Reversed-phase chromatography separation modes

Reverse-Phase Chromatography—Separation mode on bonded phase columns in which the solvent/column polarities are the opposite of normal-phase separations. Polar compounds elute before nonpolar compounds, Nonpolar columns require polar solvents. 

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.]... 

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. 

A combination of different modes of chromatography can provide excellent resolution for those components that chromatograph poorly with a single mode [108,109]. The chromatographic separation of baclofen (I) from its potential transformation product (II) with dual mode chromatography entailing ion-pair reversed-phase chromatography and... 

Obviously, the monolithic material may serve its purpose only if provided with a suitable surface chemistry, which depends on the desired application. For example, hydrophobic moieties are required for reversed phase chromatography, ionizable groups must be present for separation in the ion-exchange mode, and chiral functionalities are the prerequisite for enantioselective separations. Several methods can be used to prepare monolithic columns with a wide variety of surface chemistries. 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

Reversed-phase chromatography is the most popular mode of analytical liquid chromatography for phenolic compounds. In most cases, the reported systems for the separation of phenolics and their glycosides in foods are carried out on reversed-phase chromatography on silica-based Cl8 bonded-phase columns. Occasionally, silica columns bonded with C8 were applied in the analysis of phenolic acid standards and coumarins (7), and C6 columns for the analysis of ferulic acid in wheat straw (8). 

Reversed-phase chromatography is the most popular mode for the separation of low molecular weight (<3000), neutral species that are soluble in water or other polar solvents. It is widely used in the pharmaceutical industry for separation of species such as steroids, vitamins, and /3-blockers. It is also used in other areas for example, in clinical laboratories for analysis of catecholamines, in the chemical industry for analysis of polymer additives, in the environmental arena for analysis of pesticides and herbicides, and in the food and beverage industry for analysis of carbohydrates, sweeteners, and food additives. 

Drugs possessing a steroid structure are particularly easy to separate by CEC (see also the section on Steroids). Euerby et al. [203] published the separation of tipredane and five related compounds. A conventional capillary packed with 3 mm Spherisorb ODS-1 can be used for this purpose using acetonitrile-Tris pH 7.8 buffer (8 2) (50 mmol/1). Under these conditions it is also possible to separate the C-17 diastereoi-somer of the active compound without the addition of a chiral modifier (b-cyclodextrin is needed to achieve a comparable result in other separation modes) (Fig. 10.25). The elution order of individual compounds was exactly the same as with reversed phase chromatography, and it was concluded that with unionized species HPLC methods should be directly transferable to the CEC mode. 

At first glance the specifications of many stationary phases in a particular mode of separation may appear to be the same. For example, in reversed phase chromatography there are many C18 based media that will have the same carbon load, the same pore size distribution, and so on. However, stationary phases from different manufacturers will behave differently, so it is important... 

For small neutral organic compounds, reversed-phase chromatography usually provides much better efficiency than lEC. However, a mixed-mode separation mechanism on ion-exchange columns can offer a unique selectivity for the separation of some biopolymers such as peptides or proteins [73). 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

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