Mobile phase nonaqueous

This section discusses in detail the column types that are available for the size exclusion chromatography of both polar and nonpolar analytes. It first discusses the various columns available for standard nonaqueous size exclusion chromatography. It then reviews the columns available for general size exclusion chromatography using aqueous mobile phases. Finally, it examines the columns designed for size exclusion chromatography of proteins and peptides. 

The use of totally permeated flow markers in aqueous GPC offers similar advantages along with many of the same shortcomings that one finds in nonaqueous GPC. One problem commonly found in aqueous GPC is that salt peaks due to the on-column ion exchange of counter ions of a polyelectrolyte with dissimilar ions in the GPC mobile phase will occur at or near the total permeation volume of the column. These salt peaks will often obscure the flow marker used in the analysis. Short of preconditioning the sample to exchange... 

Two improved methods of molecular weight calibration using broad MWD standards have been proposed and some evaluation has been done experimentally for aqueous and nonaqueous SEC. The experimental evaluations indicate that both methods appear very promising and justify further experimental investigation. It is recommended that these new calibration methods be evaluated for a wide range of polymers,packings and mobile phases. 

In PLC, polar adsorbents (silica and alumina) and nonaqueous solvents of low viscosity are usually used. Chemically bonded adsorbents (silanized silica) are poorly wettable by aqueous mobile phases and are relatively expensive, thus they are not often applicable [3]. 

For example, an alumina layer with a nonaqueons mobile phase was optimized for the separation of the taxoid fraction from ballast snbstances [5]. Figure 11.3 shows the densitogram obtained for Taxus baccata cmde extract chromatographed on the alnmina layer developed with nonaqueous elnents. The nse of ethyl acetate and dichloromethane enables elntion of nonpolar fractions (chlorophylls and waxes) and purification of the starting zone (Figure 11.3a). In this system, all taxoids are strongly retained on the alumina layer. The use of a more polar mobile phase... 

Liquid-solid chromatography (LSC), sometimes referred to as normal phase or straight phase chromatography, is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase... 

Most small organic molecules are soluble in mixed organic-aqueous solvents and can be easily analyzed using RPLC. However, there are some polar compounds which are not soluble in typical RPLC solvent systems or are unstable in an aqueous mobile phase system. These compounds can be analyzed on an RPLC column with a nonaqueous solvent system. This technique is called "nonaqueous reversed phase chromatography" (NARP).20-21 The NARP technique is primarily used for the separation of lipophilic compounds having low to medium polarity and a molecular weight larger than... 

Nova-Pak C18 column in a methanol water chloroform gradient.92 Choline chloride was added to the mobile phase. One review of techniques used in the analysis of triacylglycerols lists over 300 references on separations of the triglyceride fraction of fats using nonaqueous RPLC, aqueous RPLC, argen-tation chromatography, and other chromatographic methods.93... 

Applications High-temperature liquid chromatography with packed-capillary columns, nonaqueous mobile phases, and ELSD and ICP-MS detection, has been developed specifically as a robust analytical tool for the analysis of high-MW polymer additives [731,738]. Dissolving such moderately polar, heavy compounds with low water solubility at ambient temperature usually... 

FTIR is a natural for HPLC in that it (FTIR) is a technique that has been used mostly for liquids. The speed introduced by the Fourier transform technique allows, as was mentioned for GC, the recording of the complete IR spectrum of mixture components as they elute, thus allowing the IR photograph to be taken and interpreted for qualitative analysis. Of course, the mobile phase and its accompanying absorptions are ever present in such a technique and water must be absent if the NaCl windows are used, but IR holds great potential, at least for nonaqueous systems, as a detector for HPLC in the future. 

In contrast to the various CSPs mentioned so far, but still based on covalently or at least very strongly adsorbed chiral selectors (from macromolecules to small molecules) to, usually, a silica surface, the principle of dynamically coating an achiral premodified silica to CSPs via chiral mobile phase additives (CMPA) has successfully been adapted for enantioseparation. The so-called reverse phase LC systems have predominantly been used, however, ion-pairing methods using nonaqueous mobile phases are also possible. 

With TSP, ammonium acetate has emerged as the best general-purpose electrolyte for ionizing neutral samples. Improved ionization can be obtained by the use of a filament or discharge electrode to generate reactive ions for CI (87, 88). The processes involved in filament or discharge-assisted ionization must be used when operating in the absence of a buffer with nonaqueous eluents. With ionic analytes, the mechanism of ion evaporation is supposed to be primarily operative since ions are produced spontaneously from the mobile phase (89). Ion evaporation often yields mass spectra with little structural information in order to overcome this problem, other ionization modes or tandem MS have been applied (90). 

When HPLC is used as part of the analysis, the mobile phase is typically a mixture of methanol and methyl-tert-butyl ether (i.e., 50 50, v/v), although other HPLC solvents for LC/MS using APCI (e.g., water, tetrahydrofuran) can be used. It is important to note that if combustible nonaqueous solvent systems are used, water or a halogenated solvent such as methylene chloride or chloroform should be added to the mobile phase postcolumn to suppress ignition in the ion source. In addition, the APCI source must be vented outside the laboratory and should not allow air into the ionization chamber. A scan range of m/z 300 to 1000 will include the known carotenoids and their most common esters. 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

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). 

Aqueous samples or aqueous extracts of nonaqueous samples analyzed by HPLC on a C-18 reverse phase column analyte detected by UV at 195 nm mobile phase, water, flow rate 2 mL/min pressure 38 atm. 

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