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Continuous asymmetrical flow field-flow fractionation for the purification of proteins and nanoparticles
Journal Pre-proofs Continuous asymmetrical flow field-flow fractionation for the purification of proteins and nanoparticles Maria Marioli, Wim Th. Kok PII: DOI: Reference:
S1383-5866(19)33581-6 https://doi.org/10.1016/j.seppur.2020.116744 SEPPUR 116744
To appear in:
Separation and Purification Technology
Received Date: Revised Date: Accepted Date:
22 August 2019 4 January 2020 21 February 2020
Please cite this article as: M. Marioli, W. Th. Kok, Continuous asymmetrical flow field-flow fractionation for the purification of proteins and nanoparticles, Separation and Purification Technology (2020), doi: https://doi.org/ 10.1016/j.seppur.2020.116744
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© 2020 Published by Elsevier B.V.
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Continuous asymmetrical flow field-flow fractionation for the
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purification of proteins and nanoparticles
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Maria Marioli*, Wim Th. Kok
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Analytical Chemistry Group, van’t Hoff Institute for Molecular Sciences, University of Amsterdam,
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Postbus 94157, 1090 GD Amsterdam, The Netherlands
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Keywords: Field-flow fractionation, continuous, protein, nanoparticle, purification, patterned
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membranes
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*Corresponding author:
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Email: [email protected]
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1
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Abstract
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We present a novel continuous two-dimensional asymmetrical flow field-flow fractionation (2D-
19
AF4) system that is able to fractionate a feed solution of nano-sized solutes (e.g., proteins,
20
nanoparticles) according to their size in aqueous solvents. The key component that generates the
21
continuous separation is a microstructured ultrafiltration membrane with slanted grooves on its
22
surface. The solutes are migrating over the grooves, which are causing a lateral displacement from
23
the direction of the main channel flow, and they are exiting the channel at different outlets. The
24
deflection angle depends on the mean layer thickness of the solutes and consequently on their
25
hydrodynamic radius; with a specific cross-flow, larger solutes exhibit a larger deflection angle
26
which results in a spatial separation. By adjusting the outlet flow and the cross-flow rate, the
27
system can be optimized with respect to purity, recovery and speed. A prototype device has been
28
designed and tested. A proof of principle of the continuous fractionation is demonstrated with
29
mixtures of two model proteins, apoferritin (443 kDa) and thyroglobulin (669 kDa), and of two
30
polystyrene latex standards with diameters of 34 and 102 nm.
2
31
1 Introduction
32
Size-selective separations of macromolecules and nanoparticles are of paramount importance in
33
various fields. For instance, therapeutic proteins produced by modern biotechnology require a
34
high purity of their monomeric form in the final product [1], and polymer-based or inorganic
35
nanoparticles require a narrow size distribution for their proper function [2,3]. Of the techniques
36
used for this purpose, membrane filtration suffers from low resolution and pore clogging when it
37
is used to fractionate polydisperse samples, and ultracentrifugation is time consuming and
38
requires extensive technical skills. Size exclusion chromatography (SEC) is a technique frequently
39
used in analytical and in (semi-)preparative applications but it is poorly scalable, and it may lead
40
to adsorption or shear degradation of some solutes. In downstream processing, the high cost of
41
the chromatographic supports has led to the development of continuous/multicolumn systems
42
(e.g., simulated moving bed chromatography, annular chromatography) [4,5] and membrane
43
chromatography [6] to increase productivity.
44
Asymmetrical flow field flow fractionation (AF4) is the field flow fractionation (FFF)
45
subtechnique that separates nano-sized solutes based on their hydrodynamic size in an open
46
channel by applying a cross-flow through an ultrafiltration (UF) membrane. It is applicable to
47
samples of a very broad size range and chemistry such as biomolecules [7,8], drug delivery
48
systems [9], and nanoparticles [10]. The loading capacity is inherently low (< 100 µg) as during
49
separation the analytes are concentrated very close to the membrane with a mean layer thickness
50
of a few microns. For this reason it has been used mainly as an analytical technique, although
51
commercial semi-preparative long channels with a larger breadth (maximum breadth of ~ 5cm)
52
compared to the conventional channels are occasionally used [11,12], and there have been several
53
attempts to increase sample loading further. Bria et al. [13] investigated the effect of the breadth
54
(up to a maximum breadth of 10 cm) on the sample loading, Maskos and Schupp [14] constructed
55
a circular AF4 system with twelve channels in a quasi-parallel order and Lee et al. [15] developed
56
a multiplexed hollow-fiber flow FFF system interconnecting five hollow fibers. Nevertheless,
57
although larger channel dimensions and multilane systems may increase significantly the loading
58
capacity, they also result in higher solvent consumption, larger footprint and/or more complex
59
equipment.
60
Another option to achieve higher throughput in AF4 is by developing a continuous two-
61
dimensional FFF (2D-FFF) system. As defined by Giddings [16], in continuous 2D-FFF two
62
displacements should occur simultaneously in different directions in a planar geometry, with at
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least one of them based on an FFF subtechnique. The first continuous 2D-FFF technique developed
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was a steric-sedimentation FFF system [17] and later continuous dielectrophoretic-gravitational
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FFF systems [18,19] were introduced. However, these systems were applicable only for micron3
66
sized solutes such as microbeads and cells. A continuous fractionation of macromolecules was
67
achieved by Vastamäki et al. [20,21] via a thermal field-flow fractionation system with a rotating
68
wall. Nevertheless, this system is suitable mainly for organic solvents since thermal diffusion is
69
weak in aqueous solutions. Kim and Moon [22] developed a 2D system combining isoelectric
70
focusing (IEF) in the first dimension and a multilane AF4 channel configuration in the second
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dimension. Although this 2D system is able to spatially separate proteins, the two separation
72
processes occur sequentially and therefore a continuous separation is not possible.
73
Split-flow thin cell (SPLITT) fractionation is a technique related to FFF that can be
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operated in a continuous manner with different types of fields such as gravitational [23] and
75
centrifugal [24]. It can fractionate microparticles with high resolution but for smaller components
76
(i.e., operation in the diffusion mode) the resolution is inherently poor [25]. Another fundamental
77
limitation of SPLITT fractionation is that it can only give binary (high – low) separations.
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Several other (non-FFF) continuous fractionation methods have been developed for nano-
79
sized solutes but none of them is able to provide a high throughput size-based fractionation [26].
80
Systems based on electrophoresis [27] or magnetophoresis [28] can fractionate macromolecules
81
in a continuous flow but according to their electric or magnetic properties and not to their size.
82
Continuous separations based on an acoustic field is able to separate particles only larger than
83
100 nm [29,30], and deterministic lateral displacement with pillar arrays [31] requires
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nanofabrication and it has low throughput. An example of a size-selective continuous
85
fractionation, which is related partly with our study, is a microfluidic channel with slanted grooves
86
on its surface as developed by Bernate et al. [32]. Their method was suitable for micron-sized
87
silica particles and cells, but cannot be used for nano-sized solutes since it is based on gravity and
88
inertia effects.
89
The objective of our study is to demonstrate that a continuous fractionation of mixtures of
90
proteins or nanoparticles, based on their hydrodynamic size, can be achieved using an AF4
91
channel with an UF membrane with slanted grooves (Fig. 1). A spatial separation is caused by the
92
differences in the selectivity for the solutes in two dimensions, one dimension along the grooves
93
and one dimension across the grooves. In the latter direction, the superimposition of the
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perpendicular grooves is causing an increase in selectivity as we have shown in a previous study
95
[33]. We present a “proof-of-concept” investigation conducting physical experiments with
96
microstructured (MS) membranes fabricated by hot-embossing and a prototype 2D-AF4 channel.
4
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2 Materials and Methods
98
2.1
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Polystyrene latex (PS-latex) nanoparticles with nominal diameters of 34 nm and 102 nm, and
100
narrow size distributions were purchased from Duke Scientific (Palo Alto, CA, USA). All other
101
standards and reagents were obtained by Sigma–Aldrich (St. Louis, MO, USA) and were of high
102
analytical purity grade. Hemoglobin from bovine blood, bovine serum albumin, γ-globulin from
103
bovine serum, apoferritin from equine spleen and thyroglobulin from bovine thyroid were used
104
as protein standards. Phosphate-buffer saline (PBS) with an ionic strength of 0.15M (20 mM due
105
to sodium phosphate salts) and a pH of 7.2 was used as a diluent and as a carrier liquid for the
106
proteins. A solution with 0.1% (w/v) sodium dodecyl sulfate (SDS) was used as carrier liquid and
107
diluent for the PS latex standards.
108
2.2
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The AF4 system was an Eclipse DualTec system (Wyatt Technology Europe, Dernbach, Germany)
110
connected to an Agilent HPLC 1200 system (Agilent Technologies, Waldbronn, Germany) that
111
consisted of a degasser, an isocratic pump, a UV detector and an autosampler equipped with an
112
injection loop 100 μL and a thermostat. A second isocratic HPLC pump, Spectroflow 400 (Kratos,
113
Ramsey, NJ, USA), was used to inject continuously the protein solution.
114
2.3
115
A patterned silicon (Si) wafer (LioniX BV, Enschede, The Netherlands) was used as a mold for the
116
fabrication of the microstructured (MS) membranes. The wafer had an area of diameter 15.1 cm
117
patterned with parallel grooves with a cavity width of 50 µm, a ridge width of 50 µm and a ridge
118
height of 12 µm. Polyethersulfone (PES) membranes with 10 kDa molecular weight cut-off
119
(MWCO) (Sartorius, Göttingen, Germany) were hot embossed against the Si wafer with an
120
imprinter (Obducat, Sweden) at 120 °C and 40 bar for 180 sec, and the demolding was performed
121
at 40°C, as it has been described in the literature [34]. Parts of the membrane were broken in
122
liquid nitrogen and gold-sputtered for SEM imaging (XL30 ESEM-FEG, Philips, Eindhoven, The
123
Netherlands).
Samples and carrier liquid
Instrumentation
Fabrication and characterization of the microstructured (MS) membranes
124
To assess the effect of the grooves on the retention time and selectivity, the flat
125
membranes (before hot embossing) and the MS membranes (after hot embossing) were tested on
126
a commercial AF4 channel (Wyatt technology Europe) as it is described in the Supplementary
127
Material. For this purpose, the flat membranes were cut with surgical scissors in the shape of the
128
porous frit which supports the membrane. The MS membranes were cut in the same manner with
129
the grooves aligned parallel or perpendicular to the channel flow. 5
130
2.4
Design and operation of the 2D-AF4 system
131
A commercial channel with a frit inlet (Wyatt Technology Europe) was converted into a 2D
132
channel with six outlets (Fig. 2). The upper inlay with the frit inlet was modified to create 1 mm
133
ports and internal threads for the outlets. Mylar A4 sheets (Mylar A grade) with a thickness of
134
190 and 125 µm were cut to create the spacers for the 2D-AF4 channel. Two spacers were used
135
(spacers A and B in Fig. 2a) which were positioned one over the other. The tip-to -tip length was
136
13.3 cm, the breadth 2.1 cm and the area of the accumulation wall 22.4 cm2. In Fig. 2c the assembly
137
of the 2D-AF4 channel is displayed. The MS membranes were cut with the grooves with an angle
138
of 45° to the channel flow and in the shape of the porous frit (which supports the membrane) with
139
surgical scissors.
140
The flow distribution over the outlets was controlled with PEEK tubing of a suitable length
141
and internal diameter (i.d.) for each outlet. Specifically, tubing with a nominal i.d. of 102 µm or
142
127 µm and lengths of 0.3 – 1 m were cut such that the flow rate at outlets No 1 - 4 was ~ 8% of
143
the total outlet flow rate and ~ 33% at outlets No 5 - 6. The batch mode experiments with the 2D-
144
AF4 channel were carried out using the autosampler and splitting the inlet flow stream into a flow
145
stream towards the frit inlet (𝑉𝑓) and a sample flow stream (𝑉𝑠). The ratio 𝑉𝑠: 𝑉𝑓 was 1:40
146
regulated with suitable tubing. When the 2D-AF4 system was used in the continuous mode, the
147
main HPLC pump was carrying the carrier liquid to the frit inlet (𝑉𝑓) and the second HPLC pump
148
was used to provide the continuous flow of the feed solution (𝑉𝑠).
149
The fractions were collected manually in Greiner polypropylene tubes and each fraction
150
was analyzed offline with conventional AF4 as is described in the Supplementary Material. For the
151
estimation of the recoveries, a calibration curve of each sample component was constructed
152
diluting the initial mixture at appropriate concentrations.
153
3 Results and Discussion
154
3.1
155
The SEM images of the MS membrane disks taken from the top (patterned) side and of the cross
156
section are shown in Fig. 3. The patterned grooves on the membrane surface had the same
157
dimensions with the Si wafer (i.e., a cavity width of 50 µm, a ridge width of 50 µm and a ridge
158
height of approx. 12 µm) and round corners. When the MS membranes were cut with the grooves
159
aligned parallel or perpendicular to the channel flow and tested on a conventional AF4 channel, it
160
was revealed that the recoveries of the smaller proteins decreased. In particular, BSA (66.5 kDa)
161
and γ-globulin (150 kDa) had much lower recoveries (<40%) compared to the flat membranes
Characterization of MS membranes
6
162
(>75%). Nevertheless, the larger proteins, apoferritin (443 kDa) and thyroglobulin (669 kDa), had
163
similar recoveries (>75%) when analyzed with flat or MS membranes.
164
Apoferritin was chosen as the model compound to assess the effect of the grooves on the
165
retention time, on the plate height and on the selectivity between the monomer and the dimer.
166
The experimental results obtained with a cross-flow rate of 1.5 mL/min and an outlet flow rate of
167
0.8 mL/min are displayed in Fig. 4 and in Table 1. The retention times were higher with the MS
168
membranes compared to the flat membranes and the increase was larger when the channel flow
169
was transverse to the grooves. A patterned surface with shallow grooves can be described
170
macroscopically as a surface with slip. The slip is smaller when the flow is across the grooves
171
compared to the flow along the grooves [35] which can explain the higher reduction of the zone
172
velocity over perpendicular grooves.
173
Furthermore, it was revealed that the selectivity remained virtually unchanged with MS
174
membranes with parallel grooves while it increased significantly with perpendicular grooves (Fig.
175
4, Table 1). The plate height increased in both cases as expected; for perpendicular grooves, the
176
plate height increases because the solutes need to diffuse out of the grooves, and, for parallel
177
grooves, because the velocity is lower in the edges of the grooves. For the MS membranes with
178
perpendicular grooves, the results suggest also that, although the plate height is increasing,
179
resolution appears to increase because of the higher selectivity. The potential benefits of applying
180
a wall with grooves perpendicular to the channel flow in FFF have been discussed in previous
181
studies [33,36].
182
The increase in selectivity over perpendicular grooves (Table 1), although statistically
183
significant, is relatively small (~10%). It is sufficient to demonstrate the proof-of-concept for the
184
2D separation but the difference in selectivities in the two dimensions (across and along the
185
grooves) should be higher for an efficient fractionation. In a previous study, we have presented a
186
theoretical model to solve the analytical problem of the mass transfer over perpendicular grooves,
187
along with physical experiments and simulations [33]. The results suggested that a much higher
188
selectivity can be achieved by optimizing the groove structure, for instance by patterning grooves
189
with sharper edges. As Giddings et al. [36] already suggested, any departure of the rectangular
190
shape of the grooves could lead to a lower increase in selectivity but a similar increase in retention
191
time and in plate height.
192
Furthermore, since the solutes may accumulate in the edges of the grooves, to avoid
193
overloading the optimal groove structure should be similar with the theoretical model (i.e., very
194
small ridge width, high aspect ratio of the grooves to avoid slip and groove height close to the
195
mean accumulation thickness). However, practically it is difficult to achieve very small ridge
196
width, rectangular structure and high aspect ratio of the grooves. Therefore, for practical reasons 7
197
ridge and cavity width are larger than the optimal values and subsequently groove height needs
198
also to be increased to increase retention.
199
The difference in selectivity that we observe along and across the grooves entails that a
200
continuous separation can be accomplished when the grooves are placed at an angle 45° to the
201
channel flow. This can be explained by expressing the displacement of the solutes over the slanted
202
grooves as the sum of two displacements that occur simultaneously, one along and one across the
203
grooves (Fig. 5). For a retained solute, the zone velocity along the grooves (𝑣𝑖, ∥ ) is larger than
204
across the grooves (𝑣𝑖, ⊥ ) and the deflection angle (in radians) 𝜗𝑖 can be estimated by, 𝜗𝑖 =
𝑣𝑖, ⊥ 𝜋 ― 𝑎𝑟𝑐𝑡𝑎𝑛 4 𝑣𝑖, ∥
(1) 𝑣2, ⊥
𝑣1, ⊥
205
Consequently, the deflection angle between two components is different when
206
selectivity between two sample components is 𝑎 ⊥ across the grooves and 𝑎 ∥ along the channel,
207
then 𝑣2, ⊥ 𝑣2, ∥
=
( ) 𝑎⊥ 𝑎∥
⋅
𝑣1, ⊥ 𝑣1, ∥
𝑣2, ∥
≠ 𝑣1, ∥ . If the
(2)
208
and, therefore, a separation occurs when 𝑎 ⊥ ≠ 𝑎 ∥ . From the retention times in Table 1, it can be
209
estimated that for the monomer and the dimer of apoferritin the deflection angles would be 4°
210
and 7° respectively.
211
It is important to note that, although the hot embossing method appears to be suitable to
212
show the proof-of-concept, and it is a relatively fast and low-cost technique that offers scalability,
213
it results in higher actual MWCO. In addition, the edges of the grooves become round instead of
214
rectangular. Therefore, in the future the method should be optimized to avoid these effects or
215
other techniques should be explored to fabricate grooves which retain the membrane porosity
216
and result in sharper edges.
217
3.2
218
After confirming that the selectivity between two sample components was different across and
219
along the grooves with MS membranes and, hence, a spatial separation could occur, a 2D-AF4
220
channel was designed. The AF4 channel (Wyatt Technology Europe) that was modified for this
221
purpose had a frit inlet [37] in order to achieve hydrodynamic relaxation of the sample
222
components, as the focusing process cannot be applied in continuous systems. Using the same
223
terminology as Moon et al. [38], the region of the channel beneath the frit element is referred here
2D-AF4 channel design: Injection point and channel outlets
8
224
as relaxation segment and the region from the frit element to the channel outlet as separation
225
segment.
226
The channel was formed combining two spacers (top spacer “A” and bottom spacer “B” in
227
Fig. 2a) which differed only close to the sample inlet and had otherwise identical shapes. In the
228
relaxation segment, the spacers had a narrow (2 mm) confined region after the sample inlet; this
229
spacer geometry enabled the introduction of the sample as a narrow band along the breadth of
230
the channel to avoid extra dispersion. The advantage of using two spacers instead of one is that
231
the sample can be introduced closer to the membrane and, therefore, relaxation can be achieved
232
at higher sample flow rates (~0.05 mL/min). Experiments conducted using only one spacer
233
(spacer B) of thickness 250 or 350 µm, at cross-flow rates 1-3 mL/in and at outlet flow rate 0.8
234
mL/min, showed that the hydrodynamic relaxation of the protein standards was incomplete
235
(most of the sample eluted unfractionated in the void peak within five minutes after sample
236
injection), unless very low sample flow rates (<0.01 mL/min) were used. Flow rates lower than
237
0.01 mL/min are technically impractical and close to the limit of the HPLC pump, affecting
238
precision. Furthermore, to increase sample loading at such low sample flow rates would require
239
highly concentrated feed solutions, which could cause viscosity effects.
240
In the separation segment, the spacers were modified on one side edge of the channel with
241
saw tooth slots that were cut beneath the outlets No 1 - 4 to create “traps” for the solutes and to
242
direct them towards the outlets. Similar saw tooth notches were used by Giddings et al. in the
243
continuous steric - sedimentation FFF system [17]. In the absence of those features, the solutes
244
would not exit the channel from the side outlet ports, since the ports are located at the depletion
245
wall (opposite the accumulation wall) and withdraw the liquid that flows close to this region.
246
Furthermore, the spacers were modified in the end of the separation segment to streamline the
247
flow into two exits (outlets No 5 - 6).
248
We define here the z-axis as the axis along the channel length (as it is commonly done in
249
AF4) and 𝑧 = 0 the point where the separation segment begins. The migration distances z and the
250
deflection angles that the solutes have when they exit from each outlet are given in Table 2. It is
251
however important to note that next to the lateral selective displacement of the solutes, caused by
252
the slanted grooves, the flow through outlets No 1 - 4 is causing an additional lateral bulk
253
displacement. It can be estimated that when no cross-flow is applied, the unretained solutes will
254
exit at outlet No 5, as their migration angle will be ~7° caused by this lateral bulk flow
255
displacement of the outlets No 1 - 4. For the same reason, under the action of the cross-flow, the
256
deflection angle of the solutes will be slightly larger compared to the one expected by only the
257
selective displacement caused by the slanted grooves. Taking into consideration the deflection
258
angle caused by the drift of the carrier liquid 𝜗𝑑, Eq. (1) is written as, 9
𝜗𝑖 =
𝑣𝑖, ⊥ 𝜋 + 𝜗𝑑 ― 𝑎𝑟𝑐𝑡𝑎𝑛 4 𝑣𝑖, ∥
(3)
259
3.3
Spatial separation of proteins
260
The spatial separation of proteins was demonstrated in the batch mode injecting 100 µL of a
261
mixture of apoferritin and thyroglobulin in the 2D-AF4 system and analyzing the fractions
262
collected from each outlet. The injected solution had a concentration of 1 mg/mL of each protein.
263
The thickness of the spacers for this batch-mode experiment were 190 and 125 µm for spacers A
264
and B, respectively (315 µm total channel thickness). The total outlet flow was kept constant at 0.8
265
mL/min and different cross-flow rates were applied (0/1.2/2.0 mL/min). All other flow rates
266
were dependent on these values because the total inflow rate (Vs + Vf) equals to the total outflow
267
rate (Vc + Vout) and it is distributed 1/40 to Vs and 39/40 to Vf. Furthermore, the flow rate at the
268
outlets No 1-4 and at the outlets 5-6 was ~33% and ~8% of the total channel outlet flow (Vout)
269
respectively. The exact values of all flow rates that correspond to the different applied cross-flow
270
rates are tabulated in Table 3.
271
First, the experiment was carried out without applying cross-flow and fractions were
272
collected in the time interval 0 - 30 min after the sample injection. Next, cross-flow was applied
273
(1.2 or 2.0 mL/min) and fractions were collected from each outlet in the time intervals 0 – 5 min,
274
5 – 35 min and 35 – 40 min. The first fractions (0 – 5 min) were pooled together and analyzed to
275
make sure that the sample was not eluting in the void peak and the last fractions (35 - 40 min)
276
were also pooled and analyze to make sure that all the sample had been eluted in the previous
277
time interval. In these pooled fractions, all the peaks were below the limit of detection. All
278
fractions were analyzed with conventional AF4 as it is described in the Supplementary Material.
279
The fractogram of the mixture obtained after dilution with conventional AF4 is displayed
280
in Fig. 6a. It exhibited three peaks which are designated here as peak I, II and III and correspond
281
predominantly to the apoferritin monomer, thyroglobulin monomer and thyroglobulin dimer,
282
respectively. The results of the fractions collected at 5 – 35 min are shown in Fig.6b and Fig. 6c.
283
When no cross-flow was applied, the proteins exited the channel from the outlet No 5 (Fig. 6b) at
284
a composition similar to the initial mixture. This indicates that no spatial separation occurs in the
285
2D-AF4 channel when no cross-flow is applied. When a cross-flow rate of 1.2 mL/min was applied
286
the fractions collected at the outlets 4 and 5 had a different composition than the initial mixture.
287
The fraction from outlet 4 was richer in the larger components (peak II and III) and the fraction
288
from outlet 5 richer in the smaller component (peak I). When the cross-flow rate was increased
289
to 2.0 mL/min, the sample components exited the channel from earlier outlets (larger deflection
290
angle), as expected. 10
291
The total recoveries of the peaks I, II and III were estimated as 94%, 93% and 71%,
292
respectively, for a cross-flow rate of 1.2 mL/min, and 90%, 88%, 67%, respectively, for a cross-
293
flow rate of 2.0 mL/min. The run-to-run repeatability of the recoveries had an average RSD of 2%
294
and the membrane-to-membrane reproducibility an average RSD of 8%.
295 296
3.4
Spatial separation of nanoparticles
297
To demonstrate the separation of nanoparticles, 100 µL of a mixture of PS-latex standards was
298
injected in the 2D-AF4 channel. The injected solution contained 34 and 102 nm standards at
299
concentrations of 4.6 mg/mL and 1.2 mg/mL respectively. The thickness of the spacers of the 2D-
300
AF4 channel was 125 µm for both spacers A and B (total channel thickness 250 µm). The
301
performance of the system was investigated at various combinations of cross-flow and outlet flow
302
rates. Fractions were collected in the time intervals 0 - 3 min, 3 - 43 min and 43 – 48 min. The first
303
fractions (0 – 3 min) were pooled and analyzed to make sure that the sample did not elute in the
304
void time and the last fractions (43 - 48 min) were also pooled and analyzed to make sure that all
305
the sample amount had eluted in the previous fractions.
306
The fractogram of the (diluted) initial solution analyzed with conventional AF4 is shown
307
in Fig. 7a. The results of the fractions collected at 3 – 43 min are displayed in Fig. 7b and Fig. 7c.
308
As expected, higher cross-flow rates are causing larger deflection angles. Although higher cross-
309
flow rates and higher cross-flow to outlet flow ratios seem to improve somewhat resolution, the
310
recovery decreases. In particular, the total recovery of the 34 nm and 102 nm standards was
311
estimated 97% and 91% for cross-flow 0.5 mL/min, 91% and 76% for cross-flow 1 mL/min, and
312
90% and 62% for cross-flow 2 mL/min, respectively. For comparison, the experiments were
313
repeated with a flat (non-patterned) membrane where the sample components were eluted at the
314
outlet 5 and with slightly better (within 5%) recoveries.
315
Since the recovery decreases at higher cross-flow rates (because the sample components
316
have a smaller mean layer thickness and, therefore, are more prone to interact with the
317
membrane), the resolution should be improved in other ways. For instance, the resolution could
318
increase by optimizing groove structure as it was mentioned above or by increasing channel
319
length and width (with a proportional increase in cross-flow rate to maintain the mean layer
320
thickness of the sample components).
321
3.5
322
The performance of the 2D system was subsequently tested with an uninterrupted continuous
323
sample flow. A feed solution containing apoferritin and thyroglobulin at a concentration of 0.25
Continuous fractionation of proteins
11
324
mg/mL of each protein was introduced in the 2D-AF4 system under continuous operation; the
325
secondary pump was employed to provide the feed solution at a sample flow rate (𝑉𝑠) of 0.05
326
mL/min for 160 min. Consequently, in total 8 mL of the feed solution was fractionated containing
327
2 mg of each protein. The thickness of the spacers was 190 µm and 125 µm for the spacers A and
328
B respectively. The frit inlet flow rate (𝑉𝑓) was set at 2.8 mL/min and the cross-flow rate at 2
329
mL/min. Fractions from each outlet were collected every 30 minutes (after discarding the volume
330
collected in the first 40 minutes of operation to make sure that a steady state was reached) and
331
each fraction was analyzed with conventional AF4.
332
The overlaid fractograms are displayed in Figure 8. Although the fractionation is obviously
333
not complete, it is clearly shown that the solution collected at a smaller deflection angle (outlet
334
No. 5) is strongly enriched in the low MW protein, while at a larger deflection angle (outlet No 3)
335
the larger proteins are collected. The total recoveries of the sample components that correspond
336
to the peaks I, II and III are estimated as 92%, 91% and 77%, respectively, which is similar (within
337
5%) to the recovery estimated in the batch mode. We conclude that a continuous operation is
338
possible as the composition of the solution in each outlet was different for all the fractions
339
collected in the different time intervals even after several hours of operation. However, in the
340
course of time, there is an apparent loss in resolution. Cleaning of the membrane with the carrier
341
solution by applying only channel flow for 30 min, restored partly the performance.
342
The system was stressed further increasing the protein concentration of the feed solution
343
four-fold and the operation time up to 5 hours. When the membrane was subsequently removed
344
and inspected, signs of membrane fouling (discoloration of the membrane and loss of its shiny
345
appearance) were observed at the point where the relaxation segment ends. This finding indicates
346
that fouling takes place there which is reasonable as at this area the sample reaches the maximum
347
concentration being compressed close to the membrane (with a mean layer thickness of few
348
micrometers) and confined in an area of 2 mm width.
349
Membrane fouling would be a bottleneck in the utility of this continuous 2D-AF4 system
350
but it could be prevented by several means. First, to reduce the concentration close to the
351
membrane, larger channels could be used where the confined area in which the sample is
352
introduced is wider. Secondly, lower cross-flow rate could be applied to decrease concentration
353
close to the membrane. However, in this case, in order to maintain resolution, the spacer thickness
354
and the groove height should also be increased proportionally. Thirdly, introducing periodic
355
washing steps by interrupting the continuous fractionation at regular time intervals could prevent
356
the accumulation of the sample components in the areas prone to fouling. Lastly, a small amount
357
of very large aggregates present in the sample, which is strongly retained by the grooves, might
12
358
be causing the fouling. In this case, sample filtration prior fractionation could improve the
359
performance.
360
Furthermore, membrane fouling does not only reduce separation efficiency but it also
361
increases the risk of carry-over. It is easy to replace the membrane in the channel and probably
362
less time-consuming than cleaning a fouled membrane. Therefore, microstructured membranes
363
should be disposable and this would only be possible if they are low-cost. Although low-fouling
364
UF membranes (typically used in AF4 and for other ultrafiltration purposes) are inexpensive, the
365
hot embossing step required to create the grooved pattern could increase substantially their cost.
366
High throughput hot embossing technologies, such as continuous roll-to-roll hot embossing, could
367
offer affordable microstructured membranes for commercial use.
368 369
4 Conclusion
370
A continuous 2D-AF4 system has been developed using microstructured ultrafiltration
371
membranes with slanted grooves on their surface. Τhe continuous separation was demonstrated
372
with a mixture of large proteins (apoferritin 443 kDa and thyroglobulin 669 kDa), and PS latex
373
nanoparticles (34 and 102 nm). This is the first continuous field-flow fractionation system that
374
can fractionate a feed solution of macromolecules or nanoparticles in aqueous solvents based on
375
their hydrodynamic size. The device could be scaled up for (semi-)preparative applications or
376
scaled down and integrated into lab-on-a-chip devices. Since the second dimension is generated
377
passively from the slanted grooves, the instrumentation required is very simple; the only
378
components that are indispensable are two pumps (one for the main flow and one for the sample
379
flow stream) and a flow controller to regulate the cross-flow.
380
It was shown that higher cross-flow rates may improve the resolution but they also
381
decrease recovery and, therefore, alternative solutions are required to achieve better resolution
382
e.g. by optimizing the groove shape/dimensions or by increasing the channel dimensions. Future
383
research should be focused on the fabrication of larger and low-cost microstructured membranes
384
with grooves that have sharper edges and with lower MWCO (that are able to retain smaller
385
proteins such as antibodies). The membrane fouling observed during the continuous fractionation
386
could be prevented by operating at low cross-flow rates (adjusting channel thickness and the
387
height of the grooves to maintain resolution) and by increasing the breadth of the channel,
388
particularly in the confined region where the sample is introduced. Finally, the fouling might be
389
related to a small amount of very large aggregates that are strongly retained by the grooves.
390
Therefore, sample filtration/centrifugation prior fractionation or application of periodic washing
391
steps during operation could improve the performance. 13
392
The concept of an accumulation wall with slanted grooves that we demonstrate here for
393
the AF4 system is versatile; it could be applied in any other FFF system (thermal, magnetic, etc.)
394
to transform it into a continuous 2D device. Regardless of the applied field, the component which
395
interacts stronger with it (i.e., which is more retained) would exhibit a larger deflection angle
396
resulting to a continuous fractionation. In fact, the continuous system with fabricated slanted
397
grooves for the fractionation of micron-sized microparticles developed by Bernate et al. [32] could
398
be considered as a continuous gravitational 2D-FFF system.
399
Acknowledgements
400
This work was part of the research program SmartSep with project number 11400 which was
401
financed by the Netherlands Organization for Scientific Research (NWO). The authors are grateful
402
to Ü. Bade Kavurt and prof. dr. Dimitrios Stamatialis (University of Twente) for the fabrication of
403
the microstructured membranes and the SEM images. Wyatt Technology Europe is acknowledged
404
for providing technical expertise and Udo van Hes (University of Amsterdam) for his help on the
405
construction of the 2D-AF4 channel.
14
406
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18
515
Table 1. Effect of the microstructured membranes on the retention time of the apoferritin
516
monomer (𝑡𝑅,1) and dimer (𝑡𝑅,2), on their selectivity 𝑎 and on the plate height of the monomer 𝐻. Membrane
𝑡𝑅,1 (min)
𝑡𝑅,2 (min)
𝑎
𝐻 (mm)
Flat membrane
7.0 ± 0.2
9.6 ± 0.4
1.37 ± 0.01
0.35 ± 0.02
MS membrane (parallel grooves)
9.6 ± 0.5
13.3 ± 0.8
1.38 ± 0.00
0.48 ± 0.03
MS membrane (perpendicular grooves)
11.1 ± 0.6
16.9 ± 0.8
1.51 ± 0.01 0.58 ± 0.03
517 518
19
519
Table 2. Migration distance (along the z-axis) and deflection angle that the solutes have when
520
they exit from each outlet Migration distance Deflection angle 𝑧 (cm)
𝜃
Outlet 1
0 - 1.8
> 27°
Outlet 2
1.8 - 3.3
27° - 15°
Outlet 3
3.3 - 4.8
15° - 11°
Outlet 4
4.8 - 6.3
11° - 8°
Outlet 5
6.3 - 10.0
< 8°
521
20
523
Table 3. Cross-flow rate (𝑉𝑐) and total channel outlet flow rate (𝑉𝑜𝑢𝑡) used in batch mode 2D-AF4
524
(Tables 6 and 7) to separate proteins or nanoparticles, and the corresponding frit inlet flow rate (
525
―6 𝑉𝑓), sample flow rate (𝑉𝑠), and flow rate in each outlet (𝑉1𝑜𝑢𝑡 ). All values are given in mL/min.
𝑽𝒄
𝑽𝒐𝒖𝒕
𝑉𝑓
𝑉𝑠
―4 𝑉1𝑜𝑢𝑡
𝑉5,6 𝑜𝑢𝑡
Spatial separation of proteins 0.00
0.80
0.78
0.02
0.07
0.27
1.20
0.80
1.95
0.05
0.07
0.27
2.00
0.80
2.73
0.07
0.07
0.27
Spatial separation of nanoparticles 0.50
0.50
0.97
0.03
0.04
0.17
1.00
1.00
1.95
0.05
0.08
0.33
2.00
2.00
3.90
0.10
0.17
0.66
2.00
1.00
2.92
0.08
0.08
0.33
3.00
1.50
4.39
0.11
0.13
0.50
526
21
527 528
Figure 1. Continuous separation over an accumulation wall with slanted grooves; larger solutes
529
are more retained by the grooves and exhibit a larger deflection angle.
22
530 531
Figure 2. a) Illustration of the spacer A showing the position of the frit element placed above it, of
532
the spacer B showing the separation of the sample components over the MS membrane placed
533
below it, and of the channel cross section. The colored dashed lines indicate the particle
534
trajectories. The channel thickness and the dimensions of the grooves are exaggerated for visual
535
purposes. b) Photograph of the 2D-AF4 channel displaying the sample inlet (𝑉𝑠), the frit inlet (𝑉𝑓),
536
and the outlets No 1 - 6. c) Assembly of the 2D-AF4 channel.
23
537 538
Figure 3. SEM images of the MS membranes: a) Top and b) cross-sectional view.
24
539 540
Figure 4. Overlaid fractograms of apoferritin obtained with (i) flat membranes, (ii) MS
541
membranes with the grooves parallel to the channel flow and (ii) MS membranes with grooves
542
perpendicular to the channel flow. AF4 conditions: cross-flow rate 1.5 mL/min, outlet flow rate
543
0.8 mL/min and injected mass 5 µg.
25
544 545
Figure 5. The system with slanted grooves may be considered two-dimensional: one dimension
546
is along the grooves ( ∥ ) and the other is across the grooves ( ⊥ ); for well-retained solutes the
547
selectivity is different in the two dimensions which results in their spatial separation.
26
548 549
Figure 6. Spatial separation in the 2D-AF4 system of a solution containing apoferritin and
550
thyroglobulin. The fractograms were obtained by analyzing the samples (initial mixture and
551
fractions) with conventional AF4 (with a cross-flow rate of 1.5 mL/min and an outlet flow rate of
552
0.8 mL/min). a) Fractogram of the initial mixture, b) fractograms of the fractions in every outlet
553
of the 2D-AF4 system acquired at different cross-flow rates (0, 1.2, and 2.0 mL/min) and c)
554
recovery of each component in each outlet. The flow rates mentioned in the figure correspond to
555
the cross-flow rates applied during the 2D-AF4 fractionation where the fractions were collected.
27
556 557
Figure 7. Spatial separation in the 2D-AF4 system of a solution containing PS-latex nanoparticles
558
with diameters of 34 and 102 nm. The fractograms were obtained by analyzing the samples (initial
559
mixture and fractions) with conventional AF4 (with a cross-flow rate of 0.5 mL/min and an outlet
560
flow rate of 1.0 mL/min). a) Fractogram of the initial mixture, b) fractograms of the fractions in
561
every outlet acquired under different cross-flow and outlet flow rates and c) recovery of each
562
component in each fraction. The flow rates mentioned in the figure correspond to the flow rates
563
applied during the 2D-AF4 fractionation where the fractions were collected.
28
564 565
Figure 8 Continuous fractionation of a mixture of proteins: Overlaid fractograms of the fractions
566
collected in different time intervals from the outlets 3, 4 and 5 under continuous operation of the
567
2D-AF4 system. The fractograms are normalized to the highest peak for visual comparison.
568 569
29
570
Graphical abstract
571
572
30
S1383-5866(19)33581-6 https://doi.org/10.1016/j.seppur.2020.116744 SEPPUR 116744
To appear in:
Separation and Purification Technology
Received Date: Revised Date: Accepted Date:
22 August 2019 4 January 2020 21 February 2020
Please cite this article as: M. Marioli, W. Th. Kok, Continuous asymmetrical flow field-flow fractionation for the purification of proteins and nanoparticles, Separation and Purification Technology (2020), doi: https://doi.org/ 10.1016/j.seppur.2020.116744
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© 2020 Published by Elsevier B.V.
1
Continuous asymmetrical flow field-flow fractionation for the
2
purification of proteins and nanoparticles
3 4
Maria Marioli*, Wim Th. Kok
5
Analytical Chemistry Group, van’t Hoff Institute for Molecular Sciences, University of Amsterdam,
6
Postbus 94157, 1090 GD Amsterdam, The Netherlands
7 8
Keywords: Field-flow fractionation, continuous, protein, nanoparticle, purification, patterned
9
membranes
10 11 12 13
*Corresponding author:
14
Email: [email protected]
15 16
1
17
Abstract
18
We present a novel continuous two-dimensional asymmetrical flow field-flow fractionation (2D-
19
AF4) system that is able to fractionate a feed solution of nano-sized solutes (e.g., proteins,
20
nanoparticles) according to their size in aqueous solvents. The key component that generates the
21
continuous separation is a microstructured ultrafiltration membrane with slanted grooves on its
22
surface. The solutes are migrating over the grooves, which are causing a lateral displacement from
23
the direction of the main channel flow, and they are exiting the channel at different outlets. The
24
deflection angle depends on the mean layer thickness of the solutes and consequently on their
25
hydrodynamic radius; with a specific cross-flow, larger solutes exhibit a larger deflection angle
26
which results in a spatial separation. By adjusting the outlet flow and the cross-flow rate, the
27
system can be optimized with respect to purity, recovery and speed. A prototype device has been
28
designed and tested. A proof of principle of the continuous fractionation is demonstrated with
29
mixtures of two model proteins, apoferritin (443 kDa) and thyroglobulin (669 kDa), and of two
30
polystyrene latex standards with diameters of 34 and 102 nm.
2
31
1 Introduction
32
Size-selective separations of macromolecules and nanoparticles are of paramount importance in
33
various fields. For instance, therapeutic proteins produced by modern biotechnology require a
34
high purity of their monomeric form in the final product [1], and polymer-based or inorganic
35
nanoparticles require a narrow size distribution for their proper function [2,3]. Of the techniques
36
used for this purpose, membrane filtration suffers from low resolution and pore clogging when it
37
is used to fractionate polydisperse samples, and ultracentrifugation is time consuming and
38
requires extensive technical skills. Size exclusion chromatography (SEC) is a technique frequently
39
used in analytical and in (semi-)preparative applications but it is poorly scalable, and it may lead
40
to adsorption or shear degradation of some solutes. In downstream processing, the high cost of
41
the chromatographic supports has led to the development of continuous/multicolumn systems
42
(e.g., simulated moving bed chromatography, annular chromatography) [4,5] and membrane
43
chromatography [6] to increase productivity.
44
Asymmetrical flow field flow fractionation (AF4) is the field flow fractionation (FFF)
45
subtechnique that separates nano-sized solutes based on their hydrodynamic size in an open
46
channel by applying a cross-flow through an ultrafiltration (UF) membrane. It is applicable to
47
samples of a very broad size range and chemistry such as biomolecules [7,8], drug delivery
48
systems [9], and nanoparticles [10]. The loading capacity is inherently low (< 100 µg) as during
49
separation the analytes are concentrated very close to the membrane with a mean layer thickness
50
of a few microns. For this reason it has been used mainly as an analytical technique, although
51
commercial semi-preparative long channels with a larger breadth (maximum breadth of ~ 5cm)
52
compared to the conventional channels are occasionally used [11,12], and there have been several
53
attempts to increase sample loading further. Bria et al. [13] investigated the effect of the breadth
54
(up to a maximum breadth of 10 cm) on the sample loading, Maskos and Schupp [14] constructed
55
a circular AF4 system with twelve channels in a quasi-parallel order and Lee et al. [15] developed
56
a multiplexed hollow-fiber flow FFF system interconnecting five hollow fibers. Nevertheless,
57
although larger channel dimensions and multilane systems may increase significantly the loading
58
capacity, they also result in higher solvent consumption, larger footprint and/or more complex
59
equipment.
60
Another option to achieve higher throughput in AF4 is by developing a continuous two-
61
dimensional FFF (2D-FFF) system. As defined by Giddings [16], in continuous 2D-FFF two
62
displacements should occur simultaneously in different directions in a planar geometry, with at
63
least one of them based on an FFF subtechnique. The first continuous 2D-FFF technique developed
64
was a steric-sedimentation FFF system [17] and later continuous dielectrophoretic-gravitational
65
FFF systems [18,19] were introduced. However, these systems were applicable only for micron3
66
sized solutes such as microbeads and cells. A continuous fractionation of macromolecules was
67
achieved by Vastamäki et al. [20,21] via a thermal field-flow fractionation system with a rotating
68
wall. Nevertheless, this system is suitable mainly for organic solvents since thermal diffusion is
69
weak in aqueous solutions. Kim and Moon [22] developed a 2D system combining isoelectric
70
focusing (IEF) in the first dimension and a multilane AF4 channel configuration in the second
71
dimension. Although this 2D system is able to spatially separate proteins, the two separation
72
processes occur sequentially and therefore a continuous separation is not possible.
73
Split-flow thin cell (SPLITT) fractionation is a technique related to FFF that can be
74
operated in a continuous manner with different types of fields such as gravitational [23] and
75
centrifugal [24]. It can fractionate microparticles with high resolution but for smaller components
76
(i.e., operation in the diffusion mode) the resolution is inherently poor [25]. Another fundamental
77
limitation of SPLITT fractionation is that it can only give binary (high – low) separations.
78
Several other (non-FFF) continuous fractionation methods have been developed for nano-
79
sized solutes but none of them is able to provide a high throughput size-based fractionation [26].
80
Systems based on electrophoresis [27] or magnetophoresis [28] can fractionate macromolecules
81
in a continuous flow but according to their electric or magnetic properties and not to their size.
82
Continuous separations based on an acoustic field is able to separate particles only larger than
83
100 nm [29,30], and deterministic lateral displacement with pillar arrays [31] requires
84
nanofabrication and it has low throughput. An example of a size-selective continuous
85
fractionation, which is related partly with our study, is a microfluidic channel with slanted grooves
86
on its surface as developed by Bernate et al. [32]. Their method was suitable for micron-sized
87
silica particles and cells, but cannot be used for nano-sized solutes since it is based on gravity and
88
inertia effects.
89
The objective of our study is to demonstrate that a continuous fractionation of mixtures of
90
proteins or nanoparticles, based on their hydrodynamic size, can be achieved using an AF4
91
channel with an UF membrane with slanted grooves (Fig. 1). A spatial separation is caused by the
92
differences in the selectivity for the solutes in two dimensions, one dimension along the grooves
93
and one dimension across the grooves. In the latter direction, the superimposition of the
94
perpendicular grooves is causing an increase in selectivity as we have shown in a previous study
95
[33]. We present a “proof-of-concept” investigation conducting physical experiments with
96
microstructured (MS) membranes fabricated by hot-embossing and a prototype 2D-AF4 channel.
4
97
2 Materials and Methods
98
2.1
99
Polystyrene latex (PS-latex) nanoparticles with nominal diameters of 34 nm and 102 nm, and
100
narrow size distributions were purchased from Duke Scientific (Palo Alto, CA, USA). All other
101
standards and reagents were obtained by Sigma–Aldrich (St. Louis, MO, USA) and were of high
102
analytical purity grade. Hemoglobin from bovine blood, bovine serum albumin, γ-globulin from
103
bovine serum, apoferritin from equine spleen and thyroglobulin from bovine thyroid were used
104
as protein standards. Phosphate-buffer saline (PBS) with an ionic strength of 0.15M (20 mM due
105
to sodium phosphate salts) and a pH of 7.2 was used as a diluent and as a carrier liquid for the
106
proteins. A solution with 0.1% (w/v) sodium dodecyl sulfate (SDS) was used as carrier liquid and
107
diluent for the PS latex standards.
108
2.2
109
The AF4 system was an Eclipse DualTec system (Wyatt Technology Europe, Dernbach, Germany)
110
connected to an Agilent HPLC 1200 system (Agilent Technologies, Waldbronn, Germany) that
111
consisted of a degasser, an isocratic pump, a UV detector and an autosampler equipped with an
112
injection loop 100 μL and a thermostat. A second isocratic HPLC pump, Spectroflow 400 (Kratos,
113
Ramsey, NJ, USA), was used to inject continuously the protein solution.
114
2.3
115
A patterned silicon (Si) wafer (LioniX BV, Enschede, The Netherlands) was used as a mold for the
116
fabrication of the microstructured (MS) membranes. The wafer had an area of diameter 15.1 cm
117
patterned with parallel grooves with a cavity width of 50 µm, a ridge width of 50 µm and a ridge
118
height of 12 µm. Polyethersulfone (PES) membranes with 10 kDa molecular weight cut-off
119
(MWCO) (Sartorius, Göttingen, Germany) were hot embossed against the Si wafer with an
120
imprinter (Obducat, Sweden) at 120 °C and 40 bar for 180 sec, and the demolding was performed
121
at 40°C, as it has been described in the literature [34]. Parts of the membrane were broken in
122
liquid nitrogen and gold-sputtered for SEM imaging (XL30 ESEM-FEG, Philips, Eindhoven, The
123
Netherlands).
Samples and carrier liquid
Instrumentation
Fabrication and characterization of the microstructured (MS) membranes
124
To assess the effect of the grooves on the retention time and selectivity, the flat
125
membranes (before hot embossing) and the MS membranes (after hot embossing) were tested on
126
a commercial AF4 channel (Wyatt technology Europe) as it is described in the Supplementary
127
Material. For this purpose, the flat membranes were cut with surgical scissors in the shape of the
128
porous frit which supports the membrane. The MS membranes were cut in the same manner with
129
the grooves aligned parallel or perpendicular to the channel flow. 5
130
2.4
Design and operation of the 2D-AF4 system
131
A commercial channel with a frit inlet (Wyatt Technology Europe) was converted into a 2D
132
channel with six outlets (Fig. 2). The upper inlay with the frit inlet was modified to create 1 mm
133
ports and internal threads for the outlets. Mylar A4 sheets (Mylar A grade) with a thickness of
134
190 and 125 µm were cut to create the spacers for the 2D-AF4 channel. Two spacers were used
135
(spacers A and B in Fig. 2a) which were positioned one over the other. The tip-to -tip length was
136
13.3 cm, the breadth 2.1 cm and the area of the accumulation wall 22.4 cm2. In Fig. 2c the assembly
137
of the 2D-AF4 channel is displayed. The MS membranes were cut with the grooves with an angle
138
of 45° to the channel flow and in the shape of the porous frit (which supports the membrane) with
139
surgical scissors.
140
The flow distribution over the outlets was controlled with PEEK tubing of a suitable length
141
and internal diameter (i.d.) for each outlet. Specifically, tubing with a nominal i.d. of 102 µm or
142
127 µm and lengths of 0.3 – 1 m were cut such that the flow rate at outlets No 1 - 4 was ~ 8% of
143
the total outlet flow rate and ~ 33% at outlets No 5 - 6. The batch mode experiments with the 2D-
144
AF4 channel were carried out using the autosampler and splitting the inlet flow stream into a flow
145
stream towards the frit inlet (𝑉𝑓) and a sample flow stream (𝑉𝑠). The ratio 𝑉𝑠: 𝑉𝑓 was 1:40
146
regulated with suitable tubing. When the 2D-AF4 system was used in the continuous mode, the
147
main HPLC pump was carrying the carrier liquid to the frit inlet (𝑉𝑓) and the second HPLC pump
148
was used to provide the continuous flow of the feed solution (𝑉𝑠).
149
The fractions were collected manually in Greiner polypropylene tubes and each fraction
150
was analyzed offline with conventional AF4 as is described in the Supplementary Material. For the
151
estimation of the recoveries, a calibration curve of each sample component was constructed
152
diluting the initial mixture at appropriate concentrations.
153
3 Results and Discussion
154
3.1
155
The SEM images of the MS membrane disks taken from the top (patterned) side and of the cross
156
section are shown in Fig. 3. The patterned grooves on the membrane surface had the same
157
dimensions with the Si wafer (i.e., a cavity width of 50 µm, a ridge width of 50 µm and a ridge
158
height of approx. 12 µm) and round corners. When the MS membranes were cut with the grooves
159
aligned parallel or perpendicular to the channel flow and tested on a conventional AF4 channel, it
160
was revealed that the recoveries of the smaller proteins decreased. In particular, BSA (66.5 kDa)
161
and γ-globulin (150 kDa) had much lower recoveries (<40%) compared to the flat membranes
Characterization of MS membranes
6
162
(>75%). Nevertheless, the larger proteins, apoferritin (443 kDa) and thyroglobulin (669 kDa), had
163
similar recoveries (>75%) when analyzed with flat or MS membranes.
164
Apoferritin was chosen as the model compound to assess the effect of the grooves on the
165
retention time, on the plate height and on the selectivity between the monomer and the dimer.
166
The experimental results obtained with a cross-flow rate of 1.5 mL/min and an outlet flow rate of
167
0.8 mL/min are displayed in Fig. 4 and in Table 1. The retention times were higher with the MS
168
membranes compared to the flat membranes and the increase was larger when the channel flow
169
was transverse to the grooves. A patterned surface with shallow grooves can be described
170
macroscopically as a surface with slip. The slip is smaller when the flow is across the grooves
171
compared to the flow along the grooves [35] which can explain the higher reduction of the zone
172
velocity over perpendicular grooves.
173
Furthermore, it was revealed that the selectivity remained virtually unchanged with MS
174
membranes with parallel grooves while it increased significantly with perpendicular grooves (Fig.
175
4, Table 1). The plate height increased in both cases as expected; for perpendicular grooves, the
176
plate height increases because the solutes need to diffuse out of the grooves, and, for parallel
177
grooves, because the velocity is lower in the edges of the grooves. For the MS membranes with
178
perpendicular grooves, the results suggest also that, although the plate height is increasing,
179
resolution appears to increase because of the higher selectivity. The potential benefits of applying
180
a wall with grooves perpendicular to the channel flow in FFF have been discussed in previous
181
studies [33,36].
182
The increase in selectivity over perpendicular grooves (Table 1), although statistically
183
significant, is relatively small (~10%). It is sufficient to demonstrate the proof-of-concept for the
184
2D separation but the difference in selectivities in the two dimensions (across and along the
185
grooves) should be higher for an efficient fractionation. In a previous study, we have presented a
186
theoretical model to solve the analytical problem of the mass transfer over perpendicular grooves,
187
along with physical experiments and simulations [33]. The results suggested that a much higher
188
selectivity can be achieved by optimizing the groove structure, for instance by patterning grooves
189
with sharper edges. As Giddings et al. [36] already suggested, any departure of the rectangular
190
shape of the grooves could lead to a lower increase in selectivity but a similar increase in retention
191
time and in plate height.
192
Furthermore, since the solutes may accumulate in the edges of the grooves, to avoid
193
overloading the optimal groove structure should be similar with the theoretical model (i.e., very
194
small ridge width, high aspect ratio of the grooves to avoid slip and groove height close to the
195
mean accumulation thickness). However, practically it is difficult to achieve very small ridge
196
width, rectangular structure and high aspect ratio of the grooves. Therefore, for practical reasons 7
197
ridge and cavity width are larger than the optimal values and subsequently groove height needs
198
also to be increased to increase retention.
199
The difference in selectivity that we observe along and across the grooves entails that a
200
continuous separation can be accomplished when the grooves are placed at an angle 45° to the
201
channel flow. This can be explained by expressing the displacement of the solutes over the slanted
202
grooves as the sum of two displacements that occur simultaneously, one along and one across the
203
grooves (Fig. 5). For a retained solute, the zone velocity along the grooves (𝑣𝑖, ∥ ) is larger than
204
across the grooves (𝑣𝑖, ⊥ ) and the deflection angle (in radians) 𝜗𝑖 can be estimated by, 𝜗𝑖 =
𝑣𝑖, ⊥ 𝜋 ― 𝑎𝑟𝑐𝑡𝑎𝑛 4 𝑣𝑖, ∥
(1) 𝑣2, ⊥
𝑣1, ⊥
205
Consequently, the deflection angle between two components is different when
206
selectivity between two sample components is 𝑎 ⊥ across the grooves and 𝑎 ∥ along the channel,
207
then 𝑣2, ⊥ 𝑣2, ∥
=
( ) 𝑎⊥ 𝑎∥
⋅
𝑣1, ⊥ 𝑣1, ∥
𝑣2, ∥
≠ 𝑣1, ∥ . If the
(2)
208
and, therefore, a separation occurs when 𝑎 ⊥ ≠ 𝑎 ∥ . From the retention times in Table 1, it can be
209
estimated that for the monomer and the dimer of apoferritin the deflection angles would be 4°
210
and 7° respectively.
211
It is important to note that, although the hot embossing method appears to be suitable to
212
show the proof-of-concept, and it is a relatively fast and low-cost technique that offers scalability,
213
it results in higher actual MWCO. In addition, the edges of the grooves become round instead of
214
rectangular. Therefore, in the future the method should be optimized to avoid these effects or
215
other techniques should be explored to fabricate grooves which retain the membrane porosity
216
and result in sharper edges.
217
3.2
218
After confirming that the selectivity between two sample components was different across and
219
along the grooves with MS membranes and, hence, a spatial separation could occur, a 2D-AF4
220
channel was designed. The AF4 channel (Wyatt Technology Europe) that was modified for this
221
purpose had a frit inlet [37] in order to achieve hydrodynamic relaxation of the sample
222
components, as the focusing process cannot be applied in continuous systems. Using the same
223
terminology as Moon et al. [38], the region of the channel beneath the frit element is referred here
2D-AF4 channel design: Injection point and channel outlets
8
224
as relaxation segment and the region from the frit element to the channel outlet as separation
225
segment.
226
The channel was formed combining two spacers (top spacer “A” and bottom spacer “B” in
227
Fig. 2a) which differed only close to the sample inlet and had otherwise identical shapes. In the
228
relaxation segment, the spacers had a narrow (2 mm) confined region after the sample inlet; this
229
spacer geometry enabled the introduction of the sample as a narrow band along the breadth of
230
the channel to avoid extra dispersion. The advantage of using two spacers instead of one is that
231
the sample can be introduced closer to the membrane and, therefore, relaxation can be achieved
232
at higher sample flow rates (~0.05 mL/min). Experiments conducted using only one spacer
233
(spacer B) of thickness 250 or 350 µm, at cross-flow rates 1-3 mL/in and at outlet flow rate 0.8
234
mL/min, showed that the hydrodynamic relaxation of the protein standards was incomplete
235
(most of the sample eluted unfractionated in the void peak within five minutes after sample
236
injection), unless very low sample flow rates (<0.01 mL/min) were used. Flow rates lower than
237
0.01 mL/min are technically impractical and close to the limit of the HPLC pump, affecting
238
precision. Furthermore, to increase sample loading at such low sample flow rates would require
239
highly concentrated feed solutions, which could cause viscosity effects.
240
In the separation segment, the spacers were modified on one side edge of the channel with
241
saw tooth slots that were cut beneath the outlets No 1 - 4 to create “traps” for the solutes and to
242
direct them towards the outlets. Similar saw tooth notches were used by Giddings et al. in the
243
continuous steric - sedimentation FFF system [17]. In the absence of those features, the solutes
244
would not exit the channel from the side outlet ports, since the ports are located at the depletion
245
wall (opposite the accumulation wall) and withdraw the liquid that flows close to this region.
246
Furthermore, the spacers were modified in the end of the separation segment to streamline the
247
flow into two exits (outlets No 5 - 6).
248
We define here the z-axis as the axis along the channel length (as it is commonly done in
249
AF4) and 𝑧 = 0 the point where the separation segment begins. The migration distances z and the
250
deflection angles that the solutes have when they exit from each outlet are given in Table 2. It is
251
however important to note that next to the lateral selective displacement of the solutes, caused by
252
the slanted grooves, the flow through outlets No 1 - 4 is causing an additional lateral bulk
253
displacement. It can be estimated that when no cross-flow is applied, the unretained solutes will
254
exit at outlet No 5, as their migration angle will be ~7° caused by this lateral bulk flow
255
displacement of the outlets No 1 - 4. For the same reason, under the action of the cross-flow, the
256
deflection angle of the solutes will be slightly larger compared to the one expected by only the
257
selective displacement caused by the slanted grooves. Taking into consideration the deflection
258
angle caused by the drift of the carrier liquid 𝜗𝑑, Eq. (1) is written as, 9
𝜗𝑖 =
𝑣𝑖, ⊥ 𝜋 + 𝜗𝑑 ― 𝑎𝑟𝑐𝑡𝑎𝑛 4 𝑣𝑖, ∥
(3)
259
3.3
Spatial separation of proteins
260
The spatial separation of proteins was demonstrated in the batch mode injecting 100 µL of a
261
mixture of apoferritin and thyroglobulin in the 2D-AF4 system and analyzing the fractions
262
collected from each outlet. The injected solution had a concentration of 1 mg/mL of each protein.
263
The thickness of the spacers for this batch-mode experiment were 190 and 125 µm for spacers A
264
and B, respectively (315 µm total channel thickness). The total outlet flow was kept constant at 0.8
265
mL/min and different cross-flow rates were applied (0/1.2/2.0 mL/min). All other flow rates
266
were dependent on these values because the total inflow rate (Vs + Vf) equals to the total outflow
267
rate (Vc + Vout) and it is distributed 1/40 to Vs and 39/40 to Vf. Furthermore, the flow rate at the
268
outlets No 1-4 and at the outlets 5-6 was ~33% and ~8% of the total channel outlet flow (Vout)
269
respectively. The exact values of all flow rates that correspond to the different applied cross-flow
270
rates are tabulated in Table 3.
271
First, the experiment was carried out without applying cross-flow and fractions were
272
collected in the time interval 0 - 30 min after the sample injection. Next, cross-flow was applied
273
(1.2 or 2.0 mL/min) and fractions were collected from each outlet in the time intervals 0 – 5 min,
274
5 – 35 min and 35 – 40 min. The first fractions (0 – 5 min) were pooled together and analyzed to
275
make sure that the sample was not eluting in the void peak and the last fractions (35 - 40 min)
276
were also pooled and analyze to make sure that all the sample had been eluted in the previous
277
time interval. In these pooled fractions, all the peaks were below the limit of detection. All
278
fractions were analyzed with conventional AF4 as it is described in the Supplementary Material.
279
The fractogram of the mixture obtained after dilution with conventional AF4 is displayed
280
in Fig. 6a. It exhibited three peaks which are designated here as peak I, II and III and correspond
281
predominantly to the apoferritin monomer, thyroglobulin monomer and thyroglobulin dimer,
282
respectively. The results of the fractions collected at 5 – 35 min are shown in Fig.6b and Fig. 6c.
283
When no cross-flow was applied, the proteins exited the channel from the outlet No 5 (Fig. 6b) at
284
a composition similar to the initial mixture. This indicates that no spatial separation occurs in the
285
2D-AF4 channel when no cross-flow is applied. When a cross-flow rate of 1.2 mL/min was applied
286
the fractions collected at the outlets 4 and 5 had a different composition than the initial mixture.
287
The fraction from outlet 4 was richer in the larger components (peak II and III) and the fraction
288
from outlet 5 richer in the smaller component (peak I). When the cross-flow rate was increased
289
to 2.0 mL/min, the sample components exited the channel from earlier outlets (larger deflection
290
angle), as expected. 10
291
The total recoveries of the peaks I, II and III were estimated as 94%, 93% and 71%,
292
respectively, for a cross-flow rate of 1.2 mL/min, and 90%, 88%, 67%, respectively, for a cross-
293
flow rate of 2.0 mL/min. The run-to-run repeatability of the recoveries had an average RSD of 2%
294
and the membrane-to-membrane reproducibility an average RSD of 8%.
295 296
3.4
Spatial separation of nanoparticles
297
To demonstrate the separation of nanoparticles, 100 µL of a mixture of PS-latex standards was
298
injected in the 2D-AF4 channel. The injected solution contained 34 and 102 nm standards at
299
concentrations of 4.6 mg/mL and 1.2 mg/mL respectively. The thickness of the spacers of the 2D-
300
AF4 channel was 125 µm for both spacers A and B (total channel thickness 250 µm). The
301
performance of the system was investigated at various combinations of cross-flow and outlet flow
302
rates. Fractions were collected in the time intervals 0 - 3 min, 3 - 43 min and 43 – 48 min. The first
303
fractions (0 – 3 min) were pooled and analyzed to make sure that the sample did not elute in the
304
void time and the last fractions (43 - 48 min) were also pooled and analyzed to make sure that all
305
the sample amount had eluted in the previous fractions.
306
The fractogram of the (diluted) initial solution analyzed with conventional AF4 is shown
307
in Fig. 7a. The results of the fractions collected at 3 – 43 min are displayed in Fig. 7b and Fig. 7c.
308
As expected, higher cross-flow rates are causing larger deflection angles. Although higher cross-
309
flow rates and higher cross-flow to outlet flow ratios seem to improve somewhat resolution, the
310
recovery decreases. In particular, the total recovery of the 34 nm and 102 nm standards was
311
estimated 97% and 91% for cross-flow 0.5 mL/min, 91% and 76% for cross-flow 1 mL/min, and
312
90% and 62% for cross-flow 2 mL/min, respectively. For comparison, the experiments were
313
repeated with a flat (non-patterned) membrane where the sample components were eluted at the
314
outlet 5 and with slightly better (within 5%) recoveries.
315
Since the recovery decreases at higher cross-flow rates (because the sample components
316
have a smaller mean layer thickness and, therefore, are more prone to interact with the
317
membrane), the resolution should be improved in other ways. For instance, the resolution could
318
increase by optimizing groove structure as it was mentioned above or by increasing channel
319
length and width (with a proportional increase in cross-flow rate to maintain the mean layer
320
thickness of the sample components).
321
3.5
322
The performance of the 2D system was subsequently tested with an uninterrupted continuous
323
sample flow. A feed solution containing apoferritin and thyroglobulin at a concentration of 0.25
Continuous fractionation of proteins
11
324
mg/mL of each protein was introduced in the 2D-AF4 system under continuous operation; the
325
secondary pump was employed to provide the feed solution at a sample flow rate (𝑉𝑠) of 0.05
326
mL/min for 160 min. Consequently, in total 8 mL of the feed solution was fractionated containing
327
2 mg of each protein. The thickness of the spacers was 190 µm and 125 µm for the spacers A and
328
B respectively. The frit inlet flow rate (𝑉𝑓) was set at 2.8 mL/min and the cross-flow rate at 2
329
mL/min. Fractions from each outlet were collected every 30 minutes (after discarding the volume
330
collected in the first 40 minutes of operation to make sure that a steady state was reached) and
331
each fraction was analyzed with conventional AF4.
332
The overlaid fractograms are displayed in Figure 8. Although the fractionation is obviously
333
not complete, it is clearly shown that the solution collected at a smaller deflection angle (outlet
334
No. 5) is strongly enriched in the low MW protein, while at a larger deflection angle (outlet No 3)
335
the larger proteins are collected. The total recoveries of the sample components that correspond
336
to the peaks I, II and III are estimated as 92%, 91% and 77%, respectively, which is similar (within
337
5%) to the recovery estimated in the batch mode. We conclude that a continuous operation is
338
possible as the composition of the solution in each outlet was different for all the fractions
339
collected in the different time intervals even after several hours of operation. However, in the
340
course of time, there is an apparent loss in resolution. Cleaning of the membrane with the carrier
341
solution by applying only channel flow for 30 min, restored partly the performance.
342
The system was stressed further increasing the protein concentration of the feed solution
343
four-fold and the operation time up to 5 hours. When the membrane was subsequently removed
344
and inspected, signs of membrane fouling (discoloration of the membrane and loss of its shiny
345
appearance) were observed at the point where the relaxation segment ends. This finding indicates
346
that fouling takes place there which is reasonable as at this area the sample reaches the maximum
347
concentration being compressed close to the membrane (with a mean layer thickness of few
348
micrometers) and confined in an area of 2 mm width.
349
Membrane fouling would be a bottleneck in the utility of this continuous 2D-AF4 system
350
but it could be prevented by several means. First, to reduce the concentration close to the
351
membrane, larger channels could be used where the confined area in which the sample is
352
introduced is wider. Secondly, lower cross-flow rate could be applied to decrease concentration
353
close to the membrane. However, in this case, in order to maintain resolution, the spacer thickness
354
and the groove height should also be increased proportionally. Thirdly, introducing periodic
355
washing steps by interrupting the continuous fractionation at regular time intervals could prevent
356
the accumulation of the sample components in the areas prone to fouling. Lastly, a small amount
357
of very large aggregates present in the sample, which is strongly retained by the grooves, might
12
358
be causing the fouling. In this case, sample filtration prior fractionation could improve the
359
performance.
360
Furthermore, membrane fouling does not only reduce separation efficiency but it also
361
increases the risk of carry-over. It is easy to replace the membrane in the channel and probably
362
less time-consuming than cleaning a fouled membrane. Therefore, microstructured membranes
363
should be disposable and this would only be possible if they are low-cost. Although low-fouling
364
UF membranes (typically used in AF4 and for other ultrafiltration purposes) are inexpensive, the
365
hot embossing step required to create the grooved pattern could increase substantially their cost.
366
High throughput hot embossing technologies, such as continuous roll-to-roll hot embossing, could
367
offer affordable microstructured membranes for commercial use.
368 369
4 Conclusion
370
A continuous 2D-AF4 system has been developed using microstructured ultrafiltration
371
membranes with slanted grooves on their surface. Τhe continuous separation was demonstrated
372
with a mixture of large proteins (apoferritin 443 kDa and thyroglobulin 669 kDa), and PS latex
373
nanoparticles (34 and 102 nm). This is the first continuous field-flow fractionation system that
374
can fractionate a feed solution of macromolecules or nanoparticles in aqueous solvents based on
375
their hydrodynamic size. The device could be scaled up for (semi-)preparative applications or
376
scaled down and integrated into lab-on-a-chip devices. Since the second dimension is generated
377
passively from the slanted grooves, the instrumentation required is very simple; the only
378
components that are indispensable are two pumps (one for the main flow and one for the sample
379
flow stream) and a flow controller to regulate the cross-flow.
380
It was shown that higher cross-flow rates may improve the resolution but they also
381
decrease recovery and, therefore, alternative solutions are required to achieve better resolution
382
e.g. by optimizing the groove shape/dimensions or by increasing the channel dimensions. Future
383
research should be focused on the fabrication of larger and low-cost microstructured membranes
384
with grooves that have sharper edges and with lower MWCO (that are able to retain smaller
385
proteins such as antibodies). The membrane fouling observed during the continuous fractionation
386
could be prevented by operating at low cross-flow rates (adjusting channel thickness and the
387
height of the grooves to maintain resolution) and by increasing the breadth of the channel,
388
particularly in the confined region where the sample is introduced. Finally, the fouling might be
389
related to a small amount of very large aggregates that are strongly retained by the grooves.
390
Therefore, sample filtration/centrifugation prior fractionation or application of periodic washing
391
steps during operation could improve the performance. 13
392
The concept of an accumulation wall with slanted grooves that we demonstrate here for
393
the AF4 system is versatile; it could be applied in any other FFF system (thermal, magnetic, etc.)
394
to transform it into a continuous 2D device. Regardless of the applied field, the component which
395
interacts stronger with it (i.e., which is more retained) would exhibit a larger deflection angle
396
resulting to a continuous fractionation. In fact, the continuous system with fabricated slanted
397
grooves for the fractionation of micron-sized microparticles developed by Bernate et al. [32] could
398
be considered as a continuous gravitational 2D-FFF system.
399
Acknowledgements
400
This work was part of the research program SmartSep with project number 11400 which was
401
financed by the Netherlands Organization for Scientific Research (NWO). The authors are grateful
402
to Ü. Bade Kavurt and prof. dr. Dimitrios Stamatialis (University of Twente) for the fabrication of
403
the microstructured membranes and the SEM images. Wyatt Technology Europe is acknowledged
404
for providing technical expertise and Udo van Hes (University of Amsterdam) for his help on the
405
construction of the 2D-AF4 channel.
14
406
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18
515
Table 1. Effect of the microstructured membranes on the retention time of the apoferritin
516
monomer (𝑡𝑅,1) and dimer (𝑡𝑅,2), on their selectivity 𝑎 and on the plate height of the monomer 𝐻. Membrane
𝑡𝑅,1 (min)
𝑡𝑅,2 (min)
𝑎
𝐻 (mm)
Flat membrane
7.0 ± 0.2
9.6 ± 0.4
1.37 ± 0.01
0.35 ± 0.02
MS membrane (parallel grooves)
9.6 ± 0.5
13.3 ± 0.8
1.38 ± 0.00
0.48 ± 0.03
MS membrane (perpendicular grooves)
11.1 ± 0.6
16.9 ± 0.8
1.51 ± 0.01 0.58 ± 0.03
517 518
19
519
Table 2. Migration distance (along the z-axis) and deflection angle that the solutes have when
520
they exit from each outlet Migration distance Deflection angle 𝑧 (cm)
𝜃
Outlet 1
0 - 1.8
> 27°
Outlet 2
1.8 - 3.3
27° - 15°
Outlet 3
3.3 - 4.8
15° - 11°
Outlet 4
4.8 - 6.3
11° - 8°
Outlet 5
6.3 - 10.0
< 8°
521
20
523
Table 3. Cross-flow rate (𝑉𝑐) and total channel outlet flow rate (𝑉𝑜𝑢𝑡) used in batch mode 2D-AF4
524
(Tables 6 and 7) to separate proteins or nanoparticles, and the corresponding frit inlet flow rate (
525
―6 𝑉𝑓), sample flow rate (𝑉𝑠), and flow rate in each outlet (𝑉1𝑜𝑢𝑡 ). All values are given in mL/min.
𝑽𝒄
𝑽𝒐𝒖𝒕
𝑉𝑓
𝑉𝑠
―4 𝑉1𝑜𝑢𝑡
𝑉5,6 𝑜𝑢𝑡
Spatial separation of proteins 0.00
0.80
0.78
0.02
0.07
0.27
1.20
0.80
1.95
0.05
0.07
0.27
2.00
0.80
2.73
0.07
0.07
0.27
Spatial separation of nanoparticles 0.50
0.50
0.97
0.03
0.04
0.17
1.00
1.00
1.95
0.05
0.08
0.33
2.00
2.00
3.90
0.10
0.17
0.66
2.00
1.00
2.92
0.08
0.08
0.33
3.00
1.50
4.39
0.11
0.13
0.50
526
21
527 528
Figure 1. Continuous separation over an accumulation wall with slanted grooves; larger solutes
529
are more retained by the grooves and exhibit a larger deflection angle.
22
530 531
Figure 2. a) Illustration of the spacer A showing the position of the frit element placed above it, of
532
the spacer B showing the separation of the sample components over the MS membrane placed
533
below it, and of the channel cross section. The colored dashed lines indicate the particle
534
trajectories. The channel thickness and the dimensions of the grooves are exaggerated for visual
535
purposes. b) Photograph of the 2D-AF4 channel displaying the sample inlet (𝑉𝑠), the frit inlet (𝑉𝑓),
536
and the outlets No 1 - 6. c) Assembly of the 2D-AF4 channel.
23
537 538
Figure 3. SEM images of the MS membranes: a) Top and b) cross-sectional view.
24
539 540
Figure 4. Overlaid fractograms of apoferritin obtained with (i) flat membranes, (ii) MS
541
membranes with the grooves parallel to the channel flow and (ii) MS membranes with grooves
542
perpendicular to the channel flow. AF4 conditions: cross-flow rate 1.5 mL/min, outlet flow rate
543
0.8 mL/min and injected mass 5 µg.
25
544 545
Figure 5. The system with slanted grooves may be considered two-dimensional: one dimension
546
is along the grooves ( ∥ ) and the other is across the grooves ( ⊥ ); for well-retained solutes the
547
selectivity is different in the two dimensions which results in their spatial separation.
26
548 549
Figure 6. Spatial separation in the 2D-AF4 system of a solution containing apoferritin and
550
thyroglobulin. The fractograms were obtained by analyzing the samples (initial mixture and
551
fractions) with conventional AF4 (with a cross-flow rate of 1.5 mL/min and an outlet flow rate of
552
0.8 mL/min). a) Fractogram of the initial mixture, b) fractograms of the fractions in every outlet
553
of the 2D-AF4 system acquired at different cross-flow rates (0, 1.2, and 2.0 mL/min) and c)
554
recovery of each component in each outlet. The flow rates mentioned in the figure correspond to
555
the cross-flow rates applied during the 2D-AF4 fractionation where the fractions were collected.
27
556 557
Figure 7. Spatial separation in the 2D-AF4 system of a solution containing PS-latex nanoparticles
558
with diameters of 34 and 102 nm. The fractograms were obtained by analyzing the samples (initial
559
mixture and fractions) with conventional AF4 (with a cross-flow rate of 0.5 mL/min and an outlet
560
flow rate of 1.0 mL/min). a) Fractogram of the initial mixture, b) fractograms of the fractions in
561
every outlet acquired under different cross-flow and outlet flow rates and c) recovery of each
562
component in each fraction. The flow rates mentioned in the figure correspond to the flow rates
563
applied during the 2D-AF4 fractionation where the fractions were collected.
28
564 565
Figure 8 Continuous fractionation of a mixture of proteins: Overlaid fractograms of the fractions
566
collected in different time intervals from the outlets 3, 4 and 5 under continuous operation of the
567
2D-AF4 system. The fractograms are normalized to the highest peak for visual comparison.
568 569
29
570
Graphical abstract
571
572
30