Performance characteristics of the Agilent Series preparative pump Technical Note Abstract The Agilent Series preparative pump as part of the Agilent Series purification system is especially designed for flow rates up to ml/min at a maximum backpressure of 4 bar. In this Technical Note we show the performance characteristics of the preparative pump for flow rates ranging from below ml/min up to ml/min.
Introduction The Agilent Series preparative pump is an isocratic high performance pump with two parallel pistons. It offers flow rates up to ml/min at 4 bar without the need to change pump heads. Two pumps are connected via a lowvolume mixing device to give a high-pressure gradient mixing system with an internal delay volume of only about.7 ml. Additional features of the pump include an automated seal wash using a peristaltic pump and an automated electro-magnetic purge valve. Although the pumping system is designed for flow rates up to ml/min it also shows very good performance at lower flow rates 2. In this Technical Note we show the performance characteristics of a binary gradient system over a flow rate range starting below ml/min up to ml/min. The following performance parameters were measured: composition ripple, composition precision, composition accuracy, composition linearity, flow accuracy, flow precision (isocratic and gradient performance), and precision of retention times. Note: All experiments were carried out on a system that is used for the daily work in Agilent s laboratory. Depending on the working hours of your system the results for the tests may vary slightly. Equipment All experiments were performed on an Agilent Series system containing the following modules: Two Agilent Series preparative pumps Agilent Series preparative autosampler Agilent Series diode array detector The system was controlled using the Agilent ChemStation (rev. A.9.). Results. Composition ripple, precision, accuracy and linearity To perform reproducible separations based on gradient runs it is important that the pump mixes the solvents accurately and precisely. For fast and direct method scale-up it is important that the gradient formation is consistently good over the complete flow rate range. If the preparative pumps are used in an Agilent Series purification system precise retention times from run to run are especially important for timebased fraction collection or peakbased fraction collection over a certain time interval of the purification run. Gradient composition test set-up Method and data analysis from the gradient composition test of the ChemStation OQ/PV was used to determine the parameters composition ripple, composition precision, composition accuracy and composition linearity. Solvent A was water, solvent B was water with.5 or 5 % acetone as tracer. The test was performed at flow rates of, 2, 5,, 25, 5 and ml/min. Different flow cells with pathlengths of, 3 and.3 mm were used in the diode array detector. A restriction capillary was used for all experiments to keep the backpressure at about bar. The autosampler was removed from the system for the composition ripple, composition precision, composition accuracy and composition linearity tests. Table gives an overview of the gradient composition test parameters. Flow cell [mm] Backpressure [bar] % Acetone 49.5 2 56.5 5 22.5 3 2.5 25 3 83.5 5.3 5.3 65 5 Table Test parameters for composition, ripple, precision, accuracy and linearity tests 2
Results of gradient composition test Figure shows the results for the composition ripple, accuracy, precision and linearity tests. The marked areas show the limits as specified in the ChemStation OQ/PV for the Agilent Series quaternary pump. All values are within the limits except for the composition ripple. The variances in the values are due to different pressures, flow cell pathlengths and acetone concentration. This shows that the pump stays within the limits even if these parameters are changed. The performance criterion of the composition ripple is not fulfilled due to the missing pressure damper of the gradient pumping system. A pressure damper was not built into the preparative pump to keep the delay volume as small as possible. As mentioned before, the experiments were carried out with a restriction capillary instead of a chromatographic column. A preparative column in the system works as a dampening mechanism and reduces the ripple drastically. Therefore, a composition ripple experiment was repeated using a column instead of a restriction capillary. Table 2 shows that the ripple is reduced by a factor of when using a column appropriate for the flow rate. Note: The gradient composition test must never be carried out with a column. A restriction capillary must be used. The experiment with the column was only done to show the influence of the column as a dampening mechanism in a system containing the preparative pumps. Flow rate Ripple Flow cell Backpressure % Acetone [ml/min] [% B] [mm] [bar] Restriction capillary 25 2.54 3 83.5 Column (2.2 5 mm) 25.222 3 4.5 Table 2 Composition ripple with restriction capillary and with column Limit <.5 4 3 2 Ripple [% B] Accuracy [% B] 2 Limit <.5.5.5 Limit <.5.8.6.4.2 Precision [% B] Limit <.999.9995.999.9985 Correlation Figure Gradient composition test showing composition ripple, accuracy, precision and linearity (correlation) 3
2. Flow accuracy Flow accuracy is a parameter which is not important for run-torun precision, however, it is important for proper method performance after scale-up experiments. Flow accuracy depends on the system backpressure and, on the compensation algorithms of the preparative pump firmware. 7 6 5 4 3 2 ml/min 5 ml/min 25 ml/min 5 ml/min ml/min Flow accuracy Flow accuracy test set-up The flow accuracy was measured by collecting the solvent (water) over five minutes. The collected volume was determined by weighing the collected water and dividing the amount by the density. The test was performed at flow rates of, 2, 5,, 25, 5 and ml/min. The accuracy for each flow rate was measured at three different backpressures (< bar, > and < 2 bar, > 2 bar). Restriction capillaries were used instead of a chromatographic column for all experiments. Results of flow accuracy test Figure 2 shows the results of the flow accuracy tests. The flow accuracy is below 5 % for all flow rates and pressure settings except for ml/min and high backpressure. This ensures fast and easy scale-up over the complete flow rate range without time-consuming method redevelopment as shown in an earlier Application Note 3. 5 5 2 25 3 35 3. Flow precision isocratic Flow precision is important for run-to-run precision of retention times. Since the pump flow is controlled by the compensation algorithms of the preparative pump firmware the flow precision is backpressure dependent. Flow precision test set-up The flow precision was measured by collecting the solvent (water) over 5 minutes. The collected volume was determined by weighing the collected water and dividing the amount by the density. The test was performed at a flow rate of 25 ml/min. The precision was measured at three different backpressures (< bar, > and < 2 bar, > 2 bar). Pressure [bar] Figure 2 Flow accuracy for different flow rates and backpressures Five runs were performed at each pressure to determine the precision. Restriction capillaries were used instead of a chromatographic column for all experiments. Results of flow precision test Figure 3 shows the results for the isocratic flow precision tests. The relative standard deviation of the flow precision is marked. The relative standard deviation is below.3 % for the different backpressures. 4
4. Flow precision gradient Flow precision test set-up The flow precision was measured by collecting the solvent (water, methanol) over seven minutes. The collected volume was measured. The test was performed at a flow rate of ml/min. Gradient: % water for min, % water to % MeOH in 5 min, % MeOH for min. The precision was measured at three different backpressure ranges. Five runs were performed at each pressure to determine the precision. Restriction capillaries were used for all experiments instead of a chromatographic column. Flow [ml/min] 25. 24.8 24.6 24.4 24.2 24. Flow Precision RSD.2 % RSD.2 % 5 5 2 25 3 Pressure [bar] RSD.9 % Figure 3 Relative standard deviation of the isocratic flow precision at different backpressure settings Results of flow precision test Figure 4 shows the results of the gradient flow precision tests. The relative standard deviation of the flow precision, shown with the black error bars, is below.4 % for the different backpressure settings. Flow [ml/min].2. 9.8 RSD.25 % Flow Precision RSD. % RSD.4 % 9.6 5 5 2 25 3 Max. pressure [bar] Figure 4 Relative standard deviation of the gradient flow precision at different backpressure settings 5
5. Precision of retention time Precision of retention time is an important characteristic that influences qualitative and quantitative results in analytical HPLC. In preparative HPLC this parameter is important for repetitive purification runs using time-based fraction collection. The retention times have to be stable within a certain time window. Precision of retention time test setup Precision of retention times was measured by ten repetitive injections from the same sample containing two major and one minor compound (figure 5). The precision of retention time was calculated using the Chem- Station sequence summary report. The experiments were carried out on columns with inner diameters of 3, 4.6, 9.4, 2.2 and 5 mm with flow rates of.35,.85, 3.5, 8 and ml/min, respectively. Mobile phase A was water, mobile phase B was methanol Gradient: 5 % B to 25 % B in min, 25 % B for 9.9 min, 25 % B to 5 % B in. min. Figure 6 shows the relative standard deviation of the retention times measured for consecutive runs for a sample that contained three compounds. For flow rates above ml/min and up to ml/min the relative standard deviation of the retention times is.3 % or below, which is excellent for a preparative pump. mau 5 4 3 2 Flow rate: 3.5 ml/min consecutive runs 2 4 6 8 2 4 6 Time [min] Figure 5 Precision of retention time test at 3.5 ml/min RSD [%].8.6.4.2 Precision of Retention Time. Figure 6 Relative standard deviation for consecutive runs Peak Peak 2 Peak 3 6
Conclusion In this Technical Note we showed the performance of the Agilent Series preparative pump over the flow rate range of.35 up to ml/min. We showed that the performance below 5 ml/min is almost as good as for an analytical pump, for example, the Agilent Series quaternary pump. Although the pump performance is still very good at such low flow rates, other parts of the system, especially the capillaries with a large inner diameter required for higher flow rates, will decrease the overall system performance. Therefore, Agilent Technologies offers dedicated systems from capillary to preparative scale rather than a single system covering the complete flow rate range. This ensures best performance for a specific application rather than low performance over a wide application range. References. New perspectives in purification with HPLC and HPLC/MS, Agilent Technolgies Brochure, 2, publication number 5988-3673EN 2. Performance of the Agilent Series preparative pump at low flow rates", Agilent Technologies Technical Note, 2, publication number 5988-2435EN 3. Scale-up from an analytical scale to a preparative scale method with the Agilent Series purification system PS, Agilent Technologies Application Note, 22, publication number 5988-6979EN 7
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