Reversed-phase HPLC systems

If simple sample pretreatment procedures are insufficient to simplify the complex matrix often observed in process mixtures, multidimensional chromatography may be required. Manual fraction collection from one separation mode and re-injection into a second mode are impractical, so automatic collection and reinjection techniques are preferred. For example, a programmed temperature vaporizer has been used to transfer fractions of sterols such as cholesterol and stigmasterol from a reversed phase HPLC system to a gas chromatographic system.11 Interfacing gel permeation HPLC and supercritical fluid chromatography is useful for nonvolatile or thermally unstable analytes and was demonstrated to be extremely useful for separation of compounds such as pentaerythritol tetrastearate and a C36 hydrocarbon standard.12... 

Zhu, J., Shi, Z. (2003). ESI-MS studies of polyether surfactant behaviors in reversed-phase HPLC system. Int. J. Mass Spectrom. 226(3), 369-378. 

A variety of gradient and isocratic reverse phase HPLC systems-have been used to ehromatograph dorzolamide, and the salient characteristics of these are summarized in Table 6. 

Porphyrins are separated from interfering pigments in stool by diethylether and then analyzed on a reverse-phase HPLC system with fluorescence detection using the same conditions as for urinary porphyrins. 

Inject extract into the normal- or reversed-phase HPLC system (see Basic Protocol). SAMPLE PREPARATION FROM MEATS... 

The determination of polyphenolics may result in interference due to co-elution of phenolic acids and procyanidins. This problem can be eliminated by fractionation of polyphenolics into acidic and neutral polyphenolics prior to sample injection into the HPLC system. Because the fractionation techniques effectively improve the resolution of many polyphenolic peaks in the reversed-phase HPLC system, it is suggested that further characterization and identification of unknown peaks be conducted by additional methods such as mass spectrometry and nuclear magnetic resonance. 

Miwa et al. (26) have demonstrated that both short- and long-chain fatty acids can also be converted into their 2-nitrophenylhydrazides and separated bv RP-HPLC with acetonitrile-water as the eluent. They have described a method for the direct derivatization without an extraction step and the simultaneous microanalysis of 14 kinds of C 0 0-C22 6 fatty acid hydrazides in a reverse-phase HPLC system (27). 

Cascone et al. (126) used a reverse-phase HPLC system for the determination of thaumatin. It consisted of a Vydac 218TP54 column and elution with 10% acetonitrile in 0.1% trifluoroace-tic acid. 

Synthetic dyes, because of their sulfonic and in some cases carboxylic acid functions, have short retention times in a reverse-phase HPLC system (168). Another problem encountered during reverse-phase HPLC is the tailing observed for compounds with sulfonic groups (216). Nevertheless, adequate pH and solvent composition have permitted the separation of some dyes in a reverse-phase system, as indicated on Table 7. 

The decarboxylated product of moxalactam (see Section 4) is determined by an HPLC technique. A reverse phase HPLC system consisting of 80 parts of 0.1M ammonium acetate and 20 parts of methanol is used with a Dupont Zorbax C8 or other suitably similar column to determine the decarboxylated product. In this system, the decarboxylated moxalactam should elute with a k of about 6.5. The decarboxylated moxalactam is quantitatively determined by comparing the peak response for the sample with a peak response calibration curve of the authentic decarboxylated moxalactam reference standard material. 

Building on published literature and based on empirical trials, an efficient reverse-phase HPLC system and sample preparation scheme has been developed for vitamin D3 in... 

A9 Tetrahydrocannabinol and ll-hydroxy-A -tetra-hydrocannabinol were quantitatively separable on the reverse phase HPLC system at 47% (or less) acetonitrile in water. The collection efficiencies in the chosen ranges were 98% of the recoverable radioactivities of 8H-ll-hydroxy-A8-tetrahydrocannabinol and l c-A8-tetrahydrocannabinol (Fig. 5). A8- and A8-Tetrahydro-... 

Carr et al. [47-52] used the solvophobic theory to elucidate the retention of solutes in a reversed-phased HPLC system on nonpolar stationary phases. It is assumed that the free energy of transfer of an analyte molecule from the mobile phase to the stationary phase, AG, can be regarded as a linear combination of the energies, AGj, from the interaction of various molecular subunits according to the following equation ... 

Probably the most widely studied is the solvophobic theory [12] based on the assumption of the existence of a single partitioning retention mechanism and using essentially equation (10-1) for the calculation of the analyte retention. Carr and co-workers adapted the solvophobic theory [12,13] and LSER theory [11, 14-17] to elucidate the retention of solutes in a reversed-phase HPLC system on nonpolar stationary phases. 

A comprehensive semi-empirical description of reversed-phase HPLC systems, for predicting the relative retention and selectivity within a series of analytes, has been developed by Jandera and co-workers [72,73]. The approach consists of determining the interaction indices and the structural lipophilic and polar indices. A suitable set of standard reference analytes is necessaiy to calibrate the retention (or selectivity) scale. 

Recent comparative studies by Abraham et al. [99] demonstrated that reversed-phase HPLC systems with PS-DVB phases could be used to determine water-alkane partition coefficients, whereas the modem electrostatically shielded octadecylsilica phase produces retention parameters correlating better to the standard log P from the octanol-water system. 

UV) wavelengths up to 215 nm results in compromised method selectivity and, therefore, longer and more specific detection wavelengths were applied in recent methods. The combination of the direct separation and determination using a reversed-phase HPLC system, coupled with photodiode array detection, is found to be accurate and more precise and selective than previously published methods for the determination of purine metabolites in urine. 

Abstract The determination of dichlorobenzene and naphthalene in commercial repellents used in Spain has been validated. This was done using an isocratic regime, to test the reverse -phase HPLC system with acetonitrile water 65 35 (v v) as the mobile phase, at 20 °C This technique is proposed for the modular validation of the HPLC... 

GENERAL OUTLINES OF A REVERSED-PHASE HPLC SYSTEM FOR THE SEPARATION OF BASIC COMPOUNDS... 

A summary of the properties of some of the commonly used reversed phase stationary phases is given in the appendix. The general outlines for a reversed-phase HPLC-system for basic compounds is given in Scheme 1.1. 

Reversed phase HPLC-system column, lOym Silica RP18 (250x4.0 mm I.D.), mobile phase, acetonitrile - 0.01 M ammonium carbonate (4 6), flow rate 2.5 ml/min. 

Within the last several years HPLC separations have been optimized in terms of the most appropriate mobile phase composition for a particular set of solutes by exploring the whole plane of solvent selectivities using this solvent classification scheme with a minimal number of measurements in statistically-designed experiments. For reversed phase HPLC systems, the selectivity triangle is often defined by methanol, acetonitrile, and tetrahydrofuran with water as the diluent (37). 

For HPLC-MS/MS assays, the mobile phase is an important consideration. For reversed-phase HPLC systems (the most common), mobile phase A is water plus one or more modifiers while mobile phase B is usually either acetonitrile or methanol with one or more modifiers. Modifiers have to be volatile for HPLC-MS/MS assays. Typical modifiers are acetic acid, formic acid, and ammonium acetate. Formic acid is so popular that it is now available as a premixed HPLC solvent (0.1% in water or acetonitrile). Most other modifiers are not recommended. Triethylamine (TEA) and trifluoroacetic acid (TFA) are a problem as they are known to cause ion-suppression problems. For some special HPLC-MS/MS assays that need ion-pairing reagents, hexylamine and heptafluorobutyric acid have been found to be successful [51-53], Gao et al. [54] recently evaluated a series of ion-pairing reagents in terms of their suitability for an HPLC-MS/MS assay. 

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. 

This difference could be due to the fact that the interaction of the PCB congeners in the reverse-phase HPLC system could involve surface adsorption rather than partitioning that would be affected by the nonplanar conhguration induced by ortho substitution. Correction factors have been derived to account for this anomaly. 

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