Column packing methods preparative scale

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

The same factor is used for the scale-up of the sample load. The translation of an analytical method to a preparative system is obviously dependent on the sorbent characteristics of the preparative column. Several reviews on preparative methods, column-packing techniques, theory, and equipment design have been published [132-135,145]. Specific examples of the application of preparative procedures are listed in Table 5.3. 

YMC Inc. has simplified the process of scaling up an analytical separation for preparative isolation by developing matched R D column sets. Each R D column set contains an analytical methods development column and a preparative isolation column packed from the same lot of packing material. This provides assurance that any separation developed on the analytical column will scale-up directly on the matched preparative column without further method modification. The use of these column sets eliminates potential selectivity differences caused by different types of silica and different particle and pore size packings by providing matched columns. 

Scale-up of chromatographic separations usually is done by testing at several sizes following a sequence such as analytical column. I in. (245 mm) diameter preparative column, 6 in. (15 cm) diameter eolumn. 3 ft (1 m) diameter column, with optimum injection-eJution policy determined at each step. Each scale of operation has its own distribation and packing method problems associated with it, which can ba solved only partially by resting at a smaller scale. 

Some common terms used in preparative-scale liquid chromatography are summarized in Table 11.4, The production rate, specific production, or the recovery yield provide suitable objective functions to judge the relative success of individual methods. For efficient use of the separation system, the production rate and the recovery yield should be maximized. Invariably, this results in operating the column in an overloaded condition. Unfortunately, column operation under nonlinear conditions is complex, and optimum conditions are not as easy to predict as the less demanding, although less powerful, scale-up approach. To scale up an analytical separation, the same column packing, column length, and mobile phase velocity are used, and the column diameter increased... 

Our method for the quantitative analysis of lAA consists of four major steps 1) extraction, 2) prepurification, 3) HPLC, and 4) GC-MS. The major time-consuming steps are the purification steps prior to GC-MS. The traditional prepurification [1, 11] has involved solvent partitioning steps and, in some protocols, open column liquid chromatography. Use of high resolution bonded phase capillary GC columns has allowed increased sensitivity at the mass spectral step, and the use of 3 and 5 /xm HPLC packings has made it reasonable to use shorter columns, thus reducing the time required for HPLC. The improvement in recovery afforded by the shorter HPLC columns and the improved sensitivity of capillary GC-MS suggested to us that it was possible to scale down the sample size to a level that made practical the use of Sep-Pak-Mko disposable mini-columns for sample preparation [2, 6, 7]. 

The purity of the 2-cyclohexenone may be assayed by gas chromatography on an 8 mm. x 215 cm. column heated to 125° and packed with di-(2-ethylhexyl) sebacate suspended on ground firebrick. This method of analysis indicates that the 3-cyclo-hexenone in the product amounts to no more than 3%. The fore-run from this fractional distillation contains substantial amounts of 2-cyclohexenone accompanied by ether, ethanol, and minor amounts of other lower-boiling impurities. Additional quantities of pure 2-cyclohexenone can be recovered by redistillation of this fore-run. The preparation of 2-cyclohexenone has been run on twice the scale described with no loss in yield. The ultraviolet spectrum of an ethanol solution of the 2-cyclohexenone obtained has a maximum at 226 m/i (s = 10,400). 

---