Reversed-phase mobile

A variety of detectors have been used for the HPLC determination of NOC in foods. These include UV, fluorescence, electrochemical, TEA, and various postcolumn denitrosation detectors. To be applicable for the low-ppb detection of these compounds in foods, an HPLC detector should meet the following criteria high sensitivity and specificity, responsive to all classes of NOC, linearity over a fair range of concentration, compatibility with both normal- and reversed-phase mobile phases, and minimal interference from changes in solvent composition, thereby making it amenable to solvent programming. As will be seen from the following discussion, none of the detectors currently available meet all these criteria. 

The chiral resolutions on re-acidic and re-basic CSPs were carried out under the normal phase mode. However, some reports are also available dealing with the use of reversed-phase eluents, but the prolonged use of the reversed-phase mobile phase is not recommended. With the development of the more stable and new CSPs, the use of the reversed-phase mode came into existence on these CSPs. Currently, both modes of mobile phases (i.e., normal and reversed) are in use. Therefore, the optimization of the chiral resolution on these phases can be achieved by varying the concentration of the mobile phases, including the use of organic modifiers. In addition, the temperature, structures of solutes, and CSPs are also important parameters that control the chiral resolution on these CSPs. 

Ultraviolet Absorbance of Reverse Phase Mobile Phases Ultraviolet Absorbance of Normal Phase Mobile Phases Some Useful Ion-Pairing Agents... 

ULTRAVIOLET ABSORBANCE OF REVERSE PHASE MOBILE PHASES... 

Ultraviolet Absorbance of Reverse Phase Mobile Phases... 

The reverse-phase mode is used for all the separations performed in this experiment. Reverse phase is the term used when the stationary phase is more nonpolar than the mobile phase with regard to the polarity of the sample. The isopropanol/water and isopropanol/vinegar mobile phases are typical of reverse-phase mobile phases, which generally are composed of water mixed with polar organic modifiers. The bonded Cig column used is a very nonpolar surface and is the most popular stationary phase for reverse-phase HPLC. In this experiment the silica column when used in the reverse-phase mode provides a very weak nonpolar surface in comparison to C g. Silica is normally thought of as a highly polar surface and is most commonly used in the normal-phase mode. The use of silica in the normal-phase mode, with a nonpolar mobile phase is the subject of Chapter 9 (Experiment 2). 

The basis for separation employing micellar mobile phases stems from their ability to differentially solubilize and bind structurally similar solutes. Skeptics view MLC as a fascinating example of the incorporation of secondary equilibria for control or adjustment of retention (101). However, it is the ultimate of secondary equilibria since the types of interactions possible with micellar aggregates cannot be duplicated by any single other equilibrium system, or for that matter, any one or mixture of traditional normal or reversed phase mobile phase systems. This is due to the fact that solutes can interact with the surfactant aggregates via hydrophobic, electrostatic, hydrogen bonding, and/or a combination of these factors. 

The FBI is preferentially operated at flow-rates between 0.1 and 0.5 ml/min, thus fitting the optimum flow-rates of a 2-mm-ID column. The FBI can be used in combination with both normal-phase and reversed-phase mobile phases. In reversed-phase LC, the optimum settings of most interface parameters are very much influenced by the water content of the mobile-phase. In general, the best response is obtained with a mobile phase with a high organic modifier content a 70% loss in response between pure methanol and pure water was reported for methylene blue, furosemide, spectinomycin, and 2-chloro-4-iutrobenzamide [90]. 

In the reported LC-MS schemes, both positive and negative ionization modes have been used however, positive ionization is the dominant mode. Thermospray and electrospray interfaces have been utilized for the coupling of reversed phase mobile phases to the MS. In tandem MS, fragments are obtained from the taxane ring (ions with miz 268 and 286) and the C-13 side ester chain (ions with mIz 569, 551, and 509). 

Enantioselective packings Polar or nonpolar Packings with enantioselective cages or enantioselective surfaces, microcrystalline cellulose triacetate, cellulose ester or cellulose, carbamate/sil-ica composites, optically active poly(acrylamide)/silica composites, chemically modified silicas (Pirkle phases), cydodextrine modified silicas Operated either with normal phase or reversed phase mobile phases... 

In chromatographic enantiomer separation, reversed-phase mobile phase condi-... 

Disadvantages of donor-acceptor type CSPs are their rather limited scope of application and incompatibility with polar-organic and reversed-phase mobile phase conditions. Thus, the spatially well-defined, but restricted functional group repertoires of donor-acceptor-type SOs can satisfy the chiral recognition requirements of a few classes of analytes only. In addition, to produce useful levels of enantioselectivity with donor-acceptor-type CSPs, analytes may frequently require dedicated (achiral) derivatization to attenuate basicity/acidity and/or to complement... 

Column reverse phase Mobile phase MeOH water 82 18 Injection volume 50 Detector F ex 200 em 300... 

Both hydrophobic and anionic character are exhibited on bonded silica-based anion-exchange phases. Hydrophobicity is introduced through the alkyl chain portion of the ligand which is buri under the cationic ammonium site. El Rassi and Horvath (47) have studied several different anion-exchange supports under reversed-phase mobile phase conditions of methanol and water. Compared to simple reverscd-phasc materials, similar retention behavior on a homologous series of n-alkybenzenes (CHjfCHjjaQH, where n 0- 3] was observed once phase ratios were normalized. 

When the HPLC instrument is converted from a reversed-phase mobile phase to a normal-phase mobile phase, care should be taken to flush all... 

The question is frequently asked how the pH of a reversed-phase mobile phase should be measured in the presence of the organic solvent or without the organic solvent For the purpose of a reproducible measurement, it really does not matter, as long as it is always done in the same way. But for the purpose of a consistent lab practice, and to put different data on the same basis. 

If an instrument is used for both normal-phase and revetsed-phase chromatography, special care should be taken when converting the instrument from a reversed-phase mobile phase to a normal-phase mobile phase. If any water remains in any comer of the fluid path, it can later bleed slowly into the normal-phase eluent and contaminate the column. Dead-ended T-connections in the fluid path, used, for example, to attach pressure gauges, are a common source of this problem. 

Twenty-five ecdysteroids, derived irom 20-hydroxyecdysone, were studied in both normal-phase (silica column) and reversed-phase (Cig column) modes. IPA/dichloromethane/water (30/125/2), IPA/cyclohexane/water (40/400/3), or IPA/iso-octane/water (30/100/2) was selected as NP mobile phase. Here is an instance where the water concentration in a immiscible matrix (e.g., iso-octane) is increased due to the presence of a mutually miscible solvent, IPA. Methanol/water (50/50), ethanol/water (30/70), or IPA/water (18/72), all containing 0.1% TEA, were studied as reversed-phase mobile phases [423]. Methanol proved particularly effective in the RP separation of analytes that varied by the degree of unsaturation (i.e., number of double bonds). IPA, as a RP solvent, was superior in resolving 5a-5jS pairs. IPA was extremely effective in the separation of 20-hydroxyecdysone and polypodine B mixtures. These results are not surprising since ecdysones are polyhydroxylated. 

Fig. 20 Enantioseparation of 5-methyl-5-phenylhydantoin on Chirobiotic T in (a) reversed phase (mobile phase 20/80 MeOH/10 mM NH4OAC), (b) polar organic phase (mobile phase MeOH) and (c) normal phase (mobile phase 60/40 EtOH/heptane)...

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