Comparison with reversed-phase HPLC

The application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

Fig. 2.28. Relative abundance of carotenoid pigments in zebra finch diet, plasma and tissue. The carotenoid profile of each sample was determined by conventional reversed-phase HPLC. Letters denote significant differences in carotenoid composition (within tissues only) as determined by post hoc paired comparisons. Reprinted with permission from K. J. McGraw et al. [67].

Reversed-phase HPLC uses a nonpolar stationary phase and a polar mobile phase. The characteristics are operational simplicity, high efficiency, column stability, and ability to analyze simultaneously a broad spectrum of both closely related and widely different compounds. Separation is based on hydrophobicity.33 Findlay et al. provide a comparison of chromatography methods with immunoassays (Table ll.l).27... 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

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. 

Farina, A. et al., HPTLC and reflectance mode densitometry of anthocyanins in Malva Silvestris L. a comparison with gradient-elution reversed-phase HPLC, J. Pharm. Biomed. Anal, 14, 203, 1995. 

The reverse-phase mode is used for all the separations performed in this experiment. Reverse phase is the term used when the stationary phase is more nonpolar than the mobile phase with regard to the polarity of the sample. The isopropanol/water and isopropanol/vinegar mobile phases are typical of reverse-phase mobile phases, which generally are composed of water mixed with polar organic modifiers. The bonded Cig column used is a very nonpolar surface and is the most popular stationary phase for reverse-phase HPLC. In this experiment the silica column when used in the reverse-phase mode provides a very weak nonpolar surface in comparison to C g. Silica is normally thought of as a highly polar surface and is most commonly used in the normal-phase mode. The use of silica in the normal-phase mode, with a nonpolar mobile phase is the subject of Chapter 9 (Experiment 2). 

An interesting and useful variant of reversed-phase HPLC is called ion-paired reversed-phase HPLC. In such a system the analytical columns are packed with the same material, but a compound such as tetrabutylammonium is added to the mobile phase. The separation of ATP and adenosine on such a system is shown in Figure 2.15. A comparison of this profile to that shown for the same compounds in Figure 2.14 immediately highlights the change in the elution sequence. Whereas without ion pairing, the order is ATP followed by adenosine, with ion pairing the order is adenosine followed by ATP. 

Another interesting aspect of this study was the comparison of MEKC and HPLC. While in MEKC the drugs represented in Figure 8.1 migrated in the order morphine (1), phenobarbital (2), 6-monoacetylmoprhine (6-MAM) (3), 3-monoacetyl morphine (3-MAM) (4), methaqualone (5), heroin (6), acetylcodeine (7), papaverine (8), and noscapine (9), in reversed-phase HPLC the elution pattern was substantially different and not correlated with MEKC... 

Rhodotorulic acid (RA), a dihydroxamate siderophore, forms dimeric complexes with iron, aluminium and chromium of the stoichiometry M2(RA)3 at neutral pH 36 188). The coordination chemistry of this siderophore is probably the most complicated of the siderophores. The combination of cis-trans, A and A configurations of two iron miters, connected by three RA molecules, makes 42 non-redundant isomers theoretically possible each can be simulated by molecular models. Recently three different isomers or mixtures of isomers of Cr2RA3 were separated by reversed phase HPLC-chromatography177). The visible spectrum of the most abundant fraction corresponds to the cis isomer the two other fractions are very similar to the visible spectrum of the trans Cr(men)3 isomer. The CD spectra, in comparison with the Cr(men)3 model complex, show two different optical isomers, assigned as A -trans and A -trans. The A isomer preparation seems also to contain a certain amount of the A configuration. This is the first time that two different, kinetically stable optical isomers have been isolated from the metal complexes of a siderophore 177). 

Use of the reverse-phase HPLC system is highly flexible since it can also be applied to ionizable compounds such as carboxylic acids, phenols, and amines. The partition coefficients relate to the unionized compounds that are generally assumed to be the principal forms in which these compounds are transported into biota, even though their concentration may be low in comparison with the dissociated states at physiological pH values acidic compounds such as highly chlorinated phenols or many carboxylic acids have... 

Comparison of Metabolic Profiles In Urine. Llyer aml Kldfigj The metabolic profiles of liver and kidney were compared with that of urine. The comparison was done using the analysis of the extracts on two reverse phase HPLC systems. Based on the comparison of RRTs, It was evident that the metabolites found In liver and kidney were also found In the urine. 

To extend the )plications of LC-NMR, we have further examined the compositions and blockiness of various polymer mixtures, including pBA and polybutadiene, where 1,4-butadiene, 1,2-butadiene and BA were identified by their unique H chemical shifts at tqrproximately 5.35, 4.95 and 3.98 ppm, respectively. In reverse-phase HPLC with the same solvent gradient conditions as above, homopolymer pBA and polybutadiene eluted at 21.76 and 34.20 min., respectively. TTie random copolymers of p(MMA/BA) and p(MMA/Sty) both eluted between 8 and 18 minutes. Owing to their hydrophobicity, the hi er the percentage of BA and styrene in the copolymer, the longer the retention time. Figure 5 illustrates the LC separation of pMMA, pBA, p(MMA/BA) and p(BA-b-MMA) by a reverse-phase column. A comparison of p(MMA/BA) random copolymer (retention time 13.9 min.) to p(BA-b-MMA) block copolymer (retention time 18.3 min.) with similar composition shows that the block copolymer interacted more with the C-18 stationary phase and eluted at a later time. This result demonstrated that the retention of p(MMA/BA) copolymer by reverse-phase LC is predominately influenced by die pBA portion of dre copolymer. The block copolymer, which mimics the homopolymer pBA, is mote hydrophobic and retained more on die C18 colunm than the random copolymer. The excellent LC separation permits us to quantitatively determine... 

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