Reversed-phase chromatography organic solvent

In analogy to reversed-phase chromatography, organic solvents such as acetonitrile or methanol are added to the aqueous mobile phase as organic modifiers. Because the solvent molecules are adsorbed at the surface of the stationary phase, they are in a... 

Common surfactants that have been used in MEKC, are listed in Table 3.1 with the respective critical micelle concentrations the most popular are SDS, bile salts, and hydrophobic chain quaternary ammonium salts. Selectivity can also be modulated by the addition to the aqueous buffer of organic solvents (methanol, isopropanol, acetonitrile, tetrahydrofuran, up to a concentration of 50%). These agents will reduce the hydrophobic interactions between analytes and micelles in a way similar to reversed-phase chromatography. Organic modifiers also reduce the cohesion of the hydrophobic core of the micelles, increasing the mass transfer kinetics and, consequently, efficiency. Nonionic... 

Although many water-soluble organic solvents can be used in HILIC, the preferred solvent is acetonitrile because of its low viscosity, resulting in good column efficiencies at low backpressure. This is similar to the situation in reversed-phase chromatography. Common solvent compositions vary between 50 and 90% acetonitrile. Sugars are typically separated using 70% acetonitrile 30% water. 

MLC enables to analyse drugs and active phamiaceutical substances without using special column and lai ge quantity of organic solvents. So, from the point of view of pharmaceutical analysis ecology and green chemistry conception, assay with MLC using will be better than conventional reversed-phase chromatography. 

Another example of the use of small particle silica is in the analysis of theophylline in plasma, as shown in Figure 5 (40). The clean-up procedure is simply a single extraction of the plasma with an organic solvent. This analysis has also been achieved by reverse phase chromatography (41), and this points out the fact that in some separations (e.g. with components of moderate polarity) either the adsorption or reverse phase mode can be used. 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Most small organic molecules are soluble in mixed organic-aqueous solvents and can be easily analyzed using RPLC. However, there are some polar compounds which are not soluble in typical RPLC solvent systems or are unstable in an aqueous mobile phase system. These compounds can be analyzed on an RPLC column with a nonaqueous solvent system. This technique is called "nonaqueous reversed phase chromatography" (NARP).20-21 The NARP technique is primarily used for the separation of lipophilic compounds having low to medium polarity and a molecular weight larger than... 

In reversed-phase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

Reverse-phase chromatography may also be used to separate proteins on the basis of differential hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic column to which most proteins will bind. Elution is promoted by decreasing the polarity of the mobile phase. This is normally achieved by the introduction of an organic solvent. Elution conditions are harsh and generally result in denaturation of many proteins. 

Efficiency the organic modifier can be used to adjust solvent selectivity as normally practiced in reversed-phase chromatography. Lowers mobile-phase viscosity and improves solute mass-transfer kinetics. 

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. 

In reverse-phase chromatography, the stationary phase is nonpolar (often a hydrocarbon) and the mobile phase is relatively polar (e.g., water, methanol, and acetonitrile). The most polar components elute first, and increasing the mobile phase polarity (i.e., decreasing the organic solvent concentration) increases elution time. 

The liquid liquid partition chromatography (LLPQ method involves a stationary liquid phase that is more or less immobilized on a solid support, and a mobile liquid phase. The analyte is therefore distributed between the two liquid phases. In conventional LLPC systems, the stationary liquid phase is usually a polar solvent and the mobile liquid phase is an essentially water-immiscible organic solvent. On the other hand, in reversed-phase chromatography (RPQ, the stationary liquid is usually a hydrophobic... 

Impact of the Organic Solvent and Other Mobile-Phase Properties on Peptide Separation in Reversed-Phase Chromatography... 

Elution in reversed-phase chromatography is often carried out using a gradient, produced from water and some water-miscible organic solvent. The solute components are thus distributed between the stationary and mobile phases mainly on the basis of their polarities. In reversed-phase chromatography hydrophilic compounds elute before hydrophobic ones. 

Electrospray requires low-ionic-strength solvent so that buffer ions do not overwhelm analyte ions in the mass spectrum. Low-surface-tension organic solvent is better than water. In reversed-phase chromatography (Section 25-1), it is good to use a stationary phase that strongly retains analyte so that a high fraction of organic solvent can be used. A flow rate of 0.05 to 0.4 mL/min is best for electrospray. 

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