Technical Note 702 Using the Chromeleon Summit 2 Tandem Operation Wizard 1 INTRODUCTION New Summit 2 Tandem Operation support of the Program Wizard is available in Chromeleon version 6.70. The purpose of this Program Wizard extension is to make it simpler and faster for users to setup a dualcolumn tandem operation program. This provides increased sample throughput without having to change validated methods. With dual-column tandem operation analysis, times can be shortened by performing the time-consuming wash and reequilibration steps off-line. This is accomplished via column switching and a second pump. The flow paths are automatically switched to run two columns of the same type in dual-column tandem operation with off-line column regeneration, allowing uninterrupted analysis and raising productivity by typically 50 100%. The Wizard takes users through a step-by-step guide. Basically the user is asked to define the currently used gradient pump profile and at which point to stop the active part of the run. With this information, the Wizard automatically breaks down the original method into an active (or analytical) part that is used to separate and detect the analytes of interest on one column and an off-line reconditioning part that is used to wash and recondition the second column in parallel. This functionality makes the dual-column tandem operation setup intuitive and easy and provides users with a simple way to take advantage of this new technology. 2 PREREQUISITES In order to use the Summit 2 Tandem Operation Wizard, the following items are required: Chromeleon 6.70 Two P680 gradient pumps (e.g. P680A DGP-6 Dual-Gradient Pump) ASI-100 Automated Sample Injector TCC-100 MSV 2P-10P Thermostatted Column Compartment with a motorized 2-position, 10-port switching valve Note: The P680A DGP-6 houses two independent ternary gradient pumps in a single enclosure. The pump provides the highest flexibility for all standard HPLC methods as well as for advanced chromatographic techniques requiring two pumps, such as two-column tandem operation, 2-D chromatography, or online sample preparation.
3 SUMMIT 2 TANDEM OPERATION WIZARD The Program Wizard is started either from the File\New... drop down menu or by clicking on the New icon in the toolbar. 3.1 Timebase and Program Type Selection In this dialog, the timebase can be selected (see Figure 1). The selection options provided are the standard Chromeleon options. That is, a timebase can be selected either from the local PC (My Computer) or from the network (Network Neighborhood). If the Program for Summit 2 Tandem Operation is selected, the following wizard dialogs will be accompanied by a Tandem System Schematic (see Figure 3). This schematic helps the user to understand the general setup of the dual-column tandem operation. Figure 3. Tandem System Schematic is displayed adjacent to all subsequent Wizard dialogs. Figure 1. Timebase Selection dialog. 3.2 Program Type Selection and Tandem System Schematic The next dialog (see Figure 2) is only displayed if the prerequisites listed in chapter 2 are fulfilled. If this is the case the user can choose to create either a Regular Program (default setting) or a Program for Summit 2 Tandem Operation. 3.3 Autosampler and Column Oven Temperature Options If a thermostatted autosampler is used, the appropriate settings can be defined in the dialog displayed in Figure 4. This dialog is not displayed if a non-thermostatted autosampler is used, or if the temperature control is deactivated in the server configuration. Figure 4. Thermostatted autosampler options. Figure 2. Program type selection dialog. 2 Using the Chromeleon Summit 2 Tandem Operation Wizard
Use the next dialog (see Figure 5) to set the column oven options as required by your method. Note that the column is always linked to the respective MSV position. Figure 6. Pump Options dialog with pump selection for tandem operation mode. Figure 5. In this dialog the Column Oven options can be set. 3.4 Tandem Operation Options 3.4.1 Define the Analytical and the Reconditioning Pump The Pump Options page (see Figure 6) includes the Pump Selection part where the user defines which pump is to be used for the analytical run and which is to be used for the off-line reconditioning. If you change the Analytical Pump, the Reconditioning Pump changes accordingly. Note that the gradient mode is always multistepgradient. Default pressure limits are read from the analytical pump and they are assumed to be the same for both pumps. The Pump Selection affects the SyncWithPump property (injection synchronization between the autosampler and the low-pressure gradient pump) and the PumpDevice property (defines which pump is used for injection synchronization). That is, the pump that is chosen as the active gradient pump is the one that is used for injection synchronization. Note that maximum flow deceleration is not available with P680 pumps and that this field is grayed out. 3.4.2 Single Pump Configuration Gradient Profile and Off-Line Reconditioning Start Time After the Pump Options page, you can specify the single pump gradient profile used (see Figure 7). In addition, the time when the analytes of interest have eluted must also be set ( Off-line Reconditioning Start Time ). This time is also known as the Split Point. Essentially this time defines the end of the active gradient part of the method. The time can be selected by entering a time value in the Off-line Reconditoning Start Time field or by moving the red slider (the mouse cursor changes to double arrow when over the slider). If the user does not modify the slider position or enter a split point time, this time is automatically set as 50% of the run time. Note that in case a curved gradient is defined, the curve number has to be entered in every gradient step line. Figure 7. Gradient profile for single pump configuration. Technical Note 702 3
If the flow rate is zero or not defined at the split point, the Wizard issues the message in Figure 8. If the multi-step gradient timetable contains no entry (or only one entry), the Wizard issues the message shown in Figure 9. Figure 8. Warning message if the flow rate is set to zero at the split point. Figure 9. Warning message in case the multi-step gradient timetable contains no entry (or only one entry). Void (Delay) Volume Purge. At the end of the active chromatographic run, the eluent lines between the point of gradient formation and the switching valve are not filled with the starting eluent composition. Therefore this void (delay) volume has to be purged with starting eluent composition before the off-line reconditioned second column can be switched into the analytical flow path. For this purpose purge volume, purge flow, and purge time can be specified. The default purge volume of 2.4 ml (void volume of about 0.8 ml and flush factor of 3) is valid for a standard Summit x2 Dual-Gradient HPLC System. The purge volume can be minimized by using the MicroFlow Kit for the pump, a 100-µL syringe and loop for the autosampler, and by using capillaries with a smaller internal diameter. The purge time can be shortened by applying a higher purge flow rate compared to the analytical separation. Purge time, purge volume, and purge flow are related to each other (see Equation 1): 3.4.3 Void Volume Purge and Bypass Mode Figure 10 shows the split gradient profiles with additional options. The upper gradient profile reflects the active part of the chromatographic run and is associated (in this example) with the right pump (PumpRight). The lower gradient profile reflects the wash and reconditioning phase which are performed using the alternative flow path and the second pump (in this example PumpLeft ). Figure 10. Tandem Operation Options dialog. Purge Time (min)= Purge Volume (ml) Purge Flow (ml min -1 ) Equation 1. Relationship between purge time, purge volume, and purge flow. The Wizard adds the calculated purge time to the analytical gradient and extends the cycle time accordingly. All changes are applied immediately to the gradient plots. Note: The purge volume cannot be changed once the wizard is completed. Bypass Mode. The Bypass Mode (default: deactivated) is used to switch the autosampler out of the fluidic flow path. This reduces the gradient delay volume and allows a faster response of a gradient change on the column thus providing faster elution of the analytes and hence shorter run times. A prerequisite for the bypass mode is an initial isocratic phase. If there is no initial isocratic phase the Wizard issues a warning when activating bypass mode (see Figure 11). If Bypass Mode is enabled, a marker shows the bypass switching time (see Figure 10). The bypass time can be calculated using Equation 2. 4 Using the Chromeleon Summit 2 Tandem Operation Wizard
Figure 12 shows the 2 Tandem Options page in the program editor. Figure 11. Warning message if Bypass Mode is used with no initial isocratic phase. Bypass Time (min)= (Injection Volume (µl) + Volume Needle Seat to MSV (µl)) Flush Factor Flow Rate (µl min -1 ) Equation 2. Formula for calculating the bypass time. Use the Flush Factor (default is 5) to ensure that the injected sample is quantitatively transferred to the column before the sampler flow is switched from mainpass to bypass. For example, if 10 µl is injected with a flush factor of 5, a 400 µl/min flow rate, and with a 19-µL seat capillary volume, the autosampler will be switched to bypass 21.8 sec after injection (see Equation 3). Bypass Time (min)= (10 µl + 19 µl) 5 400 µl min -1 = 0.3625 min = 21.8 sec Equation 3. Calculation of bypass time using example values. Reconditioning Flow The reconditioning flow can be set separately to the analytical and void volume purge flow. 3.5 Other Wizard Pages The other wizard dialogs (e.g., Sampler Options, ColumnOven_Temp Options and Acquisition Options ) are the same as with the regular program file wizard and not discussed here. 4 TANDEM MODE ACCESS IN THE PROGRAM EDITOR If the following prerequisites are fulfilled, the program editor contains an 2 Tandem Options page: A timebase containing Summit modules which can be used for tandem mode is detected (see chapter 2 for details) Program was created using CM 6.70 or higher. Figure 12. Activating tandem mode in the program editor. 4.1 Tandem Options Tab in the 2 Tandem Options Page The isocratic/gradient pump assignment for Analytical Pump and Reconditioning Pump depends on the ASI-100 s PumpDevice property. If the PumpDevice statement is missing, the value is read on-line from the device. Nevertheless, a warning is issued in the Consistency Check tab of the 2 Tandem Options page. All fields for Void Volume Purge are read-only. The Bypass Mode is activated if a corresponding MSVToLoad command is found in the program script. Only the fields Injection Volume and Flush Factor can be modified in this page. If the user changes one of these values, the MSVToLoad command in the script is shifted to the recalculated Bypass Time. However, a check is done to verify that the Bypass Time is within the initial isocratic phase. 4.2 Consistency Check Tab of the 2 Tandem Options Page A consistency check is performed once the user leaves the 2 Tandem Options page or if the user tries to save an edited tandem mode program. If the check fails, a dialog appears containing the check results (see Figure 13). The user then has the option to either correct the issue (by clicking on Yes ) or to accept the reported issues (by clicking on No ). Technical Note 702 5
Figure 13. Consistency Check warning. 4.3 Tandem System Schematic Tab of the 2 Tandem Options Page The tab Tandem System Schematic of the 2 Tandem Options page outlines the basic setup of a dual-column tandem operation mode (see Figure 15). The following checks are performed The properties SyncToPump and PumpDevice must be present in the initial part of the program script. The two pump profiles must have the same run time. The eluent composition must be the same at the beginning and the end of the analytical profile, as well as at the end of the reconditioning profile. There must be an isocratic part at the end of the analytical pump profile for the void volume purge. If Bypass Mode is activated, the bypass volume must be flushed within the isocratic part at the beginning of the analytical profile and within the specified bypass time. The Consistency Check tab displays the results in form of a list (see Figure 14): Figure 15. Tandem System Schematic tab of the 2 Tandem Options page. 5 LIMITATIONS The initial 2 Tandem Wizard implementation in Chromeleon 6.70 does not cover Dionex IC or Ultimate instruments. This feature will not be extended to third party instruments as it requires module data input that is only provided by Dionex instrumentation Figure 14. Consistency Check tab of the 2 Tandem Options page. ASI-100 is a trademark and Summit and Chromeleon are registered trademarks of Dionex Corporation. Dionex Corporation Dionex Corporation Dionex U.S. Regional Offices Dionex International Subsidiaries 1228 Titan Way Salt Lake City Technical Center Sunnyvale, CA (408) 737-8522 Australia 61 (2) 9420 5233 Austria (01) 616 51 25 Belgium (03) 353 42 94 Canada (905) 844-9650 P.O. Box 3603 1515 West 2200 South, Suite A Westmont, IL (630) 789-3660 China (852) 2428 3282 Denmark 36 36 90 90 France 01 39 30 01 10 Germany 06126-991-0 Sunnyvale, CA Salt Lake City, UT Houston, TX (281) 847-5652 India 91-22-28475235 Italy (06) 66 51 50 52 Japan (06) 6885-1213 Korea 82 2 2653 2580 94088-3603 84119-1484 Atlanta, GA (770) 432-8100 The Netherlands (0161) 43 43 03 Switzerland (062) 205 99 66 United Kingdom (01276) 691722 (408) 737-0700 (801) 972-9292 Marlton, NJ (856) 596-06009 * Designed, developed, and manufactured under an NSAI registered ISO 9001 Quality System. LPN 1740 PDF 11/05 2005 Dionex Corporation 6 Using the Chromeleon Summit 2 Tandem Operation Wizard