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Figure 42. Comparison of column performance at equivalent loading.1106'

even distribution. The larger the pore size of the sintered plate, however, the less efficient the system. Drawbacks of sintered plates are their tendency to adsorb substances on their very large surfaces and the possibility of fouling. These columns may also be stacked.

Whatman has developed a column with a new flow distribution system specifically designed for Whatman's cellulosic ion exchange resins. The bed height is 18 cm. The diameter of the first commercial unit was 40 cm and contained 25 liters of adsorbent. The resin bed is covered with a perforated plate with a high free surface. The slightly conical head plate covers an empty space and has a steep cone in the middle. The total empty head space is about 5% of the bed volume. The feed solution enters the steep cone tangentially in its upper part. The resulting rotary movement efficiently mixes the supernatant liquid and allows gradient elution. It is claimed that filling and equilibration take only one hour.

AMF has developed an unconventional new approach with its ZETA-PREP cartridge. The cartridges consist of concentric polymer screens which bear the ionic groups and are supported by cellulosic sheets. The flow is radial from the outer rim toward a perforated central pipe. The available nominal cartridge lengths are from 3 cm to 72 cm with a constant diameter of about 7 cm throughout. Scaling-up with this approach is quite straightforward. Single cartridges, each mounted in a housing, can be combined to a multi-cartridge system. For such a system, flow rates up to 12 liter/min and bovine serum albumin capacities of 1400 g are claimed. The present ion exchange functionalities available are DEAE, QAE and SP.

The step-wise transition from high pressure liquid chromatography to medium pressure chromatography, such as described for the preparation of pectic enzyme,[109] illustrate the progression toward large scale industrial application of the techniques developed in analytical laboratories. The pressure in these medium pressure chromatography applications is only 6 bar instead of the 100 to 150 bar associated with HPLC. The lower pressure results in longer processing times (about one hour) compared to the 5 to 20 minutes required for an analytical determination with HPLC.

Studies, such as the one by Frolik and coworkers,[110] which examine the effect and optimization of variables in HPLC of proteins, can be expected to contribute to the implementation of this type of protein resolution technique into future commercial biotechnology processes.

The first chromatographic systems capable of handling more than 100 kg/day were merely scaled up versions of laboratory chromatography.fl 11 J[1121 Even with some of these systems it was necessary to recycle a portion of the overlap region to have an economical process. A typical example of such a system would be the Techni-Sweet System of Technichem'1131 used for the separation of fructose from glucose. The unique distributors and recycle system are designed to maximize the ratio of sugar volume feed solution per unit volume of resin per cycle while at the same time minimizing the ratio of volume of water required per unit volume of resin per cycle.

The flow through the Technichem system is 0.56 m3/(hr-m3) with a column height of 3.05 m. The feed solution contains 45% dissolved solids, and a feed volume equal to 22% of the volume of the resin is added to the column each cycle. The rinse water added per cycle is equal to 36% of the volume of the resin. This is much less rinse water than the 60% volume that was required by the earlier systems.

This technique is known as the stationary port technique since the feed solution and the desorbent solution are always added at the same port and the product streams and the recycle stream are always removed from another port. Technichem and Finn Sugar manufacture chromatography systems which utilize the stationary port technique.

One of the earlier attempts'1141 at industrial chromatography used an adaptation of the Higgins contactor for the ion exclusion purification of sugar juices. The physical movement of the low cross-linked resin caused attrition as it was moved around the contactor. It was also difficult to maintain the precise control needed on flow rates because of the pressure drop changes and volume changes of the resin as it cycled from the mostly water zone to the mostly sugar solution zone.

An alternate approach'1151 utilizes moving port or pseudo-moving bed techniques. With these techniques, the positions on the column where the feed solution is added and where the product streams are removed are periodically moved to simulate the countercurrent movement ofthe adsorbent material. At any given time the resin column can be segmented into four zones (Fig. 43). Zone 1 is called the adsorption zone and is located between the point where the feed solution is added and the point where the fast or less strongly adsorbed component is removed. In this zone the slow or more strongly adsorbed component is completely adsorbed onto the ion exchange resin. The fast component may also be adsorbed, but to a much smaller extent. The second zone, Zone 2, is the purification zone and is located between the point where the fast component is removed and the point where the desorbent solution is added. Zone 3 is called the desorbent zone and is between the point where the desorbent is added and the point where the slow component is removed. In this zone the slow component is removed from the resin and exits the column. The final zone, Zone 4, is called the buffer zone and is located between the point where the slow component is removed and the point where the feed solution is added. There is a circulating pump which unites the different zones into a continuous cycle.

Figure 43. Moving port chromatographic column with four zones for continuous chromatographic separation.1115'

Different sections of the column serve as a specific zone during the cycle operation. Unlike the stationary port technique, the liquid flow is not uniform throughout the column. Because of the variations in the additions and withdrawals of the different fluid streams, the liquid flow rate in each of the zones will be different.

With such a system, one must slowly develop the chromatographic distribution pattern through the different zones. It may take from 8 to 36 hours for the pattern to be established. Other practical considerations are that the recirculation system must represent a small (< 10%) portion of a single zone to prevent unacceptable back-mixing which would alter the established chromatographic pattern.

The flow rate and the pressure drop per unit length of the chromatographic column are much lower for the stationary port compared to the moving port system. Also, the moving port system is much less capital intensive. The moving port technique, however, is calculated to require only one-third of the column volume and ion exchange volume and two-thirds of the desorbent volume compared to the stationary port technique.

After the expiration of the UOP patent covering the rotary valve, there have been several modifications to the moving port technique by Amalgamated Sugar,11161 Illinois Water Treatment,'1171 and Mitsubishi.'1181 Each manufacturer has its own proprietary approach for the establishment and control of the chromatographic pattern.

Wankat'1191 proposed a hybrid system which has some of the characteristics of both elution chromatography and the pseudo-moving bed system. During the feed pulse, the feed position was moved continuously up into the column at a velocity that lies between the two solute velocities. The eluting solvent was continuously fed into the bottom of the column. Elution development with solvent was used when the feed pulse was over. This method reduces irreversible mixing of solutes near the feed point. Wankat and Ortiz'1201 have used this system for gel permeation chromatography and claim improved resolution, narrower bands and higher feed throughputs compared to conventional systems. McGary and Wankat'1211 have had similar results applying it to preparative HPLC. This technique uses less adsorbent and produces more concentrated products compared to normal preparative chromatography, but more adsorbent and less concentrated products than pseudo-moving bed systems. Wankat'1221 has proposed that his system will be of most value for intermediate size applications or when only one product is desired.

The key items to identify when considering an industrial chromatographic project are the capital for the equipment, yield and purity of the product, the amount of dilution of the product and waste stream, the degree of flexibility the computer controls allow, the expected life of the ion exchange material and whether the equipment allows for periodic expansion of the resin.

New techniques are continuing to be developed which can be expected to be used in future specialized industrial applications. Multi-segmented columns have been demonstrated for the preparative purification of urokinase.'1231 Begovichand coworkers'1241'1251 have developed a technique for continuous spiral cylinder purifications which allow separation of the basis of electropotential in addition to the selective affinity of the adsorbent resin for the components in solution. A schematic of this device is shown in Fig. 44.

Figure 44. Schematic of the pressurized continuous annular chromatograph.'124'

Another new technology that offers promise for commercial biotechnology purifications is the use of parametric pumping with cyclic variations of pH and electric field. This has been described by Hollein and coworkers. f126' They worked with human hemoglobin and human serum albumin protein mixtures on a CM-Sepharose cation exchanger. The extensive equations they reported for parametric separations allow analysis of other systems of two or more proteins which may be candidates for this type of separation.

Applications of ion exchange and column chromatography techniques have been incorporated into the commercial purification scheme for fermentation products, biomaterials and organic chemicals. While the majority of these applications are on the small scale (less than 500 kg/month but greater than 10 g/month), several large industrial scale applications have arisen in the last decade. The extraction of sugar from molasses, the separation of glucose from fructose, the separation of polyhydric alcohols, the separation of xylene isomers and the separation of amino acids are carried out in industrial scale operations preparing thousands of metric tons of purified material each year. Two recent books t127!!128! provide extensive examples of these applications. Additional examples, mostly of laboratory studies, are available in books specifically on the HPLC of peptides and proteins.[129]t1301 LC-GC and Chromatography are two periodicals with helpful operational suggestions.

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