Gradient elution liquid chromatograph

The remainder of this section deals primarily with selectivity optimization in isocratic liquid chromatography and with gradient elution Before entering these subjects proper, however, a discussion of the relevant chromatographic properties of solvents is in order as a framework for the intuitive selection of the preferred solvent or solvent mixtures for selectivity optimization. 

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

GRADIENT ELUTION FOR THE REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF COLOUR PIGMENTS IN THE EXTRACTS OF PAPRIKA (<CAPSICUM ANNUUM). ELUENT A, METHANOL-ACETONITRILE (80 20, V/V) ELUENT B, BIDISTILLED WATER... 

The chemical structures of betanin and indicaxanthin found in the prickly pear are depicted in Fig. 2.150. Pigments were extracted by homogenizing fresh fruit flesh with methanol (1 5, w/v). The suspension was fdtered and the liquid phase was applied for spectrophotometry and RP-HPLC. Liquid chromatographic separation was performed in an ODS column (250 X 4.6 mm i.d. particle size 5 pan) at ambient temperature. Gradient elution started with 1 per cent aqueous acetic acid and changed to 12 per cent solvent B in... 

Alternatively, direct methods (syringe infusion, flow injection) can be used as a preliminary step in determining the optimal MS detector conditions for particular molecules. This is especially useful when the MS is attached to a liquid chromatograph, in which the fluid entering the MS will vary with gradient elutions (varied solvent and salt compositions) or with sample types (varied sample matrices, extraction solvents, included salts, etc.), which can affect the predominant ion type and sensitivity, or produce other matrix effects such as ion suppression or extraneous signals. 

For the optimum HPLC separation, the liquid chromatograph was connected to a mass detector. A column (250 X 5-mm ID) of SPHERISORB C8 (5-yttm particles) was eluted with a solvent gradient, with reservoir A containing methanol and reservoir B containing water-pyridine-acetic acid (98.5 1.5 0.025). The mobile phase was A B = 92 8, and this composition was held for 10 min. The flow rate was 0.75 ml/min. 

The complexity of natural TG mixtures would, however, require gradient elution to utilize fully the resolution power of liquid-liquid chromatographic systems. This is not possible to achieve in combination with IR detectors, and is difficult with UV detectors in this case due to the short wavelengths that have to be used. 

The Micromeritics 7000 liquid chromatograph (Fig.3.30) also uses a reciprocating pumping system. The pump has several unique features. It delivers pulseless flow without a pulse damper, it can operate from an unlimited reservoir, it requires only a single pump for gradient elution and it is capable of operating at constant pressure or constant flow-rate. 

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