Poly column HPLC system

HPLC system with an ultraviolet detector (254 nm) and a nonporous poly-(styrene-divinylbenzene) particle based Ci8 reversed-phase column, DNASep (Transgenomic, Omaha, NE, USA). 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Reversed phase HPLC was performed using fused silica columns and solvent delivery systems developed and built in our laboratory (5,6). All chromatographies were carried out on Vydac S itm C18 RP support with or without an SDS removal precolumn. SDS removal resin was obtained from Poly-LC Solvent A was 0.1 % trifluoroacetic acid (TFA) in water and solvent B was 0.07 % TFA, 90 % acetonitrile in water. Water was obtained from a Milli-Q system. Samples were eluted with a gradient from 2% to 92 % solvent B in 45 minutes unless otherwise noted As a standard, cytochrome C digested with Lys-C (CCKCD) was used The separation of the peptides was carried out with or without SDS. 

One of the first separations of statistical copolymers using gradient HPLC was carried out by Teramachi et al. [34]. Mixtures of poly(styrene-co-methyl acrylate)s were separated by composition on silica columns through a carbon tetrachloride/methyl acetate gradient (see Fig. 11). When increasing the content of methyl acetate in the eluent, retention increased with increasing methyl acrylate content in the copolymer. This behavior fitted the normal-phase chromatographic system used. Similar separations could be achieved on other columns as well, such as polar bonded-phase columns (diol, nitrile, amino columns) [1]. 

Samples of resin A and resin C which were shown by TMA to be the two best MF resins were analysed by gel permeation chromatography (GPC). A Waters 515 HPLC pump and GPC system were used and the resins analysed through a Styragel HRl column (for determination of Mw between 100 and 5000) at an elution rate of 1 ml/min, after the column was calibrated with poly(ethylene glycol) (PEG). The PEG samples used for calibration had Mw of 200,300,400,600,1000,2000,3400, 8000, 10 000. Each resin sample after having been disolved in dimethylformamide was tested after filtering through a 0.45 pm filter. A Waters 410 refiractometer was used as the detector. 

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