Aqueous normal-phase

Hydrophilic interaction chromatography (HB IC) or aqueous normal-phase chromatography (ANP) refers to the use of polar stationary phases (e.g., bare silica, silica, or polymeric phases with bonded zwitterionic ligands, diol phases) in combination with a mobile phase rich in organic solvent but containing a significant amount of water (typically at least 3%). Bell [97] summarized the advantages of this technique as follows ... 

An unusual but repeatedly successful measure is a typical RP eluent (e.g., buffer/ACN) plus silica. This is the aqueous normal phase chromatography known from the 1980s (in the gradient mode nowadays known as hydrophobic interaction liquid chromatography (HILIC), discussed later). 

In the case of HILIC, polar stationary phases are used with mobile phases consisting of at least one polar and one nonpolar solvent. Usually, these are acetoni-trile/water mixtures with a high proportion of acetonitrile. HILIC is, therefore, an aqueous normal phase chromatography [21, 22). As shown in Figure 4.12, the more polar part of the mobile phase accumulates on the polar stationary phase. The more polar analytes prefer to stay close to the polar surface and are, therefore, more strongly retained. 

Normal-phase liquid chromatography (NP-LC) separates analytes based on the differences in their interactions with the polar stationary phase. In this mode, the stationary phase is more polar than the mobile phase. It normally uses organic solvent as mobile phase but there exists a variant of NP-LC in which water-based mobile phases are implemented aqueous normal-phase liquid chromatography, ANP-LC). 

High performance liquid chromatography Aqueous Normal Phase Chromatography Size exclusion chromatography Micellar liquid chromatography... 

Traditionally, LC and GC are used as separate steps in the sample analysis sequence, with collection in between, and then followed by transfer. A major limitation of off-line LC-GC is that only a small aliquot of the LC fraction is injected into the GC p. (e.g. 1 - 2 p.1 from 1 ml). Therefore, increasing attention is now given to the on-line combination of LC and GC. This involves the transfer of large volumes of eluent into capillary GC. In order to achieve this, the so-called on-column interface (retention gap) or a programmed temperature vaporizor (PTV) in front of the GC column are used. Nearly all on-line LC-GC applications involve normal-phase (NP) LC, because the introduction of relatively large volumes of apolar, relatively volatile mobile phases into the GC unit is easier than for aqueous solvents. On-line LC-GC does not only increase the sensitivity but also saves time and improves precision. 

Comparisons of LC and SFC have also been performed on naphthylethylcar-bamoylated-(3-cyclodextrin CSPs. These multimodal CSPs can be used in conjunction with normal phase, reversed phase, and polar organic eluents. Discrete sets of chiral compounds tend to be resolved in each of the three mobile phase modes in LC. As demonstrated by Williams et al., separations obtained in each of the different mobile phase modes in LC could be replicated with a simple CO,-methanol eluent in SFC [54]. Separation of tropicamide enantiomers on a Cyclobond I SN CSP with a modified CO, eluent is illustrated in Fig. 12-4. An aqueous-organic mobile phase was required for enantioresolution of the same compound on the Cyclobond I SN CSP in LC. In this case, SFC offered a means of simplifying method development for the derivatized cyclodextrin CSPs. Higher resolution was also achieved in SFC. 

Oily crops such as soybeans and canola (oilseed rape) cannot be extracted with aqueous buffers, because the extraction solvent cannot permeate the hydrophobic plant tissue matrix. In these cases, homogenization in acetonitrile-hexane is recommended. This solvent mixture is able to extract sulfonylureas from these samples with a minimum of co-extracted oil. After extraction, the sulfonylureas partition into the acetonitrile phase while most of the oil stays in the hexane phase. Further cleanup is accomplished using a silica SPE cartridge and normal-phase conditions. 

The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Catalysis in Transacylation Reactions. The principal objective of the study was to evaluate 4 as an effective organic soluble lipophilic catalyst for transacylation reactions of carboxylic and phosphoric acid derivatives in aqueous and two-phase aqueous-organic solvent media. Indeed 4 catalyzes the conversion of benzoyl chloride to benzoic anhydride in well-stirred suspensions of CH2CI2 and 1.0 M aqueous NaHCC>3 (Equations 1-3). The results are summarized in Table 1 where yields of isolated acid, anhydride and recovered acid chloride are reported. The reaction is believed to involve formation of the poly(benzoyloxypyridinium) ion intermediate (5) in the organic phase (Equation 1) and 5 then quickly reacts with bicarbonate ion and/or hydroxide ion at the interphase to form benzoate ion (Equation 2 and 3). Apparently most of the benzoate ion is trapped by additional 5 in the organic layer or at the interphase to produce benzoic anhydride (Equation 4), an example of normal phase-... 

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

Fused-silica column (15 cm x 4.6 mm) of Spherisorb S5W 0.1 mL chloroform-methanol-aqueous 25% ammonia (868 125 7) [1.5 mL/min] 254 nm Normal phase HPLC analysis in whole blood and urine. [96]... 

A novel development for HPLC is something called bonded reversed-phase columns, where the stationary phase is a nonpolar hydrocarbon, chemically bonded to a solid support. You can use these with aqueous eluents, usually alcohol-water mixtures. So you have a polar eluent and a nonpolar stationary phase, something that does not usually occur for ordinary wet-column chromatography. One advantage is that you don t need to use anhydrous eluents (very small amounts of water can change the character of normal phase columns) with reversed-phase columns. 

These dyes have affinity for one or, usually, more types of hydrophobic fibre and they are normally applied by exhaustion from fine aqueous dispersion. Although pure disperse dyes have extremely low solubility in cold water, such dyes nevertheless do dissolve to a limited extent in aqueous surfactant solutions at typical dyeing temperatures. The fibre is believed to sorb dye from this dilute aqueous solution phase, which is continuously replenished by rapid dissolution of particles from suspension. Alternatively, hydrophobic fibres can absorb disperse dyes from the vapour phase. This mechanism is the basis of many continuous dyeing and printing methods of application of these dyes. The requirements and limitations of disperse dyes on cellulose acetate, triacetate, polyester, nylon and other synthetic fibres will be discussed more fully in Chapter 3. Similar products have been employed in the surface coloration of certain thermoplastics, including cellulose acetate, poly(methyl methacrylate) and polystyrene. 

The second approach was taken by practicing liquid chromatographers. They routinely dealt with thermally labile, highly polar molecules and frequently sacrificed resolution, and speed in their separations because of the aqueous mobile phases that were required. With the enhanced diffusion and decreased viscosity of supercritical fluids over liquids, chromatographic run-time and resolution could be improved when supercritical fluids were used. But solubility in pure carbon dioxide mobile phases, which has the solvating powers from hexane to methylene chloride under normal density ranges, was a problem for these polar molecules. To compensate for this, experimentalists started working with mixed mobile phases. These mixed phases were based on... 

Normal-phase HPLC has also found application in the analysis of pigments in marine sediments and water-column particulate matter. Sediments were extracted twice with methanol and twice with dichloromethane. The combined extracts were washed with water, concentrated under vacuum and redissolved in acetone. Nomal-phase separation was performed with gradient elution solvents A and B being hexane-N,N-disopropylethylamine (99.5 0.5, v/v) and hexane-2-propanol (60 40, v/v), respectively. Gradient conditions were 100 per cent A, in 0 min 50 per cent A, in 10 min 0 per cent A in 15 min isocratic, 20 min. Preparative RP-HPLC was carried out in an ODS column (100 X 4.6 mm i.d. particle size 3 jum). Solvent A was methanol-aqueous 0.5 N ammonium acetate (75 25, v/v), solvent B methanol-acetone (20 80, v/v). The gradient was as follows 0 min, 60 per cent A 40 per cent A over 2 min 0 per cent A over 28 min isocratic, 30 min. The same column and mobile phase components were applied for the analytical separation of solutes. The chemical structure and retention time of the major pigments are compiled in Table 2.96. 

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