New Products and New Areas of Bioprocess Engineering

Fig. 8. Schematic diagram indicating the swapping of liquid and flow pattern during alignment and separation of the chambers

The electrophoretic extraction runs at field strengths of 5 Vm–1 and 10 Vm–1, indicated by the bar diagrams in Fig. 9a, b, respectively.

Electrophoretic extraction results of the latex particles are shown in Fig. 10a, b. From these figures it can be noted that when the field strength was doubled the cell extraction was completed in a much lower number of transfers. For instance, in case of latex particles (size 3.4 mm) at 0.05 Vm–1 field strength, about 450 particles were extracted in 5 transfers (Fig. 10a) and when field strength was increased to 0.01Vm–1 the similar number was extracted in 3 transfers only (Fig. 10b). This can be appreciated from the fact that in Eq. (20) (in Sect. 2.2.3), the cell transport velocity increases proportional to the applied electric field strength under otherwise similar conditions. An almost equal number of cells/particles are electrophoretically extracted in each transfer step as described by the physical model in Fig. 14 (in Sect. 2.2.4) and also by Eq. 20). The exception for this was observed in case of smallest latex particles (size 2.6 mm, data not shown). This is due to the carry over of small particles by the electrode gassing at higher field strength and the extent of this effect being prominent at initial transfers where the particle concentration will be high.

The parity plot of the predicted and experimental values of the electrophoretically extracted cells/particles is shown in Fig. 11. A good agreement can be seen for fixed blood cells but not for latex particles, especially those of lower size range. Two main reasons for this situation are gassing near the cathode and heating of the buffer. Gassing results in a gas/liquid dispersion instead of liquid buffer between the electrodes, distorting the effective electric field. Further, it is causing carryover of smaller latex particles into the top chambers, causing the extracted particles to be higher than the predicted ones. Heating of the buffer causes convection current, which will have more effect on smaller particles in being swapped to the top chambers during transfers. Heating may also lower

a

b

Fig. 9. Bar diagram of electrophoretic extraction of fixed human red blood cells

a

b

Fig. 10. Bar diagram of electrophoretic extraction of latex particles

Fig. 11. Parity plot for the electrophoretic extraction of cells and particles. Latex particles (3.5 mm); latex particles (2.6 mm); fixed blood cells (4.6 mm)

the effective field strength due to the variation in its conductivity with temperature. In order to overcome this problem the strength of the phosphate buffer is reduced from 0.01 mol l–1 to 0.002 mol l–1. Note that the electrophoretic mobility of any given species increases with decreasing buffer ionic strength [74]. This enhancement at lower concentration enables the separation of cells/particles whose mobilities are only slightly different from each other. The electrophoretic extraction results of latex particles with lower ionic strength buffer are encouraging (data not show). As already discussed, the most useful application of ADSEP electrophoresis is to fractionate mixed cells/particles, exploiting the differences in their mobilities. The fractionation of a mixture containing fixed blood cells and latex particles is to be carried out.

Calculations predicted heating rates of the order of millidegrees per second for the applied current using a field that would affect motion of particles with electrophoretic mobilities around 10–4 m2 V–1 s–1 [72]. Theoretical values of temperature-increase (predicted values) were calculated using buffers of known conductivity and compared with exact measurements. In 0.01 mol l–1 phosphate buffer, kE = 3.6 S m–1 (which is considered a high conductivity buffer for use in an electrophoretic instrument) a field of approximately 0.05 V m–1 was applied [72]. The Joule-heating calculation with t in seconds is given by

The plot depicting the above relationship, indicating the variation of DT with the time of application of field at various values of electric currents, is shown in Fig. 12. The above relationship was counter checked by measuring the temperature with a thermistor probe over a 2-min period (twice the typical time period of one electrophoretic transfer) and obtaining the following linear relationship:

Fig. 12. Rise of temperature with the time of application of electric field. 2.5 mA; 5.0 mA;10.0 mA

Interestingly the observed temperature rise was less than the expected rise. This relationship scales linearly with conductivity and applied field (the product EI contains conductivity). The actual experimental temperature profile consisted of a rise from 23.0 °C to 24.2 °C in 120 s. In this extreme case a temperature rise of 0.6 °C per transfer (60 s of field application) could be expected. In a typical experiment involving 20 transfers, the total temperature rise would be 12 °C, typically from 23 °C to 35 °C. Thus when low-conductivity buffers are used there is no obvious reason to resort to thermoregulation since this temperature rise will be much lower.

One significant application of ADSEP electrophoresis is direct measurement of electrophoretic mobilities as demonstrated in the present work. Capillary zone electrophoresis (CZE) is normally used to measure the electrophoretic mobility of solutes in free solution. Several undesirable features of CZE are absent in the present version of ADSEP, namely:

1.Limited to solutes (particles can not be evaluated)

2.Need of a calibration standard due to electroosmotic backflow

3.Non-absolute mobility values as all measurements are relative

4.Small sample volume and hence lack of recoverable amounts of separands

5.No recovery of fractions is possible as both the ends of the capilllary are submerged in buffer

In the present case the cells/particles that move electrophoretically to the top chamber are collected and counted, and by using the following equation (derivation is shown in Sect. 2.2.3) the electrophoretic mobility can be estimated, as m is experimentally determined and other parameters except mE , are known:

In order to confirm the validity of this approach, the electrophoretic mobilities of fixed blood cells, which are well known from the literature, are estimated by this method. The values of estimated mobilities of different cells/particles are in

so that a bioparticle of mobility mE moves a distance y in an electric field of intensity E applied for a time of t. It is obvious that if E is increased t can be decreased to achieve the same distance of migration and vice-versa, under otherwise similar conditions.

The bioparticles are randomly distributed in space over the volume of the bottom chamber. However, the time required for any individual particle to move into the top chamber increases as its distance from the top surface of the bottom chamber increases, under a given set of experimental conditions of E and t. That is, the ratio of these heights gives the relative number of bioparticles that migrate to the top chamber at any given set of E and t. To calculate the absolute number of particles transferred to the top chamber, the ratio of the distance to the top of the chamber to the total height h, has to be multiplied by the concentration of the particles (i.e., number of particles per unit volume), since the ratio of the heights is nothing but the ratio of the volumes of the chamber corresponding to the location considered.

Therefore, when an electric field is applied to capture the particles with mobility mE located at distance y from the top surface of bottom chamber, the number of bioparticles that would migrate during a single step is

where N = (C) (prc2 h) and C is the cell concentration (cells ml–1) and prc2 h is the volume of the chamber and since y = mE Et [from Eq. (22)]. This movement of particles has already been depicted in Fig. 14

2.2.3.2

Mixed Cells/Particles

The bottom chamber contains two types of particles having electrophoretic mobilities m1E and m2E . The change in the number of type-1 particles in a stage n during step r will be equal to the number of bioparticles that migrated to the top chamber during step r. So the general equation can be written for this situation by material balance as

where (m1)n, r is number of type-1 particles with mobility mE1 that migrated from stage n during step r to the top chamber; N is the total number of bioparticles present in any of the n bottom chambers, at t = 0; N1 is the number of bioparticles with mobility m1E ; N2 is the number of bioparticles with mobility

m2E ; N = N1 + N2 and x1 = N1 /[N1 + N2 ] = N1 /N. Now using Eq. (22), Eq. (24) can be written as

Alternatively the number of type 1 particles remaining will, since N1 = x1 (N),

This is a general equation, which enables us to estimate the fraction of bioparticles having mobility m1E at any stage provided their concentration is known in the previous stage. Similar equations can be written for other particles having mobility m2E . It can be easily extended to any number of cell/particle types in the initial sample mixture in the bottom cavity of stage 1.

The bioparticles are assumed to be uniformly distributed in the chamber and they move in a plug flow under the influence of the applied electric field (Fig. 14). Therefore in each step the same number of particles migrate to the top chamber (that are contained by the slug of height y of Eq. 22), and hence the following material balance equation can be written:

where r is the step number. With this model we can predict, for example, the number of bioparticles of different mobilities that migrated during each electro-extraction step and thereby the concentration of these particles in a given chamber during the process of multistage extraction. The model calculations are already shown in the previous section and more details are given elsewhere [72].

2.2.3.3

Heat Transfer

A major problem in the scale-up of electrokinetic processes is known to be heating which in turn causes mixing. However, the major advantage of the multistage process is the speed at which it performs separations. Hence our aim is to determine design modifications (such as provision for proper heat transfer/removal) in order to reduce the adverse effects of heating, while scaling up the process based on the basic laws of heat generation and transmission. Electrical energy is dissipated as heat according to the equation

where W is the power density (kWm–3), I is the current (A), E is the electric field strength (Vm–1), and A is the area (m2) over which the field is applied. For a system that obeys Ohm’s law,

where kE is the electrical conductivity of the medium (S m–1). It may be noted that the heat generation increases as the square of the current passed. For this reason, nearly all electrokinetic applications are performed in the most resistant media compatible with the unit operation meaning that low-con- ductance solutions must be used to have low current in order to operate for

longer periods of time. In an adiabatic stagnant system the temperature gradually increases uniformly with time as

where t is the time of application of electric field, Cp is the specific heat of the media carrying the current (kJ g–1 °C–1), and Ç is the density of the system (kg m–3). The above equation represents the maximum (adiabatic) temperature increase in the electrophoretic system.

In the cases where the temperature rise is higher than a typical case, it will be necessary to calculate further how much heat needs to be removed. For this purpose, the temperature and the circulation flow rate of the coolant needs to be determined. Presuming that the system boundaries remain at the initial temperature, the Grasshof number is given as [57]

where b is coefficient of thermal expansion (°C–1), D is the diameter of the chamber (m) or distance (perpendicular to g) from the high temperature in the system to the closest lateral boundary; Ç and h are the same as defined earlier. The Rayleigh number is the product of Gr and Pr (Prandtl number), that is

where kT is the thermal conductivity of the medium (kWm–1 °C–1). In case of the jacketed slit the critical Ra was reported to be 6.1 by Ivory [75] and around 8.0 by Rhodes and Snyder [58]. In general Ra and Gr should be minimized which usually means minimizing D. However, in the present case D is practically fixed. The only way to reduce these numbers is to reduce g (by operating at low gravity) or minimize DT (close to zero using low I/A).

At normal gravity, the Nusselt number, Nu (hD/kT), can be obtained from these two dimensionless numbers, from which hT (kWm–2 °C–1), the heat transfer coefficient, can be calculated. Then the heat that is to be removed from the system can be calculated as

where DT is already available from the above calculations, and AT is the effective surface (heat transfer) area of the chamber. Then from Q (kW) the flow rate, Mc (kg s–1), of the coolant (with specific heat Cpc) that is to be circulated to remove the required heat can be calculated:

The following conclusions can be drawn based on the work carried out to date. The multistage electrophoretic extraction concept was shown to be capable of electrokinetic transport of cells and particles.Although the depth of the chambers hampers the resolution of cell extraction, studies to date have paved way for new generation equipment. Resolution can be increased by decreasing the depth of the chamber and increasing the diameter or cross sectional area of the chambers.

The heat transfer analysis presented here has led to the design of the next generation of multistage electrophoretic separators. Two types of ADSEP could be designed for the market:

1.An inexpensive version without temperature control, to be used with low conductivity buffers.

2.A more expensive, thermostated instrument that can be used with high conductivity buffers.

3

Extraction of Macromolecules

The world market for industrial enzymes has been estimated at $1.2 billion in 1995 [76]. Production methods, such as fermentation of genetically engineered microorganisms, have advanced tremendously in recent years. This has promoted work on the development of macromolecule products such as recombinant blood proteins, which require production on the multi-ton scale at low cost (only a few dollars per gram) [77].

Generally many unit operations are used in combination, such as filtration (normal or membrane), centrifugation, precipitation, crystallization, etc. for the separation and purification of macromolecules. Chromatography in its various forms (ion exchange, reversed-phase, hydrophobic interaction, affinity, and gel filtration) has proved to be a general purification technique that can achieve the desired product purity. Although individual unit operations for large-scale purification of macromolecules are generally considered to be well developed and satisfactory, the high degree of complexity (and large number) of steps involved in a complete process are often a source of excessive cost and other problems. Because of this, many of the recent developments in this field have been directed at combining and eliminating different process steps [77].

In conventional methods like centrifugation, and even modern methods like electrophoresis and column chromatography, scale-up problems are enormous, making them uneconomical or prohibitively expensive unless the product is of high value. Therefore there has been a need for alternative approaches to bioseparation problems.

Liquid-liquid extraction is one such method. This technology has been successfully used in separation of compounds in chemical and related industries for many years. However, it is only recently that liquid-liquid extraction technology has been recognized as potentially useful in biotechnology [78, 79]. Two classes of two-phase extraction system are suitable for biomolecule recovery:

1.Aqueous two-phase systems (polymer-polymer type and polymer-salt type).