3.0 NGC™ Chromatography Systems and ChromLab™ Software User Guide Version 3.
NGC™ Chromatography Systems and ChromLab™ Software User Guide Version 3.
Bio-Rad Technical Support Department The Bio-Rad Technical Support department in the U.S. is open Monday through Friday, 5:00 AM to 5:00 PM, Pacific Time. Worldwide technical support is available on the Web at www.consult.bio-rad.com. Phone: 1-800-424-6723, option 2 Fax: 1-510-741-5802 Email: LSG.TechServ.US@Bio-Rad.com (U.S.) LSG.TechServ.Intl@Bio-Rad.com (International) Web: www.consult.bio-rad.
Table of Contents Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Main NGC Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 NGC Chromatography Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Finding Out More . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Chapter 2 The Workspace . . . . . . . . . . . . . . .
Table of Contents Chromatogram View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Changing Chromatogram View Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Showing or Hiding Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Changing Trace Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Zooming In and Out. . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents Detector Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Single-Wavelength Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Multi-Wavelength Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Email Server Tab . . . . . . . . . . . . .
Table of Contents Method Settings Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Fluidic Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Column Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Detector Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Unit Selection . . . . . . . . . . . . . . . . . . .
Table of Contents Collecting Fractions in Run Queues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Collecting Fractions Using the BioFrac Fraction Collector . . . . . . . . . . . . . 174 Collecting Fractions Using Outlet Valves . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Saving a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Renaming a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents Annotating the Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Copying the Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Customizing the Trace Table Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 Changing Table Grouping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 Sorting Table Columns . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents Peak Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Peak Filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Default Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Integrate and Delete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Starting Peak Integration . . . . . . . . . . . .
Table of Contents Chapter 8 Importing and Exporting Data . . . . . . . . . . . . . . . . . . . . . 259 Importing an NGC Method or Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Importing Unicorn Data Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Importing BioLogic DuoFlow Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Exporting Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Introduction NGC™ chromatography systems provide a general purpose purification platform for purifying all forms of biomolecules using a combination of chromatography techniques. The systems are also useful for developing and optimizing purification protocols. The systems can provide highly purified proteins, peptides, nucleic acids, monoclonal antibodies, and other small molecules.
1 | Introduction ChromLab™ software enables you to set up and control an NGC instrument, run protein separations and other operations manually, program methods to automate purification runs, evaluate the results, and generate and print experiment reports. This user guide explains how to perform all these tasks.
Main NGC Features Organize the location of the modules to optimize separation performance based on method scale and complexity, and to minimize the system swept volume Minimize errors when connecting tubing using the Point-to-Plumb™ feature in ChromLab software User Guide | 13
1 | Introduction NGC Chromatography Systems All NGC chromatography systems include ChromLab software and the NGC touch screen. NGC chromatography systems are available in several combinations. Each system is equipped with either two 10 ml system pumps (the 10 series) or two 100 ml system pumps (the 100 series).
NGC Chromatography Systems The NGC Scout™ chromatography system includes the following: All modules on the Quest system pH detector valve Buffer blending valve The NGC Discover™ chromatography system includes the following: All modules on the Scout system Column switching valve Two buffer inlet valves Sample pump The NGC Discover Pro chromatography system includes the following: All modules on the Discover system Fourth expansion tier Sample inlet valve Outlet val
1 | Introduction Finding Out More After you install NGC documentation from the NGC Chromatography Systems Documentation DVD, you can access installed NGC guides and tutorials on the Help menu in any ChromLab view. More information about the NGC chromatography systems and ChromLab software is available from the following sources. The NGC Chromatography Systems and ChromLab Software Installation Guide is available on your NGC Chromatography Systems Documentation DVD as a .pdf file.
2 The Workspace ChromLab™ software provides an intuitive interface for developing chromatography methods, operating an NGC™ instrument, and analyzing data from chromatography runs. ChromLab software presents four primary workspaces. The Home window The System Control window The Method Editor window The Evaluation window Each workspace is shown and described in this chapter. The NGC instrument touch screen is also described.
2 | The Workspace The Home Window ChromLab software opens with the Home window, which displays three panes and the System Control tab. The three panes provide quick access to the system control, method editor, and evaluation workspaces. The status of connection to the NGC instrument is also displayed. Links to recently completed runs, recently created methods, and recently accessed or analyzed runs and analyses appear listed at the bottom of the window.
The Home Window Method Editor and Evaluation tabs become visible in the Home window when you select tasks in the Method Editor and Evaluation panes.
2 | The Workspace File Menu Commands Connect to System — opens a dialog box that enables you to choose an NGC chromatography system to connect to. ChromLab detects the NGC systems available on the same subnetwork or those systems that are directly connected to the computer. Their system name, network name, and IP address appear in the table. To connect to a system, you can: Select a name in the list of detected systems and click Connect. Note: If your system does not appear in the list, click Detect.
The Home Window Import — displays links from which you can import the following: NGC File — opens a dialog box in which you can import a method, a method with runs, or a run exported from ChromLab software running on another NGC system. See Importing an NGC Method or Run on page 259 for more information. Unicorn Data — opens a dialog box in which you can import a Unicorn data file into the NGC database. See Importing Unicorn Data Files on page 261 for more information.
2 | The Workspace Help Menu Commands Help — displays screen-level help topics and links to installed manuals. Export Diagnostic Logs — opens the Export Diagnostic Logs dialog box in which you can export all critical information that Bio-Rad Technical Support requires to diagnose issues. The log files and data are zipped and saved to a location that you choose. See Exporting Diagnostic Logs on page 271 for more information. About — displays ChromLab copyright and version information.
The System Control Window The System Control Window The System Control window enables you to run the instrument manually, monitor method runs while they are running, select fluidic schemes, calibrate pumps and detectors, and verify the accuracy of instrument plumbing. This window displays a chromatogram during a run. A fluidic scheme graphically depicts the flow path of all the modules on the system. A status panel appears above each module displaying its real-time status.
2 | The Workspace The Method Editor Window The Method Editor window enables you to create, open, review, edit, and run a method. You can also open and edit a method template to create a new template. Method Editor functionality is detailed in Chapter 5, Method Editor. See also Chapter 6, Creating a Method.
The Evaluation Window The Evaluation Window The Evaluation window enables you to view and compare run data, perform peak integration, and save run data as analyses. Evaluation functionality is detailed in Chapter 7, Evaluating Results.
2 | The Workspace Instrument Control Touch Screen In addition to ChromLab software running on a computer, the instrument is equipped with a touch screen that accesses system control functionality. You can use this touch screen to run, control, and monitor a run independent of ChromLab. See System Control on page 29 for more information.
Instrument Control Touch Screen Touch Screen Menu Commands Calibrate — opens the Calibration dialog box, which displays instructions and settings for selecting a module and calibrating it. See Calibrating a Module on page 64 for details. Point-to-Plumb — starts the Point-to-Plumb™ feature and simultaneously turns off instrument LED lights so you can visually verify or change instrument plumbing. Displays the current fluidic scheme.
2 | The Workspace Touch Screen Toolbar Commands Save — saves in a data file the steps executed during a manual run. Clear — deletes manual run data from the touch screen display. Autoscale — automatically scales the chromatogram’s primary y-axis to the tallest peak height during the run. Autoscaling is enabled by default. Reset Zoom — resets the view to show the full chromatogram. Annotate — adds a note to the chromatogram at points on the x-axis during a run or after the run completes.
3 System Control The ChromLab™ System Control window is the main interface to the NGC™ chromatography system instrument. This interface also appears on the instrument touch screen. System Control settings enable you to perform a manual run, monitor and control a method run, verify the device plumbing with the Point-to-Plumb™ feature, control and calibrate the system, and map two or more valves of the same type on your instrument to their position in the fluidic scheme.
3 | System Control LEGEND 30 1 The menu bar provides quick access to File, View, Manual, Tools, and Help menu commands. 2 Tabs provide quick navigation among open windows (Home, System Control, Method Editor, and Evaluation). 3 The tab toolbar provides commands to save the current run, autoscale the UV trace, change the chromatogram view, annotate the chromatogram, and delete manual run data from the display. 4 Expand buttons expand the selected pane to fill the screen.
System Control Window LEGEND 8 The Fluidic Scheme pane depicts graphically how modules are configured and plumbed for an experiment. File Menu Commands Connect to System — connects your computer to the NGC system if it is not already connected. In the dialog box that appears enter the DNS name of the NGC system and click Connect. Disconnect System — when you start ChromLab, you are automatically logged in to the NGC system to which your computer is connected.
3 | System Control View Menu Commands Show Chromatogram — displays a chromatogram of the current run data. Clearing this command hides the chromatogram from view. Show Fluidics — displays the fluidic scheme. Clearing this command hides the fluidic scheme from view. Manual Menu Commands Enter/Exit Manual Mode — toggles ChromLab between manual and automatic modes. Save Recorded Manual Run — in manual mode, saves in a data file the steps executed during a manual run.
System Control Window Map Fluidic Scheme — opens the Fluidic Scheme Mapping dialog box, which displays the location of two or more valves of the same type in the fluidic scheme, for instance two or more inlet valves or column-switching valves. You can use this dialog box to map the device on your instrument to its position in the fluidic scheme. See Fluidic Scheme Mapping on page 60 for details. Help Menu Commands Help — displays screen-level help topics and links to installed manuals.
3 | System Control Autoscale UV Trace — automatically scales the primary y-axis to the tallest peak height during the run. While enabled, UV scale in the legend cannot be manually set. Copy Chromatogram — copies the chromatogram to the clipboard so you can paste it into another application. Save Chromatogram As —saves the chromatogram in an image format you choose (.bmp, .gif, .jpeg, .png, or .tiff). Export as .csv — exports run data as a .csv file, which can be opened in spreadsheet applications.
Chromatogram View Chromatogram View A chromatogram shows the real-time acquisition of data during the run as well as its outcome. It also depicts the quality of the purification. The chromatogram is recorded only when pumps are running and data are being acquired by the detectors in the fluidic scheme. The chromatogram legend matches each colored trace to its detector type.
3 | System Control You can expand the chromatogram or the fluidic scheme by clicking the arrow in the upper-right corner of the relevant pane. This is especially useful when viewing a chromatogram on the touch screen. Events that occur during the run, such as valve changes or changes in certain parameters, appear as event markers. Error events such as overpressure appear in red. The view also shows when fractions are collected and their locations in the selected rack. The chromatogram has two y axes.
Chromatogram View Table 1.
3 | System Control Table 1. Trace Definitions, continued Trace Module Explanation Delta Col Pressure Column Switching Valve Available when at least one column switching valve is present Tip: When two or more column switching valves are present, the traces appear in different shades of blue on the chromatogram. The trace number corresponds to the valve number in the fluidic scheme. For example, Col Pressure 1 in the legend and the chromatogram corresponds to C1 in the fluidic scheme.
Chromatogram View When a manual or method run is saved, the new settings are saved with it. When you open the run in the Evaluation window, it displays the new settings. Some chromatogram view settings that are changed on the computer automatically change in the touch screen chromatogram view and vice versa.
3 | System Control Changing Trace Color To change trace color 1. Expand the legend and click the trace color in the Edit column. 2. In the Color dialog box, select a color and click OK. Tip: See Trace Settings Tab on page 74 to revert trace colors to their factory default settings. Zooming In and Out To zoom in on a section of the chromatogram While clicking in the chromatogram, drag the pointer to mark the zoom region of interest.
Chromatogram View Changing the X-Axis Units and Scale For runs performed from saved methods, the x-axis unit can be changed to Time (min), Volume (ml), or Column Volume (CV). For runs performed manually, the x-axis unit can be changed to Time (min) or Volume (ml). To change the x-axis units and scale Click the x-axis title to toggle among the available options. Changing the Y-Scale Values You can change the maximum and minimum y-scale values in the legend to set the chromatogram scale.
3 | System Control When a single UV absorbance trace is monitored with a single-wave UV you can Autoscale all UV traces to the same scale . The y-axis scale is based on the signal intensity of the UV trace with the highest intensity and all other traces are normalized to the intensity of that trace. Disable autoscaling . To autoscale the UV trace Do one of the following: Click Autoscale on the toolbar to choose a status.
Chromatogram View To add annotations 1. Click Annotate on the toolbar and drag the icon onto the chromatogram. The green annotation dialog box opens with the Location field automatically filled. Tip: You can edit the Location field if necessary. 2. Type a title for the annotation. 3. (Optional) Type a description for the annotation. 4. Click Save to save the annotation. Note: Clicking closes the dialog box without saving the annotation. To edit an annotation 1.
3 | System Control Showing and Hiding Events, Fractions, Errors, and Annotations You can choose whether to show or hide fractions, events, errors, and annotations by selecting or clearing the appropriate checkboxes at the bottom of the legend.
Chromatogram View When you pause the pointer on a trace, a tooltip displays the real-time reading at the current location in the chromatogram.
3 | System Control Fluidic Scheme Pane The Fluidic Scheme pane graphically displays each module currently in use on the instrument. Real-time status information includes buffer flow rate, sample flow rate, pressure, and valve position through the system. The fluidic scheme reflects how the instrument is plumbed and the flow path through the various modules on the system. Important: The selected fluidic scheme must match the system’s installed hardware modules.
Fluidic Scheme Pane When pumps are not running, the predicted flow path appears in a lighter color corresponding to the pump flow path (light blue for the sample pump or light green for the system pump). On the NGC Discover system, two flow paths are displayed — one for the system pump (bright green) and another for the sample pump (light blue).
3 | System Control The fluidic scheme displays the number of a valve’s active port in bright green. A grey number on a valve indicates that it is not in use. The NGC instrument supports up to four inlet valves (two buffer inlet and two sample inlet valves), three column-switching valves, and two outlet valves. Pausing on a valve displays a tooltip indicating its type.
Fluidic Scheme Pane Modules Fluidic scheme modules are more fully described in Chapter 2, Components, of the Instrument Guide. For ease of reference, Table 2 depicts each module that can appear in a fluidic scheme and describes corresponding dialog box options. Table 2. Fluidic Scheme Modules System Pumps and Mixer This module dialog box controls the NGC system’s two gradient pumps and shows the gradient status.
3 | System Control Table 2. Fluidic Scheme Modules, continued Sample Pump This module dialog box controls the sample pump and displays the sample pump status. It is used to automatically load samples into a sample loop or to directly inject samples onto a column during an experiment. It is used primarily for large volume samples.
Fluidic Scheme Pane Table 2. Fluidic Scheme Modules, continued pH Monitor and Valve This module dialog box controls the pH monitor and displays the pH. It is used to place the pH flow cell inline or to bypass it during an experiment. It also enables you to calibrate the pH probe without taking it offline. Fraction Collector This module dialog box controls fraction collection. It is used to select racks and rack location and to start and stop fraction collection.
3 | System Control Table 3. Fluidic Scheme Configuration Options, continued Sample Inject 52 System pump and inlet B Select from up to eight different buffers for B. System pump and inlet A and B Select from eight buffers each for A and B (16 total). System pump and buffer blending valve Start and stop buffer blending valve gradient formation and perform buffer blending.
Fluidic Scheme Pane Table 3. Fluidic Scheme Configuration Options, continued Sample inject valve with sample pump Automatically load a specific predetermined sample volume onto a sample loop, or directly onto the column, using the sample pump. Sample inject valve with autosampler Automatically load samples onto a column using an autosampler. Sample inject valve with sample pump and a single sample inlet valve For use as a mini autosampler, can select from eight different samples.
3 | System Control Table 3. Fluidic Scheme Configuration Options, continued Columns Detectors 54 Configuration Explanation Column Self-explanatory when column switching valve is absent. Column with column switching valve Used to select from up to five different columns, bypass the columns, or reverse the flow through the columns. Two column switching valves, each with a column attached Used to select from up to 10 different columns, bypass the columns, or reverse the flow through the columns.
Fluidic Scheme Pane Table 3. Fluidic Scheme Configuration Options, continued Single-wavelength UV with conductivity and pH probe For selection of a single-wavelength UV detector with conductivity, temperature, and pH valve. Multi-wavelength UV/Vis detector with conductivity and temperature For selection of a multi-wavelength UV/Vis detector with conductivity and temperature.
3 | System Control Table 3. Fluidic Scheme Configuration Options, continued Fraction Collection Configuration Explanation BioFrac™ fraction collector For configuring a BioFrac fraction collector. Used for starting and stopping fraction collection to determine fraction size and number of tubes to collect. Fraction collector with outlet valve For collecting a combination of large and small volume fractions.
Fluidic Scheme Pane Table 3. Fluidic Scheme Configuration Options, continued Two outlet valves For use as a fraction collector, can collect up to 22 large-volume fractions when two outlet valves are daisychained together. Select outlet ports can be plumbed to divert a fraction eluted from a column onto a sample/storage loop to be subsequently reinjected onto a second column during tandem applications.
3 | System Control 2. Click an entry in the Fluidic Scheme Selector pane. The right pane displays the fluidic scheme you selected. 3. Click Select. To create a new fluidic scheme Note: You cannot save a duplicate fluidic scheme. 1. 2. Do one of the following: In the System Control window, select Tools > Change Fluidic Scheme. On the touch screen, choose Change Fluidic Scheme on the dropdown menu. Click New.
Fluidic Scheme Pane 3. Click Edit under the group of modules to change. A spin box displays a number of modules of the type you selected. 4. Scroll up or down to view module choices. 5. Select a module and click Accept. 6. Repeat steps 3–5 to edit other modules. 7. When you have finished editing modules, click Save and type a name in the New Fluidic Scheme dialog box that appears. 8. Click OK. To change module settings 1. In the fluidic scheme, double-click a module. 2.
3 | System Control Fluidic Scheme Mapping The NGC instrument supports up to four inlet valves (two buffer and two sample inlet valves), three column-switching valves, and two outlet valves. When more than one of a specific valve type is installed, ChromLab automatically detects the valves and maps the fluidic scheme to their default positions on the NGC instrument.
Fluidic Scheme Mapping The color and labeling scheme for the Fluidic Scheme Mapping dialog box is as follows: Color Label valve Blue A and B Buffer inlet valve Blue S1 and S2 Sample inlet valve Green C1–C3 Column switching valve Orange O1 and O2 Outlet valve To map a fluidic scheme to the instrument, you first unmap the current settings and then map the valves to the new fluidic scheme.
3 | System Control To unmap valves 1. Do one of the following: In the System Control window, select Tools > Map Fluidic Scheme. On the touch screen, choose Map Fluidic Scheme on the dropdown menu. The Fluidic Scheme Mapping dialog box appears with the valves on the fluidic scheme mapped to their default positions on the instrument map. 2. Select a valve on the instrument map. The border of the valve on the fluidic scheme becomes highlighted, indicating that it is selected. 3. Click Unmap.
Fluidic Scheme Mapping To map valves 1. In the Fluidic Scheme Mapping dialog box, select a valve to map in either the upper or lower pane. 2. Select its position on the corresponding scheme. In the instrument map, a label appears on the selected valve and its border thickens, indicating that it is mapped. 3. (Optional) Continue mapping the unmapped valves. 4. Click Save to save the new mapping.
3 | System Control Calibrations The NGC instrument arrives factory calibrated. For the most part, the instrument will not require further calibration. The pH probe, however, should be recalibrated each day the instrument is used and when there are large fluctuations in temperature because pH probe settings drift with time.
Calibrations Calibrating Flow Rate of Pumps If the buffer flow seems to be incorrect, you might want to recalibrate the pumps. Important: The system must be primed with water before you calibrate the pumps’ flow rate. Otherwise the calibration will be incorrect. To calibrate the flow rate of the pumps Select Pump Flow Rate in the Calibrate dropdown list at the top of the Calibration dialog box. The Calibration dialog box displays instructions and settings for calibrating the pumps.
3 | System Control Calibrating Pressure Settings WARNING! You must remove the tubing from the pressure sensor before you calibrate it. The sensor must be at atmospheric pressure when you start. You can calibrate sample pump pressure, system pressure, and column switching valve pressure to zero. If pressure is not reading zero or close to it, you can reset the pressure values to zero. To calibrate a pressure setting 1.
Verifying Plumbing with the Point-to-Plumb Feature Verifying Plumbing with the Point-to-Plumb Feature The NGC chromatography system instrument arrives plumbed. The Point-to-Plumb feature enables you to change the plumbing or to verify that the instrument is plumbed correctly for the fluidic scheme you want to use. You can access the Point-to-Plumb feature on the touch screen menu or on the computer running ChromLab. (Select Tools > Point-to-Plumb.
3 | System Control path between them is selected. If the fluidic path includes an NGC module after the external detector, the inlet LED on that module turns on. To verify the plumbing path 1. With both the Point-to-Plumb window and the front of the instrument in view, click a segment of the gray path in the Point-to-Plumb window. The segment you selected turns green to indicate the correct plumbing path.
System Settings System Settings The System Settings dialog box enables you to customize your system.
3 | System Control When the delay volume is set, the fraction collector or outlet valves wait for the fraction peak to travel from the detector to the drophead (or port) before collection begins. Fraction collection on the instrument can be delayed relative to the collection information shown in the chromatogram and status panels.
System Settings 3. Select Synchronize with detector. 4. Specify the inner diameter of the tubing in use. Tubing Color Inner Diameter Orange 0.02" (0.5 mm) Green 0.03" (0.75 mm) Clear 0.062" (1.6 mm) 5. If your system includes the BioFrac fraction collector, select the checkbox Include BioFrac – 12 l and specify the tubing length between the UV outlet port and the fraction collector drophead. 6.
3 | System Control 7. If your system includes both the BioFrac fraction collector and an outlet valve, select both checkboxes. In the Include Outlet valve – 50 l section, specify the tubing length between the UV outlet port and the outlet valve as well as the tubing length between the outlet valve and the fraction collector drophead. 8. Select or clear the next four checkboxes, depending on whether these modules are present on your system.
System Settings When Control Flow to avoid overpressure is selected, the system lowers the flow rate to avoid exceeding the maximum pressure. Typically, Control Flow activates when the pressure reaches 80% of the maximum set pressure from the column. This limit can be changed. The flow rate is lowered to half the set flow rate. Flow rate returns to normal when the phase changes and drops only if the pressure in the next phase continues to exceed the upper pressure limit of the column.
3 | System Control 4. (Optional) Specify a minimum flow rate for the system pump and for the sample pump if the sample pump is available. Tip: When you change the pump heads on the system pumps, ChromLab software checks at instrument startup whether the most recently used flow rate setting in the System Settings dialog box Control Flow tab is compatible with the range that the new pump heads allow. If it is not, the flow rate is set to the minimum that the new pump heads allow.
System Settings To revert trace display settings to their factory defaults 1. Select File > System Settings to open the System Settings dialog box. 2. Choose the Trace Settings tab. 3. Do one or more of the following: 4. To show all traces in the System Control legend, click Show All. To reset the y-scale values to their factory default settings, click Reset. To reset all trace colors to their factory default settings, click Reset. Click OK.
3 | System Control Device Input Tab You can import analog (voltage) signal from up to two external devices and convert it to digital NGC data via the signal import module (SIM).The SIM imports and converts the signal to the appropriate trace units through its SIM channels. When enabled, the details appear in the chromatogram’s legend, the trace appears on the chromatogram, and the data can be analyzed in the Evaluation window.
System Settings 4. Choose the Device Input tab. 5. By default, the fields in this dialog box are disabled. Select the Enabled checkbox for SIM 1 and provide the following details to enable connection to the first detector: a. Type a trace name for the detector in the Trace Name field. Note: This field has a 20-character limit. b. Range (Volt) is the output from the detector. Set the minimum and maximum volt range. c. Type a unit type for the trace in the Trace Unit field.
3 | System Control 6. (Optional) Select the Enabled checkbox for SIM 2 and repeat steps 5a—5d for a second external detector. 7. Click OK. To disable the connection On the Device Input tab, clear the Enabled checkbox for the device and click OK. Note: Disabling the SIM connection does not turn off the detector. Device Output Tab You can convert digital NGC data to analog voltage via the SIM and output the voltage to an external recording device, such as a light scattering detector.
System Settings 5. 6. By default, the fields in this dialog box are disabled. Select the Enabled checkbox for Signal Output A and provide the following details: a. Choose a signal type from the Signal dropdown list. b. Scale converts the trace units to the device’s input type. Set the minimum and maximum unit range (y-axis scale). c. Range is the output to the device. The SIM’s DAC channels output 0—10 V. Recommended settings within this range are specific to the external device.
3 | System Control Air Sensors Tab The system supports up to two air sensor modules and up to eight air sensors. Each air sensor module can contain up to four air sensors. The system detects the number of air sensors that are attached to the instrument. Air sensors can be set to detect air, indicating end of buffer, in lines connected to the system pumps. When the air sensor detects end of buffer, the system stops the system pumps and the run.
System Settings 3. To activate one or more air sensors, select its checkbox and from the dropdown list choose either Sample (to detect end of sample) or Buffer (to detect end of buffer). 4. Click OK. To deactivate an air sensor On the Air Sensors tab, clear its checkbox and click OK.
3 | System Control Email Notifications Tab You can connect ChromLab to your outgoing email server to send email notifications to a list of users.
System Settings 3. Select Enable Email Notification. Note: If you have not set up a valid SMTP connection for ChromLab, an error message appears informing you that ChromLab cannot connect to the email server. Click OK. The Email Server dialog box appears. To set up the connection between ChromLab and your SMTP server see To connect ChromLab to an email server on page 90. 4. Below the Email Recipients box, click Add. The Add Email Address dialog box appears. 5.
3 | System Control All added users receive an email from DoNotReply_NGC notifying them that they are registered to receive email about NGC system events. 6. In the Event Types section, clear the checkboxes for the events about which you do not to want to receive email notifications. Note: All email recipients will receive email about all selected event types. 7. Click OK to save the changes and exit the dialog box. To edit a name in the Email Recipients section 1.
System Settings System Name Tab You can specify a unique name for the NGC instrument. ChromLab uses this name to identify the system to which the computer is connected. The system name appears in the Home window when ChromLab connects to the NGC system. It also appears in the formatted run reports to identify the system on which the run was generated. Note: The system name is limited to any combination of 10 alphanumeric and special characters.
3 | System Control System Information The System Information dialog box enables you to view general information about your system as well as information about the system components, processes, and UV and UV/Vis detectors. Note: This dialog box is accessible in manual mode and from the menu on the touch screen.
System Information Detector Tab This tab displays details about UV or UV/Vis lamp usage, from which you can determine whether the lamps need to be replaced. The system determines which detector module is installed and displays information specific to that module. Single-Wavelength Detector The system retrieves the lamp status for both lamps (255 nm and 280 nm) and displays the reference voltage, pulse-width modulation (PWM) percentage, and total lamp hours for each lamp on the Detector tab.
3 | System Control To reset the lamp hours On the Detector tab, click Reset Lamp Time. Note: A dialog box appears warning you that this cannot be undone. Click No if you do not want to reset lamp hours. Multi-Wavelength Detector The tab displays the measured signal counts at specific wavelengths. It also displays the lamp usage time (in hours) for both the deuterium and tungsten lamps. The system must have stopped running before lamp status can be determined.
Preferences Preferences The Preference dialog box enables you to select pressure units for all system and software pressure values. This is a global setting. This dialog box also enables you to connect ChromLab to your internal email server. ChromLab can then send email alerts about specific system events. Email Server Tab Note: See your system administrator to connect ChromLab to the internal email server.
3 | System Control To connect ChromLab to an email server 90 1. In ChromLab select File > Preferences. 2. Choose the Email Server Setup tab. 3. Provide the following information for your company: SMTP server name — the name of the outgoing email server at your company. Port — the port number for the SMTP server. The default is 25. Use SSL — by default Secure Socket Layer (SSL) is disabled. If you use SSL at your company, select this checkbox.
Preferences Authentication — by default, Authentication is disabled. If your site requires account authentication, select this checkbox. The Authenticated Account and Password fields become active. Authenticated Account — the name of the authenticated account. Account Password — the password for the authenticated account. 4. To verify that the SMTP server settings are correct, enter a valid email address in the Test Email Address field and click Send Test Email. 5.
3 | 92 System Control | NGC Chromatography Systems and ChromLab Software
4 Performing a Manual Run The NGC™ chromatography system instrument operates in two modes: manual mode and automated, programmed method mode. In manual mode you have full control of each NGC pump, valve, detector, and fraction collector. This mode is used primarily for nonautomated processes such as priming and cleaning or purging the system, but it can also be used to load samples, optimize chromatography parameters, and run simple experiments.
4 | Performing a Manual Run Preparing the System System preparation consists of priming the system to remove air from the pumps, tubing, and valves followed by flushing the fluidics system to remove storage buffer, cleaning solutions, or old buffer. Priming is performed in manual mode, while both purging and cleaning tasks can be performed either manually or through preprogrammed method phases. See Chapter 5, Method Editor for more information about working with methods.
Preparing the System 5. Prime the system. (See Priming and Purging the Systems in Chapter 3 of the NGC Chromatography Systems and ChromLab Software Instrument Guide for detailed instructions.) To purge the NGC Quest system pumps 1. In the fluidic scheme, touch or click the Sample Inject valve module to open its dialog box. 2. Set the sample inject valve to System Pump Waste. 3. In the fluidic scheme, double-click the System Pump module to open its dialog box.
4 | Performing a Manual Run 4. 96 In the System Pump dialog box: a. Set Flow Rate to 10 ml/min. b. (Optional) Set Duration to at least 1 min. c. If selected, clear the Enable Air Sensor checkbox. d. Click Start.
Preparing the System To purge the buffer blending valve 1. In the fluidic scheme, double-click the System Pump module to open its dialog box. 2. Select Priming in the Mode dropdown list. To purge an individual line of air bubbles, proceed to step 3 on page 98. To purge all lines of air bubbles sequentially, proceed to step 4 on page 98.
4 | Performing a Manual Run 3. 4. 5. Mode Description Gradient via Blending Valve (High Flow) Buffer blending valve ports Q1 and Q4 are used as inlets for buffers A and B. Gradients are formed by the buffer blending valve rather than by the pumps. This enables each pump to run at their full flow rate capacity which doubles the total flow rate, for example to 20 ml/min for F10 pumps. To purge an individual line of air bubbles and fill it with buffer: a. Select a port to open. b.
Preparing the System To purge the buffer inlet valves (if available) 1. Open the System Pump dialog box and select Priming on the Mode dropdown list. Note: When in Priming mode, the sample inject valve switches the system pump flow to waste (W2). After the process is completed, the sample inject valve switches the system pump to manually load the column through the loop. 2. Select the first buffer port to purge, for example Buffer A 1. 3. (Optional) Change the default flow rate.
4 | Performing a Manual Run To purge the sample pump (if available) 100 | 1. In the fluidic scheme, double-click the Sample Pump module to open its dialog box. 2. Set Flow Rate to 10 ml/min. 3. Click Change Position to set the sample inject valve to Sample Pump Waste and close the dialog box. 4. (Optional) When an air sensor is present, in the Sample Pump dialog box, select Enable Air Sensor to use end-of-sample detection to stop the pump. 5. Click Start. 6.
Preparing the System To purge sample inlet valves (if available) 1. In the fluidic scheme, double-click the Sample Pump module to open its dialog box. 2. Select a port to purge. If only one sample inlet valve is present, select S1 Port 8 from the dropdown list. If two sample inlet valves are present, click Valve 2 and select S2 Port 8 from the dropdown list. 3. Set Flow Rate to 10 ml/min. 4. Click Change Position to set the sample inject valve to Sample Pump Waste and close the dialog box.
4 | Performing a Manual Run Cleaning the System System cleaning consists of rinsing the system to remove buffers, washing with base to hydrolyze any adsorbed proteinaceous material from the fluidic system, and rinsing again to remove the base. The system can also be cleaned in method mode. See Chapter 5, Method Editor for details. To clean the system manually 102 | 1. Take the column offline. 2.
Running an Experiment Running an Experiment Important: When running an experiment manually, ensure that the valves are set in the correct positions before starting the pump. Clicking Start in the System Pump dialog box starts the run. You can change valve positions while the pump is running and your manual run is in progress. You can stop a run at any point and save it. You can then restart the pumps and continue the run with a different name.
4 | Performing a Manual Run To set up the experiment in the fluidic scheme 1. 2. 3. 104 | Double-click the Fraction Collector module and choose the following settings in the dialog box that appears. a. In the Rack Type dropdown list, select the installed rack type. b. Under Collection Scheme, select the fraction collection Start Rack and Tube, number of Tubes to Collect, and Fraction Size. c. Press Collect.
Running an Experiment 4. 5. (Optional) Double-click the Column Switching Valve module, if installed, choose the following settings, and close the dialog box. a. Select the column position or click Bypass. b. (Optional) Select the Reverse Flow Direction checkbox to cause the buffer to flow from the column bottom to its top instead of from top to bottom. Double-click the Sample Inject Valve module and set the valve to Manual Load Loop.
4 | Performing a Manual Run 6. Double-click the Sample Pump module, if installed, and choose the following settings in the dialog box that appears: a. If one or two sample inlet valves are in line, select a port from which to draw sample. b. Under Flow Control, select the flow rate. c. Under Pressure Limits, select the lower and upper pressure limits. d. (Optional) Select Control Flow to avoid overpressure.
Running an Experiment 7. Double-click the System Pump module and choose the following settings in the dialog box that appears: a. If a buffer blending module is in line, select a Mode (Isocratic or Gradient) under Flow Control. b. Under Pressure Limits, select the lower and upper pressure limits. c. (Optional) Select Control Flow to avoid overpressure. This setting causes the flow rate to decrease automatically if the system exceeds set pressure limits. d.
4 | Performing a Manual Run To run an experiment step 1. 2. 108 | Isocratic or gradient step: a. In the System Pump module dialog box, under Flow Control, set the Flow Rate, Mode (Isocratic or Gradient), %B value, Start and End values (in Gradient mode), and Duration (optional for Isocratic mode) for the run. b. Click Start to begin the run. c. Minimize the Sample Inject Valve dialog box. Sample injection step: a.
Running an Experiment Changing Module Settings Tip: For some modules, you must stop the pumps before you can change the settings. To change module settings 1. In the fluidic scheme, double-click a module. 2. In the dialog box that appears, edit module options. 3. Click Apply (where necessary). Stopping a Manual Run To stop a manual run Click Stop in the System Pump dialog box. Clearing Run Data A manual run must be cleared before you can run a method.
4 | Performing a Manual Run Saving a Manual Run on the Touch Screen To save a manual run on the touch screen 1. Click Save on the touch screen toolbar. The Save Manual Run dialog box appears. 2. 110 | Do one of the following: In the Projects pane, select a project or subproject. In the toolbar, click the appropriate button to create a new project, subproject, or root project and type a name for the project in the box that appears above the Projects pane. 3.
Running an Experiment Viewing Run Data Note: See Chapter 7, Evaluating Results, for detailed information about analyzing a run. When you select a single saved run in the Open Run/Analysis dialog box, a read-only image of its chromatogram appears in the pane at the bottom of the dialog box. Clicking the Analysis tab displays saved analysis data, if they are available. Clicking the Notes tab displays the text entered when the run was saved. To view saved run data in the Evaluation window 1.
4 | Performing a Manual Run 112 | NGC Chromatography Systems and ChromLab Software
5 Method Editor This chapter introduces ChromLab™ software Method Editor concepts and functionality and prepares you to create a method, the subject of Chapter 6. A method consists of a list of standard or custom phases that are executed in a sequence. Each phase consists of a list of method steps. All of these steps and phases are associated with elements of the fluidic scheme. When the fluidic scheme changes, Method Editor settings change to match it. The Method Editor automates chromatography runs.
5 | Method Editor Before You Begin Before you work with Method Editor features, it is important to set as the default the fluidic scheme that matches the plumbing and configuration of your NGC™ system. When you run a method, ChromLab expects the system to be plumbed exactly like the fluidic scheme. The fluidic scheme selected also determines the settings that appear in the Phase Parameters tab. See Fluidic Scheme Configurations on page 51 for more information.
Accessing Method Editor Features Opening a Method When you click Open Method in the Method Editor pane, the Open Method dialog box appears. This dialog box lists saved projects in the left pane. Saved methods in the selected project are listed in the upper-right pane. The lower-right pane displays information about the selected method in three tabs: The Gradient tab displays the gradient graph for the selected method. The Runs tab displays a list of runs in the selected method.
5 | Method Editor Method Editor Window The Method Editor enables you to create, open, import, export, review, edit, and run a method or a redefined method template. The Method Editor window presents several panes, including the fluidic scheme and a gradient graph. The selected phase is highlighted in both the method outline and the gradient graph. Note: Available phases and options depend on system configuration. See Fluidic Scheme Configurations in Chapter 3.
Method Editor Window LEGEND 6 The Method Steps tab displays all method steps in a table. Double-click a step to open its dialog box and modify settings. The Step Library appears in the right pane (not shown). 7 The fluidic scheme, for visual reference only, displays the current instrument flow path. 8 The gradient graph displays the programmed gradient and break points for flow segments with a gradient step. Move points and edit gradient duration and slope by dragging black dots to new locations.
5 | Method Editor Export Method — exports a saved regular or scouting method to the desktop or to a directory and file name you choose and appends the file extension .ngcMethod. Exported methods can be imported into another NGC system. Export Method with Runs — exports a saved method with runs to the desktop or to a directory and file name you choose and appends the file extension .ngcMethodRuns. Exported methods and their runs can be imported into another NGC system.
Method Editor Window View Menu Commands Phase Library – displays the Phase Library pane. This is the default. Clearing this command hides the Phase Library pane from the Method Editor display. Method Outline – displays the Method Outline pane. This is the default. Clearing this command hides the Method Outline pane from the Method Editor display. Help Menu Commands Help — displays screen-level help topics and links to installed manuals.
5 | Method Editor Phase Library Pane The Phase Library pane contains both standard and custom phases you can use to create methods. Each phase consists of a series of programmed steps that represent a process in chromatography. You can add or remove steps from a phase to customize it for a specific application. You can save modified phases as custom phases in the Custom tab where they will be available to all ChromLab software users. Standard phases are further described in Standard Phases on page 138.
Phase Library Pane Table 4. Standard Phases, continued Phase Explanation Column Preparation/Activation Before column use, removes the storage solution and equilibrates the column. By repeating the phase multiple times, several preparation solutions can be used one after another. Column Performance Test Tests the efficiency of a packed column in terms of height equivalent to a theoretical plate (HETP) and the peak asymmetry factor (As).
5 | Method Editor Method Outline Pane The Method Outline pane lists phases in the order in which they run. Method Settings is always the first phase in a method. As you drag additional phases into the Method Outline pane, they appear in the order in which you added them. You can reorder phases by dragging them to a new location. You can remove a phase from a method by rightclicking it and choosing Delete in the menu that appears or by selecting the phase and pressing Delete on your keyboard.
Method Steps Pane In this pane you can define the parameters of each method phase. Parameters vary depending on the phase and the devices available on the system that is to participate in that method phase. Some global method parameters defined in the Method Settings phase can be altered for each phase. Such parameters include flow rate, buffer selection, pH, and fraction volume.
5 | Method Editor Viewing Details of a Step in the Step Table To view step details Right-click the step and choose Show Step Details on the menu that appears. Adding, Modifying, and Deleting Steps in the Step Table To add a step to the step table 1. Select a step in the step table. 2. Double-click a step in the Step Library pane. The step appears in the table immediately below the step you selected.
Method Settings Parameters Method Settings Parameters Method Settings are general parameters that affect the entire method. These settings include fluidic scheme, column selection, monitor settings, unit selection, pH valve, fraction collection, and buffer selection. Fluidic Scheme The current instrument configuration appears in the bottom-left corner of the Method Editor window. Parameters available in each phase are specific to the devices in the fluidic scheme that participate in the phase.
5 | Method Editor Column Selection Under Column Selection you select the column and the column parameters for the method. The column’s volume and pressure parameters are set automatically when you select a column type from the list of predefined columns. If you have one or more column switching valves in the fluidic scheme, you can choose whether to set the parameters for a single column or for multiple columns.
Method Settings Parameters You can remove a column from the list if it is no longer used, clearing the assigned port. You can then add a different column to the port. You can add a port with no assigned column to the method using the Change Valve step from the Step Library. When you save the method, a message appears prompting you to assign pressure limits to that port. You can use the Configure Ports dialog box to assign pressure limits without assigning a column to the port.
5 | Method Editor 3. Select a column position to configure from the dropdown list. 4. (Optional) Show the columns that are available for a specific technique from the dropdown list. 5. Select a column type. Do one of the following: Choose a column type from the dropdown list. The volume and pressure parameters are set automatically for that column type. Choose Custom from the dropdown list and set the volume and pressure parameters for the column at that port. 6. Click Add. 7.
Method Settings Parameters To add user-defined columns 1. In the Column Selection section, do one of the following: Select Single Column and click . Select Multiple Columns, click Configure Ports, and click Configure Columns dialog box. in the The Add User Defined Column dialog box appears. Tip: The pressure units are based on the pressure values set in File > Preferences.
5 | Method Editor 2. Provide the requisite information for the column. A red asterisk (*) indicates the field is required. Note: The Column Name field has a 45-character limit. The Column Volume, Medium Bed Height, and Hardware Diameter fields are connected. When you enter or change values for any two fields, the system automatically calculates the value for the third field. The Default Linear Flow Rate and Max Linear Flow Rate fields are read-only.
Method Settings Parameters To remove user-defined columns 1. In the Column Selection section, do one of the following: Select Single Column and click . Select Multiple Columns, click Configure Ports, and click Configure Columns dialog box. in the The Remove User Defined Column dialog box appears. 2. Select one or more columns to remove. 3. Click Remove. Detector Settings Under Detector Settings, set single UV or multi UV/Vis wavelengths that will be used to monitor the run.
5 | Method Editor pH Valve Enable pH monitoring — if a pH valve is present, this checkbox is selected by default. To bypass the pH valve, clear this box. Fraction Collection Device Type — displays the currently selected fraction collector device.
Method Settings Parameters BioFrac Settings — by default, fraction collection begins in the first tube of the first rack. The fraction size is the default for the rack type. BioFrac Collection Pattern — by default, Serpentine is selected. For microplates, fractions can also be collected by rows or columns. Outlet Valve Settings — by default, fraction collection begins from Outlet valve 1 (O1) Port 2.
5 | Method Editor Buffer Blending — buffer acid, buffer base, water, and salt are mixed together to form a buffer of a specified pH, buffer concentration, and salt concentration. A buffer system (recipe) is selected for use, after which the acid, base, water, and salt solutions are prepared and fed to the pumps through the buffer blending valve ports, Q1–Q4. Gradients are formed by the blending valve rather than the pumps.
Method Settings Parameters Select Buffer — opens the Buffer Recipes dialog box in which you can choose a recipe for the buffer and name the buffers. To select a buffer 1. In the Buffer Recipes dialog box under Recipe Selection, you can filter the list of recipes by selecting a titration type on the dropdown list. 2. On the Sort by dropdown list, choose Name or pH to view the list of recipe names in the order you prefer. 3. Choose a recipe on the Recipe Name dropdown list.
5 | Method Editor Available Range — available ranges for the recipe concentration and pH values vary depending on the recipe you choose. The ranges provided are recommendations. You can choose values above the upper end of the range, but doing so might require other adjustments to ensure accuracy. For most recipes you can change the buffer concentration by reducing the stock concentration by 1/2 to 1/8 on Conjugate Acid/Base Pair titration and up to 1/4 on Acid or Base titration.
Phase Controls and Parameters In gradient segments, lower numbers yield increasing gradients. Higher numbers yield negative gradients. Load/Inject Sample — controls sample loop loading, sample injection, and loop washing. Fraction Collection — turns on fraction collection for the phase, sets the fraction collection scheme, enables you to select the primary collector (BioFrac fraction collector or outlet valve), sets the start tube or port, and sets the fraction size.
5 | Method Editor Standard Phases Some standard phases use identical parameters. For example, the column wash phase and the elution phase display the same phase parameters. An example of each phase’s parameters is included in its description. Note: The images in this section display the parameters and settings for the NGC Discover Pro fluidic scheme. Your parameters and settings might vary depending on the fluidic scheme and method in use.
Phase Controls and Parameters Loading the Loop with a Sample Pump When a sample pump is used to load the loop, you can define the flow rate, the volume to load into the loop, and, subsequently, the volume of sample to be injected onto the column. You also have the option to interrupt loading the loop if air is detected.
5 | Method Editor You can change the port on the sample inlet valve from which to load sample (the system defaults to Port 1). You can also choose to prime the tubing with sample before application. If you select this checkbox, you can define the flow rate and sample volume with which to prime. Finally, you can choose to interrupt loading the loop if air is detected.
Phase Controls and Parameters Injecting Sample Directly with a Sample Pump When a sample pump is present in the fluidic scheme and the sample is injected directly onto the column, you have the option to interrupt the injection if air is detected. If you select this checkbox and air is detected in the line connected to the air sensor set to detect end of sample, the system stops the sample pump and the method continues to the next step.
5 | Method Editor You can change the port on the sample inlet valve from which to load sample (the system defaults to Port 1). You can also choose to prime the tubing with sample before application. If you select this checkbox, you can define the flow rate and sample volume with which to prime. Finally, you can choose to interrupt injection if air is detected.
Phase Controls and Parameters Injecting Sample with an Autosampler If your fluidic scheme includes an autosampler and you choose to use manually prepared buffer via inlet valves in Method Settings, you have the option to use the same inlets as defined in Method Settings or to select specific buffers from each port. Column Wash Parameters This phase washes the column of impurities and unbound sample.
5 | Method Editor Although it is typically used after the sample application or elution phase, this phase can be used on its own or at any point that column washing is wanted. Elution Parameters In this phase, the sample is eluted from the column using a gradient or isocratic flow at defined salt concentrations (%B) over a defined volume, column volume, or time. You can edit the parameters in the gradient segment or add more isocratic or gradient steps to this segment.
Phase Controls and Parameters System CIP (Clean in Place) — Storage Parameters Note: System CIP must be run as the only type of phase in the method. You can add several System CIP phases to the method if multiple cleaning solutions are required. Bio-Rad recommends that you save System CIP as a separate method.
5 | Method Editor NGC Scout Systems Buffer selection: Manually Prepared Buffer via Blending Valve Components to clean Sample loop Buffer blending valve pH flow cell Wash solution is delivered through Q1 for the sample loop and pH flow cell. Ports Q1 and Q4 are washed individually with 5 ml.
Phase Controls and Parameters NGC Discover and NGC Discover Pro Systems Note: To perform CIP for the buffer blending valve and buffer inlet valves you must create separate methods, which you can queue in the Run Scheduler.
5 | Method Editor Sample inlet valves are washed by using the sample pump to pull wash solution through ports 1–8 on S1 (if only one sample inlet valve is present) or ports 1–7 on S1 and ports 1–8 on S2 (if two sample inlet valves are present). Ports Q1 and Q4 are washed individually with 5 ml. Buffer inlet ports A1–7 and B1–7 are not washed.
Phase Controls and Parameters Wash solution is delivered through buffer inlet ports for the sample loop, pH flow cell, and column switching valve. Sample inlet valves are washed by using the sample pump to pull wash solution through ports 1–8 on S1 (if only one sample inlet valve is present) or ports 1–7 on S1 and ports 1–8 on S2 (if two sample inlet valves are present). System pumps each run at 0% B and 100% B from buffer inlet ports A1–7 and B1–7. Ports Q1, Q2, Q3, and Q4 are not washed.
5 | Method Editor Column switching valve Outlet valve Sample inlet valves Wash solution is delivered through Q1 for the sample loop, pH flow cell, and column switching valve. Sample inlet valves are washed by using the sample pump to pull wash solution through ports 1–8 on S1 (if only one sample inlet valve is present) or ports 1–7 on S1 and ports 1–8 on S2 (if two sample inlet valves are present). Ports Q1, Q2, Q3, and Q4 are washed individually with 5 ml.
Phase Controls and Parameters 6. Fill a syringe with distilled water, connect the syringe to the pH valve Cal In port, and inject the distilled water. 7. Click Close in the Calibration dialog box to leave calibration mode. 8. Switch the valve back to the default position.
5 | Method Editor Column Preparation/Activation Parameters This phase is used to prepare a column before use by removing the storage solution or to activate a column to bind the sample before applying it. If activation requires the column to incubate in the activation buffer, select Pause Until Resume and define the time required for the column to incubate.
Phase Controls and Parameters Column Performance Test Parameters This test combines the three basic phases necessary for a run (equilibration, sample application, and elution). After equilibrating the column, a non-adsorbing sample, such as acetone or salt, is injected via the sample loop and eluted under isocratic conditions. After the run, calculate the column efficiency in terms of height equivalent to a theoretical plate (HETP) and the peak asymmetry factor (As).
5 | Method Editor System Preparation Parameters This phase removes storage solution and fills the tubing and system inlet line with buffer solution before a run. The inlets, outlets, and column positions to be prepared are selected, and the system is filled with the appropriate buffer solution. Because each inlet port can be plumbed to a different buffer or sample, multiple buffers and samples can be used in each phase. Note: System Preparation must be run as the only type of phase in the method.
Phase Controls and Parameters Create New Phase Parameters This phase enables you to create a new phase by adding steps from the Step Library pane in the Method Steps tab. Phases you create can be saved in the custom phase library for reuse in other methods.
5 | Method Editor Step Library Pane Visible when the Method Steps tab is selected, the Step Library pane comprises a list of steps available for creating a phase. Steps determine individual events that occur on the system; for example, changes in valve positions. Double-clicking a step name adds the step to the current phase. You can add multiple steps of the same type to a single method.
Scout Parameters Pane Zero Baseline Hold Until Pause System Wash To get more information about method steps In the Method Editor window, select Help > Method Steps and then select a step on the dropdown list that appears. Scout Parameters Pane The Scout Parameters pane displays a table of all scouting runs associated with the scout method and their settings. This pane is read-only and cannot be modified.
5 | Method Editor 158 | NGC Chromatography Systems and ChromLab Software
6 Creating a Method In the NGC™ chromatography system, methods are used to encapsulate an entire process to be run. Methods consist of phases. A phase consists of a sequence of steps in a chromatography run and the properties associated with each step. ChromLab™ software includes standard methods as well as tools for creating, editing, and managing methods. You can edit the phases within a method and define the settings within each phase. Methods are created in the Method Editor window.
6 | Creating a Method Standard Method Templates The workflow for standard method templates consists of the following phases: Equilibration Sample application Column wash Elution Column wash Re-equilibration for the next run ChromLab includes the following eight standard method templates. Table 6. Standard Method Templates Method Template Explanation Affinity Affinity chromatography is the separation of biomolecules based on highly specific interactions.
Standard Method Templates Table 6. Standard Method Templates, continued Method Template Explanation Chromatofocusing Chromatofocusing chromatography uses a charged matrix to bind protein molecules. A pH gradient is used to elute the bound proteins, which elute when the pH is the same as the pI of the protein of interest (overall charge = 0). Special buffer systems are required to perform the pH gradient over a large range. Desalting Desalting is usually used for buffer exchange.
6 | Creating a Method Table 6. Standard Method Templates, continued Method Template Explanation Multicolumn Tandem Multicolumn tandem purification uses these templates when two different chromatography techniques are combined into one method. First, samples are injected, bound to a column and washed to remove any main contaminants. Next, the target fractions are eluted and either applied directly onto another column in tandem or stored temporarily in a sample loop or container.
Creating a Method from a Template Creating a Method from a Template Standard methods are supplied as templates. You must save a template as a method before you can use it. To select a method template and save it as a method 1. Do one of the following: In the Home window, click Open Method Template. In the Method Editor window, click Open Template on the toolbar. The Open Template dialog box appears. Standard method templates are organized in folders by technique.
6 | Creating a Method 2. In the left pane, click a technique folder and select a template in the list that appears in the upper-right pane. In the lower-right pane, the Gradient tab displays the gradient graph of the selected template. The Notes tab displays explanatory text that you can edit in the Method Settings phase. When available, the Overview tab displays a graphic representation of the purification steps.
Creating a Method from Standard Phases 6. Do one of the following: Select a project folder. In the Method Name box, type a name for the method, and then click Save. Click New SubProject or New Root Project and type a name for the new project in the Enter Project Name box that appears above the Projects pane. Click Save.
6 | Creating a Method Each element of the fluidic scheme is associated with settings in the Method Editor window. When the fluidic scheme changes, Method Editor settings change to match it. To create a method Do one of the following: In the Home window Method Editor pane, select New Method. If the Method Editor tab is already in view, click New in the Method Editor toolbar. The new method opens. Its Method Settings phase appears in the Phase Parameters pane.
Creating a Method from Standard Phases 2. Choose a fluidic scheme that matches your NGC instrument configuration and click Select. Note: General settings appear in the Phase Parameters pane when you select the Method Settings phase. General settings vary depending on the fluidic scheme selected. For more information, see Fluidic Scheme on page 125. To specify general settings 1.
6 | Creating a Method 5. Depending on the method, either Single Wave UV Detector Settings or Multi-Wave UV/Vis Detector Settings can be selected. Choose the appropriate UV absorbance wavelength values to monitor the run. Tip: The single-wavelength UV detector monitors UV absorbance one wavelength at a time. You can select either 255 nm or 280 nm as the default. The multi-wavelength UV/Vis detector monitors up to four wavelengths in the UV/Vis range of 190–800 nm. 168 | 6.
Creating a Method from Standard Phases Serpentine is selected by default. Fractions can also be collected by rows or columns for microplates. Possible choices appear for the rack you select. If you have outlet valves in the fluidic scheme, choose a starting valve and port and modify the fraction size. 8. Under Flow, specify the default flow rate for the method. You can change the default flow rate within individual phases if needed.
6 | Creating a Method about priming your system. 9. Select a pump head type. To set the control parameters see Control Flow Tab on page 72. 10. Under Buffer Selection, select buffers for each inlet line or select the buffer system and pH if you are using the buffer blending module. See also Buffer Selection on page 133. 11. Click Rename Ports to change the names Buffer A and Buffer B to more descriptive terms. Names you select here appear in the method report.
Renaming Phases 3. Repeat steps 1 and 2 until you have finished editing phase settings for the method. 4. Save the method to preserve your changes. Note: Changes you make in the phase parameters to the duration of flow steps and salt gradients are graphically depicted in the Gradient Graph pane. Conversely, changes you make to the gradient graph are reflected in the phase parameters. Renaming Phases You cannot rename phases in the Phase Library pane.
6 | Creating a Method Deleting Phases Note: You cannot delete the Method Settings phase. To delete a phase from a method 1. In the Method Outline pane, select the phase to delete. 2. Select Edit > Delete Phase. 3. To confirm deleting the selected phase, click Yes in the dialog box that appears. Opening a Method To open a method 1. Do one of the following: In the Home window, click Open Method. In the Method Editor window, click Open on the toolbar. The Open Method dialog box appears.
Running a Method and Collecting Fractions Running a Method and Collecting Fractions You can schedule a method to start immediately or add it to the run queue to run at a later time. You can start a run immediately only if there are no scheduled methods in the run queue. If there are methods in the queue, new methods are added to the end of the queue. Queued methods run sequentially. You can remove a scheduled method from the queue if it is not running, but you cannot reorder the queue.
6 | Creating a Method Collecting Fractions in Run Queues When you schedule queued methods, collection begins with the tube or port defined in the first method in the queue. Collecting Fractions Using the BioFrac Fraction Collector After the first method is completed, fraction collection skips a tube and continues with the next available tube (as if the run queue is set to Append mode).
Running a Method and Collecting Fractions To run a method 1. Open the method to run. 2. On the toolbar, click Start Run. The Schedule Run dialog box appears. 3. (Optional) Type a name for the run. The run name can consist of up to 85 characters. 4. To begin the run immediately, click Start Run. To add runs to the run queue 1. In the Method Editor click Start Run.
6 | Creating a Method 3. Repeat steps 1–2 to add as many methods to the queue as needed. 4. If the first run was not selected to start immediately, click the green start arrow on the first run in the Run Queue to start running the methods in the queue. Tip: To remove a run from the Run Queue, click . To run a method multiple times in a sequence 176 | 1. In the Schedule Run dialog box, select Multiple Runs. 2.
Saving a Method Saving a Method You can modify a method — whether or not you have run it — and save it under the same name. The modifications will not apply to previously saved runs of that method. Saved runs will display the parameters that were used by the method to perform that run before it was modified. To save a method Click Save on the toolbar. The method is saved in the current project. To save a modified method or to save a template as a method 1. Click Save As on the toolbar. 2.
6 | Creating a Method 2. Type a name for the new template and click Save. Renaming a Method You can rename an open method. While Save As retains the original method and saves the modified method under a new name, renaming a method saves the original method under a new name. To rename a method 1. Open the method to rename. 2. Select File > Rename. The Rename Method dialog box opens. 3. 178 | Type a new name for the method and click Rename.
Deleting a Method Deleting a Method You can delete an open method. If the method is associated with runs, you can choose to delete only the method or both the method and its associated runs. To delete a method 1. Open the method to delete. 2. Select File > Delete. 3. (Optional) If runs are associated with the method, choose one of the following options in the dialog box that appears: Delete Method Only — the system hides the method from view.
6 | Creating a Method Scouting on Parameters within a Method Scouting is a procedure used to systematically locate parameters that most impact peak resolution and to optimize on these parameters to achieve your protein purification goals (for example, high purity, yield, stability, or activity). Molecules differ from one another in their charge, hydrophobicity, solubility, reactivity, and substrate specificity, and in their intermolecular interactions.
Scouting on Parameters within a Method The Scouting Wizard The scouting wizard guides you through three pages: Choose a Scouting Parameter, Select Method Steps to Scout, and Generate Scout Sequence. This section explains how to use each page in detail. Page 1: Choose a Scouting Parameter On this page you select the parameter on which to scout. You can select only one parameter per method to scout. Flow Rate — optimize the pump flow rate for adsorption and elution steps.
6 | Creating a Method Column — test up to five column types for each column-switching valve in use. This parameter requires at least one column-switching valve in the fluidic scheme. Note: Pages 2 and 3 of the scouting wizard differ when scouting for columns and samples. See Using the Scouting Wizard to Scout Columns on page 185 or Using the Scouting Wizard to Scout Samples on page 187 for more information.
Scouting on Parameters within a Method Depending on the parameter you chose on Page 1, you can select individual steps to scout or you can select the checkbox to scout all steps in the method in which the properties for that parameter from the method settings are enabled. All steps that you select are highlighted.
6 | Creating a Method checkbox is also cleared. The other steps remain selected and will be included in the scout. Page 3: Generate Scout Sequence On this page you set the number of runs to be performed, name each run, and set the parameters for the scouted steps. Number of runs — sets the number of runs to be performed as part of the scout experiment. Starting Value — sets the starting value for the parameter chosen to be scouted. The default is the starting value in the base method.
Scouting on Parameters within a Method Run Name — the scout run name. The autogenerated name is based on the parameter you chose on Page 1 of the scouting wizard and the scout starting value and increment value. This field is editable. Tip: To edit the run name column easily, create a list in Microsoft Word or Excel. Copy and paste the list into the column to replace the autogenerated names.
6 | Creating a Method On this page you can select up to five columns for each column-switching valve to scout. The column dropdown list comprises all columns in the column library, including user-defined columns (see the section To add user-defined columns on page 129 for information about user-defined columns). The wizard displays the values for column volume, maximum precolumn pressure, and maximum deltacolumn pressure for each selected column.
Scouting on Parameters within a Method Port — the column’s position on the column switching valve module. This field is not editable. Include in Scout Sequence — determines whether the run is included in the scout method. By default, all runs are included. This field is editable. Using the Scouting Wizard to Scout Samples This section explains how to set up pages 2 and 3 of the scouting wizard when scouting samples. Note: This section applies only if you chose to scout samples on Page 1.
6 | Creating a Method include a preinjection sample pump wash, the method is updated and you proceed to Page 3. If you click No in the message box (if, for example, you are using the same sample), the method is not updated and you proceed to Page 3. Page 3: On this page you can name each run and include or exclude a sample in the scout sequence. The scout sequence table comprises the following columns: Run # — the scout run number. This field is not editable. Run Name — the scout run name.
Scouting on Parameters within a Method Include in Scout Sequence — determines whether the run is included in the scout method. By default, all runs are included. This field is editable. Creating a Scouting Method Note: Before starting the scouting wizard, ensure that the method settings and protocol are correct. After creating a scouting method series, any changes will require you to run the wizard again. To create a scouting method 1.
6 | Creating a Method Special Considerations Rules for generating scout sequences can vary depending on the parameter chosen to scout. This section provides a list of considerations to take into account when generating a scout sequence. Generating %B Scout Sequences You can scout on two or more steps with the same starting %B values. If you select two or more steps with different starting values, you must clear the steps with the different values.
Scouting on Parameters within a Method Generating Column Scout Sequences This option is available only when the fluidic scheme includes the column switching valve. If you chose Custom as the column type and changed the default values for column volume, maximum precolumn pressure, and/or maximum deltacolumn pressure, your custom settings must be within the system limits defined by the method settings. If your custom values are outside of this range, a red box appears around the number.
6 | Creating a Method Viewing a Summary of the Scouting Method When you complete the scouting wizard, the Method Editor table displays a summary of the scouting parameters in a read-only table in a new pane, Scout Parameters. See Scout Parameters Pane on page 157 for more information. Tip: When you open the scouting wizard in a saved scouting method, the values for that scouting method persist in the wizard as well as in the method.
Scouting on Parameters within a Method When you collect fractions during scouting, the fractions get appended to the fractions collected in the previous run. The collection skips a tube between each scout run and goes to the next available tube. Tip: See Running a Method and Collecting Fractions on page 173 for more information about running methods. To run a scout method 1. Open the method to run. 2. On the toolbar, click Start Run.
6 | Creating a Method 4. Do one of the following: Click Start Run to begin the run immediately. Click Schedule Run to put the methods in the run queue and run them later. Tip: To remove a run from the run queue, click 194 | NGC Chromatography Systems and ChromLab Software .
7 Evaluating Results In the Evaluation window, you can process and analyze chromatography data. The results can be viewed in a variety of ways. A single run appears when you open a run. Multiple runs appear in separate tabs when you open multiple runs individually. Multiple runs can also be compared in a single tab view. The runs in this view can be either stacked or overlaid for comparison. There are two types of results files: runs and analyses.
7 | Evaluating Results Managing Analysis Projects You can open a run or analysis in the Open Run/Analysis dialog box and organize runs and analyses by creating projects and subprojects. You can also create a root project in which to store your projects and subprojects. Saved projects and subprojects are listed in the left pane. Runs in the selected project or subproject are listed in the right pane with start and end times and method type.
Managing Analysis Projects To group the runs list by method Right-click the runs list and choose Group by Method in the menu that appears. To display run data for a single run Select a run and click Open Run. The Evaluation window displays the run data. To display data for multiple runs 1. Hold down Ctrl or Shift and select multiple runs from the list in the right pane. The Open Runs button becomes a dropdown list. 2.
7 | Evaluating Results Evaluation Window In the Evaluation window you can display run data for a single run or multiple runs. Multiple runs can be displayed in separate tabs or in a single Analysis tab as a trace comparison. Data appear as individual traces in the chromatogram. Pausing the pointer over a trace in a chromatogram displays a tooltip with coordinate information. The trace table that appears below the chromatogram documents the details of each trace.
Evaluation Window File Menu Commands Open Run/Analysis — opens the Open Run/Analysis dialog box in which you can select runs to display in the Evaluation window. Add Run — opens the Add Runs dialog box in which you can select additional runs to display in the same tab for multiple runs trace comparison. Remove Run from Analysis — opens a dialog box in which you can choose runs to remove from a multiple runs trace comparison. Show Method — opens the method for the displayed run in the Method Editor window.
7 | Evaluating Results Import Unicorn Data — opens a dialog box in which you can import a data file into the NGC database. See Importing Unicorn Data Files on page 261 for more information. Import DuoFlow Data — opens a dialog box in which you can import a data file into the NGC database. See Importing BioLogic DuoFlow Data Files on page 263 for more information. Rename Run/Analysis — opens a dialog box in which you can rename the displayed run or analysis.
Evaluation Window View Menu Commands Overlay View — displays multiple runs in a single chromatogram window. Stack View — displays multiple runs in a stack, one above the other, sorted alphabetically by name. Show Pre-Injection — displays data collected before the injection point. Show Baseline — following peak integration, shows the baseline curves in the chromatogram. Show Peak Area —following peak integration, shows the peak areas in the chromatogram.
7 | Evaluating Results Delete Peak List — deletes previously calculated peaks. All peaks are removed from traces you selected in the traces list in the peak integration pane. Analyze Column Performance — following a column performance test run, detects the largest peak within the column performance phase and opens the Column Performance pane. Help Menu Commands Help — displays screen-level help topics and links to installed manuals.
Customizing the Chromatogram Customizing the Chromatogram You can change the color of the traces, show or hide selected traces, control the range of the y-scale for each trace, and annotate the chromatogram at specific points on the x-scale. ChromLab saves trace display and run view settings and uses them when displaying subsequent runs. Displaying Traces The available traces in each configuration appear in the following order in the System Control and Evaluation windows chromatogram legends.
7 | Evaluating Results Selecting the Active Trace When a trace is selected, it appears bolded, the row corresponding to the trace is selected in the trace table, and the primary y-axis (on the left of the chromatogram) changes to the units and scale of the trace. For example, when UV is selected, the units and scale of the y-axis display as mAU. Note: The secondary y-axis (on the right side of the chromatogram) does not change when you select the active trace.
Customizing the Chromatogram Showing or Hiding a Trace To show or hide a trace In the trace table, select or clear the Show checkbox in the trace row. Changing the Axes The chromatogram has two y-axes. The left axis is the primary axis. Its default trace is UV absorbance. The default trace of the right axis is Conductivity. You can change the units of either y-axis by clicking its label, which is a toggle, to show the next trace’s scale and units.
7 | Evaluating Results To rescale UV units individually for multiple or overlaid traces Right-click a trace in the trace table or chromatogram and clear Lock UV scales in the menu that appears. All traces are unlocked and can be rescaled individually. To lock UV units for multiple or overlaid traces Right-click a trace in the trace table or chromatogram and select Lock UV scales in the menu that appears.
Customizing the Chromatogram To revert to the default trace color Right-click the trace color in the table and choose Restore Default Color on the menu that appears. Zooming In and Out To zoom in on a section of the chromatogram While clicking in the chromatogram, drag the pointer to mark the zoom region of interest.
7 | Evaluating Results Obtaining Statistics of a Selected Region on the Chromatogram For each trace, you can view specific statistics for a selected region on the chromatogram. Selecting a region on the chromatogram creates the Region Statistics table, which displays the following data: Trace name — the type of trace. Only traces selected in the Runs/Traces table appear in the Region Statistics table. Units — the trace’s units.
Customizing the Chromatogram To select a region on the chromatogram Right-click the start point in the chromatogram and drag the pointer to the end point. A grey box appears over the selected area and the Region Statistics table opens. To resize the selected region Do one of the following: On the chromatogram, drag an edge of the grey box to the new point. In the Region Statistics dialog box, type new values in the Left X or Right X fields located above the table.
7 | Evaluating Results To clear the selected region on the chromatogram Close the Region Statistics table. Annotating the Chromatogram In the Evaluation window, you can add notes to the chromatogram to associate observations with data points. The annotation dialog box contains three fields: Location (in time, volume, or CV), Title, and Description. When an annotation is saved, the title appears at the specified location on the x-axis. The description appears in the run report. To add annotations 1.
Customizing the Chromatogram To edit an annotation 1. Double-click the annotation on the chromatogram to open its dialog box. 2. Edit the annotation and click Save to save the changes. To delete an annotation Double-click the annotation to open its dialog box and click Delete. Copying the Chromatogram A copied chromatogram image can be pasted into a document or presentation file or saved in a variety of image formats optimized for either screen or print display.
7 | Evaluating Results Customizing the Trace Table Display The details of each trace in a run appear in a row in the trace table, one row for each trace. By default, trace information is grouped by run. Use the table to select the visible traces, to change their colors, and to change the axes’ scale. You can customize the table display further by changing the table grouping, sorting the columns, and changing the column order. You can also copy the table.
Customizing the Trace Table Display Sorting Table Columns When the trace table is not grouped, traces are sorted in default order. To sort table columns Do one of the following: Select a column, right-click the column heading, and then select a Sort option on the menu that appears. Click a column heading to toggle between ascending and descending table data order. Tip: An up or down arrow near the column title indicates that the table was sorted using this column.
7 | Evaluating Results Copying a Table You can copy a table to the clipboard and then paste it into a spreadsheet or another type of application. The copied table appears in the application with the following adjustments: The color value is copied in hexadecimal format. The show/hide state is copied as a Boolean value. To copy the table to the clipboard Right-click the trace table and select Copy Table on the menu that appears. The table is copied to the clipboard.
Customizing the Trace Table Display Manually Setting the Injection Point (x Alignment) You can manually set the injection point for any run, including a run displayed in a trace comparison. The injection point is saved with the run (the x = 0 point). A change to the injection point in one view affects other views that include the run. To set the injection point for a run manually 1. Select Analysis > Set Injection Point. 2. In the Set Injection Point dialog box, type the new injection point. 3.
7 | Evaluating Results Managing Runs You can rename or delete a single run displayed in the Evaluation window. You can also choose how to display multiple runs. Note: You cannot delete a run that is part of another analysis. When you make the following changes in single run view, they are saved automatically: trace colors, y-scale range, x-axis units, and show/hide state.
Managing Runs Viewing Multiple Runs in Separate Tabs ChromLab software can display multiple runs in the Evaluation window. Each run opens in a separate tab so you can view each one individually. When you select a run that is already open, the system highlights the tab for that run. To open multiple runs in separate tabs 1. 2. Do one of the following: Click Open/Run Analysis in the Home window Evaluation pane. If the Evaluation window is already open, select File > Open Run/Analysis.
7 | Evaluating Results Comparing Traces ChromLab can display multiple runs selected in the Open Run/Analysis dialog box in a single tab view. This view enables you to compare the traces from different runs. Runs displayed in the tab can appear either stacked for individual display or overlaid for comparison. To open multiple runs in one tab 1. In the Home window, click Open Run/Analysis in the Evaluation pane. 2.
Comparing Traces Viewing Multiple Runs in One Tab In this view, a single table displays information for all traces in all the displayed runs. You can select a trace, zoom in or out, and change trace colors. You can also show or hide specific traces and spread the different runs across the y-scale, as described in Offsetting Traces — Overlay Mode on page 223. Stacked View Each run appears in a separate chromatogram. On the toolbar, the Charts in View box indicates the number of runs in the view.
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Comparing Traces Overlay View All visible traces from all selected runs appear in a single chromatogram. To switch between stacked and overlay views Click Stack or Overlay on the Run tab toolbar or on the View menu.
7 | Evaluating Results Customizing the Trace Comparison Table Multiple runs in a single tab can be grouped by run or by type. Multiple runs can also be ungrouped. In the Evaluation window, the default view shows multiple runs with traces grouped by run. Runs are sorted alphabetically. When the trace table is grouped by type, the trace types are sorted in the default order, as described in Displaying Traces on page 203. Expanding or Collapsing Groups When groups are expanded, all their data are visible.
Comparing Traces Offsetting Traces — Overlay Mode When two or more traces from different runs are too close together to distinguish, Offset can be used to shift traces so you can view them separately. Traces from the first run remain in their original locations. Traces from other runs are shifted. If more than two runs are involved, each additional run has its own offset. The last run in the list is shifted the most. Note: Offsetting the y-scale of a trace does not change trace values.
7 | Evaluating Results Managing Analyses Removing Runs from a Trace Comparison When you view multiple runs, you can remove one or more runs from the analysis. You cannot remove all runs from the view. To remove a run from a multiple run analysis 1. Do one of the following: On the Run tab toolbar, click Remove Run. Select File > Remove Run from Analysis. The Remove Run from Analysis dialog box opens with the multiple runs listed and the current run selected. 2.
Managing Analyses To save an analysis 1. Do one of the following: Click Save Analysis on the Run tab toolbar. Select File > Save Analysis. 2. Type a name for the analysis in the Save New Analysis dialog box. 3. Click Save.
7 | Evaluating Results Deleting an Analysis To delete an analysis 1. With the analysis displayed, select File > Delete Analysis. A dialog box appears, asking you to verify that you want to delete the analysis. 2. Click Yes to delete the analysis. Reports ChromLab supports three kinds of reports, including run and analysis reports. For more information, see Chapter 9, Reports.
Peak Integration Peak Integration ChromLab software uses an algorithm to find peaks in UV traces and calculate the necessary data for analysis, such as retention volume and peak area. The results appear in the Peaks table at the bottom of the Evaluation window and in the chromatogram. You can perform peak integration on a single run or on multiple runs in a single view.
7 | Evaluating Results Automatic Peak Integration Settings In the Peak Integration pane, the Auto Integration tab displays settings that affect baseline calculation and peak detection. Changing these settings enables you to optimize the way peaks are detected and recognized, specific to your data. Note: By default most tab sections are collapsed. Expanding the sections displays the options shown in the following illustration. Traces A list of shown UV traces grouped by run.
Peak Integration Peak Parameters You can specify initial values for two parameters that participate in the peak detection process: slope and filter sensitivity. Slope — slope defines the threshold for peak slope. The bigger the slope, the steeper the peaks that are filtered out. Slope values range from 1–100. Sensitivity — sensitivity determines the strength of the filter used for smoothing the curve before finding peaks.
7 | Evaluating Results Integrate and Delete Integrate — detects peaks, using current parameter values, for the selected traces and updates the Peaks table with detected values. Delete — deletes previously calculated peaks. All peaks are removed only from traces selected in the traces list. Starting Peak Integration When you select Peak Integration, a single run becomes an analysis, and the system prompts you for an analysis name when you first save it.
Peak Integration Displaying Peak Results — the Chromatogram In the chromatogram, a peak’s start, end, and apex are indicated by small vertical lines at the relevant x points. The color of each line matches the corresponding trace. The retention time, volume, or column volume appears near the line of the peak apex. Tip: The example screen displays the retention time, but you can display the column volume instead by clicking the Time title near the bottom of the chromatogram.
7 | Evaluating Results The calculated baseline appears on the chromatogram when peaks are detected. The baseline is drawn according to the selected baseline calculation in the same color as the associated trace. Pausing the pointer on a baseline displays a tooltip that provides details about the baseline and the trace. Changing Peak View Options You can choose to hide or show the baseline, peak area, or peak labels, or all of these options.
Peak Integration To hide or show peak view options Right-click in the chromatogram, choose View, and select the option or options you want in the dropdown menu. The same options also appear on the View menu. Displaying Peak Results — the Peaks Table Each peak is described in a Peaks table row. The following fields are displayed for each peak: Peak number — sequential. Run name Trace type — UV traces display wavelength value in nanometers, for example (280 nm).
7 | Evaluating Results Peak asymmetry — the asymmetry factor, defined as the distance from the center line of the peak to the back slope divided by the distance from the center line of the peak to the front slope. All measurements are made at 10% of the maximum peak height. Note: The asymmetry factor of a peak is usually similar to the tailing factor of the same peak, but the two values cannot be directly converted. Fractions — all fractions that reside between start and end.
Peak Integration Molecular mass (kDa) — the molecular mass of the protein. By default this field is empty. Molecular mass values that you enter in the Peaks table are automatically populated to the Fractions table for relevant fractions. Molecular mass values that you enter in the Fractions table are not automatically populated to the Peaks table. Molarity (M) — the calculated molar concentration of the protein for the specified peak.
7 | Evaluating Results To display a hidden column 1. Right-click a column heading and select Show Column Chooser on the menu that appears. 2. Drag the column heading you want to show from the Column Chooser to the location in the table where the column should appear. The column heading and data reappear in the table. Copying the Peaks Table To copy the table Right-click the table and select Copy Table on the menu that appears.
Peak Integration To expand or collapse groups of peaks Right-click the table and select Expand All Groups or Collapse All Groups on the menu that appears. Clearing Extinction Coefficient Values in the Peaks Table Note: Clearing a coefficient in the Peaks table also clears the values in the Extinction Coefficient and Concentration columns in the Fractions table for the fractions that contain that peak. To clear the extinction coefficient for a specific peak Select the value and click Delete.
7 | Evaluating Results Adjusting Peaks Manually You can manually adjust the peaks found in automatic peak integration. You can also add a peak to a chromatogram location in which no peak was detected. When you select the Manual Integration tab in the Peak Integration pane, grid lines appear in the chromatogram. The Manual Integration tab displays a list of runs in the current analysis and their integrated UV traces. You can select and adjust one peak at a time.
Peak Integration You can adjust peaks by selecting commands on the menu that appears when you right-click in the chromatogram or Peaks table. To add a peak Right-click a chromatogram location that does not contain a detected peak and then click Add Peak on the menu that appears. A new peak is added to the trace with a default width that you can later adjust. The system recalculates and updates Peaks table statistics. To change the start and end points of a peak in a chromatogram 1.
7 | Evaluating Results To split a peak In the chromatogram or in the Peaks table, right-click the peak to split, and then click Split Peak on the menu that appears. The peak splits into two peaks. The split X point appears where the pointer was located when you selected Split Peak. The Peaks table is updated to contain two peaks instead of one. Table statistics are also recalculated and updated.
Evaluating Fractions Evaluating Fractions When fraction collection is enabled the details of each fraction in the run appear in a row in the fraction table below the chromatogram, one row for each collected fraction. The location of the fraction appears in the rack image to the left of the fraction table. Use the fraction table, rack display, and the chromatogram to find fractions of interest. If multiple racks were used, use the rack selection area to choose the racks of interest.
7 | Evaluating Results Orange — for fractions collected via an outlet valve. The outlet valve and port number appear at the top of each block. A heat map, indicating the relative amounts or purity within each fraction tube, appears after peak integration is performed. Items you select in the chromatogram are highlighted in the rack or outlet valve display and fractions table and vice versa.
Evaluating Fractions Displaying Fraction Collection Results — the Fractions Table Like the Traces table, the Fractions table is available when the run is opened in the Evaluation window. The Fractions table is available only when a single run is open in the Evaluation window. It is not displayed for multiple run analyses or for trace comparisons. The following fields are displayed for each fraction collected: # — sequential row number. Rack/Tube — the rack and tube number of the fraction.
7 | Evaluating Results Extinction coefficient ((mg/ml)-1 cm-1) — the extinction coefficient of the protein, used to calculate the protein concentration. ChromLab automatically populates this field when the extinction coefficient is entered or changed in the Peaks table. Note: Changing a single coefficient in the Peaks table affects only coefficients for the fractions that contain that peak. Molecular mass (kDa) — the molecular mass of the protein. By default this field is empty.
Evaluating Fractions Calculating Protein Concentration for Fractions You can calculate and view the protein concentration for individual and pooled fractions after you perform peak integration. Peak integration is performed using default settings. If necessary, you can adjust the baseline by changing the Best Fit or Offset parameters and reintegrating. The baseline is used to calculate the area under the curve of the UV trace in each fraction. See Baseline Parameters on page 228 for more information.
7 | Evaluating Results Viewing Fraction Details To view details of a single fraction Do one of the following: Select the location of the tube or outlet valve port in the chromatogram. Select the row in the table that corresponds to the fraction of interest. Select the fraction of interest in the fraction display to the left of the table. Tip: If the fraction of interest is in another container, change the display by clicking the fraction’s number in the rack selection panel.
Evaluating Fractions Tube location — the location of the group of pooled tubes in the rack. Start —the beginning of the collection of the first fraction in the pool. End — the end of the collection of the last fraction in the pool. Collected volume (ml) — the total fraction volume collected in all tubes in the pool. Area (ml*mAU) — the sum of all areas in the pool. This column appears after peak integration is performed.
7 | Evaluating Results Viewing Fraction Collection Results — the Heat Map A configuration of the fraction collector (the rack display or the outlet valve) appears to the left of the Fractions table. The image displays the configuration of the selected fraction collector when the run was executed. After peak integration, a heat map of the different fraction properties appears. If a multi-wavelength UV detector was used for the run, you can select a trace for the heat map (the default trace is 280 nm).
Evaluating Fractions The color scale is based on the column chosen and is relative among all available fractions of the run. A darker color represents a greater value and tubes or valve positions that do not contain fractions appear dimmed, for example Viewing Heat Map Details To change the displayed trace Select another UV trace from the dropdown list (available only if the multi-wavelength UV detector was used in the method).
7 | Evaluating Results To hide the rack display Click the collapse arrow to the right of the fraction display. To select a rack or plate to view 250 | Click the relevant display icon in the rack selection panel. Use the up and down arrows next to the panel to scroll to the relevant rack, plate, or outlet valve if necessary.
Column Performance Analysis Column Performance Analysis Method runs that include the Column Performance Test phase can be analyzed for column performance. ChromLab software analyzes the performance of the column in use and provides quality statistics including the number of theoretical plates (N), the number of plates per meter (N/L), height equivalent to a theoretical plate (HETP), and the reduced plate height (rHETP).
7 | Evaluating Results Traces You can choose to analyze the column’s performance based on either a single UV trace or the conductivity trace. Calculate You can calculate and display the following measurements: Number of plates (N) — displays the number of theoretical plates, an indication of column efficiency. Theoretical plate numbers are an indirect measure of peak width for a peak at a specific time retention. Columns with high plate numbers are considered to be more efficient.
Column Performance Analysis Reduced HETP (rHETP) — the reduced plate height, a value used to compare the efficiency of multiple columns packed with different particle sizes. Columns with rHETP 22 are considered well packed and more efficient. This measurement requires the medium bed height and the average particle diameter (in m) for the column in use.
7 | Evaluating Results 4. In the section, enter the values for bed height and particle diameter to calculate N/L, HETP, or rHETP. Tip: For predefined columns, you can change the prepopulated values for bed height and particle diameter in this section without changing the values in the Method Editor. 5. Click Apply to calculate the selected column performance statistics and display them in the table. Tip: You can include the column performance statistics in the Run report.
Column Performance Analysis Column Performance — Manual Adjustment In the Manual Adjustment tab of the Column Settings pane you can manually adjust the start and end points of the peak within the column performance phase range. Adjustments to the start and end points appear in the relevant column of the Column Performance table in the bottom pane. You can also adjust the start and end points on the Column Performance table. These changes appear at the appropriate points on the chromatogram.
7 | Evaluating Results Displaying Column Performance Results — the Column Performance Table The results of the column performance analysis are detailed in the Column Performance table. The table displays the following fields for the column performance peak: N — displays the calculated number of theoretical plates for the column. N/L (cm-1) — displays the calculated number of theoretical plates per meter for the column.
Column Performance Analysis Width at half height (ml) — displays the width (in ml) of the peak at 50% of the maximum peak height. Peak asymmetry — displays the asymmetry factor, defined as the distance from the center line of the peak to the back slope divided by the distance from the center line of the peak to the front slope. All measurements are made at 10% of the maximum peak height. Peak type — displays the integration type for a peak.
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8 Importing and Exporting Data Data files can be imported in the Home, Method Editor, or Evaluation window when you choose Import on the File menu. You can import the following data files: NGC™ data files, including regular and scouting methods, methods with runs, and run files exported from ChromLab™ software running on another computer Unicorn (v. 5 and v.
8 | Importing and Exporting Data To import a method or run 1. In the Home, Method Editor, or Evaluation window, select File > Import and choose NGC File. The Import NGC File dialog box appears. 2. Click Select. In the Select Project dialog box, do one of the following: Choose a destination project for the method or run and click Select Project. Rename a project using the appropriate project button at the top of the dialog box and select the project to rename. Click Select Project.
Importing Unicorn Data Files 6. (Optional) Select Open on Import to open in the Evaluation window upon import. Note: This option is not available if you selected multiple methods or runs to import. 7. Click Import. During the import a status dialog box appears. When all methods and runs have successfully imported, the status displays Completed. 8. Click OK to close the dialog box. The files are imported into the project you selected.
8 | Importing and Exporting Data 3. Click Select Project. 4. In the Import Unicorn Data dialog box, click Browse to display the Select Unicorn File dialog box. 5. Select a run file and click Open. The name of the run file and the project you selected appear in the Import Unicorn Data dialog box. 6. (Optional) In the Run Name box, type another name for the run you want to import. 7. (Optional) To have the run file open in the Evaluation window upon import, select Open on Import. 8. Click Import.
Importing BioLogic DuoFlow Data Files 9. If it is not already open, select the run to display its chromatogram and trace table in the Evaluation window. Importing BioLogic DuoFlow Data Files BioLogic DuoFlow data are imported in a BIODB.txt file. Imported BioLogic DuoFlow data are added to the NGC database. To import a BioLogic DuoFlow data file 1. In the Home or Evaluation window, select File > Import > DuoFlow Data. 2.
8 | Importing and Exporting Data 4. Click Import. ChromLab imports all runs in the data file. To open an imported run 1. Select File > Open Run/Analysis and select the project into which you imported the data file. 2. Select a run in the Open Run/Analysis dialog box and click Open Run. The run you selected appears in the Evaluation window. Exporting Data You can export run data with the method used to generate it or export only the run or method itself.
Exporting Data Exporting Data as an NGC File You can export method and run information from the Home, Method Editor, or Evaluation windows. In the Home window, you can export single or multiple runs, methods alone, and methods with their associated runs at the same time. You do not need to open the runs or methods to export them. In the Method Editor window, you open the method and export all of its runs with it.
8 | Importing and Exporting Data To export a method with all of its runs 1. With the method displayed in the Method Editor window, select File > Export Method with Runs. The Save As dialog box appears, prepopulated with the method and file type. 2. Browse to the folder where you want to save the exported method and its runs and click Save. Once the method and its runs have been imported, click OK. To export only the method 1.
Exporting Data To export multiple methods and associated runs 1. In the Home window, select File > Export > Method/Method Runs. The Export Method/Method Runs dialog box appears. 2. Select the project folder that contains the methods that you want to export in the left pane. 3. Hold down Ctrl or Shift and select multiple methods from the list in the right pane. 4. Click Browse to specify a target folder into which to save the method data. 5.
8 | Importing and Exporting Data 7. Click OK to close the dialog box. To export multiple runs 1. In the Home window, select File > Export > Runs. The Export Runs dialog box appears. 268 | 2. Select the project folder that contains the runs that you want to export in the left pane. 3. Hold down Ctrl or Shift and select multiple runs from the list in the right pane. 4. Click Browse to specify a target folder into which to save the run data. 5.
Exporting Data Exporting Run Data as a .csv File Exporting run data to a .csv file enables you to employ various standard file formats, such as spreadsheet formats, so you can include the data in reports and custom process it. When you export run data, the following rules apply: Only traces marked Show in the Run table are exported. By default, all data points (y values) are exported, even if they are not currently in scale. The x-axis scale (units) is determined by the currently displayed axis.
8 | Importing and Exporting Data 2. Clear the checkbox for any run data you do not want to export. 3. To change the default settings, click Advanced to expose additional settings. 4. Under Range, select Full Scale to export the entire range of data or select Current view to export only the visible range. 5. Under Sampling, select Sample every n data points to reduce the number of data points by a factor you enter. This reduces the amount of data exported. 6.
Exporting Data Exporting Diagnostic Logs In ChromLab, you can export all critical information that Bio-Rad Technical Support requires to diagnose issues. The information includes system information, current log files, the associated runs and methods for a specified date range when the issue was noted, and a description of the issue or issues. Note: Lamp information is not included in the exported diagnostic logs.
8 | Importing and Exporting Data 272 | 3. (Optional) Clear the Include Runs and Methods in the export file checkbox to exclude all method and run data from the export file. 4. (Optional) Click Review to display the Include Runs and Methods dialog box. This dialog box lists the runs with methods and separately lists the methods performed during the selected period. For example: 5. In the Include Runs and Methods dialog box you can Change the date range.
Exporting Data 6. Do one of the following: Click OK to accept any changes and return to the Export Diagnostic Logs dialog box. Click Cancel to revert any changes and return to the Export Diagnostic Logs checkbox. In the Export Diagnostic Logs dialog box, click Export. By default, the ChromLabLog.zip file is saved to the ChromLab desktop. If necessary, you can navigate to another folder in which to save the file. 7. Click Save to save the ChromLabLog.zip file.
8 | Importing and Exporting Data 274 | NGC Chromatography Systems and ChromLab Software
9 Reports The three report formats available in ChromLab™ software make it easy to publish method, run, and analysis data in attractive, detailed reports. The single run report includes all information about a single run without peak integration analyses. The method report includes all information about the method. The analysis report includes all information in the single run report as well as a list of included runs and information about trace comparison and peak detected runs.
9 | Reports Producing a Report After you generate the data you want to include, you can easily create a formatted report with ChromLab. You can refine the report with Report Viewer dialog box commands and options. You can save or print the report, specify page size, and choose from several viewing options. You can also change the view by moving the scroll bar in the bottom-right corner of the dialog box.
Producing a Report Method Reports Note: The Scout column and Scouting Parameters table appear in the Method report only if the method is a scouting method. To generate a Method report With a run, method, or analysis displayed, select File > Method Report. ChromLab automatically generates a formatted report of all information in the method and displays the report in the Report Viewer dialog box. Note: You cannot generate a method report if multiple runs appear in an analysis.
9 | Reports Analysis Reports Note: You must first perform peak integration analysis in order to generate an Analysis report. To generate an Analysis report 1. In the Evaluation window, select File > Analysis Report. The Analysis Report Options dialog box displays a list of the columns that appear in the displayed Peaks table. 2. Select the checkboxes of the columns to include in the Analysis report. Note: You are limited to 12 columns. 3. Click OK.
Saving a Report Saving a Report To save a report With the report displayed in the Report Viewer dialog box, click Save and select a file format from the dropdown list that appears. The Export Settings dialog box appears. You can expand this dialog box to display detailed settings. Available settings depend on the print format you selected.
9 | Reports 280 | NGC Chromatography Systems and ChromLab Software
A Database Management ChromLab™ software stores all data (for example, methods, templates, and runs) in a database using Microsoft SQL Server 2008 R2. Bio-Rad highly recommends backing up this database regularly. Backing up the database on a different drive from the one on which ChromLab is running is also recommended. If preventing data loss is critical to your operation, consider installing ChromLab on a RAID 1 drive. With RAID level 1, data are written identically to two hard drives.
A | Database Management Back Up the NGC Database You cannot start the ChromLab Administration tool while ChromLab is running on the client computer. Before you back up the NGC database, ensure that ChromLab is not running on the client computer and that the NGC instrument is idle, that is, no manual or method operation is running. Note: You must have Windows administration privileges on the ChromLab computer to run the ChromLab Administration tool. Backing up the ChromLab database 1.
Restore the NGC Database 3. In the Backup section, click Browse to browse to a location into which to save the NGC backup (.bak) file. 4. Click Backup. A status bar appears displaying the backup progress. Depending on the size of your database, the backup can take some time. 5. When the backup completes, close the ChromLab Administration tool. 6. Restart ChromLab on the client computer. Tip: You cannot start ChromLab on the client computer while the ChromLab Administration tool is running.
A | Database Management 4. Click Restore. A status bar appears, displaying the restore progress. Depending on the size of your database, the restore can take some time. 5. When the restore completes, close the ChromLab Administration tool. 6. Restart the NGC instrument. 7. Restart ChromLab on the client computer. Tip: You cannot start ChromLab on the client computer while the ChromLab Administration tool is running.
B Multicolumn Purifications Protein purification can involve challenging separations of complex mixtures that might not provide sufficient resolution of target proteins from their impurities in a single chromatographic step. Such cases may require multiple discrete experiments involving multiple columns and different column chemistries. Multicolumn purifications combine a series of columns, often with different or orthogonal chemistries in sequence or in tandem, in a single separation protocol.
B | Multicolumn Purifications Multicolumn Purification Method Templates ChromLab™ software includes several multicolumn purification templates. The templates comprise preprogrammed methods for binding and eluting samples from multiple columns in an automated sequence. They also offer multidimensional chromatography strategies whereby the target fraction eluted from one column is loaded onto another for a second dimension of purification.
Multicolumn Purification Method Templates Table 7. Multicolumn sequential templates Template Name Template Description Affinity (1 ml) — Linear Gradient This method is used to purify many samples quickly. Using a sample inlet valve and the sample pump, the method loads sample onto separate affinity columns and washes unbound material off in quick succession. This process can be repeated for up to five columns. This method is designed to reduce the target protein’s exposure to proteases.
B | Multicolumn Purifications Table 7. Multicolumn sequential templates, continued Template Name Template Description Affinity (5 ml) — Step Gradient This method is used to purify many samples quickly. Using a sample inlet valve and the sample pump, the method loads sample onto separate affinity columns and washes unbound material off in quick succession. This process can be repeated for up to five columns. This method is designed to reduce the target protein’s exposure to proteases.
Multicolumn Purification Method Templates Multicolumn Tandem Purification Templates Tandem purifications are used when two different chromatography techniques are combined into one method. First, samples are injected, bound to a column and washed to remove any major contaminants. Next, the target fractions are eluted and either applied directly onto another column in tandem or stored temporarily in a sample loop or container. If stored, they are later reinjected onto a second column.
B | Multicolumn Purifications Table 8. Multicolumn tandem templates, continued Template Name Description 2-D Affinity (5 ml) > Desalting (50 ml) This method is used to purify up to four samples. Using a sample inlet valve and the sample pump, the method loads different samples onto separate affinity columns and washes unbound material off in quick succession. This process may be repeated for up to four columns. This method is designed to reduce the target protein’s exposure to proteases.
Performing Multicolumn Purification Chromatography Performing Multicolumn Purification Chromatography To perform multicolumn purification chromatography on an NGC system you must Plumb the NGC system Prime the NGC system Select and save a Method template Run the method Plumbing the NGC System How you set up and plumb the NGC system depends on the type of method you plan to run. This section provides information about plumbing the system to use the multicolumn purification templates.
B | Multicolumn Purifications Plumbing the NGC Discover Pro System to Use Multicolumn Purification Templates The tables in this section list the additional tasks for plumbing NGC Discover Pro systems to perform multicolumn purification. Ensure that you complete the plumbing tasks in the section Plumbing NGC Discover Pro Systems on page 291 before continuing. Tip: These templates require either the NGC Discover Pro or the NGC Discover Pro +1CSV (column switching valve) fluidic scheme.
Performing Multicolumn Purification Chromatography Multicolumn Tandem 2-D Affinity Templates Tasks 1. Insert a second column switching valve (referred to as the loop valve) near the sample inject valve on the NGC instrument and map as C2 2. Short length of tubing from port F on the sample inject valve to the left inlet port on the loop valve (C2) 3. Short length of tubing from port E on the sample inject valve to the right inlet port on the loop valve (C2) 4.
B | Multicolumn Purifications Priming the NGC System Important: Whenever you add or remove a module or change or upgrade your NGC system hardware configuration, you must replumb and reprime the system. To prime the system 1. Power on the NGC system and its connected computer. 2. To enter manual mode, do one of the following: 3. On the computer, click Manual Run in the ChromLab Home window or click the System Control tab and then click in the fluidic scheme.
Performing Multicolumn Purification Chromatography Selecting and Saving a Template Tip: You must save the template as a method before you can use it. To select a Method template and save it as a method 1. In ChromLab on the computer, do one of the following: In the Home window, click Open Method Template. In the Method Editor window, click Open Template on the toolbar. The Open Template dialog box appears. All supplied Method templates are organized in folders by technique. 2.
B | Multicolumn Purifications In the lower-right pane The Overview tab displays a graphic representation of the purification steps for the selected template. The Gradient tab displays the gradient graph of the selected template. The Notes tab displays explanatory text that you can edit in the Method Settings phase. 3. Double-click a template name to open the template in the Method Editor window. 4.
Bio-Rad Laboratories, Inc. Web site www.bio-rad.