Semipreparative

TSK-GEL SW columns have also been used to isolate heavy and light chains from immunoglobulins. Kast et al. (7) reported how these chains were desalted and isolated on a 21.5-mm i.d. X 60-cm TSK-GEL G4000SW semipreparative column using a 0.1 trifluoroacetic acid (TEA)/40% acetonitrile mobile phase. 

Also available are semipreparative, 21.5-mm i.d. and preparative 55-, 108-, 158-, and 210-mm i.d. stainless-steel columns packed with TSK-GEL SW parti-... 

Larger particle sizes of each group are for 21.5 mm X 60 cm semipreparative and 55 or 108 mm X 60 cm preparative columns. Maximum separation range determined from estimated exclusion limits. 

All H type analytical columns are supplied containing tetrahydrofuran, with the exception of GMH-HT columns, which are only shipped in o-dichlorobenzene. Semipreparative and preparative columns contain chloroform... 

Shodex preparative and semipreparative columns are supplied in Table 6.10 lrs< of 901 ... 

Based on this background, a selected number of preparative, semipreparative, and analytical aqueous SEC systems for the analysis of polysaccharides, summarized in Table 16.1, are presented and discussed briefly in this chapter. 

Dextran gels have been utilized since the late 1950s (1) for the separation of biopolymers. First attempts on Sephadex (2-5) and Sephadex/Sepharose (6-8) systems are documented for hydrolyzed and native starch glucans. Up until now, particularly for the preparative and semipreparative separation of polysaccharides, a range of efficient and mechanically stable Sephacryl gels (9-14) have been developped. 

A. Preparative Sephacryl S-IOOO and Semipreparative Sephacryl System S-200/S-400/S-500/S-I000... 

Therefore the a(1 6) branching linkages were hydrolized selectively. The resulting constituting chain-length distribution was determined on a semipreparative Sephacryl S-200/S-400/S-500/S-1000 (12 -I- 55 -I- 66 -I- 135 X 1.6 cm)... 

Components of a highly short chain branched (scb) waxymaize and a more long chain branched (Icb) amylomaize were separated on the semipreparative Sephacryl S-1000 system. Both samples contained high dp components that eluted in the exclusion volume, but the percentage of these components was quite different 90% for the scb waxymaize starch and approximately 10% for the Icb amylomaize starch (Fig. 16.6). The degree of polymerization averages for these samples was determined utilizing the previously established linear... 

FIGURE 16.5 Broad standard calibration (linear mode) of a semipreparative Sephacryl S-IOOO system (95 x 1.6 cm) with an aqueous mixture of Blue Dextran, Dextran T-SOO, and glucose eluent 0.005 M NaOH V, i = 75 ml. = 162 ml. 

In summary, semipreparative Sephacryl S-1000 proved to be an appropriate system for the separation of glucan components of any kind of branching... 

FIGURE 16.7 Native starch ( ) and fractions of native starch differing in their branching characteristics (nb/lcb amylose -type fraction scb amylopectin -t/pe fraction ) separated on semipreparative... 

Reproducibility of separation for a Dextran T-500 sample was tested on a semipreparative Sephacryl system S-500/S-1000 (65 + 95x1.6 cm) over a period of 6 months. The elution profiles of Dextran T-500 could be superimposed with deviations in the elution axis of 3 ml ( 1 fraction), and deviations in carbohydrate content within 5% referring to the maximum value at V,e, = 213 ml (Fig. 16.8). 

A range of preparative and semipreparative soft gel systems with an improved mechanical stability and thus the chance to run them with increased flow rates were tested for their potential on the separation of starch glucans. For each of these systems a Sephacryl S-200 precolumn proved to be a perfect shock absorber for sample application, improved reproducibility of separations, and increased lifetime of soft gel systems. 

However, for quantities substantially less than this level, 7- to 10-mm i.d. analytical columns can often be used in a semipreparative mode. By repeatedly injecting 300 to 500 ju,l of up to 1% polymer, reasonable quantities of polymer can be isolated. An autosampler and automated fraction collector can be setup to perform such injections around the clock. Although the larger injections and higher concentrations will lead to a loss of resolution, in some situations the result is quite acceptable, with a considerable savings in time being realized over other means of trying to make the same fractionation. 

Inhibited THF is problematic for semipreparative separations. Because small quantities of polymer are being collected along with larger volumes of solvent, more inhibitor, usually butylated hydroxytoluene (BHT), than sample is often collected in each fraction. Thus, one must carefully consider if the BHT will cause a problem in the subsequent analysis of the isolated fractions. If it does, uninhibited THF or other alternate solvents should be used. It must be remember that if uninhibited THF is used, the analyst must pay careful attention to the inevitable peroxide formation in the solvent/fractions. 

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

Fig. 3-2. Semipreparative RP-HPLC profile of eyelo(Arg-Lys-X-Pro-X-Ala). The erude sublibrary (160 mol) was dissolved in 0.1 % (v/v) TFA and applied to a Whatman Partisil 10 pm ODS-2 (1 x 50 em) eolumn. The peaks were eluted using a 40-min linear gradient of 0-25 % aeetonitrile in water at a flowrate of 7 mL min . Fractions were collected every 2 min and pooled in three fractions as indicated by arrows 130 pmol of peptides was reeovered (yield 81 %). (Reprinted with permission from ref. [75]. Copyright 1998, Ameriean Chemieal Soeiety.)...

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

Designed from CHIRBASE-3D, CHIRSOURCE provides 30 000 structures in terms of configurational diversity, most of them easily available by semipreparative scale on corporate installation or in dedicated companies with minor further optimization. 

For preparative or semipreparative-scale enantiomer separations, the enantiose-lectivity and column saturation capacity are the critical factors determining the throughput of pure enantiomer that can be achieved. The above-described MICSPs are stable, they can be reproducibly synthesized, and they exhibit high selectivities - all of which are attractive features for such applications. However, most MICSPs have only moderate saturation capacities, and isocratic elution leads to excessive peak tailing which precludes many preparative applications. Nevertheless, with the L-PA MICSP described above, mobile phases can be chosen leading to acceptable resolution, saturation capacities and relatively short elution times also in the isocratic mode (Fig. 6-6). 

Food (butter fat) Extract and cleanup on semipreparative HPLC column elute with methylene chloride-hexane GC/ECD No data No data Gillespie and Walters 1986... 

Figure 1. Chromatogram of the completely EPG-digested alkali extract on a semipreparative PAl column.

Figure 3. Fractionation of human serum proteins on Spherogel TSKSW 3000, The conditions were as in Figure 1. The analyses were made using (A) a 50-fiL injection loop with an analytical flow cell (B) a 100-L loop with a semipreparative flow cell or (C,D) a 500-L loop with a preparative flow cell.

The basic aim of PEC applications in clinical chemistry, apart from the recovery of standards of endogenous substances, consists of structural identification of isolated (without further separation) substances of relatively high purity. Therefore, the majority of works devoted to this topic pertain to semipreparative separation. Obtaining low amounts of analytes, achieved by coupling TEC with modem... 

Of course, preparative or semipreparative separation of substances of clinical interest is realized not only on silica gels or impregnated silica gels, but favorable results are also obtained on different stationary phases (e.g., cellulose or ion exchangers). 

Sueeessful semipreparative separations of amino aeids and peptides are usually achieved with the use of planar gel eleetrophoresis examples of PLC applieations are presented in Table 9.1. 

Raith and Neubert [57] have developed a method for the profihng of human stratum comeum ceramides. The method enables the investigation of the role of ceramides in maintaining the barrier function of stratum comeum. TLC using automated multiple development was modified for semipreparative purposes. The fractionation of complex lipid extracts using this method ensured specific, sensitive, and... 

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