Standard Data Analysis - Integration ChemStation - Level 1 - Training In this module you can learn the basics about the integration process. You will learn how to properly set up integration events and save them as part of your method. You will see that the software has many features to aid your integration including timed events auto integration and manual integration capabilities.

Integration locates the peaks in a signal and calculates their size What is Integration Integration locates the peaks in a signal and calculates their size Agilent Restricted

Integration Process The Integration Process consists of the following: construct initial baseline peak recognition – Start and end Time finds the apex of the peak baseline allocation (construction) peak area measurement Integration process: - defines the initial baseline - continuously tracks and updates the baseline - identify start and end time for each peak (peak recognition). - find the apex of each peak and prints the retention time (cardinal points) - construct baseline - calculate area, height, peak width. Agilent Restricted

Start, End and Apex of a Peak 1 Slope and Curvature within limit 2 Slope and curvature above limit: peak? 3 Slope remains above limit: peak recognized 4 Curvature becomes negative: front inflection 5 Negative slope: Apex 6 Curvature becomes positive: rear inflection point 7 Slope and curvature within limit: end of peak? 8 Slope and curvature within limit: end of peak 5 4 6 7 2 1 8 3 Start of a peak 1) as long as the slope and the curvature are within the limit - continue tracking the baseline 2) Slope and curvature are above the limit - possibility of a peak 3) Slope remains above the limit - peak is recognized, cardinal point is defined 4) Curvature becomes negative - front inflection point Peak apex 5) Slope becomes negative - apex of the peak, cardinal point defined 6) Curvature becomes positive - rear inflection point End of a peak 7) Slope and curvature with the limit - approaching end of a peak 8) Slope and curvature remain within limit - end of a peak, cardinal point defined 9) The integrator returns to the baseline tracking mode Agilent Restricted

Determining Peak Apex Fit highest data point and those on each side The peak apex is recognized as the high points in the chromatogram by constructing a parabolic fit that passes trough the highest data points. Fit highest data point and those on each side to a quadratic equation, solve for highest point Agilent Restricted

Default Baseline Construction BB BV VB Baseline Tick Marks In the simplest cases, the integrator constructs the baseline as a series of straight line segments between : - the start of baseline - the tick marks - the end of peak The default baseline is constructed as a series of straight line segments between: the start of the baseline, the tick marks, and the end of the peak. Agilent Restricted

Standard or Enhanced Integrator The ChemStation includes two integrators: the Enhanced Integrator (default) and the Standard Integrator (Genie) New methods by default use the Enhanced Integrator and can not be down graded to the Standard Integrator Old methods can be updated to use the Enhanced Integrator or maintained using the Standard Integrator so customers can continue using validated methods. The default method “defoldlc.m” has the Standard Integrator The default method “def_lc.m” has the Enhanced Integrator The ChemStation includes two integration algorithms. The standard integration algorithm was included in earlier versions of the ChemStation and is also included in most other Agilent analytical data evaluation software. The enhanced integration algorithm is the first revision of a new generation aimed at improved ruggedness, reliability and ease-of-use. When using this software, we recommend utilizing the traditional algorithm for existing validated methods and the new algorithm for new methods. The ChemStation includes two integration algorithms. The standard integration algorithm was included in earlier versions of the ChemStation. The enhanced integration algorithm is the first revision of a new generation aimed at improved ruggedness, reliability and ease-of-use. When using this software, we recommend utilizing the traditional algorithm for existing validated methods and the new algorithm for new methods.   Agilent Restricted

Standard or Enhanced Integrator Both integrators co-exist so you may continue to use validated methods The Enhanced Integrator offers better: baseline allocation on wandering baselines rejection of noise peaks through the use of an initial peak height parameter control of integration start and stop marks peak allocation on noisy signals peak shoulder allocation through second derivative calculations Agilent Restricted

Loading Signals Select or ‘Load Signal’ from File Menu Show/Hide the Signal Information Select path, data file name and channel Data File List Box This box shows the data files that can be selected from the current directory File Information Click on the File Information button to display the following details about the currently selected signal in the Signals list box: Date and time of injection, Operator, Sample Name, Barcode, Data acquisition method, Sequence line, Vial number, Injection number, and Signal. Signal Details The Signal Details button displays which signals will be loaded evaluated during a method run. Signals that are not in the list box will not be integrated or reported if all defined signals can be found in the current data file. If all specified signals are not in the current data file, all available signals are loaded. Signals List Box Shows the signal or signals for the selected data file. Choose to load all available or selected signals only Agilent Restricted

When a Signal is Loaded, Integration May Occur Automatically You may also integrate by: Selecting Integrate or Auto Integrate from the Integration menu. Selecting the Integration Tool. Running a method where the Run Time Checklist includes Data Analysis There are several ways to integrate a chromatographic signal in the Data Analysis view. When a signal is loaded using the File, Load Signal menu items, the analyst may select to automatically integrate after the signal is loaded. The currently loaded events will be used for this integration. The user may also select the Integrate or Auto Integrate items from the Integration menu. When the Integration Task Tool is selected, you will also find an integration tool. Running a method that includes Data Analysis will also produce an integrated chromatogram. Agilent Restricted

Standard Integrator - Initial Values Threshold Peak Width Area Reject Shoulders The initial values for integration events are used from the start of integration until new values are specified at a later time. There are no time values for the initial values. You can set initial values for: Threshold, Peak Width, and Area Reject. Initial values CANNOT be deleted from the integration events table. You can use the Auto Integration item to set the initial parameters for a chromatogram. Threshold The threshold parameter is used by the ChemStation to identify peaks during the integration process. The threshold value relates to the height of the signal. If the signal height exceeds the value represented by the threshold setting, the signal may qualify as a peak. The threshold can be used to set the noise rejection for integration. Peaks smaller than the threshold height will be ignored by the integrator. This prevents excessive baseline noise being interpreted as peaks. The threshold value is based on a relative scale ranging from -20 to 25. The value of -20 is the most sensitive (lowest) threshold and 25 is the least sensitive (highest) threshold value. Each change of one in the threshold changes the sensitivity of the integrator by a factor of two. For example, a value of eight is four times less sensitive than a value of six. Peak width The peak width is defined as the width of the peak in minutes at half peak height. The peak width setting controls the amount of selectivity the integrator uses to distinguish peaks from baseline chromatographic noise. To use the peak width successfully, the best setting should be chosen which provides just enough filtering to prevent noise being interpreted as peaks but not enough filtering to distort the chromatographic information. If the selected peak width is too low, noise may be interpreted as peaks. To choose a suitable peak width for a single peak of interest, use the time width of the peak in minutes at half height. The peak width is automatically updated during the integration process as the peaks are identified. The update is weighted 0.75 * existing peak width + 0.25 * width of current peak. The update is limited to a change of 25%. If a timed peak width event is used during the integration, the automatic peak width adjustment is disabled. The individual points are bunched during the integration as a function of the peak width and the data rate. The number of data points bunched is designed to give between 12 and 23 bunched points within the peak width. Area Reject The area reject sets the area of the smallest peak of interest. The integrator rejects any peaks, which are smaller than the area reject setting after baseline conversion. The rejected peaks will be plotted but not stored in the integration file or reported. Enter the area of the smallest peak expected. You can obtain this information by first integrating the data file with area reject set to zero (0). Following integration, select the Integration Results item from the Integration menu, and use the Area column to choose an appropriate area reject value. Events for Each Signal The signals combo box displays all the signals used to date. You can select a signal from this list and create or edit the integration events table for this signal. If an asterisk marks a signal in the signal combo box, this signal has its own integration events. The COPY TABLE button copies the selected integration events table to the current integration events table. The REMOVE TABLE button deletes the selected integration events table. Initial Values Agilent Restricted

Standard Integrator - Timed Events What timed events could you use for this integration? Timed events may be used to adjust the integration when the tick marks have been placed inappropriately. In addition, there are occasions when you may want to use special integration functions such as integration off or area sum. Below you will find an explanation for some of the timed events. Area Sum ON or OFF Sets points between which the integrator sums the areas of the peaks. You can set the area sum on or off at appropriate times to sum a group of peaks. All peaks between the times the area sum event is switched on and off are treated as one peak for area calculation purposes. The areas are summed, and the summed area is assigned the retention time of the first peak. Baseline All Valleys ON or OFF Sets points between which the integrator resets the baseline at every valley between peaks. This function is useful when peaks are riding on the back of a broad, low peak, and you want the baseline to be reset to all the valley points. Baseline Back Sets a point at which the integrator extends the baseline, horizontally backward from the declared baseline point to this point. Baseline Hold ON or OFF A horizontal baseline is drawn at the value of the signal from where the baseline hold event is switched on until where the baseline hold event is switched off, or a good chromatographic baseline is re-established. Baseline Next Valley Sets a point at which the integrator resets the baseline at the next valley between peaks, and then cancels this function automatically. This function is ignored during area summing. A valley is formed when the curvature, (change of absorbance or signal intensity with respect to time), passes from negative to positive. Baseline Now Sets a point (time and signal) at which the integrator resets the baseline to the current data point, if the signal is on a peak. If the signal is on the baseline, the function is ignored. Integrator OFF or ON Sets points between which the integrator stops and starts integrating. Peaks between the times the integrator is turned off and on are ignored. Negative Peak ON or OFF Sets points between which the integrator recognizes negative peaks. When negative peaks are recognized, the integrator no longer automatically resets the baseline after penetration. From now on, any penetration of the baseline will be integrated using the established baseline as zero. Areas are constructed relative to this baseline and are given an absolute value. Solvent Peak ON or OFF Sets points between which a peak is treated as a solvent peak. Peaks on the trailing edge of the solvent peak can be separated by tangent skimming between the time the solvent peak detection is set to on and off. Tangent Skim (Integrated Area) Sets a point at which the integrator sets a tangent skim on the trailing edge of the next peak. All peaks above the tangent are integrated to the reset baseline. The tangent is drawn from the valley before the small peak to that point after it where the detector signal gradient is equal to the tangent gradient. Agilent Restricted

Auto Integration - Standard Integrator Auto Integrate determines the optimum values for the initial threshold, peak width, and area reject by integrating the chromatogram twice. Threshold - based on signal range and averaged baseline noise. Peak Width - proportional average of the peak widths of the first five peaks. Area Reject - 1/1000st of the greatest area found. Auto Integrate determines the optimum values for the initial threshold, peak width, and area reject by integrating every chromatogram object twice. The initial threshold calculation is based on the signal full range and the averaged baseline noise at the beginning and end of the chromatogram. The initial peak width is calculated from the proportional average of the peak widths of the first five peaks in the chromatogram. The initial area reject is calculated as 1/1000st of the greatest area found in the chromatogram. The Auto Integrate function creates one integration event table for each signal with the optimized initial settings for each signal. Agilent Restricted

Integration Tools - Standard Integrator Auto Integrate Integrate Current Select Integration Tasks Integration Events Chromatogram Manual Integration Draw Peak Baseline Manual Integration Remove Peaks When the Integration Task Tool is selected, the integration tool set will appear. This tool set will allow you to perform the functions contained in the Integration menu including: Integrate Current Chromatogram, Auto Integrate, Select Integration Events, and the manual integration functions. Manual Integration Negative Peaks Manual Integration Split Peaks Manual Integration Tangent Skim Agilent Restricted

Enhanced Integrator The Enhanced Integrator offers better: baseline allocation on wandering baselines rejection of noise peaks through the use of an initial peak height parameter control of integration start and stop marks peak allocation on noisy signals peak shoulder allocation through second derivative calculations Agilent developed the Enhanced Integrator as a response to suggestions by customers using the standard integrator. Some of the improved capabilities of the enhanced integrator include: optimized baseline tracking, a peak height parameter to reject noise, better peak allocation on noisy signals, and ease of use.   The enhanced integrator performs the following tasks when integrating peaks: defines the baseline, tracks and updates the baseline, identifies the start time of the peak, finds the apex of the peak, identifies the end point of the peak, constructs the baseline, and calculates the area, height, and peak width for each peak. Agilent Restricted

Enhanced Integrator You may update methods developed on previous revisions of the ChemStation to the new Enhanced Integrator. Once you have updated the new method, you cannot revert back to the old integrator. This is a one way process. The integration events are automatically converted. Agilent Restricted

Enhanced Integrator UI Define integration parameters just by selecting and clicking on the chromatogram There are two sets of integration events : - intial events - time-based Initial Peak Width The peak width setting controls the selectivity of the integrator to distinguish peaks form baseline noise. The peak width should be set to correspond to the narrowest chromatographic peak width at half-height. The value is in minutes. Slope Sensitivity This parameter is used to identify peaks during the integration process. The slope sensitivity value relates to the slope of the signal. If the signal slope exceeds the value represented by the slope sensitivity setting, the integrator sets a peak start point. If the signal slope values decrease below the slope sensitivity setting a peak end point is established. Height Reject Height reject is a user parameter that sets the noise rejection for integration. Peaks smaller than this value will be ignored by the integrator. The higher the height reject setting, the greater the height above the established baseline must be before a data point can qualify as the start of a peak. Area Reject The area reject sets the area of the smallest peak of interest. The integrator rejects any peaks that are smaller than the area reject setting after baseline conversion. Shoulders When shoulder detection is on, the integrator detects shoulders using the curvature of the peak as given by the second derivative. When the curvature goes to zero, the integrator identifies this point of inflection as a possible shoulder. If the integrator identifies another point of inflection before the apex of the peak, a shoulder has been detected. When Advanced Baseline is selected, two features are added to the integration algorithm. The first feature is enhanced baseline recognition. Instead of defining the baseline in one pass, the enhanced integrator performs three passes to find the best fit. In the first pass, the baseline and peaks are defined in the traditional way. In the second pass, the baseline is adjusted to define the best integration start and stop points. The second feature is recognition of unassigned peaks. The next slide will illustrate this feature See further for Timed Events. Use: Slope Sensitivity  to define peak sensitivity Peakwidth  to set an initial sampling interval for the integrator to distinguish peaks from baseline noise Area Reject  to filter small peaks Height Reject  to set noise rejection Agilent Restricted

Advanced Baseline Peak Peak Unassigned Area Baseline Absorbance Advanced Integration Advanced Integration Options Off Options On When the Advanced Baseline option of the integration events has been selected, unassigned peak areas, peaks which do not have a distinct maximum, will be recognized as peaks. Typically, these unassigned areas are assigned to a neighboring peak. Turning the Advanced Baseline option on or assigning an Unassigned Peak timed event, will cause the integrator to split this area into the peak area and an unassigned area. This integration is probably a better representation of the true area of the peaks. The additional area is typically another eluting peak. Based upon additional information, the scientist could also decide to add the unassigned area to the peak. For example, when the peak was a weak acid in reversed-phase liquid chromatography and the pH was near the pK value. In this case, all the area does belong to the main peak. 12 13 14 15 16 17 18 19 20 Time [min.] Agilent Restricted

Using Initial Parameters Peak Width Height Reject Defined by the initial peakwidth the integrator calculates a moving average over this period of time. This average is called the baseline. This goes on until the curvature exceeds a limit determined by the threshold value. If this trend continues a peak is detected. Peak width: Calculated with following formula PW = 0,3 x (right inflection point - left inflection point) + 0,7 x Area/Height For a gaussian peak this corresponds to the width at half height. The PW controls the selectivity of the integrator. It should be the width of the narrowest peak. During integration the peakwidth is automatically updated as follows: 0,75 x existing peak width + 0,25 x width of current peak Agilent Restricted

Enhanced Integrator - Timed Events Add Timed Events Delete a Timed Event You can use timed events to customize signal baseline construction when default construction is not appropriate. These events can be useful for summing final peak areas and for correcting short and long term baseline aberrations. To add a timed event to the Integration Events table, select the Add a new line to the events table tool. Select the timed event from the drop box. Fill in the value required to complete. Available Timed Events for the Enhanced Integrator Baseline Now Sets a point at which the integrator resets the baseline to the current height of the data point, if the signal is on a peak. If the signal is on the baseline, the function is ignored and the detected baseline is used. Baseline at Valleys Sets points (On/Off) between which the integrator resets the baseline at every valley between peaks. Baseline Hold A horizontal baseline is drawn at the height of the established baseline from where the baseline hold event is switched on until where the baseline hold event is switched off. If the signal is on a baseline at the hold point his is the baseline value to hold. If the signal at the hold point is in a peak the latest established baseline point is used to hold. With Off the baseline is set back to an established baseline. Tail Tangent Skim On: Sets a point at which the integrator sets a tangent skim on the trailing edge of the next peak. All peaks above the tangent are integrated to the reset baseline. The tangent is drawn from the valley before the small peak to the point after it where the detector signal gradient is equal to the tangent gradient. The tangent skim event time can be entered anytime during the peak. Designates peak as a solvent peak. Off: Ends tangent skimming after current peak is completed or if in the designated interval no peaks are found (and a solvent will not inadvertently be designated in the next cluster). Tangent Skim Mode Sets tangent skim to a different mode: Straight, Exponential, as with Standard, Integration algorithm. Area Sum Sets points (On/Off) between which the integrator sums the areas between the area sum on and the area sum off time. The retention time of a peak created with area summing is the average of the start and end times. If an Area Sum On event occurs after the beginning of a peak but before the apex, the entire peak is included in the sum. If it occurs after the peak apex, but before the end of the peak, the peak is truncated and the area sum begins immediately. Integration Sets points (On/Off) between which the integrator stops and starts integrating. Peaks between the times the integrator is turned off and on are ignored. At the On and Off point the baseline point will be established again. Slope Sensitivity Sets the new slope sensitivity value. This value relates to the slope of the signal. If the signal slope exceeds the setting value, the integrator sets a start point. When the signal slope decreases below the setting, an end point is set. Area Reject Sets the new area of the smallest peak of interest. The integrator rejects any peaks which are smaller. Negative Peak Sets points (On/Off) between which the integrator recognizes negative peaks. When negative peaks are recognized, the integrator no longer automatically resets the baseline after penetration. From now on any penetration of the baseline will be integrated using the established baseline as zero. Areas are constructed relative to this baseline and are given an absolute value. Split Peak Specifies a point at which to split a peak with a drop line. Fixed Peak Width Sets the peak width which is defined as the peak width of the peak in minutes at half height. This function disables the automatic update of peak widths for the next peaks. Auto Peak Width Turns on the automatic update of the peak widths for the next peak widths. Detect shoulders Switches shoulder detection on and off. Shoulders Mode Sets a new shoulder detection mode: Tangent or Drop lines. Unassigned Peak Sets points (On/Off) between which the integrator treats areas between baseline and signal, but outside the normal peak start and end points, as Unassigned areas. Peaks are delimited by droplines, if required, at the peak start and peak end points. Areas between a peak end and a following peak start are reported as Unassigned, with a separation code of U and a “retention time” at the midpoint of the interval.   Agilent Restricted

Tangent Skim Solvent peak (Skim peak) Tangent Peak Straight Line Calculation Tangent skimming is a form of baseline construction done for peaks found on the downslope of a solvent peak. A solvent peak can be tangent skimmed on the following ways : 1) Straight line calculation 2) Exponential calculation 3) Combination for the best fit (default) Exponential Calculation Agilent Restricted

Shoulder Detection Shoulders occur when two peaks are so close together that no valley exists between them Peak with shoulders Are unresolved peaks on the leading or trailing edge of a larger peak. When a shoulder is present, there is no true valley in the sense of negative slope followed by positive slope. A peak can have any number of front and/or rear shoulders. The area of the shoulder is subtracted from the main peak. Tangent Shoulders Drop Line Shoulders Agilent Restricted

Enhanced Integrator - Auto Integration Examines beginning and end regions to estimate noise. Assigns initial Slope Sensitivity and Height Reject. Assigns temporary Peak Width value for first pass integration. Sets Area Reject to zero. Performs trial integration, may be repeated several times. Calculates Peak Width based on early eluting peaks. Refines Slope Sensitivity and Height Reject. Computes Area Reject from initial Peak Width and noise level. You may integrate all signals with the Auto Integrate menu item or the Auto Integrate tool. Auto Integrate for the enhanced integrator has been modified to make the selected parameters more robust and to supply user feedback about problems that Auto Integrate encounters. Auto Integrate examines the beginning and end regions of the chromatogram to estimate noise and assigns initial values of Slope Sensitivity and Height Reject. Auto Integrate also determines a temporary value for Peakwidth based on the run time and optimum detection criteria. With the Area Reject set to zero, a trial integration is performed. If few peaks are detected in the trial integration, the parameters are adjusted and the trial integration is repeated, several times if necessary.   After a suitable trial integration, Auto Integrate uses widths from the earliest peaks found to compute an initial Peakwidth. Peak symmetry is used to exclude poorly shaped peaks from the calculation. Auto Integrate searches between the detected peaks for a confirmed segment of baseline. If found, Auto Integrate uses this along with Peakwidth data to refine the values for Slope Sensitivity and Height Reject. A value of Area Reject is computed based on initial Peakwidth and noise level. Auto Integrate issues warnings in the message line when: ·        Peak symmetry requirements for the Peak Width calculation are relaxed ·        Insufficient peaks are found for accurate Peakwidth calculations ·        No peaks are found (Auto Integrate tries to provide a 10:1 signal-to-noise-ratio, even if this results in no peaks being detected) Limitations Auto Integrate is a useful starting point for most chromatograms and in the early stages of method development. Auto Integrate should not, however, be used or accepted as a substitute for good sample preparation and chromatographic techniques. Auto Integrate determines values for the initial time of the run which limits Auto Integrate’s ability to deal with unusual situations that may occur later in the chromatogram. Auto Integrate may have problems with chromatograms having disturbances or peaks occurring at the start or the end of the run. Auto Integrate will have difficulty if the chromatogram requires integration timed events to deal with disturbances such as: ·        Inverted peaks ·        Baseline upsets and transients (valve or signal switches) Agilent Restricted

Enhanced Integrator - Tool Bar Use Auto Integrate to find suitable integration events Integrate Current Chromatogram Integration Tasks Auto Integrate Integration Events Set Baseline Now Set Detect Shoulders When the Integration Task Tool is selected, the integration tool set will appear. This tool set will allow you to perform the functions contained in the Integration menu including: Integrate Current Chromatogram, Auto Integrate, Select Integration Events, and the manual integration functions Auto Integrate examines the beginning and end regions of the chromatogram to estimate noise and assigns initial values of Slope Sensitivity and Height Reject. Auto Integrate also determines a temporary value for Peakwidth based on the run time and optimum detection criteria. With the Area Reject set to zero, a trial integration is performed. If few peaks are detected in the trial integration, the parameters are adjusted and the trial integration is repeated, several times if necessary. After a suitable trial integration, Auto Integrate uses widths from the earliest peaks found to compute an initial Peakwidth. Peak symmetry is used to exclude poorly shaped peaks from the calculation. Auto Integrate searches between the detected peaks for a confirmed segment of baseline. If found, Auto Integrate uses this along with Peakwidth data to refine the values for Slope Sensitivity and Height Reject. A value of Area Reject is computed based on initial Peakwidth and noise level. Set Fixed Peak Width Set Baseline Hold Set Area Reject Set Tail Tangent Skim Only visible with integration events open Set Slope Sensitivity Set Integration Agilent Restricted

Save Integration Events as Part of a Method Save Events to Method Remember to save the integration events as part of your method before continuing. This task can be accomplished in three different ways. First, from the File menu, you may select Save, Method. You may also select the Save Method tool from the tool bar. If you are utilizing the enhanced integrator, you may use the save to method tool found just above the enhanced integrator events dialog box. Agilent Restricted

Manual Integration Manual integration events Manual integration is a useful technique for difficult integrations and allows you to integrate selected peaks or groups of peaks. Except for the initial area reject value, all the other events are ignored within the specific range. Manually integrated peaks are annotated in the reports with a M. All Valleys can be active or inactive. The default is inactive. When active, peaks will be automatically separated at all valleys within the range of manual integration when either Draw Baseline, Negative Peaks or Tangent Skim is used. Agilent Restricted

Manual Integration If your chromatography is so demanding that you can’t use the the automatic integration process  Use the manual integration tool set Zoom in using the zooming tools to better view your baseline Use Cursor tools to perform manual integration by selecting the required action and dragging a line over or clicking on the chromatogram (depending on the task) The new integration results are immediately visible in the user interface Draw Baseline Specifies where the baselines are to be drawn for a peak or set of peaks. You can also specify whether peaks in the range given should be automatically separated at all valley points. Draw Baseline is used to integrate peaks manually. Negative Peaks Specifies when to treat any areas below the baseline as negative peaks. You can also specify whether peaks in the range given should be automatically separated at all valley points. Negative Peaks is used to integrate negative peaks manually. Tangent Skim (Manual Integration) Calculates the areas of peaks that are tangent skimmed off a main peak. You can also specify whether peaks in the range given should be automatically separated at all valley points. The area of the tangent skimmed peak is subtracted from the area of the main peak. Split Peaks Specifies a point where to split a peak with a dropline. All Valleys All Valleys can be active or inactive. The default is inactive. When active, peaks will be automatically separated at all valleys within the range of manual integration when Draw Baseline, Negative Peaks, or Tangent Skim is used. Delete Peaks Deletes one or more peaks manually from a chromatogram. Draw a rectangle around the peaks you want to delete. The complete baseline of a peak must be included in the rectangle. The rectangle can be drawn by dragging the mouse while holding down the left mouse button. As the mouse is moved, a rectangle is drawn from the starting point to the point where the mouse is currently positioned. Double-click the left mouse button while still in the window to delete the selected peaks. The chromatogram is redrawn.   Agilent Restricted

Option 1: Save Manual Integration to Method Save Manual Integration - To the method Select Integration, Copy Manual Events to Method The manual integration events are now part of the method and cannot be reviewed in the events. When a signal is loaded they are only applied, when the Apply Manual Integration Events option is checked in the events box or when you select Apply Manual Events from Method in the menu. The manual integration events use ABSOLUTE time values. They do not adjust for signal drift or retention time shifts. Option 1: Save Manual Integration to Method Agilent Restricted

Save Manual Integration - To the Raw Data File Step 1: Select View, Windows Functions, Save Window You can save the applied manual integration as part of the individual data file. Agilent Restricted

Save Manual Integration - To the Raw Data File Step 2: Click OK to use default name or rename Note: manual changes are stored in the raw data file directory. Agilent Restricted

To Recall Manual Integration From Data File Select View, Windows Functions, Load Window Agilent Restricted

Lab Time !!!!! Lab 5 - Integration Agilent Restricted