Reversed-phase frequently used phases

The top two stationary phases in Figure 12 are the C g and Cg phases, which are the most frequently used phases in reversed-phase LC. Below that, three phases with an embedded polar group are shown the carbamate phases (e.g., SymmetryShield), amide phases (e.g.. Discovery RPAmide C g) and urea phases (such as Prism or Spectrum). It should... 

The great majority of CEC—MS applications is run in the reversed-phase mode using alkyl-bonded silica stationary phases [12,14,24,26,38,45,81,82,98-104], The dual functionality concept is represented in these stationary phases by the alkyl chains, most frequently octadecyl chains, that constitute the top retentive layer, and residual silanol groups on the surface, that dissociate at pH values higher than 3-4 and... 

There are also stationary phases that effectively partition solutes in either reversed-phase or normal-phase mode. These stationary phases are typically silica particles derivatized with cyano, diol, or amino functional groups. Particles with a cyano-functionality separate based on polarity utilizing nitrile interactions between the stationary phase and the solute. The amino group of typical amino stationary phases interacts primarily with anionic and organic acid portions of the solute. Diols utilize hydroxyl interactions similar to underivatized silica but offer a slightly different selectivity. These and other bonded-silica phases offer alternatives to underivatized silica, but they are used much less frequently. The mobile phases employed with these stationary phases are the same as used in standard reversed-phase or normal-phase chromatography. 

Bond and Wallace [10] described a microprocessor-based chromatographic system which they used for the simultaneous and automated determination of Pb(II), Cd(II), Hg(II), Co(II), Ni(II), and Cu(II). Reverse-phase was used to separate in situ formed di-thiocarbamate complexes, and the system could operate continuously and unattended for periods of several days using spectrophotometric detection, and slightly less time using electrochemical detection with background suppression because this mode required frequent suppressor regeneration. 

Silica gel, per se, is not so frequently used in LC as the reversed phases or the bonded phases, because silica separates substances largely by polar interactions with the silanol groups on the silica surface. In contrast, the reversed and bonded phases separate material largely by interactions with the dispersive components of the solute. As the dispersive character of substances, in general, vary more subtly than does their polar character, the reversed and bonded phases are usually preferred. In addition, silica has a significant solubility in many solvents, particularly aqueous solvents and, thus, silica columns can be less stable than those packed with bonded phases. The analytical procedure can be a little more complex and costly with silica gel columns as, in general, a wider variety of more expensive solvents are required. Reversed and bonded phases utilize blended solvents such as hexane/ethanol, methanol/water or acetonitrile/water mixtures as the mobile phase and, consequently, are considerably more economical. Nevertheless, silica gel has certain areas of application for which it is particularly useful and is very effective for separating polarizable substances such as the polynuclear aromatic hydrocarbons and substances... 

In SEC analysis of additive extracts from polymers, the effect of the extraction solvent on the mobile phase is less critical than in HPLC analysis. The extraction solvents typically employed generally do not interfere with the SEC mobile phases. Moreover, the same solvents are often used both as extraction solvent and as mobile phase. Therefore, there is no need to evaporate the extract to dryness prior to analysis and then to redissolve it in a suitable solvent. Typical extraction procedures often produce extracts that generally contain a small amount of wax. Frequently, removal of such oligomers from an extract is necessary, e.g. by means of precipitation, centrifuging, precolumn filtration or protection (use of a reversed-phase guard column). In SEC separations the presence of polyolefin wax does not usually disturb provided that the MW of the wax is higher than that of the analysed compounds. 

Modified silica with a C18 reversed-phase sorbent has historically been the most popular packing material, owing to its greater capacity compared to other bonded silicas, such as the C8 or CN types [22]. Applications of C18 sorbents include the isolation of hydrophobic species from aqueous solutions. The mechanism of interaction with such sorbents depends on van der Waals forces, and secondary interactions such as hydrogen bonding and dipole-dipole interactions. Nevertheless, the main drawbacks of such sorbents are their limited breakthrough volumes for polar analytes, and their narrow pH stability range. For these reasons, reversed-phase polymeric sorbents are also used frequently in environmental applications for the trace enrichment of soluble molecules that are not isolated by reversed-phase sorbents such as C18. 

Counter-ions which are frequently used include tetrabutylammonium phosphate for the separation of anions and hexane sulphonic acid for cations. The appropriate counter-ions are incorporated in the solvent, usually at a concentration of about 5 mmol 1" and the separation performed on the usual reverse phase media. This ability to separate ionic species as well as non-polar molecules considerably enhances the value of reverse-phase chromatography. 

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were 150 X 3.9 mm i.d. particle size 4 /jm and 250 X 4.6 mm i.d. particle size 5 //m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250 X 21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. 

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