GPC/SEC Software For OpenLab CDS - Agilent Technologies

Overview GPC/SEC Results The GPC/SEC software provides the ability to: View the table of MW averages and related information for one or a series of processed injections Overlay the chromatograms and derived plots (MW distribution & cumulative plot) for one or a series of injections The processed injections can either be within the same results set or across results sets within the loaded project. The GPC Results are stored as part of the processed injection. Reporting The OpenLab reporting tool can be used to create GPC specific report templates that provide the ability to generate: A report per sample injection A summary report for a series of injections The processed injections can either be within the same results set or across results sets within the loaded project. E.g. A table of MW averages for a series of injections Overlay of plots for a series of injections GPC report items are supplied which comprise of a series of GPC specific tables and plots. Two sets of default report templates are supplied, one set for typical GPC applications and the second for SEC applications. Compliance Storage of Parameters and Results The GPC specific processing parameters, column calibrations, GPC results and report templates are all stored within the OpenLab CDS system. The GPC specific processing parameters and GPC column calibration are stored as part of the processing method GPC/SEC Software for OpenLab CDS User Guide 13

Overview The GPC Results are stored as part of the processed injection Audit Trails The GPC/SEC software hooks into the audit trails & diagnostics features within the OpenLab CDS system. GPC Privileges & Roles The GPC/SEC software uses the compliance, roles and privileges features of OpenLab Shared Services and provides three GPC specific privileges: Ability to edit GPC Parameters in a processing method Ability to create and modify a GPC column calibration Ability to perform a GPC analysis The GPC specific privileges are assigned to default roles provided within the OpenLab CDS platform. Export Options The standard export features provided by OpenLab CDS are available within the various GPC specific windows and grids, allowing graphics (chromatograms, calibrations & distribution plots) and the contents of tables and grids to be exported to various standard formats. In addition, an option is available which allows the GPC/SEC results of a processed injection to be exported as a simple ASCII file. The generated ASCII file can be loaded directly into Excel and provides access to the full GPC results and slice by slice data of the processed peak(s) within the injection. 14 GPC/SEC Software for OpenLab CDS User Guide

Getting Started 3. Getting Started Familiarity with the OpenLab CDS platform for LC Applications This User Guide assumes a working knowledge of the OpenLab CDS platform for LC applications. Before using the GPC/SEC software it is strongly recommended that you learn the basics of using your Agilent LC system with OpenLab CDS by following the interactive curriculum supplied in the OpenLab Help & Learning tool. Home Get Started Getting Started OpenLab CDS with LC This interactive curriculum covers the following topics. User Interface Learn how to navigate the Control Panel and Acquisition interfaces to use your system effectively. Topics include launching instruments, using and creating custom layouts. LC Acquisition Methods Learn how to manipulate acquisition methods to control your Agilent LC instrument. Topics include modifying, saving, and downloading methods for instrument control. Run Submission Learn how to use your method to analyze samples to acquire data. Topics include single sample analysis submission and using the run queue. Data Analysis Learn how to create a processing method to generate results from your captured data. Topics include result package overview, integration, compound identification, and single level calibration. Reporting Learn how to reprocess data and generate reports with your processing method. Topics include using report templates to print single sample reports and make basic template modifications. GPC/SEC Software for OpenLab CDS User Guide 15

Getting Started Familiarization with the GPC/SEC Software Once you are familiar with the basics of using your Agilent LC system, the GPC Help & Learning system should be used to familiarize yourself with the various functions & features of the GPC/SEC Software. The GPC Help & Learning system can be accessed from the program item OpenLab GPC Help or using F1 when focused on a GPC specific window or dialog. The Online Help has the following structure: Getting Started Familiarization with the GPC/SEC software Introduction to the GPC/SEC software Familiarization with the Data Analysis user interface GPC/SEC Software Tutorial This tutorial consists of a series of videos which guide you through the various aspects of the GPC/SEC software using a set of GPC/SEC data. This data is supplied on the GPC install media and can be imported into a project on your system. The tutorial covers the following: Aims of the tutorial Importing the GPC tutorial data into a project Viewing the tutorial data in the project Generating a GPC processing method Calibrating the GPC method using the narrow standards Processing the samples Reviewing the processed samples Reporting Workflows This section provides information about the general workflows. 16 Data Acquisition Data Analysis GPC/SEC Software for OpenLab CDS User Guide

Getting Started Defining a GPC/SEC Processing Method Generating a GPC Column Calibration Creating a GPC Column Calibration (Narrow) Creating a GPC Column Calibration (Broad Hamielec) Creating a GPC Column Calibration (Broad Integral) Processing a Sample Injection Reviewing Data & Results A series of topics that describe the GPC specific views and the parameters available. These can be accessed directly using the F1 key when focused on a GPC specific window or dialog. The GPC/SEC Processing Method The GPC Results View The GPC Distributions View The GPC Calibrations Plot View Reporting Full information about the features and options available are described in the OpenLab Help & Learning tool. The following topics describe: o GPC specific additions to the OpenLab Reporting module. o The Default GPC & SEC Report Templates supplied. GPC/SEC Software for OpenLab CDS User Guide 17

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Workflows 4. Workflows The general workflow when using your software for GPC/SEC applications can be broadly defined as: 1 Using the Data Acquisition module to setup your instrument and collect data 2 Using the Data Analysis module to perform data analysis of the collected data Data Acquisition The GPC/SEC software is purely a data analysis module and does not provide any additional functionality to the Data Acquisition module. As the technique is an isocratic application (organic or aqueous) that uses GPC/SEC specific columns to perform the separation, any LC system supported by the OpenLab CDS platform capable of running in an isocratic configuration can be used. The Use of Sample Types for GPC/SEC applications The Sample Type ‘Cal. Std.’ is used by the software to identify injections that are GPC/SEC calibrants. This can be defined at time of data acquisition or can be set in the Injection List view in the Data Analysis program. GPC/SEC Software for OpenLab CDS User Guide 19

Workflows Data Analysis Once data is available to process in a project, the workflows for performing GPC/SEC analyses can be broadly summarized into the following: 1 Defining a processing method. 2 Generating a GPC column calibration from one or more calibrants linked to the processing method. 3 Reviewing the GPC column calibration. 4 Processing injections using the stored GPC column calibration linked to the processing method. 5 Reviewing the results of the processed injections. Defining a GPC/SEC Processing Method Defining a processing method can be broken down into the following key areas: General Data Processing Parameters The following parameters need to be defined: 20 Integrator to use A processing method can be setup to use either the ChemStation or EZChrom integrators. The default option is the ChemStation integrator. Integration Events Integration events need to be defined and fine-tuned for the acquired data from your GPC instrument. The manual integration option allows the integration of individual peaks identified by the integrator to be changed by interactively deleting the peak or editing the peak start, peak end, and peak baseline. System Suitability Optional, not required to perform GPC/SEC data analysis. Tools Optional, not required to perform GPC/SEC data analysis. Reports / Injection Report Optional. Define the report(s) required and destination for each processed injection. GPC/SEC Software for OpenLab CDS User Guide

Workflows GPC Specific Parameters General Options The following parameters on the GPC/GPC Method/General Tab need to be defined: Signal to use Multiple detector signals can be collected for injections, but only one detector signal can be processed by the GPC software at any one time. E.g. the Refractive index (RI) signal from a RI / UV combination or a single UV signal from multiple UV signals. To process sample injections. the detector signal used to generate a GPC Column calibration must be present in the sample injections. Flow Rate Correction Optional. This option allows injections to be corrected for variation in flow rate using the standard marker method. To use flow rate correction, a "marker component" must be included in all samples. Universal Calibration Optional. The Universal Calibration method can be applied to account for different polymer types between the calibrant(s) used to generate a GPC column calibration and the polymer samples to be evaluated. This method requires Mark-Houwink constants, K and α (Alpha) values for both sample and calibrants. The default values provided are for Polystyrene in THF at ambient temperature. GPC Calibration Specific Options Select the calibration method to generate the column calibration on the GPC/GPC Method/Column Calibration Tab: Column Calibration Type Narrow Standard Broad Hamielec Broad Integral Polynomial Curve Fit Select the order of fit Not applicable Select the order of fit Cal.Std. Mark-Houwink parameters Optional Optional Optional Peak Identification Tab Define this table Not applicable Not applicable Broad Hamielec Parameters Tab Not applicable Define this table Not applicable Broad Integral Tab Not applicable Not applicable Define this table GPC/SEC Software for OpenLab CDS User Guide 21

Workflows The options above are described in more detail in the next section. GPC Analysis Specific options Two post data analysis options are available. GPC/GPC Method/MW Range Tab If the percentage of material within specific MW ranges of an integrated peak are required, then setup the parameters required in this tab. GPC/GPC Method/Percentage Fractions Tab If the molecular weights at specific percentage area fractions of an integrated peak are required, then setup the parameters required in this tab. Save the processing method once all parameters have been defined. NOTE 22 Ensure all processing parameters are defined prior to processing injections either to calibrate or evaluate samples. If any changes are made to the processing method after for example, generating a GPC column calibration, then changes to the processing method will make the GPC column calibration inconsistent with the processing method and any processed injections will be marked as inconsistent. The injections of the GPC calibrants will need to be reprocessed to regenerate the GPC column calibration GPC/SEC Software for OpenLab CDS User Guide

Workflows Generating a GPC Column Calibration The software provides three options of generating a GPC column calibration. These options are defined in the GPC/GPC Method/Column Calibration Tab of the processing method and are: 1 From a series of Narrow Standard Calibrants Inputs: RT of calibrant peaks Mp of calibrant peaks Polynomial curve order to apply A series of “Narrow Standards” of known peak MW (Mp) 2 GPC Column Calibration Plot of Log(MW) –v- Peak RT Using the Broad Hamielec Method Inputs: Target Mn & Mw averages Target Tolerance Maximum Number of Iterations A “Broad Standard” of known Mn & Mw 3 GPC Column Calibration Plot of Log(MW) –v- Peak RT (curve fit order 1) Using the Broad Integral Method Inputs: Integral table (% Cumulative Weight Fraction and Molecular Weight) Polynomial curve order to apply A characterized “Broad Standard GPC Column Calibration Plot of Log(MW) –v- Peak RT The procedure to generate a column calibration depends on the selected calibration method. These procedures are described in the following section. GPC/SEC Software for OpenLab CDS User Guide 23

Workflows The Use of ‘Sample Types’ for GPC ‘Sample Type’ In all three calibration methods, the ‘Sample Type’ should be set to Cal. Std. in the Injection List window for all injections that are to be used to generate a GPC column calibration. ‘Run Type’ When using a series of Narrow Standards to generate a GPC Column calibration, the ‘Run Type’ needs to be set to ‘Clear all calibration’ for the first of the calibrant of a series. This ensures that a new GPC column calibration is created. 24 GPC/SEC Software for OpenLab CDS User Guide

Workflows 1 Creating a GPC Column Calibration (Narrow) The most common method of generating a column calibration is to use a series of Narrow Standards. A series of narrow dispersity polymers from the same homologous series are dissolved in the eluent and injected on the column set. Each of the standards should have known Peak Molecular Weight (Mp) value. These values will have been provided on the certificate of analysis of the calibrants. The process of creating a GPC Column Calibration from a series of Narrow standards is a two-step process. Step 1 – Creating the Peak Identification Table Step 2 – Using the Peak Identification Table to generate a GPC Calibration The process is summarized below. GPC/SEC Software for OpenLab CDS User Guide 25

Workflows Step 1 – Creating the Peak Identification Table The Mp values of the Narrow standards to be used for generating a GPC column calibration need to be predefined in the GPC processing method to be used by setting up a Peak Identification Table. This table needs to contain an entry for each calibrant consisting of, the expected retention time of the peak maximum and its associated Mp. Narrow Standards Require a series of “Narrow Standards” of known peak MW (Mp) Processing Method Identify the expected RT values of calibrants & assign the Mps from the certificate of analysis of the calibrants. Define the curve fit to apply Peak Identification Table Stored in the processing method This Peak Identification Table provides the ability to pre-define a calibration template of calibrants in a GPC processing method. This processing method can be stored as a master method for a specific GPC application. This master method provides the ability to generate a column calibration without the need for an operator to manually define the Mp values of the calibrant set. 26 GPC/SEC Software for OpenLab CDS User Guide

Workflows Step 2 – Using the Peak Identification Table to generate a GPC Calibration Once the Peak Identification table has been completed, this ‘template’ can be used to generate a GPC column calibration either from the set of calibration standards used to define the Peak Identification table, or from subsequent injections of the same calibrant set run under the same chromatographic conditions. Processing Method Narrow Standards Peak identification table Process the Calibrants GPC Column Calibration Plot of Log(MW) –v- Peak RT Stored in the processing method The process to generate a GPC column calibration using narrow standards is described below. Step 1a Setup the GPC processing method Step 1b Define the Peak Identification Table in the processing method Step 2a Create the column calibration Step 2b View the generated column calibration Step 2c Fine tune the column calibration GPC/SEC Software for OpenLab CDS User Guide 27

Workflows Step 1a. Setup the GPC Processing Method for Narrow Standard Calibration Setup the processing options as described above. The key GPC parameters are: GPC/GPC Method/General Tab 1 Select the default detector signal to use. 2 Decide if you want to use flow rate correction. 3 If using the Universal Calibration option, define the Mark-Houwink values for the samples. GPC/GPC Method/Column Calibration Tab 1 Select Calibration type Narrow Standard. 2 Select the curve fit to apply to the calibration data points. 3 If using the Universal Calibration option, define the Mark-Houwink values for the calibrants. Save the processing method. Ensure the calibrant injections to be used are linked to the GPC processing method to be calibrated. 28 GPC/SEC Software for OpenLab CDS User Guide

Workflows Step 1b. Defining the Peak Identification Table in the Processing Method There are two methods of defining this table. Interactively from a set of calibrants The method requires a set of injections of the narrow standards that are to be used under these conditions. The injections need to be either integrated using the automated integrator to identify the calibrant peaks or, defined using the manual integration tool. For the Calibrant set: 1 Select the Chromatogram view and define the start and end of each peak and the software will determines the peak maxima of each calibrant. The peak start and end do not need to be defined exactly as it is only the retention time at the peak maximum that is required for each peak. 2 If using flow correction, ensure the expect

Data Analysis component of the OpenLab CDS platform. The GPC/SEC software uses the existing workflows, processing options, data storage, reporting and compliance options supplied by the OpenLab CDS platform. It is able to process any signal from a concentration detector collected or imported into an OpenLab CDS project. Compatibility