S

SPLITT is a versatile family of techniques for separating macromolecules, colloids, and particles.

C. Bor Fuh
Chaoyang University of Technology (Taiwan)

plit-flow thin (SPLITT) fractionation is a new family of separation techniques for macromolecules, colloids, and particles (122). 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 <0.5 mm) flow channels with splitters at the outlet or at both inlet and outlet. The inlet splitter allows smooth merging of two inlet flows, and the outlet splitter allows smooth collection of fractionated samples into different outlets without remixing. Typically, SPLITT channels have one inlet and one outlet splitter. A driving force or gradient is applied across the separation channel perpendicular to the flow axis to send sample components selectively into various lateral positions for separation (Figure 1). To clearly understand SPLITT fractionation (SF), it is helpful to define an imaginary plane between the two inlet flows, called the inlet splitting plane (ISP), and another imaginary plane between the two outlet flows, called the outlet splitting plane (OSP). ISP and OSP positions are determined by the relative flow rates of the inlet and outlet substreams. SF driving forces include gravitational, centrifugal, electrical, and magnetic forces. As shown in Figure 2, these forces can yield various selectivities. For easy application of centrifugal forces, SPLITT channels can be made in a curved form, such as a circular basket shape (Figures 2a and 2b). SF can be performed in either transport or equilibrium mode. In the transport mode, samples in liquid carriers are introduced into one inlet stream, while carriers only are introduced into the other inlet at flow rates several times higher than the inlet stream. This procedure initially confines the samples in a small zone, which offers better resolution (Figures 1, 2a, and 2d). Samples are driven in the same direction, but at different field-induced transport rates. Samples driven at the higher transport rates (red and orange circles in the figures) move farther (past the OSP) toward the outside wall. Samples driven at low transport rates (white circles) move less distance (not past the OSP). Samples are distributed differentially in laminae as they pass along the channel and are divided by the outlet splitter into different outlets. Samples with transport rates fast enough to cross the OSP exit at one outlet and the slower samples exit at the other outlet. An inlet splitter is also needed in transport mode. In the equilibrium mode, samples can start from any cross-sectional position and are soon driven by the field to various equilibrium positions where they occupy different laminae before separation by the outlet splitter (Figures 2b and 2c). The inlet splitter is optional in equilibrium mode. Overall, SF features simple laminar flows, perpendicularly applied forces, high separation speeds, and simple cell geometries using unpacked columns. Separation is fast

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because of the short separation distances, which are in the 100-m range. Fractionation of samples through each exit is calculable from the force equation and Stokes law using known transport coefficients (applied forces and sample physical parameters). Conversely, one sample physical parameter can be determined using measured sample retrievals and known applied forces. Therefore, SF is also useful for measuring some sample physical properties (11, 13).

Sample inlet

Field

Outlet a

ISP

OSP

Carrier inlet Injection valve Pump Field Detector a

Outlet b

Sample or carrier

Computer Carrier Pump Detector b

SF complements LC

SF belongs to the closely related family of field-flow fractionation (FFF) techniques (2325). SF and FIGURE 1. Schematic of a SPLITT system. FFF both use laminar flows in White circles represent samples with a low transport rate; red circles have a high transport rate. thin unpacked channels and apply perpendicular fields for separation; however, their separation mechanisms differ. do not cause flow hydrodynamic mixing at fixed total flow The SF separation axis is the thin dimension, whereas rates, have been reported (16). (Fixed total flow rate is the the FFF separation axis is the flow axis. FFF does not use sum of two inlet flow rates.) These ratios are described in splitters and is not readily adaptable to continuous separathe form of a universal reference plot, which shows how flow hydrodynamic mixing can be avoided in the separation tions. Nevertheless, FFF and SF can both be used for sepprocess. Hydrodynamic mixing can cause separation failure. arating macromolecules, colloids, and particles. FFF is SF throughput is directly proportional to the following mainly used for analytical applications that measure privariables: channel length, channel breadth, volumetric flow mary and secondary properties of colloids and polymers rate of the sample stream, and sample concentration in the (23), whereas SF works best for preparative applications. feed stream. Clearly, tradeoffs between resolution and Primary properties (e.g., mass, density, molecular weight throughput are required depending on specific experimen[MW], diameter) are those that can be measured directly from FFF data with little or no prior knowledge about the tal criteria. For example, resolution requirements differ from experiment to experiment. Maintaining acceptable species. Secondary properties (e.g., polymer composition, resolutions, which also differ from experiment to experisurface composition, adsorbed films) generally require ment, while maximizing throughputs is the best choice for outside information, assumptions, or a model describing the retained components for both FFF and SF. LC, preparative SF applications. on the other hand, is mainly used for small molecules (MW < 106) and analytical applications; SF handles primaRetrieval calculation rily large molecules (MW > 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 (b)A(b) applications. SF is mainly used for preparative separations V with throughputs in grams- or subgrams-per-hour, dependFb = V (1) (b)A(b)+V (a)A(a) ing on the fields applied. The resolution of SF is proportional to the ratio be (b)/V (a)] for samples tween the two inlet flow rates [V 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

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(a)

OSP Outlet a Sample inlet ISP

Outlet b (b)

Carrier inlet

equal to 1Fb when a single outlet splitter is usedwhich 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 rela (a), in which a refers to inlet a, and V (a). The tive to V 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 > (a) (high field-induced flow rate), then F = 1. If all samV b ple components cannot pass the OSP and exit at outlet a (a)V (a) (low field-induced flow rate), when bLU < V then Fb = 0. In between those two extremes, when (a)V (a) < bLU < V (a), only fractional amounts of the V sample components pass the OSP and exit at outlet b. In this case, the equation is (a)+V (a) bLUV (a) V

Fb =

(2)

Outlet a

Sample inlet

Single inlets are usually used in the equilibrium mode (Figures 2b and 2c). Fb of the positively charged species in (a) Figure 2c can be calculated (5). As before, if bLUd > V (Ud is the field-induced velocity in the downward direc (a), then tion), then Fb = 1. If bLUd < V Fb = bLUd+V(b) V(a)+V(b)

(3)

Outlet b (c) Sample inlet + + + + + + + + + Outlet a

Outlet b (d) Carrier inlet 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-

Magnetic field

FIGURE 2. Various applied fields in transport and equilibrium modes. ISP OSP
(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.

Sample inlet

Gravitational field

Outlet b

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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 m

Outlet a (b)

Outlet b (c)

SF and physical parameters of samples

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 (bLUmbLUg) 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) Fwt 2kT

Signal

1.0 2.0 Time (min)

3.0

Signal
0

1.0 2.0 Time (min)

3.0

FIGURE 3. Separation of oversized particles (dc > 5 m) for cubic boron nitride abrasive material.
(a) Unfractionated boron nitride sample. (b) Fraction with dc < 5 m versus (c) the (mL/min): a = 0.4, b = 2.0, a = 1.4, b = 1.0. fraction with dc > 5 m. 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 of a narrow pulse caused by diffusion should be <0.1 wt for high resolution. Therefore, we generally require N 100 because N = (wt/)2. Each type of macromolecule, colloid, or particle needs to be checked against this requirement to ensure achievable resolution. Resolution can be expressed

Table 1. Field-induced velocity for various applied forces

Force Gravitationally induced velocity Symbol Us Calculation

rd 2

Centrifugally induced velocity

Uc

rd 2

r v2

Magnetically induced velocity

Um

dxH 2

Electrically induced velocity

Ue

N=

(4)

*r is the density difference between samples and carriers, h is carrier viscosity, d is the diameter of a spherical particle, r is the radius of rotation, v is angular velocity, x is the difference in magnetic susceptibilities between samples and carriers, H is the drop in magnetic field strength, is the eletrophoretic mobility of sample components, and E is the electrical field.

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(a) Flow FFF fractograms 150 mg

Signal

Signal

Albumin IgG

(b)

20

40

60

Time (min)

150 mg

100

200

300 Time (min)

400

500

FIGURE 4. Continuous separation of human IgG and albumin by isoelectrical SPLITT fractionation.
(a) Human albumin, pI = 4.9, 150 mg. (b) Human IgG, pI = 7 .57 .8, 150 mg. Channel (mL/min): a = 0.45, thickness = 0.0635 cm, and 0.01 M acetate buffer at pH = 5.6. V b = 0, a = 0.29, b = 0.16, and applied voltage of 30 V.

as m1/(m1m0); m1 and m0 are the generalized mobilities for retrievals equal to 1 and 0, respectively (15). Resolution can possibly be improved by finite, particle-size effects, the influence of hydrodynamic lift forces, and transport effects in the splitter region (15). Transport effects in the splitter region are particle sedimentation effects after particles are introduced into the inlet and before entering the separation region.

Applications
Analytical SF has been used for measuring oversized particles (those exceeding certain diameter criteria), particlesize distributions, and protein diffusion coefficients (11, 13)physical parameters that are very useful in process analysis and quality control. Sample injection loops with fixed volumes, as in LC, are used for sample introduction in analytical SF. The detection and measurement of oversized particles are very promising applications of analytical SF. Abrasive materials (boron nitride) and starch granules have both been used to demonstrate the separation and measurement of this type of particle (13). Figure 3 shows the result of setting dc = 5 m for boron nitride abrasives to remove oversized particles (13): Separation is fast with most run times at < 2 min. The elimination of oversized, abrasive material particles is very important in industrial polishing applications because these particles can cause scratching during surface polishing. Removing oversized starch granules is also important in food manufacturing because these granules can lead to taste variations. In addition, SF can make one or more determinations per minute in an ongoing analysis with the addition of an autoinjector, can be run continuously by tapping into a process feed stream, and can provide a continuous report on the oversized particle content in the stream. The rapid response of SF can

almost immediately detect undesirable shifts. Measuring particle-size distributions is another application of analytical SF. Oversized-particle amounts can be (a) (inlet) and determined by systematically changing V (outlet) for each run to establish different dc values V(a) (13). Size-distribution curves can be constructed by combining all the results obtained from using different dc values. For example, standard reference materials for quartz (BCR 67) and glass beads (NIST) have been shown to agree well with particle-size distributions from analytical SF measurements. However, analytical SF using gravity for particle-size distribution is limited to large and high-density particles, generally > 10 m 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 (b)/V (a)] and varying outlet fixed inlet flow ratios [V 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, 1921). 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 m, were separated into small- and large-sized fractions with diameters below and above 0.15 m, respectively. Oversized palladium particles could cause electrical short-circuits. In another example, polydisperse nematic curvilinear align-phase liquidcrystal emulsions (110 m) were separated into large- and small-size fractions with a cutoff size of 2.8 m (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

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Original

Outlet a

Relative mass

Outlet b

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.
This work was supported by the National Science Council of Taiwan, Republic of China (NSC-882113-M-324003).

References
(1) (2) 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.

0.0

0.2

0.4 0.6 Diameter (m)

0.8

1.0
(3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)

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-

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 (cbfuh@mail.cyut.edu.tw).

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