
(170 m2 g−1, 10% carbon, 12 nm pore size).
All chemicals used were of analytical reagent grade. Water was purified
using a Milli-Q water (Millipore, Bedford, MA, USA) system.
Electrolytes were prepared from a 500 mM Tris stock solution, titrated
to pH 8.0 with hydrochloric acid and diluted with a mixture of water and
acetonitrile or pure water. All electrolytes were filtered through a 0.45
μm membrane filter (Millipore) and degassed before use. Acetone was used
as a marker for the determination of the EOF.
Procedures
Preparation of the frits.. Pretreated fused silica capillaries were packed using a slurry packing
technique similar to that described by Hilder et al.26 Frits were generated by heating a narrow band of
the packing 25 cm from the inlet end of the capillary at 430 °C for 15
s (unless otherwise stated) using a device similar to the one described by
van den Bosch et al.16 consisting
of a heating element in the shape of a ribbon 0.3 mm thick with a 0.5 mm
hole in it. During the heating, the capillary was flushed with water at 300
bar. After formation of the frit, the excess packing was flushed out of the
capillary and the length of the formed frit was measured using a
microscope. A detection window was fabricated 10 mm behind the frit using
hot sulfuric acid with the frit protected from the heating element during
this process. Finally, the capillary was cut to a total length of 34.5 cm,
mounted in the Hewlett-Packard capillary cartridge and conditioned with
mobile phase before use. Measurement of electro-osmotic and pressure-driven flow
properties of open capillaries and capillaries with frits.. This was accomplished using a procedure similar to that previously
described by Dittmann et al.21
First, the magnitude of the EOF at +20 kV (−20 kV in the case of the
SAX packing) as well as the pressure resistance was measured for each empty
capillary (75 or 100 μm id) using a pH 8.0 electrolyte consisting of 50
mM Tris–acetonitrile (1∶4) or 10 mM Tris and employing acetone
as a neutral marker. After the formation of frits using the procedure
described above, the EOF and the pressure resistance were measured again
for each frit. Finally, to include the influence of the length of the
packed bed into these considerations, the measured difference in EOF
(ΔEOF) was normalised to a 1 mm frit length by dividing the
ΔEOF value by the frit length in mm. Results
Properties of frits made from Hypersil ODS
Table 1 summarises the experimentally
obtained linear velocities characterising the electro-osmotic and
pressure-driven flow properties of open capillaries and the same
capillaries with frits made from Hypersil ODS. The following points can be
made from these results. First, the pressure resistance data for the frits
show that only low frit-to-frit reproducibility could be achieved although
the length of the sintered zone was kept within a narrow range
(1.8–2.1 mm). Second, for the electro-osmotically driven flow, very
similar linear velocities were encountered for all three frits. This leads
to the conclusion that within certain constraints (i.e., as long
as no significant blockage of the capillary occurs) the correlation between
the pressure resistance of a frit and its influence on the EOF is likely to
be rather low or insignificant. Third, an increased EOF could be observed
for all capillaries with a frit compared to the open capillaries. It is
reasonable to suggest that the surface charge on the open capillary wall
and inside the frit channels will predetermine the EOF in each region, but
it is somewhat difficult to explain why for example a frit prepared from an
ODS material (as in Table 1) which is
likely to have a relatively low surface charge density also increases the
EOF velocity compared to an open fused silica capillary. One possible
explanation is that substantial additional ionisable silanol groups are
created during the heating used for the sintering process. It might also be
considered that the differences in EOF between open capillaries and the
same capillaries containing frits may be in part due to the differences in
the nature of the electro-osmotically driven flow depending on whether it
is generated only on the walls of an open capillary or in a multitude of
channels in a frit. In the latter case the frit could work as an EOF pump
which could push the liquid through the whole cross-section of the
capillary, as discussed in greater detail later.
Table 1 Electro-osmotic and pressure-driven flow properties of 3 open
capillaries compared with the same capillaries containing frits prepared
from Hypersil ODSa
Linear flow
velocity/mm s−1b |
---|
|
---|
Open
capillary | Capillary with
frit |
---|
|
---|
Pressure-driven (20 mbar) | Voltage-driven (20 kV) | Pressure-driven (1500 mbar) | Voltage-driven (20 kV) |
---|
Conditions: capillary, 100 μm id; electrolyte, 50 mM
Tris–acetonitrile (1∶4), pH 8.0; for other conditions see
Experimental. Each data point in the Table is the mean (± standard deviation)
of at least three determinations. |
---|
7.55 ± 0.03 | 1.75 ± 0.01 | 1.50 ± 0.06 | 2.45 ± 0.01 |
7.98 ± 0.03 | 1.86 ± 0.01 | 2.79 ± 0.08 | 2.40 ± 0.01 |
7.76 ± 0.05 | 2.02 ± 0.02 | 1.82 ± 0.02 | 2.44 ± 0.01 |
Influence of the packing functionality on the EOF
The contribution of EOF of frits made from different packings, all
obtained by functionalisation of the same silica base material, was
determined by measuring the EOF velocity before and after the frit was
created. Table 2 shows the total EOF
values obtained for the open capillaries as well as the contribution to EOF
of the frit (i.e., the total EOF minus the EOF obtained for the
empty capillary), standardised to a frit length of 1 mm. As can be seen
from these data, ODS1, silica and SCX stationary phases increased the
magnitude of the EOF, with the increase being in proportion to the amount
of negatively charged sites available in the corresponding packing
material. The fact that the ODS material provided the smallest and the SCX
the largest change demonstrates that the functional groups of these packing
materials persisted after the high temperature treatment applied during the
frit making process. This is in agreement with the observations of Dittmann
et al. for an ODS packing heated by an element at 580 °C and
moved at up to 2 mm s−1.21
At this point it should be noted that the temperature given as the heating
temperature is measured at the outside of the capillary and it can be
expected that the inside temperature will be somewhat lower, but it is
difficult to judge the significance of this difference and no estimations
of this error have been offered in the CEC literature.
Table 2 EOF obtained for capillaries equipped with frits made using packing
materials with different functionalitesa
Material | EOF/10−8 m2
V−1 s−1b | ΔEOF/10−8
m2 V−1 s−1 per mm frit
length |
---|
Conditions: capillary, 75 μm id; other conditions as in Table 1. Each data point in the Table is the mean (± standard deviation)
of at least five determinations. |
---|
No frit | 3.40 ± 0.04 | — |
Silica | 4.26 ± 0.02 | 0.16 |
ODS1 | 3.59 ± 0.04 | 0.08 |
SCX | 4.68 ± 0.02 | 0.32 |
SAX | −1.88 ± 0.02 | −1.32 |
The most dramatic effect was observed in the case of the frit made from
the SAX material, which provided a reversed EOF. Previously there have been
two reports of a reversed (anodic) total EOF in a capillary packed with at
least 25 cm of a SAX packing26,27 but
there has been no report of significant alteration or reversal of the total
EOF by a frit or short length of packing material housed in a much longer
open capillary. Additionally, there has been no explanation offered for the
effects on EOF of different segments of the capillary having opposite
surface charge and consequently different local EOF values. A possible
rationalisation of the observed results is depicted in Fig. 1 and is based on the assumption that there
still exists a cathodic EOF at the negatively charged open capillary wall.
However, the bulk liquid is drawn through the capillary towards the anode
by the high anodic EOF generated in the multitude of channels in the frit
(which have positive charge arising from the residual quaternary ammonium
functionalities) through the whole cross-section of the capillary. That is,
the EOF is governed by a pumping effect arising from the frit. The flow
vector of the cathodic EOF generated by the wall becomes smaller with
increasing distance from the capillary wall and turns into an anodic EOF
further away from the wall. The resulting flow profile can be expected to
adopt a parabolic shape away from the frit (i.e., either before or
after the frit) because the flow of the bulk liquid is effectively a
pressure-driven flow caused by the frit working as an
‘EOF-pump’. This is a reasonable model since it is a well
established fact that when an EOF and a higher pressure-driven flow are
superimposed in a capillary, the pressure-driven flow moves easily through
the open capillary regardless of the direction of the EOF at the
wall.28 That suggests that the frit, even
when very short compared with the length of the entire column, is the
element which defines the total observed EOF by overriding the EOF
generated by the wall.
 |
| Fig. 1 Schematic representation of electro-osmotically generated flow in an
open capillary containing a segment of frit constructed from SAX material.
It is assumed that the frit contains residual positively charged functional
groups. The shaded area represents the frit. For explanation see Results
and Discussion. | |
One method for verifying the above model is to examine the effect on
peak shape caused by the flow profile in the capillary. Fig. 2 shows a comparison of the peak shapes
obtained for benzoate in an open capillary using an electrolyte containing
10 mM Tris (titrated to pH 8 with hydrochloric acid) and 0.5 mM
tetradecyltrimethylammonium bromide (TTAB) for EOF reversal [Fig. 2(a)], and in a capillary equipped with a frit
made from the SAX material [Fig. 2(b)] using
the same Tris electrolyte but without TTAB. EOF reversal in the latter case
was caused by the frit alone. Since the frit was positioned on the outlet
side of the detection window, at the point of detection there will be no
dispersion of the analyte band caused by passage through the frit and the
peak shape will reflect the shape of the flow profile carrying the analyte
to the detector. As can be seen from Fig. 2,
the peak shape obtained with the plug-like EOF profile of the open
capillary was superior to that in the capillary with the frit, suggesting
that there is parabolic flow induced by the SAX frit, as indicated in
Fig. 1.
 |
| Fig. 2 Peak shapes obtained for benzoate in an open capillary (a) and a
capillary equipped with a SAX frit situated directly behind the detection
window on the outlet side (b). Conditions: capillary, 75 μm id;
electrolyte, (a): 10 mM Tris, 0.5 mM TTAB pH 8; (b) 10 mM Tris, pH 8; for
other conditions see Experimental. | |
Influence of the heating time
As the frits prepared from the SAX material cause the most dramatic
change in the EOF flow, the SAX packing was chosen for experiments in which
the heating time during manufacture of the frit was varied in order to
estimate the progress of the decomposition of the functional groups of the
packing. Sintering times between 12 and 20 s were used since heating times
below 12 s did not result in a durable frit. When a sintering time less
than 12 s was used it was likely that the temperature (particularly in the
centre of the capillary) was not sufficiently high to fuse the silica
particles. Fig. 3 depicts the contribution
of the frit to the total EOF, plotted as the standardised ΔEOF values
and shows that only frits generated using heating times of less that 15 s
still carried sufficient residual positively charged functional sites to
produce a reversed EOF. Similar behaviour can be expected for other
functionalised silicas, although the rate of the packing decomposition may
differ. |
| Fig. 3 Dependence of the contribution to the EOF obtained in a fused silica
capillary (standardised to a frit length of 1 mm) of a frit made from SAX
packing material on the heating time during the frit making process.
Conditions: capillary, 75 μm id; electrolyte, 10 mM Tris, pH 8; for
other conditions see Experimental. | |
Conclusions
The results presented in this paper demonstrate the following features
for frits prepared by sintering a short section of functionalised silica
based packing. (i) A frit 4 mm in length which is short compared to the
overall length of the capillary (34.5 cm) can significantly alter or even
reverse the total observed EOF. An explanation is offered in which the frit
is considered to be the element that defines the total observed EOF. (ii)
Under appropriate conditions (heating at 430 °C for 15 s), sufficient
residual functional groups remain on the packing to significantly influence
the total EOF observed in a 35 cm long capillary using a moderately basic
carrier electrolye. (iii) The amount of residual functional groups
remaining on the packing decreases with increasing heating time and for the
SAX packing used, heating times longer than 15 s led to insufficient
residual positive charge to produce reversal of EOF.Acknowledgements
The authors wish to thank Professor Richard Cassidy and Professor
Wolfgang Buchberger for comments on Fig. 1.
Christian Klampfl would like to acknowledge support from the Austrian
Science Fund (FWF) project number J 1622 CHE.References
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