Normal-phase chromatography

The development of high performance stationary phases for amino acid analysis has been vigorously pursued since the introduction of the first amino acid analyser (Spackman et al., 1950). The low UV extinction coefficient of most amino acids means that current detection methods depend upon the reaction of the primary amino group of the amino acid to yield a coloured or fluorescent derivative. The most popular reagents, referred to by their most common nomenclature, are the following  

Phenyl isothiocyanate Dansyl chloride Dabsyl chloride 

Other reagents are available but have not yet found widespread acceptance. For example, fluorenylmethyl chloroformate, which is commonly used as a blocking agent in peptide synthesis, can be used in pre-column labelling with sensitivity in the femtomole range (Varian). 

The majority of the commercially available amino acid analysers employ an ion-exchange resin for the separation of amino acids with ninhydrin as the detection reagent (Fig. 11.2.9), although OPA and fluram can also be utilised. Despite recent developments (Section 11.2.4.3), such a dedicated instrument is often preferred in routine analysis for reasons of ease of sample handling and reproducibihty. 

The increasing popularity of RPC has prompted many researchers and commercial laboratories to develop separations which can be 

The IUPAC Compendium of Chemical Technology defines Normal Phase as an elution procedure in which the stationary phase is more polar than the mobile phase . In practice, the most widely used stationary phases for preparative HPLC are based on silica and the polarity of the underlying silyl ether and silanol provides the required hydrophilic surface. Amino and cyano bonded silica are also commonly used in normal phase mode though the latter also has some reversed phase properties. The predominant mechanism of interaction is hydrogen bonding. However, the silanol is mildly acidic so the silica surface will also have mild cation exchange properties. 

I have known the performance of irregular silica particles to improve with time and it has been postulated that this is due to erosion of the media to such an extent that the stationary phase has eventually become spherical. 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

It is also useful to note that the hydrophilic alcohol coated stationary phases originally designed for size exclusion chromatography such as Zorbax GF250 and PL-aquagel also work well as normal phase media. The advantage of these lies in the increased stability of the stationary phases at high pH. 

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Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

The results confirmed that the chloroheptane/n-heptane mixture behaves in an identical manner to carbon tetrachloride and all the points were on the same straight line as that produced using a mixture of carbon tetrachloride and toluene. These experiments are similar to normal phase chromatography using pure water instead of... 

When bnffer is not present, i.e. when normal-phase chromatography is being nsed, thermospray ionization is not possible and a filament or discharge electrode is nsed to generate a plasma in which Cl-type processes can occnr. In addition to allowing ionization nnder these conditions, it is found that the ionization of compounds may be enhanced under conditions in which true thermospray can operate. 

The mobile phase should wet the layer, thus cos 0 5 0. In normal phase chromatography, the adsorbent is completely wetted by all solvents and thus cos 0=1. 

The choice of the chromatographic system depends on the chemical character of the extracts being separated. The mobile phase should accomplish all requirements for PLC determined by volatility and low viscosity, because nonvolatile components (e.g., ion association reagents and most buffers) should be avoided. It means that, for PLC of plant extracts, normal phase chromatography is much more preferable than reversed-phase systems. In the latter situation, mixtures such as methanol-ace-tonitrile-water are mostly used. If buffers and acids have to be added to either the... 

Eluent components should be volatile. Solvents such as ethyl acetate, isopropyl ether, diethylketone, chloroform, dichloromethane, and toluene as modifiers and n-hexane as diluent are recommended for normal phase chromatography. For reversed-phase systems, methanol or acetonitrile are used as modifiers. Such components as acetic acid or buffers, as well as ion association reagents, should be avoided. 

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. 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

Normal-phase chromatography is still widely used for the determination of nonpolar additives in a variety of commercial products and pharmaceutical formulations, e.g. the separation of nonpolar components in the nonionic surfactant Triton X-100. Most of the NPLC analyses of polymer additives have been performed in isocratic mode [576]. However, isocratic HPLC methods are incapable of separating a substantial number of industrially used additives [605,608,612-616], Normal-phase chromatography of Irgafos 168, Irganox 1010/1076/3114 was shown [240]. NPLC-UV has been used for quantitative analysis of additives in PP/(Irganox 1010/1076, Irgafos 168) after Soxhlet extraction (88%... 

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