S
SPLITT is a versatile family of techniques for separating macromolecules, colloids, and particles.
C. Bor Fuh Chaoyang University of Technology (Taiwan)
266 A
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 0
plit-flow thin (SPLITT) fractionation is a new family of separation techniques for macromolecules, colloids, and particles (1–22). In this article, the basic operation and theory of SPLITT fractionation are discussed and several representative applications are described. SPLITT channels are ribbonlike, unpacked, thin (usually 106) and preparative applicaThe retrieval at outlet b (Fb) is the percentage of samples tions. Therefore, SF complements LC in many applications. exiting at that outlet. Fb can be used to compare theoretiSF is considered “continuous SF” when it is used for cal predictions with experimental results and to predict preparative applications. Samples are continuously pumped separation results, and it acts as a good experimental into channels for separation in such operations. SF is conguideline. The experimental retrieval Fb is calculated by sidered “analytical SF” when it is used to characterize sam- the equation ples (11, 13). Pulsed sample injection is used in these applications. SF is mainly used for preparative separations V˙ (b)A(b) with throughputs in grams- or subgrams-per-hour, dependFb = V˙ (b)A(b)+V˙ (a)A(a) (1) ing on the fields applied. The resolution of SF is proportional to the ratio between the two inlet flow rates [V˙ (b´)/V˙ (a´)] for samples in which A(a) and A(b) are the peak areas of the detector introduced at inlet a´. Optimal inlet flow-rate ratios, which responses at outlets a and b, and retrieval at outlet a (Fa) is
A P R I L 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
267 A
equal to 1–Fb when a single outlet splitter is used—which is the case in most applications. The theoretical retrieval Fb in transport mode (Figures 1, 2a, and 2d) can be calculated from equations (2, 3, 13). In this article, I assume that the particles entering the inlet are randomly distributed over the initial sample zone and have a constant field-induced velocity (U). Fb, thus, depends on the magnitude of field-induced flow rate relative to V˙ (a´), in which a´ refers to inlet a, and V˙ (a). The field-induced flow rate is equal to bLU, in which b is the channel breadth and L is the channel length. Fb can then be calculated according to the following three conditions for specified inlet and outlet flow rates. If all sample components pass the OSP and exit at outlet b when bLU > V˙ (a) (high field-induced flow rate), then Fb = 1. If all sample components cannot pass the OSP and exit at outlet a when bLU < V˙ (a)–V˙ (a´) (low field-induced flow rate), then Fb = 0. In between those two extremes, when V˙ (a)–V˙ (a´) < bLU < V˙ (a), only fractional amounts of the sample components pass the OSP and exit at outlet b. In this case, the equation is
(a)
OSP Outlet a
Sample inlet ISP
Carrier inlet
Outlet b (b)
Fb =
Outlet a
bLU–V˙ (a)+V˙ (a´)
(2)
V˙ (a´)
Single inlets are usually used in the equilibrium mode (Figures 2b and 2c). Fb of the positively charged species in Figure 2c can be calculated (5). As before, if bLUd > V˙ (a) (Ud is the field-induced velocity in the downward direction), then Fb = 1. If bLUd < V˙ (a), then
Sample inlet
˙ Fb = bLUd+V(b) ˙ ˙ V(a)+V(b)
(3)
Outlet b (c) Outlet a
Sample inlet +
+
+ + +
+ +
– –
– – –
–
+ +
– – –
Outlet b (d) Carrier inlet
Magnetic field
Outlet a
Fb for negatively charged species (Figure 2c) can also be similarly calculated (5). The theoretical retrieval calculations apply to both analytical and preparative applications. Diffusion is negligible for the separation of micrometer and submicrometer-sized particles, but becomes important with nano-sized particles (3, 11). In the latter case, diffusion will increase the difference between theoretically calculated and experimentally measured retrievals. Hydrodynamic lift forces are highly nonuniform forces that drive particles away from nearby elements of stationary wall (4) and are only important in separating large particles, which are close to the channel wall and under high flow-rate conditions (generally > 10 mL/min). Hydrody-
FIGURE 2. Various applied fields in transport and equilibrium modes. ISP
Sample inlet
268 A
OSP
Gravitational field
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 0
Outlet b
(a) Centrifugal SPLITT system operated in transport mode. White circles represent a low transport rate; red circles are at a high transport rate. (b) Centrifugal SPLITT system operated in equilibrium mode. Purple are low-density samples; green are highdensity samples. (c) Isoelectrical SPLITT system operated in equilibrium model. Yellow are negatively charged samples; black are positively charged. (d) Magnetic SPLITT system operated in transport mode. Violet are particles with a high magnetic susceptibility; orange are low magnetic susceptibility particles.
namic lift forces are largest when particles are near the channel wall, and they drop off exponentially as particles move away from the channel wall. This suggests that high resolutions for large particle separation cannot be achieved in SF by simply increasing the inlet flow ratio. Hydrodynamic lift forces are believed to combine with other forces to form concentrated thin layers (hyperlayers) along the flow axis for useful separation (4).
(a) 5 µm
Outlet a (b)
Fwt 2kT
(4)
Signal
Various fields exert forces with unique dependencies and thus yield unique sample selectivities in SF. U is the most important factor in determining the final lateral positions of separated sample components under conditions of fixed flow rates and can be calculated for various applied forces (Table 1). Various sample physical parameters (e.g., density, size, charge, magnetic susceptibility, electrophoretic mobility) are related to the applied forces used in determining separation selectivities. The cutoff diameter (dc) is a specified diameter for SF reference. In principle, all particles smaller than dc exit one outlet, and larger particles exit from another outlet. The cutoff diameter, based on Fb = 0.5, can be calculated with Equation 2. The results of fractionation are predictable for known sample physical parameters and applied fields. Theoretical models provide good guidelines for determining successful separations and good starting points for separation experiments. Selectivity in SF arises from the application of various fields and controllable flow rates at inlets and outlets. Field selection depends on the physical properties of samples to be separated. Combining two fields for better separation is possible for certain applications (10). In Figure 2d, magnetic and gravitational forces are applied in opposite directions to facilitate separation of magnetic particles from nonmagnetic particles with large diameters and/or high densities. The magnetic force drives particles with high magnetic susceptibility (violet in the figure) upward toward the top wall, while other samples pass along the channel below. Particles with low magnetic susceptibility (orange) settle toward the bottom wall because of gravity. The high magnetic susceptibility particles exit at outlet a, and particles with low magnetic susceptibility exit at outlet b. The resulting Fb can be calculated using the combined fieldinduced flow rate (bLUm–bLUg) in similar fashion (10). Gravity is economical and easy to use but is effective only with large and/or high-density particles. Centrifugal, electrical, and magnetic forces offer greater resolution than gravity when used with samples that differ in size, charge, electrophoretic mobility, or magnetic susceptibility. The SF efficiency, just as in chromatography, can be evaluated by the number (N) of theoretical plate generated during transport. The effective N is given by the ratio of two energies (15)
(c)
Signal
SF and physical parameters of samples
N=
Outlet b
0
1.0 2.0 Time (min)
3.0
0
1.0 2.0 Time (min)
3.0
FIGURE 3. Separation of oversized particles (dc > 5 µm) for cubic boron nitride abrasive material. (a) Unfractionated boron nitride sample. (b) Fraction with dc < 5 µm versus (c) the ˙ (mL/min): a´ = 0.4, b´ = 2.0, a = 1.4, b = 1.0. fraction with dc > 5 µm. V
in which F is the force on the particle inducing its transport, wt is the length of the transport path, k is the Boltzmann constant, and T is the temperature in Kelvin (kT is thermal energy). The standard deviation σ of a narrow pulse caused by diffusion should be 10 µm with a density of > 1.5 g/mL. Rapid diffusion-coefficient measurements for proteins using analytical SF are also a promising application. The sample diffusion through SPLITT cells can be calculated from first principles using parabolic flows, controlled flow rates, and simple cell geometries. The relative sample concentrations at two outlet streams are mathematically related to diffusion coefficients in retrieval plots. Sample diffusion coefficients can then be obtained from retrieval plots at fixed inlet flow ratios [V˙ (b´)/V˙ (a´)] and varying outlet sample fractions. For example, diffusion coefficient measurements for eight proteins using analytical SF were consistent with results from light-scattering measurements (11). SF has also been used for many industrial and environmental applications (3, 8, 10, 19–21). These applications include the separation of metal particles, sediments, various polymer latexes, magnetic particles, glass beads, and liquid crystals. They were primarily done by preparative separation, in which samples were continuously pumped into the inlet after adjustment of the applied forces and inlet and outlet flow rates. Many industrial applications require the removal of oversized particles, which would otherwise cause adverse effects. Gravity has been used to separate glass beads and coastal marine sediments as well as abrasives and starch granules (13, 19). Centrifugal force has been applied to the separation of submicrometer particles, such as colloidal palladium, PVC latex, and liquid crystals (3). For example, palladium particles, ranging in size from 0.03 to 0.3 µm, were separated into small- and large-sized fractions with diameters below and above 0.15 µm, respectively. Oversized palladium particles could cause electrical short-circuits. In another example, polydisperse nematic curvilinear align-phase liquidcrystal emulsions (1–10 µm) were separated into large- and small-size fractions with a cutoff size of 2.8 µm (3). Oversized emulsions of these liquid crystals can cause inconsistent electrical responses. Centrifugal SF extends applications of gravitational SF to colloidal and low-density particles. Optimal operating conditions and the factors for resolution deterioration with stable and unstable density gradients in centrifugal SF have been published (3, 14, 22). Preparative separations are also becoming increasingly important in many biological and biomedical applications. Protein separation and blood cell separation are two examples. Protein separation using electrical fields in equilibrium mode has been demonstrated with throughputs of
tinuous separation of submicrometer-sized emulsions with controllable cutoff sizes. SF is still in its infancy, but, as this article has demonstrated, it has many potential applications. The main obstacles to its widespread use are that the method is not well known and that commercial SF systems are not yet available. However, SF offers great potential, especially because it complements LC in many applications. As a result, SF should become a very useful technique for separating macromolecules, colloids, and particles.
Original
Relative mass
Outlet a
Outlet b
This work was supported by the National Science Council of Taiwan, Republic of China (NSC-88–2113-M-324–003).
References (1) (2)
0.0
0.2
0.4 0.6 Diameter (µm)
0.8
1.0
FIGURE 5. Particle-size analysis of original and fractionated pharmaceutical emulsions (Fluosol) by sedimentation FFF. Theoretical (green) and experimental (black) particle-size distributions for fractionated emulsions are shown. Channel thickness = 0.0381 cm; applied centrifugal force = 111.4 gravities. ˙V (mL/min): a´ =2.5, b´ = 7.5, a = 6.0, and b = 4.0.
~15 mg/h (5). The minimal difference in protein isoelectric points (pI) that could be successfully separated was around two units. Diffusion effects played important roles in limiting the pI difference in this case. Several protein mixtures, including human albumin and IgG, have been used to illustrate successful separations (Figure 4). Fractionated human IgG and albumin were collected after 8-h runs, and their purities were confirmed via flow FFF using pure samples for reference (5). Blood cell separation was demonstrated by isolating human blood cells, platelets, and plasma proteins using centrifugal force (6). SF successfully purified the subsets of all major white blood cells, including lymphocytes, monocytes, and neutrophils. Transport and equilibrium modes were used in series to overcome overlaps in sizes and densities of the blood cell subsets. The viabilities of the purified cells were >97%, as determined by dye exclusion testing. Throughput was ~1010 cells/h. Emulsion size has been considered to be related to emulsion effectiveness, toxicity, and stability. As a result, narrowing the size distributions of manufactured emulsions is very important to the pharmaceutical industry. Pharmaceutical perfluorochemical emulsions, which are used for blood substitutes, have been separated into different size distributions using centrifugal SF (Figure 5) (18). The particle-size distributions, as predicted by theory, reasonably matched experimental results. Theory provided good predictions of fractionating results based on known samples and experimental parameters. Throughput was ~1.5 g/h. Thus, SF could be a valuable technique for con-
(3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)
Giddings, J. C. Sep. Sci. Technol. 1985, 20, 749. Springston, S. R.; Myers, M. N.; Giddings, J. C. Anal. Chem. 1987, 59, 344. Fuh, C. B.; Myers, M. N.; Giddings, J. C. Ind. Eng. Chem. Res. 1994, 33, 355. Giddings, J. C. Sep. Sci. Technol. 1988, 23, 119. Fuh, C. B.; Giddings, J. C. Sep. Sci. Technol. 1997, 32, 2945. Fuh, C. B.; Giddings, J. C. Biotechnol. Progr. 1995, 11, 14. Giddings, J. C. Sep. Sci. Technol. 1988, 23, 931. Jiang, Y.; Kummerow, A.; Hansen, M. J. Microcolumn Sep. 1997, 9, 261. Williams, P. S. Sep. Sci. Technol. 1994, 29, 11. Fuh, C. B.; Chen, S. Y. J. Chromatogr., A 1998, 813, 313. Fuh, C. B.; Levins, S.; Giddings, J. C. Anal. Biochem. 1993, 208, 80. Levin, S.; Giddings, J. C. Sep. Sci. Technol. 1989, 84, 1245. Fuh, C. B.; Myers, M. N.; Giddings, J. C. Anal. Chem. 1992, 64, 3125. Gupta, S.; Ligrani, P. M.; Myers, M. N.; Giddings, J. C. J. Microcolumn Sep. 1997, 9, 261. Giddings, J. C. Sep. Sci. Technol. 1992, 27, 1489. Fuh, C. B.; Trujillo, E. M.; Giddings, J. C. Sep. Sci. Technol. 1995, 30, 3861. Williams, P. S.; Levin, S.; Lenczycki, T.; Giddings, J. C. Ind. Eng. Chem. Res. 1992, 31, 2172. Fuh, C. B.; Giddings, J. C. J. Microcolumn Sep. 1997, 9, 205. Keil, R. G.; Tsamakis, E.; Fuh, C. B.; Giddings, J. C.; Hedges, J. I. Geochim. Cosmochim. Acta 1994, 58, 879. Gao, Y.; Myers, M. N.; Barman, B. N.; Giddings, J. C. Part. Sci. Technol. 1991, 9, 105. Contado, C.; Dondi, F.; Beckett, R.; Giddings, J. C. Anal. Chim. Acta 1997, 345, 99. Gupta, S.; Ligrani, P. M.; Myers, M. N.; Giddings, J. C. J. Microcolumn Sep. 1997, 9, 307. Giddings, J. C. Science 1993, 260, 1456. Giddings, J. C. Chem. Eng. News 1988, 66, 34. Caldwell, K. D. Anal. Chem. 1988, 60, 959.
C. Bor Fuh is an associate professor at Chaoyang University of Technology (Taiwan). His research interests include developing instrumentation and methodology for separating macromolecules, colloids, and particles. Address correspondence about this article to Fuh at the Department of Applied Chemistry, Chaoyang University of Technology, 168 Gifeng East Rd., Wufeng, Taichung County 413, Taiwan (
[email protected]).
A P R I L 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
271 A
