Column/system backpressure

Figure 6.7 depicts an autosampler employed in a jtPLC system. Figure 6.8 details the autosampler component. Samples are transferred from the desired well in the microtiter plate into the columns of the Brio cartridge. If a 384-well plate is employed, the autosampler will carry out 3 sets of 8 injections into the columns, for a total of 24 columns. The solvent (mobile phase) does not circulate in the cartridge but is diverted into a backpressure regulator located in the waste line (Figure 6.2). This process of injection is known as stop-flow injection. After all samples are placed into the injection pits of the 24 columns in the cartridge (Figure 6.5), a clamp containing a seal...

Although HPLC column technology is considered to be a mature field now, improvements and new developments are being made continuously in the stationary phases. One of the improvements has been the reduction in particle sizes. Smaller particles help to improve mass transfer and provide better efficiency. Manufacturers are producing particles down to 1.5 J,m in diameter, although 3- and 5- J,m particles are still the most popular. Because of the smaller particle sizes, the backpressure increases proportionally to the inverse of the square of the particle size. Most commercially available HPLC systems cannot accommodate the pressures required to operate these columns at optimum flow rates. This has led to the introduction of systems that run at high pressures. 

From both theory and experimental evidence, raising the temperature by 10°C decreases the retention time by about 20% in isocratic chromatography and decreases the backpressure by 10% to 20% because of a reduction in the viscosity of the mobile phase. This can help to overcome the instrument limitations associated with running shorter columns packed with smaller particles, i.e., the pressure limitations of current HPLC systems. However, since the majority of reversed-phase columns available are silica-based, operating at temperatures above... 

Although a flow rate of 50 ml/mln was used in these preparative runs (typical column backpressure was 20 psi), it Is acceptable to use flow rates as high as 100 ml/mln with the 57 mm I.D. Styragel columns. Such Increased flow rates would afford reduced analysis times, especially when using several columns in series for improved fractionation. Although the pumping system used for the preparative work had a maximum flow rate of 80 ml/mln, other systems are commercially available if higher flow rates are desired. 

Column equilibration ( 10 column vol. recommended) ensures baseline stability, good peak shape, and reproducible retention times. For the specific column chosen in this unit for isoflavone analysis, at least 18 ml total is needed to equilibrate the system with mobile phase. The common practice is to purge the pump system and connect the inlet end of the column to the injector outlet. The initial pump flow should be set at 0.1 ml/min and increased to 0.6 ml/min in 0.1 ml/min increments. Once a steady backpressure and baseline have been achieved, the column is ready to use. Before injecting samples, it is suggested to run a blank gradient first to clean the column and help check for the possibility of impurity peaks. 

Very detailed separations have been obtained by numerous authors (61-66) based upon the method originally developed by Christie (67). This method is based mainly on iso-octane (similar to hexane), 2-P, water containing 500 /jlM serine adjusted to pH 7.5 with ethylamine, and trace amounts of tetrahydrofuran (THF) as a mobile-phase modifier. Lutzke and Braughler modified slightly the mobile-phase system proposed by Christie by including a flow rate gradient to maintain low column backpressure (62). According to the authors, this positively affected detector response to PLs. Markello et al. used the procedure described by Christie, albeit without the addition of serine or ethylamine (65). Melton proposed the use of two solvent mixtures only, but they included exactly the same solvents as proposed by Christie (66). However, PI and PA were not resolved. 

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