Reversed-phase HPLC semipreparative

HPLC and Isolation of Mutagenic Fractions. Analytical and semipreparative reverse-phase HPLC separations were performed by using a water-to-acetonitrile linear gradient (J2). Separations were carried out on a Hewlett Packard Model 10084 B equipped with an automatic sampling device, a solvent programmer, a variable absorbance detector, and an automatically steered fraction collector. The instrument was fitted with a 3.9-mm X 30-cm prepacked analytical column of 10-/zm silica particles bonded with octadecylsilane (Bondapack-Cis) for analytical scale. For semipreparative scale separations, the HPLC was fitted with a 7.8-mm X 30-cm prepacked column packed with 10-/xm silica particles bonded with octadecylsilane. Samples for HPLC were injected at volumes of 20 /xL (flow rate 1 mL/min) and 80 /zL (flow rate 4 mL/min), and the absorption was measured at 254 nm. Fractions... 

Mattila et al. (205) described a two-dimensional HPLC procedure for determining vitamin D3 and 25-hydroxyvitamin D3 in meat and milk products. Samples were saponified in the presence of vitamin D2 and 25-hydroxyvitamin D2 as internal standards, and the extracted un-saponifiable matter was subjected to normal-phase semipreparative HPLC to obtain a fraction containing 25-hydroxyvitamin D2 + 25-hydroxyvitamin D3 and a fraction containing vitamin D2 + vitamin D3. The collected fractions were evaporated and purified by reversed-phase HPLC. Fractions were again collected, after which vitamin D3 was quantified by tandem-column reversed-phase HPLC and 25-hydroxyvitamin D3 by tandem-column normal-phase HPLC. Analytical chromatograms of a purified extract of chicken are shown in Fig. 12. 

Colored compounds were isolated and characterised from the reaction products of xylose (1M) and glycine (1M), refluxed for 2 h, initial pH 6. The solubles in light petroleum ether (b.p. 60-80°) were fractionated into at least 13 peaks by semipreparative reverse-phase HPLC, using a water-methanol gradient. 

Pearce and Lushnikova [78] used semipreparative HPLC method for the isolation of the three omeprazole metabolites produced by the fungi. Incubation of Cunninghamella elegans ATCC 9245 and omeprazole allowed putative fungal metabolite to be isolated in sufficient quantities for structural elucidation. The metabolites structures were identified by a combination of LC/MS and NMR spectrometric experiments. These isolates are used as reference standards in the confirmatory analysis of mammalian metabolites of omeprazole. In the LC/MS and LC/MS/MS analysis, components were separated by reversed-phase HPLC on a Hypersyl HyPurity (15 cm x 4.6 mm, 5 /im) column. The mobile phase consisted... 

Purify the resulting crude CPP-PNA construct by reversed phase HPLC on a semipreparative C1 g column. Elute constructs using a 50 min gradient (solvent A 0.1 % TEA in water solvent B 0.1% TEA in acetonitrile) from 20% to 100% solvent B. Record the absorbance at both 260 and 220 nm (see Note 4). 

NMR-guided fractionation yielded benzomalvin C (7) from P. raistrickii in our study. The methylene chloride extract of H10BA2 was resolved by LH-20 size exclusion chromatography (chloroform/methanol, 1 1) into ten fractions. Fraction 2 was further resolved by reverse-phase HPLC (Rainin C8 semipreparative, 50% methanol/water - 100% methanol), yielding eighteen fractions. Fraction 13 of this separation exhibited most of the aromatic peaks of interest in the HNMR, so it was applied to a silica gel HPLC colunm (10% isopropanol/hexane) and was ultimately purified by HPLC, using a bonded phase cyanopropyl column (Rainin CNPr, 20% isopropanol/hexane). 

Another substance P inhibitor was isolated from the mycelial extract — fiscalin B (10), which was originally isolated from Neosartorya fischeri by Sterling -Winthrop labs [46]. The fiscalins are structurally similar to the tremorgenic tryptoquivaline mycotoxins produced by Aspergillus clavatus [47] and A. fumigatus [48]. Fiscalin B was purified by reverse phase HPLC (Rainin C8 semipreparative, 50% methanol/water - 100% methanol) of the initial silica gel fraction 12. 

Penitrems A and B were isolated from the initial silica gel fractions 10 and 11, which were combined, then further resolved by reverse phase HPLC (Rainin C-8 semipreparative, 50% methanol/water - 100% methanol). Forty fractions eluted fraction 22 was resolved by silica gel HPLC (30% ethyl acetate/hexane) to yield penitrem A, and fraction 28 was resolved in similar fashion to yield penitrem B. 

NMR active . Reverse phase HPLC (Rainin C-18 semipreparative, 50% methanol/water - 100% water) resulted in the isolation of a pure, antifungal metabolite which was identified spectroscopically as oxaline... 

The mycelial methylene chloride extract was also active against C. albicans. It was also applied to Sephadex LH-20 (chloroform/methanol, 1 1). Fraction 5 was bioactive, but Fraction 4 was NMR active - it exhibited several peaks typical of a diterpene like taxol. It was further resolved by reverse phase HPLC (Rainin C8 semipreparative, 60% methanol/water - 100% methanol) to yield pebrolide (24) a sesquiterpene benzoate from P. brevicompactum. 

Reversed-phase HPLC (RP-HPLC) is widely used in the purification of MCs with most isolation protocols containing at least one RP-HPLC step. The literature typically reports the use of Cjg bonded to silica as the stationary phase and using 1 cm ID columns. Table 40.4B gives a selection of references for semipreparative/preparative HPLC columns used in the purification of microcystins. 

After hydrolysis of triglycerides, denaturation (ethanol) and extraction (hexane) of milk samples are similar to those procedures used in preparation of plasma samples. EHie to high concentrations of coextracted lipophilic compounds, a semipreparative HPLC cleanup step often is used in these preparations. Indyk et al. (92) and Lambert et al. (99) used adsorption HPLC and hexane/isopropanol mixtures for cleanup and a reversed-phase HPLC for detection, whereas Isshiki et al. (94) used a Cig reversed-phase system with a methanol/acetonitrile mixture for cleanup and a C2 or C3 reversed phase for detection. Canfield et al. (95,96) worked with two open-column chromatography systems (silica) to isolate vitamin K compounds prior to HPLC detection, whereas Schneiderman et al. (98) introduced a completely different method for isolation of these compounds. They used supercritical fluid extraction (SFE) with CO2 as solvent to determine VKl in powdered infant formulas. Details of this particular method will be discussed later in this chapter. 

PET that exhibited inhibitory activity in cholesterol biosynthesis in cultured Hep G2 cells was again prepared in a larger scale. The powder was extracted with ethyl acetate (1 10, w/v). The ethyl acetate layer was concentrated and partially purified by silica gel colunm chromatography and preparative TLC (silica gel plates, 20x20-cm, 2-mm thickness n-hexane ethyl acetate=l l, v/v). The silica gel, in the Rf range corresponding to lovastatin, was collected and extracted with ethyl acetate. Further purification was carried out by semipreparative reversed-phase HPLC (C 8, 8.0x250-mm) (22). Final identification of lovastatin in PET was carried out by mass spectrometry (Micromass Platform System Manchester, UK). 

Semipreparative HPLC has been employed to obtain a vitamin D-rich fraction of the unsaponifiable matter for subsequent quantitative HPLC. Combinations of chromatographic modes used for offline semipreparative and quantitative analysis have included polar bonded-phase/adsorption (211,212), reversed-phase/adsorption (194,213), and adsorption/reversed-phase (70,125). An online two-dimensional HPLC technique using two polar bonded-phase columns has also been described (214). 

Normal-phase/reversed-phase chromatography is the ideal combination for semipreparative and quantitative separations in two-dimensional HPLC. Vitamins D2 and D3 coelute during the semipreparative stage, allowing a narrow retention window to be collected for analysis using internal standardization. By this means, Johnsson et al. (201) obtained a vitamin D3 detection limit of 0.1 yug/kg for milk and milk products. 

We have developed a new photoreactive analogue of paclitaxel, 3 -N-BzDC-3 -N-debenzoylpaclitaxel (109) and its ditritiated derivative ([3H]-109) has been evaluated for its ability to photolabel tubulin and P-glycoprotein.66 Radiolabeled photoreactive analogue [3H]-109 was synthesized by N-acylation of 3 -N-deben-zoyl-2, 7-bis(0-TES)paclitaxel (108) with N-(2,3-ditritio-3-(4-benzoyl-phenyl)propanoyloxy)succinimide ([3H]-107), followed by purification on a reversed phase semipreparative HPLC using a C-18 column (Scheme 21).66 Photoaffinity label [3H]-109 was assessed to possess >99.9% radiochemical purity and a high specific radioactivity (34 Ci/mmol). 

Reversed-phase high-performance liquid chromatography (RP-HPLC) columns (C8, C18) from semipreparative (7 mm internal 0) down to capillary (<1 mm). Phase porosity of 300 A and granulometry of 7 p,m are preferred. 

A sequence (using P-388 guided bioassay) of steric exclusion and partition-type chromatographic separations of the active butanolic fraction by reversed-phase semipreparative HPLC led to the isolation of cephalo-statin 12 (22), CaHyeNzOjz, [a]D 157° (CH3OH), (M + H)+ m/z 945.5444, and cephalostatin 13 (23), [a]D 108° (CH3OH), (M + H)+... 

Following the isolation of fungal taxol from H10BA2, we attempted to isolate and identify some of the compounds that cross-reacted with the taxane specific mAb. All of our H10BA2 extracts showed consistently high taxane titers by CIEIA. CIEIA was used to guide separation of two of these compounds by C-18 reverse phase flash chromatography (acetonitrile-chloroform) and semipreparative HPLC (Taxsil, methanol-water, 66 34) with photodiode array detection. 

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