Susan J. Kohler

May 7, 1998

Introduction: These instructions are intended to give a brief introduction to the use of the Chrom Perfect software as used on the Waters LC Module Iplus chromatography instrument. As it states in the Chrom Perfect User’s Manual, "Chrom Perfect is designed for the casual user as well as the power user." These instructions are intended for the casual user; power users are referred to the Chrom Perfect User’s Manual (CPUM).

Software Overview: The Chrom Perfect software package is designed to acquire data from up to sixteen chromatographs. In our case, the system is configured to acquire data from the two detectors of the Waters LC Module Iplus. Data acquisition is automatically triggered from the Waters LC upon sample injection. Following data acquisition, the chromatographic peaks are automatically detected and integrated, then identified by retention time. Amounts are calculated, reports are generated, and results are plotted. The peak identification and amount calculations are based on results in user-generated calibration files. The entire process is automated, but manual control may be exercised to redo any of the steps which occur after the initial data acquisition.

File Structure (CPUM p 4-2): The data acquisition and subsequent processing are controlled by a Method file with parameters for acquisition rates, integration control, and report generation. The Method file also contains the name of the Calibration file to be used, and the base name for the Data file. If multiple samples are to be run in one experiment, a Sequence file may be used to run the experiment. Sequence files enable the user to specify individual names, method files, and sample information for each

sample in the run. These various types of files have been placed in separate directories (by SJK) for ease of organization. The directories are:

c:\Cpwin\Data_uv Data from UV detector

c:\Cpwin\Data_cd Data from Conductivity Detector

c:\Cpwin\Method Method files - general purpose

c:\Cpwin\Calib Calibration files - general purpose

c:\Cpwin\Seq Sequence files - general purpose

These directories are accessed from the Configuration Editor (CPUM Ch. 6). The first two directories in the list from the editor are the destinations of the data acquired from the two detectors on the Waters LC. It is suggested that you substitute directories of your own for the two data directories, so that your results will be conveniently located in your directory. If you write your own methods, sequence, and calibration files, you may save them in the same directory with your data so that all the information pertaining to your specific experiments is together.

Getting Started: After the PC is turned on, double click on the Chrom Perfect icon. The main Chrom Perfect menu will appear, with buttons for each separate function. The most important buttons for now are the ones labeled Methods, Sequences, Acquire DT and Analyze.

The Method button will start the Method Editor and bring up templates for generating an new method (CPUM Ch. 7). Note that there are tabs for setting up data acquisition, integration, and plotting. It is only necessary to use this editor if you need to create a new method or examine the details of a method you wish to use.

The Sequence button starts the Sequence Editor (CPUM Ch. 13). Use this whenever you will be running a series of samples in the same experiment so that you can supply individual sample names and other relevant information. If you choose New under the File menu, you will be asked how you want to generate the new sequence. Choose Template to get a template to fill in with the information you wish to include. You may also start by calling up an existing sequence, modifying it, and using Save As to store it with a new name.

The Acquire DT button is used to set up a run to record data either from a single sample or from a series of samples. Click on the button, then choose the Download menu item. You will see only one choice, Chrom 1 - Waters IC. When you select it, you will get a template where you choose your Method file or Sequence file (click the Use Sequence checkbox to activate this choice) and fill in other pertinent information about your run. If you need to a select a new method or sequence, you may either type in the name (complete path is not necessary), or choose Browse to find it. Once the method file is chosen, fill in a Base Name for the data file and a Cycle Number. The cycle number appears as an extension on the final data file name, i.e. MYDATA.02R, where 2 is the cycle number and MYNAME is the base name. Then fill in the rest of the information and click on OKAY. The system will now automatically acquire data when triggered from the Waters IC. A real time plot will appear on the screen while the acquisition is in progress. Do not worry if the data go off scale; this is not the final plot! At the end of the run a report will be printed with a plot of the data and results of the integration.

The Analyze button can be used to view data which has already been acquired and to reprocess and replot it (CPUM Ch. 8). The options for reprocessing the data are enormous; the user is referred to the CPUM for more information, especially Chapters 8 (Analysis), 9 (Optimizing Integration) and 10 (Manual Integration).