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a 40-min run, with 15-20 min of post run time, 1 -2 min is hardly significant. However, with run times as short as 5 min, the injection overhead can add 20% or more to the cycle-to-cycle inject time. Another parameter that must be considered in the cycle-to-cycle inject time is the time it takes to reequilibrate the column and the system following the gradient. Each of these functions is illustrated in Fig. 5 for an LC system operated in the traditional ‘‘sequential’’ mode. Obviously, anything that can be done to reduce the time it takes to accomplish these tasks will also improve sample throughput. As shown in Fig. 6, a separation of an 11-component mix in the sequential mode with a 5-min run time can consume an additional 1.2 min due to injection overhead, for a total cycle to cycle inject time of 6.2 min. If, however, some of these functions can be performed in a ‘‘parallel’’ mode, as illustrated in Fig. 7 (p. 122), additional time savings could be realized. In the parallel mode of operation, sample-manager needle and inject port washes, and aspiration of the next sample takes place during the actual run, saving considerable time.
Figure 5 Schematic of traditional sequential mode injection to injection cycle time (injection overhead) operation of an LC system.
Figure 6 Traditional sequential mode injection showing cycle to cycle inject time. Separation conditions and peak identification are identical to those reported in Figure 2B. Data system run time was extended to 20 minutes to capture LC instrument timing.
Equilibrating the system separately from the column can also save additional time. Equilibration time is an essential part of gradient chromatography. Both the LC system and the column must be returned to initial mobile phase conditions prior to the next run to ensure repeatability. Traditionally, equilibration times of 5-10 column volumes have been used (9). However, with the use of smaller diameter and shorter LC columns, a 10-column volume is not always enough to equilibrate both the LC system and the column. To compensate for this situation, the reequilibration volume has been divided into two parts: the volume required for system equilibration and the volume required for column equilibration (10). Total equilibration time is then given by the formula:
Tr = (3Vt + 5Vc)/F
where Tr is the equilibration time (min), Vt is the total system volume, Vc is the column volume (mL), and F is the flow rate (mL/min). As the column gets smaller (with a correspondingly lower flow rate), more equilibration time is taken up by returning the system volume of the LC to initial conditions. In advanced LC systems with integrated fluidics and control, a purge step can be added post run at high flow rates (5-7 mL/min) with the column off-line to significantly reduce system equilibration times. By starting the gradient but holding off on the injection for the amount of time proportional to the system volume, the volume of the system can also be used to aid in column equilibration—a technique referred to as a ‘‘just-in-time gradient.’’ Loading the sample loop, as illustrated in Fig. 8 (p. 123), during the equilibration saves additional time by further reducing injection overhead. Comparing Figs. 6 and 8, the parallel mode with rapid equilibration increases throughput by about 30% without sacrificing chromatographic information or integrity.
Rapid equilibration techniques are particularly helpful to save time as the flow rate decreases, in, for example, microbore applications. Figure 9 (p. 124) shows a separation on a 1 X 50 mm column, at 0.3 mL/min, with a cycle-to-cycle inject time of 5.5 min using the parallel mode with rapid equilibration. Run in the traditional sequential manner, the corresponding cy-cle-to-cycle inject time would be in excess of 7.5 min. At lower flows, the time saving during reequilibration would be even more significant.
Some general notes on operating LCs in a high-throughput mode are as follows:
With the trend toward smaller columns, extra care should be taken to minimize extra column band broadening by using reduced diameter tubing, smaller volume detector cells, and properly fitting connections (9).
Start Start End Stop
Draw next sample
Gradient to column
**equilibrate system at 5 ml/min
Figure 7 Schematic of parallel mode operation with rapid equilibration.
Photolabile or temperature-sensitive compounds may require special handling. LC systems are available that maintain samples in the dark and/or chilled if required.
The small peak volumes and resolution demands from high-throughput analyses require faster data collection rates for accurate results. Adequate detector time constants should also be used.
III. ADVANCED HIGH-THROUGHPUT ANALYSIS TECHNIQUES