Phenyl bonded phase, HPLC

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

The most satisfactory HPLC conditions for analysis of VI was ultimately achieved using a phenyl-bonded phase column in the reverse phase mode. Based upon the experience described above for polymeric octadecylsi-lane, water acetonitrile was exclusively used as mobile phase in the development work. 

Figure 6. HPLC analysis of human plasma from a non-marijuana smoker to which 100 ng/mL of VI has been added. A gradient (superimposed with dashed line) beginning with 60 40, water-.acetonitrile was used on a phenyl-bonded phase...

Early PG analysis using HPLC techniques was carried out as adsorption chromatography on normal-phase (NP) columns packed with silica or alumina. The nonpolar mobile phase comprizing of organic solvents (hexane, toluene, ethyl acetate, and HOAc) allows separation of PGs which are unstable in aqueous media (e.g., PGH2 on cyano- or phenyl-bonded phases). Usually, the injection medium must be fairly polar to dissolve the PGs. This is achieved by the addition of... 

HPLC has also used to determine taxol and related compounds in biological fluids [13]. After evaluating C8-, cyanopropyl-, and phenyl-bonded phases, Riley and his co-workers selected a C8-bonded phase column with a mobile phase of MeOH NaOAc buffer (0.02 M, pH 4.5, 35 65 v/v) as their system for analysis of taxol in human plasma and urine. 

The nomenclature of the RP is not consequent. The RP most often used contains octyl (RP C8) or octadecyl (RP C18) groups. There is no differentiation even when two methyl groups are introduced additionally with the silane (as with monofunctional silanes) or only one (difunctional) or none (trifunctional silane). Some manufacturer use silanes with bulky side groups (e.g., isopropyl groups) to improve the hydrolytic stability of the bonded phases, but here also, only the longest alkyl group is used in nomenclature. RP C8 and RP C18 are the work horses in HPLC. Shorter chains (RP4) are used in protein separations, and special selectivity can be obtained with bonded phenyl, cyano, amino or fluoro groups. 

Since the 1970s numerous HPLC methods using lEC, RP and ion-pair chromatography have been proposed. In the last years, RP chromatography has become the most used method, thanks to its simplicity, sensitivity, and compatibility with different detection techniques. The stationary phases usually used are C18 or phenyl-bonded silica-based phases. More recently, alternative stationary phases, such as polar-embedded, polar endcapped, and perfluorinated phases, have been successfully tested for folate analysis [577]. The mobile phase is usually a mixture of phosphate or acetate buffer and acetonitrile or methanol. 

The anthocyanins exist in solution as various structural forms in equilibrium, depending on the pH and temperature. In order to obtain reproducible results in HPLC, it is essential to control the pH of the mobile phase and to work with thermostatically controlled columns. For the best resolution, anthocyanin equilibria have to be displaced toward their flavylium forms — peak tailing is thus minimized and peak sharpness improved. Flavylium cations are colored and can be selectively detected in the visible region at about 520 nm, avoiding the interference of other phenolics and flavonoids that may be present in the same extracts. Typically, the pH of elution should be lower than 2. A comparison of reversed-phase columns (Ci8, Ci2, and phenyl-bonded) for the separation of 20 wine anthocyanins, including mono-glucosides, diglucosides, and acylated derivatives was made by Berente et al. It was found that the best results were obtained with a C12 4 p,m column, with acetonitrile-phosphate buffer as mobile phase, at pH 1.6 and 50°C. 

Phenyl-Type Phases. Phenyl-type phases have been studied for a long time [58,59]. The presence of a phenyl ring on the surface of a bonded phase introduces so-called n-n interactions with some analytes that are capable of these types of interactions. This introduces an additional specificity for HPLC separations on these stationary phases. Compared to common alkyl-type phases, phenyl columns show lower methylene selectivity in other words, the separation of members of homologous series will be less selective on phenyl columns than on alkyl-modified phases. 

Phenyl Bonded Reversed Phase HPLC. Employing a simple and rapid system of sample preparation, Pettitt and Damon (1982) developed a reversed phase HPLC for straw analysis. This method operates under the following conditions of LC ... 

Examples of reversed-phase HPLC bonded phases are given in Table 1. These include alkyl bonded silica, where the R group is an alkyl chain, the length of which determines the degree of hydrophobicity (C18, C8, C4, etc.) the phenyl stationary phase. 

Lambert and DeLeenheer (1992) contributed a review on the TLC analysis of K vitamins. They noted that although HPLC is usually the method of choice for analysis, there is some interest in HPTLC-densitometric analysis of the K vitamins. Their review covers isolation, extraction, and cleanup methods uses of polar inorganic solvents uses of modified silica (i.e., nonpolar bonded phases such as C2, Cg, C18, and phenyl) uses of mobile phases and methods of detection and quantification of the K vitamins. Madden and Stahr (1993) assayed vitamin K in bovine liver by reversed-phase TLC with dichloromethane-methanol (7 3)... 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is more popular for analysis of carotenoids than is normal-phase HPLC because (1) retention is very little affected by small variations in the mobile-phase composition, and (2) the risk of artifact formation on passage through the column is minimal as solute-support interactions on non-polar-bonded phases only involve weak forces. A variety of stationary phases of various polarities are available, such as C18, C8, C4, C2, Cl, phenyl, and cyano derivatives the C18 phase is the most popular. 

---