Reversed-phase HPLC isolation

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

In the current era many medicinal chemists are unaware of the very important role of compound soUd state properties on aqueous solubility and therefore to oral absorption. In many organizations compound purification by crystallization has disappeared being replaced by automated reverse-phase HPLC purification. If medicinal chemists isolate a compound as a white powder from evaporation of... 

In more recent studies the use of HPLC allowed isolation and counting of individual sterols after administration of labelled precursors. The sterols isolated from mantles and viscera of the nudibranch Doris verrucosa were identified as cholestanol, cholesterol, 24-dehydrocholesterol and 7-dehydrocholesterol [103]. After injection of dl-[2-14C]-mevalonic acid DBED salt, cholesterol (57) and 7-dehydrocholesterol (58) were isolated as the acetates by reversed phase HPLC. Both sterols were found significantly labelled specific radioactivity associated with 7-dehydrocholesterol was higher by one order of magnitude than that associated with cholesterol. This fact would indicate either that the reduction of the A1 double bond of 7-dehydrocholesterol to afford cholesterol occurs at a low rate, or that the cholesterol found in D. verrucosa comes partly from a dietary source. 

BA 3,4-dihydrodiol metabolites were isolated by a reversed-phase HPLC using a Vydac C18 column (Chiu et al., unpublished results). DMBA dihydrodiol metabolites were isolated as described (42). The enantiomeric composition was determined either by CD spectral data or by CSP-HPLC (7.19.20). 

Another cultured cell line of Catharanthus roseus (EU4A), which does not produce detectable amounts of vinblastine and other bisindole alkaloids, was also examined for its ability to transform 78 (183). Cell-free extracts of the culture line were prepared, and the 35,000 X g supernatant solution was used. Incubations with 2r-tritioanhydiovinblastine yielded a mixture from which radioactive vinblastine (52) was isolated. The labeled vinblastine was reisolated after unlabeled carrier was added and rigorously purified by successive thin-layer chromatography, reversed-phase HPLC, and crystallization to constant specific activity. Boiled extracts could not produce labeled 52, thus supporting the involvement of enzymes in the conversion process. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Scammonin VII (62), as a minor ether-soluble resin glycoside, was isolated by reversed-phase HPLC from Convulvulus scammonia L. (47). Under basic hydrolysis, it gave orizabic acid A as weU as 2-methyIbutyric and tiglic acids. Compound 62 exhibits lactonization at C-3 of the rhamnose unit and is acylated by a (25)-methylbutanoyl residue at C-2 of the rhairmose unit and a tigloyl residue at C-4 of the terminal quinovose. 

Figure 2 (right). Reverse-phase HPLC elution profile of the tumor-localizing fraction of HPD isolated by non-aqueous gel exclusion chromatography. (B) Hydrolysis of this material in 50% aqueous THF containing IM HCl, at 37 °C for 24 hours. (C) After hydrolysis in IM NaOH, 50% aqueous THF under the same condition. The major porphyrins resulted are hematoporphyrin (HP), hydroxyvinyl deutero-porphyrin (HVD), and protoporphyrin (PP). 

When 13 and 17 were sialylated on a larger scale (2.0 and 3.5 mg, respectively) glycopeptides 20 and 21 could be isolated in 94 and 64% yields, respectively, after purihcation by reversed-phase HPLC. (From George et ah, 2001)... 

The sample extracts that show either toxicity or no dose response on initial testing should be fractionated. An aliquot of the extract is solvent exchanged to acetonitrile, and an initial analytical scale separation is made to assess the distribution of constituents in the sample. This separation is accomplished by using a Qg reversed-phase system eluted for 45 min with a linear gradient of 0-100% acetonitrile in water. If >75% of the sample elutes after the solvent composition of 80% and 20% acetonitrile, then the fractions are isolated by preparative reversed-phase HPLC. Fraction A is eluted with 100% water fraction B is eluted with a linear mobile-phase gradient from 100% to 75% water and 25% acetonitrile fractions C, D, and E are eluted with gradients with final compositions of 50%, 75%, and 100% acetonitrile. 

HPLC Separations. SAMPLE PREPARATION FOR INJECTION. Isolated residue organics were dissolved in water/acetonitrile solvent mixtures for reverse-phase HPLC separations as follows sample was dissolved in a minimum volume of acetonitrile and diluted with water until... 

Figure 2. Absorbance (254 nm) profile of the analytical-scale HPLC separation of 25 pg of residue organics isolated from an industrially impacted influent waste water. The separations were accomplished via reverse-phase HPLC (top) and normal-phase HPLC (bottom). Mobile phases used in these separations were water (fyO), acetonitrile (CH3CN)> methylene chloride (MECL), hexane (HX), and isopropyl alcohol (IPROH), as indicated.

Figure 3. Absorbance (254 nm) profile of the analytical-scale reverse-phase HPLC separation of 2.5-L equivalents of residue organics isolated from finished drinking water L...

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... 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

In this protocol, polyphenolics isolated as nonanthocyanin and anthocyanin fractions after a sample cleanup are analyzed by reversed-phase HPLC in order to obtain an accurate measurement of individual polyphenolic constituents. 

Phenolic acids are ionized at pH 7.0 and are un-ionized at pH 2.0. This property allows for solid-phase extraction of neutral polyphenolics at pH 7.0 and acidic polyphenolics at pH 2.0 to prevent interference. In this protocol, polyphenolics isolated as neutral and acidic fractions using pH adjustments are analyzed by reversed-phase HPLC in a thermostatically controlled environment. 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

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