Eluent reversed-phase HPLC

Moore and Jorgenson eombined the rapid two-dimensional separation aehieved by LC-CZE with SEC to make the first eomprehensive three-dimensional separation involving an eleetrodriven eomponent in 1995. Size exelusion ehromatography separated the analytes over a period of several hours while the reverse phase HPLC-CZE eombination separated eomponents in only 7 min. A sehematie diagram of the three-dimensional SEC-reverse phase HPLC-CZE instrument is shown in Eigure 9.9 (18). A dilution tee was plaeed between the SEC eolumn and the reverse phase HPLC injeetion loop in order to dilute the eluent from the SEC eolumn, sinee it eon-tained more methanol than was optimal for the reverse phase HPLC eolumn. 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Castro and Canselier [114] similarly used reverse phase HPLC with methanol-water containing a low concentration of nitric acid as eluent. Quantification was made possible by using a moving-wire flame ionization detector. 

The sulfanes are soluble in carbon disulfide, benzene, tetrachloromethane, and dry diethylether (decreasingly so in that order) while alcohols and aqueous systems initiate rapid decomposition. For this reason a report on the chromatographic separation of the sulfanes H2S by reversed-phase HPLC using methanol as an eluent [35] was shown to be in error The peaks observed in the chromatogram have to be assigned to bismethoxy oligosulfanes... 

Separation of Extracts from Chlorination of Tyrosine and Phenylalanine. Separation was by reversed-phase HPLC by using Spherisorb-ODS (25 cm X 4.6 mm i.d.) with an eluent of 35 methanol in water for the chlorinated tyrosine extract and 55 methanol in water for the chlorinated phenylalanine extract. 

Figure 6. Reverse-phase HPLC chromatograms from XAD-2/ethyl ether extracts of (a) chlorinated phenylalanine and (b) chlorinated tyrosine. An eluent of 55% methanol in water was used in (a), and an eluent of 35% methanol in water was used in (b).

Miwa et al. (26) have demonstrated that both short- and long-chain fatty acids can also be converted into their 2-nitrophenylhydrazides and separated bv RP-HPLC with acetonitrile-water as the eluent. They have described a method for the direct derivatization without an extraction step and the simultaneous microanalysis of 14 kinds of C 0 0-C22 6 fatty acid hydrazides in a reverse-phase HPLC system (27). 

Sivasubramanian and Anilkumar [81] described a simple reversed-phase HPLC method for the determination of omeprazole and domperi-done from tablet formulations. The analysis was carried out on a Hypersil ODS Ci8 (15 cm x 4.6 mm, 5 /jm) column using a mobile phase of methanol- 0.1 M ammonium acetate, pH 4.9 (60 40). The flow-rate and rim time were 1 ml/min and 10 min, respectively. The eluent was monitored at 280 nm. The method was reproducible, with good resolution between omeprazole and domperidone. The detector response was linear in the concentration range of 10-60 /[Pg.222]

The application of indirect methods in biomedical analysis has been reviewed by Schill and Arvidsson.23 Reversed-phase HPLC is the main field of application for indirect detection, and both charged and uncharged species can be visualized, although sensitivity is better for ionic solutes. With indirect UV detection for reversed-phase HPLC, the eluent contains a chromophoric group (probe), and peaks are obtained for injected solutes as well as for the mobile phase additives (system peaks). For solutes that... 

The eluents most commonly used in peptide purification by reversed phase HPLC are water and acetonitrile. These are often buffered with trifluoroacetic acid (0.1% v/v, TFA), ammonium acetate (0.05-0.1 mol/dm3 at pH 4-8) or phosphate (0.05-0.1 mol/dm3 sodium or potassium salt at pH 2-8). In addition, polymeric reversed phase media also performs well at high pH and is often buffered with ammonium hydroxide or ammonium bicarbonate (0.05-0.1 mol/dm3 at pH 8-9). 

Figure 2.15 Separation of ATP and ADO on ion-paired, reversed-phase HPLC. The column was Ci8 (/iBondapak), and the mobile phase was 65 mM potassium phosphate (pH 3.7) with 5% methanol and 1 mM n-tetrabutylammonium phosphate. The column was eluted isocratically, and the eluent was monitored at 254 nm.

In one method, the Dopa formed during the reaction was partially purified by ion-exchange and aluminum oxide chromatography and the amount present quantified by reversed-phase HPLC (ODS column). The mobile phase consisted of 0.1 M potassium phosphate buffer at pH 3.5. The column was eluted isocratically and the eluent monitored by means of an electrochemical detector. 

In this assay, the substrate, Dopa, and the reaction product were separated by reversed-phase HPLC and eluted isocratically with 0.1 M potassium phosphate buffer at pH 3.0. The eluent was monitored with an electrochemical detector. The separation obtained with this procedure is shown in Figure 9.4B, together with results obtained after incubation of L-Dopa with the enzyme from rat cerebral cortex for 20 minutes at 37°C (Fig. 9.44). Using a calibration curve of the type shown in Figure 9.5, it was possible to show that 1.55 nmol... 

In this study (not shown), the compounds vanillin and isovanillin together with p-hydroxyacetanilide, added as an internal standard, were separated by reversed-phase HPLC (LiChrosorb) with a methanol-50 mM phosphate buffer (pH 7.2) (3 7, v/v) as the mobile phase. The compounds were eluted isocratically and the eluent monitored by an electrochemical detector. 

The reactant was separated from the product as the fluorescamine derivatives on a reversed-phase HPLC column (Partisil ODS) and eluted isocratically using a two-solvent system. To elute the dipeptide, the solvent was 60% acetonitrile in water diluted 9 1 with 1 M acetic acid, at a final pH of 3.5 (Fig. 9.24). To elute the angiotensin compounds, 38% acetonitrile in 1 M ammonium acetate (pH 4.0) was used (Fig. 9.25). The eluent was monitored with a fluorometer. 

The activity is found in erythrocytes, platelets, and lymphocytes, and determination of its value aids in diagnosis of some blood disorders. In this assay, which can readily be used for purine and pyrimidine 5 - and 3 -nucleotidase activities, the nucleoside monophosphate (the substrate) was separated from the nucleoside (the product) using ion-pair reversed-phase HPLC with a mobile phase of 5% methanol-5 mAf potassium dihydrogen phosphate 0.25 mAf 1-decanesulfonic acid was also added to the mobile phase. The elution was carried out at room temperature and the eluent monitored at 254 nm. 

Adenosine kinase catalyzes the transfer of phosphate from ATP to adenosine (Ado) to form AMP and ADP. The separation of the reactants, Ado and ATP, from the products, AMP and ADP, can be accomplished by reversed-phase HPLC (Ci8) with isocratic elution with a mobile phase of 0.1 M potassium phosphate (pH 5.5) and 10% methanol. Detection depends on the substrate. In this assay, it is useful to replace the substrate adenosine with the fluorescent analog formycin A (FoA) and to monitor the column eluent with a fluorescence detector. Thus, ATP and any of its hydrolytic products will not be detected. 

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