Taylor Dispersion Analysis: Determination of the Diffusion

Coefficient of Blue Dye

Jose Muñetón Dı́az
May 19, 2019

Abstract
The diffusion of blue dye CI42090 through capillary tubes filled with distilled water was
investigated. Taylor Dispersion Analysis was employed to determine a diffusion coefficient
of D = (1.4 ± 0.1) × 10−9 m2 /s, corresponding to a hydrodynamic radius of σ = 1.7 ± 0.3 nm.
No other measurements were found in the literature.

1 Introduction
The diffusion coefficient, D, is a key parameter in a number of chemical and biological processes
that involve the dispersion of fluids flowing under laminar flow. Diffusion of proteins through
hydro-gels is of particular importance in chromatography[1, 2] and drug delivery mechanisms[3,
4]. The movement of solutes and macromolecules in cellular processes such as metabolism and
protein-protein interactions is regulated by diffusion through the cytoplasm[5, 6]. The study of
the magnitude of diffusion of water molecules within tissue gave birth to a revolutionary method
of clinical imaging used to investigate neurological disorders[7].
The knowledge of diffusion coefficients allows to infer the hydrodynamic radii of the molecules
under study (the Stokes radius)[8, 9], which can be either peptides[10], proteins[10, 11] or
nanoparticles[9, 12]. Measuring D and the radii of molecules is of significant relevance in the phar-
maceutical sector as the size can be used as a quality control measure[13], and also in biochemical
studies as a great majority of biomolecular processes involve the recognition of molecules.
Methods for the determination of D, and therefore molecule size, can be classified into two
categories[9]. The first category comprises those techniques that calculate the average diffusion
coefficient of the whole sample, either from separation processes or from measurements of D at
different positions. In this first group are included sedimentation rate[14], Nanoparticle Tracking
Analysis[15], Dynamic Light Scattering (DLS)[16] and Taylor Dispersion Analysis (TDA). Meth-
ods that use a retention-diffusion model to determine D of the analyte in a solvent fall into the
second category. These include Size Exclusion Chromatography(SEC)[17] and Hydrodynamic
Chromatography (HDC)[18]. In practical terms DLS is the most popular technique employed to
determine diffusion coefficients, as it is broadly accessible in pharmaceutical labs, not difficult
to use, and it permits high accuracy measurements[10]. However it encounters difficulties when
investigating small peptides or protein monomers, as they are not as large molecules as the ones
DLS is usually implemented on. This is because the intensity of the scattered light is related to
the sixth power of the particle diameter, which results in DLS measurements tending to have
bias towards larger size particles[15, 19]. Notably, other methods that allow fast analysis with
high accuracy and require smaller sample vlumes are greatly advantageous. TDA has been a
promising technique for obtaining diffusion coefficients since it was proposed by sir Taylor in

1
1953[20], and then further developed by Aris[21]. TDA is a rapid and absolute (absolute as
it does not require prior calibration) method that can efficiently determine average D values,
only requiring small volumes of solution (fractions of nanoliters); it is applicable to particles of
virtually any mass, and it does not need fancy equipment, but commonly available instruments.
In the present study we performed a set of measurements whose aim was to determine the
diffusion coefficient, D, of blue dye utilising TDA along with capillary tubes and an optical
microscope. The value for D was expected to be in the order of ∼ 10−9 m2 s−1 , which measured
at 20°C corresponds to hydrodynamic radii of σ ∼ 10−10 m. Particles of this size such as sucrose
[13] and small proteins[22] have been studied employing DLS. The high reproducibility of TDA
was first demonstrated by Bello et al, who calculated diffusion coefficients of small ions and
proteins (σ ∼ 10−8 m) with reported accuracies of 2%[8]. Ostergaard et al. also determined
hydrodynamic radii of small proteins employing TDA[11], showing good accuracy with other
measurements[22]. Other studies[12] employing both TDA and DLS have determined radii of
nanoparticles in the order σ ∼ 10−9 m, where DLS results have been found to be slightly larger
because of the intrinsic polydispersity of the sample, which simply means the largest particles
of a given population contribute the most to a higher light scattering intensity[23]. In other
words this demonstrates the aforementioned larger size bias DLS possess. Additional evidence of
this difficulty has been shown, which resumes in DLS being inaccurate for polydisperse samples
and the results being seriously compromised by large particles[15, 19]. TDA, however, does not
either display an advantage for he study polydisperse fluids. A comparison study by Mes et al.
reported the characteristics of different methods (TDA, DLS, SEC, HDC) for determining D
of synthetic polymers[24]. High polydispersity appears to pose trouble for all methods, causing
important differences in their results. TDA was the only method of the four that did not
require calibration. Another advantage evidenced was that TDA is concentration independent.
It encountered problems, nonetheless, with the presence of low mass molecules.
The most common method that TDA can be applied on is the investigation of elution profiles
with the aid of UV detectors at two different positions along the capillary, which then allows to
find D from a Gaussian fit of the concentration profile[8, 10]. Capillary electrophoresis is the
common method to pump the fluid through the capillaries. Our experiment was different from
these in the sense that an optical microscope was used for the observations, where changes in
concentration were measured at only one position instead of elution profiles from UV absorbance
at two positions, and the pumping device was simply a syringe pump. Specifically we followed
case B2 in Taylor’s paper, where dissolved material of constant concentration C0 is allowed to
flow at a set speed through a tube that is previously filled with a solvent. There is not much
literature available that accounts for the study of diffusion of blue dye. The only paper found
studied a different dye (CI 52015), and employed an adsorption method to calculate values for
D in the order of 10−4 m2 s−1 [25]. This is somewhat distant from our results, probably because
they used a different dye.

2 Theory
The velocities of the particles of a fluid flowing under laminar flow through a capillary tube of
small bore vary over the cross section of the tube. For the case of a cylindrical tube of circular
cross section, the velocity distribution follows a parabolic form having a maximum value in the
centre of the cross section and decreasing radially to a minimum in the walls of the tube. In
this way molecular diffusion spreads the particles outwards from the tube axis at the front of
the solute, which is where the highest concentration resides. The combined effects of the non
uniform speeds across the cross section of the fluid and of molecular diffusion result in the special

2
mechanism of fluid motion called “Taylor dispersion”.
A key limiting condition imposed by Taylor on his theory was that the time for convective
transport should be longer than the time it takes molecular diffusion to reduce the radial vari-
ations of concentration. If this longitudinal transport by convection happened as fast as the
radial spreading due to diffusion, we would be left with plain turbulent flow. In other words, the
capillary tubes should have narrow bores and the flow rates should be slow in order to achieve
good precision upon measurements and to keep these assumptions holding true. The diffusion
coefficient, D, is related to the virtual dispersion coefficient, k, by:

a2 u20
k= , (1)
192D
where a is the tube radius and u0 is the maximum speed flow.
It is seen therefore that the fluid moving through the tube at velocity 12 u0 disperses along its
cross section following the same laws of diffusion but with another diffusion coefficient, namely
k. This is the radial molecular diffusion that has been aforementioned. The equation governing
this longitudinal dispersion is

∂2C ∂C
k = . (2)
∂x21 ∂t
For the specific case here which corresponds to a dissolved substance of uniform concentration
flowing through the capillary at a constant speed, equation(2) has the following solution
1 1
C/C0 = 1
2 + 12 erf( 21 x1 k − 2 t− 2 ), (3)
1 Rz 2
where erf z = 2π − 2 0 e−z dz.
C shifts from 0.9C0 to 0.1C0 at a point x1 along the tube and over an area of length L, which
is then given by
1 1
L = 3.62k 2 t12 , (4)
t1 here is the time it has taken for the solute to reach the silicon sheet moving at speed u0 /2,
i.e. t1 = tubeu0length
/2 . Finally, the length L of the study area can be related to the concentration
profile by

L = 12 τ u0 , (5)
where τ is the time difference between the moments when the concentration of the fluid is 0.9C0
and 0.1C0 . Going backwards, experimental measurements of τ allow to determine k which then
allows the determination of D. The hydrodynamic radius σ of a particle is given by the Stokes-
Einstein relation:
kB T
σ = 6πηD , (6)
where kB is Boltzmann’s constant, T is temperature and η is the viscosity of the solvent.

3 Method
A set of three capillary tubes were joined together by a Y-junction as shown in figure 1, where
one of the two on the right side was blocked. The end of the tube on the left hand side was
connected to the syringe pump, and the end of the one on the right hand side was swapped
between a reservoir with blue dye and another with distilled water. The Y-junction, which is

3
a microfluidic device made with plasma treatment, was placed under the microscope to allow
observation. Special attention had to be taken when choosing the magnification as no area
outside the tube should appear in the pictures.

Figure 1: Microfluidic device with the shape of a Y-junction, made from plasma treatment. The tube on the left hand
side was connected to the syringe pump, one of the capillary tubes on the right side was blocked and the other had a
free end which was swapped between the reservoirs containing blue dye and distilled water.

Prior to each measurement the capillaries had to be cleaned of impurities which could interfere
with the measurement; in doing so the two tubes were completely filled with distilled water. Then,
the free end of the capillary was put into the reservoir containing blue dye, and the syringe was
set to pull at a certain pump rate. When inserting the tube into the reservoir, the front of the
dye had to be in contact with the front of the distilled water as any space between these could
easily spoil the intensity analysis. This process was crucial in preventing bubbles from entering
the tube: this actually happened for the measurement at the second pump rate, which had to be
repeated. The syringe pumped the blue dye through the tube in the silicon sheet where images
were captured with the microscope. After the concentration reached its maximum, the dye was
expelled and the tube was filled with distilled water to proceed with the next measurement.
The bore of the tubes was 2.55 × 10−4 m, which can considered to be small enough to satisfy
the limiting conditions as stated before. The camera was set to capture one image per second.
With the aid of Fiji software each image was converted into an intensity, which is equivalent
to concentration. Finally, the change in concentration was plotted against the change in time,
which allows to find time τ . This is shown in figure 2.
The aforementioned flushing of the capillaries prior to each measurement was unwittingly
omitted for the first pump rate. Also, the second pump rate had to be repeated after spotting a
bubble, and the flushing was not thoroughly performed for the repeat measurement. Both of these
pump rates showed discrepancies in the final results. Unfortunately, no repeat measurements
were performed for each pump rate, which is of key importance in order to reduce statistical
errors and increase the preciseness of the experiment. For reference the name of the dye was
Blue CI 42090.

4 Results and discussion

Each pump rate measurement aimed to obtain a value for τ . For each subsequent increment in
pump rate, time τ is expected to drop as a fluid moving faster takes less time to diffuse. From
the measurements of time τ , diffusion coefficients for eight different pump rates were obtained.

4
 1.0
190
Concentration[arbitrary units]

180 0.8

170 0.6

C/C0
160
0.4
150
140 0.2
130
0.0
0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160
Time[s] Time[s]

(a) (b)

Figure 2: Graphs of concentration against time at a flow rate of 6 ml/h. The line in figure (a)
is the data after the image processing analysis, each point in the graph is a calculated average
intensity of the photo taken in that second. Figure (b) shows the data after scaling it from 0
to 1 with the respective maximum and minimum. τ is the time difference between the values of
concentration 0.9C0 and 0.1C0 .

The average value was D = (1.4 ± 0.1) × 10−9 m2 /s, with a corresponding hydrodynamic radius
of σ = 1.7 ± 0.3 nm. The radius was calculated using a temperature of T = 20°C and a viscosity
of η = 1.002 mPa · s for distilled water. These results are summarised in table 1.

Pump rate τ L k D σ
(ml/h) (s) (m) (10−5 ) (10−9 m2 s−1 ) (nm)
1.2 142 0.116 0.0737 0.165 1.81
2.4 137 0.224 0.5762 6.260 3.38
3.6 75 0.184 0.5828 1.392 1.52
4.8 61 0.199 0.9138 1.579 1.34
6 61 0.249 1.7848 1.263 1.67
7.2 47 0.230 1.8309 1.773 1.19
8.4 44 0.251 2.5481 1.734 1.22
9.6 41 0.268 3.3026 1.747 1.21

Table 1: Experimental measurements of the diffusion coefficient of blue dye flowing at different pump rates utilising
Taylor Dispersion Analysis with the aid of an optical microscope. τ is the time difference between the moments when
the concentration of the fluid is 0.9C0 and 0.1C0 , L is the length of the area covered during the diffusion process, k is
the dispersion coefficient and D is the diffusion coefficient. The first two measurements show a slight variation. For the
first measurement the discrepancy probably originated from the fact that flushing of the capillary tube was unwittingly
skipped. An unwanted bubble brought the need for a new measurement for the second pump rate, and the capillary was
also not thoroughly flushed before this repetition.

Values for the diffusion coefficient of blue dye were difficult to come across in literature. Only
one paper that studied a different dye was found[25]. A rising dispersion coefficient k with rising
pump rate is observed, indicating that a fluid moving at a higher velocity also disperses radially
in a faster manner. This is of course not the case for the diffusion coefficient because, even
though the solute also diffuses faster when moving at higher velocities, it has virtually all the

5
space it needs to diffuse along the axis of the tube. On the other hand, the solute does not
have as much space in the radial direction where it encounters the walls of the tube. The three
higher pump rates can be interpreted to be of higher quality in regards to preciseness, however
no specific reason can be immediately suggested for this behaviour. An interesting suggestion
that can be made is to investigate the efficacy of TDA with different varying conditions such as
various capillary diameters, utilising more solvents or studying several analytes instead of only
one.

5 Conclusion
An educational investigation was accomplished utilising Taylor Dispersion Analysis along with
capillary tubes and an optical microscope. Blue dye was pumped through capillaries of small
bore radii. Measurements of concentration profiles and subsequent analysis allowed to determine
values for diffusion coefficient, D, at different pump rates, resulting in an average of D = (1.4 ±
0.1) × 10−9 m2 s−1 , which corresponds to a hydrodynamic radius of σ = 1.7 ± 0.3 nm. TDA can
be found to be a simple, effective and inexpensive way of determining diffusion coefficients and
hydrodynamic radii of small molecules.

6 Acknowledgements
Thanks to my lab partners Ruth, Harry and Louis for all the support and help in conducting
the experiment. Thanks to Mack for directions and suggestions.

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