Phenyl columns

F W. Wainer and R. M. Stifhn, Direct resolution of the stereoisomers of leucovorin and 5-methylteti ahydrofolate using a bovine serum albumin liigh-performance liquid cliromatographic chiral stationary phase coupled to an acliiral phenyl column , 7. Chromatogr. 424 158-162 (1988). 

All reagents and solvents were used as received (Sigma-Aldrich) if not stated otherwise. NOL-130 stab initiation mix and preformed, 19 mg graphite-blended l,3,5-trinitro-l,3,5-triazacyclohexane (RDX) pellets were purchased from Day Zimmerman (DZI). Samples prepared for LC-MS analysis were dissolved in ammonium hydroxide and run utilizing a Phenomenex Gemini C6-Phenyl column (4.6 mm X 150 mm) and a mobile phase of 20 mM ammonium acetate in water pH 7.02 at 0.75 ml,-min The HPLC analysis is done using... 

The minor (R,S) diastereomer is present in the crude reaction mixture to the extent of approximately 0.9% as determined by capillary gas-liquid chromatographic analysis performed on a Hewlett-Packard 5790A gas chromatograph equipped with a Hewlett-Packard 3390A integrator and HP-1 methylsilicone gum column (25 m x 0.2 mm x 0.33 pm film thickness). The checkers found that HPLC analysis (Zorbax SB-Phenyl column 25 cm x 4.6 mm, 40 60 MeCN/0.1% aqueous phorphoric acid, 1.5 mL/min, 250 nm detection) provided satisfactory resolution of the R,R- and R,S-diastereomers. The minor diastereomer is hardly discernible by 1H NMR (500 MHz) after purification by flash chromatography. 

Water and waste water Phenols separated on a Nova-Pak Phenyl column eluted with ammonium acetate acetonitrile LC-ED 0.5 mg/L 91-100% Paterson et al. 1996... 

Phenyl columns represent a third mode of selectivity. Pi-Pi bonding, properly speaking, is independent from hydrophobic interactions, but it has been shown to exert substantial influence on retention. Solutes with accessible ring structures are retained much more strongly than they are on similarly hydrophobic nonphenyl columns.5,13... 

These selectivity differences provide a rational basis for a three-column initial screening strategy a strongly hydrophobic column, a weakly hydrophobic column, and a phenyl column. Commercially available columns are not indexed in a manner that easily allows column selection based on hydrophobicity however, butyl columns are good candidates for strongly hydrophobic columns. Columns with names like ether-, iso-, and isopropyl are good candidates for weakly hydrophobic columns. 

Table 7 HPLC Methods for Quantitating Total Thiamine in Foods (Phenyl Columns Fluorescence Detection)... 

Carbaryl Reversed-phase-phenyl column, methanol-water UV 3 ng, 0.01 mg 52,117... 

Ion-pair high-performance liquid chromatography using phenyl columns [473]... 

Are the primary differences in polarity Partition columns are available that vary in polarity from nonpolar (octyldecyl), through intermediate polarity (octyl and cyanopropyl), to polar (silica). Some columns have similar polarities, but differ in their specificity. Qg and the phenyl column have similar polarities, but Ci8 separates on carbon chain length, while phenyl separates fatty acids on both carbon number and number of double bonds. Phenyl columns also resolve aromatic compounds from aliphatic compounds of similar carbon number. In another example of similar polarities, C8 is a carbon number separator while cyanopropyl selects for functional groups. 

The first grouping is a mix of fat-soluble compounds that function as hormones, co-factors, and membrane components. Fat-soluble vitamins separate on a Cis column in 80% acetonitrile/water and are usually detected at UV, 280 nm, or with fluorescence. Triglycerides are slightly less nonpolar than fat-soluble vitamins and require 60% acetonitrile/water to run on Ci8. They have poor extinction coefficients, and detection at UV, 220 nm, competes with refractive index detection in sensitivity. A phenyl column run in 50%... 

Free fatty acids can be separated on a Ci8 column based on carbon number using 50% MeOH/water pH 2.5 at UV, 280 nm a fatty acid column (actually a phenyl column) will also separate them based on the number of double-bonds. Fatty acids have also been analyzed at UV, 210 nm, or by refractive index. For high-sensitivity work, they are derivatized with bromophenacylbromide and separated on Ci8 in a 15-80% AN/water gradient at 254 nm. Increase in early running C2 and C4 fatty acids measured by HPLC is used as an indicator of bacterial action. Krebs cycle acids are di- and tricarboxylic acids involved in metab-... 

Table 5 shows that the amount of BSA eluted with GFPuv from the methyl and butyl HIC columns was half that recovered in the samples eluted from the octyl and phenyl columns, for a range of respective mean concentrations of GFPuv from 27.92 (methyl column) to 28.66 pg/mL (phenyl support), corresponding to a total average of 28.25 0.36 pg of GFPuv/mL. 

Fig. 2. Polyacrylamide gel run by SDS-PAGE (PhastSystem). Protein was stained with silver nitrate. Gel visualization was altered by nitrate oxidation. The eluted samples from HIC columns applied to the wells were as follows lane 1, mass molecular weight markers, 18-200 kDa (Gibco) lane 2, sample eluted from methyl column (0.085 pg) lane 3, sample eluted from butyl column (0.080 pg) lane 4, sample eluted from octyl column (0.077 pg) lane 5, sample eluted from phenyl column (0.069 pg).

Extract powder in low actinic flask with aqueous solution containing phosphate buffer-citric acid and metabisulfite. Condition sorbent with methanol, water, and extraction solvent. Fit SAX column on top of phenyl column. After applying sample, wash with extraction solvent. After removing the SAX column, phenyl column is washed with water, air dried, followed by hexane, methylene chloride, acetonitrile, and acetonitrile-methanol (95 5). Sample eluted with methanol-water (9 1). 

Scalia and Games developed a packed column SFC method for the analysis of free bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) [32]. The baseline separation of all five bile acids was achieved on a packed phenyl column with a methanol-modified carbon dioxide in less than 4 min. The elution order showed a normal-phase mechanism because the solutes eluted in the order of increasing polarity following the number of hydroxyl groups on the steroid nucleus. The method was also applied to the assay of UDCA and CDCA in capsule and tablet formulations. The method was found to be linear in the range 1.5-7.5 ng/ml (r > 0.99, n = 6). The average recoveries (n= 10) for UDCA and CDCA were 100.2% with a RSD of 1.7% and 101.5% with a RSD of 2.2%, respectively. The reproducibility of the method was less than 1.5% (n = 10) for both UDCA and CDCA. 

Miscellaneous steroids were separated on a packed phenyl column (Spherisorb, 10 cm x 4.6 mm ID) using UV detection at 254 nm and a... 

Holtzapple et al. developed an immunoassay method for determination of four fluoroquinolone compounds (including ciprofloxacin) in liver extracts [64]. In this method, an immunoaffinity capture SPE column was used, that contained anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix compounds had been washed away, the bound ciprofloxacin was eluted directly onto the HPLC column. The HPLC system used a 5 pm Inertsil phenyl column (15 cm x 4.6 mm i.d.), with 0.1 M-glycine hydrochloride/acetonitrile (17 3) as the mobile phase (eluted at a rate of 0.7 mL/min). Fluorimetric detection at 444 nm was used after excitation at 280 nm. The recovery of ciprofloxacin ranged from 85.7 to 93.5%, and the detection limit was... 

Nimodipine, tarcine, and their metabolites were determined by HPLC in the plasma of patients with Alzheimer s disease [23], After extraction and evaporation under nitrogen to dryness, the residue was reconstituted into acetonitrile/H20 (1 3), and analyzed on a 5-pm Hypersil phenyl column (25 cm x 4.6 mm i.d.) equipped with a Nova Pak phenyl guard column and operated with acetonitrile/4.4 mM KH2P04 buffer (39 61) as the mobile phase (flow rate of 1.5 mL/min). Detection was effected either fluorimetrically at 330 nm excitation and 360 nm emission, or by its UV... 

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. 

Final purification was accomplished on an 1-125 protein separation column operated in a normal-phase mode. The C-1 column provided the breakaway step as more than 95% of the UV-214 absorbing material in the phenyl column fractions was retained less than 24 min but all of the myotropins were eluted after 26 min postinjection. 

Additionally, the C-1 column separated some of the myotropins that would have been quite difficult to resolve on C-18. For example, leucokinin I (10) and leucopyrokinin (11) were isolated in the same 2-min fraction from the phenyl column. On the C-18 column, their retention times differed by 36 sec and the two peaks were only partially resolved (Holman, unpublished observation). However, the problem of separating leucokinin I (LK-I) and leucopyrokinin (LPK) never materialized as LPK was retained 20 min longer on C-1 than LK-I. Similarly, leucokinin VIII (LK-VIII) and leucosulfakinin (LSK) were collected in the same phenyl column fraction (12.13 and exhibited very similar retention times (51.4 min and 51.0 min, respectively) on C-18. However, the problem of separation never materialized because LK-VIII and LSK were collected from C-1 in fractions that were separated by 15 min. 

The following examples will be used to demonstrate that medium-polarity reversed phases have great potential for protein separations. In Fig. 7 the separation of the C apolipoproteins is shown on a phenyl and on a Ci8 column. The Cig column gave almost no separation of the protein mixture, despite the use of a variety of mobile-phase conditions, whereas the phenyl column gave an excellent separation. Lewis et al. (34) found that the columns of intermediate polarity were effective in the separation of tyrosinase, collagen i, cytochrome c, and bovine serum albumin. In this study the diphenyl column was more retentive than the octyl or nitrile... 

Fig. 9. Separation of porcine despenta-insulin (des-B -j,-insulin) (2) from insulin (1) on a /i-Bondapak alkyl phenyl column using a 60-min linear gradient of 25-50 f acetonitrile containing 5 mM dodecylammonium phosphate. pH 4.0 flow rate 2 ml/min. Reproduced from Hearn et cil. (70).

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 phenyl group offers a difference in selectivity to ODS for many compounds in reversed phase HPLC, and has been used in the analysis of polycyclic aromatic hydrocarbons. For most compounds, retention on a phenyl column is similar to that obtained on a C8 packing material. 

In spite of these efforts, silica based modified columns are still widely used. Many column-manufacturers have introduced quality control procedures for the synthesis and characterization of silica based columns. Therefore, lot to lot variations from the same manufacturers have been considerably reduced. However, identical phases from different manufacturers can yield different separation behaviors of the same analyte under identical instrumental and mobile phase conditions. (Of 13 phenyl columns investigated for an oncolytics, we found only three columns providing similar separation profile for a 13-component impurity mixture.)... 

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