Reversed phase, chromatography

Reversed-phase chromatography is the term used to describe the state in which the stationary phase is less polar than the mobile phase. Chemically bonded octadecylsilane (ODS), an -al kane with 18 carbon atoms, is the most frequently used stationary phase. Cg and shorter alkyl chains and also cyclohexyl and phenyl groups provide other alternatives. Phenyl groups are more polar than alkyl groups. 

Sample compounds are better retained by the reversed-phase surface the less water soluble (i.e. the more nonpolar) they are. The retention decreases in the following order aliphatics induced dipoles (e.g. CCI4) permanent dipoles (e.g. CHCI3) weak Lewis bases (ethers, aldehydes, ketones) strong Lewis 

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R. Meyer 

Branched-chain compounds are eluted more rapidly than their corresponding normal isomers. 

Yet the retention mechanisms on reversed-phase are complex and not easy to understand.  

The chromatogram depicted in Fig. 10.1 demonstrates this point. Benzene (2), chlorobenzene (3), o-dichlorobenzene (4) and iodobenzene (5) were eluted on a reversed-phase column by various methanol-water mixtures (1 being the refractive index peak of the solvent used for sample dissolution). 

Sample compounds are better retained by the reversed-phase surface the less water soluble (i.e. the more non-polar) they are. The retention decreases in the 

Practical High-Performance liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

the retention time increases as the number of carbon atoms increases, as shown in Fig. 10.2 for the separation of dec-l-ene (1), undec-l-ene (2), dodec-1-ene (3), tridec-l-ene (4) and tetradec-l-ene (5) on an ODS column. As a general rule the retention increases with increasing contact area between sample molecule and stationary phase, i.e. with increasing number of water molecules which are released during the adsorption of a compound. 

In reversed phase chromatography, the stationary phase is normally silica coated with covalently attached nonpolar hydrocarbonaceous ligands (normally alkyl 

C X chains). Consequently, the mobile phases need to be more polar (e.g., CH3CN—H20, MeOH—H20) than the coated surface. During elution, separation of constituents of an applied sample mainly relies upon the differences in distribution coefficients between the mobile phase and the hydrocarbon coatings of the stationary phase. For all these reasons, the more polar constituents of a mixture in reversed phase chromatography are eluted before nonpolar components. Note also that although water is more polar than acetonitrile or methanol, it is a poorer eluent in reversed phase chromatography because of the alkyl surface character of the stationary phase. 

Normal-phase separations have occasionally been combined off-line with reversed-phase chromatography to separate a wider range of species than could be accomplished by either technique alone.1 The feasibility of such a system, however, is contingent on the compatibility of the normal-phase eluent with that of the reversed-phase column. 

The IUPAC definition understandably states that reversed phase chromatography is an elution procedure in which the mobile phase is significantly more polar than the stationary phase . This is a somewhat simplistic statement that covers a wealth of 

There have been many developments over the years, including improvements in the wettability of the surface by introducing polar groups between the alkyl chain and the silica. There is also a range of alkyl chain lengths available but the most popular remain to be C8 and C18. 

Silica based stationary phases are unstable to alkaline conditions due to loss of the alkyl chain and dissolution of the silica 

The exact mechanism(s) of solute retention in reversed-phase high-performance liquid chromatography (RPLC) is not presently well understood. The lack of a clear understanding of the mechanics of solute retention has led to a myriad of proposals, including the following partition (K21, L6, S16) adsorption (C9, CIO, H3, H15, H16, K13, L3, T2, U2) dispersive interaction (K2) solubility in the mobile phase (L7) solvophobic effects (H26, K6, M5) combined solvophobic and silanophilic interaction (B9, M12, Nl) and a mechanism based upon compulsary absorption (B5). 

From these investigations, it is apparent that no single retention mechanism is operative in RPLC rather solute selectivity is based upon mixedmode mechanisms. At present it appears that solvophobicity (hydropho-bicity) is the primary mechanism for solute retention [Horvath et al. (B9, H23, H25, M12, M15, Nl) and Kargerer al. (K6, M5)]. 

The principle of solvophobicity as presented by Horvath et al. is based upon the tendency of the mobile phase to minimize the site of the cavity occupied by the solute molecules in the hydroorganic mobile phase. This can be viewed as a reversible association of the solute molecules with the hydrocarbonaceous stationary phase. The magnitude of the solvophobic effect for a given solute is due largely to the following four properties of the hydrooi anic solvent system (H23)  

Of these solvent properties, the dielectric constant and surface tension play important roles in governing solute retention. 

The interaction of the solute with the mobile phase can bring about forces opposing those of the hydrophobic effect. In addition to van der Waals forces, which are dependent upon the size of the molecule involved, electrostatic interactions play a key role in solute retention. Solutes which have polar substituents can interact more strongly with the polar hydroorganic mobile phase, leading to a decrease in retention compared to similar compounds with no polar moiety. The ionization of a solute molecule under the appropriate mobile-phase conditions, results in an increase in electrostatic attraction between solute and eluent and ultimately to a decreased c q acity for chromatographic retention. 

The most common liquid chromatography employed for analysis of antibiotics is reversed phase liquid chromatography (RPLC). RPLC stationary phases (3.5- or 5.0-p,m particles) consist of non-polar or hydrophobic organic species 

For optimum functionality in automated systems designed primarily for reversed phase chromatography and other gradient techniques, the LKB advanced-gradient system is recommended (Fig 1.1(c)). Key features include the following  

This system is ideal for automatic method for development and gradient optimisation. 

By a combination of the use of inert materials (glass, titanium and inert polymers) this system offers totally inert fluidics. Primary features of the system include (Fig. 1.1(d))  

This is the method of choice when corrosive buffers, eg those containing chloride or aggressive solvents, are used. 

The most commonly-used detectors are those based on spectrophotometry in the region 184-400nm, visible ultraviolet spectroscopy in the region 185-900nm, post-column derivativisation with fluorescence detection (see below), conductivity and those based on the relatively new technique of multiple wavelength ultraviolet detectors using a diode array system detector (described below). Other types of detectors available are those based on electrochemical principles, refractive index, differential viscosity and mass detection. 

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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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