Reverse-phase HPLC separations, residual

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 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...

Figure 1. Reverse phase HPLC separation of an in gel tryptic digest of 34 pmol (2.1 pg) of a 62 kD protein (lower panel). The upper panel shows the profile that resulted from incubating an equal size slice of polyacrylamide gel that did not contain protein. The 4 peaks in the 96-102 min region are due to residual Cooraassie Blue. The digest and HPLC separation were carried out as described in Materials and Methods.

Acetochlor and its metabolites are extracted from plant and animal materials with aqueous acetonitrile. After filtration and evaporation of the solvent, the extracted residue is hydrolyzed with base, and the hydrolysis products, EMA and HEMA (Figure 1), are steam distilled into dilute acid. The distillate is adjusted to a basic pH, and EMA and HEMA are extracted with dichloromethane. EMA and HEMA are partitioned into aqueous-methanolic HCl solution. Following separation from dichloromethane, additional methanol is added, and HEMA is converted to methylated HEMA (MEMA) over 12 h. The pH of the sample solution is adjusted to the range of the HPLC mobile phase, and EMA and MEMA are separated by reversed phase HPLC and quantitated using electrochemical detection. 

HPLC with thermospray MS was reported by Hurst et al.40 where residues from an archeological site were analyzed for caffeine and theobromine using reversed phase HPLC coupled to a thermospray MS interface. Samples were extracted in water and separated on a reversed phase column. The presence of theobromine in this sample was confirmed by monitoring the MH+ ion at 181 for theobromine. 

Reverse-phase HPLC can be used for the separation of peptides and proteins. Smaller peptides (less than 50 amino acid residues) may be satisfactorily separated on octadecylsilane (C-18) bonded phases whereas for adequate recovery of larger molecules, tetrylsilane (C-4) or octylsilane (C-8) is recommended. Porous column packing with gel permeation and reverse phase properties is usually required for proteins with relative molecular masses greater than 50 000. 

Analytical-Scale HPLC Separations. Reverse-phase HPLC chromatography favors the distribution of the semi- and nonpolar constituents of a sample of residue organics, whereas normal-phase HPLC chromatography favors the distribution of semipolar constituents (32). This approach is illustrated in Figure 2 by the chromatograms of residue organics from a waste water sample separated by both reverse-... 

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.

Air drawn through a midget impinger containing 10 mL of 2 N I ICl/0.5% DNPH and 10 mL isooctane the stable DNPH derivative formed partitions into isooctane layer, isooctane layer separated aqueous layer further extracted with 10 mL 70/30 hexane/methylene chloride the latter combined with isooctane the combined organic layer evaporated under a steam of N2 residue dissolved in methanol the DNPH derivative determined by reversed phase HPLC using UV detector at 370 nm (U.S. EPA Method TO-5, 1988). 

A solution of 2-iodoadenosine (56 mg, 0.142 mmol) in HjO (75 mL) was placed in a quartz reaction vessel and photolyzed for 7.5 h with a Rayonet photochemical reactor using light with the principal wavelength of 2537 A. The solvent was then removed under reduced pressure, and the residue was purified by reverse-phase HPLC (Amberlite XAD-4 HjO/EiOH 90 10). The separated product was lyophilized and the residue crystallized (HjO) yield 22 mg (55%) colorless crystals mp 237-241 C. 

Recombinant human leptin was recently cloned (8) and expressed in E. coli, and demonstrated to effectively regulate adiposity in mice through modulation of appetite and metabolism (9, 10). The molecule contains four methionine residues at positions 1, 54, 68, and 136. In this paper, we report the separation and characterization of three norleucine-incorporated recombinant human leptins which were uniformly labeled with 15n isotope or double labeled with and isotopes. The extent of incorporation at each methionine residue can be determined by reverse-phase HPLC and amino acid analysis methods. The norleucine incorporation was observed preferentially occurring at the internal Met residues. 

Isofonns F (Met68->Nle68) and G (Met54->Nle54) were not separated from semi-preparative reverse phase HPLC, and were analyzed together for amino acid contents, b The typical hydrolysis yield for methionine residue is approximately 80%. 

An azobenzene-modified oligonucleotide 5 -AAAXAAAA-3 [15, X is the residue having an azobenzene moiety in the side chain (Fig. 4A)] was prepared and further separated into two diastereomers (15a and 15b) based on the chirality of the stereogenic carbon atom of 15 by reversed-phase HPLC [53]. The melting temperature (7m) of the duplex of each diastereomer of 15 with its complementary oligonucleotide counterpart (5 -TTTTTTTT-3 ) was photoregulated by the trans-... 

AEO can be sensitively determined in the form of these corresponding UV-active phenylisocyanate derivatives by UV detection. In this case, the residue of the extraction of a water sample or a solid matrix is dissolved in dichloromethane or dichloroethane. This solution is mixed with phenylisocyanate as well as 1-octanol and/or 1-eicosanol as internal standards and heated to 55 to 60°C for 45 to 120 min. Then the AEO derivatives are separated either by reversed-phase HPLC with regard to different alkyl chain lengths or by normal phase HPLC with regard to different ethoxylate oligomers.The addition of the internal standard is imperative for quantitative determination because derivatization is not completed even after 2 h. ... 

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