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 (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 <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
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
dependFb = V (1) ˙ (b)A(b)+V ˙ (a)A(a) ing on the fields applied. FFF does not use sum of two inlet flow rates.g. LC. diameter) are those that can be measured directly from FFF data with little or no prior knowledge about the tal criteria. SF is mainly used for preparative separations V with throughputs in grams.. 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. assumptions. SF handles primaRetrieval calculation rily large molecules (MW > 106) and preparative applicaThe retrieval at outlet b (Fb) is the percentage of samples tions.g. SF and FIGURE 1. arating macromolecules. For example. colloids. which shows how flow hydrodynamic mixing can be avoided in the separation tions. FFF and SF can both be used for sepprocess. Pulsed sample injection is used in these ˙ (b)A(b) applications. The experimental retrieval Fb is calculated by sidered “analytical SF” when it is used to characterize sam. have been reported (16). is mainly used for small molecules (MW < 106) and analytical applications. density. volumetric flow mary and secondary properties of colloids and polymers rate of the sample stream. whereas SF works best for preparative applications. which are in the 100-µm range. FFF is SF throughput is directly proportional to the following mainly used for analytical applications that measure privariables: channel length. their separation mechanisms differ. on the other hand. and retrieval at outlet a (Fa) is
A P R I L 1 .or subgrams-per-hour. FFF both use laminar flows in White circles represent samples with a low transport rate. and particles. polymer composition. whereas rates. feed stream. thin unpacked channels and apply perpendicular fields for separation. 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´.) These ratios are described in splitters and is not readily adaptable to continuous separathe form of a universal reference plot. SF is conguideline. while maximizing throughputs is the best choice for outside information. and sample concentration in the (23). 13). Hydrodynamic mixing can cause separation failure. Secondary properties (e. mass. resolutions. Optimal inlet flow-rate ratios. molecular weight throughput are required depending on specific experimen[MW]. do not cause flow hydrodynamic mixing at fixed total flow The SF separation axis is the thin dimension. Clearly. which responses at outlets a and b. Therefore.
Carrier inlet Injection valve Pump Field Detector a
Sample or carrier
Computer Carrier Pump Detector b
SF complements LC
SF belongs to the closely related family of field-flow fractionation (FFF) techniques (23–25). Therefore. SF is also useful for measuring some sample physical properties (11. Nevertheless. Conversely. exiting at that outlet.because of the short separation distances. tradeoffs between resolution and Primary properties (e. however. red circles have a high transport rate. Schematic of a SPLITT system..the equation ples (11. resolution requirements differ from experiment to experiment. Maintaining acceptable species. SF complements LC in many applications. (Fixed total flow rate is the the FFF separation axis is the flow axis. 13). and it acts as a good experimental into channels for separation in such operations. 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
. channel breadth. adsorbed films) generally require ment. Samples are continuously pumped separation results. or a model describing the retained components for both FFF and SF. preparative SF applications. 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). one sample physical parameter can be determined using measured sample retrievals and known applied forces. which also differ from experiment to experisurface composition.
only fractional amounts of the V sample components pass the OSP and exit at outlet b. Fb. Fb of the positively charged species in ˙ (a) Figure 2c can be calculated (5). thus. In this case. 2a. If all sample components pass the OSP and exit at outlet b when bLU > ˙ (a) (high field-induced flow rate). but becomes important with nano-sized particles (3. In this article. 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. in which a´ refers to inlet a. The theoretical retrieval calculations apply to both analytical and preparative applications. Diffusion is negligible for the separation of micrometer and submicrometer-sized particles. Fb can then be calculated according to the following three conditions for specified inlet and outlet flow rates. and V ˙ (a). Purple are low-density samples. orange are low magnetic susceptibility particles. White circles represent a low transport rate. Hydrody-
FIGURE 2. then tion). (b) Centrifugal SPLITT system operated in equilibrium mode.
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 . Violet are particles with a high magnetic susceptibility. red circles are at a high transport rate. The tive to V field-induced flow rate is equal to bLU.(a)
OSP Outlet a Sample inlet ISP
Outlet b (b)
equal to 1–Fb when a single outlet splitter is used—which is the case in most applications. and 2d) can be calculated from equations (2. I assume that the particles entering the inlet are randomly distributed over the initial sample zone and have a constant field-induced velocity (U ). when ˙ (a)–V ˙ (a´) < bLU < V ˙ (a). black are positively charged. when bLU < V then Fb = 0. In between those two extremes. If all samV b ple components cannot pass the OSP and exit at outlet a ˙ (a)–V ˙ (a´) (low field-induced flow rate). Yellow are negatively charged samples. the equation is ˙ (a)+V ˙ (a´) bLU–V ˙ (a´) V
Single inlets are usually used in the equilibrium mode (Figures 2b and 2c). 13). 2 0 0 0
. in which b is the channel breadth and L is the channel length. If bLUd < V ˙ Fb = bLUd+V(b) ˙ ˙ V(a)+V(b)
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). which are close to the channel wall and under high flow-rate conditions (generally > 10 mL/min). The theoretical retrieval Fb in transport mode (Figures 1. depends on the magnitude of field-induced flow rate rela˙ (a´). (d) Magnetic SPLITT system operated in transport mode. ISP OSP
(a) Centrifugal SPLITT system operated in transport mode. Various applied fields in transport and equilibrium modes. green are highdensity samples. then F = 1. As before. (c) Isoelectrical SPLITT system operated in equilibrium model. 3. then Fb = 1. 11). if bLUd > V (Ud is the field-induced velocity in the downward direc˙ (a). diffusion will increase the difference between theoretically calculated and experimentally measured retrievals. In the latter case.
Each type of macromolecule. k is the Boltzmann constant. Field-induced velocity for various applied forces
Force Gravitationally induced velocity Symbol Us Calculation
Centrifugally induced velocity
Magnetically induced velocity
Electrically induced velocity
*∆r is the density difference between samples and carriers. electrophoretic mobility. b´ = 2. charge.0. and magnetic forces offer greater resolution than gravity when used with samples that differ in size. while other samples pass along the channel below.0
1.g. The SF efficiency. and T is the temperature in Kelvin (kT is thermal energy). fraction with dc > 5 µm. ∆x is the difference in magnetic susceptibilities between samples and carriers. and they drop off exponentially as particles move away from the channel wall. Theoretical models provide good guidelines for determining successful separations and good starting points for separation experiments. r is the radius of rotation. electrical. wt is the length of the transport path. b = 1. h is carrier viscosity. and larger particles exit from another outlet. The high magnetic susceptibility particles exit at outlet a. can be calculated with Equation 2. d is the diameter of a spherical particle.namic lift forces are largest when particles are near the channel wall.4. can be evaluated by the number (N) of theoretical plate generated during transport. (b) Fraction with dc < 5 µm versus (c) the ˙ (mL/min): a´ = 0.
(a) Unfractionated boron nitride sample. v is angular velocity. ∆H is the drop in magnetic field strength. based on Fb = 0. µ is the eletrophoretic mobility of sample components. magnetic susceptibility. Resolution can be expressed
Table 1. Combining two fields for better separation is possible for certain applications (10). 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
.0 Time (min)
3. Various sample physical parameters (e. 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).. The effective N is given by the ratio of two energies (15) Fwt 2kT
1. Field selection depends on the physical properties of samples to be separated. Hydrodynamic lift forces are believed to combine with other forces to form concentrated thin layers (hyperlayers) along the flow axis for useful separation (4). Gravity is economical and easy to use but is effective only with large and/or high-density particles. just as in chromatography. The cutoff diameter. colloid.5. The magnetic force drives particles with high magnetic susceptibility (violet in the figure) upward toward the top wall. we generally require N ≥ 100 because N = (wt/σ)2. V
in which F is the force on the particle inducing its transport. or magnetic susceptibility.
(a) 5 µm
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. electrophoretic mobility) are related to the applied forces used in determining separation selectivities.0 Time (min)
3.0. In Figure 2d. Separation of oversized particles (dc > 5 µm) for cubic boron nitride abrasive material. 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. or particle needs to be checked against this requirement to ensure achievable resolution.
A P R I L 1 . Particles with low magnetic susceptibility (orange) settle toward the bottom wall because of gravity. In principle. This suggests that high resolutions for large particle separation cannot be achieved in SF by simply increasing the inlet flow ratio. and E is the electrical field. Selectivity in SF arises from the application of various fields and controllable flow rates at inlets and outlets. Centrifugal.0 2. The resulting Fb can be calculated using the combined fieldinduced flow rate (bLUm–bLUg) in similar fashion (10). all particles smaller than dc exit one outlet. a = 1. and particles with low magnetic susceptibility exit at outlet b. Therefore. The standard deviation σ of a narrow pulse caused by diffusion should be <0. density. The cutoff diameter (dc) is a specified diameter for SF reference.0
FIGURE 3. size. charge. The results of fractionation are predictable for known sample physical parameters and applied fields.4.1 wt for high resolution.0 2.
pI = 7 . pI = 4. The elimination of oversized. sediments. thickness = 0. However. 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. which would otherwise cause adverse effects. In another example. and transport effects in the splitter region (15).9. Size-distribution curves can be constructed by combining all the results obtained from using different dc values. Sample diffusion coefficients can then be obtained from retrieval plots at ˙ (b´)/V ˙ (a´)] and varying outlet fixed inlet flow ratios [V sample fractions. Continuous separation of human IgG and albumin by isoelectrical SPLITT fractionation. can be run continuously by tapping into a process feed stream.and small-size fractions with a cutoff size of 2. PVC latex. polydisperse nematic curvilinear align-phase liquidcrystal emulsions (1–10 µm) were separated into large. palladium particles. were separated into small.5 g/mL. generally > 10 µm with a density of > 1. Oversized palladium particles could cause electrical short-circuits. in which samples were continuously pumped into the inlet after adjustment of the applied forces and inlet and outlet flow rates. ranging in size from 0.45.8. m1 and m0 are the generalized mobilities for retrievals equal to 1 and 0. These applications include the separation of metal particles. 8. such as colloidal palladium. 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).and large-sized fractions with diameters below and above 0. and simple cell geometries. 10.15 µm. various polymer latexes. and liquid crystals. Channel ˙ (mL/min): a´ = 0. particle-size effects. b = 0.(a) Flow FFF fractograms 150 mg
300 Time (min)
FIGURE 4.16. Rapid diffusion-coefficient measurements for proteins using analytical SF are also a promising application. controlled flow rates.29. and can provide a continuous report on the oversized particle content in the stream. Sample injection loops with fixed volumes. Removing oversized starch granules is also important in food manufacturing because these granules can lead to taste variations. are used for sample introduction in analytical SF.
Analytical SF has been used for measuring oversized particles (those exceeding certain diameter criteria). respectively. 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 µm for boron nitride abrasives to remove oversized particles (13): Separation is fast with most run times at < 2 min. as in LC. Protein separation and blood cell separation are two examples.03 to 0. SF can make one or more determinations per minute in an ongoing analysis with the addition of an autoinjector. 13)—physical parameters that are very useful in process analysis and quality control.
as m1/(m1–m0). Optimal operating conditions and the factors for resolution deterioration with stable and unstable density gradients in centrifugal SF have been published (3. and protein diffusion coefficients (11. respectively (15).0635 cm. Measuring particle-size distributions is another application of analytical SF. particlesize distributions. abrasive material particles is very important in industrial polishing applications because these particles can cause scratching during surface polishing. SF has also been used for many industrial and environmental applications (3. the influence of hydrodynamic lift forces. For example. Protein separation using electrical fields in equilibrium mode has been demonstrated with throughputs of
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 . The rapid response of SF can
almost immediately detect undesirable shifts. Transport effects in the splitter region are particle sedimentation effects after particles are introduced into the inlet and before entering the separation region. The relative sample concentrations at two outlet streams are mathematically related to diffusion coefficients in retrieval plots. 14. magnetic particles. a = 0. Many industrial applications require the removal of oversized particles. V b´ = 0.8 µm (3).01 M acetate buffer at pH = 5. Gravity has been used to separate glass beads and coastal marine sediments as well as abrasives and starch granules (13. 19–21). In addition. Oversized emulsions of these liquid crystals can cause inconsistent electrical responses. and liquid crystals (3).5–7 . and 0. For example. 2 0 0 0
. Centrifugal SF extends applications of gravitational SF to colloidal and low-density particles. They were primarily done by preparative separation. Preparative separations are also becoming increasingly important in many biological and biomedical applications.
(a) Human albumin. and applied voltage of 30 V. (b) Human IgG. diffusion coefficient measurements for eight proteins using analytical SF were consistent with results from light-scattering measurements (11). 150 mg. Resolution can possibly be improved by finite. Centrifugal force has been applied to the separation of submicrometer particles. 22).3 µm. glass beads. For example. 19). The detection and measurement of oversized particles are very promising applications of analytical SF. The sample diffusion through SPLITT cells can be calculated from first principles using parabolic flows. analytical SF using gravity for particle-size distribution is limited to large and high-density particles. 150 mg.6.
1988. G. Hedges. 29. 1997. Bor Fuh is an associate professor at Chaoyang University of Technology (Taiwan). J.4 0. Giddings.. C. J. Giddings. B. Blood cell separation was demonstrated by isolating human blood cells. Fuh. Technol... 355. SF successfully purified the subsets of all major white blood cells. Sep..Original
tinuous separation of submicrometer-sized emulsions with controllable cutoff sizes. 1993. Acta 1994. monocytes. Technol. 1985. Giddings. Anal. C. Sep. B. J.. Technol. J. 1991. Microcolumn Sep. Geochim. 313. The minimal difference in protein isoelectric points (pI) that could be successfully separated was around two units. S. S. Eng. J. C. Sep.. J. Thus. 34. C. Anal. Giddings. B. C. C.. 31. Jiang.edu. The particle-size distributions. Gupta. Lenczycki. narrowing the size distributions of manufactured emulsions is very important to the pharmaceutical industry... C. Myers.. Giddings. Williams. it has many potential applications. J. His research interests include developing instrumentation and methodology for separating macromolecules. Progr.. 1988. C. Fuh.. Giddings. Kummerow. P. Giddings. As a result. 58.. Taichung County 413. C. C. 23. Caldwell. Ind.
This work was supported by the National Science Council of Taiwan. 66. Beckett. 9.. 1245. and particles... Dondi. C. Fuh.
Theoretical (green) and experimental (black) particle-size distributions for fractionated emulsions are shown. b´ = 7. E.. Channel thickness = 0. Chem. SF offers great potential. 1997. N. Fuh. which are used for blood substitutes. 344. ˙ V (mL/min): a´ =2. M. 261. have been used to illustrate successful separations (Figure 4). P. Fuh. Acta 1997. 99. P. 205. K. Several protein mixtures. R. Technol. J. Technol. colloids. platelets. Sep. C. SF could be a valuable technique for con-
C. Sci. Technol. 3861. 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
.6 Diameter (µm)
(1) (2) Giddings.. B. B. a = 6. Chen. and b = 4. Trujillo. Throughput was ~1010 cells/h. Fuh. 1989. M. 1988. 1456. and their purities were confirmed via flow FFF using pure samples for reference (5). Biotechnol. Sci. Myers. as determined by dye exclusion testing.. S. but. B. Giddings. J. C.. J. Republic of China (NSC-88–2113-M-324–003). S. Giddings. Chim. Giddings. 60. 59. M.0
(3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)
0. C.. M. However. Sep. Chem. D. J.. Springston. Hansen. J. Fuh. E. S. Sci. 9. C. and plasma proteins using centrifugal force (6). S. 32. P. T. R. C. Giddings. 33. C. Technol.. Giddings.5. Chaoyang University of Technology. Science 1993.0381 cm. 64.5 g/h. Ligrani. Address correspondence about this article to Fuh at the Department of Applied Chemistry.5. I. N. Microcolumn Sep. Giddings.. 11. 105. 931. Giddings. Williams. B. J. Fractionated human IgG and albumin were collected after 8-h runs. 80. J. N. Eng. N. J.. 168 Gifeng East Rd. 1997. Part.. Myers. Myers.. Sci. Sep. J. Anal. as this article has demonstrated.0.. 14. SF should become a very useful technique for separating macromolecules. Gao. 749. S. M. have been separated into different size distributions using centrifugal SF (Figure 5) (18).. 1987.. 879. including lymphocytes. J. J. C.. J. 1992. Cosmochim. Pharmaceutical perfluorochemical emulsions. Theory provided good predictions of fractionating results based on known samples and experimental parameters. 30. B. 261. and particles. SF is still in its infancy. 3125. C. 9. J. 9. F. J. A 1998. 208. N. and stability. Giddings. Fuh. A. Keil. Myers. Giddings.0. 20. Levin. and neutrophils. 1992. 1992. The viabilities of the purified cells were >97%.. especially because it complements LC in many applications. Giddings. Sci. J. Anal. N. J.cyut. B. Res. 1995. Sci. applied centrifugal force = 111. J. 1995. Chem. Tsamakis. 1997. Ligrani. Res. 307. Technol. Eng. J. Contado.
A P R I L 1 . 11. C. M. C. C. Chem. 1994. 260. 9. M. 2172. Transport and equilibrium modes were used in series to overcome overlaps in sizes and densities of the blood cell subsets. C. Biochem. B. Chem. 84. Giddings. Y. 27. M. Emulsion size has been considered to be related to emulsion effectiveness. The main obstacles to its widespread use are that the method is not well known and that commercial SF systems are not yet available. C. 1997. including human albumin and IgG. Y. Throughput was ~1. S. Anal. Chem. 959.4 gravities. Sci. Gupta. C. 2945.
0. J. Taiwan (cbfuh@mail. C. Y. Microcolumn Sep. C. 119. M.. Technol. N.. S. 813. colloids.tw). Giddings. C. as predicted by theory. 345. Microcolumn Sep. Chromatogr. Barman. Myers. J. Sep..0
0.. Giddings. Fuh. C. 1994. Ind. reasonably matched experimental results. Wufeng. Diffusion effects played important roles in limiting the pI difference in this case. Sci.8
1. Levin. C.
~15 mg/h (5). Particle-size analysis of original and fractionated pharmaceutical emulsions (Fluosol) by sedimentation FFF.. As a result. 23. Sep. 1489.. News 1988. Sci. R. M. C. toxicity. Levins.