Anal. Chem. 2003, 75, 6105-6108
Circular Asymmetrical Flow Field-Flow Fractionation for the Semipreparative Separation of Particles Michael Maskos*,† and Wolfgang Schupp‡
Institut fu¨r Physikalische Chemie, Universita¨t Mainz, Welder Weg 11, D-55099 Mainz, Germany, and ConSenxus GmbH, Binger Strasse 17, D-55437 Ober-Hilbersheim, Germany
A new technique for the separation and characterization of particles and polymers based on asymmetrical flow field-flow fractionation was developed. The new circular asymmetrical flow field-flow fractionation instrument (CAFFFE) resembles a quasi-parallel arrangement of 12 individual flow channels. As compared to the classical asymmetrical flow field-flow fractionation (AF-FFF), which can be used so far only for analytical separation and characterization of particles and polymers, the CAFFFE allows the introduction of higher amounts of sample into the channel in a single run so that semipreparative to preparative separation becomes possible. This was demonstrated by the separation of polymer latex standards. The separation and characterization of particles and polymers in solution is sometimes a great challenge, especially in solutions of complex composition. For example, Ma¨chtle reported the characterization of PBA particles with grafted SAN by ultracentrifugation with high experimental effort.1 Asymmetrical flow fieldflow fractionation (AF-FFF) on the other hand, is a powerful tool for the separation and characterization of particles, polymers, and even complex mixtures.2-7 The centerpiece is a small ribbonlike channel with a semipermeable membrane as one of the two walls, as shown in Scheme 1. Between these two walls, a laminar flow is established in the elution mode. During sample introduction, the loaded material is accumulated at the membrane. The particle drift with the solvent cross-flow toward the membrane is counterbalanced by the diffusion of the particles or polymers in the opposite direction. This leads to the formation of a quasi-exponential concentration profile for each particle species, which is transported during elution by the parabolic flow through the channel to the outlet. * Corresponding author. Phone: 49-6131-39-24190. Fax: 49-6131-39-23768. E-mail:
[email protected]. † Universita¨t Mainz. ‡ ConSenxus GmbH. (1) Ma¨chtle, W. In Analytical Ultracentrifugation in Biochemistry and Polymer Science; Harding, S. E., Rowe, A. J., Horton, J. C., Eds.; The Royal Society: Cambridge, U.K., 1992. (2) Wahlund, K.-G.; Giddings, J. C. Anal. Chem. 1987, 59, 1332. (3) Wahlund, K.-G.; Litze´n, A. J. Chromatogr. 1991, 548, 393. (4) Litze´n, A.; Wahlund, K.-G. Anal. Chem. 1991, 63, 1001. (5) Tank, C.; Antonietti, M. Macromol Chem. Phys. 1996, 197, 2943. (6) Co ¨lfen, H.; Antonietti, M. New Dev. Polym. Anal. 2000, 150, 67. (7) Jungmann, N.; Schmidt, M.; Maskos, M. Macromolecules 2001, 34, 8347. 10.1021/ac034394z CCC: $25.00 Published on Web 10/04/2003
© 2003 American Chemical Society
For particles larger than the ones analyzed here, additional separation mechanisms have to be taken into account.8-10 In a given experimental setup, the separation is mainly influenced by the ratio of the cross-flow V˙ c to the channel outlet flow V˙ and the diffusion coefficient D of the analyte. In a simplified approach, the retention time tR is related to the diffusion coefficient by2
tR ) t0w2V˙ c/V06D
where t0 is the void time, V0 is the void volume, and w is the channel height. The sample load is crucial in AF-FFF, because an overload will severely disturb the separation and a characterization becomes impossible.11-13 A typical sample load is on the order of 0.5-50 µg of particles or polymers per run, depending on the size and the properties of the analyte. Here we introduce a new instrument based on the AF-FFF separation principle that allows the separation of substantially higher amounts of material so that the successful analytical separations already shown to be possible by AF-FFF, for example, the separation and characterization of proteins and aggregates,14 ribosomes,15 polysaccharides,16 or colloids,7 can be performed in a larger scale for semipreparative to preparative fractionation. EXPERIMENTAL SECTION Materials. Water was purified with a Milli-Q deionizing system (Waters). Sodium azide, blue dextran, the surfactant Tween 20 (Fluka), and the dispersions of latex standards with narrow size distribution (solid content 1%, Duke Scientific) were used as received. (8) Giddings, J. C.; Chen, X.; Wahlund, K.-G.; Myers, M. N. Anal. Chem. 1987, 59, 1957. (9) Martin, M. In Field-Flow Fractionation Handbook; Schimpf, M., Caldwell, K., Giddings, J. C., Eds; Wiley: New York, 2000. (10) Martin, M. Adv. Chromatogr. 1998, 39, 1. (11) Wahlund, K.-G.; Litze´n, A. J. Chromatogr. 1989, 461, 73. (12) Litze´n, A.; Wahlund, K.-G. J. Chromatogr. 1989, 476, 413. (13) Wittgren, B.; Wahlund, K.-G. J. Chromatogr., A 1997, 791, 135. (14) Wahlund, K. G. In Field-Flow Fractionation Handbook; Schimpf, M., Caldwell, K., Giddings, J. C., Eds.; Wiley: New York, 2000. (15) Arfvidsson, C.; Wahlund, K.-G. Anal. Biochem. 2003, 313, 76. (16) Wittgren, B.; Wahlund, K.-G.; Andersson, M.; Arfvidsson, C. Int. J. Polym. Anal. Charact. 2002, 7, 19.
Analytical Chemistry, Vol. 75, No. 22, November 15, 2003 6105
Scheme 1. Simplified Scheme of the Separation in AF-FFFa
Figure 1. Picture of the CAFFFE channel. The syringe placed on top of the channel contains a solution of blue dextran.
a (Top) x, direction perpendicular to the membrane; z, channel flow direction; li, idealized characteristic transport zone distance (assuming a quasi-exponential concentration profile) from the membrane for species i. The scheme shows the accumulation of a simplified trimodal particle mixture and the schematized establishment of a quasi-exponential concentration profile (focus, relaxation) and the separation of the mixture (elution). Flow directions in focusing mode (A); in elution mode (B)
Circular Asymmetrical Flow Field-Flow Fractionation (CAFFFE) System. CAFFFE measurements were performed using a ConSenxus Flowbox 2, a Bronckhorst Hi-Tec Liqui-Flow delivering the cross-flow, a SFD degasser GT-152, a valve box, and a controller. A SFD analytical UV detector UV/Vis 3240 operating at 254 nm monitored the eluting particles. A bypass is needed in front of the detector in order to reduce the flow through the detector and avoid a high back pressure induced by the detector. Regenerated cellulose membranes were utilized as semipermeable walls (MWCO 10 000, ConSenxus). Degassed Milli-Q water with NaN3 (200 mg/L) and Tween 20 (100 mg/L) was used as eluent. The circular channel was constructed in the following way.17 The circular membrane (diameter 20 cm) was placed on top of a 6106 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
metal frit in a steel manifold. The PMMA cover plate has a circular cutout of 20 cm in diameter and 500 µm in height and is mounted on the steel ground plate with the cutout facing the membrane. The system is sealed with an O-ring. The PMMA plate has 24 holes with capillaries on the outer diameter (9.5 cm from the center) in equal distance, forming the channel inlet flows. The capillaries are connected with a system of distributors, which itself is connected to the valve box. At 8.5 cm from the center, 12 equidistant holes with interconnected capillaries form the injection system (directly in line with every second of the inlet holes). In the center, a hole with connected capillary is used to collect the channel outlet flow to the detector. At the outer rim of the cover plate, two opposite holes with capillaries in connection with the side of the metal frit are directly attached to the liquiflow forming the cross-flow. Each individual “subchannel” is defined and confined by the neighboring solvent flow. The individual length of the capillary connections were chosen to ensure equal distribution of the solvent flow, which was checked both optically by injection of blue dextran and by UV measurement. A mismatch of the solvent flows generates broad, asymmetric, or multiple void volume and analyte peaks. The flow conditions were 12 mL/min for the channel outlet flow and the cross-flow was decreased from 3 to 0 mL/min. For an individual subchannel, this corresponds to a channel outlet flow of 0.5 mL/min and a cross-flow decreasing from 0.125 to 0 mL/ min. A scheme of the channel and of the flow connections can be found in Figure 1 and Scheme 2. AF-FFF System. In addition to CAFFFE, an analytical AFFFF 2.0 system (ConSenxus) was employed to measure the samples. Except for the channel, the same kind of equipment, solvent, and membrane as compared to the CAFFFE system was used. In this system, a trapezoidal spacer with a thickness of 190 µm was used to form the channel. The channel outlet flow was 1.0 mL/min and the cross-flow was decreased from 0.5 to 0 mL/min. (17) Pat. Pend. DE10117863.8, April 10, 2001.
Scheme 2. Flow Schematics of the CAFFFE System
Figure 2. Elution of blue dextran in the CAFFFE channel.
DLS. Dynamic light scattering was performed with an argon ion laser (Stabilite, λ ) 514 nm, Spectra-Physics) with SP-125 goniometer and ALV-5000 multiple-tau digital correlator (ALV) at 293 K and angles between 50° and 130° in steps of 20°. The solutions were filtered with Millex filters (0.45 µm, Millipore). The correlation functions were analyzed by a cumulant fit to determine the angular dependent diffusion coefficient Dq. The extrapolation to zero scattering angle yields the apparent diffusion coefficient D, which can be translated into the appropriate hydrodynamic radius via the Stokes-Einstein relation.18 RESULTS AND DISCUSSION To visualize the flow path within the circular channel, first experiments were performed by injection of blue dextran into the instrument. After focusing, the sample was eluted in the 12 channels. A picture of this process is shown in Figure 2. Care was taken that the individual streams were eluted parallel in time. In all examples, we used a programmed field (linear decay of the cross-flow rate, constant channel outlet flow rate) during the elution to ensure complete elution of the sample. First, we injected three different latex standards, which were characterized by dynamic light scattering in addition to the data provided by the manufacturer. The diameters of the samples were found to be 50 (sample code PS50), 102 (PS100), and 199 nm (PS200). All samples showed second cumulants smaller than 0.05, indicating low size polydispersities. A mixture of all three latex samples with a total analyte load of 2.5 mg was injected to demonstrate the separation power of the CAFFFE channel. Following the void volume or system peak at the beginning of the measurement, the corresponding peaks (18) Berne, B. J.; Pecora, R. Dynamic Light Scattering; J. Wiley: New York, 1976.
Figure 3. CAFFFE fractogram of a mixture of PS50, PS100, and PS200 (3:2:1 in volume); linear decay of the cross-flow rate and constant channel flow rate.
for the individual latex standard are detected. The fractogram is shown in Figure 3. We obtained separated peaks for each latex standard, although the void volume peak contains ∼9% of unretained sample. Measurements of the individual latex samples resulted in identical fractograms for the corresponding latex sample (not shown). In addition, for PS50 and PS100, nearly no increase in the intensity of the void volume peak as compared to the injection of pure solvent was observed. Only PS200 showed an increase in intensity of the void volume peak corresponding to ∼9% of unretained sample. This indicates that the time of the sample injection and the flow conditions during the sample injection were not optimal, especially for the largest sample PS200. This is also observed in the classical AF-FFF system (see below). Nevertheless, for the measured analyte loads between 0.5 and 2.5 mg, no sample overloading effect and no significant adsorption of the sample on the membrane was observed, which would be detected by, for example, a distorted analyte peak or a shift in the position of the peak maximum.11-13 Employing an analyte load of 10 mg of the Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
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Figure 4. AF-FFF fractograms of the fractions collected after CAFFFE separation of PS50 (a), PS100 (b), and PS200 (c); linear decay of the cross-flow rate and constant channel flow rate.
latex mixture results in a comparable fractogram, although with poorer baseline separation of the analyte peaks. The peak area observed in Figure 3 for the three different lattices corresponds approximately to the amount of analyte injected. From the width in elution time of an individual analyte peak, the dilution factor can roughly be estimated. For example, the elution of PS100 takes ∼8.5 min, which corresponds to 102 mL at a channel outlet flow rate of 12 mL/min. Comparing this to the injected volume of 1 mL yields a dilution of ∼1:100. This value is comparable to measurements in a classical AF-FFF channel. To estimate the effectiveness of the separation of the latex mixture, we collected fractions at the outlet for 1 min around each peak maximum and re-injected 1 mL of each fraction in a classical AF-FFF (Figure 4). The amount of sample injected (∼40 µg) corresponds to the typical sample load used in AF-FFF. The fractograms of the fractions are comparable to the fractograms of the corresponding individual latex and do not contain other latex standards. Here, as already detected in the CAFFFE system, we also observe an
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increased void volume peak due to unretained sample of ∼15% for sample PS200. In the case of PS50 and PS100, the intensity of the void volume peak corresponds to the one obtained when only solvent was injected. This problem obviously occurring in both systems is currently being further investigated. In addition to the analysis with AF-FFF, DLS of the fractions from the CAFFFE system yield the same hydrodynamic diameters as for the corresponding individual latex sample. The fact that the separation with the CAFFFE system takes ∼2-3 times the time of the AF-FFF system is due to the lower channel outlet flow of 0.5 mL/min per subchannel and the slightly different cross-flow conditions in the CAFFFE system. Changing the flow pump in the Flowbox 2, which has a maximum solvent flow of 15 mL/min, to a preparative flow pump should result in faster separation times for the CAFFFE system. CONCLUSIONS The new CAFFFE channel allows us to separate and characterize particles and polymers according to the well-known mechanisms of AF-FFF in semipreparative to preparative amounts. The instrumental setup corresponds to 12 quasi-parallel AF-FFF channels running simultaneously. As compared to a classical AFFFF instrument, the analyte mass loading in CAFFFE was increased by a factor of ∼50 in a single run, although at first glance a factor of 12 would be anticipated. This was successfully demonstrated with polystyrene latex standards. Nevertheless, the reason for the observed increase in sample loading is so far unknown. Ongoing work is related to the increase of channels per plate. First experiments demonstrate that the total sample load can even be increased by increasing the number of subchannels, and the practical limit is going to be investigated. ACKNOWLEDGMENT M.M. thanks Manfred Schmidt for his support. Received for review April 16, 2003. Accepted September 2, 2003. AC034394Z
