Separation reversed-phase chromatography

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. 

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

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.]... 

To retain solutes selectively by dispersive interactions, the stationary phase must contain no polar or ionic substances, but only hydrocarbon-type materials such as the reverse-bonded phases, now so popular in LC. Reiterating the previous argument, to ensure that dispersive selectivity dominates in the stationary phase, and dispersive interactions in the mobile phase are minimized, the mobile phase must now be strongly polar. Hence the use of methanol-water and acetonitrile-water mixtures as mobile phases in reverse-phase chromatography systems. An example of the separation of some antimicrobial agents on Partisil ODS 3, particle diameter 5p is shown in figure 5. 

HPLC requires a mobile phase in which the analytes are soluble. The majority of HPLC separations which are carried out utilize reversed-phase chromatography, i.e. the mobile phase is more polar then the stationary phase. In these systems, the more polar analytes elute more rapidly than the less polar ones. 

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. 

Chapter 3 through Chapter 8 deal with the basic aspects of the practical uses of PLC. Chapter 3 describes sorbent materials and precoated layers for normal or straight phase (adsorption) chromatography (silica gel and aluminum oxide 60) and partition chromatography (silica gel, aluminum oxide 150, and cellulose), and precoated layers for reversed-phase chromatography (RP-18 or C-18). Properties of the bulk sorbents and precoated layers, a survey of commercial products, and examples of substance classes that can be separated are given. 

Separation and detection methods There exists an array of methods for the separation of the Sb species, ranging from liquid-liquid extraction to the formation of complexes with immobilized reagents, followed by HPLC or reversed phase chromatography, and eletroanalytical methods. Detection is based on spectrophotometric measurement, AAS, ICP-OES and ICP-MS. 

Chromatographic fractions, glucuronide fractions A, B, and C were separated by reversed-phase chromatography. 

To assess the trapping of biological nucleophiles, the pyrido[l,2-a]indole cyclopropyl quinone methide was generated in the presence of 5 -dGMP. The reaction afforded a mixture of phosphate adducts that could not be separated by reverse-phase chromatography (Fig. 7.16). The 13C-NMR spectrum of the purified mixture shown in Fig. 7.16 reveals that the pyrido [1,2-a] indole was the major product with trace amounts of azepino[l,2-a] indole present. Since the stereoelec-tronic effect favors either product, steric effects must dictate nucleophilic attack at the least hindered cyclopropane carbon to afford the pyrido[l,2-a]indole product. Both adducts were stable with elimination and aromatization not observed. In fact, the pyrido [1,2-a] indole precursor (structure shown in Scheme 7.14) to the pyrido [l,2-a]indole cyclopropyl quinone methide possesses cytotoxic and cytostatic properties not observed with the pyrrolo [1,2-a] indole precursor.47... 

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. 

Hirabayashi, J. and Kasai, K.-I., Applied slalom chromatography. Improved DNA separations by the use of columns developed for reversed-phase chromatography, /. Chromatogr. A, 722, 135, 1996. 

Superheated water at 100°-240 °C, with its obvious benefits of low cost and low toxicity, was proposed as a solvent for reversed-phase chromatography.59 Hydrophobic compounds such as parabens, sulfonamides, and barbiturates were separated rapidly on poly(styrene-divinyl benzene) and graphitic phases. Elution of simple aromatic compounds with acetonitrile-water heated at 30°-130 °C was studied on coupled colums of zirconia coated with polybutadiene and carbon.60 The retention order on the polybutadiene phase is essentially uncorrelated to that on the carbon phase, so adjusting the temperature of one of the columns allows the resolution of critical pairs of... 

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... 

IonPac KC-811 column separated the labile compounds N-acetylneuraminic acid and N-glycolylneuraminic acid released by mild acid hydrolysis of bovine vitronectin.245 Sialic acid is extremely labile to conditions of handling and must be released by mild acid hydrolysis.246 Derivatization with phe-nylisothiocyanate and separation by reversed phase chromatography was found useful in analysis of hexosamines from gastric mucosa.247 A review on separation of sugars and other carbohydrates which covers many important aspects is available.248... 

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