ChromGraph REPORT data processing Methods are concerned only with the analysis of acquired data. We will call this analysis PROCESSING. Data collection and instrument control are the provinces of ChromGraph CONTROL. To distinguish the two types of Methods, the extensions .MTH (CONTROL) and .MET (REPORT) are automatically appended to the Method names.
All the information necessary to process a Data file is contained within a REPORT Method. This information includes the sensitivity for discriminating peaks from baseline, the times during which to search for peaks, which set of standards to use, what items to include in the report, etc. We will go through the development of an entire Method here. This will rarely be needed, since in most cases an existing Method can be edited slightly to create new Methods.
Begin creating a Method by loading an existing Method which we will modify. (Loading is not strictly necessary: a temporary Method is always loaded into RAM at startup, so a Method is always present. But this is a good place to learn how to load a Method.)
Select the Method drop-down menu by clicking on the METHOD section of the MAIN MENU:
Select the LOAD METHOD option, and a dialog box containing a directory of existing Methods in the default data location will appear. To load a Method, either double click on its name or highlight it and click the OK button.
Methods can be loaded from other locations by browsing through the 'Look in' section of the dialog box until you find the correct location. All the examples in this manual were taken from Methods and Data files in the C:\BAS\DATA directory.
If you load a file from a different directory, this new directory automatically becomes the default data location. The default data location will remain in effect throughout the session, unless changed by another LOAD or SAVE operation. When you finish your session and QUIT, saving the REPORT.INI file will save the new default data location for subsequent sessions. If you don't save the REPORT.INI file, the new data location will be forgotten.
To edit or review the Method you must open the Method screens (technically they are called dialog boxes). Start by clicking on the METHOD section of the MAIN MENU. A list of options will appear:
Except for the LOAD, LIST and SAVE options, each option will open a section of the Method. Simply click on the desired section with the mouse.
At the bottom of each Method screen are five pushbuttons for subsequent action:
OK accepts any changes and saves them to RAM. These changes are then used in subsequent runs of the Method. To permanently save these changes to disk, use the SAVE option.
<< (destination) and (destination) >>. These have the same function as OK, except they automatically bring up the previous (<<) or next (>>) screen of the Method.
CANCEL closes the dialog box without accepting any changes that were made in the box. The original information is thus preserved.
SAVE opens a dialog to save the Method with a name and location of your choice. Valid Method names are discussed here.
Choosing the DATA DESCRIPTION option under METHOD brings up the edit screen for the first section of the Method. The Data Description section shows information about the Data file currently loaded; as a consequence, it is not a permanent part of the Method and will change as new Data files are loaded.
OPERATOR, CONDITIONS and RUN NOTES are user-inserted comments entered into the ChromGraph Control software Method at the time of data collection. This information is saved along with every Data file generated by CONTROL. REPORT imports this information and includes it in the final processing report that it generates. By using these fields when you collect data, you will always be able to identify any particular Data file. You can fill out a table of information that will be imported to the RUN NOTES section during automatic operation: click here for details.
A SLASH (/), followed by a number, in the RUN NOTES field indicates a volume or mass adjustment for the sample. Adjustments are generally attached during data collection by ChromGraph Control (details here). Adjustments allow the results to be reported on a per mass or per volume basis when the weight or volume of the original sample is known. When a number appears here, the amount reported for this sample in the final report will be the calculated amount divided by the number.
The following items are entered automatically by Control at the time of data collection:
TIME and DATE of run indicate when the data were collected.
VIAL# refers to the position of the sample in the BASi Sample Sentinel autosampler, if used.
DATA NAME is the name of the data file.
RUN# is the run's number in the sequence of samples.
DETECTOR is the detector code letter, A-D.
The Data file name begins with the data name, which can have up to four numbers and letters (the last position must be a letter, to avoid confusion with the run number, which follows). Run numbers (three digits, up to 999) and a letter (A through D, for detector 1 through 4) are automatically appended to the data name by the data-acquisition software. When saved to disk, the name is given the extension .DAT. Thus, CATS003A.DAT is the third run in a series called cats, and contains data from detector A.
The information above will print out with the report. Three remaining items do not print out:
START and END minutes indicate the time from the start of the run during which data were collected.
POINTS PER MINUTE is the rate of data collection.
This next section of the Method can be accessed by either choosing it from the Method drop-down menu or by clicking the << or >> buttons from other screens. The options in this screen define how peaks are detected and specify some aspects of the report. Try the OPTIMIZE VARIABLES option initially. Optimize will decide on appropriate values for all the processing variables. You can then observe the peaks and modify the values if necessary.
IMPORTANT NOTE REGARDING THE PREBUNCH AND PREFILTER PARAMETERS: These parameters are intended for advanced users who have a firm understanding of peak-finding algorithms. If you use them, keep the following points in mind:
PREBUNCH attempts to reduce the number of data points to some optimum number for peak detection. It does this by averaging every x number of points, with x selectable from 2-16. An optimum sampling rate would produce at least 20 data points for the narrowest peak and no more than 500 data points for the widest peak. In most cases the sampling rate automatically set by ChromGraph Control will provide excellent peak detection.
PREFILTER applies additional filtering to the data. Filter rates are in Hertz, with smaller numbers providing greater filtering than larger numbers. As filtering increases, small details become smoothed over and unresolved peaks tend to merge.
SMOOTHING WIDTH compensates for noisy baselines. Use a small value for smooth baselines, and a larger value if baseline noise is being detected as peaks. Peak detection takes longer with large values than with small values.
INITIAL SLOPE THRESHOLD is the sensitivity of the peak-finding algorithm. A small value is very sensitive, whereas a large value is very insensitive. Set this option so the important peaks are detected, while small extraneous peaks are not.
MINIMUM PEAK AREA enables you to reject peaks below a certain area during peak detection. Use this option to exclude small peaks that are of no interest, but still large enough to be detected. Unless you are thoroughly familiar with your data, use a small value so that all detected peaks appear in the report.
TIME UNITS selects minutes or seconds as time units in tables and graphs of the report.
LIST PEAKS allows one of the intermediate steps of peak detection, the finding of start and stop points for the peaks, to be listed to the screen during interactive data processing.
REVIEW BASELINE causes a graph of the peaks, with their start and stop points marked, to appear on the screen during data processing. Processing halts until the user reviews the graph and chooses to cancel or continue.
The SAVE BASELINE checkbox causes a copy of the chromatogram to be saved to the default data location each time a run is processed. The file will have a name composed of the data name and run number, followed by a .BLN extension. These files take up a lot of disk space, and are redundant in that the chromatogram can be easily redrawn at a later time from the raw data. However, if the regulations you work under require the archiving of the original chromatogram, check this box.
Indicate the time during which your peaks elute, and the number of peaks expected. Then check one or more of the variables to be optimized. Click the OPTIMIZE button and the software will calculate the values and insert all except Prebunch and Prefilter into the Method. Click REVIEW PEAKS to see the results of peak detection with the optimized variables. Prebunch and Prefilter are excluded here because they alter the data. Optimized values for these two parameters are calculated, but the user must decide whether to use them.
This section of the Method can be reached by taking the PROCESSING EVENTS option from the Method drop-down menu or by clicking the << or >> buttons from other screens:
The left side of this section contains a listbox of instructions that determine how peaks are detected. These instructions are called events because they can be activated or disabled at specified times. (In this manual, time will always refer to elapsed time from the start of a chromatographic run.) The following events are available:
DETECT PEAKS (ON or OFF) determines the times during which ChromGraph searches for peaks. All Methods must have at least one Peak Detect ON (usually at time 0.0) in order to process data. A more sophisticated use is to turn Peak Detect OFF at time 0.0 to ignore the baseline disturbance at the beginning of the run, and turn it ON before the first peak of interest.
NEGATIVE PEAK (ON or OFF) may be useful in vacancy chromatography. It serves to reverse a negative-going peak so it can be processed like a normal peak. Most users will not use this option.
PERPENDICULAR DROP (ON or OFF) determines how the baseline is drawn under two peaks that are not completely resolved:
ON draws a straight baseline between the start of the first peak and the end of the second, and drops a perpendicular divider from the valley between the two peaks to the baseline. In this figure the green lines indicate the area boundaries of peaks 13 and 14 when perpendicular drop is on:
OFF draws the baseline for each peak independently, resulting in a 'tangent skimming' approach that is appropriate for quantifying small peaks on the shoulders of larger ones. The figure below shows this approach. Had perpendicular drop been on in this case, the area under peak 18 would have nearly doubled.
The default state for Perpendicular Drop is ON.
FIX BASELINE forces the software to accept the baseline value at any given time as a true baseline. Occasionally the baseline at the beginning of a run has such a steep slope that the software won't recognize it as a baseline, and therefore won't recognize the first peak. Inserting a Fix Baseline command before the first peak will solve this problem.
THRESHOLD VALUE allows the Initial Slope Threshold to be changed at a given time. This feature can be used to increase the sensitivity for late-eluting, broad peaks. In most cases, however, the Initial Slope Threshold will be sufficient to detect all the peaks.
To edit an existing event, click on it so its values appear in the edit boxes on the right side of the screen. Make the appropriate changes and click the CHANGE button. To insert a new event, enter the time and event in the edit boxes, then click the INSERT button. To delete an event, highlight it and click the DELETE button. In addition to this editing menu, Processing Events can be edited graphically, using the Event Cursor.
The REPORT OPTIONS screen is accessed through the Method drop-down menu or by clicking the << or >> buttons from other screens. This screen customizes the report of the analysis, detailing which output devices are to be used and which items are to be included:
Output can be routed to the SCREEN and the PRINTER. The remaining output options are intended to transfer reports to spreadsheets or word processors:
PRN, CSV and TAB files are ASCII files, formatted so their information can be readily imported into spreadsheets . As ASCII files, they contain only text (not graphs). To select PRN, comma-delimited (CSV) or tab-delimited (TAB) files, see the SETUP OPTIONS dialog.
In INTERACTIVE MODE, requesting a PRN, CSV or TAB file will generate one ASCII file for each Data file (each run). The file will have the Data file name followed by .PRN, .CSV or .TAB (e.g., CATS005A.PRN). This is not very useful, but the ASCII files were designed to be used in automatic mode.
In AUTOMATIC MODE, you can specify one file to which reports for all Data files will be sent. Thus, the results from hundreds of runs can be put in a format that's easily manipulated by a spreadsheet.
REP files also are ASCII files. These are exact images of the reports that are generated on the screen or printer. If selected, one REP file will be generated per Data file, and named with the data file name followed by .REP (e.g., CATS002A.REP). Only text can be stored in a .REP file. REP files are recommended only for those who's regulatory environment requires permanent storage of the original results for each data file.
CLIPBOARD sends an image of the report that is generated on the screen or printer to Clipboard, Windows' internal message board. From Clipboard the image can be pasted to other Windows programs. Since Clipboard can hold only one report or image at a time, reports from subsequent Data files will overwrite those of earlier Data files.
On the remainder of this screen, check the items you wish printed in the final report. The screen and printer have only a finite width; if more items than can fit on a line are selected, the line will wrap around and print on a second line. For neatness, we suggest you select only the items that are important for your analysis. The available items include the following:
DATA DESCRIPTION is the identifying information about the chromatographic run.
GRAPH OF RUN plots the chromatogram.
PEAK NUMBER is a sequential numbering of the peaks in the chromatogram, ordered by retention time.
RETENTION TIME is the elution time of the apex of each peak. Time units are minutes or seconds, as selected in Processing Options.
PEAK TYPE describes how well a peak was resolved from its neighbors. The first letter of this two-letter code indicates whether the peak started at the baseline (B) or in a valley (V) between peaks, and the second letter indicates the same for the end of the peak. Thus, a BV peak is one that began at the baseline, but was not well separated from the following peak.
SKEW is a number from 0 to 2 that represents the symmetry of the peak. A symmetrical peak has a skew of 1. Peaks with skews below 1 have a majority of their area before the apex, while those with skews greater than 1 have a majority of their area after the apex.
WIDTH is the peak width at half height, in the time units selected in Processing Options.
HEIGHT is the height of the peak.
% HEIGHT is the percent contribution made by any one peak to the sum of the heights of all peaks in the chromatogram.
AREA is the area delimited by the peak and the baseline drawn under it.
% AREA is the percent contribution made by any one peak to the sum of the areas of all peaks in the chromatogram.
OFFSCALE Y VALUE can be used under certain circumstances when a detector has a lower full-scale range than does ChromGraph. In this rare situation, an offscale peak on the detector might appear flat topped in ChromGraph, but since it doesn't go off the ChromGraph scale it is not reported as offscale. To remedy this situation, enter a maximum peak height here ” peaks having a greater height will be reported as offscale. For most uses, enter a zero here to disable this feature.
The STANDARDS OPTIONS screen is reached through the Method drop-down menu or by clicking the << or >> buttons from other Method screens. STANDARDS OPTIONS define how the peaks are to be quantitated and which items will appear in the report. Creation and use of standards files is discussed in the section on QUANTITATION.
STANDARDS FILENAME is the name of the Standards file to which the sample results are to be compared. When a new Standards file is created and saved, its name will automatically be inserted here. To skip the standards-comparison step of data processing, delete the name from this box.
TIME ZERO minutes (or seconds) compensates for data whose injection time do not correspond to the injection time of the standard. Time units are those specified in Processing Options This option will rarely be used, and usually should be set to 0.
TIME SCALE FACTOR compensates for runs in which elution was faster or slower than that of the standard. The Time Zero minutes is subtracted from the retention time of each peak, and the result is multiplied by the Time Scale Factor, to make the correction. This option will rarely be used, and usually should be set to 1.
DILUTION MULTIPLE corrects calculated amounts for any known dilutions of the samples. Thus, if you diluted your samples in half before analyzing, a dilution multiple of 2.0 will double the calculated amounts so they are corrected to the original sample. The default value of 1.0 eliminates the correction. The correction is applied equally to all samples. To apply corrections when individual samples differ in volume or mass, see the discussion above and in the section about importing sample information into ChromGraph Control.
The STANDARD ADDITIONS checkbox indicates whether you have used standard additions (endogenous) calibration. This occurs when a pooled matrix (such as plasma or urine) containing an unknown but constant amount of an endogenous analyte is spiked with known amounts of the analyte to create a series of calibrators. In this situation the size of the peak is determined by both the endogenous amount and the spiked amount. The endogenous amount is represented by the y-intercept of the regression equation, and the spiked amounts by the slope. When you check this box the y-intercept will be dropped from the formula, thereby correcting for the endogenous component of the calibrators.
TYPE OF STANDARD CURVE: Choose MULTILINE (point-to-point), LINEAR, QUADRATIC, CUBIC or QUARTIC. Most standard curves are linear (if there are three or more levels of standards) or multiline (two or fewer levels).
The following items will be included in the report if checked. Since there are too many items to fit neatly across the width of a standard page, we suggest that you select only those that are useful in the report.
PEAK NAME is the name assigned to a peak with a given retention time. Names are entered when creating a Standards file.
CALCULATED AMOUNT is the result of comparison of a sample component to internal and/or external standards.
STANDARD ERROR is the standard error of the calculated amount.
STANDARD # is the sequential number of the peak with this retention time in the Standards file.
RET. TIME ERROR is the difference in time between the unknown peak and the standard peak. Time units are those selected in Processing Options.
GROUP NUMBER allows the standards to be assigned to categories for pooled area reports. Standard peaks are assigned to categories when the Standards file is created.
DEGREE OF POLYNOMIAL (0 = multiline, 1 = linear, etc.) is the coefficient of the standard curve that was actually used for quantification. This may be lower than the degree requested above (Type of Standard Curve) if some standard peaks or levels are missing.
UNIDENTIFIED PEAKS are those that do not match any peak in the Standards file. They may be included in the report by checking this option.
A complete list of the files created by both ChromGraph Control and ChromGraph Report is available here.
To review the Method, you may scroll forward or back through all the screens using the << and >> buttons, or choose LIST METHOD from the Method drop-down menu:
Methods may be listed to the screen, printer, disk and Clipboard. If listed to the screen, they scroll along the rearmost section of the screen, the OUTPUT WINDOW. After the listing is finished, you may scroll through the Method by using the scroll bars on the right side of the Output Window:
Choose the SAVE button from any of the Method screens, or SAVE METHOD from the Method drop-down menu. A SAVE screen will appear, showing the default Method name and the location to which it will be saved:
A listbox will show the existing Methods in the default data location. Change the Method name or data location as appropriate and click on the OK button to save it. The extension .MET will automatically be appended to the name you choose. If a file of the same name already exists, a warning will appear.
Method names can theoretically be up to 127 characters long, including the path (thus, C:\BAS2\DATA\mymethod.met = 25 characters). Some advanced procedures may require shorter names, so we recommend using names with total lengths (including the path) of less than 33 characters.
For display purposes, a Method name may be truncated somewhat in various places in the program.
ChromGraph CONTROL Methods have the extension .MTH to distinguish them from ChromGraph REPORT Methods. They can therefore be given the same name, to associate them. For example, Methods to run and process catecholamine samples can be named CATS.MTH and CATS.MET, respectively, while those to run and process amino acids can be named AMINO.MTH and AMINO.MET.