Reversed-phase HPLC solvents

The polarity index or the solubility parameter may be used as a measure of solvent strength, which would be a measure of polarity in those cases. For reversed phase HPLC, solvent strength parameters have been proposed for the four most common solvents used, i.e. water (Si = 0), methanol (Si = 2.6), acetonitrile (Si = 3.2) and THF (Si = 4.5). Using these values water makes no contribution to the eluting power of the mobile phase and the solvent strength is measured by the volume fraction of organic modifier. 

Anisole (methylphenylether) can be regarded as methylated phenol and has been proposed (Kauppda 2004a) as a suitable dopant for APPI of analytes with low proton affinity (PA) values in reverse phase HPLC solvents in which benzene and toluene are unsuitable. Previously only acetone was used in these conditions but its fairly high PA (812kJ.mop ) meant that it was suitable for only analytes with even higher PA values. It was shown... 

Analysis of DCLs by a combination of high-performance liquid chromatography (HPLC) and MS (LC-MS) has proven very powerful, provided the library members are stable enough to allow chromatographic separation. LC-MS analysis can yield both the quantity and the identity of individual library members. Care should be taken that the exchange between library members is slow on the timescale of the experiment. This is usually the case for hydrazone and disulfide libraries, without requiring special precautions. It has recently been reported that even the composition of DCLs of hydrolytically labile imines can be analyzed directly by HPLC, provided an acidic mobile phase is used. Beau et al. successfully used a common reversed-phase HPLC solvent system based on acetonitrile/water mixtures containing 0.1% trifluoroacetic acid [10]. Imine hydrolysis is slow under these conditions because at low pH the zwitterionic hemi-aminal intermediate that is believed to be important in the hydrolysis process is more difficult to attain (Scheme 2.1). 

Solvent triangle for optimizing reverse-phase HPLC separations. Binary and ternary mixtures contain equal volumes of each of the aqueous mobile phases making up the vertices of the triangle. 

The teehniques of membrane extraetion permit an effieient and modern applieation of elassieal liquid-liquid extraetion (LLE) ehemistry to instmmental and automated operation. Various shorteomings of LLE are overeome by membrane extraetion teehniques as they use none or very little organie solvents, high enriehment faetors ean be obtained and there ai e no problems with emulsions. A three phase SLM system (aq/org/aq), where analytes are extraeted from the aqueous sample into an organie liquid, immobilized in a porous hydrophobie membrane support, and further to a seeond aqueous phase, is suitable for the extraetion of polar eompounds (aeidie or basie, ehai ged, metals, ete.) and it is eompatible with reversed phase HPLC. A two-phase system (aq/org) where analytes ai e extraeted into an organie solvent sepai ated from the aqueous sample by a hydrophobie porous membrane is more suitable for hydrophobie analytes and is eompatible with gas ehromatography. 

T. Hyotylainen, K. Grob, M. Biedermann and M-L. Riekkola, Reversed phase HPLC coupled on-line to GC by the vaporizer/precolumn solvent split/gas dischar ge analysis of phthalates in water , 7. High Resolut. Chromatogr. 20 410-416 (1997). 

Purification of the activation products (PMs). The methylamine activation product dissolved in methanol is purified by chromatography, first on a column of silica gel using a mixed solvent of chloroform/ethanol, followed by reversed-phase HPLC on a column of divinylbenzene resin (such as Jordi Reversed-Phase and Hamilton PRP-1) using various solvent systems suitable for the target substance (for example, acetonitrile/water containing 0.15% acetic acid). 

Dansyl chloride does have some drawbacks when used with reverse-phase HPLC. One of these is that the quantum yield of fluorescence varies greatly with the polarity of the solvent for dansyl derivatives (10,11). As the polarity of the solvent increases, the... 

Acetochlor and its metabolites are extracted from plant and animal materials with aqueous acetonitrile. After filtration and evaporation of the solvent, the extracted residue is hydrolyzed with base, and the hydrolysis products, EMA and HEMA (Figure 1), are steam distilled into dilute acid. The distillate is adjusted to a basic pH, and EMA and HEMA are extracted with dichloromethane. EMA and HEMA are partitioned into aqueous-methanolic HCl solution. Following separation from dichloromethane, additional methanol is added, and HEMA is converted to methylated HEMA (MEMA) over 12 h. The pH of the sample solution is adjusted to the range of the HPLC mobile phase, and EMA and MEMA are separated by reversed phase HPLC and quantitated using electrochemical detection. 

Snyder L.R., Dolan J.W., Molnar I., and Djordjevic, N.M., Selectivity control in reversed-phase HPLC methods development varying temperature and solvent strength to optimize separations, LC-GC, 15 (2), 136, 1997. 

Discussed below are various modes of separations in HPLC. Included here is brief coverage of mobile-phase selection for various modes of chromatography and elementary information on mechanism, choice of solvents and columns, and other practical considerations. It should come as no surprise that reversed-phase HPLC is discussed at greater length in this section because it is the most commonly used technique in HPLC (more detailed discussion is provided in Section 15.8). Clearly,... 

The k values in HPLC can be controlled by means of solvent strength. When it is necessary to increase k value, a weaker solvent is used. For example, in reversed-phase separations, solvent strength is greater for pure methanol than for pure water. The right proportionality of these solvents has to be found to get optimum separation. 

The term polarity refers to the ability of a sample or solvent molecule to interact by combination of dispersion, dipole, hydrogen bonding, and dielectric interactions (see Chapter 2 in reference 5). The combination of these four intermolecular attractive forces constitutes the solvent polarity, which is a measure of the strength of the solvent. Solvent strength increases with polarity in normal phase, and adsorption HPLC decreases with polarity in reversed-phase HPLC. Thus, polar solvents preferentially attract and dissolve polar solute molecules. 

In this experiment, a mixture of methyl, propyl, and butyl paraben (structures shown in Figure 13.14) in methanol solvent will be separated by reverse phase HPLC. Mobile phase compositions of varying polarities will first be tested to see which one gives the optimum resolution of this mixture, and following this, a standard curve for methyl paraben will be constructed and its concentration in this solution determined. 

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 peptides generated by proteolysis are separated using reverse-phase HPLC to minimize mass overlap and ionization suppression caused by ion competition in the electrospray source [40]. The optimized LC gradient parameters efficiently separate peptides while minimizing loss of deuterium through back exchange with solvent. Increased sensitivity can be achieved by using capillary HPLC columns and nanoelectrospray methods [47]. 

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