HPIEC

Instrumentation. A Pharmacia BioPilot Column Chromatography system was used to perform large-scale size exclusion chromatography (SEC) with an 11.3 x 90 cm BioProcess column packed with Sephacryl S-200 HR gel. High performance size exclusion (HPSEC) and ion exchange chromatography (HPIEC) were conducted with Pharmacia Superose 6 and 12 (HR 10/30) and Mono-Q (HR 5/5) columns respectively, equipped with Beckman model 520 system controller and Beckman model HOB HPLC pumps. 

Hig purity /l-D-g uoosidase. The high purity em me grade for kinetic and DSC studies was prepared by a sequential HPSEC and HPIEC procedure, previously reported by Baker et iX. (77), using the Sephacryl S-200 and Mono-Q columns, respectively. This approach yields a -D-glucosidase preparation of approximately 99% purity by SDS-PAGE. 

High-Performance Ion Exchange Chromatography (HPIEC) of Mono- and Oligosaccharides... 

FIGURE 2 Purification of a 7.5 kD natural product peptide1 la (a) CE of peptide purified by reversed-phase HPLC, (b) preparative HPIEC of the peptide purified by reversed-phase HPLC, and (c) CE of the peptide purified by HPIEC. 

The differences between CIEC and HPIEC are that microsize resin beads are used as the stable phase fluids are forced to flow through the ion-... 

In industrial CIEC processes, when the linear flow rate is less than 1.0 cm/min, the band can only migrate at a speed of 0.02 cm/min, but the HETP reaches a value as high as 0.86 cm. In HPIEC processes, the linear flow rate can be greatly increased because of the use of microsize resin beads. When the concentration of the chelate displacer (e.g., EDTA) is 0.075 mol/L, and the linear flow rate is 10 cm/min, the band can migrate 0.86 cm/min, while the HETP is only 0.40 cm. This observation illustrates the advantages of HPIEC, namely in high speed and high efficiency. 

Increasing the temperature in HPIEC lowers the pressure buildup in a column. Table 1 indicates that at TO C it is half that experienced at 30 C. Because higher pressure is employed in HPIEC, the gas cannot... 

The advantages of using HPIEC for the separation of RE elements are that it shortens the separation time, increases the recovery of products, minimizes the need for complicated equipment, and makes the process amenable to automatic control. To illustrate this the separations of Tm-Yb and Lu-Yb are presented as examples. The results obtained with high pressure and classical chromatography are compared in Tables 2 and 3, respectively, for this purpose. 

From the results obtained we can see that when the concentration of the eluate is identical and the flow rate in HPIEC is 5-10 times greater than that employed in CIEC for the separation of Tm-Yb the recovery of Yb in HPIEC is considerably higher than in CIEC. The Tm can be obtained as product in HPIEC but not in CIEC as well to demonstrate the separation advantage afforded by HPIEC. 

Table 2 Results of Separating Tm and Yb with CIEC and HPIEC...

The equipment used in HPIEC is different from that used in CIEC. The columns, valves, and pipes are exposed to high pressures. This requires them to be not only corrosion resistant to acids, bases, and other chemical agents, but they also need to be pressure resistant. Because of this, the materials used in their construction are required to be of higher quality than the material used to construct the CIEC devices. The devices, e.g., the column, valves, pipes, sieves, pressure vessels, and pressure gauges used by the authors, were primarily made of special stainless steel. Some junctions were made of polytetrafluoroethylene plastic. Usually the last column of a system of columns was made of glass so as to permit observation of the separation affected. 

Table 4 Column Parameters and Functions of HPIEC Three-Column System...

The five-column HPIEC system, because of the sizable scope of its functions, was used by the authors to study those rare earth separations that were too difficult to examine with the three-column system. The largest rare earth oxide sample employed with it was 50 g. 

When use of the ion-exchange separation method developed in this study was considered it was determined from the separation factor calculated from the stability constants 4] of their complexes with EDTA that a column ratio greater than 40 would be needed to separate them. Experiments showed that a column ratio nearly 10 times larger would be needed to affect their separation with CIEC. Theoretically based studies [7-9] led to success in separation Eu and Gd by the use of HPIEC and a binary displacer. This technique has made the separation of Gd and Eu simple and useful enough to warrant its use as an industrial process. 

The introduction of HPIEC has overcome many disadvantages such as longer period of production, lower efficiency, and higher cost, that are encountered with CIEC. Under the conditions studied in this chapter, production efficiency can be increased by a factor of hundreds. HPIEC has provided new and more useful ways of separating the RE elements. Once separated, the purities of RE products can reach 99.9-99-99%. The problem of separating Gd-Eu without prior reduction of Eu has been solved. This development is not only of fundamental significance, but is also important to industrial practice. 

L. Q. Chen et al., A New Technical Process of Separating Heavy RE Elements by HPIEC, A monograph of Journal of Lanzhou University (III), 1984. 

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