HPLC stationary phases nonpolar bonded

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. 

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. 

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. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

Carotenoid separations can be accomplished by both normal- and reversed-phase HPLC. Normal-phase HPLC (NPLC) utilizes columns with adsorptive phases (i.e., silica) and polar bonded phases (i.e., alkylamine) in combination with nonpolar mobile phases. In this situation, the polar sites of the carotenoid molecules compete with the modifiers present in the solvent for the polar sites on the stationary phase therefore, the least polar compounds... 

The analytes are separated by adsorption to a polar or nonpolar support surface or by partition into a stationary liquid phase. Silica is the most common polar adsorbent. HPLC basically involves the separation of compounds by partition on a stationary liquid phase, bonded to a support. The support, such as silica, is derivatized with a functional group that is covalently attached to the surface and is more stable than ary coated phase. Such bonded phases can be used with most solvents and buffers. A mobile liquid phase transports the sample into the column where individual compounds are selectively retained on the stationary liquid phase and thus separated. 

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). 

We now have a fairly adequate understanding of the different properties, including the particle diameter i/p, the pore size, the degree of permeability, and the chemical composition of the surface of the support matrix, to know which type of stationary phase can be successfully used with a particular class of peptides. Most of the HPLC packing materials now in use for peptide separations are based on the wide pore microparticulate silica gels with polar or nonpolar carbonaceous phases chemically bonded to the surface of the matrix. Methods for the preparation of these chemically bonded stationary phases, their available sources of supply. 

If these small silica particles are used, then the chromatography is called normal phase, and the polarity of the stationary phase is higher than that of the mobile phase this is what happens, for example, when silica is used in adsorption chromatography. However, almost all the work in analytical HPLC is now carried out with chemically modified silica, which is the bonded phase. In a bonded phase, the highly polar surface of silica is modified by the chemical attachment of various functional groups. Bonded-phase chromatography is experimentally much easier, more versatile, and quicker it also has better reproducibility than the older modes. When a nonpolar-bonded phase is used, the operation is performed in an RP mode, which means that the polarity of the stationary phase is less than that of the mobile phase. These columns, contrary to normal silica columns, elute polar compounds more rapidly than nonpolar compounds. 

The interaction between mobile phase and stationary phase should not be overlooked. For example, many of the very widely used HPLC columns consist of silica particles coated with chemically bonded hydrocarbons to give a nonpolar reverse phase stationary phase. However, there are generally a number of free silanol (-Si-OH) groups that can play a significant role in the adsorption of solutes. The addition of various mobile phase modifiers (e.g., inorganic buffer ions) can lead to interaction with these groups and alter the nature of the solutes adsorption. This is particularly important in the elution of... 

Over 500 HPLC packings have been described in the Hterature. Nevertheless, as the result of years of development, only a limited number of types of stationary phases remain on the market. Most of the conventional HPLC separations today are performed using monodisperse silica gel 3 or 5 Xm microbeads, especially those grafted with C4, C8, or C18 alkyl chains, as well as with cyano-propyl or amino-propyl groups. The last two bonded silicas and bare silica are used in normal phase (NP) HPLC, where the mobile phase (usually hexane with small amounts of isopropyl alcohol) is less polar than the stationary phase. Even more popular is the reversed phase (RP) mode, which uses polar eluents (mosdy water or methanol with such additives as acetonitrile, methanol, or tetrahydrofuran (THE)) in combination with nonpolar alkyl-bonded stationary phases. 

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. 

Amphetamine (see Figure 6.4) is a weak base with a pKa of 10.1. It is basic in nature due to the -NHj group and is nonpolar due to the predominance of nonpolar C-C and C-H bonds. If we were to leave the pH and mobile phase composition aside for the moment, we would expect amphetamine to have an affinity for a nonpolar stationary phase and we would expect this affinity to increase with column packing chain length. Therefore, we can say that we would expect to achieve satisfactory chromatography using RP-HPLC (refer to Chapter 4). 

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