Mobile phase RP-HPLC

The mode of separation in the HPLC depends on the selection of the stationary and mobile phases. In HPLC of lipids, normal- and reversed-phase modes are primarily used, with the reverse phase being more common than the normal phase. Separation in the re-versed-phase mode is mainly by partition chromatography, whereas separation in the normal phase mode is primarily by adsorption chromatography. Normal-phase HPLC is used for the separation of the lipids into classes of Upids [1,F]. Reversed-phase HPLC (RP-HPLC), on the other hand, is mainly used to separate each lipid class into individual species [2,B1]. For example, several triglycerides were separated from each other via nonaqueous reversed-phase HPLC, involving an octadecyl (ODS) column and a nonpolar (non-aqueous) mobile phase. RP-HPLC alone can be used to separate the fat molecules into classes and species [2,B1]. 

Reverse-phase (RP) HPLC has been used widely for FFA analysis. The stationary phase is almost always the octadecylsilyl (ODS) type. The mobile phase is typically acetonitrile or methanol in water and detection is by UV between 205 and 210 nm. FFAs are separated on the basis of both chain length and degree of unsaturation (Christie, 1997). An example of a modern HPLC system is shown in Figure 19.2. 

Reverse-phase (RP)-HPLC is probably the best system for purifying triterpenoids, principally when mixtures of isomers are present [35]. Gunther and Wagner in 1996 [36] carried out the separation and quantification of active triterpenes from Centella asiatica employing an RP system with acetonitrile-water as mobile phase. Recently, Gaspar et al. [37] described the complete separation of a mixture of triterpenoid isomers from the fruit of Arbutus unedo by HPLC coupled to a mass spectrophotometer by means of a particle beam interface (HPLC-PBMS). The separation of different quassinoids from crude bark of Quassia amara was developed by Vitanyi et al. [38] using a reverse-phase HPLC-MS... 

CD-containing mobile phases in HPLC have been successfully used for the separation of various isomers such as structural isomers, diastereomers and enantiomers. For example, ortho, meta, para isomers of cresol, xylene and aU six isomers of nitrocinnamic acid were separated on the Lichrosorb RP-C18 column with jS-CD solution as mobile phase [41]. Similar results were also obtained for ortho, meta and para isomers of nitrophenol, nitroaniline, fluoronitrobenzene. 

A method for determining valproic acid in patients plasma has been described [98]. The plasma from patients receiving valproate therapy was separated within 2h of blood collection. The internal standard cyclohexane-carboxylic acid and 2 ml of pentane were added to 0.25 ml of plasma. After separation, the aqueous solution was acidified with 0.25 ml of 1 M sulphuric acid, and the valproic acid was extracted with 4 ml of pentane and derivatized at 50—55 °C for Ih with 20/il of phenacyl bromide and 20 /il of triethylamine. After evaporation of the solvent, the residue was dissolved in 250 //I of the mobile phase for HPLC separation on a Lichrosorb RP-18 column with methanol/water (3 1 v/v) or acetonitrile/water (2 1 v/v) as eluent. The derivatized valproate... 

The purity of heptakis(2,3,6-tri-0-methyl)-P-CD preparations was checked by reversed-phase (RP)-HPLC using the isocratic mobile phase, methanol-water 85/15, v/v. Analytes were detected by atmospheric pressure chemical ionization-mass spectrometry (APCl-MS). The measurements illustrated that the purity of the CD derivatives markedly influences the enantioselectivity of the preparation. 

Another approach in taxoid preparative separation included solid-phase extraction (alumina, silica, or RP-8 cartridges) followed by preparative TLC on silica gel plates with quaternary mobile phase consisting of dichloromethane-dioxane-acetone-methanol (83 5 10 2, v/v). In this way, 10-DAB III, paclitaxel, and cephalomannine as well as two further taxoids could be easily isolated with relatively high efficiencies from yew materials (Fig. 2). Multiple development technique or fiuther separation of the isolated taxoid fractions (especially less polar ones) on RP-2 silica bond stationary phase with methanol-water mixtures as mobile phases was applied for purification of the compounds isolated. 10-DAB III isolated in this way was relatively pure, as was shown in reversed-phase (RP)-HPLC analysis (Fig. 3). 

C18 layers have been found to have clo,se correlation with HP liquid chromatography reversed-phase columns (Gonnet and Marichy, 1979), and TLC is used widely to scout appropriate mobile phases for HPLC (see Chapter 6). Very polar substances (basic and acidic pharmaceuticals) can be separated on RP layers by ion pair chromatography. 

When one develops new reversed-phase (RP)-HPLC methods, one usually uses the selectivity of the mobile phase as the primary method development tool. The chromatographic separation can be influenced by the choice of the organic solvent (mainly methanol and acetonitrile), or by variation of pH or buffer type. Schemes for method development using these parameters have been described in the literature [1,2]. Most important are the selectivity changes caused by pH changes, which are well-understood and easily predictable (3). It is well known that the stationary phase influences the selectivity as well, but this effect is often not very well understood. The primary reason for this is the fact that reliable methods for the description of the stationary phase selectivity have only become available fairly recently. In the last few years, several papers have been published that deal with the subject of selectivity in a fimdamental way [4—9] or represent a data collection based on older methods [10-15]. In this chapter, we describe in detail the method used in our laboratory. We then look at our selectivity charts and discuss our results. It needs to be pointed out in advance that selectivity charts only accurately represent the properties of a stationary phase under the conditions of the measurement. If we depart from the mobile phase composition of the test, the relationships between different columns will change, since selectivity arises from a combined effect of the mobile phase and the stationary phase. 

Analytical screens are performed with both reverse-phase RP-HPLC and SFC isolation techniques. Analytical SFC should be screened first unless instrumentation availability or project background specifics dictate otherwise. Screening achiral column bonded phases varying in polarity and functionality against different mobile-phase solvent choices is effective for identifying analytical methods for the purpose of impurity isolation. There are currently many unique achiral SFC bonded phase column choices commercially available (2-ethyl pyridine, diethyl amino, dinitrophenyl, pyridine urea, diol, cyano, etc.). SFC column choice provides the most impact in manipulation of relative selectivity for individual... 

The composition of PPG—PEG blends has been determined using gpc with coupled density and RI detectors. PEG and PPG have different response factors for the density and RI detectors which were exploited (173). An hplc system with CHROMPAC RP-18C2g column at 298°C and acetonitrile—water or methanol—water as the mobile phase has been used to gather information about the functionaUty of PPO (174). 

As a method of research, has been used high-performance liquid chromatography in reversed - phase regime (RP HPLC). The advantage of the present method is the following the additional information about AIST and FAS composition (homologous distribution) simple preparation of samples (dilution of a CS sample of in a mobile phase). 

Early [1, 2] it was reported about RP-HPLC the separation of amino derivatives of 3-chloro-l,4-naphtoquinone with methanol mobile phase. In some cases changing organic modificator in eluent leads to the progress in effectiveness of sepai ation. In present work the compaiison was performed for separation of some amino derivatives of 3-chloro-I,4-naphtoquinone by RP-HPLC with methanol and acetonitrile eluent. It has been shown that certain differences exist for vaiious derivatives mentioned above. 

Similarly to the methods used to characterize natural chlorophylls, RP-HPLC has been chosen by several authors to identify the individual components in Cn chlorophyllin preparations and in foods. The same ODS columns, mobile phase and ion pairing or ion suppressing techniques coupled to online photodiode UV-Vis and/or fluorescence detectors have been used. ° ... 

Girod, L., Martel, S., Carrupt, P. A. The lipophilicity of zwitterionic compounds by RP-HPLC the marked influence of 1-octanol in mobile phase. Unpublished results. 

Valko et al. [37] developed a fast-gradient RP-HPLC method for the determination of a chromatographic hydrophobicity index (CHI). An octadecylsilane (ODS) column and 50 mM aqueous ammonium acetate (pH 7.4) mobile phase with acetonitrile as an organic modifier (0-100%) were used. The system calibration and quality control were performed periodically by measuring retention for 10 standards unionized at pH 7.4. The CHI could then be used as an independent measure of hydrophobicity. In addition, its correlation with linear free-energy parameters explained some molecular descriptors, including H-bond basicity/ acidity and dipolarity/polarizability. It is noted [27] that there are significant differences between CHI values and octanol-water log D values. 

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