Reverse-Phased Chromatography

In contrast, by entering the oil, a compound leaves the mobile phase and interacts with the beads Its rate of passage through the system is in effect slowed. The compound will be retained, and it will have a longer retention time than a compound that does not interact. 

A great deal of time and effort has been spent in trying to predict the retention time of compounds in the reversed-phase system. While some rules have emerged and some generalizations have been made, to date the best approach remains a few trial runs. 

The most useful parameters to consider when developing a feel for the operation of this type of chromatography are polarity and the related parameter solubility. Values of both parameters have been published for many of the compounds used in biological systems. 

In what follows, I introduce the notion of polarity and, after differentiating it from the net charge of a molecule, use it to explain the retention time for some classes of compounds. 

Polar molecules are generally more soluble in water than nonpolar molecules, and therefore solubility values can also be useful in predicting retention times. For example, some amino acids (e.g., glycine, alanine, and others containing nonpolar side chains) are not very soluble in water thus, on reversed-phase columns washed with only aqueous buffers, such compounds would be expected to interact with the nonpolar packing and be retained. Elution would be promoted by increasing the organic composition of the elution buffer. 

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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. 

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). 

Whereas recombinant proteins produced as inclusion bodies in bacterial fermentations may be amenable to reversed-phase chromatography (42), the use of reversed-phase process chromatography does not appear to be widespread for higher molecular weight proteins. 

Reversed-phase chromatography rehes on significantly stronger-hydrophobic interactions than in HIC, which can result in unfolding and exposure of the interior hydrophobic residues, i.e., leads to protein denaturation and irreversible inactivation as such, RPC depends... 

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. 

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.]... 

M. Stromqvist, Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary electrophoresis , 7. Chromatogr. 667 304-310(1994). 

Figure 3 Reversed-phase chromatography of products after alkaline hydrolysis of /3-poly(L-malate), Discrete polymer products are formed, which differ in length by several units of L-malate. The absorbance at 220-nm wavelength was measured, (a) /3-Poly(L-malate) before hydrolysis, (b) After 10-min incubation in 20 mM NaOH at 37°C. (c) After 15 h in 20 mM NaOH at 37°C. (d) After I h in 500 mM NaOH at 100°C. High pressure chromatography (HPLC) on Waters reversed-phase Ci8- i-Bondapak. The methanol gradient (in water-trifluoro acetic acid, pH 3.0) was programmed as follows 0-40 min 0.3-23%, 40-47 min 23-40%, 47-49 min 40%, 49-54 min 40-0%. (d) Inset size exclusion chromatography after 3-min alkaline hydrolysis at pH 10.2. BioSil SEC 250 column of 300 mm x 7.8 mm size, 0.2 M potassium phosphate buffer pH 7.0.

Table 8.1 Typical stationary and mobile phases for normal and reverse phase chromatography ...

Recently, new approaches of sorbent construction for reversed-phase chromatography have been developed. Silicas modified with hydrocarbon chains have been investigated the most and broadly utilized for these aims. Silica-based materials possess sufficient stability only in the pH 2-8 range. Polymeric HPLC sorbents remove these limitations. Tweeten et al. [108] demonstrated the application of stroongly crosslinked styrene-divinylbenzene resins for reversed-phase chromatography of peptides. 

These sorbents may be used either for selective fixation of biological molecules, which must be isolated and purified, or for selective retention of contaminants. Selective fixation of biopolymers may be easily attained by regulation of eluent polarity on the basis of reversed-phase chromatography methods. Effective isolation of different nucleic acids (RNA, DNA-plasmid) was carried out [115, 116]. Adsorption of nucleosides, nucleotides, tRN A and DNA was investigated. It was shown that nucleosides and nucleotides were reversibly adsorbed on... 

Other endgroups can indirectly be quantified by first hydrolyzing the polymer in diluted chloric acid solution and then determining the composition of the compound by HPFC, reverse-phase chromatography, or gas chromatography (GC).45 48... 

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. 

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... 

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