Column bonded-phase

Terpene chemists use mainly gas chromatography in dealing with terpene mixtures in research and development as weU as in quahty control. Capillary gas chromatography with stable bonded-phase columns, the primary analytical method, is also being used more frequendy in the 1990s in product quahty control because its greater resolution is helpful in producing consistent products. 

Appllca.tlons. The first widely appHcable Ic separation of enantiomeric metallocene compounds was demonstrated on P-CD bonded-phase columns. Thirteen enantiomeric derivatives of ferrocene, mthenocene, and osmocene were resolved (7). Retention data for several of these compounds are listed in Table 2, and Figure 2a shows the Ic separation of three metallocene enantiomeric pairs. P-Cyclodextrin bonded phases were used to resolve several racemic and diastereomeric 2,2-binaphthyldiyl crown ethers (9). These compounds do not contain a chiral carbon but stiU exist as enantiomers because of the staggered position of adjacent naphthyl rings, and a high degree of chiral recognition was attained for most of these compounds (9). 

Fairly reproducible and stable bonded-phase columns... 

Ionic solutes can be separated by ion-exchange chromatography using microparticulate resins or bonded ion-exchangers based on microparticulate silica. Such separations are often achieved more easily by ion-suppression or ion-pairing techniques, which use bonded phase columns in the reverse phase mode. 

In order to measure k for the solutes we have to know the retention distance or volume of an unretained solute. The accurate determination of this quantity is not an easy problem with bonded phase columns and reverse phase operation. A number of different methods have been used ... 

Distilled or deionised water contains small amounts of organic impurities which can cause problems in long term use with bonded phase columns in the reverse phase mode. The non-polar stationary phase will collect these organics, which can alter the nature of the stationary phase or sometimes produce spurious peaks (Fig. 4.3c is an example of this). Water purification can be done by distillation from permanganate, by passage of the water through bonded phase columns, or by means of commercial systems, eg the Milli-... 

If the performance of a column is no longer satisfactory it can sometimes be reconditioned by washing with a suitable solvent, or series of solvents. Some bonded phase columns, C-18 for instance, tend to collect non-polar impurities, which can sometimes be removed by washing the column with a non-polar solvent, eg heptane. Assuming the mobile phase normally used with the column is CH3OH/H2O 50 50, we cannot wash directly with heptane because of miscibility problems, we have to get to heptane via a miscible solvent or series of solvents. 

Katz, E.D. and Ogan, K.L., Selectivity factors for several PAH pairs on Cl8 bonded phase columns, J. Liq. Chromatogn, 3, 1151, 1980. 

The pH of a buffered mobile phase is significant not only because retention of ionizable compounds is dependent upon pH but because bonded phase columns are made via an acid catalyzed reaction. Manufacturers recommendations are to avoid any system with a pH lower than 2 or accept the risk of reversing the bonding reaction. However, the effective pH experienced by a column in a water organic mixture is difficult to predict. We have operated at a pH of 1.5 in this laboratory with no adverse effects to the column, but exercise the simple precaution of not allowing the acidic mobile phase to remain on the column overnight or static for long periods of time (1 hr). We have particularly noticed that the CN column is stable at low pH s. 

The pKa of the imidazole ring is near 6 (16) so histamine would only exist as an ion in the acidic (pH = 2-3) mobile phase. One would predict no retention on a bonded phase column under this condition however, it does occur. Figure 3 is the simplest way to account for this retention. Here, the mineral acid acts as the counter-ion, as well as the buffer. All of the histamine in the mobile phase is in the ionic form and is in equilibrium with the ion-pair which is only soluble in the stationary phase chemically bonded to silica. Histamine only elutes in the ionic form and is then derivatized for detection. A sharp peak in the chromatogram with good shape and no change in retention time with variation in sample concentration indicates a working system. However, if the paired ion has some solubility in the mobile phase, peak tailing occurs. 

The HPLC method presented is based upon the separation of histamine itself and not a derivative of unknown structure. An ion-pair model is proposed to control retention of histamine on bonded phase columns. This method and model should be applicable to a wide range of biosystems, as well as other aqueous plant extracts. Our results determined by HPLC analysis agree closely with those obtained previously using biological methods but provide geater reproducibility and simplicity. 

Riggin and Howard (1979, 1982), Matsui et al. (1983), Fabre et al. (1984), and Ahuja et al. (1988) reported that High Performance Liquid Chromatography (HPLC) with UV or electrochemical detection is capable of analyzing 1,2-diphenylhydrazine. Reversed phase chromatographic columns have been used most often (Ahuja et al. 1988 Fabre et al. 1984 Riggin and Howard 1979, 1982). Cyano-amino polar bonded phase columns also have been used (Matsui et al. 1983). Using a reversed phase and UV detection, the minimum amount detected (on column amounts) is approximately 6-7 ng and the minimum amount quantifiable is less than 1 pg (Ahuja et al. 1988 ... 

Practical Operation of Bonded-Phase Columns in High-Performance Liquid Chromatography... 

J. B. Adams, Jr., HPLC of Basic Compounds at High pH with a Silica-Based Bidentate-C18 Bonded-Phase Column, Am. Lab., November 1999, p. 22 ... 

Both the highly active adsorbent and the moderate eluent seem to be necessary for the separation of oligostyrenes into stereoisomers. Elution of another sample on a nitrile bonded-phase column with iso-octane/dichloromethane could not reveal stereoisomers but only well-separated oligomers 48). 

Earlier work in the HPLC analysis of TGs used a differential refractometer as the detector a number of papers have detailed isocratic systems combined with refractive index (RI) detectors, often with acetonitrile/acetone mobile phases. Although aqueous mobile phases were generally used with alkyl-bonded phase columns, due to the lipophilicity of TGs, water could not be used in the mobile phase for this particular application therefore the mobile phases generally employed consisted of mixtures of acetone and acetonitrile and occasionally tetrahydrofuran, methylene chloride, or hexane (the conspicuous absence of water in the mobile phase prompted the term nonaqueous reverse phase, or NARP, to describe these systems). 

MA Kaluzny, LA Duncan, MV Merritt, DE Epps. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res 26 135-140, 1985. 

Bonded-phase column packings for use in normal-phase chromatography are available in which the stationary phase is a polar functional group chemically bonded onto the silica surface. One... 

Table 10 Properties of Polymeric ODS Bonded-Phase Column Packings...

Semipreparative HPLC has been employed to obtain a vitamin D-rich fraction of the unsaponifiable matter for subsequent quantitative HPLC. Combinations of chromatographic modes used for offline semipreparative and quantitative analysis have included polar bonded-phase/adsorption (211,212), reversed-phase/adsorption (194,213), and adsorption/reversed-phase (70,125). An online two-dimensional HPLC technique using two polar bonded-phase columns has also been described (214). 

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). 

Most cases of isocratic HPLC reported were carried out on reversed-phase chromatography on silica-based ODS (C,8 bonded phase) columns (1) for citrus juices. For mobile phases utilizing C,8 columns, isocratic solvents of 80-82% aqueous acetic acid with acetonitrile were commonly used. A ternary mobile-phase system of water-acetonitrile-glacial acetic acid... 

---