Gradient Elution Technique

The three main reasons for using gradient elution are  

1) Separation of sample components showing a wide range of k  

2) Fast evaluation of optimum eluent composition for subsequent isocralic analysis 

In addition, the technique can be applied to remove strongly retained solutes or impurities from the column, thus enhancing sensitivity and column life. 

Compared to the isocratic elution mode, the equipment necessary for gradient elution is extended by a second solvent reservoir, an additional pump (in the case of a high-pressure-mixing gradient system), a mixing chamber, and a proper- 

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The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Fluorescence detectors can also be used and while their sensitivity may be greater, they are less widely applicable owing to the smaller number of fluorescent compounds. Differential refractometers will detect changes in the refractive index of the solvent due to the presence of solutes and, while they are less sensitive than the other detectors and often cannot be used effectively with gradient elution techniques, they are capable of detecting the presence of any solute. 

Gradient-elution techniques can be combined with elevated temperature operation or temperature programs [13-17], flow-rate programming [18,19], column switching, and two-dimensional (2D) operation [20-24] to get full advantage of separation selectivity and to separate complex samples in as short a time as possible. 

Resin capture can be faster and more efficient than classical methods of purification (e.g., chromatography). Chemoselective sequestration requires minimal amounts of solvent for separating reactants from solution-phase products. Gradient elution techniques, common in chromatographic separations, are avoided, saving time and solvent. Additionally, concurrent use... 

Offline precolumn derivatization is the most common alternative in this respect it involves separating the esters obtained from the organic acids by reversed-phase chromatography, which amply surpasses solvophobic chromatography (i.e., the use of undissociated acids as such) and allows gradient elution techniques to be applied, thanks to the wider lipophilicity range covered by the derivatized compounds. 

A method for the determination of ethoxyquin in paprika that avoided the previous separation steps from other colored substances was proposed by Vinas (133). Analysis is carried out by reverse-phase HPLC using the gradient elution technique and UV detection at 270 nm. Using fluorimetric detection with excitation at 311 nm and emission at 444 nm, a detection limit of 0.2 jig /ml was reached. The method can be applied to the determination of ethoxyquin in commercial samples in the presence of paprika (Capsicum annuum) carotenoids. 

This is a simple upgrade of the isocratic system with the facility for gradient elution techniques and greater functionality (Fig. 1.1(b)). The basic system provides for manual operating gradient techniques such as reversed phase, ion exchange and hydrophobic interaction chromatography. Any of the detectors listed above under the isocratic system can be used. 

Individual oligomeric BPA/DC cyclotrimerizates were separated by the HPLC gradient-elution technique in THF/H20 [13], The step-growth mechanism of the polycyclotrimerization was supported by quantitative analysis of the results. 

To many analysts the major limitation of electrochemical detection for liquid chromatography (LCEC) is its limited applicability to gradient elution techniques. Amperometric electrochemical detectors exhibit both the best and the worst characteristics of solute property and bulk property detectors. While the Faradaic current arises only from the solute, the non-Faradaic current arises from... 

The reversed-phase analysis of oligoribonucleotides has be reported by McFarland and Borer (M6). Employing linear gradient elution techniques, they have separated many dinucleotide monophosphates, homooligonucleotide mixtures, and block co-polymers. Crowther and... 

In this context it should further be pointed out that a commercial method development computer software package (DRYLAB) has been successfully applied to optimize enantiomeric separations of a multi-component amino acid sample utilizing isocratic and gradient elution techniques 389. ... 

Using anion exchange chromatography with pulsed amperometric detection, polymers up to DP70 may be analyzed. The necessary gradient elution technique is based on the combination of sodium hydroxide and sodium acetate eluents described above. 

Gradient Elution Techniques in Anion Exchange Chromatography... 

These problems have been circumvented by both the introduction of modern micromembrane suppressors and the development of short clean-up columns for eliminating inorganic impurities in the eluent [130]. Therefore, the gradient elution technique in ion chromatography today is as common as in the area of conventional HPLC. 

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