Nonpolar stationary phase

Environmental Analysis One of the most important environmental applications of gas chromatography is for the analysis of numerous organic pollutants in air, water, and wastewater. The analysis of volatile organics in drinking water, for example, is accomplished by a purge and trap, followed by their separation on a capillary column with a nonpolar stationary phase. A flame ionization, electron capture, or... 

Liquid chromatography using a nonpolar stationary phase and a polar mobile phase. 

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

For most samples liquid-solid chromatography does not offer any special advantages over liquid-liquid chromatography (LLC). One exception is for the analysis of isomers, where LLC excels. Figure 12.32 shows a typical LSC separation of two amphetamines on a silica column using an 80 20 mixture of methylene chloride and methanol containing 1% NH4OH as a mobile phase. Nonpolar stationary phases, such as charcoal-based absorbents, also may be used. 

Capillary Electrochromatography Another approach to separating neutral species is capillary electrochromatography (CEC). In this technique the capillary tubing is packed with 1.5-3-pm silica particles coated with a bonded, nonpolar stationary phase. Neutral species separate based on their ability to partition between the stationary phase and the buffer solution (which, due to electroosmotic flow, is the mobile phase). Separations are similar to the analogous HPLC separation, but without the need for high-pressure pumps, furthermore, efficiency in CEC is better than in HPLC, with shorter analysis times. 

Method f2.i describes the analysis of the trihalomethanes CHCI3, CHBr3, CHChBr, and CHClBr2 in drinking water using a packed column with a nonpolar stationary phase. Predict the order in which these four trihalomethanes will elute. 

A mixture of n-heptane, tetrahydrofuran, 2-butanone, and n-propanol elutes in this order when using a polar stationary phase such as Carbowax. The elution order is exactly the opposite when using a nonpolar stationary phase such as polydimethyl siloxane. Explain the order of elution in each case. 

Zhou and colleagues determined the %w/w H2O in methanol by GG, using a capillary column coated with a nonpolar stationary phase and a thermal conductivity detector. A series of calibration standards gave the following results. 

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... 

Gas-Liquid Chromatography. In gas-liquid chromatography (GLC) the stationary phase is a liquid. GLC capillary columns are coated internally with a liquid (WCOT columns) stationary phase. As discussed above, in GC the interaction of the sample molecules with the mobile phase is very weak. Therefore, the primary means of creating differential adsorption is through the choice of the particular liquid stationary phase to be used. The basic principle is that analytes selectively interact with stationary phases of similar chemical nature. For example, a mixture of nonpolar components of the same chemical type, such as hydrocarbons in most petroleum fractions, often separates well on a column with a nonpolar stationary phase, while samples with polar or polarizable compounds often resolve well on the more polar and/or polarizable stationary phases. Reference 7 is a metabolomics example of capillary GC-MS. 

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. 

Reverse phase HPLC describes methods that utilize a polar mobile phase in combination with a nonpolar stationary phase. As stated above, the nonpolar stationary phase structure is a bonded phase—a structure that is chemically bonded to the silica particles. Here, typical column names often have the carbon number designation indicating the length of a carbon chain to which the nonpolar nature is attributed. Typical designations are C8, C18 (or ODS, meaning octadecyl silane), etc. Common mobile phase liquids are water, methanol, acetonitrile (CH3CN), and acetic acid buffered solutions. 

Filter and degas a part of the sample. Prepare the instrument as you have done before, choosing a particular stationary and mobile phase system (such as a reverse phase system using a methanol-water mixture for the mobile phase and a nonpolar stationary phase) and flow rate that you will use as a first trial. 

In reverse phase chromatography, the polar mixture components would elute first since they would be attracted by the polar mobile phase and repelled by the nonpolar stationary phase. In normal phase chromatography, nonpolar mixture components would elute first since they would be attracted by the nonpolar mobile phase and repelled by the polar stationary phase. 

Adsorption LC uses a nonpolar mobile phase and a polar stationary phase, while the RP separation mode is characterized by the application of a polar mobile phase and a nonpolar stationary phase. 

Reversed-phase chromatography employs a nonpolar stationary phase and a polar aqueous-organic mobile phase. The stationary phase may be a nonpolar ligand, such as an alkyl hydrocarbon, bonded to a support matrix such as microparticulate silica, or it may be a microparticulate polymeric resin such as cross-linked polystyrene-divinylbenzene. The mobile phase is typically a binary mixture of a weak solvent, such as water or an aqueous buffer, and a strong solvent such as acetonitrile or a short-chain alcohol. Retention is modulated by changing the relative proportion of the weak and strong solvents. Additives may be incorporated into the mobile phase to modulate chromatographic selectivity, to suppress undesirable interactions of the analyte with the matrix, or to promote analyte solubility or stability. 

Reversed-phase HPLC uses a nonpolar stationary phase and a polar mobile phase. The characteristics are operational simplicity, high efficiency, column stability, and ability to analyze simultaneously a broad spectrum of both closely related and widely different compounds. Separation is based on hydrophobicity.33 Findlay et al. provide a comparison of chromatography methods with immunoassays (Table ll.l).27... 

Horvath, C.S., Melander, W.R., and Molnar, L, Solvophobic interactions in liquid chromatography with nonpolar stationary phases, J. Chromatogr., 125, 129, 1976. 

In selecting columns, the general rule is that columns with a polar stationary phase are used to separate polar compounds, whereas columns with nonpolar stationary phases are used to separate nonpolar... 

Reversed-phase chromatography is the predominant technique in HPLC, and chemically bonded silica gel supports are made specifically for the nonpolar stationary phase. In the last decade, as many as 60% of the published LLPC techniques refer to RPC. The reasons for this involve the significantly lower cost of the mobile liquid phase and a favorable elution order that is easily predictable based on the hydrophobicity of the eluate. 

Adsorption chromatography on polar stationary phases is especially suited for the separation of nonpolar to medium polar substances that have some solubility in solvents immiscible with water. More polar substances, which are soluble in pojar solvents such as alcohols or water, of course, can be, and have been, separated on polar stationary phases, too. However, as nonpolar stationary phases are now easily available and widely used in reversed phase chromatography, such substances are preferentially separated on such phases because of better reproducibility and the convenience offered by this technique. (/)... 

Besides hydrocarbonaceous bonded phases, other nonpolar stationary phases can also be employed in RPC. Although some of them will be... 

The solvophobic theory has been successfully applied to treat the effect of solute ionizatiqn as well as the effect of salts on the retention of both neutral and ionized species. There is ample experimental evidence that retention of a spfeies decreases upon ionization according to the theoretical prediction. Addition of salts to aqueous eluents increases surface tension and consequently. the retention of neutral eluites on nonpolar stationary phases. With ioni d solutes, however, the solvophobic theory predicts a minimum at low ionic strength in plots of retention versus ionic strength and this phenomenon has also been experimentally demonstrated. 

The idea of using a nonpolar stationary phase and a polar mobile phase was first put forward in the literature by Boscott ( ), who advocated the use of cellulose acetate as the stationary phase. Shortly thereafter Bol-dingh (4) separated Cg-Cu fatty acids by using moderately vulcanized rubber saturated with benzene as the stationary phase and water-methanol as the mobile phase. This was rapidly followed by other work (5-7) and the method was widely practiced by lipid chemists until the advent of HPLC. The main problem with the use of rubber for the stationary phase is that the degree of swelling is extremely critical in determining the... 

Eluents used in reversed-phase chromatography with bonded nonpolar stationary phases are genei ly polar solvents or mixtures) of polar solvents, such as acetonitrile, with water. The properties of numerous neat solvents of interest, their sources, and their virtues in teversed-phase chromatography have been reviewed (128). Properties of pure solvents which may be of value as eluents are summiuized in Table. VII. The most significant properties are surface tension, dielectric constant, viscosity, and eluotropic value. Horvath e/ al. 107) adapted a theory of solvent effects to consider the role of the mobile phase in determinmg the absolute retention and the selectivity found in reversed-phase chromatography. 

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