Reversed-phase chromatography nonpolar bonded phases

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. 

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. 

The mechanism of reversed-phase chromatography arises from the tendency of water molecules in the aqueous-organic mobile phase to self-associate by hydrogen bonding. This ordering is perturbed by the presence of nonpolar solute molecules. As a result, solute molecules tend to be excluded from the mobile phase and are bound by the hydrophobic stationary phase. This solvophobic... 

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. 

If solutes dissolve only in nonpolar or weakly polar solvents, the decision tree suggests that we try reversed-phase chromatography. Our choices include bonded phases containing octadecyl (CJg), octyl, butyl, ethyl, methyl, phenyl, and cyano groups. 

Reverse-Phase Chromatography—Separation mode on bonded phase columns in which the solvent/column polarities are the opposite of normal-phase separations. Polar compounds elute before nonpolar compounds, Nonpolar columns require polar solvents. 

Unlike the more popular reversed-phase chromatographic mode, normal-phase chromatography employs polar stationary phases, and retention is modulated mainly with nonpolar eluents. The stationary phase is either (a) an inorganic adsorbent like silica or alumina or (b) a polar bonded phase containing cyano, diol, or amino functional groups on a silica support. Tlie mobile phase is usually a nonaqueous mixture of organic solvents. As the polarity of the mobile phase decreases, retention in normal-phase chromatography... 

Typical applications of reversed-phase chromatography are shown in Table 2. Beyond analytical apphca-tions, RP-TLC on bonded phases is also a tool for physicochemical measurements, particularly for molecular hpophilicity determination of biologically active compounds. Hydrophobicity can be measured by partition between an immiscible polar and nonpolar solvent pair, particularly in the reference system n-oc-tanol-water. The partition coefficient, P, is frequently used to interpret quantitative structure-activity relationships (QSAR studies). 

The most conunon nonpolar bonded phases (for reversed-phase chromatography) are Cjg and Cg (shown above), with Cig the most popular (known as ODS for octadecylsilane) Cg is intermediate in hydrophobicity, and Cig is very nonpolar. Phenyl groups are also useful [R = (CH2)3C6H5]. C5 particles are used for... 

In reversed-phase chromatography (RPC), a relatively nonpolar stationary phase is used, with a polar mobile phase such as methanol, acetonitrile, tetrahydro-furan, water, or usually a mixture of water with one of the organic solvents. The organic solvent is called the modifier, and acetonitrile is the most common one. The water content is varied for adjusting the polarity. Methanol is used for acidic compounds and acetonitrile for basic compounds. Tetrahydrofuran is used for those with large dipoles. These solvents are UV transparent and have low viscosity. The most common bonded phases are n-octyldecyl (Cig) or n-decyl (Cg) chains, or phenyl groups. Polar reversed-phase columns such as polyethylene glycol (PEG)... 

What are some commonly used nonpolar bonded phases for reversed-phase HPLC What are bonded polar phases for normal-phase chromatography ... 

Silica gel may be chemically modified in a number of ways that alter both its chromatographic and physical properties. As shown in Fig. 4, the reactive silanol groups of silica gel may be blocked with a variety of silylchlorides to produce a nonpolar (reverse phase) or polar (bonded normal phase) chromatography support. The most commonly employed bonded phase silica gels are the reverse phase class although the use of bonded normal phase silica has increased with improved materials. 

In reversed-phase chromatography, a nonpolar stationary phase is used in conjunction with polar, largely aqueous mobile phases. Between 70 and 80% of all HPLC applications utilize this technique. Its popularity is based largely on its ease of use equilibration is fast, retention times are reproducible, and the basic principles of the retention mechanism can be understood easily. Most stationary phases are silica-based bonded phases, but polymeric phases, phases based on inorganic substrates other than silica, and graphitized carbon have found their place as well. 

Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surfooe of the stationary phase, which is strongest when nonpolar solvents are used as mobile phase. Previously, it was also called adsorption chromatography. However, the technique has expanded from the exclusive application of metal oxide adsorbents such as silica and alumina as stationary phases to the use of polar bonded phases. Thus the name adsorption chromatography has become too narrow. 

Figure 7 SPE based on reverse-phase chromatography. 1. Solvate the bonded phase with six to ten cartridge holdup volumes of methanol or acetonitrile. Flush the cartridge with six to ten holdup volumes of water or buffer. Do not allow the cartridge to dry out. 2. Load the sample dissolved in a strongly polar solvent. 3. Elute interfering impurities with the strongly polar solvent. 4. Elute weakly held analytes (Analyte 1) of interest with a less polar solvent. 5. Elute more tightly bound analytes (Analyte 2) with progressively more nonpolar solvents.

REVERSE- or REVERSED-PHASE CHROMATOGRAPHY. Liquid-liquid partition TLC in which the stationary phase is nonpolar compared to the mobile phase. The layer can be impregnated or bonded in nature. 

Reversed-phase chromatography is perhaps the most widely used chromatographic method. It was first developed by Howard and Martin (1950) to separate fatty acids by using a polar eluent (mobile phase) and a nonpolar stationary phase that consisted of paraffin oil and octane. Today the stationary phase consists of a liquid that is chemically bonded to a support. For example, the column packings contain octadecylsilyl (Cig), octylsilyl (Cg), butylsilyl (C4), or propylsUyl (C3), which are bonded to silica supports having various pore sizes (e.g., 100, 300, and 500 A) and particle sizes (e.g., 5 and 10 pm). The extent of retention of a molecule depends on the number, size, and stereochemistry of its hydrophobic (e.g., alkyl) and hydrophilic (e.g., ionic) groups. 

There are variations in a given technique identified in Table 7.1.5. For example, in liquid-liquid chromatography (LLC), normaiiy the stationary-phase liquid is polar and the mobile phase is nonplolar, both being immiscible with each other, in reversed-phase LLC, the stationary-phase liquid is nonpoiar, whereas the mobile phase is polar the polar phase can even be water. In many cases, the stationary liquid phase may be a monomoiecular iayer chemically bonded to the surface groups of the porous support material. This is often achieved with a nonpolar hydrocarbon liquid phase. 

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. 

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. 

Bonded phase chromatography is a type of liquid-liquid chromatography in which the liquid stationary phase is chemically bonded to the support material (as opposed to being simply adsorbed). The stationary phase can be either polar or nonpolar, and thus both normal phase and reverse phase are possible. 

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

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