Representative reverse phase conditions

TABLE I I Representative Reversed Phase LC Column Screening Conditions... 

Figure 12.12 Coupled SEC-RPLC separation of Plioflex rubber stock (a) SEC (b) RPLC ti ace of fraction 1, Wingstay 100 (Eive-peak pattern is representative of diarylphenylenedi-amine isomers) (c) RPLC ti ace of fraction 2, mixed disulfide and MBTS (2,2 -thiobis (ben-zothiazole)). Obtained under the same conditions as given for Eigure 12.11. Reprinted from Journal of Chromatography, 149, E. L. Johnson et al, Coupled column chromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

Figure 13.9 Coupled-column RPLC-UV (215 nm) analysis of 100 p.1 of an extract of a spiked soil sample (fenpropimoiph, 0.052 mg Kg ). LC conditions C-1, 5 p.m Hypersil SAS (60 m X 4.6 mm i.d.) C-2, 5 p.m Hypersil ODS (150 m X 4.6 mm i.d.) M-1, acetonitrile-0.5 % ammonia in water (50 50, v/v) M-2, acetonitrile-0.5 % ammonia in water (90 10, v/v) flow-rate, 1 ml min clean-up volume, 5.9 ml transfer volume, 0.45 ml. The dashed line represents the cliromatogram obtained when using the two columns connected in series without column switcliing. Reprinted from Journal of Chromatography A, 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in envir onmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

Figure 11.6.3 Gradient HPLC separation of isoflavone standards (see Basic Protocol 3). Peaks 1, daidzin 2, glycitin 3, genistin 4, malonyldaidzin 5, malonylglycitin 6, acetyldaidzin 7, acetylglycitin 8, malonylgenistin 9, daidzein 10, glycitein 11, acetylgenistin 12, genistein. Conditions Waters Nova-Pak C18 reversed-phase column (150 x 3.9 mm 4-pm i.d. 60 A pore size) mobile phase 1% acetic acid in water (solvent A) and acetonitrile (solvent B) flow rate 0.60 ml/min UV detector 260 nm column temperature 25°C. The dotted line represents the gradient of solvent B.

Desalting by reversed-phase HPLC (see Subheading 2.4.) or SEC or micro-SPE onto Ci 8 Zip-Tip (used the conditions recommended by the manufacturer Millipore). The recently developed magnetic beads from Bruker may represent a fast and economical alternative. 

Micelles and cyclodextrins are the most common reagents used for this technique. Micellar electrokinetic capillary chromatography (MECC or MEKC) is generally used for the separation of small molecules [6], Sodium dodecyl sulfate at concentrations from 20 to 150 mM in conjunction with 20 mM borate buffer (pH 9.3) or phosphate buffer (pH 7.0) represent the most common operating conditions. The mechanism of separation is related to reversed-phase liquid chromatography, at least for neutral solutes. Organic solvents such as 5-20% methanol or acetonitrile are useful to modify selectivity when there is too much retention in the system. Alternative surfactants such as bile salts (sodium cholate), cationic surfactants (cetyltrimethy-lammonium bromide), nonionic surfactants (poly-oxyethylene-23-lauryl ether), and alkyl glucosides can be used as well. 

Fig. 11.5.2. HPLC separation of prostaglandins. Chromatographic conditions column, pBondapak fatty acid reversed phase column mobile phase, water-acetonitrile-benzene-acetic acid (76.7 23.0 0.2 0.1) flow rate, 2 ml/min detection, one-minute fractions were collected and assayed for radioactivity. The peaks shown represent 1-2 Jig or 0.05-1.0 jiCi of tritiated compound. I5KH2E2, 15-keto-13,14-dihydro PGEj.

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