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Quantification – Concepts and Integration

Quantification – Concepts and Integration. In This Section, We Will Discuss:. Quantification calculations. When to use peak area or peak height. The difference between precision and accuracy. How to properly integrate a chromatogram. Beer’s Law and Quantification.

cassandra-brady
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Quantification – Concepts and Integration

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  1. Quantification – Concepts and Integration

  2. In This Section, We Will Discuss: • Quantification calculations. • When to use peak area or peak height. • The difference between precision and accuracy. • How to properly integrate a chromatogram.

  3. Beer’s Law and Quantification

  4. External Standard Calibration Equation: Absolute Amt of x = [(Responsex x RF) - yintercept] x M x D Responsex = peak area or height RFx = Amountx Responsex M = Multiplier D= Dilution Factor

  5. Equation: Actual Amt of x = [(RFx x ResponseRatiox x AmountISTD) - yintercept]x M xD Response Ratio = Responsex Responseistd Increase the Precision with an Internal Standard

  6. Choice of Internal Standard

  7. Peak Height or Peak Area for Calculations

  8. Precision and Accuracy

  9. Building a Calibration Table • Inject standards with concentrations bracketing yourunknowns. • Set-up appropriate integration events for the low standard. • Integrate to obtain the peak height or area. Save theintegration events to the method. • Begin a new calibration table with level one, the lowestlevel. • Integrate each additional standard level and add to thecalibration table. • Examine the calibration curve and save it to the method.

  10. To Integrate a Chromatogram, the Integrator: 1) Defines the initial baseline. 2) Continuously tracks and updates the baseline. 3) Identifies the start time for a peak and marks this point with a vertical tick mark. 4) Finds the apex of each peak, creates a parabolic fit for the peak top, and stores the retention time. 5) Identifies the end time for the peak, and marks this point with a vertical tick mark. 6) Constructs a baseline. 7) Calculates the area, height, and peak width for each peak.

  11. When a Signal is Loaded, Integration May Occur Automatically • Integrate by: • Selecting Integrate after load in Load • Signal dialog box orPreferences dialog box. • Selecting Integrate orAuto Integrate from the menu. • Selecting the Integration or Auto Integration Tool. • Running a method where the Run • Time Checklist includes Data Analysis.

  12. Set Up Integration Loads Integration Events Set up integration for your method in the Data Analysis mode using a representative chromatogram.

  13. 0.1 0.05 Peak Width – controls the ability of the integrator to distinguishpeaks from baseline noise. In general, increasing the peak width will result in broader peaks. Initial Settings - Events Area reject- All peaks whose areas are below this value will not be reported. Height reject- All peaks whose heights are below this value willnot be reported. Slope sensitivity – decreasing slope sensitivity will result in detecting smaller and broader peaks. Shoulder Detection Mode – shoulders detected using the second derivative of peak

  14. Initial Settings – For All Signals Some Events in this table are dependenton other events in this table. Tangent Skim Mode – only applies whenconditions for the following settings are met: Tail Peak Skim Height Ratio, or Front Peak Skim Height Ratio, and Skim Valley Ratio Peak To Valley Ratio Baseline Correction must beAdvanced

  15. Tangent Skim Mode • Tangent Skim Modes • New Exponential • Exponential • Straight • Standard The Tail Peak Skim Height RatioandSkim Valley Ratio will be used to determine whether a tangent skim will be applied to calculate the area of a child peak on the trailing edge of a parent peak.

  16. Tail Peak Skim Height Ratio Hp Hc Ratio must be greater than set value to be skimmed Hp Hc Setting the value to zero disables tangent skimming

  17. Skim Valley Ratio Height Child Peak Height Valley Hv Hc When the ratio is less than the set value the child will be skimmed.

  18. Tail Peak Skim Height Ratio Tangent Skimmed No Tangent Skimming Tail Peak Skim Height Ratio = 0 Skim Valley Ratio = 20 Tail Peak Skim Height Ratio = 3 Skim Valley Ratio = 20

  19. Hp 449.7947 New Exponential Area = 209.3318 Hc  Set Front Skim Height Ratio Hc  Set Valley Height Ratio Hv 176.9880 483.1385 Front Peak Skim Height Ratio Very similar to Tail Peak Skimming Straight

  20. Advanced Baseline Integrator tries to improvethe start and end pointsof a peak, re-establish thebaseline for a cluster of peaks and removebaseline penetrations.Uses Peak to ValleyRatio Classical Advanced Baseline tracks the curvature of baselines better Advanced

  21. Peak to Valley Ratio LOWER than User Setting Peak to Valley Ratio HIGHER than User Setting H1>=H2 PtoVRatio = H2 / Hv H2>H1 PtoVRatio = H1 / Hv Advanced Baseline Peak to Valley Ratio

  22. Integration Example - Default Parameters Default integration parameters may not always be the best for your analysis • Possible Problems • Noise selected as peaks • Baseline tracking difficulties • Drop lines inappropriate

  23. Practical Integration Advice – Starting Point • If all peaks of interest were notintegrated, lower the slope sensitivityuntil all real peaks are integrated. • If there are still peaks that cannot beintegrated, lower the peak width setting. • Use timed events if necessary. • Remove undesired peaks with the heightor area reject. • Set the slope sensitivity to 50. • Estimate the peak width from the initial integration. Use the smallest peak width from a realchromatographic peak, not noise. Set initial height and area rejectto zero. • Set the Tail Peak Skim Height Ratioto 3, the Front Peak Skim Height Ratioto 6, and the Skim Valley Ratio to 20. • Baseline correction is Advanced withTangent Skim Mode New Exponential. • Integrate and view the results.

  24. Example – Initial Current Integration from DEF_LC.M default integration values Set initial values, then re-integrate Pick narrowest real chromatographic peak for peak width.

  25. First Integration Results Desired peaks not Integrated Better, but still needs work. Baseline problems

  26. Adjust Initial Parameters • Lower the slope sensitivity • to detect smaller, broader peaks. • Here the slope sensitivity was • changed from 50 to 20. • The smaller, broader peaks • were detected.

  27. Insert Timed Events • Use drop box to select desired event. • Click on chromatogram at the desired time. • Or, use the insert timed event tool. • Use the delete timed events tool to remove an event.

  28. Insert Timed Events Before After timed event inserted and integration

  29. Finish Integration Use area and height reject to ignore unwanted peaks

  30. Save and Close

  31. Save Integration Events as Part of a Method When finished creating the integration events, save them to the method.

  32. Auto Integrate Can be a good starting pointfor integration events. • 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 as 90% area of most symmetrical peak. Autointegrates based on your settings in For All Signals.

  33. Manual Integration • Draw Peak Baseline • Negative Peak • Tangent Skim Peak • Split peak • Delete Peak(s)

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