Talanta 56 (2002) 365– 373

www.elsevier.com/locate/talanta

Diffusion coefficient measurements in microfluidic devices

Christopher T. Culbertson, Stephen C. Jacobson, J. Michael Ramsey *
Oak Ridge National Laboratory, Chemical Sciences Di6ision, P.O. Box 2008, Oak Ridge, TN 37831 -6142, USA

Received 25 May 2001; received in revised form 7 September 2001; accepted 18 September 2001

Abstract

Four methods for measuring diffusion coefficients were compared on a microfabricated fluidic device using
rhodamine 6G as the analyte. The measurements were made using a static imaging method and three dynamic
methods—stopped flow, varying the applied potential (E-field method), and varying the detection length (length
method). Under conditions where analyte–wall interactions (adsorption) are minimized, e.g. in a 50/50 (v/v)
methanol/aqueous buffer, the stopped flow (2.71 9 0.09× 10 − 6 cm2 s − 1), E-field (2.684 90.005× 10 − 6 cm2 s − 1) and
the static imaging (2.69 9 0.02× 10 − 6 cm2 s − 1) measurements were all within experimental error of one another and
previously reported values. Under 100% aqueous conditions, however, the diffusion coefficient measured dynamically
was 11% larger than that measured statically. Diffusion coefficients for rhodamine B, fluorescein, 2%,7%dichloro-fluores-
cein (DCF), rhodamine 6G, tetramethylrhodamine labeled glutamic acid and isoleucine, and fluorescein conjugated
bovine serum albumin and ovalbumin were also measured using the static imaging method. © 2002 Elsevier Science
B.V. All rights reserved.

Keywords: Diffusion coefficient; Microfluidic device; Rhodamine; Fluorescein

1. Introduction theoretical plates generated (N), is inversely pro-

The ability to quickly and accurately measure
diffusion coefficients is important to assessing and
interpreting the quality of experimental results
obtained from liquid chromatography and capil-
lary/microchannel electrophoresis (CE). For ex-
ample, in CE under ideal conditions, the only
significant source of band broadening is longitudi-
nal (molecular) diffusion. The separation effi-
ciency, therefore, as measured by the number of
* Corresponding author. Tel.: +1-865-574-5662; fax: + 1-
865-574-8363.
E-mail address: ramseyjm@ornl.gov (J. Michael Ramsey).
portional to the diffusion coefficient (D) for a
given applied electric field strength (E) and migra-
tion distance (l) as shown in Eq. (1),
vekEl
N= (1)
2D
where vek is the electrokinetic mobility of an
analyte. This equation is generally used to assess
how well a particular separation method is func-
tioning, but often requires an estimation of an
analyte’s diffusion coefficient which gives rise to
errors and uncertainties in the results. The actual
analyte diffusion coefficients are generally not
measured because of the time and effort involved.

0039-9140/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 6 0 2 - 6
366 C.T. Culbertson et al. / Talanta 56 (2002) 365–373
Three methods, however, for measuring diffusion
coefficients in CE have been reported: ‘on-the-fly-
by-electrophoresis’ [1,2], ‘stopped flow’ [1,2], and
the E-field method [2]. These methods can be
considered dynamic methods because the analyte
is moving under the influence of an electric field
while the measurement is being made. In the
on-the-fly-by-electrophoresis method the peak
width, measured as the temporal peak variance, is
first converted to a spatial peak variance (| 2) and
then using the Einstein–Smoluchowski equation
(Eq. (2)),
| 2 =2Dt (2)
and the run time (t), a diffusion coefficient is
calculated. If the peak variance due to the injec-
tion is significant, it can be calculated from the
injection parameters and subtracted from the total
peak variance prior to calculating the diffusion
coefficient [2]. For the stopped flow method, two
consecutive electrophoretic runs are made. In the
first run the analyte is injected and electromi-
grated through the detection window. In the next
run the analyte is injected and migrated part of
the way to the detector at which point the electric
field is removed for a specified period of time.
After this time the electric field is reapplied, and
the analyte is electromigrated through the detec-
tion window. The difference in the spatial peak
variances between the two runs and the stopped
flow time are used to calculate the diffusion coeffi-
cient using the Einstein equation (Eq. (2)). In the
E-field method, a series of electrophoretic runs is
made between which the field strength is varied
but kept low to minimize any Joule heating ef-
fects. The analyte spatial peak variance for each
of these runs is then plotted against the migration
time. The slope of this plot is equal to 2D.
For all of the dynamic methods described
above, the assumption is made that the only
source of peak variance is longitudinal diffusion.
This is often not the case as the initial analyte
injection width, analyte– wall interactions (ad-
sorption), Joule heating and electrodispersion are
also frequently significant contributors. These
methods, therefore, may not reliably determine
the actual longitudinal diffusion coefficient. Mea-
suring the diffusion coefficient in the absence of
an electric field, i.e. statically, by actually imaging
the analyte band in the capillary column over time
would remove the contribution of these four
sources to the peak variance assuming that the
equilibration time for any adsorption–desorption
process is small compared to the diffusion coeffi-
cient measurement time. One static diffusion co-
efficient measurement method has been developed
in a capillary [3]; however, given the spatial extent
of the typical initial plug width (determined, in
large part, by the injection plug length), it is
difficult to evenly illuminate and image a large
enough section of capillary to perform this type of
measurement. Performing static diffusion coeffi-
cient measurements in channels on microfabri-
cated devices, however, is easier as the injection
plug lengths are shorter, and therefore, the area
on the chip that needs to be illuminated and
imaged is smaller. In addition, the planar surfaces
on the chip also make the imaging optics simpler.
The ability to perform rapid diffusion coeffi-
cient measurements on microchips is also crucial
to continuing the development of microfluidics
technology. While microfabricated fluidic devices
have been successfully demonstrated for a wide
variety of electrokinetic separations [4–7], the
separations must perform at or near their theoret-
ical limit to be successful given the short channel
lengths and run times. Being able to quickly mea-
sure analyte diffusion coefficients provides the
rapid feedback necessary to make such
assessments.
In this paper, we compare three dynamic meth-
ods with a static imaging method for measuring
diffusion coefficients on microfabricated devices.
These comparisons are made using the fluorescent
dye rhodamine 6G under conditions where ana-
lyte–wall interactions are minimized by using a
methanol/aqueous buffer and under aqueous con-
ditions where significant analyte–wall interactions
occur. The precision and accuracy of the measure-
ments are evaluated and show that the static
diffusion coefficient measurement method is in-
deed accurate, fast and robust. Seven other com-
pounds— fluorescein, 2%,7%-dichlorofluorescein
(DCF), rhodamine B, tetramethylrhodamine
derivatized isoleucine and glutamic acid, and
fluorescein conjugated ovalbumin and bovine
 C.T. Culbertson et al. / Talanta 56 (2002) 365–373 367

serum albumin—were also measured using the 2.3. Microchip operation

static method under aqueous conditions and their
diffusion coefficients are reported.
2. Experimental
2.1. Reagents
2%,7%-Dichlorofluorescein was obtained from
Aldrich (Milwaukee, WI). Bovine serum albumin
fluorescein conjugate (BSA; 5.5 mol dye mol − 1),
ovalbumin fluorescein conjugate (Ova; 2.8 mol
dye mol − 1) and tetramethylrhodamine isothio-
cyanate (TRITC) were obtained from Molecular
Probes Inc. (Eugene, OR). Fluorescein (Fl), glu-
tamic acid (Glu) and isoleucine (Ile) were ob-
tained from Sigma (St. Louis, MO). Rhodamine B
(RhB) and rhodamine 6G (R6G) were obtained
from Eastman Chemical Co. (Kingsport, TN). All
chemicals were used as received. The amino acids
were individually derivatized with TRITC as pre-
viously reported [8]. All solutions were made us-
ing distilled deionized water from a Barnstead
Nanopure System (Dubuque, IA) and then
filtered through 0.45 mm Acrodiscs (Gelman Sci-
ences, Ann Arbor, MI).
The electrokinetic runs and constant volume
injections [9] were performed using four indepen-
dent and remotely programmable high voltage
(0–10 kV) power sources from a multiple source
supply (2866, Bertan, Hicksville, NY). The proper
potentials to apply at each reservoir were deter-
mined using Kirchhoff’s rules and Ohm’s Law.
The high voltage control and data acquisition
software were written in-house using LABVIEW
(National Instruments). For the dynamic diffu-
sion coefficient measurements the analyte was
simply electromigrated through the detection
zone. For the static diffusion coefficient measure-
ments the analyte was electromigrated into a sec-
tion of the channel imaged by a CCD camera
(Fig. 1), and when in the center of this region the
potentials were removed until the static measure-
ments were complete.

2.2. Microchip design

A simple cross chip design was used in both the

static and dynamic diffusion coefficient measure-
ments (Fig. 1). The chips were fabricated on
soda-lime glass (Telic Company, Santa Monica,
CA) using standard photolithographic, wet chemi-
cal etching, and cover plate bonding techniques
[9]. Access holes to the channels were ultrasoni-
cally drilled (Sonic-Mill, Albuquerque, NM) into
one of the slides prior to bonding. Small fluid
reservoirs (140 ml capacity) were attached with
Epo-tek 353ND epoxy (Epoxy Technologies, Inc.,
Billerica, MA) at points where the access holes
were drilled. Channel widths were measured using
a stylus-based surface profiler (P-10, Tencor,
Mountain View, CA) and are reported as the
width at half the channel depth. Seven chips were
Fig. 1. Schematic of microchip used for diffusion coefficient
used in the experiments reported below and had measurements. The detection points and the imaged area for
depths ranging from 10 to 15 mm and widths at the various methods are shown in the schematic. The reported
half-depth ranging from 30 to 46 mm. detection distances were measured from the cross intersection.
368 C.T. Culbertson et al. / Talanta 56 (2002) 365–373
2.4. Single point detection for dynamic diffusion
coefficient measurements
All of the dynamic diffusion coefficient experi-
ments were performed using a single point detec-
tion system similar to that previously described
[10]. Laser induced fluorescence (LIF) detection of
the analytes was performed using either the 488
(Fl, BSA, Ova) or 514 nm (DCF, RhB, R6G,
TRITC labeled amino acids) line of an Innova 90
Argon ion laser (Coherent, Inc., Palo Alto, CA)
as the excitation source at a power of 8 mW. The
laser beam was focused through a 300 mm focal
distance planoconvex lens to create an estimated
excitation spot size of 50 mm on the chip. The
fluorescence was collected using a 40× micro-
scope objective (CD-240-M40X; Creative Devices,
Neshanic Station, NJ), and the image was focused
onto a 800 mm pinhole. The signal passing
through the pinhole was then spectrally filtered
using a 488 or 514 nm notch filter (Kaiser Optical
Systems, Inc., Ann Arbor, MI) and a 530 or 580
nm bandpass filter (530df30 or 580df30; Omega
Optical, Brattleboro, VT) before being measured
by a photomultiplier tube (PMT, 77348; Oriel
Instruments, Inc., Stratford, CT). The signal from
the PMT was amplified using an SR570 low noise
current preamplifier (Stanford Research Systems,
Inc., Sunnyvale, CA) with a 100 Hz lowpass filter.
The signal from the amplifier was sampled at 100
Hz using a PCI-MIO-16XE50 multifunction I/O
card (National Instruments, Inc., Austin, TX) in a
G3-300 Power Macintosh. All data analysis was
performed using Igor Pro from Wavemetrics, Inc.
(Lake Oswego, OR).
2.5. Dynamic diffusion coefficient measurements
2.5.1. Stopped flow measurements
For the stopped flow method two consecutive
runs were made. In the first run R6G was injected
and electromigrated past the detection window.
Immediately, thereafter, a second run was made.
This time, however, the R6G was electromigrated
half way to the detection window at which point
the potential was removed for anywhere between
4 and 8 min —the ‘stop time’. The ‘stop time’ was
set such that the ratio of the first to second run
variance was B0.1.
2.5.2. E-field method
In the E-field method several consecutive runs
were made in which the field strength was varied
from 46 to 370 V cm − 1, and the detection dis-
tance was set at 0.5 cm (Fig. 1). The peak vari-
ance was again plotted against the peak migration
time to obtain the diffusion coefficient from the
slope of the plot.
2.5.3. Length method
In the length method several consecutive runs
were made with detection lengths of 0.2, 0.4, 0.6
and 0.8 cm (Fig. 1). The peak variance was plot-
ted against the peak migration time to obtain the
diffusion coefficient from the slope of the plot.
2.6. Imaging for static diffusion coefficient
measurements
The static diffusion coefficient measurements
were made using a TE300 inverted microscope
outfitted with an epifluorescence attachment from
Nikon, a Micromax 512 BFT CCD camera from
Princeton Instruments (Trenton, NJ), and a Unib-
litz electronic shutter for the high pressure mer-
cury arc lamp from Vincent Associates
(Rochester, NY). A BV1 filter cube which con-
tained the excitation, dichroic, and emission filters
(Nikon) was used for the RhB, R6G, and TRITC
labeled amino acid experiments, and a BV2 filter
cube (Nikon) was used for the Fl, DCF, and
protein measurements. Twenty-five images of the
sample plug in the channel were taken one every 5
s for RhB, DCF, R6G, Fl, TRITC labeled amino
acids or one every 20 s for the proteins. The
exposure time for the small molecules was 200 ms
per image and for the proteins was 1000 ms per
image. The sample was only illuminated during
the time that the image was taken. The data was
acquired using IPLab Spectrum (Scanalytics,
Fairfax, VA) and analyzed using scripts created in
IPLab and macros developed using IGOR Pro.
To obtain accurate temperature measurements,
a thermistor (ON-402-PP with an HH42 digital
thermometer; Omega Stamford, CT) was used for
both the static and dynamic methods. For the
dynamic methods, the microchip was contained in
 C.T. Culbertson et al. / Talanta 56 (2002) 365–373 369

Fig. 2. (A) Fluorescence images of R6G diffusing over time in a microchip obtained for use with the static imaging method. (B)
Profiles obtained from part A by summing across the width of the channel. Each profile was fitted by a Gaussian equation (gray
dotted lines) to extract the peak variance.

a light tight box which also served to reduce air
circulation, and the thermistor was placed in the
box on the surface of the chip. For the static
method the thermistor was also placed on the
surface of the chip. In addition, for both methods
the reservoirs on the microchip were covered with
parafilm to reduce solvent evaporation.
3. Results and discussion
The static diffusion coefficients were measured
in the absence of fluid flow, i.e. no electrical or
hydrodynamic potential was applied. Fig. 2A
shows the static diffusion of rhodamine 6G over
time in a series of images. These images were flat
field corrected for light intensity variations, and
axial concentration profiles of the analyte were
extracted by summing across the width of the
channel (Fig. 2B). The variance of each peak was
then determined by fitting a Gaussian function to
the peak profile. Finally, the peak variance (| 2)
was plotted against the diffusion time (t). The
slope of the linear regression through this data is
equal to two times the diffusion coefficient (D),
assuming that diffusion obeys the Einstein–
370 C.T. Culbertson et al. / Talanta 56 (2002) 365–373
Smoluchowski relation (Eq. (2)) (Fig. 3A). Good
linearity was obtained from the linear regression
with correlation coefficients (r) ranging from
0.99993 to 0.99998. The diffusion coefficients
measured at temperature X were all corrected to
25 °C using Eq. (3).
T25°C pX °C
D25°C = DX °C (3)
TX °C p25°C
which is based on the Stokes– Einstein relation
[11], where T is the absolute temperature and p is
the solution viscosity. The corrected values are
reported in Table 1. For all of the static diffusion
coefficient measurements the excitation light was
Fig. 3. Peak variance vs diffusion time for (A) static; (B)
E-field; and (C) length methods. R6G was the sample. The
lines represent the best least-squares fit to the data which are
represented by their respective error bars. The error bars are
drawn at 9 1.35% which assumes a temperature measurement
uncertainty of 9 0.5 °C. The errors reported in the diffusion
coefficient values on the plots are due to the error in the
least-squares fitting. The diffusion coefficient values reported
on the plots have not been corrected to 25 °C as have the
values in the tables. In (A) the peak areas are represented by
the triangles.
shuttered when data were not being taken so no
photobleaching was observed as seen by the con-
stant value of the peak area over the time course
of the measurement (Fig. 3A).
The results for the three dynamic diffusion
coefficient measurements are shown in Table 1.
For the stopped flow measurements the ‘stop
time’ was set such that the ratio of the first to
second run variance was B 0.1 to reduce the
effects of measurement errors. In the E-field
method low field strengths were used to minimize
any effects from Joule heating. At 370 V cm − 1,
the highest field used, the power dissipation was
only 0.29 W m − 1 which is well below the 28 W
m − 1 power dissipation threshold, where Joule
heating was detected under similar conditions
[12]. A representative plot of the peak variance vs
migration time is shown in Fig. 3B. Good linear-
ity was obtained from the regression analyses for
all of the trials with correlation coefficients rang-
ing from 0.999 to 0.99995. For the length method,
a field strength of only 183 V cm − 1 was used to
minimize any effects from Joule heating. A typical
plot of the peak variance vs migration time is
shown in Fig. 3C. Good linearity was observed
from the regression analyses for all of the trials
with correlation coefficients ranging from 0.9995
to 0.99995.
The values obtained from the three dynamic
diffusion coefficient measurements vary by 7.5%
with the E-field method giving the smallest mea-
sured values and the length method giving the
largest values. The reason for the discrepancy in
the two results is unknown. The most likely rea-
son would be a systematic error in adjusting the
detection length; however, the micrometers used
to move the chip were checked and found to be
properly calibrated. Another possibility is that the
channel width, height, or surface roughness varied
along the length of the channel. The result, how-
ever, was not device specific as it was repeated on
several different microchips. The E-field method,
stopped flow, and static diffusion coefficient mea-
surements were within experimental error of one
another. More importantly, however, all of the
results were within the experimental error of two
other results reported in the literature using spec-
troscopic methods [13,14]. The variation in ap-
 C.T. Culbertson et al. / Talanta 56 (2002) 365–373
Table 1
R6G diffusion coefficients measured in both a 50/50 (v/v) methanol/aqueous solution and an aqueous solution
Method 50/50 Methanol/aqueous
Diffusion coefficient (×10−6 cm2 s−1) at
25 °C
Static 2.69 90.02
Dynamic
Stopped flow 2.71 90.09
E-field 2.684 90.005
Length 2.88 90.17
Fister [13] 2.7 90.1
Hansen [14] 2.5 90.3
NA, not available.
a
Percent relative standard deviation.
b
Number of trials.
plied field and the static measurements were the
most reproducible; both gave relative standard
deviations (RSDs) of B1%. These results show
that, under ideal circumstances, electrophoretic
separations on microfluidic devices are diffusion
limited.
While the static and best dynamic diffusion
coefficient measurements for R6G in the
methanol/aqueous buffer were within experimen-
tal error of each other, under many circumstances
the longitudinal diffusion coefficient of an analyte
cannot be obtained from dynamic measurements
as the analyte may interact with the wall. Static
diffusion coefficients, however, can be obtained as
long as the equilibration time for adsorption– des-
orption processes is small compared to the diffu-
sion coefficient measurement time. To examine
the effect of analyte– wall interaction on the mea-
surement of the diffusion coefficient, we measured
the dynamic diffusion coefficient for R6G in a 20
mM boric acid, 100 mM Tris solution using the
E-field method and compared the results to those
obtained from static measurements (Table 1). The
diffusion coefficient from the dynamic measure-
ment is 11% larger than the static measurement
indicating that there is a significant amount of
analyte –wall interaction as the R6G migrates
through the channel. For the rest of our diffusion
coefficient measurements, therefore, we used the
371
Aqueous
% RSDa n b Diffusion coefficient (×10−6 cm2 s−1) at nb
25 °C
0.74 6 4.14 90.1 6
3.3 11
0.19 6 4.59 90.06 6
5.9 8
3.7 NA
12 NA
static method to ensure that only longitudinal
diffusion was being measured.
Additional static diffusion coefficient measure-
ments were made of seven other fluorescent dyes
or molecules conjugated to fluorescent dyes—
fluorescein, rhodamine B, 2%,7%-dichlorofluores-
cein, TRITC-Glu, TRITC-Ile, fluorescein
conjugated ovalbumin and fluorescein conjugated
bovine serum albumin (Table 2). For the rho-
damine and fluorescein families of dyes and the
proteins, the diffusion coefficient decreased with
increasing molecular weight as expected. This,
however, was not the case for the TRITC labeled
amino acids as the diffusion coefficient for
TRITC-Glu (MW 589) was larger than for
TRITC-Ile (MW 573). The reason for this appar-
ent discrepancy is that the van der Waals volume
for Ile (124 A, 3) is larger than for Glu (109 A, 3)
[15]. Diffusion coefficient values for the two
proteins have been reported previously in the
literature and are also shown on the table. The
static diffusion coefficient measurements for oval-
bumin (at an ionic strength of 0.015 M) are 13%
less than the average reported literature value.
The diffusion coefficient measurements, however,
reported in the literature were performed in solu-
tions of different ionic strength which tends to
change the diffusion coefficient, in some cases
more than 20%, by changing the magnitude of the
372 C.T. Culbertson et al. / Talanta 56 (2002) 365–373

Table 2
Diffusion coefficients measured by the static imaging method in an aqueous solution

Analyte MWa Diffusion coefficient (×10−6 cm2 s−1) at 25 °C % RSD n Literature values

Rhodamine 6G 471 4.14 9 0.01 0.24 6

Rhodamine B 444 4.279 0.04 0.94 13
Fluorescein 332 4.25 9 0.01 0.24 6
2%,7% Dichlorofluorescein 401 4.10 9 0.07 1.7 12
TRITC-Glu 589 3.609 0.03 0.83 6
TRITC-Ile 573 3.43 9 0.04 1.2 5
Ovalbumin 45 000 0.6759 0.006b 0.9 6 0.789 [16]
0.776 [17]
0.8139 0.009c 1.1 16 0.759 90.014 [18]
BSA 65 000 0.638 9 0.015 2.4 5 0.63 90.02 [19]
0.62 90.01 [20]

a
Molecular weight.
b
Ionic strength 0.015 M.
c
Ionic strength 0.040 M.

Coulombic repulsions among the protein
molecules. For our static measurements we in-
creased the ionic strength of the 20 mM boric
acid/100 mM Tris buffer by adding 25 mM NaCl
to the solution. This increased the diffusion coeffi-
cient by 20% from 6.75×10 − 7 cm2 s − 1 at an ionic
strength (I) of 0.015 M– 8.13 ×10 − 7 cm2 s − 1 at
an ionic strength of  0.040 M. These values
bracket those reported in the literature. The mea-
surements for BSA are within experimental error of
those reported in the literature. Molecular weight
(  2%) and, therefore, molecular volume changes
due to dye conjugation to the proteins— Ova (2.8
mol dye mol − 1 protein) and BSA (5 mol dye mol − 1
protein)—does not change the diffusion coefficient
measured within the error of the experiment.
At present the precision and accuracy of the
static measurements is limited by our ability to
accurately measure and control the temperature of
the chip. The temperature at the surface of the chip
was observed to vary by as much as 0.5 °C over
the time course of an experiment. Such fluctuations
indicate that diffusion coefficient measurement er-
rors in terms of both accuracy and precision of up
to 1.35% might be expected. This is considerably
larger, however, than most of the RSDs (measure-
ment precision) reported in Tables 1 and 2 above
indicating that the temperature inside the channels
remains relatively constant over the time course of
the experiment even as the ambient air temperature
changes. This is problematic, however, in terms of
accurately measuring the temperature inside of the
channels. Significantly improved accuracy and pre-
cision should be realized, however, with better
thermostatting of the chip.
The static coefficient measurements reported
above were all performed on microscopy setups in
our lab which we generally use to observe fluid
movement on all of the microfabricated devices
that we develop. As such, these measurements can
be performed with the typical instrumentation
found in a lab equipped for microfluidics research.
In addition, the measurements themselves only take
2–8 min to perform depending upon the size of the
molecule, and the post-data acquisition processing
is less than 2 min. This technique, therefore, can be
performed rapidly and accurately with minimal
equipment investment.
Acknowledgements
This research is sponsored by The National
Cancer Institute, The National Institutes of Health,
under grant number CA83238. Oak Ridge National
Laboratory is managed and operated by UT-Bat-
telle, LLC under contract DE-AC05-00OR22725
with the U.S. Department of Energy. The authors
wish to thank Christopher D. Thomas for fabrica-
tion of the microchips.
 C.T. Culbertson et al. / Talanta 56 (2002) 365–373
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