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Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of Ionic strength In Suk Woo, Euo Chang Jung, Seungho Lee
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Received date: 27 April 2014 Revised date: 28 May 2014 Accepted date: 29 May 2014 Cite this article as: In Suk Woo, Euo Chang Jung, Seungho Lee, Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of Ionic strength, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.05.061 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of ionic strength In Suk Wooa, Euo Chang Jungb and Seungho Leea* a
b
Department of Chemistry, Hannam University, Daejeon, 305-811, Korea Republic
Nuclear Chemistry Research Center, Korea Atomic Energy Research Institute, Daejeon 305-353, Korea Republic
*
To whom correspondence should be addressed (e-mail: [email protected]; telephone +82-42-
629-8822; fax +82-42-629-8811)
ABSTRACT
Retention behavior of micron-sized particles in gravitational field-flow fractionation (GrFFF) was studied in this study. Effects of ionic strength and flow rate as well as the viscosity of the GrFFF carrier liquid was investigated on the size-based selectivity (Sd), retention ratio (R), and plate height (H) of micron-sized particles using polystyrene latex beads as model particles. It was found that the retention ratio of microparticles increases with increasing flow rate or the viscosity of the carrier liquid as the particles are forced away from the accumulation wall by increased hydrodynamic lift forces (HLF). On the other hand, the retention time increases (retention ratio decreases) with increasing ionic strength of the carrier liquid at the same flow rate, due to decreased repulsive interaction between the particles and the channel accumulation wall (glass in this study) allowing the particles approach closer to the wall.
Results suggest the ionic strength of the carrier liquid plays a
critical role in determining retention of microparticles in GrFFF as well as the viscosity or the flow rate of the carrier liquid. It was found that the resolution and the separation time could
be improved by increasing the carrier viscosity and by carefully adjusting the ionic strength of the carrier liquid.
Keywords: Gravitational field-flow fractionation (GrFFF), Microparticles, Ionic strength, Viscosity, Resolution
1. Introduction
Field-flow fractionation (FFF) is a family of elution-based separation techniques that utilize a thin flow channel, where an external field is applied vertically to the direction of the carrier flow [1-3]. The external field forces the particles move across the channel, impelling them to approach the channel accumulation wall [4]. Particles of different sizes are separated as they migrate down the channel at different rates by the channel flow having a parabolic profile [5-7]. _ENREF_1FFF is capable of providing separation and characterization of macromolecules and particles which are in sizes from a few nanometers to a few tens of microns [8-10] including blood cells [11-13]. Gravitational field-flow fractionation (_ENREF_4_ENREF_4_ENREF_8GrFFF) is a member of FFF family, and employs the Earth’s gravity as the external field. GrFFF is usually applied for separation of micron-sized particles where larger particles are eluted earlier than smaller ones. In GrFFF, particles are eluted by the “lift-hyperlayer” mode [13-21], where the particles are lifted up from the accumulation wall by the HLF, and, as a result, the particles are focused into a narrow zone (‘hyperlayer’) at a distance away from the accumulation wall. The hyperlayer is located at the position where the effective particle weight FG equals HLF. The ‘lift-hyperlayer’ mode is also called ‘focusing’ mode.
Various ways of changing the external field force in GrFFF have been suggested [5], which includes changing of the channel angle [10, 22], the density of the carrier liquid [6, 23], and the carrier viscosity [4, 22, 23]. In our previous report [22], effect of the carrier viscosity and the channel angle on the retention behavior of microparticles in GrFFF was investigated. It has been reported that the retention in FFF may be affected by the particle-wall and particle-particle interactions (electrostatic repulsion and van der Waals attraction) depending upon the carrier composition (mainly the ionic strength) and experimental conditions (e.g., flow rate, field strength, and channel dimension) [24, 25]. This work focuses on the effect of the ionic strength and the flow rate of the carrier liquid. Effect of the ionic strength and flow rate as well as the viscosity of the carrier liquid on various retention (and thus the resolutionrelated) parameters such as the retention ratio (R), plate height (H), distance of particles from the accumulation wall (), size-based selectivity (Sd), and resolution (Rs) was investigated using micron-sized polystyrene (PS) latex beads as model particles. Hydroxypropyl methyl cellulose (HPMC) was added to the carrier liquid to change the viscosity of the carrier liquid without affecting density as in our previous report [22].
2. Theory 2.1. Retention in GrFFF In GrFFF theory, the retention ratio (R) is given by [7] d (1) w , where d is the particle diameter and w is the channel thickness. The is the ‘steric correction factor’ that is known to vary with experimental conditions such as the field strength, carrier flow rate and the particle size [26-28]. As the carrier flow rate increases or the field strength decrease, increases [18, 29]. Experimentally, R can be measured from to (2) R tr R
3J
, where t o is the channel void time and tr is the sample retention time. can thus be determined from Eq. (1) by measuring R of particles of known diameter using Eq. (2). As
suggested by Eq (1) and (2), tr increases as the particle size decreases in GrFFF, providing a size-based separation. The size-based selectivity ( S d ) is the absolute value of the slope of a GrFFF calibration plot ( log tr vs. log d ) and is given by [27, 30, 31]
d log tr d log d
Sd
(3)
2.2. Near-wall lift force ( FNW ) The near-wall lift force FNw is the force exerted on particles in the region near the wall, and is given by [18, 26, 32] a3 (4) FNw 6CK u wG
, where C is an empirical dimensionless coefficient, is the viscosity of the carrier liquid, u is the average linear flow velocity of the carrier liquid, is the particle radius, and G is the closest distance between the particle surface and the channel bottom (accumulation wall). The FNW plays an important role in determining the equilibrium position of a particle in a FFF channel [21]. It can be seen from Eq. (4) that FNw increases with particle size, flow rate, viscosity of carrier liquid, and with decreasing channel thickness [4, 6, 20]. At some point, the lift force in Eq. (4) becomes suƥcient to counter the gravitational force and result in a particle equilibrium position established above the wall (>0). If the inertial contribution to lift force is negligible, then the net force Fnet on a particle in the direction away from the wall is given by [22] 6Ca 3K Q FG wG
Fnet
(5)
, where FG is the force on the particle due to gravity (equal to (4/3)a3G, where is the diơerence between the particle density and uid density and G is the acceleration due to gravity or 9.81 m/s2). The particle equilibrium position is given when the net force becomes zero. Rearranging Eq. (5) with Fnet = 0 gives:
G eq
9 C Q K 2 S w'U G
It is noted that that eq is independent of the particle size.
(6)
Electrostatic repulsion between the negatively charged accumulation wall (glass) and negatively charged particles may also contribute to elevation of the particle zone [33]. Thus the difference between the retention behaviors of the particles in different carrier liquid compositions could be explained by a combination of the hydrodynamic lift forces (HLF) and the electrostatic force [6].
2.3. Plate height (H)
Plate height H is a measure of band broadening, and is usually consisted of several contributing factors including the non-equilibrium effect ( H n ), instrumental effect ( H i ), sample polydispersity ( H p ) and the diffusion of particles ( H D ). The total plate height H is thus given by H
H n H i H p H D [34]. In GrFFF, particles are eluted either by the
steric or the lift-hyperlayer mode, and thus it can be assumed that H n and H i are similar for particles of different sizes. If the particle polydispersity (and thus H p ) remains same for all samples, the band broadening would be mainly caused by H D , the diffusion of particles [34, 35]. Diffusion increases with decreasing particle size, and thus H is expected to increases as the particle size decreases. H can be measured from the well-known relationship, H L , where L is the GrFFF N channel length and N is the number of the theoretical plate that can be obtained from the 2
half peak width ( w1/ 2 ) and the elution volume of the peak (A) by N
· [35]. 5.54 §¨ A ¸ © w1/ 2 ¹
2.4. Resolution
As a measure of resolution ( Rs ), a resolution factor Rs * was defined as [34] Sd (7) Rs* ¦H , where
¦H
is sum of plate height of each particle sample.
3. Experimental 3.1. Materials
Polystyrene (PS) latex beads having nominal diameters of 6, 12, and 20 m were obtained from Fluka AG (Steinheim, Germany), and 40 m beads from Duke Scientific (Palo Alto, CA, USA). All PS beads have density of 1.05 g/mL. Two HPMC products (HPMC H7509 and HPMC H-8384) were obtained from Sigma Aldrich (St. Louis, MO, USA). HPMC7509 provides higher viscosity than HPMC-8384 at the same concentration. All chemicals were used without further purification. FL-70, a mixture of anionic and non-ionic compounds, was purchased from Fisher Scientific (Fair Lawn, NJ, USA), and sodium azide (NaN3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PS latex bead samples were diluted with the GrFFF carrier liquid before being injected into the channel. The GrFFF carrier liquid was de-ionized and distilled water having various concentrations of FL-70, sodium azide, and hydroxyl propyl methyl cellulose (HPMC). HPMC was added to adjust the viscosity of the carrier liquid without affecting density of the liquid. Carrier liquids of seventeen different compositions (denoted ‘A-1’, ‘A2’, ‘A-3’, ‘A-4’, ‘B-1’, etc.) were prepared in this study, and are listed in Table 1. The ionic strength is also shown for each carrier liquid. It can be seen that the ionic strengths of the carrier liquids with HPMC added (Carrier liquid-C’s and D’s) are in a relatively narrow range between 4.28 and 4.32. The viscosity of the carrier liquid was measured with a VIBRO SV10 viscometer (A&D Company, Tokyo, Japan).
3.2. Gravitational field-flow fractionation (GrFFF)
The GrFFF channel was cut in a 200 P m -thick spacer. The dimensions of the channel were 2 and 50 cm in width and length, respectively. Particle suspensions were injected directly into the channel using a 50 μL syringe (Hamilton Co., Reno, NV, USA) through a rubber septum. After the injection, the particles were allowed to settle by stopping the channel flow for 3 minutes [4, 36]. The sample would be eluted on resumption of the flow. The carrier liquid was pumped by a Young-Lin SP930D HPLC pump (Seoul, Korea). The elution of the particles was monitored by Young-Lin M720 UV detector at the operating wavelength of 254 nm.
4. Result and Discussion 4.1. Effect of flow rate
As mentioned earlier, flow rate affects the hydrodynamic lift forces (HLF). GrFFF retention ratios of 40, 20, 12, and 6 P m PS latex beads obtained at various flow rates in carrier liquids of seventeen different compositions are shown in Fig. 1-(a), (b), (c), and (d), respectively. As expected, for all samples, R generally increases with increasing flow rate. And R is always higher for larger particles at the same flow rate, showing GrFFF provides size-based separation of particles. Dotted lines in Fig. (1) represent R values calculated from Eq. (1) with J 1 . It can be seen that, as the flow rate increases, the particles are forced away from the accumulation wall by increased HLF, resulting in a gradual increase in R with the flow rate. It is noted that, for 40 P m particles with the carrier liquid of pure water (Fig. 1-(a)), R does not follow the general trend at the flow rate lower than 2 mL/min (those in circled area), where the R values are much higher than those in other carrier compositions, and decreases as the flow rate increases. At the flow rate higher than 2 mL/min, R follows the general trend (increases with increasing flow rate) again. Those high R’s are probably due to electrostatic repulsion between particles and the glass wall (used as the channel accumulation wall) at low ionic strengths. As the flow rate increases, particles are lifted up by stronger HLF, and effect of the electrostatic interaction becomes insignificant. This phenomenon was not observed for smaller particles, suggesting the electrostatic repulsion is insignificant for smaller particles at the same conditions. The size-based selectivity S d was determined at various channel flow rates using Eq. (3), and the results obtained with carrier liquids containing HPMC (carrier liquid C’s and D’s) are listed in Table 2 and Fig. 2. It can be seen that no general trend in S d with flow rate was observed at the flow rate below about 2 mL/min. At the flow rate above about 2 mL/min, S d tends to increase as the flow rate increases. It is seen that all Sd 's in the carrier liquids with HPMC-7509 (Carrier C’s) are lower than those in the carrier liquids with HPMC-8384 (Carrier D’s). However no general trends in S d with the carrier viscosity nor the ionic strength were observed, suggesting that S d may be determined by a combination of some
other factors as well as the carrier viscosity and ionic strength. It is interesting that the highest S d was obtained with the carrier liquid ‘B-2’ (water with 0.1% FL-70 and 0.02% NaN3), which is the carrier composition generally used in FFF. As shown in Eq. (3), only the retention time difference ( d log tr ) is taken into account in S d calculation. If the degree of band broadening remains constant, higher S d will result in higher resolution ( Rs ). Usually the band broadening is measured by the plate height H. Fig. 3 shows plate heights measured at various flow rates for 40, 20, 12 and 6 P m PS beads, respectively, in various carrier compositions. It can be seen that, for the same size particles, H tends to slowly increase as the flow rate increases, which agrees with the results reported previously [12, 37].
4.2. Effect of ionic strength.
Table 3 shows retention time tr of PS latex beads measured at the flow rate of 1 mL/min with various carrier liquid compositions. tr ' s in Table 3 are averages of two measurements. As shown in Table 3, at all conditions, tr decreases with increasing diameter as expected (size-based separation). The data shown in Table 3 are plotted in Fig 4. It can be seen in Fig. 4 that, for each PS bead sample, tr increases with increasing ionic strength. As mentioned earlier, the retention behaviors of particles in various carrier liquid compositions could be explained by a combination of the lift force and the electrostatic force [6]. At a constant flow rate, the lift forces will be the same for particles for particles of the same size, and the difference in retention time shown in Fig. 4 can only be explained by the difference in the electrostatic interaction. Effects of ionic strength in FFF have been extensively studied in previous reports [21, 25, 38]. Usually the repulsive interaction between particles and between particles and the channel wall is stronger in pure water than in a solution, and decreases as the ionic strength increases [38] allowing particles approach closer to the channel bottom, which results in increasing retention with increasing ionic strength as shown in Fig. 4. The retention time tends to level off after the ionic strength reaches about 4 mM, and the retention times obtained with the carrier liquids of B-2, 3, and 4 (last three data points for each particle size) are similar.
This suggests water with 0.1% FL-70 and 0.02% NaN3 (Carrier B-2) is
appropriate as the carrier liquid for GrFFF separation of PS latex beads (or may be for many other types of particles). The in Eq. (4) is the closest distance between the particle surface and the channel bottom
(accumulation wall), and is zero when the particle is on the channel bottom, and will increases as the particle is lifted away from the channel bottom. The eq can be determined experimentally for each particle from its retention ratio R. The effect of the ionic strength on eq is expected to be the same as that on R (shown in Fig. 4) as R is proportional to eq. Fig. 5 (a) and (b) are plots of eq vs. ionic strength obtained at various flow rates for 20 P m PS latex beads and for PS beads of various sizes at a fixed flow rate of 2 mL/min, respectively. In Fig. 5(a), at the same flow rate, eq decreases as the ionic strength increases due to reduced repulsive interaction between particles and the channel bottom, and then levels off. It can be seen that, unlike at higher flow rates, the changes in eq with ionic strength at lower flow rates (0.5 and 1 mL/min) are insignificant, and eq stays close to 0, suggesting particles stay close to the channel bottom due to relatively lower (or insignificant) lift force. The lift force increases with increasing flow rate, and it seems higher lift force multiply the influence of the ionic strength on eq. It can also be seen in Fig. 5(a) that, at the same ionic strength, eq increases as the flow rate increases due to increased lift forces. In Fig. 5(b), eq is plotted against particle diameter, and no general trend in eq with particle diameter was observed as expected from Eq. (6). Fig. 6 shows effect of ionic strength on plate height, H, for PS latex beads of various sizes. No general trend in H with ionic strength was observed. It seems there is no correlation between H and the ionic strength of the carrier liquid in the tested range.
4.3. Effect of viscosity
In accordance with Eq. (4), lift force increases with increasing viscosity of the carrier liquid, and thus the retention ratio is also expected to increases. Fig. 7 shows the effect of the carrier viscosity on retention ratio of PS latex beads at various flow rates. It can be seen that, at all flow rates, the retention ratio generally increases with increasing carrier viscosity, as expected. At the carrier viscosity near 1, retention ratios are high due to repulsive interaction between particles and the channel bottom as mentioned earlier. Fig. 8 shows eq determined for 20 m PS latex beads from the data in Fig. 7. As expected, eq tends to increases with the carrier viscosity at relatively higher range of the carrier viscosity. Fig. 9 shows again that there is no general trend in H with the carrier viscosity. Fig. 10(a) and (b) show effects of the carrier viscosity on selectivity (Sd) and resolution (Rs*), respectively. As shown in Fig 10(a), the selectivity increases with increasing viscosity
of the carrier liquid in the flow rate range of 2-4 mL/min. At flow rates lower than 2 mL/min, the trend was not clear (data are not shown).
In Fig. 10(b), the resolution
(calculated from eq. (7)) also increases with increasing carrier viscosity at relatively higher range of the carrier viscosity. From Figs. 9 and 10, it can be concluded that improved resolution with increasing viscosity observed in this study is caused mainly by the improvement in selectivity with the carrier viscosity (not by the plate height).
5. Conclusion
Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF) was studied in this study. It seems the ionic strength of the carrier liquid plays a critical role in determining the size-based selectivity, retention ratio, and plate height, thus the resolution of microparticles in GrFFF as well as the viscosity or the flow rate of the carrier liquid. The repulsive interaction between particles and between particles and the channel wall decreases as the ionic strength increases, allowing particles approach closer to the channel bottom, which results in increasing retention with increasing ionic strength.
Results suggest that the
resolution and the separation time could be improved in GrFFF by increasing the viscosity and by carefully adjusting the ionic strength of the carrier liquid.
Acknowledgment
The authors acknowledge the support provided by the National Research Foundation (NRF) of Korea.
References
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Figure captions.
Fig. 1. Effect of flow rate on R of 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions. Dotted lines are for cases of = 1. Fig. 2. Effect of flow rate on Sd in GrFFF with various carrier liquid compositions. Fig. 3. Effect of flow rate on H for 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions. Fig. 4. Effect of ionic strength of carrier liquid on GrFFF retention of 6, 12, 20, and 40 P m latex beads in various carrier liquid compositions. Flow rate was fixed at 1 mL/min. Fig. 5. Effect of ionic strength on G eq in GrFFF observed at various flow rates for 20 P m PS latex beads (a) and for PS beads of various sizes at 2 mL/min (b). Fig. 6. Effect of ionic strength on plate height for PS latex beads in GrFFF at flow rate of 1 mL/min. Fig. 7. Effect of viscosity of carrier liquid on retention ratio (R) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively. Fig. 8. Effect of viscosity on eq of 20 m PS latex beads in GrFFF at various flow rates. Fig. 9. Effect of viscosity of carrier liquid on plate height (H) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively. Fig. 10. Effect of viscosity on selectivity (Sd) and resolution (Rs*) of PS latex beads in GrFFF observed at various flow rates.
Table 1
GrFFF carrier liquids used in this study. Carrier liquid
Composition
Viscosity a (cP)
Ionic strength I (mM)
W
Water
1.000
0.0001
A-1
0.05% FL-70
1.023
0.600
A-2
0.1% FL-70
1.033
1.200
A-3
0.2% FL-70
1.027
2.400
A-4
0.3% FL-70
1.033
3.600
B-1
0.1% FL-70+0.01% NaN3
1.047
2.738
B-2
0.1% FL-70+0.02% NaN3
1.057
4.277
B-3
0.1% FL-70+0.03% NaN3
1.070
5.815
B-4
0.1% FL-70+0.04% NaN3
1.077
7.353
1.207
4.282
1.417
4.286
1.700
4.288
1.897
4.289
1.160
4.289
1.273
4.303
1.300
4.312
1.343
4.321
C-1 C-2 C-3 C-4 D-1 D-2 D-3 D-4 a
0.1% FL-70+0.02% NaN3 +0.02% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.05% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.07% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.09% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.02% HPMC-8384 0.1% FL-70+0.02% NaN3 +0.05% HPMC-8384 0.1% FL-70+0.02% NaN3 +0.07% HPMC-8384 0.1% FL-70+0.02% NaN3 +0.09% HPMC-8384
Viscosities of carrier liquids are averages of three measurements.
Table 2
Effects of channel flow rate on size-based selectivity, S d , in GrFFF. Carrier liquid
S d at flow rate (mL/min) of 0.5
1.0
2.0
3.0
4.0
C-1
0.703
0.678
0.629
0.655
0.771
C-2
0.685
0.568
0.505
0.553
0.615
C-3
0.660
0.630
0.670
0.764
0.824
C-4
0.645
0.632
0.685
0.773
0.863
D-1
0.689
0.695
0.739
0.869
0.960
D-2
0.697
0.704
0.772
0.936
1.021
D-3
0.697
0.707
0.758
0.858
0.982
D-4
0.698
0.718
0.682
0.811
0.926
Table 3
Effects of ionic strength on retention time of PS latex beads in GrFFF.
Carrier liquid
6
12
20
40
W
16.00
10.28
7.32
2.83
A-1
21.60
10.90
7.26
4.53
A-2
20.80
11.03
7.36
4.72
A-3
23.18
11.96
8.05
5.08
A-4
25.33
13.33
9.23
5.90
B-1
25.68
13.31
9.00
5.60
B-2
26.50
15.33
10.20
6.06
B-3
25.10
14.12
9.85
5.25
B-4
25.28
14.75
10.10
6.20
Retention time tr is average of two measurements made at flow rate of 1 mL/min.
Retention ratio, R
(a)
(b)
1.2
1.0
0.8
W
C-1 C-2 C-3 C-4 D-1 D-2 D-3 D-4
A-1 A-2 A-3 A-4 B-1 B-2 B-3 B-4
0.9 0.8
0.6
J 1
0.4
Retention ratio, R
a
tr a (min) measured for PS beads having nominal diameter ( P m ) of
0.7 0.6 0.5 0.4 0.3 0.2 0.1
0.2 0
1
2
3
Flow rate (mL/min)
4
0
1
2
3
Flow rate (mL/min)
4
(c)
(d)
0.50
0.27
Retention ratio, R
0.45
Retention ratio, R
0.30
0.40 0.35 0.30 0.25 0.20 0.15 0.10
0.24 0.21 0.18 0.15 0.12 0.09 0.06
0
1
2
3
0
4
1
2
3
4
Flow rate (mL/min)
Flow rate (mL/min)
Fig. 1. Effect of flow rate on R of 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions. Dotted lines are for cases of = 1.
1.1 D-2 D-3 D-1 D-4
Selectivity, Sd
1.0 0.9
C-4 C-3
0.8
C-1
0.7 C-2
0.6 0.5 0.4 0
1
2
3
Flow rate (mL/min) Fig. 2. Effect of flow rate on Sd in GrFFF with various carrier liquid compositions.
4
(b)
1.0
C-1 C-2 C-3 C-4 D-1 D-2 D-3 D-4
A-1 A-2 A-3 A-4 B-1 B-2 B-3 B-4
0.8
0.6
Plate height, H (cm)
Plate height, H (cm)
(a)
0.4
0.2
0.0
0.5
0.4
0.3
0.2
0.1
0.0
0
1
2
3
0
4
1
Flow rate (mL/min) (d)
1.2
Plate height, H (cm)
Plate height, H (cm)
(c)
2
3
4
Flow rate (mL/min)
1.0
0.8
0.6
0.4
0.2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0
1
2
3
Flow rate (mL/min)
4
0
1
2
3
4
Flow rate (mL/min)
Fig. 3. Effect of flow rate on H for 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions.
Retention time tr (min)
30 6 Pm
25
20
15
12 Pm
10
20 Pm 40 Pm
5
0 0
1
2
3
4
5
6
7
8
9
Ionic strength, ,(mM) Fig. 4. Effect of ionic strength of carrier liquid on GrFFF retention of 6, 12, 20, and 40 P m latex beads in various carrier liquid compositions. Flow rate was fixed at 1 mL/min.
(a)
14 1.0 mL/min 2.0 3.0 4.0
12
Geq (Pm)
10 8 6 4 2 0 0
1
2
3
4
5
6
Ionic strength (mM)
7
8
(b)
4.5 A-1
4.0 3.5
W A-2
Geq(Pm)
3.0 2.5
A-3 B-2 B-1
2.0 1.5
A-4 B-4 B-3
1.0 0.5 0.0 6
12
20
40
Diameter (Pm) Fig. 5. Effect of ionic strength on G eq in GrFFF observed at various flow rates for 20 P m PS latex beads (a) and for PS beads of various sizes at 2 mL/min (b).
Plate height, H (cm)
0.4 40 mm 20 12 6
0.3
0.2
0.1
0.0 0
1
2
3
4
5
6
7
8
Ionic strength, , (mM) Fig. 6. Effect of ionic strength on plate height for PS latex beads in GrFFF at flow rate of 1 mL/min.
(a) 0.8
(b)
Retention ratio, R
0.7 0.6 0.5 0.4 0.3 0.2
0.7 0.6
Retention ratio, R
40 Pm 20 12 6
0.5 0.4 0.3 0.2 0.1
0.1 0.0
0.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
(d)
0.7
0.8
0.6
Retention ratio, R
Retention ratio, R
(c)
0.5 0.4 0.3 0.2
0.6
0.4
0.2
0.1 0.0
0.0
1.0
1.2
1.4
1.6
1.8
2.0
1.0
1.2
Viscosity (cP)
1.4
1.6
1.8
2.0
Viscosity (cP)
Retention ratio, R
(e) 1.0
0.8
0.6
0.4
0.2
0.0 1.0
1.2
1.4
1.6
1.8
2.0
Viscosity (cP)
Fig. 7. Effect of viscosity of carrier liquid on retention ratio (R) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively.
18 0.5 mL/min 1.0 2.0 3.0 4.0
16 14
Geq (Pm)
12 10 8 6 4 2 0 1.0
1.2
1.4
1.6
1.8
2.0
Viscosity (cP) Fig. 8. Effect of viscosity on eq of 20 m PS latex beads in GrFFF at various flow rates.
(b)
0.25 40 Pm 20 12 6
0.20
Plate height, H (cm)
Plate height, H (cm)
(a)
0.15
0.10
0.05
0.00
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
(c)
(d) Plate height, H (cm)
Plate height, H (cm)
0.7 0.6 0.5 0.4 0.3 0.2 0.1
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0 1.0
1.2
1.4
1.6
1.8
2.0
1.0
1.2
Plate height, H (cm)
(e)
1.4
1.6
1.8
2.0
Viscosity (cP)
Viscosity (cP) 1.0
0.8
0.6
0.4
0.2
0.0 1.0
1.2
1.4
1.6
1.8
2.0
Viscosity (cP)
Fig. 9. Effect of viscosity of carrier liquid on plate height (H) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively.
(a) 1.0 2.0 mL/min 3.0 4.0
Selectivity, Sd
0.9
0.8
0.7
0.6
0.5 1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
1.8
1.9
2.0
Viscosity (cP) (b)
Resolution, Rs*
3.0 2.0 mL/min 3.0 4.0
2.5
2.0
1.5
1.0
0.5 1.3
1.4
1.5
1.6
1.7
Viscosity (cP) Fig. 10. Effect of viscosity on selectivity (Sd) and resolution (Rs*) of PS latex beads in GrFFF observed at various flow rates.
Highlights { Effects of ionic strength on Sd, R, and H in GrFFF was investigated. { Ionic strength plays a critical role in determining retention of microparticles. { Retention increases with increasing ionic strength due to electrostatic interaction { Resolution can be improved by adjusting ionic strength of the carrier liquid.
Geq (Pm) 0
2
4
6
8
10
12
14
1
3
4
5
6
Ionic strength (mM)
2
7
eq decreases with increasing ionic strength.
0
1.0 mL/min 2.0 3.0 4.0
Effect of ionic strength on eq in GrFFF
8
Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of Ionic strength In Suk Woo, Euo Chang Jung, Seungho Lee
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PII: DOI: Reference:
S0039-9140(14)00457-3 http://dx.doi.org/10.1016/j.talanta.2014.05.061 TAL14822
To appear in:
Talanta
Received date: 27 April 2014 Revised date: 28 May 2014 Accepted date: 29 May 2014 Cite this article as: In Suk Woo, Euo Chang Jung, Seungho Lee, Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of Ionic strength, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.05.061 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of ionic strength In Suk Wooa, Euo Chang Jungb and Seungho Leea* a
b
Department of Chemistry, Hannam University, Daejeon, 305-811, Korea Republic
Nuclear Chemistry Research Center, Korea Atomic Energy Research Institute, Daejeon 305-353, Korea Republic
*
To whom correspondence should be addressed (e-mail: [email protected]; telephone +82-42-
629-8822; fax +82-42-629-8811)
ABSTRACT
Retention behavior of micron-sized particles in gravitational field-flow fractionation (GrFFF) was studied in this study. Effects of ionic strength and flow rate as well as the viscosity of the GrFFF carrier liquid was investigated on the size-based selectivity (Sd), retention ratio (R), and plate height (H) of micron-sized particles using polystyrene latex beads as model particles. It was found that the retention ratio of microparticles increases with increasing flow rate or the viscosity of the carrier liquid as the particles are forced away from the accumulation wall by increased hydrodynamic lift forces (HLF). On the other hand, the retention time increases (retention ratio decreases) with increasing ionic strength of the carrier liquid at the same flow rate, due to decreased repulsive interaction between the particles and the channel accumulation wall (glass in this study) allowing the particles approach closer to the wall.
Results suggest the ionic strength of the carrier liquid plays a
critical role in determining retention of microparticles in GrFFF as well as the viscosity or the flow rate of the carrier liquid. It was found that the resolution and the separation time could
be improved by increasing the carrier viscosity and by carefully adjusting the ionic strength of the carrier liquid.
Keywords: Gravitational field-flow fractionation (GrFFF), Microparticles, Ionic strength, Viscosity, Resolution
1. Introduction
Field-flow fractionation (FFF) is a family of elution-based separation techniques that utilize a thin flow channel, where an external field is applied vertically to the direction of the carrier flow [1-3]. The external field forces the particles move across the channel, impelling them to approach the channel accumulation wall [4]. Particles of different sizes are separated as they migrate down the channel at different rates by the channel flow having a parabolic profile [5-7]. _ENREF_1FFF is capable of providing separation and characterization of macromolecules and particles which are in sizes from a few nanometers to a few tens of microns [8-10] including blood cells [11-13]. Gravitational field-flow fractionation (_ENREF_4_ENREF_4_ENREF_8GrFFF) is a member of FFF family, and employs the Earth’s gravity as the external field. GrFFF is usually applied for separation of micron-sized particles where larger particles are eluted earlier than smaller ones. In GrFFF, particles are eluted by the “lift-hyperlayer” mode [13-21], where the particles are lifted up from the accumulation wall by the HLF, and, as a result, the particles are focused into a narrow zone (‘hyperlayer’) at a distance away from the accumulation wall. The hyperlayer is located at the position where the effective particle weight FG equals HLF. The ‘lift-hyperlayer’ mode is also called ‘focusing’ mode.
Various ways of changing the external field force in GrFFF have been suggested [5], which includes changing of the channel angle [10, 22], the density of the carrier liquid [6, 23], and the carrier viscosity [4, 22, 23]. In our previous report [22], effect of the carrier viscosity and the channel angle on the retention behavior of microparticles in GrFFF was investigated. It has been reported that the retention in FFF may be affected by the particle-wall and particle-particle interactions (electrostatic repulsion and van der Waals attraction) depending upon the carrier composition (mainly the ionic strength) and experimental conditions (e.g., flow rate, field strength, and channel dimension) [24, 25]. This work focuses on the effect of the ionic strength and the flow rate of the carrier liquid. Effect of the ionic strength and flow rate as well as the viscosity of the carrier liquid on various retention (and thus the resolutionrelated) parameters such as the retention ratio (R), plate height (H), distance of particles from the accumulation wall (), size-based selectivity (Sd), and resolution (Rs) was investigated using micron-sized polystyrene (PS) latex beads as model particles. Hydroxypropyl methyl cellulose (HPMC) was added to the carrier liquid to change the viscosity of the carrier liquid without affecting density as in our previous report [22].
2. Theory 2.1. Retention in GrFFF In GrFFF theory, the retention ratio (R) is given by [7] d (1) w , where d is the particle diameter and w is the channel thickness. The is the ‘steric correction factor’ that is known to vary with experimental conditions such as the field strength, carrier flow rate and the particle size [26-28]. As the carrier flow rate increases or the field strength decrease, increases [18, 29]. Experimentally, R can be measured from to (2) R tr R
3J
, where t o is the channel void time and tr is the sample retention time. can thus be determined from Eq. (1) by measuring R of particles of known diameter using Eq. (2). As
suggested by Eq (1) and (2), tr increases as the particle size decreases in GrFFF, providing a size-based separation. The size-based selectivity ( S d ) is the absolute value of the slope of a GrFFF calibration plot ( log tr vs. log d ) and is given by [27, 30, 31]
d log tr d log d
Sd
(3)
2.2. Near-wall lift force ( FNW ) The near-wall lift force FNw is the force exerted on particles in the region near the wall, and is given by [18, 26, 32] a3 (4) FNw 6CK u wG
, where C is an empirical dimensionless coefficient, is the viscosity of the carrier liquid, u is the average linear flow velocity of the carrier liquid, is the particle radius, and G is the closest distance between the particle surface and the channel bottom (accumulation wall). The FNW plays an important role in determining the equilibrium position of a particle in a FFF channel [21]. It can be seen from Eq. (4) that FNw increases with particle size, flow rate, viscosity of carrier liquid, and with decreasing channel thickness [4, 6, 20]. At some point, the lift force in Eq. (4) becomes suƥcient to counter the gravitational force and result in a particle equilibrium position established above the wall (>0). If the inertial contribution to lift force is negligible, then the net force Fnet on a particle in the direction away from the wall is given by [22] 6Ca 3K Q FG wG
Fnet
(5)
, where FG is the force on the particle due to gravity (equal to (4/3)a3G, where is the diơerence between the particle density and uid density and G is the acceleration due to gravity or 9.81 m/s2). The particle equilibrium position is given when the net force becomes zero. Rearranging Eq. (5) with Fnet = 0 gives:
G eq
9 C Q K 2 S w'U G
It is noted that that eq is independent of the particle size.
(6)
Electrostatic repulsion between the negatively charged accumulation wall (glass) and negatively charged particles may also contribute to elevation of the particle zone [33]. Thus the difference between the retention behaviors of the particles in different carrier liquid compositions could be explained by a combination of the hydrodynamic lift forces (HLF) and the electrostatic force [6].
2.3. Plate height (H)
Plate height H is a measure of band broadening, and is usually consisted of several contributing factors including the non-equilibrium effect ( H n ), instrumental effect ( H i ), sample polydispersity ( H p ) and the diffusion of particles ( H D ). The total plate height H is thus given by H
H n H i H p H D [34]. In GrFFF, particles are eluted either by the
steric or the lift-hyperlayer mode, and thus it can be assumed that H n and H i are similar for particles of different sizes. If the particle polydispersity (and thus H p ) remains same for all samples, the band broadening would be mainly caused by H D , the diffusion of particles [34, 35]. Diffusion increases with decreasing particle size, and thus H is expected to increases as the particle size decreases. H can be measured from the well-known relationship, H L , where L is the GrFFF N channel length and N is the number of the theoretical plate that can be obtained from the 2
half peak width ( w1/ 2 ) and the elution volume of the peak (A) by N
· [35]. 5.54 §¨ A ¸ © w1/ 2 ¹
2.4. Resolution
As a measure of resolution ( Rs ), a resolution factor Rs * was defined as [34] Sd (7) Rs* ¦H , where
¦H
is sum of plate height of each particle sample.
3. Experimental 3.1. Materials
Polystyrene (PS) latex beads having nominal diameters of 6, 12, and 20 m were obtained from Fluka AG (Steinheim, Germany), and 40 m beads from Duke Scientific (Palo Alto, CA, USA). All PS beads have density of 1.05 g/mL. Two HPMC products (HPMC H7509 and HPMC H-8384) were obtained from Sigma Aldrich (St. Louis, MO, USA). HPMC7509 provides higher viscosity than HPMC-8384 at the same concentration. All chemicals were used without further purification. FL-70, a mixture of anionic and non-ionic compounds, was purchased from Fisher Scientific (Fair Lawn, NJ, USA), and sodium azide (NaN3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PS latex bead samples were diluted with the GrFFF carrier liquid before being injected into the channel. The GrFFF carrier liquid was de-ionized and distilled water having various concentrations of FL-70, sodium azide, and hydroxyl propyl methyl cellulose (HPMC). HPMC was added to adjust the viscosity of the carrier liquid without affecting density of the liquid. Carrier liquids of seventeen different compositions (denoted ‘A-1’, ‘A2’, ‘A-3’, ‘A-4’, ‘B-1’, etc.) were prepared in this study, and are listed in Table 1. The ionic strength is also shown for each carrier liquid. It can be seen that the ionic strengths of the carrier liquids with HPMC added (Carrier liquid-C’s and D’s) are in a relatively narrow range between 4.28 and 4.32. The viscosity of the carrier liquid was measured with a VIBRO SV10 viscometer (A&D Company, Tokyo, Japan).
3.2. Gravitational field-flow fractionation (GrFFF)
The GrFFF channel was cut in a 200 P m -thick spacer. The dimensions of the channel were 2 and 50 cm in width and length, respectively. Particle suspensions were injected directly into the channel using a 50 μL syringe (Hamilton Co., Reno, NV, USA) through a rubber septum. After the injection, the particles were allowed to settle by stopping the channel flow for 3 minutes [4, 36]. The sample would be eluted on resumption of the flow. The carrier liquid was pumped by a Young-Lin SP930D HPLC pump (Seoul, Korea). The elution of the particles was monitored by Young-Lin M720 UV detector at the operating wavelength of 254 nm.
4. Result and Discussion 4.1. Effect of flow rate
As mentioned earlier, flow rate affects the hydrodynamic lift forces (HLF). GrFFF retention ratios of 40, 20, 12, and 6 P m PS latex beads obtained at various flow rates in carrier liquids of seventeen different compositions are shown in Fig. 1-(a), (b), (c), and (d), respectively. As expected, for all samples, R generally increases with increasing flow rate. And R is always higher for larger particles at the same flow rate, showing GrFFF provides size-based separation of particles. Dotted lines in Fig. (1) represent R values calculated from Eq. (1) with J 1 . It can be seen that, as the flow rate increases, the particles are forced away from the accumulation wall by increased HLF, resulting in a gradual increase in R with the flow rate. It is noted that, for 40 P m particles with the carrier liquid of pure water (Fig. 1-(a)), R does not follow the general trend at the flow rate lower than 2 mL/min (those in circled area), where the R values are much higher than those in other carrier compositions, and decreases as the flow rate increases. At the flow rate higher than 2 mL/min, R follows the general trend (increases with increasing flow rate) again. Those high R’s are probably due to electrostatic repulsion between particles and the glass wall (used as the channel accumulation wall) at low ionic strengths. As the flow rate increases, particles are lifted up by stronger HLF, and effect of the electrostatic interaction becomes insignificant. This phenomenon was not observed for smaller particles, suggesting the electrostatic repulsion is insignificant for smaller particles at the same conditions. The size-based selectivity S d was determined at various channel flow rates using Eq. (3), and the results obtained with carrier liquids containing HPMC (carrier liquid C’s and D’s) are listed in Table 2 and Fig. 2. It can be seen that no general trend in S d with flow rate was observed at the flow rate below about 2 mL/min. At the flow rate above about 2 mL/min, S d tends to increase as the flow rate increases. It is seen that all Sd 's in the carrier liquids with HPMC-7509 (Carrier C’s) are lower than those in the carrier liquids with HPMC-8384 (Carrier D’s). However no general trends in S d with the carrier viscosity nor the ionic strength were observed, suggesting that S d may be determined by a combination of some
other factors as well as the carrier viscosity and ionic strength. It is interesting that the highest S d was obtained with the carrier liquid ‘B-2’ (water with 0.1% FL-70 and 0.02% NaN3), which is the carrier composition generally used in FFF. As shown in Eq. (3), only the retention time difference ( d log tr ) is taken into account in S d calculation. If the degree of band broadening remains constant, higher S d will result in higher resolution ( Rs ). Usually the band broadening is measured by the plate height H. Fig. 3 shows plate heights measured at various flow rates for 40, 20, 12 and 6 P m PS beads, respectively, in various carrier compositions. It can be seen that, for the same size particles, H tends to slowly increase as the flow rate increases, which agrees with the results reported previously [12, 37].
4.2. Effect of ionic strength.
Table 3 shows retention time tr of PS latex beads measured at the flow rate of 1 mL/min with various carrier liquid compositions. tr ' s in Table 3 are averages of two measurements. As shown in Table 3, at all conditions, tr decreases with increasing diameter as expected (size-based separation). The data shown in Table 3 are plotted in Fig 4. It can be seen in Fig. 4 that, for each PS bead sample, tr increases with increasing ionic strength. As mentioned earlier, the retention behaviors of particles in various carrier liquid compositions could be explained by a combination of the lift force and the electrostatic force [6]. At a constant flow rate, the lift forces will be the same for particles for particles of the same size, and the difference in retention time shown in Fig. 4 can only be explained by the difference in the electrostatic interaction. Effects of ionic strength in FFF have been extensively studied in previous reports [21, 25, 38]. Usually the repulsive interaction between particles and between particles and the channel wall is stronger in pure water than in a solution, and decreases as the ionic strength increases [38] allowing particles approach closer to the channel bottom, which results in increasing retention with increasing ionic strength as shown in Fig. 4. The retention time tends to level off after the ionic strength reaches about 4 mM, and the retention times obtained with the carrier liquids of B-2, 3, and 4 (last three data points for each particle size) are similar.
This suggests water with 0.1% FL-70 and 0.02% NaN3 (Carrier B-2) is
appropriate as the carrier liquid for GrFFF separation of PS latex beads (or may be for many other types of particles). The in Eq. (4) is the closest distance between the particle surface and the channel bottom
(accumulation wall), and is zero when the particle is on the channel bottom, and will increases as the particle is lifted away from the channel bottom. The eq can be determined experimentally for each particle from its retention ratio R. The effect of the ionic strength on eq is expected to be the same as that on R (shown in Fig. 4) as R is proportional to eq. Fig. 5 (a) and (b) are plots of eq vs. ionic strength obtained at various flow rates for 20 P m PS latex beads and for PS beads of various sizes at a fixed flow rate of 2 mL/min, respectively. In Fig. 5(a), at the same flow rate, eq decreases as the ionic strength increases due to reduced repulsive interaction between particles and the channel bottom, and then levels off. It can be seen that, unlike at higher flow rates, the changes in eq with ionic strength at lower flow rates (0.5 and 1 mL/min) are insignificant, and eq stays close to 0, suggesting particles stay close to the channel bottom due to relatively lower (or insignificant) lift force. The lift force increases with increasing flow rate, and it seems higher lift force multiply the influence of the ionic strength on eq. It can also be seen in Fig. 5(a) that, at the same ionic strength, eq increases as the flow rate increases due to increased lift forces. In Fig. 5(b), eq is plotted against particle diameter, and no general trend in eq with particle diameter was observed as expected from Eq. (6). Fig. 6 shows effect of ionic strength on plate height, H, for PS latex beads of various sizes. No general trend in H with ionic strength was observed. It seems there is no correlation between H and the ionic strength of the carrier liquid in the tested range.
4.3. Effect of viscosity
In accordance with Eq. (4), lift force increases with increasing viscosity of the carrier liquid, and thus the retention ratio is also expected to increases. Fig. 7 shows the effect of the carrier viscosity on retention ratio of PS latex beads at various flow rates. It can be seen that, at all flow rates, the retention ratio generally increases with increasing carrier viscosity, as expected. At the carrier viscosity near 1, retention ratios are high due to repulsive interaction between particles and the channel bottom as mentioned earlier. Fig. 8 shows eq determined for 20 m PS latex beads from the data in Fig. 7. As expected, eq tends to increases with the carrier viscosity at relatively higher range of the carrier viscosity. Fig. 9 shows again that there is no general trend in H with the carrier viscosity. Fig. 10(a) and (b) show effects of the carrier viscosity on selectivity (Sd) and resolution (Rs*), respectively. As shown in Fig 10(a), the selectivity increases with increasing viscosity
of the carrier liquid in the flow rate range of 2-4 mL/min. At flow rates lower than 2 mL/min, the trend was not clear (data are not shown).
In Fig. 10(b), the resolution
(calculated from eq. (7)) also increases with increasing carrier viscosity at relatively higher range of the carrier viscosity. From Figs. 9 and 10, it can be concluded that improved resolution with increasing viscosity observed in this study is caused mainly by the improvement in selectivity with the carrier viscosity (not by the plate height).
5. Conclusion
Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF) was studied in this study. It seems the ionic strength of the carrier liquid plays a critical role in determining the size-based selectivity, retention ratio, and plate height, thus the resolution of microparticles in GrFFF as well as the viscosity or the flow rate of the carrier liquid. The repulsive interaction between particles and between particles and the channel wall decreases as the ionic strength increases, allowing particles approach closer to the channel bottom, which results in increasing retention with increasing ionic strength.
Results suggest that the
resolution and the separation time could be improved in GrFFF by increasing the viscosity and by carefully adjusting the ionic strength of the carrier liquid.
Acknowledgment
The authors acknowledge the support provided by the National Research Foundation (NRF) of Korea.
References
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Figure captions.
Fig. 1. Effect of flow rate on R of 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions. Dotted lines are for cases of = 1. Fig. 2. Effect of flow rate on Sd in GrFFF with various carrier liquid compositions. Fig. 3. Effect of flow rate on H for 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions. Fig. 4. Effect of ionic strength of carrier liquid on GrFFF retention of 6, 12, 20, and 40 P m latex beads in various carrier liquid compositions. Flow rate was fixed at 1 mL/min. Fig. 5. Effect of ionic strength on G eq in GrFFF observed at various flow rates for 20 P m PS latex beads (a) and for PS beads of various sizes at 2 mL/min (b). Fig. 6. Effect of ionic strength on plate height for PS latex beads in GrFFF at flow rate of 1 mL/min. Fig. 7. Effect of viscosity of carrier liquid on retention ratio (R) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively. Fig. 8. Effect of viscosity on eq of 20 m PS latex beads in GrFFF at various flow rates. Fig. 9. Effect of viscosity of carrier liquid on plate height (H) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively. Fig. 10. Effect of viscosity on selectivity (Sd) and resolution (Rs*) of PS latex beads in GrFFF observed at various flow rates.
Table 1
GrFFF carrier liquids used in this study. Carrier liquid
Composition
Viscosity a (cP)
Ionic strength I (mM)
W
Water
1.000
0.0001
A-1
0.05% FL-70
1.023
0.600
A-2
0.1% FL-70
1.033
1.200
A-3
0.2% FL-70
1.027
2.400
A-4
0.3% FL-70
1.033
3.600
B-1
0.1% FL-70+0.01% NaN3
1.047
2.738
B-2
0.1% FL-70+0.02% NaN3
1.057
4.277
B-3
0.1% FL-70+0.03% NaN3
1.070
5.815
B-4
0.1% FL-70+0.04% NaN3
1.077
7.353
1.207
4.282
1.417
4.286
1.700
4.288
1.897
4.289
1.160
4.289
1.273
4.303
1.300
4.312
1.343
4.321
C-1 C-2 C-3 C-4 D-1 D-2 D-3 D-4 a
0.1% FL-70+0.02% NaN3 +0.02% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.05% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.07% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.09% HPMC-7509 0.1% FL-70+0.02% NaN3 +0.02% HPMC-8384 0.1% FL-70+0.02% NaN3 +0.05% HPMC-8384 0.1% FL-70+0.02% NaN3 +0.07% HPMC-8384 0.1% FL-70+0.02% NaN3 +0.09% HPMC-8384
Viscosities of carrier liquids are averages of three measurements.
Table 2
Effects of channel flow rate on size-based selectivity, S d , in GrFFF. Carrier liquid
S d at flow rate (mL/min) of 0.5
1.0
2.0
3.0
4.0
C-1
0.703
0.678
0.629
0.655
0.771
C-2
0.685
0.568
0.505
0.553
0.615
C-3
0.660
0.630
0.670
0.764
0.824
C-4
0.645
0.632
0.685
0.773
0.863
D-1
0.689
0.695
0.739
0.869
0.960
D-2
0.697
0.704
0.772
0.936
1.021
D-3
0.697
0.707
0.758
0.858
0.982
D-4
0.698
0.718
0.682
0.811
0.926
Table 3
Effects of ionic strength on retention time of PS latex beads in GrFFF.
Carrier liquid
6
12
20
40
W
16.00
10.28
7.32
2.83
A-1
21.60
10.90
7.26
4.53
A-2
20.80
11.03
7.36
4.72
A-3
23.18
11.96
8.05
5.08
A-4
25.33
13.33
9.23
5.90
B-1
25.68
13.31
9.00
5.60
B-2
26.50
15.33
10.20
6.06
B-3
25.10
14.12
9.85
5.25
B-4
25.28
14.75
10.10
6.20
Retention time tr is average of two measurements made at flow rate of 1 mL/min.
Retention ratio, R
(a)
(b)
1.2
1.0
0.8
W
C-1 C-2 C-3 C-4 D-1 D-2 D-3 D-4
A-1 A-2 A-3 A-4 B-1 B-2 B-3 B-4
0.9 0.8
0.6
J 1
0.4
Retention ratio, R
a
tr a (min) measured for PS beads having nominal diameter ( P m ) of
0.7 0.6 0.5 0.4 0.3 0.2 0.1
0.2 0
1
2
3
Flow rate (mL/min)
4
0
1
2
3
Flow rate (mL/min)
4
(c)
(d)
0.50
0.27
Retention ratio, R
0.45
Retention ratio, R
0.30
0.40 0.35 0.30 0.25 0.20 0.15 0.10
0.24 0.21 0.18 0.15 0.12 0.09 0.06
0
1
2
3
0
4
1
2
3
4
Flow rate (mL/min)
Flow rate (mL/min)
Fig. 1. Effect of flow rate on R of 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions. Dotted lines are for cases of = 1.
1.1 D-2 D-3 D-1 D-4
Selectivity, Sd
1.0 0.9
C-4 C-3
0.8
C-1
0.7 C-2
0.6 0.5 0.4 0
1
2
3
Flow rate (mL/min) Fig. 2. Effect of flow rate on Sd in GrFFF with various carrier liquid compositions.
4
(b)
1.0
C-1 C-2 C-3 C-4 D-1 D-2 D-3 D-4
A-1 A-2 A-3 A-4 B-1 B-2 B-3 B-4
0.8
0.6
Plate height, H (cm)
Plate height, H (cm)
(a)
0.4
0.2
0.0
0.5
0.4
0.3
0.2
0.1
0.0
0
1
2
3
0
4
1
Flow rate (mL/min) (d)
1.2
Plate height, H (cm)
Plate height, H (cm)
(c)
2
3
4
Flow rate (mL/min)
1.0
0.8
0.6
0.4
0.2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0
1
2
3
Flow rate (mL/min)
4
0
1
2
3
4
Flow rate (mL/min)
Fig. 3. Effect of flow rate on H for 40 (a), 20 (b), 12 (c) and 6 P m (d) PS beads in GrFFF with various carrier liquid compositions.
Retention time tr (min)
30 6 Pm
25
20
15
12 Pm
10
20 Pm 40 Pm
5
0 0
1
2
3
4
5
6
7
8
9
Ionic strength, ,(mM) Fig. 4. Effect of ionic strength of carrier liquid on GrFFF retention of 6, 12, 20, and 40 P m latex beads in various carrier liquid compositions. Flow rate was fixed at 1 mL/min.
(a)
14 1.0 mL/min 2.0 3.0 4.0
12
Geq (Pm)
10 8 6 4 2 0 0
1
2
3
4
5
6
Ionic strength (mM)
7
8
(b)
4.5 A-1
4.0 3.5
W A-2
Geq(Pm)
3.0 2.5
A-3 B-2 B-1
2.0 1.5
A-4 B-4 B-3
1.0 0.5 0.0 6
12
20
40
Diameter (Pm) Fig. 5. Effect of ionic strength on G eq in GrFFF observed at various flow rates for 20 P m PS latex beads (a) and for PS beads of various sizes at 2 mL/min (b).
Plate height, H (cm)
0.4 40 mm 20 12 6
0.3
0.2
0.1
0.0 0
1
2
3
4
5
6
7
8
Ionic strength, , (mM) Fig. 6. Effect of ionic strength on plate height for PS latex beads in GrFFF at flow rate of 1 mL/min.
(a) 0.8
(b)
Retention ratio, R
0.7 0.6 0.5 0.4 0.3 0.2
0.7 0.6
Retention ratio, R
40 Pm 20 12 6
0.5 0.4 0.3 0.2 0.1
0.1 0.0
0.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
(d)
0.7
0.8
0.6
Retention ratio, R
Retention ratio, R
(c)
0.5 0.4 0.3 0.2
0.6
0.4
0.2
0.1 0.0
0.0
1.0
1.2
1.4
1.6
1.8
2.0
1.0
1.2
Viscosity (cP)
1.4
1.6
1.8
2.0
Viscosity (cP)
Retention ratio, R
(e) 1.0
0.8
0.6
0.4
0.2
0.0 1.0
1.2
1.4
1.6
1.8
2.0
Viscosity (cP)
Fig. 7. Effect of viscosity of carrier liquid on retention ratio (R) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively.
18 0.5 mL/min 1.0 2.0 3.0 4.0
16 14
Geq (Pm)
12 10 8 6 4 2 0 1.0
1.2
1.4
1.6
1.8
2.0
Viscosity (cP) Fig. 8. Effect of viscosity on eq of 20 m PS latex beads in GrFFF at various flow rates.
(b)
0.25 40 Pm 20 12 6
0.20
Plate height, H (cm)
Plate height, H (cm)
(a)
0.15
0.10
0.05
0.00
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
1.0
1.2
1.4
1.6
Viscosity (cP)
1.8
2.0
(c)
(d) Plate height, H (cm)
Plate height, H (cm)
0.7 0.6 0.5 0.4 0.3 0.2 0.1
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0 1.0
1.2
1.4
1.6
1.8
2.0
1.0
1.2
Plate height, H (cm)
(e)
1.4
1.6
1.8
2.0
Viscosity (cP)
Viscosity (cP) 1.0
0.8
0.6
0.4
0.2
0.0 1.0
1.2
1.4
1.6
1.8
2.0
Viscosity (cP)
Fig. 9. Effect of viscosity of carrier liquid on plate height (H) of PS beads in GrFFF at flow rate of 0.5 (a), 1 (b), 2 (c), 3 (d) and 4 mL/min (e), respectively.
(a) 1.0 2.0 mL/min 3.0 4.0
Selectivity, Sd
0.9
0.8
0.7
0.6
0.5 1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
1.8
1.9
2.0
Viscosity (cP) (b)
Resolution, Rs*
3.0 2.0 mL/min 3.0 4.0
2.5
2.0
1.5
1.0
0.5 1.3
1.4
1.5
1.6
1.7
Viscosity (cP) Fig. 10. Effect of viscosity on selectivity (Sd) and resolution (Rs*) of PS latex beads in GrFFF observed at various flow rates.
Highlights { Effects of ionic strength on Sd, R, and H in GrFFF was investigated. { Ionic strength plays a critical role in determining retention of microparticles. { Retention increases with increasing ionic strength due to electrostatic interaction { Resolution can be improved by adjusting ionic strength of the carrier liquid.
Geq (Pm) 0
2
4
6
8
10
12
14
1
3
4
5
6
Ionic strength (mM)
2
7
eq decreases with increasing ionic strength.
0
1.0 mL/min 2.0 3.0 4.0
Effect of ionic strength on eq in GrFFF
8
