Evaluation of Novel Electrode Materials

for Electrical Field-Flow Fractionation

(ElFFF) of Biopolymers

ALEXANDRA BERGMAN

Masters Degree Project

Stockholm, Sweden 2004

TRITA-NA-E04072

Numerisk analys och datalogi

KTH
100 44 Stockholm

Department of Numerical Analysis

and Computer Science
Royal Institute of Technology
SE-100 44 Stockholm, Sweden

Evaluation of Novel Electrode Materials

for Electrical Field-Flow Fractionation (ElFFF)
of Biopolymers

ALEXANDRA BERGMAN

TRITA-NA-E04072

Masters Thesis in Biomedical Engineering (20 credits)

at the School of Electrical Engineering,
Royal Institute of Technology year 2004
Supervisor at Nada was Erik Fransn
Examiner was Anders Lansner

EVALUATION OF NOVEL ELECTRODE MATERIALS FOR ELECTRICAL FIELDFLOW FRACTIONATION (ELFFF) OF BIOPOLYMERS
The electrical field flow fractionation (ElFFF) is a relatively new separation technique,
especially well suited for handling macromolecules and particles. The technique separates
based on the product of size and electrophoretic mobility. Colloidal particles, emulsions and
bacteria have been separated with good resolution. One problem has been the polarization
of the electrodes during separation. An applied electric field will polarize the material by
orienting the dipole moments of polar molecules. This reduces the effective field and, hence,
the systems ability to perform satisfactorily. The project will pioneer the use of electrodes
consisting of indium-tin-oxide (ITO). Preliminary results with these electrodes have shown
interesting resolutions of proteins in mixture. The results from experiments on ITO and
titanium channels show that plate height value for ITO is better than titanium.

UTVRDERING AV NYA ELEKTRODMATERIAL FR ELEKTRISK FLT-FLDES

FRAKTIONERING (ELFFF) AV BIOPOLYMERER
Elektrisk flt- och fldes fraktionering (ElFFF) r en relativt ny separationsteknik som lmpar
sig fr att separera stora molekyler och partiklar. ElFFF separerar molekyler genom ett
elektriskt flt som lggs ver en tunn kanal dr mnet passerar. Stora partiklar, emulsioner
och bakterier har tidigare separerats med ett bra resultat. Ett problem med ElFFF r att
elektrodytorna ltt blir polariserade vilket innebr att joner lgger sig p ytan fr att utjmna
attraktion mellan materialet och omgivningen. Detta innebr att det r svrt att separera
effektivt efter en tid. Denna rapport kommer att titta nrmare p kanaler gjorda av titan och
indium-tin-oxid (ITO). Ett teoretiskt vrde p kanalens separationseffektivitet har tagits fram
och mtningar gjorda p hur effektivt det plagda fltet r i varje kanal har gjorts. Resultaten
visar att ITO-kanalen r bttre i vissa aspekter.

Table of Contents
1

Introduction...............................................................................................................1

Background and theory.................................................................................................2

2.1 Chromatography..................................................................................................2
2.2 Field Flow Fractionation (FFF)...........................................................................3
2.2.1 The separation process...............................................................................3
2.2.2 General theory of FFF................................................................................4
2.2.3 Different field options................................................................................8
2.2.3.1 Sedimentation FFF (SdFFF) ..........................................................8
2.2.3.2 Thermal FFF (ThFFF)....................................................................9
2.2.3.3 Flow FFF (FlFFF) ..........................................................................9
2.2.3.4 Magnetic FFF (MgFFF)...............................................................10
2.2.3.5 Other FFF techniques...................................................................10
2.3 Electrical Field-Flow Fractionation (ElFFF).....................................................10
2.3.1 Micro channels in ElFFF .........................................................................11

Method... .............................................................................................................12
3.1 Plate height determination.................................................................................13
3.2 I/V curves ..........................................................................................................13

Results.............................................................................................................16
4.1 Titanium channel...............................................................................................16
4.2 Indium-tin-oxide (ITO) channel........................................................................19
4.3 Micro channel experiments ...............................................................................22

Discussion and conclusions ........................................................................................23

List of references ........................................................................................................24

Introduction

The need for reliable methods to separate and analyze different compounds with different
types and sizes of molecules is a constant one. The appearance of the field-flow fractionation
concept and its imposing growth, resulting in a consistent methodology, represents a critical
breakthrough in analytical separations.
Field flow fractionation (FFF) is a set of high-resolution techniques for separation and
characterization of a wide range of macromolecules, colloids and particles with sizes varying
from 1 m to 10 nm. All FFF systems consist of a pump, an injection valve, a channel and a
detector. Separation takes place in the thin ribbon-like channel with smooth walls.
FFF has shown to be a very accurate method for particle separation and measurement.
Particles can be separated with high resolution based on size, density or electrophoretic
mobility. This last separation process is the motion of charged particles in a colloid under the
influence of an electric field. Particles with a positive charge go to the cathode and negative to
the anode. Applications of FFF are diverse. Together the different sub-techniques provide a
useful tool for characterization of a broad range of particles and biomaterials. Electrical field
flow fractionation (ElFFF) is one of the sub techniques which have evolved lately as an
interesting tool of separation.
The future research in this area will be in optimizing FFF in different ways.
This report will address the theory of FFF in general and ElFFF in specific. The experiments
with novel electrode materials in ElFFF will be analysed and discussed. There is also a quick
look at micro channels in ElFFF.

Background and theory

2.1

Chromatography

Chromatography is a broad range of physical methods used to separate and analyze different
compounds and mixtures. A mixture of various components enters a chromatography process,
and the different components are flushed through the system at different rates. These different
rates of migration as the mixture moves over adsorptive materials provide separation.
Repeated sorption/desorption acts that take place during the movement of the sample over the
stationary bed determine the rates. The smaller the affinity a molecule has for the stationary
phase, the shorter the time spent in a column. Affinity means that the molecule wants to attach
itself to another surface or molecule. A special type of affinity is the ability to separate a
porous material. Chromatographic processes based on this mechanism are referred to as
Size-Exclusion Chromatography or Gel Filtration. The process can be viewed in figure 1.

Figure 1 Size-exclusion chromatography [6]

All molecules larger than the pore size are unretained and elute, i.e. come out, together.
Molecules that can enter the pores will have an average time in the particles that depends on
the molecules size and shape. Different molecules therefore have different total transit times
through the column [6].
The signals collected by a detector after injecting a sample should be Gaussian in shape. The
concentration profile of an analyte migrating through a separation channel under conditions of
lateral equilibrium is governed by Ficks second law and the analyte solution after a sufficient
time in the channel has a Gaussian concentration profile in the direction of migration [1]. In
figure 2 below there are examples of these curves. Obtaining these good signals in electrical

field-flow fractionation is something which is still dependant on a large number of factors.

This will be addressed in the chapter concerning electrical field-flow fractionation.

Figure 2 An example of gaussian shaped curves. Component A is here one of several

compounds being separated. [11]

2.2

Field Flow Fractionation (FFF)

2.2.1

The separation process

The particles injected onto the column without the field or flow turned on are evenly
distributed across the column. Figure 3

Figure 3 The sample is injected into the channel from the

left and migrating towards the right [6].
When a field is applied the solute zone is compressed into a narrow layer against one wall.
See figure 4.

Figure 4 The applied field pushes the sample closer to one wall [6].
When the liquid flow is initiated, the solute zone is carried downstream at a rate depending on
the layer thickness, l, which can be related to the particle size, density, diffusion coefficient or
electrophoretic mobility. Figure 5.

Figure 5 The different molecules are migrating at a different rate

out of the channel due to the applied field [6].

2.2.2

General theory of FFF

Field-flow fractionation is basically a separation technique with much of the same

characteristics as chromatography. There are a lot of variations of this technique and I will
address some of them in this report. Important work on the theory regarding FFF started more
or less with a communication by J. Calvin Giddings of the University of Utah, Salt Lake City,
published in 1966. In this short but significant paper he described a new separation concept
based on a coupling of fractionated sample (solute) concentration distribution and fluid flow
nonuniformities inside a narrow channel which causes differential migration of the solute
species and thus separation [6].
FFF is worked by injecting a sample or solution to be analysed in a thin, long channel. The
walls are commonly parallel. In theory, the channel is thought of being bounded by infinite
parallel plates. A solution of some sort flows through the channel as a carrier for the injected
sample. This solution could be degassed water or water containing ionic solutes of a certain
concentration. A field is then applied perpendicular to the flow and retention is achieved. The
velocity of the flow of the fluid inside the channel varies in the direction across the channel.
This velocity profile is caused by the laminar flow.
Laminar flow in a thin, unobstructed channel means that the flow has a parabolic or nearparabolic profile. The velocity of the flow stream varies with the distance from the walls of
4

the channel. The velocity at any wall is zero because of the frictional drag of the wall on the
fluid. As one moves away from a wall, and thus away from the region where fluid motion is
stagnated by friction, the velocity gradually increases. In FFF this increase continues to the
centre of the channel, after which there is a gradual decrease in velocity until stagnation again
occurs on contact with the opposite wall. The velocity at different points is represented by
flow velocity vectors as shown in figure 6. A plot of velocity versus distance from a wall has
a parabolic shape and is described as a parabolic flow profile. In FFF, the increase in flow
velocity v from zero to a maximum (vmax) and back to zero occurs abruptly over the space of
50-250 m, the typical thickness of the FFF channel [2].

Figure 6 The flow profile in a thin channel [16].

In FFF, as mentioned earlier, a field is applied across the narrow channel in a direction
perpendicular to the flow axis. The purpose of the field is to drive different kinds of particles
and macromolecules to different localized regions or positions between the channel walls.
Particles confined in localized regions will be displaced downstream at a velocity equal to the
parabolic flow velocity in that region. Because different particles are now held by the field in
different regions with different speed, this will mean that the particles will exit at different
times. This is the main idea behind FFF. With this you can achieve separation.
Retention theory involves an understanding of what causes different particles to be retained to
different degrees, i.e., move through the channel at different rates. The governing retention
equation for all FFF systems is given by
R = 6 [coth(1 / 2 ) 2 ]

where R is the retention ratio or the ratio between void time (volume) and elution time
(volume). is a dimensionless parameter related to height, w, of the channel and the physical
properties of the particles being retained and is given by

= D / Uw ( =

l
, where l is the layer thickness indicated in figures 5 and 6)
w

Where D is the diffusion coefficient of the particles and U, the drift velocity, is dependent on
the applied field strength as shown by

U = S / f
S is the applied field strength, is the field susceptibility of the particles and f is the sample
friction coefficient. Therefore, the average thickness of the particle cloud will be determined
by a balance between dispersive and concentrating forces. The diffusion coefficient, D, can be
calculated using the modified Einstein equation
5

D = kT / 3d
where k is the Boltzmann constant, T is the absolute temperature, is the viscosity, and d is
the particle diameter.
At high retention levels (as and R approach zero), the Retention formula is typically
approximated by the simpler expression
R = 6

Another assumption with possible bearing on the scaling analysis presented here revolves
around the assumption that the particle distribution in an ELFFF system remains exponential.
Since the voltage drop across the ELFFF channel is clearly not constant, there is some
likelihood that the assumption of an exponentially distributed particle cloud is not entirely
correct.
The level of separation efficiency generated by a particular instrument can be quantified using
the plate theory from chemical engineering, and also used in chromatography. In plate theory,
the length, L, of a separation column can be broken down into N theoretical plates of height H
H=L/N
The plate height, H, is a measure of variance () that has been created by the separation
system while the band of particles being separated moves through the channel. N therefore
becomes a measure of the separation efficiency of the system and indicates the number of
times a certain separation level is accomplished in a channel. H can be closely approximated
by
H=/L
H=Hd+Hn+Hi(+Hp)
Hn=

1 w 2 v

105
D

The factor

1
is specific for infinite parallel plates.
105

The total plate height can be thought of as the sum of several contributing factors, one of
which is a linear function of flow velocity. One group of factors, known as instrumental
factors, Hi, can be minimized by good instrument design and operation procedures. Once the
instrumental factors have been minimized, the largest contributor to band broadening will be
the nonequilibrium effects, Hn. These nonequilibrium effects are caused by the inherent
distribution of the sample over a number of volume elements and the slow movement of
particles between volume elements. A third group of factors, Hp, is related to the
polydispersity, or variation, in the sample being tested or separated. The fourth factor, Hd, is
caused by the diffusion of particles in the system. The terms Hp and Hd are not related to
system parameters or are small enough to be neglected and can be ignored for optimization of
FFF systems [6].

When evaluating an ElFFF channel and the plate height you want to flow something through
the channel that is not retained in the channel and is not polydispersed.
Unlike the situation in other FFF systems, the field strength in ElFFF, and thus the resolution,
varies inversely with channel height for a given applied voltage. This is an indication that
miniaturization of ELFFF systems is potentially beneficial. A closer look indicates that
reducing channel heights is nearly the only way to improve the resolution in ELFFF systems.
An important effect encountered in FFF systems is the steric transition that occurs when
particles elution times begin to reverse once the radius of the particles being separated
exceeds the average particle layer thickness in figure 5. In normal FFF modes, smaller
particles will elute ahead of larger particles. However, beyond the steric transition point, this
elution order is reversed and larger particles elute ahead of smaller particles. The steric
inversion diameter, di, can be found using
d i = S d 2kT / 3U

where is a dimensionless number of order unity used for complications originating from
wall repulsion and other effects. Sd is the size selectivity index for the system. In some
systems, Sd is close to 1 and of little importance to estimates of resolution or retention [3].
This steric mode of operation has been accounted for by the modified retention equation
a a 2
1 2a / w
2
2a

R = 6 + 6 1 coth
w w
w
2
1 2a / w

which for small particles of radius a coincides with the original formula for retention, while
for large particles it predicts an increased elution volume with a decrease in size [4].
For general FFF systems, there is no effect from channel height on the steric transition point.
The only parameter that determines the steric inversion point is the sample drift velocity,
which is a function of the applied field for most systems and is not sensitive to channel
dimensions [3].
The steric transition point for ElFFF systems, where the field is the applied voltage divided by
the distance between electrodes, is by definition dependent upon channel height. Thus, by
miniaturizing the system, it becomes possible to significantly reduce the steric transition point
and make available the high speed separations possible using the steric mode of separation
[3].

2.2.3

Different field options

A field can be defined as any external influence extending through space capable of causing
the relative displacement of components with respect to their surroundings. To use a field
effectively in FFF it must have three properties. First it must have enough strength to
forcefully drive the components of interest into highly localized regions of the parabolic flow
profile. Secondly it is necessary to have adequate selectivity such that different components
are driven to different parts of the flow profile to achieve separation and finally sufficient ease
of implementation to make instrument development practical, acquisition economical, and
operation simple and reliable. The reason that different fields are used in FFF instead of a
single universal field is that different field types have different ranges and areas of
applicability [1].
For applying different types of fields one needs different instrumentation and this should
come as no surprise after the run-through of the field types. There have been experiments
where the channel has been operated with one instrument applying different fields. This must
however be seen as an exception. It would be effective to have an instrument in the future
where the different fields could be switched off and on but for now there will be a different
instrument for each field [1].

2.2.3.1 Sedimentation FFF (SdFFF)

This technique, utilizing a sedimentation field at right angles to channel flow, has become the
classical FFF method for the separation and characterization of colloidal and larger particles.
The instrumentation for sedimentation (centrifugal) FFF is rather complex and expensive
because of the need for a centrifugal apparatus with special rotating seals. However, because
of its broad applicability and high effectiveness for particle separation and characterization,
this technique is likely to play a major role in the foreseeable future. The companion
technique, sedimentation (gravitational) FFF, by contrast, utilizes a very simple and
inexpensive channel structure, but the weak field (1 g) severely limits its applicability [1].
SdFFF separations are performed within a flat open channel, usually having a rectangular
cross-section and triangular end pieces where the sample and carrier fluid enters and leaves.
SdFFF has excellent resolution but can only process small quantities (<1 mg) of sample in a
single run.
The mechanism for particle separation involves only physical interactions. The sample is
introduced into the channel through a septum or injection valve, and then the flow is turned
off. A centrifugal field is then applied at right angles to the flat face of the ribbon-like
channel. This flat channel sits within a centrifuge basket and the centrifugal field drives the
particles towards the accumulation wall. There they form equilibrium clouds whose average
thickness or elevation above the accumulation wall depends on how strongly the particles
interact with the field and also their diffusivity.
When the carrier liquid flow is turned on at the end of the stop flow (relaxation) period, the
run begins. The carrier flow in the thin flat channel is laminar with the linear fluid velocity
8

being zero at the channel walls and increasing with distance away from each wall, thus
approaching a maximum at the centre of the channel. The particles with a larger effective
mass will have more compressed sample clouds and will consequently be swept down the
channel by the flow at a lower average velocity than the smaller particles. In this normal mode
of SdFFF the smallest particles will elute first [6].

2.2.3.2 Thermal FFF (ThFFF)

Thermal FFF is a sub technique of FFF in which a temperature gradient dT/dx is employed as
the external field. The temperature gradient is generated by heating one plate of the channel
while cooling the other plate. Molecules migrate towards the cold plate which thus becomes
the accumulation wall.
The technique is much more selective than size-exclusion chromatography and is especially
advantageous for ultrahigh-molecular-weight analysis. Recently, the scope of the technique
has been expanded to include micro gels and particles. The ThFFF apparatus must be
designed for a substantial input and removal of heat to maintain temperature gradients as high
as 104 C/cm. This instrument is on the other hand less complex and expensive than that used
for sedimentation (centrifugal) FFF [1].
Thermal FFF is the preferred sub technique for characterizing synthetic polymers which can
be dissolved in an organic solvent. Applications of thermal FFF for a wide range of organic
soluble synthetic polymers and copolymers include the determination of the average
molecular weight and molecular weight distributions, polydispersity especially for ultranarrow polymer samples, and Brownian diffusion coefficient. In addition, thermal FFF has
been a powerful tool for studies of thermal diffusion since it provides one of the few methods
for determining the thermal diffusion coefficient of polymer molecules. Expansion of thermal
FFF to a limited number of water-soluble polymers has also been conducted successfully [6].

2.2.3.3 Flow FFF (FlFFF)

In flow field-flow fractionation (FlFFF), the field is a cross flow of carrier liquid
perpendicular to the usual channel flow. The field strength is determined by the flow rate of
this cross flow. It is the most universal of all FFF techniques. To move objects, or in this
example molecules and particles, has historically been done with a flow of some sort. The
force of a flow is not to be underestimated, just look at floods and avalanches. Flow FFF is
presently applicable in the range 0,001-50m. The technique is applicable to synthetic
polymers and biological macromolecules as well as particles. Although most work has been
done in aqueous media, nonaqueous solvents have been used as well. The selectivity of flow
FFF for polymers is relatively high, comparable to that of thermal FFF. The flow FFF
apparatus is relatively simple although it requires multiple pumps or pump heads to supply the
different flow streams. The only negative aspect of this technique is the fact that the semipermeable membrane is not sufficiently stable compared to other channels [1].

2.2.3.4 Magnetic FFF (MgFFF)

This technique has been shown to be applicable based on differences in magnetic properties.
However, very little work has been done to exploit magnetic FFF or even define its range of
applicability [1].

2.2.3.5 Other FFF techniques

FFF techniques based on other fields are yet to be developed into practical tools. There is not
enough work and experiments done with this technique to substantiate a reliable conclusion
about its applicability [1].

2.3

Electrical Field-Flow Fractionation (ElFFF)

This technique is highly promising because separation is based on the difference in electrical
charge on particles or macromolecules, a charge that can be readily modulated by pH
changes. Although the feasibility of electrical FFF was demonstrated as early as 1972, the
procedure, usually complicated by the generation of electrolysis gases, has not been widely
used. In earlier work, electrode chambers (separated by membranes from the FFF channel)
were used for isolating electrolysis products. Recent work has been directed at overcoming
this instrumental complexity. Electrical FFF is likely to be an important technique in future
applications, particularly for the analysis of biological materials [1].
The main application of ELFFF systems to date has been for the characterization of polymer
suspensions, colloids, sugars and clays. Other applications for the system parallel applications
for other FFF systems and include such diverse biological materials as cells, bacteria, viruses,
proteins, DNA, starches, lipid emulsions, liposomes, micelles and vesicles [3].
Although the applications for ELFFF are significant and increasing, the performance of
current ELFFF systems has been limited by factors such as fabrication technology. In order
for ELFFF to reach its full potential and prove successful commercially, more powerful
ELFFF systems will need to be developed. Miniaturization of the ELFFF system appears to
be one route for development of an improved ELFFF system [3].
In ELFFF, an electric field, E, is applied across the channel and particles are subjected to the
applied field according to their electrophoretic mobility, . To a first look, E might be
expressed as V/w, where V is the applied voltage and w is the distance between electrodes.
This assumption does not work for ELFFF, since most of the applied voltage is dropped
across the interface and polarization layer between the electrode and the bulk carrier solution.
If a material contains polar molecules, they will generally be in random orientations when no
electric field is applied. An applied electric field will polarize the material by orienting the
dipole moments of polar molecules. This decreases the effective electric field between the
plates and will increase the capacitance of the parallel plate structure [9].

10

Thus, the actual effective field for retention in the channel is much less, typically around 1%
of the applied field. This loss of effective voltage is caused by the slow kinetics of the
electrode reaction that is needed to sustain a current over the channel height.
Another difficulty associated with ELFFF concerns the aqueous carrier solutions used in most
ELFFF systems. Aqueous carrier solutions, which are especially of interest for biological
separations, break down into hydrogen and oxygen at relatively low applied voltages (1.7 V
and above). This electrolysis can eliminate retention in the system due to disruption of the
flow characteristics by bubble formation [3]. As with the electric field strength, the effective
voltage, Veff, in the channel is much less than the applied voltage and will be defined as

Veff E eff w

2.3.1

Micro channels in ElFFF

Most of the performance gains associated with miniaturised FFF systems are connected to two
phenomena. First, the field strength for a given system increases as the system is miniaturised.
This means that a higher field is available to drive the separation over a shorter distance
which reduces analysis times and time-dependent plate height contributions. Second, the
smaller channel dimensions reduce spatially related contributions to plate height [3].
A miniaturised ElFFF system would potentially gain all the advantages associated with
miniaturisation of general chromatography systems as well as a few unique advantages such
as reduced time constants, reduced solvent consumption, reduced steric transition point, and
reduced relaxation and equilibrium times. This miniaturised ElFFF system would be available
to provide analysis in a number of different fields and for a range of applications.
The plate height equations for ElFFF are the same as for general FFF systems, but the effect
of miniaturisation on the systems is somewhat different. Reductions in channel height
decrease plate heights in ElFFF systems in a quadratic manner.

11

Method

The basic setup is the same for all channels (figure 7). First the electrodes are prepared with
entering holes for the tubing leading the fluid and electrical contacts. These preparations are
made in a workshop. The following actions are performed by the handler. A Mylar spacer is
placed in between the electrodes to create the channel height. Mylar is a thin plastic film. The
electrodes are then placed between two holding blocks. In the tests of the titanium channel the
blocks are made of an unknown plastic, and in the tests of ITO they are made of see-through
Plexiglas. These blocks are held together by 30 steel bolts and placed along the edges of the
polymer blocks, well removed from contact with the electrodes. After manually tightening the
bolts holding the electrodes in place, the channel is placed in a chamber to ensure its stability.
The channel is then connected to a pump and an injection valve. The sample and carrier enter
and exit the channel via Teflon tubing, friction fit to holes through one of the blocks. An
injection port is mounted to a three-way Hamilton valve which is placed in close proximity to
the inlet end of the assembly. Through this port loading of a sample, normally 1-10 l in size
is made with a syringe. The injection is then made manually by switching a shunt on the
injection valve and thereby forcing the sample in to the flow from the pump and into the
channel. This injection is recognised by the computer and a flow lag of 0,125 ml is taken into
account before monitoring the signal. At the outlet of the channel the tubing is connected to a
Linear UV-detector, which monitors extinction at 254 mm. This signal is monitored by the
computer as well as the current and voltage affecting the channel.

Figure 7 Set-up of the experiment in the lab [13].

12

Tests have included establishing plate height and effective field in the channel. After these
initial tests on the channel there will be attempts of retaining small molecules and proteins.
The detector used in this setup is working with a UV-lamp.
This report was supposed to include tests on three macro channels. These being titanium, ITO
and palladium. Unfortunately, the Palladium channel was not delivered in time and is not
included in this report. Now follows a description of the initial experiments done on the
channels.

3.1

Plate height determination

The theory behind the plate height is discussed earlier in this paper. The practical aspects of
the determination are quite straightforward. You start the flow throw the channel. No voltage
is applied. Then you start a series of data collections. The flow rate is typically started at 0,5
ml/min. At each flow rate, three injections of 5 l acetone are made in the channel and
recorded by the detector. The curves of the acetone should have a Gaussian form and not be
skewed at the end if possible. A comparison with curves collected in sedimentation FFF
(figure 2) gives an idea of the results wanted. The flow rates are introduced in the order of 0.3
0.5 0.7 0.9 1.1 ml/min. The number of theoretical plates is then estimated using the
formula
N = 5.54 (VR/w1/2)2.
VR being the retention volume and w1/2, the width at half height, are experimentally
determined parameters, taken from a fractogram. The more plates the better. It is however
important to remember that the concept of the theoretical plate is an imaginary way to better
understand the process in the channel (or column), such plates do not exist in reality. The
number of plates, N, increases with an increase in column length, L. To compare the inherent
efficiency between columns, it is therefore desirable to compare the amount of zone
broadening incurred per unit length of column, as described by the plate height H [11]. This is
discussed in more detailed in the general theory behind FFF.

3.2

I/V curves

Although the nominal field applied to the ELFFF channel is easily calculated as the applied
voltage divided by the distance w between the electrode walls, this field is far from constant
across the narrow gap. Due to the limited reaction rate at the electrode surfaces, the actual
working field responsible for particle migration to the appropriate channel wall, and the
resulting retention, are both significantly weaker than indicated by the nominal value. By
increasing the ionic strength of the carrier, the conductivity in the channel will increase while
the reaction rate at the electrode surface will remain constant [4].
Just as in the generation of streaming potentials, the shearing action of the carrier flow will
perturb the polarization layer. In view of the many factors affecting the actual strength of this
field, it is expected that any determination of its magnitude will have to involve the use of
standard particles whose mobility has been separately determined in the same fluid as that
used as a carrier in the ELFFF analysis [4].

13

The second test to be made is the one where the efficiency to distribute different voltages over
the channel is determined. The way this is made is by flowing degassed water to begin with
and then introducing different salt solutions in the channel. These different ion-levels are
made by diluting (NH4) HCO3 in degassed water. Experiments have also been made with
NaCl. The different concentrations are introduced beginning with the lowest one. Here
follows the order of solutions:
Degassed water
Degassed water after one day
10 M (NH4) HCO3
50 M
100 M
500 M
1 mM
5 mM
10 mM
It is important to start measuring immediately after preparing the different solutions.
Degassed water that is left in the open for a couple of hours will contain more ions and thus
be of a different concentration and will lead to a different expected result. The flow rate for
this measurement was 0.5 ml/min. Each solution is flowed through the channel for about 15
min before starting the measurements and after that introducing the next concentration into
the system. This 15 min period in the beginning of each new solution is made with the
electrodes not connected to any voltage source at all. Then the electrodes are connected and
the current is set to 0 with the computer and another period of 5 min is observed. The
measurements then starts with 0,5 mA and continues with 1;1,5;2;3;4;5 and finally 10 mA. A
stabilization period of 1 min is held between measuring and changing the current.
These data are useful in determining the range of voltages that may be applied to produce a
useful separation. Low voltages will not produce sufficient effective field for a separation,
while voltages that are too high will generate bubbles from electrolysis (typically between 2.6
and 3.2 V) [3].
The results will be presented as I/ [Conc] against voltage attained. The relationship in this
experiment can be looked upon as several resistances coupled in series (figure 8). The relation
between the different components is
I =V

1
V
Conc

This factor out the effect of the increase in conductivity and will give an indication of the
effectivity of the field.

14

Electrode

Interface

Liquid

Interface

Electrode

Figure 8 Resistances in series.

As can be seen later in figure 13, at low concentrations the electrode reaction rate can keep up
with the current. When the concentration is increased the current is not increasing with
concentration (as expected). The current is limited by the slow electrode reaction.

15

Results

4.1

Titanium-channel

The first channel to get tested in this project was the titanium channel. There were initial
problems with the setup for the channel. The detector was not working and had to be replaced.
There were also problems with leakage in the outflow tubing that needed to be resolved. At
first, the channel is run with DI water to get a calibrated base line. This particular channel was
mounted in between two plastic holding blocks and could not be seen through. Further on, the
holding blocks on the ITO channel are of a see-thru material and this enables the handler to
easier see problems like air bubbles and so on in the channel. This process has been a learning
curve in itself and these initial preparations of the channel have improved in speed and
understanding. Starting with the titanium meant that a lot of time was spent on just flowing it
through to get rid of possible air bubbles. The curves resulting after the initial injections of
acetone were quite poor and needed to be adjusted. Changing the filter in the UV detector
gave much better results.
Problems concerning the measurements on the titanium channel were persistent during a
couple of days. The detector was not working at full potential and that was partially due to
problems with air bubbles in the channel and therefore also in the detector. There was also a
problem with relying on the apparatus at the lab site. The pump controlling the flow rate
through the channel was remotely controlled via the computer and there was no evidence
pointing to the fact that this process was not working properly. Not verifying this process was
a mistake and set the project back in time. The suspicion arose from another person at the lab
and looking at the process more closely we together concluded that the computer did not
control the pump. Following this conclusion, the pump was set to be controlled manually in
the future. The measurements were done again and the results for H quickly improved from
an adjusted value of 2.6 mm to 1.6 mm. The following figures 9, 10 and 11 show this
development.
Titanium - all collected data
y = 0.000562x + 0.002754
0.005

H (m)

0.004
0.003
0.002
0.001
0
0

0.5

1.5

v(mL/min)
H

Linear (H)

Figure 9 All measurements on H value at different flows. A linear equation is adjusted to the
values in order to get an intercept with the y-axis.

16

Titanium - adjusted curve

y = 0.000538x + 0.002631
0.004
0.0035

H(m)

0.003
0.0025
0.002
0.0015
0.001
0.0005
0
0

0.5

1.5

v(mL/min)
Series1

Linear (Series1)

Figure 10 In this figure the H values that were very off the expected result were removed. The
adjusted equation now gives a better H value.

H (m)

Titanium - all data working pump

y = 0.002196x + 0.001586

0.006
0.005
0.004
0.003
0.002
0.001
0
0

0.5

1.5

v(mL/min)

Linear (H)

Figure 11 H-value measurements now with a working pump

Taken together, the result was a plate height value of 1.6 mm for the titanium channel.
Next in line of measurements were the I/V curves. The carrier solution was (NH4) HCO3 and
the result can be seen in figure 12.

17

Titanium I vs V (NH4)HCO3
120000

I/[Conc] (mA/mM)

100000
80000

degass MQ
old MQ
10uM
50um
100uM
500um
1mM
5mM
10mM

60000
40000
20000
0
0

10

12

Volts (V)

Figure 12 I/V curves for titanium. All concentrations.

Titanium I vs V (NH4)HCO3 selected data

250

I/[Conc] (mA/mM)

200

50um
100uM

150

500um
1mM

100

5mM
10mM

50

0
0

Volts (V)

Figure 13 I/V curves for titanium. Degassed water curve removed.

In figure 13, the degassed water curve is removed for clarity.

18

4.2

Indium-tin-oxide (ITO) channel

ITO films have many positive properties as substrates for optical devices, such as a high
luminous transparency, a good electrical conductivity, and a good infrared reflectivity. They
are widely adopted as transparent anodes in modern organic light-emitting diodes (OLEDs),
liquid crystal displays, solar cells and other micro devices. It is known that surface treatment
of the ITO can affect its electrode function. One current model suggests that organization of a
dipole at the ITO electrode surface can increase its work function and hence effect changes in
its hole injection behaviour in a device. Surface modifications can help enhance the efficiency
of carrier injection into organic layers. Characterization and control of commercially available
ITO substrates is necessary for successful fabrication of different micro machines. [11, 12]
The huge amount of work done to ITO in other scientific areas has been a reason to test it in
ElFFF. Could these surface modifications give ITO an advantage as an electrode in ElFFF?
With the ITO channel, some improvements regarding the apparatus and the fact that the
holding blocks were made of a see-thru material, made the preparations and measurements
easier to observe and control. The channel was injected with ethanol in a pushing fashion to
reduce surface tension in the fluid and thereby making it easier to get air bubbles out. The
channel was visible with the naked eye and this made the process run quicker and smoother
compared with the titanium.
Because the flatness of its electrodes, the expected result on the ITO channel was that it
should have a better H value than the titanium system, whose electrodes were somewhat
rough. Looking at the H-curve for ITO in figure 14 this was also the result. The H value was
0.08 mm.
ITO
y = 0.1119x + 0.0812
0.25

H (mm)

0.2

0.15

0.1

0.05

0
0

0.2

0.4

0.6

rate (ml/m)

Figure 14 H value measurements.

19

0.8

1.2

The I vs. V measurements were made with solutions of (NH4) HCO3. In figure 15 all data is
depicted. To be able to look more closely at the curves that appear superimposed, a special
plot is made for these and can be studied in figure 16.
ITO I vs V (NH4)HCO3
120000

I/[Conc] (mA/mM)

100000
80000

degass MQ
old MQ
10uM
50um
100uM
500um
1mM
5mM
10mM

60000
40000
20000
0
0

Volts (V)

Figure 15 I/V curves for ITO. All concentrations.

ITO I vs V (NH4)HCO3 selected data

250

I/[Conc] (mA/mM)

200

50um
100uM

150

500um
1mM

100

5mM
10mM

50

0
0

Volts (V)

Figure 16 I/V curves for ITO. Degassed water curve removed.

Another test of I vs. V was made with solutions of NaCl. The different concentrations were 10
M, 100 M, 1 mM and 10 mM. The results can be seen in figure 17.
20

ITO I vs V NaCl
1200

I/[Conc](mA/mM)

1000
800

10uM
100uM

600

1mM
10mM

400
200
0
0

Volts (V)

Figure 17 I/V curves for ITO. Selected concentrations for carrier NaCl.
Comparison NaCl and (NH4)HCO3 in ITO
45

I/[Conc] (mA/mM)

40
35
30
25

100uM (NH4)HCO3

20

100uM NaCl

15
10
5
0
0

Volts (V)

Figure 18 Comparison between two carrier solutions. Same concentration. ITO channel.
Regarding the differences between solutions of the two electrolytes (NH4) HCO3 and NaCl, a
comparison between the two carriers were made (figure 18). Concerns that NaCl would work
poorly compared to (NH4) HCO3 were not substantiated and could be dismissed. The two
carriers worked more or less identically.

21

Attempts to get retention in the ITO channel were made. Proteins ConA and ConA+Dextran
complex were injected and were indeed retained during applied voltage. A separation,
however, did not occur.

4.3

Micro channel experiments

Experiments on micro channels made of ITO and palladium have been made and results have
varied. These micro channels are made by hand and have a varied quality. There have been
problems with leakage and the channel being short circuited. The principles behind the
channels are basically the same as with the macro ones. Obviously, the mere size of the
channel constitutes a problem with manufacturing and handling. The upside to having these
smaller channels is that the very small channel height 30-60 m means that the given field,
i.e. retention can be achieved with a lesser amount of voltage applied. The theory of FFF also
implies that the efficiency of a smaller channel would be greater. The first experiments on the
micro ITO channel involved I/V- measurements. Tests on a palladium-channel concluded that
the channel was short-circuited after a short period of time. The channel was taken apart and
we could see that the palladium-layer had begun to flake off. An experiment with an open
palladium-channel in a salt solution gave further evidence to the fact that the voltage over the
electrodes fed a chemical process leading to the palladium-layer flaking off. Further
investigation under microscope showed pin holes in the palladium-layer and gas forming at
the negative electrode. This chemical process is as following:
1
( H + + e H 2 )
2
These figures reveal the pin holes.

Figure 19 Micro channel under microscope reveals pinholes in layers.

The layers on these electrodes are 200 nm palladium on top of 20 nm titanium. The flow
through these channels was also investigated with a microscope. Fluorescent beads were
injected in the channel and the flow was caught on camera. The flow was good and the only
restriction in the channel was the fact that it leaked.

22

Discussion and conclusions

The aim of this report was to examine novel electrode materials in ElFFF, in particular
titanium, ITO and palladium. Due to an unfortunate delivery failure of the palladium
electrodes, the only electrodes being compared were the titanium and the ITO. The results
obtained were plate height values for both channels. The plate height value for titanium was
1.6 mm and for ITO 0.08 mm. Results were also obtained regarding the effective field in both
channels.
Regarding the plate height measurements on the titanium and ITO, one could argue that the
value achieved for the titanium should have been lower. The fact that the channel flow is not
to be seen during the experiment will undoubtedly affect the value in a negative way. The
advantage of seeing the channel in the ITO channel, being able to spot air bubbles and other
discrepancies makes an important difference in these hands-on experiments. The surface of
ITO should present a better plate height value and this was indeed true. The plate height value
for the ITO was an expected one and will be used mainly as a reference for further
measurements on the micro ITO channels.
A comparison of figure 12 and 15 shows that the ITO channel gives a more efficient field and
therefore a better separation. This result was expected. At low voltages, the currents are
relatively small and indicate a lack of sufficient field strength to perform adequate
separations. For both titanium and ITO channels there exists a turn-on voltage where the
current rises quickly in an approximately linear fashion. This turn-on voltage is
characteristic of electrode systems of this type [2]. The value of the turn-on voltage is about
2.5 V for both electrode materials.
Concerning the attempted retention of proteins ConA and ConA+Dextran complex in the ITO
channel, it is suspected that the samples are too easily stuck to the surface of the electrode.
This has not yet been verified.
The measurements of the micro channel in ITO were furthermore an interesting path of this
project. The flow through a micro channel has been studied and the potential for this
instrument should be great in the future. Something else being investigated now is which type
of material should be used as a spacer in the micro channel. The micro channels are, however,
still produced one by one, and a lot of questions regarding the manufacturing process still
remain.
Finally these experiments have shown how insignificant and practical problems during the
work will seriously affect the measurements. The problems arising will also seriously affect
the time frame for doing such an experiment. It is however, very rewarding, to have this
hands-on feel for the work being done.
In the nearest future, measurements will be done to test new electrodes with a palladiumlayer.

23

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24

16. Young, Lee Jae. Analytical chemistry homepage. Parabolic flow figure.
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25