Reversed phase ion-pair HPLC

A comparison of ion-exchange, reversed phase and reversed phase ion-pair chromatography in the separation of nucleotides from bacterial cells concluded that the latter method was preferable for analytical separations (Fig. 11.1.10). The resolution obtained allowed 

Optimal mobile phase conditions for the separation of nucleotides using reversed phase ion-pair chromatography have been found to be a phosphate buffer which contains tetrabutylammonium phosphate (TBAP) as the counter-ion (Darwish and Prichard, 1981). Phosphate concentrations between 50 mM and 120 mM gave the best resolution, whilst the addition of TBAP at concentrations between 0.5 mM and 

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A method developed to analyze alkylbenzenesulfonates by reverse phase ion pair HPLC with UV detection has also been applied to the determination of alcohol ether sulfates [286]. 

We have developed reverse-phase ion-pairing HPLC separations of substituted EDTA metal chelates of several transition metals (including Cd, Zn, Fb, and Hg) and several lanthanides (La, Ce, Eu, Dy, Er, Yb, Lu). Detection levels of these chelates are currently being assessed. A sensitive metal ion analysis employing an inherently fluorescent EDTA seems feasible. 

Since the order of increasing CL intensity for alkyl amines reacted with Ru(bpy)32+ is tertiary amines > secondary amines > primary amines, pharmaceutical compounds bearing a tertiary amine function (e.g., antihistamine drugs [99], anticholinergic drugs [100], erythromycin [101], and its derivatives [102]) have been sensitively determined after HPLC separation (Table 3). The method was applied to the detection of d- and L-tryptophan (Trp) after separation by a ligand-exchange HPLC [103], The detection limits for d- and L-Trp were both 0.2 pmol per injection. Oxalate in urine and blood plasma samples has also been determined by a reversed-phase ion-pair HPLC (Fig. 18) [104], Direct addition of... 

Crystal structures are available for many (N)4Co-amino acid complexes (Table I). Many of the diastereomers (AS, AS) in the bis-en series have been resolved using classic crystallization (usually via bromocamphor sulfonate, arsenyl-, or antimonyl-tartrate salts) or ion exchange methods (Table II). Reversed-phase ion-pair HPLC, using aryl phosphate or aryl/alkyl sulfonate ion pairing reagents in MeOH/ H20 eluent, has allowed diastereomer separations to be carried out on analytical amounts (28) (Table II). 

Reverse-phase and ion-exchange HPLC have been used for the determination of intermediates and subsidiaries of synthetic dyes. However, reverse-phase ion-pair HPLC has been found particularly useful for the separation and detection of these compounds (195). 

CR Warner, DH Daniels, MC Fitzgerald, FL Joe Jr, GW Diachenko. Determination of free and reversibly bound sulphite in foods by reverse-phase, ion pairing HPLC. Food Addit Contam 7 515-581, 1990. 

Dopamine /3-hydroxylase is a monoxygenase that catalyzes the hydroxylation of dopamine to form norepinephrine. This enzyme is localized in the chromaffin granules of the adrenal medulla and in the storage vesicles of central and peripheral catecholaminergic neurons. Since these compounds are unstable, this activity is often assayed by following the formation of octopamine from tyramine. For example, in the assay developed by Feilchenfeld et al. (1982), the reactant tyramine was separated from the product octopamine by reversed-phase, ion-paired HPLC (/uBondapak C18 using a mobile phase of 17% (v/v)... 

The assay for oxidation of these amines involves the use of any of the compounds listed above as the substrate and their separation from their respective products, aminochromes, by reversed-phase, ion-paired HPLC (ODS-Hypersil). The column was eluted isocratically using a mobile phase containing 50 mAf potassium phosphate buffer (pH 5.5), 2 mAf sodium heptanesulfonate, and methanol, at concentrations of 7.5 to 17.5% depending on the substrate. The detection was at 300 nm. 

Figure 9.149 Separation of CMP (peak 1) and CMP-NeuAc (peak 2) by reversed-phase, ion-paired HPLC. (From Spiegel et al., 1992.)...

Richardson, D.D., Sadi, B.B.M., and Caruso, J.A. Reversed phase ion-pairing HPLC-ICR-MS for analysis of oiganophosphorus chemical warfare agent degradation products. J. Anal. Atom. Spectrom. 2006, 21, 396-A03. 

A reverse phase ion-pairing HPLC method was developed by the submitters for analysis. Chromatographic conditions A 10-pL sample (0.1 mg/mL in acetonitrile) is injected onto a suitable liquid chromatograph equipped with a Waters Symmetry Shield RP18 column, 250 x 4.6 mm, 5 pm particle size at 40°C with a mobile phase of 0.404 g/L heptanesulfonic acid, sodium salt -i- 0.1% phosphoric acid (Component A, pH 2.2) and acetonitrile (Component B) at a flow rate of 1.0 mL/min, programmed with a linear gradient from 95 5 A B (v/v) to 30 70 A B (v/v) over 20 min. Detection is achieved by UV at 300 nm. The retention time is approximately 10 min. 

The general outlines of a reversed-phase ion-pair HPLC-system are summarized in Scheme 1.1. 

Berberine Phenyl ethyl ami nes,neuroleptic amines,quaternary ammonium compounds Analysis in coptis species Separation by reversed-phase ion-pair HPLC... 

Quaternary ammonium derivatives and basic drugs Atropi ne.scopolami ne,meth-ylatropine,various drugs Pyri dos ti gmine,neosti gmi-ne,edrophonium and their 3-hydroxy metabolites Separation by ion-pair adsorption chromatography Separation by reversed-phase ion--pair HPLC(Fig.4.7) Analysis in biological fluids... 

E.C.Y. Chan, P.Y. Wee and PC. Ho, Evaluation of degradation of urinary catecholamines and metanephrines and deconjugation of their sulfoconjugates using stability-indicating reversed-phase ion-pair HPLC with electrochemical detection, J. Pharm. Biomed. Anal., 22, 515-526 (2000). 

Fig. 11.1.4. Separation of uracil and 5-fluorouracil bases, nucleosides and nucleotides by reversed phase ion-pair HPLC. Chromatographic conditions column, Bondapak Cig (300 x 4 mm) mobile phase, (from 0-30 min) 0.1 mM tetrabutylammonium hydrogen sulphate (Cjg), 2.5 mM tetraethylammonium bromide (Cg) and 2% methanol in 2 mM sodium acetate, 1.5 mM phosphate buffer, pH 6.0 (Buffer A) (from 30-50 min) Buffer A-i-30 mM phosphate detection, UV at 254 nm. Peaks FU, fluorouracil FUR, fluorouracU riboside/ FUdR, fluorouracil deoxyriboside FUMP, fluorouridine 5 -monophosphate 5 dFUR, 5 -deoxyfluorouracil riboside FdUMP, deoxyfluorouri-dine monophosphate UDPG, uridine diphosphoglucose UDP, uridine diphosphate dUDP, deoxyuridine monophosphate UTP, uridine triphosphate. Reproduced from Au et al. (1982), with permission.

The relative merits of ion-exchange and reversed phase modes are discussed in a study of the nucleotide pools from mammalian tissues (Brown et al., 1982). The tissue extracts were initially purified on a Cu -loaded Chelex 100 resin to maximise both resolution and sensitivity during the main HPLC separations. It was concluded that the best routine method was on the anion-exchange column containing jaBondapak NH2 (Fig. 11.1.7), but where necessary this could be complemented with reversed phase or reversed phase ion-pair HPLC. 

The extensive use of reversed phase ion-pair HPLC in peptides has meant that some aspects of ion-exchange HPLC are not being exploited, e.g. for peptide mapping. The isocratic separation of the two desired components from a tryptic digest of an epidermal factor fusion peptide can be achieved using a cation-exchange stationary phase in combination with a step-wise gradient of sodium chloride in... 

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