Chromatography organic mobile phases

There is no other facet where thin-layer chromatography reveals its paper-chromatographic ancestry more clearly than in the question of development chambers (Fig. 56). Scaled-down paper-chromatographic chambers are still used for development to this day. From the beginning these possessed a vapor space, to allow an equilibration of the whole system for partition-chromatographic separations. The organic mobile phase was placed in the upper trough after the internal space of the chamber and, hence, the paper had been saturated, via the vapor phase, with the hydrophilic lower phase on the base of the chamber. 

Efficiency the organic modifier can be used to adjust solvent selectivity as normally practiced in reversed-phase chromatography. Lowers mobile-phase viscosity and improves solute mass-transfer kinetics. 

Reversed-phase chromatography employs a nonpolar stationary phase and a polar aqueous-organic mobile phase. The stationary phase may be a nonpolar ligand, such as an alkyl hydrocarbon, bonded to a support matrix such as microparticulate silica, or it may be a microparticulate polymeric resin such as cross-linked polystyrene-divinylbenzene. The mobile phase is typically a binary mixture of a weak solvent, such as water or an aqueous buffer, and a strong solvent such as acetonitrile or a short-chain alcohol. Retention is modulated by changing the relative proportion of the weak and strong solvents. Additives may be incorporated into the mobile phase to modulate chromatographic selectivity, to suppress undesirable interactions of the analyte with the matrix, or to promote analyte solubility or stability. 

The mechanism of reversed-phase chromatography arises from the tendency of water molecules in the aqueous-organic mobile phase to self-associate by hydrogen bonding. This ordering is perturbed by the presence of nonpolar solute molecules. As a result, solute molecules tend to be excluded from the mobile phase and are bound by the hydrophobic stationary phase. This solvophobic... 

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

This phenomenon can be exploited for separation and concentration of solutes. If one solute has certain affinity for the micellar entity in solution then, by altering the conditions of the solution to ensure separation of the micellar solution into two phases, it is possible to separate and concentrate the solute in the surfactant-rich phase. This technique is known as cloud point extraction (CPE) or micelle-mediated extraction (ME). The ratio of the concentrations of the solute in the surfactant-rich phase to that in the dilute phase can exceed 500 with phase volume ratios exceeding 20, which indicates the high efficiency of this technique. Moreover, the surfactant-rich phase is compatible with the micellar and aqueous-organic mobile phases in liquid chromatography and thus facilitates the determination of chemical species by different analytical methods [104]. 

Recently, Tai, and Gohda [546] have reported a method using a novel technique named hydrophilic interaction chromatography, which use a polar stationary phase with aqueous-organic mobile phase. It enables to quantify acciffately AA and related compounds, to run at high flow rate thanks to the low back pressiffe, and to be easily coupled with an MS. 

Kim H, Guiochon G. Thermodynamic studies of the solvent effects in chromatography on molecularly imprinted polymers. 3. Nature of the organic mobile phase. Anal Chem 2005a 77 2496-2504. 

Thin-layer chromatography (TLC), sometimes also called planar chromatography, employ a stationary phase immobilized on a glass or plastic plate and an organic mobile phase. It is a rather old technique whose application in residue analysis has been limited in the past by poor chromatographic resolution, inadequate selectivity, and insufficient sensitivity (49). This was due to inherent problems in the quality of the available stationary phase materials and in the uniformity of the layers prepared. Today, the availability of affordable, precoated plates with acceptable performance and consistency has led to the general acceptance of TLC as an efficient procedure for residue analysis (50). The method is used preferentially when analysts must process large numbers of samples in a short period of time (51). 

In adsorption chromatography the mobile phase is usually a liquid and the stationary phase is a finely-divided solid adsorbent (liquid-solid chromatography). Separation here depends on the selective adsorption of the components of a mixture on the surface of the solid. Separations based on gas-solid chromatographic processes are of limited application to organic mixtures. The use of ion-exchange resins as the solid phase constitutes a special example of liquid-solid chromatography in which electrostatic forces augment the relatively weak adsorption forces. 

Naidong, W., Shou, W. Z., Addison, T., Maleki, S., and Jiang, X. (2002b). Liquid chromatography /tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/ organic mobile phases A novel approach of eliminating evaporation and reconstitution steps in 96-well SPE. Rapid Common. Mass Spectrom. 16 1965-1975. 

Sample preparation for complex formulations, such as creams, can frequently be as simple as dissolving the cream in the totally organic mobile phase such as the ones typically used in normal-phase chromatography. Organic solutions of flurometholone [3, p. 677] and hydrocortisone acetate [3, p. 758] creams were assayed by HPLC and hydroquinone cream by TLC [3, p. 769]. A similiar approach has been applied to sample preparation of ointments. 

C. J. C. M. Laurent, H. A. H. Billiet, and L. de Galan, High performance liquid chromatography of heroin samples on alumina by ion exchange in mixed aqueous organic mobile phases, Chromatography, 255 161 (1984). 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

The main disadvantages of micellar chromatography are the observed diminished chromatographic efficiency, higher column back pressure, and in preparative work, the need to separate the final resolved analyte from the surfactant (95) (a later section of this review will discuss this latter problem and its resolution in further detail). The higher column back pressure and part of the decreased efficiency stem from the fact that surfactant-containing mobile phases are more viscous compared to the usual hydro-organic mobile phases employed in conventional RP-HPLC (refer to viscosity data in Table X)... 

Gorse J, Balchunas AT, Swaile DF, Sepaniak MJ. Effects of organic mobile phase modifiers in micellar electrokinetic capillary chromatography. J High Resolut Chromatogr 1988 11 554. 

Waichigo, M.M. et al. AUcylammonium formate ionic liquids as organic mobile phase replacements for reversed-phase liquid chromatography. J. Liq. Chromatogr. Rel. Technol. 2007, 30, 165-184. 

Onyewuenyi, N., and Hawkins, P. 1996. Separation of toxic peptides (microcystins) in capillary electrophoresis, with the aid of organic mobile phase modifiers. Journal of Chromatography A 749 271-278. 

The main complication with using aqueous pKa values in chromatography lies in the profoundly nonaqueous nature of most reversed-phase systems today. The presence of organic mobile-phase modifiers affects both the pKa of the analyte and the effective pH of the buffer (see Chapter 4). 

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