Open column gradient elution

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

The rise in popularity of HPLC is due in large part to its high-performance nature and the advantages offered over the older, noninstrumental open-column method described in Chapter 11. Separation and quantitation procedures that require hours and sometimes days with the open-column method can be completed in a matter of minutes, or even seconds, with HPLC. Modern column technology and gradient solvent elution systems, which will be described, have contributed significantly to this advantage in that extremely complex samples can be resolved with ease in a very short time. 

Equipment for supercritical fluid chromatography is similar to that for HPLC with packed columns12 or open tubular columns. Eluent strength is increased in HPLC by gradient elution and in gas chromatography by raising the temperature. In supercritical... 

Figure 9.152 Profile of separation of relaxed DNA from supercoiled plasmid pBR329 on a DEAE-NPR column. The reaction was carried out with 1 unit of calf thymus topoisomerase I. Twenty microliters of the reaction mixture was applied on the column and eluted with a linear gradient of 0.5 to 0.65 M NaCl for 30 minutes. DNAs from peaks a and b were collected manually and analyzed by electrophoresis on a 1% agarose gel after ethanol precipitation. Inset O.C., open circular S.C., supercoiled. (From Onishi et al., 1993.)...

Enzymatic hydrolysis (trypsine) carried out for fauna samples sediments were treated with 1 mol L H3PO4 in an open focused MW system separation on anion-exchange column (Hamilton PRP-XlOO) step gradient elution with a phosphate-based mobile phase, pH 6-7.5 on-line ICP-MS... 

High-performetnce micro packed fused-silica and open-tubular glass capillary columns were prepared and applied to separations of complex mixtures. Solvent-gradient elution was quite useful for the separation of solutes with wide polarity. Instruments and some applications are described. 

Micro packed fused-silica columns showed high resolution by resolving a lot of components of complex mixtures. Solutes with wide poleurity were sepeirated on high-resolution micro packed fused-silica columns with the help of gradient elution. Chemically bonded open-tubular capillary columns with 31 ym i.d. could resolve epoxy resin oligomers although their performance was somewhat poorer than that of packed columns. 

An example of a separation where both sorption and precipitation-redissolution can be seen for the same sample is shown in Fig. 43 the separation of a SO,000-Da polystyrene sample by gradient elution (water/ tetrahydrofuran gradient, Cig column). The heavy, dashed curve and solid data points represent the solubility curve for this sample, showing the saturation-concentration (Cnn, x axis) plotted versus mobile-phase composition 0. The light curve and open dides are retention data, plotted as mobUo-phase composition at elation (4> versus sample mass (equivalent to... 

Fig. 7. Fractionation by anion exchange chromatography of 1 ml portions of a 20% aqueous solution of ampicillin sodium (initial pH 8.5) kept at 22 °C for the specified periods. The DEAE-Sephadex A-25 column was eluted with 0.05 M phosphate (pH 7.4), with a linear sodium chloride gradient. Peak identities A, ampicillin B, a-aminobenzylpenicilloic acid C, dimer D, mixture of a trimer having a closed -lactam ring and a dimer having an open j -lactam ring E, tetramer /% mixture of a tetramer having an open j -lactam ring and a pen-tamer with an intact j -lactam ring G, hexamer H, octamer. (Bundgaard and Larsen 1977)...

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