Reverse-phase chromatography mobile phases

Figure 7 Liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) chromatogram of a standard mixture of organolead and inorganic lead compounds (Pb2+, TML, and TEL) using reversed-phase HPLC. Mobile phase, 0.1 M ammonium acetate, and 0.1 M acetic acid at pH 4.6, 30% ethanol. Flow rate 1 mL/min. (From Ref. 26.)...

Figure 4.10. Plot of the retention factor as a function of the volume fraction (% v/v) of organic solvent in reversed-phase chromatography. Stationary phase is an octadecylsiloxane-bonded silica sorbent with methanol-water as the mobile phase. Solute identification 1 = naphthalene 2 = bromobenzene 3 = acetophenone 4 = 2-phenylethanol and 5 = benzamide.

Figure 3 Chromatogram of a plasma extract showing separation of the anticonvulsants primidone, phenobarbitone, phenytoin, and carbamazepine by isocratic reversed-phase chromatography. Solid-phase extraction with a CIS column was used for sample preparation. Methanol (45%) in dilute acid is used as the mobile phase. Flow rate 1.0 ml min"h Detection UV absorption at 225 nm. Column CDS Hypersil 5 pm, 15 cm x 4.6 mm. Two internal standards are used IS 1 is 4-methyl primidone and IS 2 is 5-ethyl-5-p-tolybarbituric acid.

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.]... 

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. 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

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... 

Microparticulate silica can be used in a number of modes for hplc of these, reverse phase chromatography using bonded phases is the most widely used. In normal and reverse phase chromatography the retention times and selectivities of solutes can be altered by adjustment of the nature and composition of the mobile phase. 

Analysis using reversed-phase chromatography at low pH was carried out under conditions typically used for 2DLC-MS/MS analysis of peptides. The mobile phases for other chromatographic conditions were also chosen to be compatible with MS detection, including SCX LC, where the peptides were eluted with volatile ammonium formate buffer. 

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. 

The term chemistry in interphases was first introduced in the field of reverse-phase chromatography [41], In 1995 Lindner et al. transferred the concept to the area of transition metal catalysis [42] and in a recent review the concept is explained in detail [43], The interphase is defined as a region within a system in which the stationary and a mobile component penetrate on a molecular level without the formation of a homogeneous mixture. In these regions the reactive centre on the stationary phase... 

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. 

Honigberg et al (78) tested reversed-phase chromatography for separation of a number of drugs, including hydralazine. The columns contained either octadecyltrichlorosilane or diphenyldichlorosilane, bonded to 37 to 50 Jjn pellicular silica packing. Of the numerous mobile phases tested, the best for separating hydralazine, hydrochlorothiazide, and reserpine was acetonitrile/0.1% ammonium acetate (20 80), pH 7 35. The columns were 1.22 m x 2 3 mm (ID) and the flow rate was 1.4 ml per minute. Detection was by ultraviolet absorption at 25 nm. 

The extent to which the ions compete with B for the charged sites (X) will determine their retention. In general, this type of chromatography may be used to separate ionic species, such as organic acids or bases, which can be ionized under certain pH conditions. Besides the reaction with ionic sites on the stationary phase, retention may also be affected by the partitioning of solutes between the mobile and stationary phases, as in reversed-phase chromatography. Thus, even nonionized solutes may be retained on ion-exchange columns. 

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. 

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. 

Reverse phase chromatography describes a system with a non-polar stationary phase and a polar mobile phase. 

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