Phenyl-bonded phase column

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. 

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... 

RSP,methylreserpate Post-column reactor for Phenyl bonded phase not ACN-0.005M NaH.PO. buffer(pH 6) ... 

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 CGC analysis of the volatile degradation products were performed using a Perkin-Elmer Sigma 2000 capillary gas chromatograph. The column used was either a fused silica 0.25 micron, bonded methyl silicone (10 m, 0.25 mm I.D.) or a methyl/5% phenyl silicone (15 m 0.25 mm I.D.) bonded phase. The carrier gas was helium and the capillary column head pressure was maintained at 20 psi. The make-up gas for the pulsed electron capture detector (ECD) was 95% Ar/5% methane supplied at a flow rate of 60 ml/min. 

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 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. 

Liquid chromatographic separation of sedatives and -blockers is usually performed using reversed-phase columns. The preferred type of reversed-phase material is Cig-bonded silica (Table 29.16), but phenyl-bonded silica has also been employed for separation of xylazine and its major metabolite (525). Ion-pair liquid chromatography has also been suggested for separation of carazolol and xylazine residues, by addition to the mobile phase of dodecyl sulfate (522) or heptanesulfonate (520) pairing ions, respectively. 

FIGURE 4-7. Comparison of relative retention due to the polarity (hydrophobicity) of Cj8, phenyl, and cyano (CN) bonded phases. Column Nova Pak family (4/a) 3.9 mm ID x 15 cm. (a) C, phase, (b) Phenyl phase, (c) CN phase. Mobile phase— acetonitrile water, 35 65 flowrate 2 mL/min detection 254 nm sample 1. benzyl alcohol, 2. acetophenone, 3. p-tolualdehyde, 4. anisole. Nova-Pak CN is the least hydrophobic and therefore the least retentive, followed by the more hydrophobic Nova-Pak Phenyl, and Nova-Pak C18 with the most hydrophobicity and the longest retention times. 

FIGURE 4-8. Comparison of optimized separation on phenyl and cyano (CN) phases compared to a C18 phase. Columns 8 mm ID x 10 cm Radial Pak cartridges containing Nova-Pak bonded phase, (a) Nova-Pak C18 using a mobile phase of acetonitrile water (35 65), and Nova-Pak phenyl using a mobile phase of methanol ace-tonitrile water (33 8 59). (b) Nova-Pak C18 using a mobile phase the same as in a and Nova-Pak CN using a mobile phase of tetrahydrofuran water (10 90). Flow rate 2 mL/min. Detection 254 nm. Sample 1. benzyl alcohol 2. 2-phenoxyethanol 3. anisaldehyde 4. acetophenone 5. p-tolualdehyde 6. p-methylacetophenone, 7. anis-ole 8. phenetole. 

FIGURE 4-10. Comparison of retention on different phases. Columns 8 mm x 10 cm Radial Pak containing Nova-Pak bonded phases, (a) The mobile phase for the C,8 column is acetonitrile water (17.5 82.5) with 5 mM tetrabutylamine (PIC A) the mobile phase for the phenyl column is acetonitrile water (17.5 82.5) with 5 mM tetrabutylamine (PIC A). Flow rate 4 mL/min. Detection 254 nm. Sample—cephalosporin antibiotics 1. cephradine 2. cefotaxime 3. cefazolin 4. cefuroxime 5. cefoxitin 6. cefoperazone 7. cefamondole 8. cephalothin. (b) The mobile phase for the C)8 column is tetrahydrofuran methanol water (10 27.5 62.5) with 5 mM octane-sulfonic acid (PIC B8) the mobile phase for the CN column is tetrahydrofuran methanol water (5 22 73) with 5 mM octanesulfonic acid (PIC B8). Flow rate 2 mL/ min. Detection 254 nm. Sample—Beta-adrenergic blockers 1. atenolol 2. nadolol 3. pindolol 4. metoprolol 5. timolol. 

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. 

The separation of chiral compounds will be discussed in Chapter 22. However, the separation of diastereomers can be accomplished using achiral stationary phases. Another alternative is the use of chiral columns for the separation of diastereomers in either the reversed-phase or normal-phase mode. The use of achiral bonded phases without chiral additives, such as phenyl and alkyl bonded phases for the separation of diastereomeric pharmaceutical compounds, is acceptable. Different selectivities can be obtained by employing stationary phases containing varying functionalities (phenyl, polar embedded moieties). The effect of aqueous mobile-phase pH, temperature, and type of organic eluent (acetonitrile versus methanol) can also play a dramatic role on the separation selectivity of diastereomeric compounds. 

The efficiency of chiral stationary phase (CSP) is crucial in chromatographic technique. Recently, a new p-cyclodextrin phenyl isocyanate bonded chiral stationary phase (CSP) was developed. This CSP is quite stable and can be used in most of HPLC solvents. Many drug enantiomers that do not have enantioseparation effect on native P-cyclodextrin column in reversed phase were separated very well on this new CSP. 

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