Hidden Peak Fitting

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Hidden Peak Fitting

Launching PeakFit

Click on the PeakFit icon or Start Menu shortcut to launch the program. Hidden Peak Data This tutorial uses a data set designed specifically to illustrate the fitting of hidden peaks with noisy data. The data consists of fifteen peaks total, four of which are hidden since they produce no local maximum in the data. This synthetic data set consists of peaks placed at 0.5 intervals from x=1.0 to 8.5, with the exception of there being no peak at x=4.5. Five percent random Gaussian noise has been added to simulate particularly noisy data. Importing SAMPLE.XLS All PeakFit sample data is in a single Excel worksheet, SAMPLE.XLS. Each data set is on its own sheet or page in the overall worksheet file. We will import the data set in the first sheet. Use the File menus Import option, or click on the first toolbar icon. Change the file type to Excel and select sample.xls. First click on column A!A to select the X variable, and then on A!B to select the Y variable. PeakFit uses an Excel-like nomenclature to specify the different sheets or pages of the worksheet.

Click on OK to accept the selections. The next dialog offers an opportunity to modify the basic titles. Simply click on OK to accept the titles imported from the spreadsheet. Automated Fitting Using Residuals Select the AutoFit menus AutoFit Peaks I Residuals option, or click on the toolbar button with the peak and Roman numeral I. Because of the high amount of noise within this sample set, the default smoothing level and amplitude rejection thresholds are insufficient. Initially you will see 100 placed Gaussian peaks.

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Click on the AI Expert button next to the smoothing level. PeakFit contains an algorithm that seeks to determine an optimum smoothing level. For this data set, you will see that the Savitzky-Golay smoothing window was increased almost five-fold.

The upper plot of the upper graph contains three elements. These consist of the raw data minus the current estimate for the baseline, a smoothed copy of this data, and the sum curve consisting of all peaks currently placed and active. The lower plot of the upper graph contains the individual component peaks. The upper plot of the lower graph consists of residuals, the y-difference between the smoothed data and sum curve. The lower plot consists of the raw data with points turned on or off to reflect PeakFits automated baseline fitting. Setting the Amplitude Rejection Threshold Note that two peaks were detected in the noise at the two extremes of the data. To clear these out, change the Amp% (amplitude threshold) to 8%. You can enter this value directly, use the spin buttons, or right click the field or spin buttons and select 8% from the popup menu. Note that the amplitude threshold is represented by a dotted line in the components and residuals plots.

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Adding Hidden Residuals Peaks At this point, you should be looking at the 11 local maxima peaks (the status display indicates peak count). The residuals graph should show four very clear and quite substantial hidden peaks, well above this 8% threshold.

Check the Add Residuals box to automatically add these four peaks to the overall model. You should now have 15 peaks total:

For this data set, we will fit the default linear baseline, Gaussian amplitude peaks, and accept the constant width setting. When Vary Widths is turned off, all peaks will be fitted to single width. Visual Fitting For this data set we will visually fit the data. This means there is a graphical update for each iteration where there is an improvement in the overall fit merit function. Click on the Full Peak Fit with Graphical Update button located near the bottom of the control panel. You will see the fit progress graphically. After 9 iterations, the fit will be complete with an r goodness of fit of 0.99087.

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Click on Review Fit to proceed to PeakFits Review. PeakFits Review uses a desktop metaphor. You may have the main peak fit graph, the residuals graph, the numeric summary, and a data summary all simultaneously displayed if you wish. At this stage, we will explore a few of the Review options. Fitted points are colored by standard error by default. In the pre-defined color schemes with black graph backgrounds, points that are red in color are outside 3 standard errors and those outside 2 standard errors are yellow.

Click on the Select Function Labels button near the far right of the graphs toolbar. Select Area Parameter for the label type.

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Even though we fitted a Gaussian amplitude model, an analytic area is computed using the parameters from the fit. You should now see this analytic area reported directly above each peak. Click on the Show Confidence/Prediction Intervals button at the far right of the toolbar. You should see the default 95% prediction intervals forming a confidence band about the fitted curve.

The Set Confidence/Prediction Intervals, % Confidence button offers the means to set either or both types of intervals, and 90, 95, or 99% confidence levels. After observing the intervals, simply click once again on the Show Confidence/Prediction Intervals button to toggle off the intervals. In peak-fitting, the numeric analysis is very often the primary objective. PeakFit offers a comprehensive numeric summary. Click on the Numeric button.
Fitted Parameters r2 Coef Det DF Adj r2 Fit Std Err F-value 0.99087103 0.99022455 5.16445263 1584.02528 Peak Type a0 a1 a2 1 Gauss Amp 101.350053 0.99977381 0.20087369 2 Gauss Amp 120.777814 1.50036843 0.20087369 3 Gauss Amp 150.244205 2.00059637 0.20087369 4 Gauss Amp 76.3834430 2.49163548 0.20087369 5 Gauss Amp 176.712313 2.99643241 0.20087369 6 Gauss Amp 91.2243268 3.49117978 0.20087369 7 Gauss Amp 121.844896 3.99335264 0.20087369 8 Gauss Amp 99.1953657 4.99687327 0.20087369 9 Gauss Amp 120.274614 5.49962035 0.20087369 10 Gauss Amp 151.709148 6.00378274 0.20087369 11 Gauss Amp 81.9743916 6.51785997 0.20087369 12 Gauss Amp 172.036096 7.00799405 0.20087369 13 Gauss Amp 88.5446586 7.49967219 0.20087369 14 Gauss Amp 115.977346 7.99063147 0.20087369 15 Gauss Amp 102.449403 8.49286836 0.20087369

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Hidden Peak Fitting

B Linear Bg -1.1308483 0.08609085

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The Fitted Parameters section contains the goodness of fit statistics and the estimates of the fitted parameters for the background, if any, and for all of the peaks in the model.
Measured Values Peak Type 1 Gauss 2 Gauss 3 Gauss 4 Gauss 5 Gauss 6 Gauss 7 Gauss 8 Gauss 9 Gauss 10 Gauss 11 Gauss 12 Gauss 13 Gauss 14 Gauss 15 Gauss Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Type Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Total

Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp

Amplitude 101.350053 120.777814 150.244205 76.3834430 176.712313 91.2243268 121.844896 99.1953657 120.274614 151.709148 81.9743916 172.036096 88.5446586 115.977346 102.449403 Anlytc Area 51.0313391 60.8135210 75.6503105 38.4602600 88.9774175 45.9328775 61.3508139 49.9464205 60.5601521 76.3879326 41.2753902 86.6228693 44.5836226 58.3964107 51.5848791 891.574217

Center FWHM 0.99977381 1.50036843 2.00059638 2.49163547 2.99643241 3.49117978 3.99335264 4.99687329 5.49962035 6.00378274 6.51785997 7.00799404 7.49967219 7.99063147 8.49286836

Asym50 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139 0.47302139

FW Base 1.00000000 1.00000000 0.99999994 1.00000004 1.00000001 1.00000000 1.00000003 0.99999988 1.00000000 0.99999999 1.00000000 1.00000004 1.00000000 1.00000007 1.00000000

Asym10 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 Moment2 0.99977436 1.50036843 2.00059637 2.49163548 2.99643241 3.49117978 3.99335264 4.99687327 5.49962035 6.00378274 6.51785997 7.00799405 7.49967219 7.99063147 8.49286836

1.00000000 1.00000000 0.99999996 1.00000002 1.00000001 1.00000000 1.00000002 0.99999993 1.00000001 1.00000000 1.00000000 1.00000002 1.00000000 1.00000003 1.00000000

Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp

% Area Int Area % Area Centroid 5.72373428 51.0313117 5.72373138 6.82091517 60.8135210 6.82091538 8.48502672 75.6503105 8.48502698 4.31374745 38.4602600 4.31374758 9.97981053 88.9774175 9.97981083 5.15188491 45.9328775 5.15188507 6.88117857 61.3508139 6.88117878 5.60204855 49.9464205 5.60204872 6.79249702 60.5601521 6.79249723 8.56775927 76.3879326 8.56775953 4.62949572 41.2753902 4.62949587 9.71572166 86.6228693 9.71572196 5.00055091 44.5836226 5.00055107 6.54980927 58.3964107 6.54980947 5.78581997 51.5848791 5.78582015 100.000000 891.574189 100.000000

0.04034970 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024 0.04035024

The Measured Values section contains the properties for each peak. The first portion of the table contains the amplitude, center, full width at half-maximum, the asymmetry at half maximum, the estimated full width at the peak base, and the asymmetry at 10% maximum. The second portion of the table contains an analytic area, if one exists, an integrated area from Xmin to Xmax, and the first and second moments.
Parameter Statistics Peak 1 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 101.350053 1.77558396 57.0798425 97.8609294 Ctr 0.99977381 0.00506103 197.543354 0.98982859 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 2 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 120.777814 1.87410471 64.4456060 117.095091 Ctr 1.50036843 0.00570802 262.852866 1.48915186 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 3 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 150.244205 1.87528190 80.1181970 146.559169 Ctr 2.00059637 0.00495961 403.377433 1.99085045 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 4 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 76.3834430 1.91610147 39.8639864 72.6181948 Ctr 2.49163548 0.00976476 255.166068 2.47244717 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 5 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 176.712313 1.80993172 97.6347953 173.155694 Ctr 2.99643241 0.00413044 725.451006 2.98831586 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 6 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 91.2243268 1.84041666 49.5672143 87.6078036 Ctr 3.49117978 0.00753689 463.212403 3.47636937 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 7 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 121.844896 1.66637381 73.1197858 118.570377 Ctr 3.99335264 0.00403720 989.138615 3.98541931 Wid 0.20087369 0.00119183 168.542173 0.19853167

P>|t| 104.839176 1.00971904 0.20321570

0.00000 0.00000 0.00000

P>|t| 124.460536 1.51158501 0.20321570

0.00000 0.00000 0.00000

P>|t| 153.929240 2.01034230 0.20321570

0.00000 0.00000 0.00000

P>|t| 80.1486911 2.51082379 0.20321570

0.00000 0.00000 0.00000

P>|t| 180.268931 3.00454896 0.20321570

0.00000 0.00000 0.00000

P>|t| 94.8408500 3.50599019 0.20321570

0.00000 0.00000 0.00000

P>|t| 125.119415 4.00128598 0.20321570

0.00000 0.00000 0.00000

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Peak 8 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 99.1953657 1.64780414 60.1985171 95.9573371 Ctr 4.99687327 0.00495603 1008.24147 4.98713440 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 9 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 120.274614 1.75036101 68.7141757 116.835055 Ctr 5.49962035 0.00554121 992.494622 5.48873156 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 10 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 151.709148 1.73873455 87.2526218 148.292436 Ctr 6.00378274 0.00457343 1312.75156 5.99479568 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 11 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 81.9743916 1.85692454 44.1452465 78.3254295 Ctr 6.51785997 0.00892161 730.570310 6.50032851 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 12 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 172.036096 1.82456277 94.2889434 168.450727 Ctr 7.00799405 0.00450819 1554.50368 6.99913520 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 13 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 88.5446586 1.89762209 46.6608495 84.8157235 Ctr 7.49967219 0.00869268 862.757644 7.48259059 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 14 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 115.977346 1.80989280 64.0796772 112.420804 Ctr 7.99063147 0.00605167 1320.40183 7.97873961 Wid 0.20087369 0.00119183 168.542173 0.19853167 Peak 15 Gauss Amp Parm Value Std Error t-value 95% Confidence Limits Amp 102.449403 1.68544868 60.7846467 99.1374004 Ctr 8.49286836 0.00486634 1745.22662 8.48330572 Wid 0.20087369 0.00119183 168.542173 0.19853167 Baseline Linear Bg Parm Value Std Error t-value 95% Confidence Limits a0 -1.1308483 0.99060944 -1.1415682 -3.0774521 a1 0.08609085 0.12892738 0.66774683 -0.1672588

P>|t| 102.433394 5.00661215 0.20321570

0.00000 0.00000 0.00000

P>|t| 123.714173 5.51050914 0.20321570

0.00000 0.00000 0.00000

P>|t| 155.125860 6.01276980 0.20321570

0.00000 0.00000 0.00000

P>|t| 85.6233537 6.53539143 0.20321570

0.00000 0.00000 0.00000

P>|t| 175.621466 7.01685289 0.20321570

0.00000 0.00000 0.00000

P>|t| 92.2735938 7.51675379 0.20321570

0.00000 0.00000 0.00000

P>|t| 119.533889 8.00252334 0.20321570

0.00000 0.00000 0.00000

P>|t| 105.761405 8.50243100 0.20321570

0.00000 0.00000 0.00000

P>|t| 0.81575550 0.33944047

0.25422 0.50463

The Parameter Statistics section lists the individual fit statistics. By default, the 95% confidence limits about the parameters are reported. The probabilities are optional, and a useful way to detect parameters that cannot be statistically judged non-zero. For this fit, only the background parameters are higher than p=0.05, a limit traditionally used for significance testing. As such, the background is suspect. The probabilities can often detect overspecified models containing extraneous peaks or peaks that cannot be judges to be statistically valid in the model.
Chromatographic Analysis Peak Type Nmoment NGauss FW Base 1 Gauss Amp 24.7721472 24.7717937 2 Gauss Amp 55.7891485 55.7891541 3 Gauss Amp 99.1911328 99.1911434 4 Gauss Amp 153.859001 153.859016 5 Gauss Amp 222.516831 222.516853 6 Gauss Amp 302.063550 302.063581 7 Gauss Amp 395.211181 395.211220 8 Gauss Amp 618.800373 618.800438 9 Gauss Amp 749.582290 749.582365 10 Gauss Amp 893.313366 893.313455 11 Gauss Amp 1052.84381 1052.84391 12 Gauss Amp 1217.14227 1217.14239 13 Gauss Amp 1393.92195 1393.92209 14 Gauss Amp 1582.39936 1582.39952 15 Gauss Amp 1787.56845 1787.56863

Asym10 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080 0.94685080

Resolution 1.00000000 1.00000000 0.99999996 1.00000002 1.00000001 1.00000000 1.00000002 0.99999993 1.00000001 1.00000000 1.00000000 1.00000002 1.00000000 1.00000003 1.00000000

0.52869430 0.52830704 0.51860240 0.53313250 0.52251883 0.53036113 1.05985087 0.53096757 0.53246234 0.54293372 0.51764657 0.51927732 0.51851810 0.53042876

The Chromatographic Analysis section reports the theoretical plates based upon the moment and Gaussian methods, as well as the estimated full width at the base, the asymmetry at 10% maximum, and the resolution between adjacent peaks.
Overlap Areas Peak Type 1 Gauss 2 Gauss 3 Gauss 4 Gauss 5 Gauss Peak 1 Peak 2 Peak 3 51.0313117 11.8343019 11.8343019 60.8135210 0.78910348 14.4198009 0.00905249 0.65594567 4.5065e-05 0.01441377 Peak 4 0.78910348 14.4198009 75.6503105 11.6865533 1.08119255 Peak 5 0.00905249 0.65594567 11.6865533 38.4602600 11.8213825 Peak 6 4.5065e-05 0.01441377 1.08119255 11.8213825 88.9774175

Amp Amp Amp Amp Amp

2.7089e-08 3.8139e-05 0.01218085 0.53967880 13.6509232

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6 7 8 9 10 11 12 13 14 15 Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Peak 1 2 3 4 5 Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Type Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Gauss Type Gauss Gauss Gauss Gauss Gauss Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp 2.7089e-08 5.1674e-12 1.2823e-21 2.2491e-27 8.1374e-34 2.867e-41 9.573e-49 3.3798e-57 4.4159e-66 6.3281e-76 3.8139e-05 3.3335e-08 1.7775e-16 1.4606e-21 2.4943e-27 4.2852e-34 6.493e-41 1.0462e-48 6.2318e-57 4.2186e-66 0.01218085 4.796e-05 5.3932e-12 2.0655e-16 1.658e-21 1.3841e-27 9.4927e-34 6.9639e-41 1.8872e-48 6.0233e-57 Peak 10 2.2491e-27 1.4606e-21 2.0655e-16 3.3927e-12 3.406e-08 3.0342e-05 0.01080932 11.5723415 60.5601521 14.2130937 0.56165294 0.01256557 3.3302e-05 3.3484e-08 5.1941e-12 0.53967880 0.00899588 1.9689e-08 3.3927e-12 1.2384e-16 4.8719e-22 1.4662e-27 4.7599e-34 5.6987e-41 8.3277e-49 Peak 11 8.1374e-34 2.4943e-27 1.658e-21 1.2384e-16 5.868e-12 2.3651e-08 3.839e-05 0.75134845 14.2130937 76.3879326 11.0730036 1.01110315 0.01144140 5.0703e-05 3.6419e-08 13.6509232 0.96455508 4.2464e-05 3.406e-08 5.868e-12 1.1281e-16 1.5521e-21 2.3153e-27 1.2755e-33 8.8923e-41 Peak 12 2.867e-41 4.2852e-34 1.3841e-27 4.8719e-22 1.1281e-16 2.1461e-12 1.6616e-08 0.00695107 0.56165294 11.0730036 41.2753902 12.9451578 0.62329518 0.01208778 4.0739e-05

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45.9328775 11.1724976 0.00854201 3.0342e-05 2.3651e-08 2.1461e-12 1.3004e-16 8.6409e-22 2.1186e-27 6.8212e-34

Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp

Peak 7 Peak 8 Peak 9 5.1674e-12 1.2823e-21 3.3335e-08 1.7775e-16 4.796e-05 5.3932e-12 0.00899588 1.9689e-08 0.96455508 4.2464e-05 11.1724976 0.00854201 61.3508139 0.69111425 0.69111425 49.9464205 0.01080932 11.5723415 3.839e-05 0.75134845 1.6616e-08 0.00695107 4.5174e-12 3.6515e-05 1.3594e-16 2.204e-08 1.5135e-21 4.9708e-12 2.2972e-27 1.6556e-16

9.573e-49 6.493e-41 9.4927e-34 1.4662e-27 1.5521e-21 1.3004e-16 4.5174e-12 3.6515e-05 0.01256557 1.01110315 12.9451578 86.6228693 13.4391174 1.02496555 0.01460661

Amp Amp Amp Amp Amp

Peak 13 Peak 14 Peak 15 3.3798e-57 4.4159e-66 6.3281e-76 1.0462e-48 6.2318e-57 4.2186e-66 6.9639e-41 1.8872e-48 6.0233e-57 4.7599e-34 5.6987e-41 8.3277e-49 2.3153e-27 1.2755e-33 8.8923e-41

The Overlap Areas section is a matrix that displays the % overlap in area between specific peaks.
Analysis of Variance r2 Coef Det DF Adj r2 0.99087103 0.99022455 Source Sum of Squares Regr 1351950.2 32 Error 12455.624 467 Total 1364405.8 499 Fit Std Err 5.16445263 DF Mean Square F 42248.443 1584.0253 26.671571

The Analysis of Variance section contains the ANOVA from the fit. The F-statistic is useful for comparing models with a fewer or greater number of peaks. The model with the highest F-statistic may be the most statistically valid.
Details of Fit Set Convergence State Iterations Minimization 1E-6 Converged 9 Least Squares 1/1 Curvature Matrix Constraints Violated Sparse-Roots 25.0000-5.00000-50.0000- None - None Extent

The Details of Fit section summarizes the preferences used to generate the fit. This numeric summary can be printed, copied to the clipboard, saved to file, or edited within PeakFit. Note that the Options menu enables you to turn on and off specific sections of this summary. We will explore additional Review options at the conclusion of the next fit. Click on Numeric or close the Numeric Summary window directly. Click on OK (the green check mark) to close the Review. This saves the current settings. The Cancel (the red X) does not save the current settings on exit. Automated Fitting Using the Second Derivative Select the AutoFit menus AutoFit Peaks II Second Derivative option, or click on the main toolbar button with the peak and Roman numeral II. Again, due to the high amount of noise within this sample set, the default smoothing level and amplitude rejection thresholds are insufficient. Here PeakFits limit of 100 peaks is depleted even earlier in the data. The smoothed second derivative is in the upper plot of the lower graph. Click on the AI Expert button next to the smoothing level. Note that AI Expert smoothing level produces a smooth second derivative curve and a smooth raw data stream in the uppermost plot. Set the Amp% value to 10% to clear out the peaks appearing in the noise close to the baseline. A second derivative produces minima at peak locations. Very often, peaks which evidence no local maxima in the original data, do show up as local minima in a smoothed second derivative. Here you will note that we directly have the 15 peaks we seek to fit. The second derivative procedure directly found the 11 local maxima peaks as well as the 4 hidden peaks.

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Fitting Preferences Before initiating the fit, we will check the current preferences for the fitting procedure. Use the Modify Peak Fit Preferences button to open the fit options dialog:

Be sure the a0, a1, and a2 parameter constraints are set and locked at their default 25%, 5%, and 50% values. Constraints The constraints are relative, and apply only to the initial peak placement (the adjustable parameters set when the fitting begins). If the constraints are too tight, or the initial estimates are more than these percentages out of range, the fit will be bound by these constraints. In order to achieve the optimal fit, it may be necessary to repeat the fitting process one or more times. Numeric Fitting For this data set we will numerically fit the data. This is a very fast way to complete a fit when you can be reasonably assured nothing will go wrong. The constraints for the built-in functions can go a long way toward this assurance, but they may also lock the fit into a suboptimal state. Click the Fast Peak Fit with Numerical Update button. The fit should take over fifty iterations to converge and the Constraints item will oscillate between 0 and 1, an indicator that one of the parameters is up against a constraint.

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Note that the final r value is 0.99039 as compared to the 0.99087 fit using the Residuals method. The fit is suboptimal because one of the parameters ran up against a constraint bound. Click on the Addl Adjust button and again click on the Fast Peak Fit with Numerical Update button in order to restart the fit. In this case, the relative constraints begin with the estimates from this initial fit. Because these are now more accurate, no constraint violations occur during the fit and the results now match those from the Residuals method.

Note that the optimal fit could have been achieved in a single step by widening or eliminating the constraints. The danger in doing this is that one or more of the parameters, unconstrained or less constrained, might assume an unrealistic value in the fit iterations thus producing a invalid or inaccurate fit. A good strategy is to accept PeakFIt's default constraint settings and initiate additional fit steps if constraints are noted in the fitting. If any of the peaks assume unrealistic values in the fitting process, tighter constraints may correct the problem. For assuring the optimum fit, visual or numeric peak placement is available. Manual peak placement is demonstrated in the Deconvolution method that follows later in the tutorial. Click on Review Fit when the fit is completed. If you would like to create a printed graph, click on the Print button in the graphs toolbar. Print preview is automatic and font scaling can be set prior to printing. Normally-Distributed Errors In least-squares fitting, the standard errors and confidence limits for parameters can only be judged valid when the assumption of normal errors is verified. This means that the residuals profile must be Gaussian. Click on Residuals to open a standard residuals plot.

Click on the Display Residuals Distribution button to see a histogram of the residuals. You will note it is Gaussian in appearance.

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Click on the Display Residuals in Stabilized Normal Probability Plot button to see a Monte-Carlo based critical limit test for normality.

The four lines on each side of the data represent 90, 95, 99, and 99.9% critical limits. A 99% critical limit means that in only 1 out of 100 data sets should even a single point fall outside this limit. For this fit, all values are safely within the 90% critical limits. Close the Residuals window directly or click on the Residuals button. Click on OK to close the Review. Again, this preserves the current settings. Automated Fitting Using Deconvolution Select the AutoFit menus AutoFit Peaks III Deconvolution option, or click on the main toolbar button with the peak and Roman numeral III. When data is deconvolved with a Gaussian response function, it is sharpened in the sense that peaks become narrower and of greater amplitude. Deconvolution often produces local maxima in what were previously hidden peaks. The deconvolved data is in the upper plot of the lower graph. Deconvolution is extremely sensitive to the width of the response function (it must always be less than the smallest peak width) and to the level of Fourier domain noise filtration. Note that Gaussian deconvolution is generally only useful for data containing symmetric peaks, particularly those that are close to Gaussian. This means that chromatographic peaks with a significant skew, or spectral peaks with a significant Lorentzian character, are not appropriate for Gaussian deconvolution.

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Here PeakFits initial estimate for a response function width and the default noise filtration produce the 15 desired peaks as well as a some small ones within the baseline noise. Click on the AI Expert button next to the filter level. Note that even this small change produces a significant change in the deconvolved data. Set the Amp% value to 8% to clear out the peaks appearing in the noise close to the baseline. Note that the 11 local maxima and 4 hidden peaks in the original data are quite clearly 15 local maxima peaks in the deconvolved data stream. Visual Adjustment Beyond automated placement, PeakFit offers a graphical or visual placement capability as well as a numerical parameter adjustment. At present, each peak has a single primary adjustment anchor which defines its amplitude and center. A peak can thus be dragged to any location desired simply by clicking and holding down the left mouse button while dragging the peak to a new position. Left clicking a primary peak anchor without motion toggles a peak on and off. At this point, experiment with moving and toggling peaks. A peak is added simply by left clicking, with no movement, where there is currently no peak. Move the mouse to the gap in the peaks (to about 4.5,150) and left click the mouse. A new peak appears.

Zoom-in is simply a matter of left clicking (other than on a peak anchor) with movement. The standard Windows type of zoom-in band is shown as the x-y region is boxed. Zoom-in on the peak just added. Afterwards, left click the mouse anywhere except on a peak anchor. This restores default scaling. Note that you can also use the Reset Scaling graph toolbar button to restore automatic scaling. At this point, check the Vary Widths checkbox. Until now, we have fit a single width for all peaks. By checking this box, PeakFits autoscan offers one additional degree of peak refinement and additional anchors are now shown on each peak. These are used to adjust peak widths.

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Use the half-height anchors to adjust one or more peak widths. Right clicking a primary anchor opens a function popup dialog. This dialog can remain up when selecting different peaks. When it is up, peaks can be selected by left or right clicking the primary peak anchors. This dialog is used to delete a peak, to select a different function for this particular peak, or to individually adjust parameters and their states. A locked parameter is fixed at its current value and is not fitted. A shared parameter is shared with all other parameters at this particular parameter position who also have a shared state. Right click the peak that was added.

Change this peak to a Lorentz Amp model. Adjust the peak graphically and note the update in parameter values. Afterward, click on the Delete Peak icon to permanently remove this peak. To restore the autoscan conditions, click on the Reset AutoScan button. It is among the buttons at the top of the control panel. Click on the Full Peak Fit with Graphical Update button located near the bottom of the control panel.

With the widths varying across peaks, there is a greater potential for running up against a constraint, as discussed previously in this tutorial. In this case, one of the parameters is running up against a constraint bound. To achieve the optimum fit, we will again rerun the fitting using the new parameter estimates. Clicking on Addl Adjust and again select the Full Peak Fit with Graphical Update option. To insure that the optimum fit is achieved, rerun the fit one additional time.

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Note that the r value of 0.99114 is higher than the r value of the fixed width fits (0.99087). The least-squares goodness of fit is thus improved by allowing the 15 peaks to have different widths. But is this actually a better fit? The F-statistic for this variable width fit is 1101. For the fixed width fit, the F-statistic is 1584. By this particular information criterion, there is no evidence that variable widths exist. Click on Review Fit once the fit has converged. The Copy button in the graph's toolbar is used to copy the graph to either clipboard or file based metafiles or bitmaps. You can also copy all of the numeric information used for the graph to a large spreadsheet block.

The Data option in the Review dialog offers a point by point data summary with predicted values, residuals, confidence limits, and prediction limits. To explore this option, click on the Data button.

When finished, either click again on the Data button or close the Data window directly. The Eval option offers an interactive evaluation of the fit model. In addition to function evaluations, this option also offers first and second derivatives, cumulative and region areas, and the means to generate tables based upon the fitted model.

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Click on Eval and practice entering various function, derivative, and area evaluations of the model. You can use this procedure to compute the energy between any two spectral frequencies. You may also want to experiment with generating a table, a means to create a discrete data representation of the model, this at any data range and density desired.

The Export option is used to create ASCII, Excel, Lotus, Quattro, or SigmaPlot files containing the raw data as well as the predicted values for the overall model and each component peak. You may specify the actual range and increments of the X values used for generating the model and component values. If you wish to create an exported file, click on Export, select the file and type, and click on OK and then enter the desired file name. Click on OK to close the Review and then click on the OK (green check mark) button in the AutoFit option to preserve the current settings. Exit PeakFit by closing its main window or via the File menus Exit command.

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