Studies in reversed-phase ion-pair chromatography

Bartha, Ákos; Vigh, Gy.; Billiet, H.A.H.; Galan, L. de

doi:10.1016/s0021-9673(01)96042-x

Cited by 58 publications

(6 citation statements)

References 15 publications

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“…This phenomenon has been ascribed to the formation in the mobile phase of micelles and to competitive adsorption when the concentration of the interaction reagent exceeds the critical micelle concentration. 9,10 For nitrate, the retention increased continuously over the entire range of 1 -10 mM octylammonium orthophosphate. This may be attributed to the increasing ionogenic behavior of the stationary phase due to the adsorption of octylammonium ions and the overall energy increases due to ion-ion interaction.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous Determination of Nitrite and Nitrate Ions in Culture Media by Ion Interaction Chromatography

Threeprom

Watanesk

et al. 2002

ANAL. SCI.

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The ion interaction chromatographic technique has been widely used for the determination of inorganic anions. [1][2][3][4][5] This technique has high flexibility because the parameters can be readily varied to achieve suitable separation. Moreover, this powerful technique permits the separation of inorganic anions on commercially available reversed stationary columns and conventional HPLC instrumentation. 6 Culture media samples are complex because they contain inorganic salts, organic compounds, proteins and large particles (which make the solutions turbid). 7 Therefore, good sample preparation and optimization of the chromatographic conditions are needed for determining nitrite and nitrate ions.In this work, the determination of nitrite and nitrate ions in culture media (Staphyllococcus enrichment broth) including Staphyllococcus epidermis were studied. Most strains reduce nitrate ion to nitrite ion and/or ammonia as a result of nitratase and nitritase activities, respectively. 8 The goal was to find the optimum conditions for the simultaneous determination of nitrite and nitrate ions in the culture media by varying the ion interaction reagent concentration. Experimental InstrumentsThe chromatographic apparatus was a Hewlett Packard Series 1100 instrument, equipped with a quaternary pump, a diode array detector, a column thermostat and an autosampler. The chromatograms were recorded and analyzed with Microsoft Windows NT workstation version 4.0 and HP Chemstation for the LC 3D system. A Varian Microsorb MV column (3 µm, 4.6 × 50 mm) was used throughout. An Orion digital pH meter was used for pH measurements. ReagentsUltrapure water from a Millipore Milli-Q system was used to prepare all solutions. All reagents were of analytical grade. Octylamine and zinc sulfate were obtained from Aldrich, potassium hexacynoferrate was obtained from Alfa, and nitrite and nitrate ions in the form of sodium salts were obtained from Baker. Bacto m Staphyllococcus broth was prepared following a method of from the Difco manual. Chromatographic conditionsSolutions of octylammonium orthophosphate at pH 6.3 were used as the mobile phase. They were prepared by adjusting octylamine solutions (1 to 10 mM in concentrations) with orthophosphoric acid until pH 6.3 was obtained. Therefore, the concentration of the mobile phase is denoted by the concentration of the octylamine solution. No organic modifier was present. The chromatographic system was conditioned by passing the eluent through the column until a stable baseline signal was obtained. Usually, about 30 min were necessary. Then, 5 µl of the standard or sample were injected and detected at 210 nm. Sample preparationAfter 100 ml of a culture broth (Staphyllococcus enrichment broth) was prepared based on the method of Difco, Inc., the addition of the starting nitrate followed. The culture broth was sterilized in an autoclave for 15 min at a pressure of 15 pounds (121˚C). After cooling the broth to room temperature, the required amount of S. epidermis (5%v/v) was added into the sterili...

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Section: Resultsmentioning

confidence: 99%

Simultaneous Determination of Nitrite and Nitrate Ions in Culture Media by Ion Interaction Chromatography

Threeprom

Watanesk

et al. 2002

ANAL. SCI.

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“…In the first case, protonated bases and anionic additives (inorganic as well as organic) provide the formation of neutral adducts and can be resolved by a reversed phase mechanism. In the second case, the anionic additives, especially those bearing a hydrophobic portion, can be adsorbed onto the stationary phase, promoting an electrostatic interaction similarly to a strong or weak anion exchange stationary phase [37][38][39][40][41]. In these elution conditions, the anionic silanols are not shielded by counterions; however, their effect on the peak distortion can be negligible.…”

Section: Dynamic Errp-hplcmentioning

confidence: 99%

Static vs. Dynamic Electrostatic Repulsion Reversed Phase Liquid Chromatography: Solutions for Pharmaceutical and Biopharmaceutical Basic Compounds

et al. 2021

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Many efforts have been made to separate basic compounds, which are challenging to resolve in reversed phase liquid chromatography. In this process, they are strongly retained and the peak shape undergoes significant distortion. The principal origin of this has been identified with the non-negligible interaction with residual deprotonated silanols. Consequently, all solutions that efficiently shield silanols are being sought. This review is an upgrade on the use of the electrostatic repulsion reversed phase (ERRP) approach: retention of bases, in protonated form, can be achieved by modulating the charge repulsion caused by the presence of positive charges in the chromatographic system. This study successfully (i) introduced fixed positive charges in the structure of stationary phases, (ii) used cationic and hydrophobic additives in the mobile phase, and (iii) used the ERRP-like approach employed at the preparative level for peptide purification.

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“…To select a counterion for a particular separation, the most important consideration is charge compatibility. Generally, the retention of oppositely charged ionic solutes increases with increasing hydrophobicity (increasing chain length) of the pairing ions when used at identical mobile phase concentrations [370][371][372]. The reversed-phase separation of small or polar cations requires the use of hydrophobic counterions, such as alkyl-and arylsulfonates or sulfates.…”

Section: Retentionmentioning

confidence: 99%

The Column in Liquid Chromatography

Poole

1991

Chromatography Today

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Studies in reversed-phase ion-pair chromatography

Cited by 58 publications

References 15 publications

Simultaneous Determination of Nitrite and Nitrate Ions in Culture Media by Ion Interaction Chromatography

Simultaneous Determination of Nitrite and Nitrate Ions in Culture Media by Ion Interaction Chromatography

Static vs. Dynamic Electrostatic Repulsion Reversed Phase Liquid Chromatography: Solutions for Pharmaceutical and Biopharmaceutical Basic Compounds

The Column in Liquid Chromatography

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