Apparatus

Devices that today might be called countercurrent chromatographs were described as early as the 1930s and were used to separate lipophilic substances such as oil-soluble vitamins. However, the modern era of CCC began in the mid-1960s when Yoichiro Ito observed that if two immiscible liquids were placed in opposite halves of a closed helical coil and the coil was rotated on its axis, the liquids flowed into one another in countercurrent fashion (Figure 1). When placed at the initial interface, the individual components of a soluble sample migrate at different rates determined by their relative partition coefficients, K, in the two-phase system. If K, for example, is defined as concentration in the shaded phase divided by concentration in the unshaded phase shown in Figure 1 then components with K higher than unity will migrate to the right, while those with K less than unity will migrate to the left, and those with K equal to one will concentrate in a band at the initial interface. Ito's first CCC apparatus produced true countercurrent flow and was demonstrated to separate soluble mixtures of dyes and proteins in organic-aqueous systems, and also erythrocytes as an example of particulates that partition in two-phase aqueous polymer systems.

When the ends of the coil are opened, the system resembles an Archimedes' screw pump and both phases migrate in the same direction towards what is called the head end of the helix. The actual direction of flow depends on the handedness, left or right, of the coil and its direction of rotation, and will be towards the right side of the example shown in Figure 1. Now, if either one of the phases is pumped into the right end of the rotating coil in Figure 1, creating head-to-tail flow of the mobile phase, the other phase, attempting to flow towards the head, will be retarded by viscous forces and a very stable equilibrium will be established in which 40 to 60% of the unpumped phase will remain in the column as a stationary phase. Components of a sample introduced as a bolus in the mobile phase will partition between the mobile phase and the stationary phase and be eluted from the coil at the tail end in order of their partition coefficients. This process is exactly analogous to conventional column partition chromato-graphy. As in other forms of chromatography, it is desirable to define the partition coefficient as

K = Q/ Cm where C represents concentration in the stationary (Cs) and mobile (Cm) phases, respectively. Then substances with higher partition coefficients will be retained longer in the column.

Instrument geometries

Ito and colleagues have devised many ways in which a helical or modified helical coil of tubing can be rotated with respect to gravitational and inertial fields to achieve good retention of a stationary phase while minimizing band spreading and promoting efficient mass transfer of solutes. A few will be described here based on their historical or practical significance.

Horizontal flow-through coil planet centrifuge The behaviour just described in 'Apparatus' above is that produced by the horizontal flow through coil planet centrifuge (HFTCPC), first described in the late 1970s. Its characteristic motion is shown in Figure 2B. The device typically consisted of several single-layer helixes formed by winding PTFE tubing, measuring a few millimetres internal diameter, on rods about 12 mm in diameter and about 40 cm long, and mounting several of these, connected in series, on a cylindrical column holder. The holder was geared to rotate on its own axis, twice in each orbit, as it revolved around a central or solar shaft. Separations

typically required 10 or more hours. The HFTCPC was not a commercial success at the time, but the configuration has reappeared as an optional column configuration in some recently described high-speed CCC apparatus.

Droplet countercurrent chromatography Earlier in the 1970s, Ito and colleagues described a simple non-centrifugal CCC technique called droplet countercur-rent chromatography (DCCC). As illustrated in Figure 2A with a mobile heavy phase, it consisted of some 300 glass tubes, 1.8 mm i.d., connected in series with narrow-bore PTFE tubing. Either phase could be mobile. The technique, however, was limited to solvent systems that formed droplets. It relied on gravity for phase separation and was very slow, requiring 60 or more hours for a typical separation. In spite of these limitations, it was successfully marketed and was widely employed by natural product chemists; the technique is still available today.

Multilayer coil planet centrifuge The multilayer coil planet centrifuge (MLCPC) described by Ito and colleagues in the early 1980s is one of the most versatile and widely used instruments. Versions of it are available worldwide from several companies. It is shown schematically in Figure 2C, where the helical coil is seen to consist of several layers of tubing wound concentrically on the column holder, which again rotates twice on its axis in each orbit. The forcefield acting on the coil is quite different here from that obtained in the eccentric configuration (Figure 2B) just described. The orbital radius, R, is typically about 10 cm and the orbital frequency is typically about 800 r.p.m. The coil consists of about 10 to 16 layers of PTFE tubing, 2-3 mm i.d., wound on a spool 5 or more cm wide, starting at a radius r of about 5 cm (half of R) to a maximum of 8 or 9 cm. The winding is not actually a spiral as shown in Figure 2C, but consists of the multiple back-and-forth helical layers typically obtained by winding flexible tubing on a spool. A spool 5 cm wide and 17 cm in diameter might contain about 130 m of tubing 1.68 cm with i.d., with a total volume of about 300 mL.

The forcefield obtained in the MLCPC produces a unique mixing pattern in which the phases in the outward portions of the coil are separated as concentric layers of moving phase and stationary phase, the heavier layer being outwardly directed. On the other hand, in a segment of the inner portion of the coil, comprising about one-third of the coil volume, the phases are quite vigorously mixed. This dynamic mixing pattern is independent of the mobile phase flow rate and since the entire coil rotates through the mixing zone some 13 times each second, very good solute mass transfer is obtained. Typical separations are obtained in from 2 to 6 hours in 300-mL coils and

Figure 2 Schematic illustrations of (A) droplet countercurrent chromatography, (B) the horizontal flow-through coil planet centrifuge, (C) the multilayer coil planet centrifuge, (D) the centrifugal droplet countercurrent chromatograph or centrifugal partition chromatograph, (E) the cross-axis CCC and (F) the laterally displaced cross-axis CCC.

Figure 2 Schematic illustrations of (A) droplet countercurrent chromatography, (B) the horizontal flow-through coil planet centrifuge, (C) the multilayer coil planet centrifuge, (D) the centrifugal droplet countercurrent chromatograph or centrifugal partition chromatograph, (E) the cross-axis CCC and (F) the laterally displaced cross-axis CCC.

much faster separations are obtained, with some loss of resolution, in smaller coils. Resolution is echanced in the MLCPC by its very high retention of stationary phase, often 80% of the coil volume. A unique feature of all Ito-style CCC apparatus developed since about 1980 is the lack of a rotating seal. Because of the gear systems employed to rotate the columns, influent and effluent streams convey liquids from the instrument exterior to the rotating coil using a so-called antitwisting scheme, which does not involve a rotating seal, thereby avoiding leakage, local heating and contamination associated with such seals. Apparatus is available containing one, two or three spools on a single rotor and in some instruments multiple coils are available in a single spool. These may be operated independently or in series making the MLCPC one of the most versatile types of CCC apparatus.

Certrifugal partition chromatography One of the few types of modern CCC instrumentation developed outside Ito's laboratory was designed by Murayama and colleagues at Sanki Engineering (Japan) and was introduced in the early 1980s. It was designated simply as a centrifugal countercurrent chromatograph and today is usually referred to as a centrifugal partition chromatograph (CPC). Some confusion results from use of the CPC acronym since it is widely used to represent the coil planet centrifuge in Ito-style chromatographs. Conceptually the technique has been described as centrifugal droplet countercurrent chromatography (CDCCC, see Figure 2D). This description is based on the early models of the apparatus, which consisted of a series of cartridges of fluoroplastic in which a series of drilled cylindrical chambers were connected by small grooves, superficially resembling the larger gravitational DCCC apparatus. Cartridges were connected in series and spun at high speed in a rotor, to which inlet and outlet lines were connected through rotating seals. The apparatus could be filled with one phase and, when rotated, the other phase could be pumped through. Either the heavy (descending mode) or lighter (ascending mode) phase could be pumped through. In later versions of the apparatus, the chambers and passageways are formed by etching them in the surface of a sandwiched stack of rotor plates. Although described as a form of droplet chromatograph, actual droplet formation may not play a major role in mass transfer since many solvent systems function well in the CPC that do not form droplets in the gravitational apparatus. The apparatus is sometimes characterized as a static system, as opposed to a dynamic system, since mixing and mass transfer are dependent on the chamber design and on the mobile phase flow rate; mixing efficiency increases with increasing flow rate. An advantage of the CPC is that units of very large volume can be constructed since it is not subject to the problems of vibration associated with coil planet centrifuges.

Cross axis and laterally displaced cross axis CCC

Since about 1989, Ito and colleagues have published many papers on chromatographs described as cross-axis, X-axis or laterally displaced cross-axis chromatographs (XL). It is difficult to illustrate these without three-dimensional models, but an attempt has been made to show them schematically in Figure 2E and F. In the X-axis unit, the coil axis is simply tilted 90°, or crossed with respect to the solar axis. In the earlier CCC units (Figure 2B and C), these axes are parallel. In the XL unit, the coil is further displaced a distance L along the planet axis from the central X position, where the solar axis lies in the rotational plane of the coil.

The effect of tilting the coil is to introduce a third dimensional component to the forcefield. In the parallel shaft units (Figure 2B and C), the forcefield vectors all lie in the two-dimensional plane of rotation of the coil. But the X and XL displacements introduce a force vector in the vertical or solar axis plane. These changes have a significant effect in improving phase mixing phenomena and column efficiency. This is particularly important for viscous solvent systems such as aqueous two-phase polymer systems of the polyethylene glycol-salt and polyethylene glycol-dextran type. These systems function poorly in the HFTCPC and even less well in the MLCPC systems. The aqueous two-phase polymer systems are important for the separation of proteins, enzymes and biological particulates. Unfortunately, the X and XL chromatographs are mechanically more complex than previous instruments and are not yet commercially available.

Monitors

Many CCC technicians simply collect fractions and monitor their composition by thin-layer chromato-graphy (TLC). The column effluent can often be continuously monitored by UV absorption spectro-photometry. The main interference with monitoring is the tendency for droplets of stationary phase carried into the flow cell to cause noise spikes or to adhere to the cell windows. The problem is minimized by using a flow cell with a vertical flow path and by flowing a lighter mobile phase in the downward direction, and a heavier mobile phase in the upward direction. This tends to flush droplets of stationary phase through the cell quickly. Other methods used include warming the column effluent just before it enters the monitor cell, or bleeding a stream of a miscible solvent, such as methanol, into the column effluent. Another approach uses a diode array detector to monitor the droplet noise in an area of the spectrum where the analyte does not absorb, such as the visible region, and to subtract the noise, on line, from the absorbance at the wavelength used for monitoring. Fluorescence may be used for monitoring and presents similar problems to absorptiometry.

Evaporative light-scattering detectors are excellent for monitoring compounds that lack chromophores and work with other compounds as well. The volatile solvents employed in CCC are quite compatible with evaporative light scattering as long as no non-volatile constituents like salts or buffers are incorporated into the solvent system. Several publications have appeared linking CCC with mass spectrometry and with nuclear magnetic resonance (NMR).

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

Get My Free Ebook


Post a comment