Reversed-phase HPLC analysis

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 X 1 mm Cig column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TEA) to 0.1% TEA in acetonitrile, over a period of 55 min, at a flow... 

Figure 9. Reversed-phase HPLC analysis of PAH s extracted from SRM 1649, urban dust/organics, with UV detection, not preceded by normal-phase HPLC clean-up, (Reprinted from reference 72.

Compounds that were included in the pharmacologic profile of [ H]MDA binding were subjected to reverse-phase HPLC analysis to assess their relative lipophilicity. Briefly, each compound (10 pg) was injected onto a Waters Nova-Pak C18 column and eluted with a linear gradient from 95 percent buffer A 5 percent buffer B to 20 percent buffer A 80 percent buffer B (buffer A=95 percent water, 5 percent acetonitrile, 0.1 percent ammonium acetate buffer B=20 percent water, 80 percent acetonitrile,... 

Fig. 2 Reversed phase HPLC analysis of a solution containing the oligonucleotide 7 during irradiation (0-11 min). The dimer containing DNA strand elutes at a retention time of about 16.5 min. The DNA strand after cycloreversion of the dimer elutes at about 19 min. Clearly evident is the clean formation of the product DNA strand upon irradiation...

Jiang, S.X., Liu, X. (1997). Reverse phase HPLC analysis of alkyl sulfonates with nonsuppression conductivity detection. J. Liq. Chromatogr. Related Technol. 20(13) 2053-2061. 

Stainless steel column (25 cm x 4.6 mm) of YWG-C18H37 (10 pm) Aqueous 85% methanol [1.5 mL/min] 267 nm Reversed-phase HPLC analysis in rabbit serum. [97]... 

During the reversed phase HPLC analysis of the tritiated echinocandin 77 it has been observed that the radioactivity of 77 has been detected prior to the UV absorbance of the... 

As an example, Table 4.3.1 presents the results calculated from reversed-phase and normal-phase HPLC analysis, respectively, of samples taken from an industrial WWTP [16]. Concentrations were expressed in microgram per gram of material as received. In the water sample, reversed-phase HPLC analysis showed the presence of NP and NPEO (Fig. 4.3.7(A)) with levels of 0.008 and 0.383 p,gg 1 (sum of NPEO), respectively. Normal-phase analysis of NPEO oligomers... 

Fig. 2.55. Gradient reversed-phase HPLC analysis of flavonoids in white onions (a) and celery (b). ODS column of 150 X 3.9mm i.d particle size 5pm. Mobile phase 20min gradient of 15-35 per cent acetonitrile in water adjusted to pH 2.5 with TFA. Fowrate lml/min. Upper and lower traces represent samples before and after hydrolysis, respectively. Detection wavelength 365 nm. IS = internal standard Qc = quercetin Ap = apigenin Lt = luteolin. Reprinted with permission from A. Crozier et al. [159],...

This method is used for anthocyanin samples of lesser complexity that do not contain acylated pigments. It is the most common method used for anthocyanin analysis and is effective for most commodities the major exceptions being red grape and cabbage containing products. This protocol describes the dilution, filtration, and reversed-phase HPLC analysis of these samples. 

HPLC Separation of Polyphenolics Basic Protocol Reversed-Phase HPLC Analysis of Polyphenolics Separated 11.3.1... 

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. 

REVERSED-PHASE HPLC ANALYSIS OF POLYPHENOLICS SEPARATED INTO NONANTHOCYANIN AND ANTHOCYANIN FRACTIONS... 

Table 11.3.2 Solvent Gradient for Reversed-Phase HPLC Analysis of Polyphenolics3...

Reversed-phase HPLC can separate polyphenolics of extracts on the basis of polarity. HPLC easily produces better resolution among chemically similar compounds in extracts than conventional chromatographic methods. The operating temperature of the column during reversed-phase HPLC analysis should be controlled for data reproducibility. A change in temperature produces only a minor effect, however, on band spacing in reversed-phase HPLC and produces essentially no effect in normal-phase HPLC (Lee and Widmer, 1996). A range of ambient temperatures is widely used, and elevated temperatures are often applied. The retention times of the peaks are dependent upon the type of column and the combination of various solvents used in the method. 

Fig. 44 Reverse-phase HPLC analysis of purified regioselective product triacylglycerols. Sample size 0.5-1.0 mg 5 fi C-18 column (0.49 X 50 cm) 120-min solvent gradient acetonitrile/methylene chloride (70 30 to 40 60, v/v) flow rate, 0.8 ml/min flame ionization detector. Column cleaned with methylene chloride after analysis. L, linoleic Ln, linolenic O, oleic P, palmitic S, stearic.

After renaturation, the majority of the recombinant chemokines are easily quantified by UV spectroscopy, although there are examples of chemokines such as NAP-2, which do not possess an aromatic amino acid that serve as chromophores. In this case, quantification is achieved by comparison of the peak height on reverse phase HPLC analysis to that of a known concentration of another chemokine. They can then be lyophilized after a change of buffer into a trifluoroacetic acid or acetic acid solution, which facilitates their storage as lyophilized powders. It is important that they are redissolved in water, before dilution into buffer or medium. Their handling is easy and rapid, as they are instantly soluble at concentrations as high as 1 mM if necessary, in aqueous solutions. 

Polyphenol Analyses. Skin and seed extracts were prepared as described elsewhere (4,15). Flavonol and anthocyanin composition of grape skin extracts and wines were determined by direct reversed-phase HPLC analysis with diode array detection. The chromatographic conditions were the same as described earlier (16) but the formic acid concentration in the elution solvents was raised to 5% to improve anthocyanin resolution. Quantitations were based on peak areas, using mdvidin-3-glucoside (at 530nm) and quercetin-3-glucoside (at 360 nm) response factors, respectively, for anthocyanins and flavonols. 

Solubilize the peptide in acetonitrile/water/TFA 50 50 0.1 and lyophilize. Solvent used for this step can be changed to increase solubility. Lyophilization can be repeated twice when scavengers are detected after reverse phase HPLC analysis of the peptide. 

Reverse phase HPLC analysis was performed using a Zorbax C3 analytical column. The oven temperature was maintained at 40°C. Solvent A = H20/0.1 % TFA and Solvent B = 100% acetonitrile/0.1% TFA. Flow rate = ImVmin. The column was equilibrated in 35% solvent B. Following sample injection the column was maintained for 5 minutes with 35% solvent B then ramped to 49% solvent B over 45 minutes. Samples were prepared by precipitation with ice-cold acid/acetone (Witkowska et al., 1993) and solubilization of the pellet in 0.1% TFA/20% acetonitrile. 

Figure 2 Reverse phase HPLC analysis of dehydroascorbate modified hemoglobin.

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