Reversed-phase HPLC buffers

Reverse phase HPLC buffers Buffer A 0.1% trifluoroacetic acid. Buffer B Buffer A containing 90% acetonitrile. 

Since it is necessary to remove solvent from the product, the mobile phase buffer must be considered. Some popular reverse-phase HPLC buffers, such as phosphates or zwitterion organic buffers, are nonvolatile. They must be replaced by a volatile buffer such as formic acid or ammonium acetate. Otherwise, a desalting step must be added. Trifuouroacetic add is another common buffer. Although it is fairly volatile, it forms a salt with the basic product and therefore cannot be completely removed from the final product. 

Berzas Nevado, J.J. et al., A reverse phase HPLC method to determine six food dyes using buffered mobile phase, Anal. Lett., 31, 2513, 1998. 

Addition of 4-nitrophenyl (3-D-glucopyranoside to a solution of the 20 kinds of primary alcohols dissolved in phosphate buffer (pH 5) containing (3-glucosidase was carried out over a period of 16-32 h. The reaction can be easily monitored by reverse-phase HPLC and terminated when the formation of the desired product is at a maximum. The results are summarized in Table 1. 

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,... 

The disassembly rate of dendritic molecules 18 and 19 was evaluated in phosphate buffered saline (PBS, pH 7.4) in the presence and absence of PGA. The release of tryptophan was monitored by a reverse-phase HPLC at a wavelength of 320 nm. The results are presented in Figs. 5.11 and 5.12. No disassembly of either system was observed in the buffer without PGA (data not shown). In the presence of PGA, dendritic molecule 18 disassembled to release tryptophan within approximately 4 days (Fig. 5.11), whereas dendritic molecule 19 released its tryptophan tail units within 40 min (Fig. 5.12). 

Tissue Weigh sample homogenize in aqueous zinc acetate using a rotostator at 18,000 rpm for 20 seconds dilute with horate buffer convert to methylene blue. Ion-interaction reversed-phase HPLC nmol g 1 NR Mitchell et al. 1993... 

Reverse phase HPLC describes methods that utilize a polar mobile phase in combination with a nonpolar stationary phase. As stated above, the nonpolar stationary phase structure is a bonded phase—a structure that is chemically bonded to the silica particles. Here, typical column names often have the carbon number designation indicating the length of a carbon chain to which the nonpolar nature is attributed. Typical designations are C8, C18 (or ODS, meaning octadecyl silane), etc. Common mobile phase liquids are water, methanol, acetonitrile (CH3CN), and acetic acid buffered solutions. 

Reverse phase HPLC of FMOC-amines. Amines were derivatized with 9-flu-orenylmethyl chloroformate (FMOC-Cl) at pH 8. Products were extracted with pentane, diluted with 25% (v/v) acetonitrile in buffer pH 8, and analyzed by reverse phase (RP) HPLC on a Varian ODS-80TM column af 40°C wifh gradienf elution and fluorescence defection (X, nm) as described elsewhere (Bank ef al., 1996). 

The most commonly used stationary phase for the separation of aspartame from synthesis intermediates, stereoisomers, and degradation products is the reverse-phase Cl8 column. As can be seen on Table 2, the main type of the mobile phase used is a phosphate buffer at pH ranging from 2.5 to 5.0 associated with acetonitrile (14,55,80,83). Reverse-phase HPLC with gradient elution of acetonitrile in phosphate buffer has also been used (16,78). 

Automated HPLC methods were developed for the determination of subsidiary dyes, intermediates, and side reaction products of erythrosine. Peeples and Heitz (154) described a reverse-phase HPLC method to monitor the purity of erythrosine and a series of xanthene dyes involving a yuBondapak Cl 8 column and mixtures of methanol and ammonium acetate buffer. 

Lower-sulfonated subsidiary colors of sunset yellow, among them 5-(phenylazo)-6-hydroxy-naphthalene-2-sulfonic acid (ANSC) and 4-[(2-hydroxynaphthalene-l-yl)azo]benzenesulfonic acid (BNSC), were determined by reverse-phase HPLC using Novapak Cl 8 and gradient elution with a water-tetrahydrofuran solvent system buffered with ammonium acetate (192). 

Flak and Schaber (5,62) used reverse-phase HPLC for the quantitative and simultaneous determination of benzoic acid and sorbic acid, as well as 4-hydroxybenzoic acid, salicylic, 5-nitrofurylacrylic, and p-chlorobenzoic acid and the EsHBA (methyl, ethyl, propyl) in wines and beverages. The first five compounds can be determined by isocratic elution from a Clg column using 0.12 M acetate, pH 3.8 acetonitrile (85 15), and all may be separated with gradient elution (increasing acetonitrile from 10 to 60%, with a simultaneous decrease of the pH of the acetate buffer from 3.9 initially to 3.3). 

Ethoxyquin, a synthetic antioxidant, is not generally allowed for human consumption in foods, but it is being added to animal feed and to fruits as an antiscald agent (94,143). Ethoxyquin is also used in the spice industry to prevent carotenoid loss during postharvest handling. However, ethoxyquin-treated paprika is unacceptable for some markets and some consumers (129). Perfetti et al. (130) described a method for determination of ethoxyquin in paprika and chili powder. Ethoxyquin was extracted from the spice with hexane and partitioned into 0.3 N HC1. After adjusting the solution to pH 13-14, ethoxyquin was extracted into hexane, and the hexane layer was evaporated to dryness. An acetonitrile solution of the residue was then analyzed by reversed-phase HPLC, with detection at 254 nm. The mobile phase was water/acetonitrile with ammonium acetate buffer. Recoveries from samples fortified at 50, 100, and 200 ppm averaged 92%, with a coefficient of variation of 2.3%. The method was applied to a number of commercial samples of paprika and chili powder. Ethoxyquin was found in paprika samples at levels up to 63 ppm and in chili powder samples at levels up to 20 ppm. 

The determination of OXO in Japanese oyster was realized using reversed-phase HPLC. Samples were extracted with LLE and SPE recoveries were 88.3% (193). Oyster samples were homogenized with a phosphate buffer adjusted to pH 7. After centrifugation, supernatants were concentrated using an SPE C-18 cartridge. Before use, the cartridge was activated with MeOH and phosphate buffer. After the sample had been passed, the cartridge was flushed with water and the analytes were eluted with MeOH-orthophosphoric acid (9 1). The eluate was evaporated, and the residues were dissolved in the mobile phase. The method developed was validated and the study of OXO stability was performed. The limits of detection and determination were 10 and 40 ng/ml, respectively. 

Although a great variety of analytical techniques have been applied to the simultaneous determination of methylxanthines in various matrices, HPLC is the one most frequently used nowadays. Most of the methods are based on reversed-phase HPLC, using ACN, MeOH, or THF in acetate or phosphate buffer as mobile phase and UV spectrophotometric detection (256 -270). Some RP-HPLC methods were proposed in combination with solid-surface room-temperature phosphori-metric detection (271), mass spectrometry (272), or amperometric (273) detection. The separation can also be achieved by RP ion-pair or ion-interaction HPLC (274-277) or micellar HPLC (278). In contrast, in recent years few normal-phase HPLC methods (279) were reported (see Table 5). 

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