Gradient elution equipment, HPLC

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

Equipment for supercritical fluid chromatography is similar to that for HPLC with packed columns12 or open tubular columns. Eluent strength is increased in HPLC by gradient elution and in gas chromatography by raising the temperature. In supercritical... 

The choice of solvents for gradient elution is still somewhat empirical however, using the data from Table 3.5 narrows the choices. Modern HPLC instruments are equipped with solvent programming units that control gradient elution in a stepwise or continuous manner. 

At the end of the reaction, the mixture was dissolved into ethanol and analysed with an HPLC equipped with a light scattering detector and an apolar column (Licrospher). The separation of different products was done by a gradient elution. The percentage of each compound was determined by using standardisation methods with methyl laurate as an internal standard. 

The HPLC analyses were conducted with a Water 600 Pump apparatus. This apparatus was equipped with a quaternary solvent delivery system, a Rheodyne injector with 20qL sample loop and a UV detector Waters 486 Tunable which was fixed at 280 nm. Throughout this study. Alltech Intertsil ODS-5 C18 reversed phase column (150 mm, 4.6 mm, 5qm particle size) was used. The flow rate of the mobile phase was of 1 mL / min and the gradient elution was adapted from (Nakatani et al., 2000 Bouayed et al, 2007). The solvent composition and the gradient elution program are reported in the table 1. 

An elution with a single solvent or solvent mixture of constant composition is termed an isocratic elution. In gradient elution, two (and sometimes more) solvent systems that differ significantly in polarity are used and varied in composition during the separation. The ratio of the two solvents is varied in a preprogrammed way, sometimes continuously and sometimes in a series of steps. Modern HPLC instruments are often equipped with proportioning valves that introduce liquids from two or more reservoirs at ratios that can be varied continuously (Figure 28-3). The volume ratio of the solvents can be altered linearly or exponentially with time. 

High pressure liquid chromatography (HPLC). A Hewlett Packard Model 1084 liquid chromatograph equipped with an UV detector (254 nm) was used. Analyses were carried out at pressure of 30 atm using gradient elution with 5 to 70% of acetonitrile water, at a flow of 1 ml/min. 

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