Mobile Phases for Reversed-Phase Chromatography

The mobile phases used in reversed-phase chromatography are based on a polar solvent, typically water, to which a less polar solvent such as acetonitrile or methanol is added. Solvent selectivity is controlled by the nature of the added solvent in the same way as was described for normal-phase chromatography solvents with large dipole moments, such as methylene chloride and 1,2-dichloroethane, interact preferentially with solutes that have large dipole moments, such as nitro- compounds, nitriles, amines, and sulfoxides. Solvents that are good proton donors, such as chloroform, m-cresol, and water, interact preferentially with basic solutes such as amines 

Chapter 2 Separations in High-Performance Liquid Chromatography 

The eluting strength of the solvent is inversely related to its polarity, as indicated in Table 2.3 where P is the solvent polarity parameter. A solvent with a low P is chosen initially, and quantities of a second solvent with a greater P are added until the desired separation is achieved. More details on the development of mobile phases can be found elsewhere.1,6,7 

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Why are methanol or acetonitrile preferred to other water miscible solvents for the preparation of mobile phases for reverse phase chromatography ... 

M. Roses, Determination of the pH of binary mobile phases for reversed phase chromatography, J. Chromatogr. A 1037 (2004), 283-298. 

The stationary phase is usually Cg or Cig, but the use of Spherisorb CN or Nucleosil columns was also reported. The mobile phase for reversed-phase chromatography is aqueous, while, for normal-phase chromatography, it generally contains pentane or hexane. The elution can be isocratic or with a gradient. The detection is usually UV in the range 116-254 nm. 

The importance of sample preparation for HPLC cannot be overemphasized. This is particularly true for ionizable metabolites whose retention under reverse phase conditions will vary with pH and for polar metabolites whose retention is relatively poor. The nearer the solvent composition of the sample is to that of the mobile phase, the greater the chance of success in a preparative HPLC experiment. If possible, the proportion of organic solvent in the sample should be kept to less than, or equal to, its proportion in the mobile phase for reverse phase chromatography. 

In all these calculations we need to know the viscosity of the mobile phase. Table 2.2 contains the data for the solvents commonly used in HPLC. Most mobile phases, however, are mixtures of solvents. For nonpolar solvents, one can estimate the viscosity of a mixture by linear interpolation between the viscosities of the component solvents. This is not true for aqueous mixtures due to the strong association of polar solvents. Since aqueous mixtures are the mobile phases for reversed-phase chromatography, it is important to know their viscosity. Figure 2.9 ows the viscosity of the aqueous mixtures of the polar organic solvents commonly used in reversed-phase chromatography. Note that the water-alcohol mixtures exhibit a pronounced viscosity... 

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. 

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

One of the first examples of the application of reverse-phase liquid chromatography-gas chromatography for this type of analysis was applied to atrazine (98). This method used a loop-type interface. The mobile phase was the most important parameter because retention in the LC column must be sufficient (there must be a high percentage of water), although a low percentage of water is only possible when the loop-type interface is used to transfer the LC fraction. The authors solved this problem by using methanol/water (60 40) with 5% 1-propanol and a precolumn. The experimental conditions employed are shown in Table 13.2. 

The ionic or polar substances can be seperated without any reaction on specially treated chromatographic columns and detected refractometrically. This is necessary because alkyl sulfosuccinates show only small absorption in the UV-visible region no sensitive photometric detection can be obtained. Separation problems can arise when common steel columns filled with reverse phase material (or sometimes silica gel) are used. This problem can be solved by adding a suitable counterion (e.g., tetrabutylammonium) to the mobile phase ( ion pair chromatography ). This way it is possible to get good separation performance. For an explanation of separation mechanism see Ref. 65-67. A broad review of the whole method and its possibilities in use is given in an excellent monograph [68]. 

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. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

In reversed-phase thin-layer chromatography (RP-TLC), the choice of solvents for the mobile phase is carried out in a reversed order of strength, comparing with the classical TLC, which determines a reversed order of values of compounds. The reversed order of separation assumes that water is the main component of the mobile phase. Aqueous mixmres of some organic solvents (diethyl ether, methanol, acetone, acetonitrile, dioxane, i-propanol, etc.) are used with good results. 

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... 

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. 

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