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1. ee ren an gg egn Figure 75 Setup Identity Test Method Group statistics If you use the confidence level to calculate the threshold the Outlier column is very helpful to see at once the number of spectra per group which are outside the threshold To have the Hit Quality and File Name displayed click on the button of the respective Group Name line 92 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Validate 7 5 Setup Identity Test Method Validate Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentTuta Load Method Reference Spectra Parameters Threshold Validate Store Method I Selectivity _ ay a a ee g 1 da pe E Detailed report Ident_Demo 00 Jetalled repor Validation Report File name Ident_Demodata IP1 IP3 class_en Path C ProgrammeSOPUS6_O Data Extend Stored date time 5410 2004 6 58 32 Operator name Default Library Ident_Demo Sub libraries Carbonhydrates Diclofenac amp Nifedipin Sugar Lactose Nifedipin Always use lowest IP level No Parameters of library Ident_Demo Algorithm standard Vector normalized spectra Na From 10502 4 annan AA Figure 76 Setup Identity Test Method Validate tab Start validation by clicking on the Validate button The following menu pops alidate x Validate This Library Validate This Library and All Sub Libraries Below Figu
2. Load Method Reference Spectra Parameters Threshold Validate Store Method Load Method Rename Library ay Ident_Demo B Carbonhydrates Lactose E Diclofenac amp Nifedipin m General information of selected library Nifedipin Sugar Name Ident_Demo Groups 18 Spectra 726 Frequency ranges 1 Data preprocessing First derivative Algorithm Standard Sub libraries B Library Tree as Shown Printer Library Tree as Shown R PDF Whole Library Tree Whole Library Tree and Group Statistics Print Whole Library Tree and Statistics for Each Spectrum Figure 59 Setup Identity Test Method Load Method tab Use the Load Method button to load an existing IDENT method IDENT method files have the extension F AA It is also possible to load IDENT method files created by OPUS OS 2 However if you store such an OS 2 method using OPUS IDENT you will not be able to load the method by OPUS OS 2 IDENT again To solve this problem store the modified OPUS OS 2 IDENT file by using a different file name The General information of selected library group field provides statistical information on the existing method file The number of spectra used for the method and the number of frequency ranges included are displayed Bruker Optik GmbH OPUS IDENT 77 Reference Section 7 2 Add Spectra for New Group Add Spectra to Sel Group You will get additional informa
3. Sprungantwort 11228 o Testmischung Testmischung Sprungantwort 1230 0 Testmischung D Temp Conformity Mixing_TU_Munich Spec Sprungantweort 11234 0 Testmischung D TempiConformityMixing_TU_MunichiSpec Sprungantwort 232 0 Testmischung D Temp Conformity Mixing_TU_Munich Spec Sprungantwort 233 0 Testmischung D TempConformity Mixing_TU_MunichiSpec Sprungantwort 234 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 235 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 236 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 237 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 238 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 1239 0 Testmischung D iTemp Conformity Mixing_TU_MunichiSpec Sprungantwort 1240 0 Testmischung D iTemp Conformity Mixing_TU_MunichiSpec Sprungantwort 1241 0 Testmischung D iTemp Conformity Mixing_TU_MunichiSpec Sprungantyort 242 0 Testmischung D iTemp Conformity Mixing_TU_MunichiSpec Sprungantywort 243 0 Testmischung D iTemp Conformity Mixing_TU_MunichiSpec Sprungantwort 1244 0 Testmischung b E d x o D TempiConformityMixing_TU_MunichiSpec Sprungantwort 245 0 Testmischung DAADAAAAADAAAAAAAAAAA AAA AA aAl A DiTammiCan aemidRMivina TLL Mu eteten Crenimammtiain
4. Threshold 0 011451 0 079217 0 036291 0 028581 0 025936 0 067953 0 081288 0 023606 0 053122 0 029839 0 031277 0 029364 0 016202 0 092360 0 026632 72 OPUS IDENT Bruker Optik GmbH Class Test class1 FAA Signed by Bruker Optik 2002 02 28 1 Method file D itest42 Validation Ident methods class1 FAA From date 4 03 03 time 9 29 25 Description CAN BE CONFUSED WITH 11 OTHER HITS 1 Hit quality with expected reference 0 339112 Threshold for expected reference 0 694965 Threshold calculation depends on each ref spectrum Algorithm Standard vector normalized spectra Yes Order of Derivative 0 Smoothing points 1 No of used factor sp 0 15 hits of 15 x Ranges d Class Name HANS Class Test NOT OK 2 Using residuals No Order of Internal Derivative 0 Smoothing Points for Internal Derivative 1 Reduction Factor 1 Hit No Hit Quality Sample Name Group _ Threshold 1 0 339112 000001 Sample L Leucin Ay of 11 000001 0 694965 d 0 339817 000015 Sample L Methionin Av of 11 000015 0 883473 a 0 390038 000004 Sample DL Alanin Av of 11 000004 0 793804 4 0 453472 000007 Sample L Tryptophan Ay of 11 000007 0 995592 5 0 456483 000002 Sample DL Isoleucin Av of 11 000002 0 991531 6 0 514776 000014 Sample DL Methionin Av of 11 000014 0 885595 7 0 599937 000005 Sample L Alanin Ay of 11 000005 0 819521 3 0 624271 000006 Sample DL Tryptophan Av of 11 000006
5. OPUS Spectroscopy Software User Manual IDENT gt lt BRUKER LL 2006 BRUKER OPTIK GmbH Rudolf Plank StraBe 27 D 76275 Ettlingen www brukeroptics com All rights reserved No part of this manual may be reproduced or transmitted in any form or by any means including printing photocopying microfilm electronic systems etc without our prior written permission Brand names registered trademarks etc used in this manual even if not explicitly marked as such are not to be considered unprotected by trademarks law They are the property of their respective owner The following publication has been worked out with utmost care However Bruker Optik GmbH does not accept any liability for the correctness of the information Bruker Optik GmbH reserves the right to make changes to the products described in this manual without notice This manual is the original documentation for the OPUS spectroscopic software Table of Contents AbDOUCOPUS SU B MKO 1 Mmtroduction egiek sors eRe eee ee SS eb eae Wee eraren gero 1 1 Setting Up an Identity Test Method ce eee eee 3 1 1 Loading Existing Method aa 3 1 1 1 Methods created by prior OPUS releases a z 6 1 2 Creating New Method say Ea E E EE E e E enere 6 1 3 Setting Parameters Za sass AE e e Pd Ge aye Eate aria ee 10 1 4 Identity Vest Limit sis Zt Ori Oi E E ae SEO SE ere ee 13 1 5 Validating KLAU 14 Lovk Validation Report atea E pa Soe tek een ka
6. ow Nifedipin high Nifedipin slow Ident_Demo 0 379696 0 020507 d Nifedipin slow Nifedipin high Ident_Demo 0 379696 0 008811 HEA Nitrofurantoin po Diclofenac sodium P1 Ident Demo 2 338191 0 004601 E Pharmacoat Corn starch Ident_Demo 2 064307 0 003043 ek N Raspberry aroma Sugar Ident_Demo 1 203249 0 007993 EO Ident Demo 1 203249 0 018253 Sugar Raspberry aroma Figure 79 Selectivity Report The selectivity report displays the result in different colors Red spectra with a selectivity of lt 1 Green spectra with a selectivity of gt 2 Black spectra with a selectivity between 1 and 2 Gray spectra with a selectivity of lt 1 overlappings in the validation report without single spectra Note Generally single spectra are not relevant in case of selectivity however they are indicated in the validation report If there are no single spectra overlappings in this report the respective group is displayed in gray in the selectivity report It may occur that in case of libraries with reference spectra not any of these spectra is within the intersection of two clusters Therefore the validation would yield to a non overlapping result However the selectivity does indicate the geometric overlapping as exemplified in figure 80 96 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Validate Material 1 Material 2 Se
7. 3d File Filelist Parameters Report Store Method Options Score Plot Window EO Diagnosis for 5 classes 1 mlana ham AC errekun a Figure 94 Cluster Analysis Score Plot button Select Factorization from the Method drop down list on the Parameters tab and click on the Start Calculation button Define at least 3 factors from the Factor Spectra dialog which will serve as a basis for the 3D factor view Subsequently select Diagnosis from the drop down list and define the number of classes you want to see in the score plot by using the Options button For details on the Options dialog see chapter 7 11 2 Click on the Score Plot button PC 4 Figure 95 Cluster analysis 3D factor view Bruker Optik GmbH OPUS IDENT 111 Reference Section If you position the mouse on one specific spectrum the file name and group name will be displayed To improve the factor view you can rotate the box If you position the mouse on the edge of the box the cursor changes into EA To rotate the box press the left mouse button and move the mouse to the position desired Right clicking somewhere on the 3D view pops up the Properties button If you click on this button the View properties dialog is displayed which allows further plot settings 7 11 2 Options Click on the Options button to open the Cluster Analysis Options dialog You can define the algorithm used to calculate spectral distances between differ
8. Bruker Optik GmbH OPUS IDENT 19 Performing an IDENT Analysis If an identity test method has already been loaded e g if you have created a method prior to starting the analysis the path and name of this method name will be indicated in the Loaded Identity Test Method field To load or change an IDENT method click on the Load Ident Method button and select the desired method from the dialog box that opens The analysis uses the No Reference Defined function for an IDENT method if you have not defined an expected reference Click on the Change button to modify this default setting The Change button will be disabled if the operator has no right to change parameters of this kind For details on this dialog see section 7 7 1 Click on the dentity Test button to start the test The result of the analysis is appended to the respective file in the form of an IDENT report block If you click on this report block a report window opens automatically and displays the results 20 OPUS IDENT Bruker Optik GmbH Identity Test Reports IDENT Report Both the results of an identity test and the averaging of original spectra to generate reference files for an IDENT library are stored in report blocks You can open these reports like any other OPUS report by double clicking on the report block in the OPUS browser window 3 1 Identity Test Reports The results of the spectrum comparison between test spectrum and reference spectra ar
9. C Program OPUS6_0 Data Extended Demodata IdentTutorial lc Load Method Reference Spectra Parameters Threshold Validate Store Method piz Threshold DI Print List Maximum hit X SDev m Confidence Level 0 25 99 99 Set Set emane Secta elu HEE Testad A Acetylcystein 100 0 017578 2 Cellulose PH101 99 9988 0 0061317 3 Cellulose PH102 99 9968 0 0038525 A Cellulose PH112 99 8456 0 0040854 5 Corn starch 99 9999 0 018445 6 Diclofenac potassi 99 9849 0 011674 Diclofenac sodium i 99 5257 0 020664 Eudragit 99 9614 0 0099051 9 Glucose i 99 9996 0 0072312 10 Isosorbide dinitrat E 99 9998 0 0052611 41 Lactose 80mesh 100 0 0078193 Lactose 200mesh 99 938 0 011821 143 Nifedipin high 99 9862 0 028052 Nifedipin slow 100 0 01876 15 Nitrofurantoin pow 99 9833 0 005713 i Pharmacoat 100 0 0047947 amp Figure 11 Setup Identity Test Method Threshold tab 1 5 Validating Library When creating a library first check whether the IDENT parameters selected for the IDENT method are optimized for all reference spectra and whether an unambiguous assignment of test spectra to a group can be ensured This is done by a validation procedure which compares each original spectrum with the average spectra of all groups To validate the library click on the Validate tab The following dialog opens 14 OPUS IDE
10. Store Method Number of classes p J Number 0 for the largest class Store Trace Figure 99 3d File Filelist Defining number of classes Note If you activate the Jgnore Rest check box the biggest cluster will get the number 0 in the trace report 116 OPUS IDENT Bruker Optik GmbH 3D File Filelist The TRC data block zegi is added to the file displayed in the OPUS browser window Right click onto this data block to have the corresponding report displayed C Programme OPUS6_O Data Mehrschichtfolie 0 f Micron 1490_1420B 1700_1500B 1800 17008 ClusterList ClusterList ClusterList a Data parameters 4B Multiple 0 000000 0 418906 0 120632 0 173669 1 000000 1 000000 1 000000 4B Multiple 1 000000 0 278322 0 196559 0 116866 3 000000 3 000000 1 000000 Data parameters ScSmjMultiple 2 000000 0 151217 1 032597 0 395969 3 000000 3 000000 1 000000 ScSm Multiple 3 000000 0 101227 0 717422 0 241355 3 000000 3 000000 1 000000 4 000000 0 277226 0 700929 0 020516 3 000000 3 000000 1 000000 5 000000 0 547875 0 505013 0 018080 3 000000 3 000000 1 000000 6 000000 0 644494 0 847821 0 129880 3 000000 3 000000 1 000000 7 000000 0 671650 1 464416 0 143565 3 000000 3 000000 1 000000 8 000000 1 109604 1 766877 0 075675 3 000000 3 000000 1 000000 9 000000 1 526494 2 598635 0 043315 3 000000 3 000000 1 000000 10 000000 2 988753 2 730210 0 076485 3 000000 3 000000 1 000000 11 000000 4 816987 2 387223 0 199955
11. a k a k a 6 8 dig eea 6 9 Lay Zar 1 6 10 If vector normalized spectra are represented in n dimensional space and n being the number of selected data points all spectra are on the unit sphere n dimensional sphere around the coordinate origin with radius 1 see figure 46 The maximum distance between two spectra is the diameter of the unit sphere L e 2 Figure 46 Two vector normalized spectra on the unit sphere To explain this in more detail create a new spectrum Invert one reference spectrum from the example library i e multiply the spectrum by 1 using the OPUS Spectrum Calculator Compare the inverted spectrum with the reference spectra Select an IDENT method that preprocesses data by Vector Normalization Figure 47 shows the original spectrum top and the inverted spectrum down Figure 48 shows the identity test result The last hit in the result list is the original reference spectrum with a spectral distance of 2 compared to the test spectrum 62 OPUS IDENT Bruker Optik GmbH Data Preprocessing 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 Figure 47 Original and inverted spectrum One advantage of Vector Normalization is that the range of values for Hit Quality is known from 0 to 2 This simplifies the interpretation of the identity test result Additionally using Vector Normalization as data preprocessing provides an even more important aspect Bruker Optik
12. 0 identical spectra to 2 maximum spectral difference This does not apply to D when using the Factorization method If all factor spectra are used the spectral distances between reference spectra are constant i e D J2 To calculate spectral distances the elements of L Eigen vector matrix are used This matrix consists of orthogonal unit vectors in s dimensional space The distance between two orthogonal unit vectors always has to be doe However when using the Factorization method not all factor spectra are used because the higher factor spectra mainly increase the noise level of the reconstructed spectrum The Factorization method also allows to calculate spectral distances by using residuals For details see chapter 7 3 The spectral residual is calculated from the difference between the original and reconstructed spectrum To calculate SpecRes spectral residual u being an arbitrary spectrum the equation is as follows SpecRes IE u k ky fiK ky H k k fK 6 21 k The summation is performed for all selected k data points D spectral distance between u spectrum and d reference spectrum is calculated as follows D J La hy Lgy kp Lg SpecRes pestea 6 22 Figure 45 shows a scheme representing the Factorization method 58 OPUS IDENT Bruker Optik GmbH Factorization Theory C Reference Spectra D J Factorization Factor Spectra F Coefficients Eigen
13. 0 01146 Starch s 5 Starch from wheat n 0 20849 0 00602 Starch f02 6 Starch from potato n 0 22265 0 02762 Starch f00 f Lactose monohydrate n 0 25061 0 00951 Lactose 01 8 Lactose 80 mesh_110250_01_KFO 0 26170 0 04676 Lactose 00 9 Lactose 200 mesh_110251_02_KFO 0 29578 0 04622 Lactose 10 Glucose monohydrate n 0 29989 0 00736 Glucose IDENTIFIED AS Cellulos VY EK Figure 85 IDENT Evaluation Result display Result OK In the lower part of the display the Identity Test result is indicated A green check mark and the description OK would indicate that the test has passed i e the product has been identified A red cross and the description NOT OK would indicate that the comparison has failed i e the product has not been identified 102 OPUS IDENT Bruker Optik GmbH Identity Test Result of IDENT Evaluation Sample C OPUS Daten_Ident_Kurs Cellulose microcryst Cellulose microcryst Cellulose 5 Method File C OPUS Daten_Ident_Kurs DemoMethod FAA Date and Time 20 08 2001 14 43 52 Hit No Sample Name Hit Qual Threshold Group 1 Cellulose microcryst n 0 05012 0 01046 Cellulos 2 Starch from rice n 0 13808 0 01013 Starch f01 3 Starch from corn n 0 16486 0 00905 Starch f 4 Starch soluble n 0 17751 0 01146 Starch s 5 Starch from wheat n 0 19599 0 00602 Starch f02 6 Starch from potato n 0 20626 0 02762 Starch f00 ZO Lactose monohydrate n 0 24137 0 00951 Lactose 01 8 Lactose 80 mesh_110250_01_KFO 0 25294 0 04676 Lactose 00 9 Lactos
14. 0 904797 9 0 679802 000003 Sample L Isoleucin Av of 11 000003 0 920193 10 0 757945 000011 Sample Xylit Av of 11 000011 0 725310 11 0 760448 000010 Sample Fructose Av of 11 000010 0 775521 12 0 922144 000008 Sample Glucose H2Ofrei Av of 11 000008 1 091812 13 0 934589 000012 Sample Sorbit Av of 11 000012 0 791596 14 1 023140 000013 Sample Mannit Ay of 11 000013 1 032565 15 1 061457 000009 Sample Glucose H20 Ay of 11 000009 0 976118 Figure 55 IDENT Report with class test performed Test Not OK Code numbers are assigned to the individual class test results This causes the results to be easily evaluated in an OPUS macro Table 3 Code Numbers of Class Test Class Test Result Code Class Test OK 1 Class Test not Performed 0 Class Test not OK 2 Bruker Optik GmbH OPUS IDENT 73 IDENT Theory 6 7 Validation When setting up an IDENT library you have to check whether the IDENT method parameters are optimized for all reference spectra in one library This can be done by Validation which compares original spectra with average spectra Figure 56 shows a validation report using the Standard method All original spectra have been compared with the average spectra of the library The Abs Threshold values are used as confidence region in the validation process The results are either Uniquely Identified Not Identified and Can Be Confused With similar to the Identity Test see chapt
15. 1 GO S a 1 5 2 GU oa 2 ESE 3 Data preprocessing Vector normalization Ward s algorithm Frequency Ranges Standard 4000 11995 cm Method File TEMPVIEW CLA Date 17 11 2005 Figure 24 Cluster Analysis Options to modify report display To return to the report click on the Back to Report button The Options button on the Report tab also enables to modify the report display and to define the algorithm and matrix parameter in more detail You can select Sample Name from the Dendrogram drop down list in the Cluster Analysis dialog to change the dendrogram labeling accordingly You can also add file names sample names or file numbers to the dendrogram or have the dendrogram displayed without any labeling 36 OPUS IDENT Bruker Optik GmbH 3D Files Filelist Cluster Analysis Options x Ward s algorithm DI r Dendrogram Sample Name ss m Diagnosis Number of classes 2 Histogram Which part of the matrix do you want Whole matrix DI Coordinates for rectangle fi fi fi fi Width for classes of distances 0 0808758 Cancel Figure 25 Cluster Analysis Options For further details on the Cluster Analysis dialog see section 7 11 4 3 3D Files Filelist Instead of single spectra it is also possible to use 3D files or file lists in connection with the cluster analysis Note that not more than one 3d file or file list can be loaded t
16. 5257 0 020664 a Eudragit E 99 9614 0 0099051 99 9996 0 0072312 IU Isosorbide dinitrat 5 99 9998 0 0052611 11 Lactose 80mesh 100 0 0078193 Lactose 200mesh i 99 938 0 011821 Nifedipin high d 99 9862 0 028052 d4 Nifedipin slow 100 0 01876 OS Nitrofurantoin pow f 99 9833 0 005713 16 Pharmacoat E 100 0 0047947 Figure 74 Setup Identity Test Method Threshold tab The threshold of a reference spectrum is the sum calculated by the maximum distance maximum Hit Quality listed in Group Statistics plus the amount resulting from standard deviation SDev and a user defined x factor The threshold values listed in the Threshold column are indicated for each reference spectrum 7 4 1 Maximum Hit X SDev This formula is used to calculate the threshold value You can enter any value for X in the entry field with 0 25 being set as default To confirm your entry click on the Set button This causes the new value to be set for all reference spectra and the Threshold values will be updated 7 4 2 Confidence Level Two parameters are derived from the spectral distance to define the confidence level You can enter any factor between 95 and 99 9999 into the entry field 99 99 is set by default To confirm your entry click on the Set button See also chapter 1 4 90 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Threshold 7 4 3 Set If you click
17. Ay of 11 Figure 48 Ident Result of inverted spectrum searching 4N000009 100 4N000008 100 AN000013 100 AN000012 100 AN000010 100 AN000011 100 AN000007 100 ANO00006 100 A4N00001 4 100 ANO00005 100 ANOO0004 100 4N000015 100 AN000002 100 ANOO0003 100 4N000001 100 0 026233 0 090445 0 017529 0 029502 0 030262 0 032523 0 051395 0 022966 0 072976 0 073910 0 011690 0 061757 0 033536 0 023866 0 021304 Vector Normalization also reduces the differences between each single measurement of the same sample Figure 49 shows 11 spectra acquired from one single sample As the substance has been powder the single spectra differ substantially from each other These differences can be considerably reduced by using Vector Normalization Note the different scaling of the ordinate y axis in figure 49 Spectra derived from the same sample have to show only very small differences Therefore Vector Normalization is highly recommended in these cases Further data preprocessing methods are First and 2nd Derivative 64 OPUS IDENT Bruker Optik GmbH Data Preprocessing 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 Figure 49 Original and vector normalized spectra Bruker Optik GmbH OPUS IDENT 65 IDENT Theory Repeat the identity test for the first analytical example and select Vector Nor malization to preprocess data Figure 50 shows th
18. Bruker Optik GmbH OPUS IDENT 17 Setting Up an Identity Test Method 18 OPUS IDENT Bruker Optik GmbH Performing an IDENT Analysis Compared to the setup of an IDENT method the analysis of unknown samples is easy Before you start the analysis load the spectra of your unknown samples into the OPUS browser window The analysis compares the test spectrum with all reference spectra The result of a comparison between spectrum A and B results in the spectral distance D which is also called Hit Quality The better two spectra match the smaller the spectral distance The Hit Quality for identical spectra is 0 i e a reference spectrum is compared with itself To start an IDENT analysis select the Identity Test command from the Evaluate menu The dentity Test dialog box opens Identity Test E EA Select File s m File s for identity test m Expected reference as a m Loaded identity test method C Program OPUS6_O Data Extended Demodata IdentT utorial Ident_Demodata IP1 IP3 class_en FAA Load Ident Method Change m Output options M Show results immediately Print results automatically Identity Test Cancel Help Figure 14 Identity Test Select File s Drag and drop the absorption block of the test spectra from the OPUS browser window into the File s for Identity Test field If you release the left mouse button the spectra are added to this entry field
19. CI Limit Use Sum I Limit or Use Sum 2 Limit option button on the Store Method tab Bruker Optik GmbH OPUS IDENT 47 Conformity Test Setup Conformity Test C Program OPUS6_0 Konformitatstest Mixing_TU_Munich Mixing_ TU Munich 1stD21ptCrT Figure 36 Setup Conformity Test Store Method tab 5 2 Performing Conformity Test Start the conformity test by clicking on the Conformity Test command The following dialog opens 48 OPUS IDENT Bruker Optik GmbH Performing Conformity Test Conformity Test x Select Files m Files for Conformity Test Ci Programmet OPUS Konformit tstesti Mix Loaded Conformity Test Method Programme OPUS Konformitatstest Mix Load Conformity Test Method Figure 37 Conformity Test Select Files tab Drag amp drop the file s to be evaluated from the OPUS browser window into the File s for Conformity selection field Click on the Load Conformity Method button and load the particular method which path is displayed above this button The Conformity Test results will be stored in a CONF data block cone and displayed in a specific report view C Programme OPUS Konformitatstest Conformity Test Repo Conformity Test Report AB Method File mixing_tu_munich_1std21pt cft 2003 10 08 07 44 36 GMT 2 Conformity Test Report Conformity Test Mode CI Limit Conformity Index Limit 3 0 Sum Limit 0 30 Use Signed CI Values
20. D 1 6 5 After this transformation the spectral distances of the second spectral range have the same values as the distances belonging to the first spec tral range As the scaling has referred to the first spectral range it does matter which spectral range is selected first Make sure to select the correct spectral range as first range If spectral distances are sorted according to ascending values this order directly results from the spectral ranges selected For example when comparing a test spectrum with a reference spectrum using an IDENT 60 OPUS IDENT Bruker Optik GmbH Data Preprocessing test the spectral distance may have the lowest value to the fourth refer ence spectrum best Hit Quality based on the first spectral range If you consider the second spectral range the spectral distance may have the lowest value to the eleventh reference spectrum 4b When using the Normalize to Reprolevel method you have to define a reproduction level see figure 71 for each spectral range The spectral distances will be divided by this reproduction level This is the reason why the spectral distances are indicated as reproduction level units which means that you can set a threshold for the identity test For exam ple if the Hit Quality is below 1 in case of a test spectrum the sample is regarded as being dentified If however the spectral distance is above 1 the spectrum cannot be assigned to any reference spectrum 5
21. GmbH OPUS IDENT 63 IDENT Theory Report of Correlation Search W S Ident example2 S tandard FAA Method file from date time Description 24 05 00 15 10 08 IDENTITY NOT CHECKED 0 Hit quality with expected reference 0 000000 No Threshold avail Threshold calculation Algorithm 0 000000 depends on each ref spectrum Standard Vector normalized spectra Yes Order of Derivative Smoothing points No of used factor sp 15 hits of 15 x Ranges Class Name AU 1 0 Class Test NOT PERFORMED Using residuals Smoothing Points for Internal Derivative 0 N Order of Internal Derivative 0 1 1 Reduction Factor _Hit No Hit Quality Sample Name FileName Threshold 1 812099 1 849116 1 854669 1 877448 1 883937 1 885355 1 978973 1 982136 1 992922 1 993975 1 994812 1 997453 1 997778 1 999440 2 000000 oon ZO GI b GA Dk zi 000009 Sample Glucose H20 Ay of 11 000008 Sample Glucose H20frei Av of 11 000013 Sample Mannit Av of 11 000012 Sample Sorbit Ay of 11 000010 Sample Fructose Av of 11 000011 Sample lt ylit Av of 11 000007 Sample L Tryptophan Ay of 11 000006 Sample DL Tryptophan Av of 11 000014 Sample DL Methionin Ay of 11 000005 Sample L Alanin Av of 11 000004 Sample DL Alanin Av of 11 000015 Sample L Methionin Ay of 11 000002 Sample DL Isoleucin Av of 11 000003 Sample L Isoleucin Ay of 11 000001 Sample L Leucin
22. I A 1000 1500 2000 2500 3000 3500 0 1000 2000 3000 400C X Wavenumber cm 1 Z ClusterList Figure 97 3D view For further details on the 3D window settings in OPUS refer to the 3D manual Now select the Cluster Analysis command from the Evaluate menu and click on the 3d File Filelist tab The following dialog opens Bruker Optik GmbH OPUS IDENT 115 Reference Section Cluster Analysis New i x Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Number of classes B F Number G for the largest class A E Te C Programme OPUS6_O Data Mehrschichtfolie 0 Figure 98 Cluster Analysis 3d File Filelist tab Drag amp drop the spectra data block of the 3D file into the entry field A in figure 98 You cannot load more than one 3D file for a cluster analysis To remove the spectra data block select the spectra file in the entry field and click on the x button Now click on the Parameters tab define the frequency regions and select an identification method Click on the Start Calculation button Depending on the number of spectra the calculation procedure can take quite some time If the calculation has been finished click on the 3D File Filelist tab again Define the number of classes and click on the Store Trace button which is now enabled Cluster Analysis C Program OPUS6_O0 IDENT 3d_4_Groups CLA Load Method Reference Spectra 3d File Filelist Parameters Report
23. IDENT 7 Setting Up an Identity Test Method x Astign IP2 Library Delete Sub Library Carbonhydrates v Enter name for new sub library Select groups for Carbonhydrates Nitrofurantoin powder Pharmacoat Cancel Figure 6 Set Sub Libraries As the spectra of the Carbonhydrates group have already been assigned as a sub library they are automatically highlighted figure 6 The indicator field shows the defined sub library IP2 level in green You can always set sub libraries exactly one level below the current library All available groups defined for the main method and which have not yet been assigned to any sub library on this level are displayed in the Select groups for field Select the New option from the upper right drop down list and enter a unique name for the new sub library Then click on the group s you want to assign If you define a new sub library name OPUS automatically checks whether the new library name does already exist to avoid double naming If a name already exists a dialog pops up requiring unique naming Click on the Assign button To delete sub libraries select them in the selection field and click on the Delete Sub Library button A menu pops up and asks you whether you want to continue or cancel the deleting operation Groups which cannot be separated into further sub libraries can however be assigned to common classes if class identity is sufficient as analysis result 8 OPU
24. Irrespective of the method used spectral distances can be weighted for each single spectral range see chapter 7 3 according to the following equation 2w D Zun Spectral distances calculated by the Normalize to Reprolevel algorithm may be above 2000 if reproduction levels are other than 1 When using the Scaling to Ist Range method the values have to be between 0 and 2000 D 6 6 6 3 Data Preprocessing OPUS provides several data preprocessing methods 6 3 1 Vector Normalization The maximum value of the Hit Quality has to be defined only if Vector Normalization was used to preprocess data If you use a preprocessing method other than Vector Normalization no upper limit for the Hit Quality has to be defined i e you can use any numerical value The maximum spectral distance is 2 maximum difference of the spectra in case of Vector Normalization provided you have selected Standard method Vector Normalization first calculates the average y value of spectra and only uses data points within the selected spectral ranges The average value calculated will then be subtracted from the spectrum which causes the spectrum to be centered at around y 0 This is followed by calculating the sum of squares of all y values and the respective spectrum is divided by the square root of this sum The vector norm of the result spectrum is 1 gt alk k 6 7 an be 6 7 Bruker Optik GmbH OPUS IDENT 61 IDENT Theory
25. MgO 71 7 2 Setup Identity Test Method Reference Spectra u z 78 7 2 1 Sorting Reference Spectra ausaz 79 7 2 2 Missing Reference Spectra sc5 vein Meas as en eet ee eet yes 79 J239 Options spor bade os thie zaie arte e eed Se Lae etz 81 T24 aba BAO 81 720 ssiom Classes OO Sea a e EE A EE a 83 73 Setup Identity Test Method Parameters a z 84 TL A da AAA 84 GO JE berd gaitz ia ba Desiree pb iE aiuta 85 7 3 3 Interactive Region Selection agarra erreen erare na eaas 85 7 3 4 Clear Selected Regions a ega ia atu ive 048 87 Tom Method a SEE EEE Sue Sc EES a HES ORS Ea ebate 87 F36 Calculat Thresholds aar ce yas e gaia RE aS 88 7 4 Setup Identity Test Method Threshold assa 90 LGA ERLE ED der dA AAE 90 TAZ Confidence bevel a det Gg tine teks ee aa ay AT Ge de E a 90 TAS bia de ez Egitate ebiden baga E Obs ease acy aa ame 91 TAA Group Statistics lt biat Sg sreco Pad ota Pace RPE eee wares aed 91 7 5 Setup Identity Test Method Validate u z 93 T Validation REport atte Eb eta Ek e E ee aN 94 GN ATE 99 7 6 Setup Identity Test Method Store Method a z 100 7 7 beg AAE 101 dias No Reference Defined A bra Kt ea Aedes aie Para 103 7 8 Cluster Analysis Load Method 0 0 c cece cece neces 105 Lel Load Method argiga sr e Ze a eS yea eee eS 105 7 8 2 General Information gots dibi d dE El waa Bee ed EEE 106 7 9 Cluster Analysis Reference Spectra 000 e cece eee eee 106 7 10 Cluster Analysi
26. No Conformity Test Max CI Value Std Dev Sum1 Sum2 Data Points gt CI Limit Failed 0 22 25 4906 15 2 58E 005 9 14 10 7 537 Figure 38 Conformity Test Report Bruker Optik GmbH OPUS IDENT 49 Conformity Test 50 OPUS IDENT Bruker Optik GmbH Algorithms IDENT Theory The aim of an IDENT analysis is to determine the differences between a test spectrum and the reference spectra of a library You have to define a method to test the similarity of spectra and a threshold This threshold determines whether a spectrum is only similar or even identical to the reference spectrum 6 1 Algorithms Basically there are two algorithms to perform the IDENT analysis the Standard and Factorization method During the analysis both methods compare the test spectrum with all reference spectra The result of a comparison between two spectra is the Hit Quality also referred to as spectral distance D The better two spectra match the smaller the spectral distance The Hit Quality for identical spectra is 0 i e if a reference spectrum is compared with itself 6 1 1 Standard Method Figure 39 shows two spectra a and b one test and one reference spectrum The spectral distance D is proportional to the area between these two curves The following formula for the so called Euclidean distance is used in the Standard method pe IE atu KEI 6 2 k where a k and b k are the ordinate values of the a and b spectra The sum in
27. Reference Spectra tab and load the respective reference spectra by clicking on the Add Reference Spectra button A dialog pops up and displays a browser window which you have to use to search for and select the spectra Click on the Open button to load the spectra 40 OPUS IDENT Bruker Optik GmbH Setting up Conformity Test Load Method Reference Spectra Test Spectra Parameter Validate Graph Report Store Method Add Reference Spectra Change Path D Temp Conformity Mixing_TU_MunichiSpec Setup Conformity Test C Program OPUS Konformitatstest Mixing_TU_Munich Mixing_TU_Munich_1stD2ip Sprungantyvort 1220 0 Set Data Set I Sera Sample Hame Testmischung D TempiConformityMixing_TU_MunichiSpec Sprungantwort 221 0 Testmischung D Temp ConformityMixing_TU_MunichiSpec Sprungantwort 222 0 Testmischung D Temp ConformityMixing_TU_MunichiSpec Sprungantwort 223 0 Testmischung D Temp ConformityMixing_TU_MunichiSpec Sprungantwort 224 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 225 0 Testmischung D Temp ConformityMixing_TU_MunichiSpec Sprungantwort 226 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 227 0 Testmischung D Temp Conformity Mixing_TU_Munich Spec D Temp Conformity Mixing_TU_Munich Spec D Temp Conformity Mixing_TU_Munich Spec Sprungantwort 1I229 0
28. To calculate spectral distances spectra have to be vector normalized first for each frequency range Then the r Pearson correlation coefficient is calculated This kind of coefficient defines the correlation between a and b spectra r GUE b k 6 2 This correlation is calculated separately for each frequency range The summation covers all k data points of a frequency range a and 5 are the normalized spectral intensities The normalization yields a k am bp Ck bm r a a a 6 3 S Ca lk an JEK am and b are the mean spectral intensities within the spectral range while a k and b k are the original spectral intensities The value range of r correlation coefficient reaches from 1 inverse spectra to 1 identical spectra The correlation coefficient is transformed into D spectral distance by the following equation D 1 r 1000 6 4 D spectral distance can be between 0 identical spectra and 2000 inverse spectra The Scaling to Ist Range determines the minimum and maximum value of spectral distances for the first spectral range Then the distances of all the other spectral ranges are calculated and scaled to the same range of values i e the same minima and maxima like the first spectral range Example The spectral distances in the first spectral range are between 2 and 10 in the second between 6 and 22 The distances of the second spectral range are transformed as follows D gt 0 5
29. enabled on the very first library level as this a global setting for the entire library structure and is deactivated by default See also chapter 1 IDENT methods which have been stored created and stored by previous OPUS without this algorithm can be loaded as well to perform this kind of analysis Depending on the method selected additional columns are added to the Regions table e g Weight and Reprolevel Regions rdi eio ee 3999 8 ji Figure 71 Regions list with weight and reprolevel columns Bruker Optik GmbH OPUS IDENT 87 Reference Section You can select between the following identification methods e Standard The Standard method calculates the Euclidean distance between the test and reference spectra e Factorization The factorization is performed on average spectra of the respective groups The spectra are first represented as linear combination of the factor spectra and the resulting coefficients are used to calculate the spectral distance e Factorization orig specs The factorization is performed on all original spectra of the respective groups e Scaling to 1st range Performs the Scaling to Ist range algorithm For details see section 6 2 1 e Normalize to Reprolevel Performs the Normalize to Reprolevel algorithm For details see section 6 2 1 7 3 6 Calculate Thresholds If you click on the Start Calculation button you start the calculation If you select the Facto
30. from 0 to 1 the second class includes spectral distances from 1 to 2 etc Each class is represented by a bar in the histogram This bar indicates the percentage frequency of spectral distances compared to the total number of distances considered In this context Class means something different than in case of clustering where cluster can also be referred to as Class or Group Besides graphical representation the Histogram includes statistical information Cluster Analysis C Programme OPUS6_0 IDENT Study _5 CLA E x Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Histogram Window IO Histogram for d values al ez ez ea Figure 93 Cluster Analysis Histogram e Diagnosis This view produces a horizontal cross section of the dendrogram Specify the number of classes to create a list which includes the members of each single class The spectral distance of the last clustering will be displayed for each cluster 110 OPUS IDENT Bruker Optik GmbH Cluster Analysis Report 7 11 1 Score Plot The cluster analysis report can also be read out as score plots in 3D format which is indicated by the B Factor view tab The additional Score Plot button will only be displayed if you have selected Factorization as analysis method and Diagnosis as report before Cluster Analysis C Program OPUS6_0 IDENT Factor_scoreCoeff CLa Load Method Reference Spectra
31. on the Set button all changes made will be displayed on the table list If you have selected only part of the column only the values of the lines selected will be changed 7 4 4 Group Statistics The Group Statistics parameter includes detailed information on the Group and File Name Hit Quality Standard Deviation and Mean Distance Two parameters are derived from the spectral distances between original spectra and the average spectrum to define the confidence region for a group Dy mean distance Dy 20 7 1 7 2 with n being the number of original spectra Select the Group Statistics option from the selection box The following dialog opens Bruker Optik GmbH OPUS IDENT 91 Reference Section Setup Identity Test Method C Programme OPUS6_0 Data Extended Demodata IdentTutorial de it Load Method Reference Spectra Parameters Threshold Validate Store Method Maximum hit SDev Confidence Level 0 25 EGON do Set Set amp 1_ Acetylcystein Acetylcystein Cellulose PH101 0 001855 b 01475 Cellulose PH102 0 001577 0 001441 Cellulose PH112 0 001916 0 001714 Cellulose_ 0 003606 Cellulose_ 0 003286 Cellulose_ 0 002961 Cellulose_ 0 002953 Cellulose_ 0 002598 Cellulose_ 0 002477 Cellulose_ 0 002147 Cellulose_ 0 002049 Cellulose_ 0 002047 Cellulose_ 0 001987 Cellulose_ 0 001971 Cellulose_ 0 001952
32. reference The code number 2 is used if all reference spectra with a Hit Quality less than the threshold have the same Sample Name or Sub Sample Name as the query spectrum The value of 2 may only occur if the Selected automatically from Sample Name option button on the Expected Reference dialog figure 87 page 104 has been activated Bruker Optik GmbH OPUS IDENT 69 IDENT Theory Table 2 Code Numbers of Identity Test Identity Test Result Code Identity not Checked no threshold available 0 Identical To Identified As l Can be Confused with No Unique Identification Possible 1 Not Identical Not Identified 2 Can be confused with lt N gt Other Hits 2 ER D lt Dr D gt Dr Identified Not Identified Figure 51 Schematic representation of Identity Test Results Test Spectrum TS Expected Reference ER 6 6 Class Test Ideally all reference spectra are well distinguishable from each other and the thresholds are so small that their confidence regions do not overlap This situation is shown in figure 52 The reference spectra are dots in a mathematical sense in the n dimensional space n being the number of data points selected The confidence regions can be depicted by spheres which centers represent the reference spectra 70 OPUS IDENT Bruker Optik GmbH Class Test O Da oe Figure 52 Library with well distinguishable reference spectra However it may oc
33. right click onto the window and select the respective cluster list from the Select trace drop down list on the Mapping tab For all the other plot options refer to the 3D manual 118 OPUS IDENT Bruker Optik GmbH 3D File Filelist 24450 24350 Rotation E Inclination 20 E x 47949 29 x 48007 92 Z 24361 29 47900 47950 48000 48050 x Micron 4000 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 z 24419 92 zi Figure 102 3D plot of TRC data block with clusters 7 12 1 File List If you use a file list several spectra of a particular spectrum type are combined into one common file list First create a file list by the Setup File List command in the Edit menu and store it Drag amp drop the LIST data block Aust into the entry field A in figure 98 Now click on the Parameters tab define the frequency regions and select an identification method Click on the Start Calculation button You can also store traces before you have defined the number of classes on the 3d File Filelist tab To open the TRC data block right click on the file list name in the OPUS browser window and select Show Parameters from the pop up menu The TRC data block is now added to the file list in the browser window Click onto the data block to be able to see the trace results Bruker Optik GmbH OPUS IDENT 119 Reference Section 7 13 Cluster Analysis Store Method Cluste
34. spectrum is equal to the square root of the corresponding Eigen value Using L orthogonality D data matrix can be as follows T D D 1 D L L F L 6 15 The reference spectra are represented as linear combinations of the factor spectra and the coefficients are contained in the columns of L matrix Based on the 6 15 equation the following applies to the first reference spectrum T T T T di Lii fitti htl At tL h 6 16 The score coefficients are the coordinates of the reference spectra in the factor spectra system Any u spectrum can be represented as linear combination of the factor spectra u F k e 6 17 Bruker Optik GmbH OPUS IDENT 57 IDENT Theory The unknown k column vector corresponds to the column elements of L matrix E error spectrum is the difference between the u spectrum and reconstructed spectrum The Least Squares solution for k which minimizes the error is as follows T 1 T Egea OR N oe 6 18 If only the first r factor spectra are taken into account D spectral distance between one u spectrum and one d reference spectrum is GARE S E ada 6 19 Instead of using the column vectors of L matrix you can use L row vectors The equation for D spectral distance in r dimensional factor space is then Ge iba AE ebb thee Lee 6 20 When using the Standard method you can specify a range of values for D distances Select Vector Normalization preprocessing The range of values reaches from
35. standard deviation 0 00100 0 00000 y Z x 1S 8 f y 9 1 Y A y OR g A NIO s d 5 W b l i y 8 y 8 6 9 9000 8500 8260 8000 7750 7500 7250 7000 6750 6500 6250 6000 5750 5500 5250 5000 4750 4600 4250 Mixing_TU_Munich_1stD21p Mixing_TU_Munich_1stD21p E M First derivati E Miing_TU_Munich_1stD21p diera R E Sprungantwort 1220 0 e 2 8 gt Interactive Region Selection EA Figure 31 Preprocessed spectra plot Use the selection box on the lower part of the dialog figure 31 to have the spectra selectively displayed Deactivate the Show check box of the spectra which you do not want to have displayed If you click on the Interactive Region Selection button the standard Select Frequency Range s dialog opens which you can use to interactively set the frequency range To continue with the conformity test click on the Go to Setup Conformity Test button If you have defined all the parameters required click on the Validate tab This tab only includes the Validate button Click on this button and you will automatically be transferred to the Graph tab 44 OPUS IDENT Bruker Optik GmbH Setting up Conformity Test C Programme OPUS6_0 Konformitatstest Mixing_TU_Munich Mixing_TU_Munich_1stD 2 1p I x Setup Conformity Test Load Method Reference Spectra Test Spectra Parameters Validate Graph Report Store Method E Conformity Index x Refe
36. the smallest spectral distance is 21 2 in this matrix the D and E spectra are merged into the DE cluster Then the distances between this new cluster and all other spectra will be calculated again Example The spectral distance between AC and D is 99 5 and between AC and E is 117 1 Based on these values the distance between AC and DE will be 108 3 The third matrix in figure 19 includes these new distance values Bruker Optik GmbH OPUS IDENT 27 Cluster Analysis In the next step the B spectrum is merged with the AC cluster into a new ABC cluster The distance between the ABC and DE cluster is 94 2 Finally the ABC and DE clusters are merged into the ABCDE cluster The y axis of the dendrogram shows the spectral distances between different clusters The horizontal lines indicate the fusion levels which are the spectral distances of the different clusters and spectra prior to new clustering Table 1 Clustering Process Number of Clusters Clusters 5 A B C D E 4 AC B D E 3 AC B DE 2 ABC DE 1 ABCDE You have to generate a cluster analysis method before a dendrogram can be graphically displayed While you generate the method the spectral distances between the different spectra are calculated The clustering is repeated until all spectra are merged in one single cluster Sometimes the intermediate states are of great interest for the user If you use the Make Diagnosis function you ca
37. to create a printout Note It is recommended to select a small font in Windows Notepad to avoid extraordinary long reports A proportional font may lead to a confusing display of the results Therefore it is advisable to use a monospace font e g Courier New 10 Bruker Optik GmbH OPUS IDENT 99 Reference Section 7 6 Setup Identity Test Method Store Method Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata Ide n Load Method Reference Spectra Parameters Threshold Validate Store Method Store Method All sub libraries will be stored simultaneously r Settings Number of hits in Ident report fio IV Store Average Spectra Figure 83 Setup Identity Test Method Store Method tab This dialog allows to store a method file created The parameter you can define is the number of Hits to be stored in the IDENT report By default the Store Average Spectra is activated Click on the Store Method button to open the standard Save File dialog box The method file has the extension FAA All sub libraries will be stored simultaneously 100 OPUS IDENT Bruker Optik GmbH Identity Test 7 7 Identity Test To start an IDENT analysis select the dentity Test command from the Evaluate menu The following dialog box opens CK x Select File s ke m File s for identity test m Expected reference _ m Loaded identity test method C Program OPUS6_O Da
38. to limit the IDENT analysis to certain frequency regions enter them into the Regions table Alternatively you can also define these regions interactively Bruker Optik GmbH OPUS IDENT 11 Setting Up an Identity Test Method Click on the Interactive Region Selection button A separate window opens which displays mean spectra Select Frequency Range s E 12000 11000 10000 9000 8000 7000 6000 5000 4000 Cancel Figure 9 Interactive Frequency Range Selection The gray areas indicate the selected frequency limits and the white spectral range is the basis for the subsequent evaluation To move spectral regions place the cursor on the respective edge between the white and gray area hold down the left mouse button and move the regions To delete a spectral region right click on the white area and select Remove from the pop up menu Click on the OK button to confirm the settings and the Parameters tab will be displayed again You can also add a new frequency region by a right click on the left or right window side Select the Add Region option from the pop up menu figure 10 12 OPUS IDENT Bruker Optik GmbH Identity Test Limit Select Frequency Range s 2 00 Zoom A Scale All Spectra gt Shift Curve gt Crosshair 175 1 50 4dd Annotation 1 26 Cop Copy All Paste 075 Properties 0 60 0 26 0 00 12000 11000 10000 9000 8000 7000 6000 50
39. values A LT Spectral Distance Coefficients k Test Spectrum u gt Figure 45 Factorization Spectral distance calculation 6 2 1 Scaling to 1st Range and Normalize to Reprolevel Scaling to Ist Range and Normalize to Reprolevel are algorithms that can be used to identify microorganisms Contrary to the Standard and Factorization method overlapping spectral ranges are not merged For example if you set 1500 1200cm as first frequency range and 1500 1400cm as second frequency range they will not be combined into one frequency range and the data points in the 1500 1400cm range will be considered twice to calculate spectral distances The single spectral ranges can be weighted by different factors These factors are defined in the Weight column see chapter 7 3 The Vector Normalization preprocessing is not available in combination with the Scaling to Ist Range and Normalization to Reprolevel algorithms as a vec tor normalization will be automatically performed in this case Contrary to the Bruker Optik GmbH OPUS IDENT 59 IDENT Theory Standard and Factorization algorithm the vector normalization is calculated separately for each spectral range The resulting values are used to determine a mean distance The vector normalization considers all data points selected when using the Standard and Factorization algorithm Calculating spectral distances 1 2 3 4a
40. values T The selectivity will be calculated as follows A with A being the and T cluster radii This results in the following e S lt 1 overlapping e S 1 cluster in contact e S gt 1 cluster separated Figure 79 exemplifies a selectivity report Bruker Optik GmbH OPUS IDENT 95 Reference Section Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata Ident Tutorial ident x Load Method Reference Spectra Parameters Threshold Validate d Store Method Gb rS Sk de t 1 Ident_Demo 00 E Selectivity report Diclofenac sodium Ident_Demo 1 945901 0 011937 Acetylcystein Cellulose PH101 Cellulose PH102 Ident_Demo 0 139743 0 004042 Cellulose PH102 Cellulose PH101 Ident_Demo 0 139743 0 002844 Cellulose PH112 Cellulose PH102 Ident_Demo 0 206656 0 003579 Corn starch Cellulose PH101 Ident_Demo 1 136996 0 010709 Diclofenac potassi Diclofenac sodium IP1 Ident_Demo 0 694469 0 008801 Diclofenac sodium Diclofenac potassi Ident_Demo 0 694469 0 019407 CO wN E On d LA b e Eudragit Nifedipin high Ident_Demo 1 494229 0 007812 wo Glucose Diclofenac sodium IP1 Ident_Demo 1 900884 0 004944 EO Isosorbide dinitrat Lactose 200mesh Ident_Demo 1 459602 0 003390 pk Lactose 80mesh Lactose 200mesh Ident_Demo 0 777014 0 004036 N Lactose 200mesh Lactose 80mesh 0 777014 0 009502 Ident_Demo
41. vector normalization The lowest spectral distance is 0 009 the next 0 33 is higher i e by about a factor of 30 This factor is higher than the one obtained without using Vector Normalization factor 6 The reference spectrum of Hit No 1 is L Leucin This identification is correct as the test spectrum has been measured from the same sample In general however the question is how far the spectral distance Hit Quality may increase to be still within an acceptable threshold to correctly identify the test spectrum To define such a threshold it is not sufficient to measure only one single spectrum per reference substance You must measure several spectra and determine this threshold from spectral fluctuations spectral differences The 66 OPUS IDENT Bruker Optik GmbH Determining Threshold Value for Identity Test average spectrum calculated from each measurement is then added to the IDENT library as reference spectrum 6 4 Determining Threshold Value for Identity Test There are two possibilities to define the limit value for an IDENT group 1 2 Fixed Algorithm Maximum Hit x SDev The threshold is calculated from the worst hit i e the largest Hit Quality value in the average report and the standard deviation S Threshold Dyr Dmax bd 6 11 whereas the default x value is 0 25 The threshold is selected so that all original spectra used to create the reference spectrum average spectrum
42. 00 4000 Figure 10 Interactive Frequency Range Selection with pop up menu Select Standard as method on the Parameter tab and click on the Start Calculation button to calculate the spectral distances 1 4 Identity Test Limit Click on the Threshold tab to have the identity test limits displayed The threshold of a reference spectrum is the sum calculated from the maximum distance listed in the average report plus the product resulting from the standard deviation SDev and any x factor For each group the threshold values are listed in the Threshold column figure 11 You can enter any factor into the entry field 0 25 is set by default Click on the Set button to confirm your entry The factor set is valid for all reference spectra and the Threshold column is updated accordingly You can also print the list Click on the Print List button For further details see chapter 7 4 In a similar way you can set the value for the Confidence Level You can enter any factor between 95 and 99 9999 into the entry field 99 99 is set by default If you click on the Set button to confirm your entry the Threshold column is updated accordingly The Outlier column displays the number of spectra which are outside the threshold If you e g set the confidence level to 95 then 5 of the total number of spectra will be identified as outliers Bruker Optik GmbH OPUS IDENT 13 Setting Up an Identity Test Method Setup Identity Test Method
43. 00 4095 000000 4095 000000 4095 000000 4095 000000 dMin 1 450400 8 559641 9 014781 1 000000 0 000000 1 000000 dMax 26 899309 181 258301 16 887852 4 000000 3 000000 4 000000 dSCF 1 000000 1 000000 1 000000 1 000000 1 000000 1 000000 dpka_fw 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 D gt I dpka bw 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 xl Figure 100 Cluster analysis TRC data report As the TRC data report in figure 100 exemplifies the Cluster List column includes the allocation to the classes defined before To have the traces scored in a 3D plot open the Map Vid Spec window by the New Registered Window command from the Window menu Drag amp drop the TRC data block into the first sub window Bruker Optik GmbH OPUS IDENT 117 Reference Section 24450 24400 2 Z Mic 24350 24450 3D Properties 3D Properties Contour Selection Mapping 47950 48000 X Micron 47900 Rotation 30 H Inclination 20 x 47949 23 x 48007 92 Se Z 24361 29 zZ 24419 92 Figure 101 3D plot of TRC data block 24350 Select rectangle Spectrum block 24400 Select trace 5 ClusterList 7 1490_14208 Select image 1700_15008 1800 17008 ClusterList ClusterList 5 ClusterList AB Multiple E 48050 47900 47950 48000 X Micron 1000 750 500 1750 1500 1250 To have the clusters displayed in the second sub window
44. 1 Cluster Analysis Report Click on the Report tab to have the analysis results displayed Cluster Analysis C Program OPUS6_O0 IDENT Study SOILA Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Dendrogram Window EI 000001 10 o00002 12 000001 ZU 000001 14 o00002 14 000001 12 000001 IE 000001 14 000001 15 000001 17 000002 20 000001 18 o00002 13 000002 17 000001 19 000001 13 o00002 10 000002 18 DUO I o00002 19 o00002 15 DUO IE ao Cc GO E D c oa ee o x Figure 92 Cluster Analysis Report tab Use the drop down list to define the form of report You can have the results displayed in the form of a dendrogram histogram or diagnosis Dendrogram is set by default e Dendrogram A dendrogram includes the spectral distances of all reference spectra Right click on the dendrogram and a menu pops up displaying different options e Histogram This kind of report is not intended to be used to analyze clustering Instead the spectrum to spectrum distances between reference spectra are analyzed Such distances can be represented in the form of a symmetrical nxn matrix n being the number of reference spectra The mean value and standard deviation are calculated and the distance values are displayed in the form of a histogram and divided Bruker Optik GmbH OPUS IDENT 109 Reference Section into classes The first class e g ranges
45. 15 IDENT Report Query spectrum identical 0 033536 0 061757 0 011690 0 073910 0 072976 0 022966 0 051395 0 032523 0 030262 0 029502 0 017529 0 090445 0 026233 Figure 16 shows a test report for the same query spectrum But this time DL Methionin has been selected as expected reference Now the result is NOT IDENTICAL to the expected spectrum The spectral distance to this reference spectrum is 0 172990 and Hit No 7 This value exceeds the threshold of 0 072976 and therefore the test spectrum is classified as not being identical 22 OPUS IDENT Bruker Optik GmbH Identity Test Reports E C OPUS IDENT examples 000001 5000C Values E Reports AB Method file C OPUS IDENT examples S TANDARD FAA Compare Spectra from date 16 06 00 Ese Compare Spectra time 10 15 01 Description gt gt NOT IDENTICAL lt lt to 000014 Sample DL Methionin Av of 11 Hit quality with expected reference Threshold for expected reference 0 072976 reshold calculation depends on each ref spectrum Algorithm Standard Vector normalized spectra Yes Order of Derivative 0 Smoothing points 1 No of used factor sp 0 15 hits of 15 x Ranges 1 Class Name Class Test NOT PERFORMED 0 Using residuals No Order of Internal Derivative 0 Smoothing Points for Internal Derivative 1 Reduction Factor 1 _Hit No Hit Quality Sample Name Group _ Threshold 1 0 010556 000001 Sample L Leuci
46. 4 000000 0 000000 1 000000 12 000000 8 534338 2 149328 1 021528 4 000000 0 000000 2 000000 13 000000 11 658361 2 408520 1 096437 4 000000 0 000000 2 000000 14 000000 13 718537 1 306824 1 135268 4 000000 0 000000 2 000000 15 000000 14 933686 0 474837 1 368319 4 000000 0 000000 2 000000 16 000000 16 100191 0 333270 2 150514 4 000000 0 000000 2 000000 17 000000 16 300209 0 140034 2 622877 4 000000 0 000000 2 000000 18 000000 16 168112 0 241585 2 715865 4 000000 0 000000 2 000000 19 000000 16 493189 0 654120 3 486097 4 000000 0 000000 2 000000 20 000000 17 045324 0 416201 3 244622 4 000000 0 000000 2 000000 xl Data parameters Trace Multiple Optic Parameters FT Parameters Acquisition parameters Sample Parameters Instrument parameters Annotation Graphics Report Datafile History __ 149014208 1700_15008_ 1800 17008 ClusterList Clustertist Clustertist l NSS 0 0 0 0 0 D INSR 0 0 0 0 0 D Run 0 0 0 0 0 D NPT 4096 4096 4096 4096 4096 4096 INoGoodFWw 0 0 0 0 0 D INoGoodBW 0 0 0 0 0 D INoBadFW 0 0 0 0 0 0 INoBadBW 0 0 0 0 0 0 dHFL 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 dLFL 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 dHFFL 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 dLFFL 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 FilterSize 0 0 0 0 0 0 FilterType 0 a 0 0 0 0 dFFP 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 dFLP 4095 000000 4095 0000
47. 7 Frequency Regions 11 60 85 107 G Group Statistics 91 H Histogram 28 109 Hit Quality 19 29 51 61 87 I IDENT Analysis 51 IDENT Report 21 Identified As 68 Identity Test 19 Result Display 102 Identity Test Limit 13 Identity Test Method 20 Identity Test Report 21 Interactive Region Selection 12 85 L Labels 36 Load Method 105 M Main Library 4 Maximum Distance 90 Maximum Hit 90 Mean Distance 67 91 Median Algorithm 113 Median Technique 30 N No Reference Defined 104 Normalization to Reprolevel 28 59 88 Not Identified 69 74 O Only Pairs 113 Only Reference 114 Only Triplets 113 P Parameters 10 32 Pearson s Correlation Coefficient 60 R Reference Spectra 6 Reference Spectrum 31 51 106 Regions 85 Report 34 109 Reprolevel 87 Result Report 94 S Scaling to First Range 28 59 88 Score Coefficients 57 Score Plot 111 Second Derivative 85 Selectivity 95 Selectivity Histogram 95 Selectivity Report 94 Setup Identity Test Method 31 77 Single Linkage 29 113 Spectral Distance 25 51 60 62 Spectral Residuals 54 Standard Deviation 67 90 91 Standard Method 21 28 51 54 88 Store Method 17 100 Sub Library 4 81 Setting 7 Summary Report 94 T Threshold 21 67 74 90 TRC Data Block 117 U Uniquely Identified 68 74 Vv Validation 14 74 94 Report 15 Vector Normalization 61 84 W Ward s Technique 29 30 113 Weight 59 87 Weighted Average Linkag
48. A From date 3 03 03 time 16 54 37 Description CAN BE CONFUSED WITH 1 OTHER HITS 1 Hit quality with expected reference 0 002891 Threshold for expected reference 0 011451 depends on each ref spectrum Algorithm Standard vector normalized spectra Yes Order of Derivative 0 Smoothing points 1 No of used factor sp 0 15 hits of 15 x Ranges 1 Class Name KLARA Class Test OK 1 Using residuals No Order of Internal Derivative 0 Smoothing Points for Internal Derivative 1 Reduction Factor 1 Hit No Hit Quality SampleName Group 1 0 002891 000004 Sample DL Alanin Ay of 11 000004 2 0 063071 000005 Sample L Alanin Ay of 11 000005 3 0 110633 000002 Sample DL Isoleucin Av of 11 000002 4 0 132710 000003 Sample L Isoleucin Ay of 11 000003 5 0 135557 000001 Sample L Leucin Av of 11 000001 6 0 154416 000015 Sample L Methionin Av of 11 000015 z 0 219237 000014 Sample DL Methionin Av of 11 000014 8 0 227688 000006 Sample DL Tryptophan Av of 11 000006 9 0 248521 000007 Sample L Tryptophan Av of 11 000007 10 0 593082 000010 Sample Fructose Av of 11 000010 11 0 611039 000011 Sample Xylit Av of 11 000011 12 0 613600 000012 Sample Sorbit Av of 11 000012 13 0 679362 000013 Sample Mannit Av of 11 000013 14 0 690536 000008 Sample Glucose H2Ofrei Av of 11 000008 15 0 733616 000009 Sample Glucose H20 Av of 11 000009 Figure 54 IDENT Report with class test performed Test OK
49. Cluster Analysis Load Method Cluster Analysis New xj Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Load Method M General information Spectra 0 Frequency ranges 1 Data preprocessing Vector normalization Algorithm Standard Preparing dendrogram Ward s algorithm Distance matrix not made yet Figure 88 Cluster Analysis Load Method tab 7 8 1 Load Method Use the Load Method button to load an existing cluster analysis method Cluster analysis method files have the extension CZA It is also possible to load cluster analysis method files created by OPUS OS 2 IDENT However if you store such a method using OPUS IDENT you will not be able to load the method by OPUS OS 2 IDENT To avoid this store the modified OPUS OS 2 IDENT file by using a different file name Bruker Optik GmbH OPUS IDENT 105 Reference Section 7 8 2 General Information The General information group field provides statistical information on the existing method file The number of spectra used for the method and the number of frequency ranges included are displayed You will get additional information on the data preprocessing method the algorithm used for the identity test and dendrogram and whether a distance matrix has been generated 7 9 Cluster Analysis Reference Spectra Cluster Analysis New Load Method Reference Spectra 3d File Filelist Paramet
50. EZE Vector normalized spectra if standard x Ranges No of Ref Spectra Factorization Yes 6 d 10 No 15 3 857202 0 000000 3999 918628 10001 725171 No 1454 1 4111 0 99 000000 H ident example2 Frequency of First Point 10001 725171 Fr 000001 Sample L Leucin Av of 11 3999 9186 000002 Sample DL Isoleucin Ay of 11 10001 725171 3999 9186 000003 Sample L Isoleucin Av of 11 10001 725171 3999 91 86 000004 Sample DL Alanin Av of 11 10001 725171 3999 9186 000005 Sample L Alanin Av of 11 10001 725171 3999 9186 000006 Sample DL Tryptophan IA ZI 10001 725171 3999 9186 000007 Sample L Tryptophan Av of 11 10001 725171 3999 9186 000008 Sample Glucose H2Ofrei Av of 11 10001 725171 3999 9186 000009 Sample Glucose H20 Av of 11 10001 725171 3999 9186 000010 Sample Fructose Av of 11 10001 725171 3999 9186 000011 Sample Xylit Av of 11 10001 725171 3999 9186 000012 Sample Sorbit Av of 11 10001 725171 3999 9186 000013 Sample Mannit Ay of 11 10001 725171 3999 9186 000014 Sample DL Methionin Av of 11 10001 725171 3999 9186 000015 Sample L Methionin Ay of 11 10001 725171 3999 9186 a 24 OPUS IDENT Bruker Optik GmbH Theory Cluster Analysis The cluster analysis tests FT IR spectra for their similarity In contrast to the identity test no input information is required The cluster analysis divide similar spectra into groups These gr
51. Konformitatstest Mixing_TU_Munich Mixing TU Munich 1stD2ipt Load Method Reference Spectra Test Spectra Parameters Validate Graph Report Store Method Mark samples which exceed the limit e Cl Value x Print Datapoints gt Cl Aa Sprungantwort 221 0 Testmischung 2 85 7868 36 6 28E 006 0 0 0 r Sprungantwort 1222 0 Testmischung 2 00 5160 72 1 07E 005 0 0 0 4 R Sprungantwort 1223 0 Testmischung 1 99 6448 97 5 71E 006 0 0 0 SS _ Sprungantwort 1224 0 Testmischung 274 5407 57 3 98E 006 0 0 0 B R_ Sprungantwort 1225 0 Testmischung 2 21 4227 31 6 92E 005 0 0 0 Z R Sprungantwort 1226 0 Testmischung 2 86 8956 04 9 75E 006 0 0 0 a R_ Sprungantwort 1227 0 Testmischung 2 84 8886 61 8 37E 006 0 0 0 a R_ Sprungantwort 1228 0 Testmischung 2 56 8014 92 5 74E 006 0 0 o OG R_ Sprungantwort 1229 0 Testmischung 229 8354 34 648E 006 0 0 0 Sprungantwort 1230 0 Testmisch II 273 6387 25 6 53E 006 D ol oO OZ R_ Sprungantweort 1231 0 Testmischung _ 3 08 7621 51 5576 006 0 000168 0 0529 2 13 R_ Sprungantwort 1232 0 Testmischung 2 26 6587 82 8 54E 006 0 0 0 14 R _ Sprungantwort 1233 0 Testmischung 3 23 6904 10 _9 64E 006 0 00119 015 5 DS R_ Sprungantwort 1234 0 Testmischung 219 8346 63 6 16E 006 0 0 0 DS R_ Sprungantwort 1235 0 Testmischung 2 46 4273 60 4 65E 005 0 0 0 i7 R_ Sprungantwort 1236 0 Testm
52. NT Bruker Optik GmbH Validating Library Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentT utoria A x Load Method Reference Spectra Parameters Threshold Validate Store Method Ident_Demo VO0 bf File name Path stored Library Sub libraries date time Operator name Selectivity ay Gk garrek arte ra Set Print Detailed report Validation Report Ident_Demodata IP1 IP3 class_en C ProgrammeSOPUS6_O NData Extend 5 10 2004 6 58 32 Default Ident_Demoa Carhbonhydrates Diclofenac amp Nifedipin sugar Lactose Nifedipin Always use lowest IP level No Parameters of library Ident_Demo Algorithm Vector normalized spectra From Figure 12 Setup Identity Test Method Validate tab standard No 10502 4 anno OO Click on the Validate button to start validation A dialog pops up and asks you whether to validate this library and all sub libraries For further details see chapter 7 5 1 5 1 Validation Report The validation result is displayed in the form of a report and stored in a file which uses the extension VAL Validation reports can also be created for a single reference library sub library or even for the entire library data structure beginning on the level from where the validation starts For further details on the single reports see section 7 5 1 If you perform more than one validation the r
53. S IDENT Bruker Optik GmbH Creating New Method To assign groups to a class click on the Assign Classes button on the Reference Spectra tab The following dialog opens Assign Classes x Delete Class New v Enter name for new class Select groups for New te ade Assign Cancel Asin Figure 7 Assign Classes The Select groups for selection field includes only groups which have not yet been a member of a sub library or class on this level Select the New option from the upper right drop down list and enter a unique name for the new class Then select the group s you want to assign and click on the Assign button A menu pops up and asks you whether you want to continue or cancel the assigning operation Click on the Options button on the Reference Spectra tab if you want to change the path of the original and averaged spectra The Add selected spectra into one group check box is checked by default For further details see chapter 7 2 3 Bruker Optik GmbH OPUS IDENT 9 Setting Up an Identity Test Method 1 3 Setting Parameters The quality of an IDENT analysis substantially depends on the data preprocessing method and spectral regions of each spectrum which both have been selected for the samples and IDENT method For the main library IP1 it is recommended to use the Standard method and define a large spectral region as the spectral noise will be substantially smoothed In case of sub lib
54. TU_MunichiSpec Sprungantwort 1248 0 Testmischung D Temp ConformityMixing_TU_MunichiSpec Sprungantwort 1249 0 Sprungantyvort 1250 0 Testmischung Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 1251 0 Testmischung D TempiConformity Mixing_TU_MunichiSpec Sprungantwort 1252 0 Testmischung D TempiConformity Mixing_TU_MunichiSpec Sprungantyort 1253 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 1254 0 Testmischung D Temp Conformity Mixing_TU_MunichiSpec Sprungantwort 1255 0 Testmischung D Temp Conformity Mixing_TU_Munich Spec Sprungantwort 1256 0 Testmischung D TempiContormity Mixing_TU_MunichiSpec Sprungantwort IZOZ D Testmischung D TempiConformity Mixing_TU_MunichiSpec Sprungantwort 1256 0 Testmischung m ZO GO GOZO GO GO GOGO am D m Sprungantwort 1259 0 D Temp Conformity Mixing TU_MunichiSpec Sprungantwort 1260 0 Testmischung Figure 29 Setup Conformity Test Spectra list with changed data set On the Parameters tab you define the spectral regions to be used for the conformity test and select a data preprocessing method 42 OPUS IDENT Bruker Optik GmbH Setting up Conformity Test C Program OPUS6_0 Konformitatstest Mixing_TU_Munich Mixing_TU_Munich_1 Setup Conformity Test Load Method Reference Spectra Test Spec
55. a Extended Demodata Ident tutorial Test tae I ee Ial Confidence Level 9 x ad Print List Threshold 0 2500 98 4729 0 069158 0 2500 99 8287 0 042641 3 Klucel H 0 2500 99 6592 0 048041 A Cellulos 11 0 2500 99 3323 0 032821 5 Corn sta 11 0 2500 99 9566 0 034313 6 Trimetho 33 0 2500 99 9921 0 16966 Cyanocob 11 0 2500 99 8936 0 05329 8 Eudragit 27 0 2500 99 9713 0 053262 3 Syloids 2 0 2500 100 0 022084 Figure 63 Total number of spectra within one group 80 OPU S IDENT Bruker Optik GmbH Setup Identity Test Method Reference Spectra 7 2 3 Options It is also possible to add average spectra generated by previous OPUS IDENT versions without requiring to reconstruct the original spectra which the average has been generated from Each average spectrum loaded represents one group To switch between original and average spectra generated by previous OPUS versions first click on the Options button The following dialog opens x m Add Spectra to new group Add selected spectra into one group Add selected spectra into separate groups m Path of original spectra Change Path Change Path Av Determine the common path of all subpaths of original files m Suggest name of new group First character PI length 3 of sample name Cancel Figure 64 Options The Add select
56. al ranges of the measured spectrum The artificial spectrum is used for the calculation of the spectral distances and the data preprocessing and includes numerous data points of the overlapping frequency regions The Scaling to Ist Range and Normalize to Reprolevel algorithms separately calculate the spectral distances for each frequency range Then an average value is calculated and each frequency range can be weighted differently If you use Normalize to Reprolevel you can additionally specify a reproduction level for each frequency range This level can be determined by the Make Histogram function The calculated spectrum to spectrum distances of the cluster analysis are equal to those calculated by the identity test if you use the Normalize to Reprolevel method This however does not apply to Scaling to Ist Range The identity test uses the spectral distances between the test spectrum and n reference spectra to determine extrema This means that n distance values have to be taken into account per each frequency range The cluster analysis however uses the distances between all reference spectra to determine extrema These are n n 1 2 distances for n reference spectra as the spectral distance of a reference spectrum to itself is not considered To be able to compare the results achieved by the identity test and cluster analysis using the Normalize to Reprolevel and Scaling to Ist Range methods the parameters required for the ident
57. apter 1 Now click on the Parameters tab Define the spectral regions to be considered for cluster analysis and select a data preprocessing method as well as the cluster analys is algorithm In case of data preprocessing you can select between Vector Normalization First and 2nd Derivative as well as combinations Normalization is set by default of both Vector 32 OPUS IDENT Bruker Optik GmbH Performing a Cluster Analysis Cluster Analysis New x Load Method Reference Spectra 3d File Filelist Parameters Report Store Method m Preprocessing Vector normalization DI Regions Interactive Region Selection Clear Selected Regions Method m Making distance matrix Standard Calculate Distances Figure 21 Cluster Analysis Parameters tab Define the frequency regions you want to use see chapter 1 Select the Standard method to calculate the spectral distance Click on the Calculate Distances button After the calculation of the distances you first have to save the method Click on the Store Method tab and on the Store Method button to open the standard Save File dialog box Enter a new file name for the method file and click on the Save button Bruker Optik GmbH OPUS IDENT 33 Cluster Analysis Cluster Analysis New Sos Figure 22 Cluster Analysis Store Method tab Now click on the Report tab to
58. ar Figure 60 Setup Identity Test Method Reference Spectra tab The spectra table lists the spectra groups and each single spectra including the sample ID Path File Name Sample Name Group Name and Sub Library You can have the groups displayed as well as each single spectra of one group Click on the sign in the first column The respective line with the group selected opens and shows the single spectra To close the list again click on the q sign Click on the numbered tiles on the left side of the table to select one spectrum or several spectra Select the whole table by clicking on the tile on the left side of the table header see mark in figure 60 Remove spectra from the table by selecting one spectrum or more spectra and pressing the DEL key on the PC keyboard 78 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Reference Spectra Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata iden If you click on the Add Spec for New Group button a dialog box opens to be used to load one or more files into the spectra list Spectra loaded simultaneously will be merged into one group Use the Add Spec to Sel Group button to add a spectrum to a group selected This is useful if you want to add a spectrum to a group later 7 2 1 Sorting Reference Spectra When working with IDENT methods consisting of a large number of groups it takes quite some time to find a particular group Therefo
59. ber of coefficients These T coefficients are also called scores The differences between the original and reconstructed spectrum are known as spectral residuals figure 41 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 Figure 41 Reference spectrum reconstructed reference spectrum and difference spectrum The Standard method directly uses the spectral intensities to calculate the spectral distance and the summation is performed for all data points within the specified frequency regions which could be more than 1000 points How many factor spectra or score coefficients have to be included in an IDENT library is a very important aspect and will be explained in the following When factorizing an IDENT library s average spectra are transformed into s factor spectra These factor spectra are orthogonal to each other The effect a certain factor has on the reproduction of reference spectra is indicated by the respective Eigen value The factor spectra are sorted according to these Eigen values The first factor spectrum is the most important one and thus has the highest Eigen value The more the Eigen value decreases the lower the spectral intensities ordinate values of the factor spectra and the more intensive the noise Factor spectra which mainly consist of noise must not be used for an IDENT method Factor spectra are stored in the IDENT method directory using the OPUS file format They have the same file name a
60. ch are closest equivalent to the test spectrum and determines the deviations between these spectra and the test spectrum This allows IDENT to identify unknown substances e g polymers and to evaluate the conformity degree of a substance with a reference standard The latter application is a typical task found in quality control To perform an identity test you first need to have a reference library which you compare the test spectrum with If no suitable library exists you have to measure a set of reference spectra i e at least one spectrum per substance However it is recommended to measure several batches of the same substance to enable the program to get more information on possible allowed variations Before the samples required have to be classified as identified using common reference analytics If you have already measured a spectrum which you want to identify the next step will be to generate an IDENT method To perform an identity test you can select several algorithms and define identification parameters by using the IDENT software The results of an identity test are stored in a report which includes the analysis result the method used as well as the parameters defined Bruker Optik GmbH OPUS IDENT 1 OPUS IDENT Bruker Optik GmbH Loading Existing Method Setting Up an Identity Test Method This chapter describes step by step how to set up a reference spectra library and how to generate an IDENT method Yo
61. corporates all selected k data points In the current IDENT report see figure 40 the smallest spectral distance which has been determined by spectrum comparison is 0 96 In this case one of the sample spectra previously used to generate an average spectrum for the reference library serves as a test sample which explains the extremely small spectral distance As this example shows the first two hits are well separated from each other The spectral distance for Hit No 2 is 6 19 which is about 6 times the smallest spectral distance Bruker Optik GmbH OPUS IDENT 51 IDENT Theory 0 40 0 60 0 80 1 00 1 20 1 40 1 60 1 80 0 20 8750 8250 8000 7750 7500 7250 7000 6750 6500 6250 6000 5750 5500 5250 5000 4750 4600 8250 8000 7750 7500 7250 7000 6750 6500 6250 6000 5750 5500 5250 5000 4750 4600 Figure 39 Two spectra and their spectral distance 52 OPUS IDENT Bruker Optik GmbH Algorithms Report of Correlation Search Values Method file MATE ident examole2 N ew FAA from date 29 05 00 time 12 11 17 Description IDENTITY NOT CHECKED 0 Hit quality with expected reference 0 000000 No Threshold avail 0 000000 Threshold calculation depends on each ref spectrum Algorithm Standard Vector normalized spectra No Order of Derivative 0 Smoothing points 1 No of used factor sp 0 10 hits of 15 ad PEAR 1 Class Name Class Test NOT PERFORMED JU Using residuals N Order of Internal De
62. ctors or Eigen Values these values will be displayed in the report window The T values see section 6 1 2 are listed in the Eigen vectors sub directory Bruker Optik GmbH OPUS IDENT 23 IDENT Report C OPUSSIDENT examples FACTOR FAA 1 1024 Reports Algorithm For Threshold Individual Delta x of x raster EO or LXV mod Delta x min x of x raster max x of x raster Whole x range No of x Points in all x Ranges No of x Ranges BlockID Order of Derivative Description For Threshold Info Entry Constant conf level Path of origin reference spectra Subpath from name first character Subpath from name length Smoothing points Using residuals File Name H Sident example24n000001 100 H Sident example2 4n000002 100 H ident example244n000003 100 H Sident example2 4n000004 100 H Sident example244n000005 100 H Sident example2 4n000006 100 H Sident example2 4n000007 100 H Sident example2 4n000008 100 H ident example2 4n000009 100 H Sident example2 4n00001 0 100 H Sident example244n000011 100 H Sident example2 4n00001 2 100 H Sident example244n00001 3 100 H ident example2 4n00001 4 100 H ident example2 4n00001 5 100 Figure 17 Report file of a method file using factorization Identity Search Method Se Identity Search Method GO x de ive a or Threshold Info Entry d bee No of Hits to be Listed IOAN
63. cur that the confidence regions of some reference spectra do overlap see figure 53 As you can see the confidence regions of the 4 B and C reference spectra clearly overlap Oc Figure 53 Three reference spectra with overlapping confidence regions It is possible to define several reference spectra which are members of one class and to perform a class test during the IDENT analysis Both in case of an expected reference as well as in case of an analysis with no reference defined the class test determines whether all reference spectra with their Hit Quality below the corresponding threshold are members of the same class If so the result will be Class Test OK Otherwise the result will be Class Test NOT OK If the expected reference spectrum is not part of any class the IDENT report says Class Test NOT PERFORMED Bruker Optik GmbH OPUS IDENT 71 IDENT Theory Make sure to define only one class name for one group in the IDENT method If you load a previously used method including extraordinary members only the first class name will be considered All class members use their individual thresholds If you load a previously used method when using the Setup Identity Test Method command from the Evaluate menu the exclamation mark next to the old class name is automatically deleted Method file Threshold calculation class FA4 Signed by Bruker O tik 2002 02 28 D test42 Validation Ident methods class FA
64. d DAS N Tanhminni ine Figure 27 Setup Conformity Test Reference Spectra tab The Reference Spectra and Test Spectra tab are based on the same principle Figure 27 exemplifies a method which consists of a large number of reference spectra The Data Set column specifies the type of spectra in this case R indicates reference T test Further spectra features are the path file and sample name which are the same for both tabs If you want to change the path for the conformity test spectra click on the Change Path tab A dialog opens which you use to define the new path It is also possible to modify the data set Select the respective reference or test spectra and click on the Set Data Set button Bruker Optik GmbH OPUS IDENT 41 Conformity Test Set Data Set Set selected spectra on Reference Exit Figure 28 Setup Conformity Test Set Data Set option You can temporarily exclude some spectra or change them from reference to test spectra or vice versa or restrict the data set to a few spectra Excluded option only and create a new method As the quality of a method depends on the reference and test spectra make sure that the data sets are carefully assembled Select one of the options from the drop down list and click on the Set Data Set button To continue click on the Exit button The spectra excluded will be marked in gray see figure 29 D TempiContormityMixing_
65. dentity Test Method Reference Spectra 7 2 5 Assign Classes To assign classes to an existing method click on the Assign Classes button The following dialog opens Assign Classes x Delete Class New X Enter name for new class Select groups for New Cancel Figure 66 Assign Classes Select the New option from the drop down list to enter a unique class name for the spectra groups defined Choose the group s which have to be assigned to this new class from the Select groups for selection field There are only groups available which have not yet been assigned to another class on the same library level Click on the Assign button To delete classes select them first and click on the Delete Class button Bruker Optik GmbH OPUS IDENT 83 Reference Section 7 3 Setup Identity Test Method Parameters Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata de ful x Load Method Reference Spectra Parameters Threshold Validate Store Method EEE i m Preprocessing Interactive Region Selection Clear Selected Regions Method r Calculate thresholds Standard E Start Calculation I Always use lowest IP level Figure 67 Setup Identity Test Method Parameters tab 7 3 1 Preprocessing You can select several data processing methods from the drop down list Vector Normalization First and 2nd Derivative as
66. e 200 mesh_110251_02_KFO 0 28417 0 04622 Lactose 10 Glucose monohydrate n 0 28957 0 00736 Glucose NOT IDENTIFIED NOT OK Figure 86 IDENT Evaluation Result display Result not OK To print the evaluation result display of the identity test activate the Print Results Automatically check box in the Output Options group field 7 7 1 No Reference Defined If there is no expected reference defined click on the Change button There are three possibilities to define an expected reference Bruker Optik GmbH OPUS IDENT 103 Reference Section Expected Reference x Fig No reference defined Selected automatically from sample name 1 _lenath 12 C User selected from table below First character JB GroupName Samplename lui 1 Acetylcystein 2 Cellulose PH101 E Cellulose PH102 4 Cellulose PH112 5 Corn starch 6 Diclofenac potassium ee Diclofenac sodium 8 Eudragit g Glucose 10 Isosorbide dinitrate 11 Lactose 80mesh 12 Lactose 200mesh ana Allo etze lei ale Sort Sample name First character 1 d IO BI E Group name ure 87 Expected Reference a No reference defined Acetylcystein Cellulose microcrystalline PH 101 Cellulose microcrystalline PH 102 Cellulose microcrystalline PH 112 Corn starch Diclofenac potassium Diclofenac sodium Eudragit RS PO Glucose dried Isosorbide dinitrate Lactose 80 mesh Lactose 200 mesh gt Rie eis locale OK Cancel Help T
67. e 30 113 Whole Matrix 113
68. e PH 101 108349 IDO KK C Progra Cellulose_microcrys Cellulose microcrystalline PH 102 C Progra Cellulose_microcrys Microcist Cellulose PH 102 108232 01 KK EO wN E ON d BA bi e C Progra Cellulose_microcrys Microcrist Cellulose PH 102_ 108232 D NM wo C Progra Cellulose_microcrys Microcrist Cellulose PH 102_108232_03 K EO C Progra Cellulose_microcrys Microcist Cellulose PH 102_108232_03_KK ek C Progra Cellulose_microcrys Cellulose microcrystalline PH 112 N C Progra Cellulose_microcrys Microcist Cellulose PH 112_ 105845 01 KK oo C Progra Cellulose_microcrys Microcrist Cellulose PH 112 Avicel _1058 d C Progra Cellulose_microcrys Microcrist Cellulose PH 112 Avicel _ 1058 n C Progra Cellulose_microcrys Microcist Cellulose PH 112_ 105845 02 KK E C Progra Cellulose_microcrys Cellulose microcrystalline RC 581 N C Progra Cellulose_microcrys Microcrist Cellulose RC 581_ 109200 01 _K EO C Progra Cellulose_microcrys Microcrist Cellulose RC 581_109200 01 M C Progra Cellulose_microcrys Microcrist Cellulose RC 581_109200_03_K an CoM wt mmol ME eia Ol Meena ANNANN OO ow Figure 20 Cluster Analysis Reference Spectra tab To create a list first define the spectra to be used on the Reference Spectra tab Add these spectra as described in ch
69. e result of the new analysis Compare these results with the ones shown in figure 40 Values Method file WW S Ident example2 New FAA from date 29 05 00 time 12 15 57 Description IDENTITY NOT CHECKED 0 Hit quality with expected reference 0 000000 No Threshold avail 0 000000 Threshold calculation depends on each ref spectrum Algorithm Standard Vector normalized spectra Yes Order of Derivative 0 Smoothing points 1 No of used factor sp 0 10 hits of 15 x Ranges 1 Class Name Class Test NOT PERFORMED 0 Using residuals N Order of Internal Derivative 0 Smoothing Points for Internal Derivative 1 Reduction Factor 1 Hit No Hit Quality SampleName File Name __ Threshold E 0 009404 000001 Sample L Leucin Av of 11 An000001 100 0 694965 2 0 324822 000004 Sample DL Alanin Av of 11 An000004 100 0 952755 3 0 338249 000015 Sample L Methionin Av of 11 4n000015 100 0 883473 4 0 350834 000002 Sample DL Isoleucin Av of 11 A4n000002 100 0 751950 5 0 355342 000007 Sample L Tryptophan Ayv of 11 AnO00007 100 0 995592 6 0 414823 000014 Sample DL Methionin Ayv of 11 An000014 100 0 834952 7 0 477705 000005 Sample L Alanin Av of 11 An000005 100 0 819521 8 0 493052 000003 Sample L Isoleucin Av of 11 An000003 100 0 949495 9 0 527658 000006 Sample DL Tryptophan Av of 11 An000006 100 0 904797 10 0 608630 000010 Sample Fructose Av of 11 An000010 100 0 775521 Figure 50 Ident Report example of
70. e written into an IDENT report This report is stored in the test spectrum file The content of the report depends on the parameters and algorithms selected to run the identity test For details see section 6 4 If you click on the REPORT data block a text window will open and show the results 3 1 1 Standard Method The IDENT report contains detailed information on the method and a list of spectral distances between the test spectrum and the reference spectra This list includes distances in ascending order i e Hit No 1 is the reference spectrum which is most similar to the test spectrum The number of listed distances can be defined when creating the IDENT method see chapter 7 6 Figure 15 shows an IDENT report using the Standard algorithm in combination with an identity test on the first reference spectrum L Leucin of the library An original individual spectrum of the reference library has been used as test spectrum The threshold of the selected reference spectrum Threshold for expected reference see figure 15 is 0 023866 The spectral distance between the test spectrum and this reference spectrum is 0 010556 Hit quality with expected reference i e it is smaller than the threshold Since no further hit can be found below this threshold Hit No 2 with 0 047006 is higher the result is IDENTICAL TO to the expected spectrum Bruker Optik GmbH OPUS IDENT 21 IDENT Report E C OPUS IDENT Sexamples O00007 O00 Value
71. ed spectra into one group option button is activated by default In this case all spectra commonly selected during loading are added to one group In the Options dialog box you define the paths for the original and average spectra Additionally you can define the group name which is derived from the respective sample name Enter the position of the first character as well as the length of the sample name Note Once defined the group name should not be changed any more to avoid confusion 7 2 4 Set Sub Library The Set Sub Library button on the Reference Spectra tab enables you to add sub libraries to the current main method Select the New option from the drop down list to enter a unique sub library name for the spectra groups defined Choose the group s which have to be assigned to this new sub library from the Select groups for selection field There are only groups available which have not yet been assigned to another sub library on the same library level Click on the Assign button Bruker Optik GmbH OPUS IDENT 81 Reference Section To delete sub libraries select them first and click on the Delete Sub Library button Set sub libraries x perre Delete Sub Library Carbonhydrates 7 Enter name for new sub library Select groups for Carbonhydrates Isosorbide dinitrate E Om nesh Nitrofurantoin powder Pharmacoat Cancel Figure 65 Set Sub Library 82 OPUS IDENT Bruker Optik GmbH Setup I
72. ent clusters In addition you specify the Number of Classes used in the diagnosis and the parameters required for the histogram Cluster Analysis Options x Ward s algorithm DI m Dendrogram Sample Name m Diagnosis Number of classes 2 m Histogram Which part of the matrix do you want Whole matrix DI oordinates for rectangle fi fi f fi Width for classes of distances 0 0508758 Cancel Figure 96 Cluster Analysis Options 112 OPUS IDENT Bruker Optik GmbH Cluster Analysis Report You can select between 7 different algorithms to calculate spectral distances between clusters Single Linkage Complete Linkage Average Linkage Weighted Average Linkage Median Algorithm Centroid Algorithm Ward s Algorithm For details see section 4 1 2 Use the Dendrogram drop down list to define the kind of labeling Dendrograms are labeled vertically There are 4 possibilities File Name of the reference spectra Sample Name of the reference spectra File Number file sequence in the list of reference spectra No Name Markers no labelling at all A text file is automatically created for each dendrogram This file has the same name as the cluster analysis method and the extension DEN The file includes the dendrogram and exact clustering levels Specify the number of classes you want to test If you test e g original spectra used to generate average spec
73. er 6 5 The number of spectra which belong to the respective class is indicated at the end of the validation report The total of all spectra has to be equal to the total of the original spectra tested In case of overlappings the Detailed and Result reports provide additional information on which groups should be assigned to a new common sub library Load Method Reference Spectra Parameters Threshold Validate Store Method Validate Selectivity uy lve 3 4 5 Jetailed report bur Ident_Demo VO2 DI Hi Check Original Spectra li Spectra from the following groups can be confused with other groups in t Groups which should be assigned to a new common sub library are listed t Cellulose PH101 Cellulose PH102 Cellulose PH112 Lactose 80mesh Figure 56 Validation Report showing assignment recommendation An original spectrum is Uniquely Identified if the spectral distances between this spectrum and the corresponding average spectrum is smaller than the threshold while the spectral distances between the original spectrum and all other average spectra are larger than the corresponding confidence region The original spectra which are Uniquely Identified are not listed in the report 74 OPUS IDENT Bruker Optik GmbH Validation If a spectrum is Not Identified the spectral distance between the original spectrum and average spectrum is larger than the threshold In thi
74. ers Report Store Method Add Spectra Change Path lei i ee arro C Progra Cellulose_microcrys Cellulose microcrystalline PH 101 C Progra Cellulose_microcrys Microcist Cellulose PH 101_108349_02_KK C Progra Cellulose_microcrys Microcrist Cellulose PH 101_108349_02_M C Progra Cellulose_microcrys Microcrist Cellulose PH 101_108349_05_K C Progra Cellulose_microcrys Microcist Cellulose PH 101_108349_05_KK C Progra Cellulose_microcrys Cellulose microcrystalline PH 102 C Progra Cellulose_microcrys Microcist Cellulose PH 102_108232_01_KK EO wN EI ON d BA bi e C Progra Cellulose_microcrys Microcrist Cellulose PH 102_108232_01_M wo C Progra Cellulose_microcrys Microcrist Cellulose PH 102_108232_03_K 10 C Progra Cellulose_microcrys Microcist Cellulose PH 102_108232_03_KK 11 C Progra Cellulose_microcrys Cellulose microcrystalline PH 112 12 C Progra Cellulose_microcrys Microcist Cellulose PH 112 105845 01 KK 13 C Progra Cellulose_microcrys Microcrist Cellulose PH 112 Avicel _1058 14 C Progra Cellulose_microcrys Microcrist Cellulose PH 112 Avicel _1058 15 C Progra Cellulose_microcrys Microcist Cellulose PH 112_105845_02_KK 16 C Progra Cellulose_microcrys Cellulose microcrystalline RC 581 17 C Progra Cellulose_microcrys Microcrist Cellulose RC 581_109200_01_K 18 C P
75. esult files will be consecutively numbered v00 v01 You can compare different validation results with each other by selecting the respective file from the drop down list You can also print the report by clicking on the Print button This starts the Windows Notepad program which you can use to reformat the text if desired Use the Notepad print function to create a printout Bruker Optik GmbH OPUS IDENT 15 Setting Up an Identity Test Method Note It is recommended to select a small font in Windows Notepad to avoid extraordinary long reports A proportional font may lead to a confusing display of the results Therefore it is advisable to use a monospace font e g Courier New 10 The Threshold values listed on the Threshold tab are regarded as confidence region during validation The results are classified in three categories uniquely identified not identified and can be confused with In case of results belonging to the first category the spectral distance between the original and average spectrum is within the threshold value The spectral distance is higher than the threshold value in case of results belonging to the second category The Can be confused category indicates that the spectral distance of an original spectrum is smaller than the confidence level compared to at least one different average spectrum For further details see section 6 7 1 6 Storing Method Files Click on the Store Method tab to store the m
76. etailed Report The Detailed Report includes additional information on the algorithm used and frequency ranges defined of all sub libraries the order of internal derivative and smoothing points for internal derivative Furthermore this reports specifies all thresholds of the overlapping groups as well as the distances of all single spectra which overlap The report provides additional information on which groups should be assigned to a new common sub library If you have activated the Always use lowest IP level check box on the Parameters tab the Detailed Report will include only the results of the lowest IP level for all spectra of each group e Selectivity Report This report is based on average spectra If you use the selectivity slider on top you can get a more detailed report For example if you set the slider to a selectivity of 3 all spectra are shown in the spectral distance between 1 and 3 94 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Validate e Selectivity Histogram The histogram is a summary of the selectivity report In general validation reports directly compare one spectra group with the adjacent spectrum to see which clusters overlap and where The selectivity report compares Material 1 and Material 2 as explained in the chart in figure 78 Material 1 Material 2 Figure 78 Calculating selectivity ee eet Ti T ratio of distance D between average spectra and the sum of threshold
77. ethod files you have created The following dialog opens Setup Identity Test Method C Program OPUS6_0 Data Extended Demod d x Load Method Reference Spectra Parameters Threshold Validate Store Method Store Method All sub libraries will be stored simultaneously Settings Number of hits in Ident report fio IV Store Average Spectra Figure 13 Setup Identity Test Method Store Method tab 16 OPUS IDENT Bruker Optik GmbH Storing Method Files The parameter you can define in this tab is the Number of Hits in Ident Report i e you enter the number of hits that have to be stored together with the IDENT report By default the Store Average Spectra check box is activated This means that you can store all average spectra of a library IP1 level in one separate directory which is a sub directory of the directory in which the IDENT method has been stored The average spectra will be stored without data pre processing and have an AVERAGE iitsead data block appended If you repeatedly store a particular IDENT method the average spectra will NOT be overwritten Instead the file extension will be incremented Click on the Store Method button The standard Save File dialog box opens to be used to save the method The method file uses the extension F4A and all sub libraries will be stored simultaneously For special details on method protection refer to the OPUS QUANT manual chapter 9
78. f smoothing points You can select between 5 and 25 points The optimal number of smoothing points however has to be evaluated empirically Preprocessing First derivative Smoothing points g SG Figure 68 Defining smoothing points 7 3 2 Regions The Regions table allows to limit data to one or several spectral regions to be considered for identification The frequency limits for the spectral regions can either be entered manually or selected interactively 7 3 3 Interactive Region Selection The spectral region shown on the white background will be processed and evaluated You can also modify the spectral regions displayed Place the cursor on the boundary between the gray and white area Press the left mouse button and move the regions It is also possible to move the entire spectral region If you position the cursor on the white area the cursor changes from b into Press the left mouse button and move the spectral region To delete a region right click on the white area and select Remove from the pop up menu Bruker Optik GmbH OPUS IDENT 85 Reference Section Select Frequency Range s E 12000 11000 10000 9000 8000 7000 6000 5000 4000 Figure 69 Select Frequency Range s If you click on the Interactive Region Selection button a separate window opens and displays the reference spectra You can add a new spectral region by right clicking on the window and selecting the Add Region option fro
79. have a lower distance than this threshold to the reference spectrum If the analysis of a sample spectrum produces a spectral distance which is larger than this threshold the sample spectrum will be defined as not being identical Confidence Level Two parameters are derived from the spectral distances see above to define the confidence region for the average spectrum The mean distance Djy Di Dy gt gd 6 12 The standard deviation Sy So 6 13 with n being the number of original spectra Note that S is the standard deviation from zero and not the standard deviation bu from the mean value The standard deviation from the mean distance Au can be calculated from Dy and So OW Dy Hel 2 Dyn Hel Sy LEE 6 14 Bruker Optik GmbH OPUS IDENT 67 IDENT Theory The threshold is calculated by multiplying the standard deviation bu with a factor f and adding the mean distance Dy aiz 6 15 The factor fis calculated from the probability which can be chosen between 95 and 99 9999 Example if you choose 97 7 factor f will be 2 The spectral distances are assumed to be distributed accord ing to a normal distribution Note that the probability value is calculated for a single sided limit If you select P 95 then 5 of the original spectra are outside the confidence region the spectral distance to the average spectrum is larger than the threshold in question Whethe
80. have the results displayed You can select between different display views 34 OPUS IDENT Bruker Optik GmbH Performing a Cluster Analysis Cluster Analysis C Program OPUS6_O IDENT Study _5 CLA f x Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Dendrogram Window E 000001 10 o00002 12 000001 20 000001 14 o00002 14 000001 12 000001 16 000001 14 000001 15 000001 17 000002 ZO 000001 16 o00002 13 o00002 17 000001 19 000001 13 DUO ID o00002 16 000002 11 DUO IO o00002 15 DUO IE gt a c a D a eo EO Figure 23 Cluster Analysis Report tab with dendrogram If you click on the Window button you can modify the report display A new dialog will open which allows to change the algorithm and the kind of labeling Bruker Optik GmbH OPUS IDENT 35 Cluster Analysis Dendrogram x BERE wad s algorithm 7 File Name Sample Name File number No name markers A JZ a Oo E E O E Oo N Or FO OF E E E E E ZI EZ dz Se d Ea o ss GO GO GO E G G G G db SZ SES S 8 8 5 B E gt gt gt gt GO N GO ee t Lo Cr E TE E dE M EEE GE EEE M RA o IRA E TE GS ZEZ d ude dE GET dE dE Eu gd e medi urek dei redu aei GO B 00 0 D F G EZ Y G SS ES EZ EE GZ E E Q Q GO Q Q 2 2 2 2 E 2 i i SG SG Eo ed tf d ed OO OF OF OF OGA 8G 8 A GB GO 0 b broker 0 5
81. he identity test analysis works without a pre defined reference spectrum b Selected automatically from sample name The reference spectrum is determined by comparing the sample name of the test spectrum with the sample names of the library spectra Usually the sample name will not be completely used but partially First character Indicates at which sample name character the character comparison will start In this example the comparison starts at character 1 Length The Length indicates how many characters will be taken into account during comparison Example the sample name of the test spectrum is 000002 Sample DL Isoleucin It is sufficient to use the first six charac ters for a definite selection First 1 and Length 6 c User selected from table below If you activate this option button all reference spectra will be listed Each line contains the sample name e g 000002 Sample DL Isoleucin the number of the reference spectrum in the library e g ID 2 and the group name Select the spectrum which you expect to match the test spectrum The test spectrum can have any sample name 104 OPUS IDENT Bruker Optik GmbH Cluster Analysis Load Method In the Sort group field you define how to sort the list You either sort according to Sample name ID or Group Name If you check Sample name you can additionally define the character number of the sample name which you start sorting with 7 8
82. he library structure displayed in the browser window Activate the Printer option button to have the library structure printed on a connected printer The drop down list includes the following printing options e Library tree as shown e Whole library tree e Whole library tree and group statistics e Whole library tree and statistics for each spectrum Select one of the printing options and click on the Print button If you activate the PDF option button the library structure will be printed as a PDF file Bruker Optik GmbH OPUS IDENT 5 Setting Up an Identity Test Method Clicking on the Print button opens the following dialog Save as PDF File x At v Portrait Landscape File name C Program OPUS6_O Data Extended Demodata IdentTutorial Test gt gt ce Figure 4 Printing as PDF file Define the appropriate format the file name and path of the PDF file Click on the gt gt button to open the Define PDF File Name dialog 1 1 1 Methods created by prior OPUS releases IDENT methods created by prior OPUS releases and which also include sub methods are automatically converted to OPUS 5 0 if you load them into the IDENT setup If one of these sub methods cannot be found in the path defined OPUS tries to search in the path where the reference method FAA is stored If the method cannot be found there either OPUS writes a remark in the og ASCIU file Sub methods containing spectra which are no me
83. ified The Can Be Confused With result indicates that the spectral distance of an original spectrum to the corresponding average spectrum is smaller than the confidence region while one or more spectral distances between the original spectrum and other average spectra are even smaller than the corresponding confidence regions If an original spectrum is tested to be Can Be Confused With other references first its average spectrum sample name and confidence region are listed in the report followed by the name of the original spectrum and the threshold In addition the name of the average spectrum the sample name and the spectral distance Hit between this average spectrum and the original spectrum are listed under the Overlapping With definition The spectral distance is smaller than the threshold confidence region If this original spectrum overlaps several average spectra they will all be listed in the report Bruker Optik GmbH OPUS IDENT 75 IDENT Theory Setup Identity Test Method C Prograne OPUS6_0 Data Extended Demodata IdentTutor Load Method Reference Spectra Parameters Threshold Validate Store Method eS Detailed report D Ident_Demo 02 Jetailed repor Selectivity Gabi eba Ge berr Set Spectra from the following groups can be confused with other groups in Groups which should be assigned to a new common sub library are listed Cellulose PH101 Cellulose PH102 Cel
84. ischung 2 35 8824 90 6 82E 006 0 0 0 DS R_ Sprungantwort 1237 0 Testmischung 2 35 6641 82 8 12E 006 0 0 0 19 R_ Sprungantwort 1238 0 Testmischung 2 38 7899 21 5 20E 006 0 0 0 20 IK Sprungantwort 1239 0 Testmischung 2 87 7706 36 5 11E 006 0 0 0 GK _ Sprungantwort 1240 0 Testmischung 2 20 4751 87 8 57E 006 0 0 0 ZZ R_ Sprungantweort 11241 0 Testmischung 3 37 8261 77 _8 12E 006 0 00299 0 118 16 23 R_ Sprungantwort 1242 0 Testmischung 2 63 5407 57 3 98E 006 0 0 0 24 R_ Sprungantwort 1243 0 Testmischung 2 26 7783 50 4 88E 006 0 0 0 25 R_ Sprungantwort 1244 0 Testmischung 2 82 8161 49 8 53E 006 0 0 0 25 ___ R_ Sprungantwort 1245 0 Testmischung 3 04 6780 67 _6 35E 006 _6 88E 005 0 0434 1 ZIK Sprungantwort 1246 0 Testmischung 2 38 5777 84 5 68E 006 0 0 0 x Figure 35 Setup Conformity Test Report tab Depending on the algorithm selected from the drop down list the corresponding outliers are marked in grey color The Data Points gt CI Limit column refers to the data points above the CI limit which is important to analyze the integral The values displayed in the Sum 1 column represent the integrals divided by all data points and the values displayed in the Sum 2 column represent the integrals divided by the data points which are above the CI limit If you want to print the report click on the Print button Before you store the method you have to define the analysis method Either activate the Use
85. ition the mouse on the edge of the box the cursor changes into EA To rotate the box press the left mouse button and move the mouse to the position desired Right clicking somewhere on the 3D display pops up the Properties button If you click on this button the View properties dialog is displayed which allows further plot settings To optimize the 3D factor view you can select additional factors by means of the Factor drop down list Always the next 3 factors of the original selection can be used for each dimension 98 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Validate iew properties x Displayed Data Rotation E Elevation 30 MV Show box MV Show points IV Show axes V Show ellipsoides Factor Text Color Factor Y E z Background Color Factor Z 3 z 1234567890 OK Cancel EEE Figure 82 View properties Apply Sometimes the clusters may be displayed as ellipsoides This is due to the scaling of the axes and not a result of the original calculation To zoom the box displayed press the left mouse button and draw a frame around the position desired Double click into the zoomed area to undo the zoom setting To return to the IDENT setup close the factor view by clicking on the X icon 7 5 2 Print To print the report click on the Print button This starts the Windows Notepad program which you can also use to reformat the text if desired Use the Notepad print function
86. ity test and cluster analysis must be identical same reference spectra same frequency ranges etc Then start the identity test Use the first or last reference spectrum as test spectrum Compare the Hit Qualities of this IDENT test report with the spectrum to spectrum distances of the cluster analysis 4 1 2 Cluster Algorithms There are 7 methods available to calculate spectral distances between a newly created cluster and all the other spectra or clusters The algorithms most frequently used are Average Linkage and Ward s Algorithm Single Linkage The p and q clusters are merged to the new r cluster D p i is the spectral distance between the p and i clusters while D q i is the spectral distance between the q and i clusters The D r i distance between the new r cluster and the 7 cluster is the smaller one of the two original distances Dur i min D p i D q i 4 1 This method can be used to create large clusters Bruker Optik GmbH OPUS IDENT 29 Cluster Analysis Complete Linkage The new distance is the larger one of the two original distances D r i max D p i D q 1 4 2 This method prefers to create small groups Average Linkage The arithmetic mean value is calculated as follows Pz Gen e POG 4 3 Weighted Average Linkage n p is the number of spectra which are merged in the p cluster and n q is the number of spectra which are merged in the q cluster The spectral distance betwee
87. k occa Ea 15 1 6 Storing Method Files 23 04 5405 oe eae a Polak oe Eda pre 16 2 Performing an IDENT Analysis 0 cc eee cece eee eee 19 3 IDENT R port 20 44 140 ise eae ewe eee ese BAe wd as aA eS 21 3 1 Identity OEE EK E a a ant gees ens Ea 21 3 1 1 Standard Method de dE ten POs E dE eN dard 21 3 1 2 Factorization Method oie eee adc ae ike nd Be 23 4 SUGE Analysis e AA E ea E Ei aate o 25 4 1 AAE 25 4 1 1 Methods to Calculate Spectral Distances assa 28 41 2 Cluster aA 29 4 2 Performing a Cluster Analysis dE idea Wag Wat ants lerria Zeta 31 4 3 SADA STEVA St A E EE A EE ETE 37 5 Conformity NO TE 39 5 1 Setting up Conformity Test 22 katie ata Mees GOES EAEg 39 5 2 Performing Conformity Test ter oad aie tae cana dew ba etek 48 6 IDENT BIGUN 51 6 1 Alg rthmS ox AE ta bb a E rka abe 51 6 1 1 Standard Method er egizta zunei gt eina giketa eta 51 6 1 2 ZEGON osc bait date Ei ein ke peta ggb Caemea eee uA 53 6 2 Factorization Theory E cas e eea arik eta 56 6 2 1 Scaling to 1st Range and Normalize to Reprolevel 59 6 3 Data NUA 61 6 3 1 Vector Normalization dug patente dar ee ga akar 61 6 4 Determining Threshold Value for Identity ISS 67 iii 6 5 Identity Test ccs a a sae a wer ea ete Baran rae ak oa ea as EE 68 6 6 Class LOSE aaa d oa eh buat oe Daman ed oa aan E 70 6 7 AGUD ERA 74 Reference Section sss rg ea Gr ete Erde eez bert 77 7 1 Setup Identity Test Method Load
88. k on the Load Method button and select the respective method Setup Identity Test Method C Program OPUS Data Extended Demodata IdentT al x Load Method Reference Spectra Parameters Threshold Validate Store Method Load Method Rename Library 3 Ident_Demo zi Carbonhydrates Lactose Diclofenac amp Nifedipin m General information of selected library Nifedipin Sugar Name Ident_Demo Groups 18 Spectra 726 Frequency ranges 1 Data preprocessing First derivative Algorithm Standard Sub libraries 5 Library Tree as Shown Printer Library Tree as Shown R C PDF Whole Library Tree Whole Library Tree and Group Statistics Print Whole Library Tree and Statistics for Each Spectrum Figure 1 Setup Identity Test Method Load Method tab existing method Figure 1 exemplifies the exact description of the main library selected including the data structure and the specific parameters in the General Information of Selected Library group field The name of the main library dent_Demo is shown in the blue indicator field on the upper left side If you click on one of the sub libraries in the browser window i e if you move to a different library level the indicator field changes its color and description In general you can define as many sub libraries as you like Each library level will have a different color e IPI Blue e IP2 Green e WPS Yel
89. lectivity lt 1 Figure 80 Selectivity Geometric overlapping The selectivity report can also be read out as score plots in 3D format which is indicated by the WFactorview tab This kind of representation is based on the factorization of single reference spectra or average spectra shows the distribution of spectra and supports the selecting of meaningful factor spectra Select Factorization from the Method drop down list on the Parameters tab and click on the Start Calculation button Define at least 3 factors from the Factor Spectra dialog which will serve as a basis for the 3D factor view Subsequently validate the library by clicking on the Validate button on the Validate tab To display the cluster of one group select the respective group from the Selectivity Report list The number of neighboring groups can be set by means of the Selectivity slider You can activate or deactivate the Opaque check box see figure 79 In both cases the real threshold of the cluster is shown in all dimensions i e x y and z axis If you deactivate the Opaque check box the spectra A in figure 81 of each cluster can be seen as the clusters will be transparent Bruker Optik GmbH OPUS IDENT 97 Reference Section Figure 81 3D factor view Transparent clouds and data points If you position the mouse on one specific spectra the file name and group name will be displayed To improve the factor view you can rotate the box If you pos
90. llowing algorithms e Max Conformity Index The maximum value will be calculated based on the frequency ranges selected e Sum 1 Sum over CI gt Limit N total All y values above the CI limit are added up and divided by the total number of data points within the frequency ranges selected e Sum 2 Sum over CI gt Limit N over Limit All y values above the CI limit are added up and divided by the number data points which are above the CI limit Depending on your specific quality control problem you select either one of these algorithms General recommendations cannot be made you have to empirically find out which procedure would be the best for your specific requirements If you click on the Display CI Spectra button the CI spectra and the CI limit will be displayed Oo Mixing_TU_Munich_1stD21p a Mixing_TU_Munich_1stD21p ZEATE K Cl_Sprungantwort 11220 0 First derivative A Cl_Sprungantwort 1221 0 E Interactive Reaion Selection Figure 34 Setup Conformity Test Cl spectra The reference spectra are displayed in blue and the test spectra are displayed in green To localize a specific spectrum and analyze the standard deviation deactivate and re activate the Show check box Click on the Go to Setup Conformity Test button to return 46 OPUS IDENT Bruker Optik GmbH Setting up Conformity Test To display the spectra report click on the Report tab Setup Conformity Test C Program OPUS6_0
91. low e IP4 Orange e IP5 Pink e IP6 Magenta If you defined IP7 as the next library level the color cycle would start from the beginning i e IP7 would be blue 4 OPUS IDENT Bruker Optik GmbH Loading Existing Method The library level you are currently working with is always displayed on the upper left side of each property tab except for the Store Method tab If you use a method which has not been properly calculated an error message pops up and the library levels not yet re calculated will be displayed in bold in the browser window 1 library not ready for use see entry with bold text in the tree Figure 2 Error message on the Load Method tab To rename the library description click on the Rename Library button The following menu pops up x Name fident D emo Cancel Figure 3 Rename reference library Enter the new name of the current library and confirm it by clicking on the OK button The new description is automatically displayed in the browser window on the right and in the General information group field The renaming procedure applies to each library level You can also delete libraries Note that all sub libraries assigned to a specific library level will be deleted as well Select the particular library in the browser window and press the DEL key on the PC keyboard A menu pops up and asks you whether you want to continue or cancel this deleting operation There are two possibilities to print t
92. lulose PH112 HH HH HHH HHH HH HHH HHH HH HH HHH HH HHH HHH HH HHH HHH HHH HHH HHH HHH HHH OK E HHH OE d A OE A A OE A OE dA HH HH HHH HHH HH HHH HH HHH HH HHH HHH HH HHH HH HH HHH HHH HH HHH HHH OE d A OE A OK d A OE d OE d A OE A A OE A OE dA Details Confused Distance Cellulose PH101 Cellulose_microcrysta lline_PH101 01 0 017566 Figure 58 Validation Report spectra can be confused with Threshold Cellulose PH102 0 029757 If all reference spectra are selected each original spectrum will be compared with all average spectra Even if you only select some of the reference spectra the original spectra belonging to these reference spectra are tested against all average spectra This option is extremely useful if an existing library is to be extended by new reference spectra and only these new spectra have to be tested If you have activated the Always use lowest IP level check box on the Parameters tab the detailed report will only list the results of the lowest IP level for all spectra of each group 76 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Load Method Reference Section Before being able to identify spectra by means of OPUS IDENT you have to create an IDENT method first Select the Setup Identity Test Method command from the OPUS Evaluate menu 7 1 Setup Identity Test Method Load Method Setup Identity Test Method C Program OPUS Data Extended Demodata Id u x
93. luster are merged into a new cluster This procedure will be repeated until only one big cluster will be left Figure 19 shows this procedure in more detail The spectral distances between any two spectra of a set of n spectra can be represented in a n x n matrix This matrix is symmetrical and the main diagonal elements are 0 Subsequently it is sufficient to test only a triangle of this matrix Five spectra A B C D and E are used in the example The triangular matrix which contains the spectral distances between these 5 spectra is shown in the upper part of figure 19 The A and C spectra are mostly similar to each other The spectral distance is 11 0 Both spectra are merged into the AC cluster 26 OPUS IDENT Bruker Optik GmbH Theory A B C D E A 0 B 440 0 C 11 0 54 4 0 D 1016 68 1 97 4 0 E 118 3 92 1 115 9 21 2 0 AC B D E AC 0 B 49 2 0 D 995 68 1 0 E 117 1 92 1 21 2 0 AC B DE AC 0 B 49 2 0 DE 108 3 80 1 0 ABC DE ABC 0 DE 94 2 0 Figure 19 Spectral distances calculated using a hierarchical cluster method Now the distances between AC and the other spectra have to be calculated This time the Average Linkage algorithm has been used The distance between A and D is 101 6 and the distance between C and D is 97 4 The distance between the AC cluster and D spectrum is the mean value of the two original distances 101 6 97 4 2 99 5 The new distance values can be seen in the second matrix of figure 19 As
94. m the pop up menu The pop up menu also includes the Zoom and Crosshair option These options allow to easily define the value of a specific data point Select Frequency Range s 2 00 Zoom A 175 Scale All Spectra gt Shift Curve gt E Crosshair gt Add Annotation 1 25 Copy Copy All Paste Properties 1 00 0 75 0 50 0 25 0 00 12000 11000 10000 9000 8000 7000 6000 5000 4000 Cancel Figure 70 Select Frequency Range s with pop up menu 86 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Parameters 7 3 4 Clear Selected Regions You can delete an entry from the Regions table by selecting the specific entry and clicking on the Clear Selected Regions button or pressing the DEL button on your keyboard 7 3 5 Method Use this drop down list to select an identification method During the identity test the test spectrum is compared with all reference spectra The result of this comparison is the spectral distance which is also called Hit Quality The more similar two spectra are the smaller the spectral distance Four methods are available to calculate the spectral distance Use the Method drop down list to select one The basic theory of each method has been described in chapter 6 To enforce an IDENT analysis on the lowest IP level during the identity test the Always use lowest IP level check box has to be activated This check box is only
95. mber of the main library IP1 will not be considered for the set up of the converted OPUS 5 0 method If spectra are assigned to more than one sub method on the same library level they will be considered only once In both cases the library conversion log file includes an appropriate remark At the end of the conversion a dialog pops up indicating that the conversion has been finished but not completed Confirm this dialog by clicking on the OK button 1 2 Creating New Method If you want to create a new IDENT method click on the Reference Spectra tab To create a reference library define the spectra to be used for the library Click on the Add Spec for New Group button to add single spectra to a new product group Click on Add Spec to Sel Group to add single spectra to a selected product group 6 OPUS IDENT Bruker Optik GmbH Creating New Method Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentTutorial I d lent Load Method Reference Spectra Parameters Threshold Validate Store Method piz Set Sub Libraries ay Add Spectra for New Group Add Spectra to Sel Group Assign Classes Acetylcystein Acetylcystein Cellulose microcryst Cellulose PH101 Cellulose microcryst Cellulose PH102 Cellulose microcryst Cellulose PH112 Corn starch Corn starch Diclofenac potassiu Diclofenac potassi Diclofenac sodium Diclofenac sodium Eudragit RS PO Eudragit Glucose dried Gluco
96. n Av of 11 000001 0 021304 2 0 047006 000003 Sample L Isoleucin Ay of 11 000003 0 023866 3 0 095210 000002 Sample DL Isoleucin Ay of 11 000002 0 033536 4 0 104591 000015 Sample L Methionin Ay of 11 000015 0 061757 5 0 146645 000004 Sample DL Alanin Av of 11 000004 0 011690 6 0 158939 000005 Sample L Alanin Av of 11 000005 0 073910 Ell 0 172990 000014 Sample DL Methionin Ay of 11 000014 0 072976 0 266797 000006 Sample DL Tryptophan Av of 11 000006 0 022966 9 0 288849 000007 Sample L Tryptophan Ay of 11 000007 0 051395 10 0 668990 000011 Sample Xylit Av of 11 000011 0 032523 11 0 671159 000010 Sample Fructose Av of 11 000010 0 030262 12 0 690747 000012 Sample Sorbit Av of 11 000012 0 029502 13 0 750100 000013 Sample Mannit Ay of 11 000013 0 017529 14 0 763355 000008 Sample Glucose H20frei Av of 11 000008 0 090445 15 0 848422 000009 Sample Glucose H20 Av of 11 000009 0 026233 Figure 16 Ident Report Query spectrum not identical 3 1 2 Factorization Method If you use the Factorization algorithm on the Parameters tab Eigen values and Eigen vectors see section 6 1 2 may also be interesting for you The factor values can be retrieved from the report file of the IDENT method file Load the method file extension F 4A into the OPUS browser window and click on the REPORT data block to open the report Open the Identity Search Method subdirectory as shown in figure 17 If you click on Eigen Ve
97. n get a cross section of the dendrogram Simply enter the number of classes and a list including the components of each single class will be generated Additionally the spectral distance for each cluster is indicated which has recently been merged As already mentioned the spectral distances between different spectra can be represented by a symmetrical matrix If you use the Make Histogram function the whole matrix or part of it can be statistically tested The results will be shown in the form of a histogram However this part of the program does not include clustering in general but calculates the distances between the different spectra only 4 1 1 Methods to Calculate Spectral Distances Four different methods can be used to calculate spectral distances e Standard algorithm e Factorization e Scaling to Ist Range e Normalize to Reprolevel These methods are an integral part of the IDENT software For details see chapter 6 and 7 The Standard algorithm uses the Euclidian distance to determine spectral distances while Scaling to Ist Range and Normalize to Reprolevel use the correlation coefficient 28 OPUS IDENT Bruker Optik GmbH Theory Using the Standard or Factorization method the spectral distances calculated by the cluster analysis differ from those calculated by the identity test Overlapping frequency ranges will not be merged when using cluster analysis An artificial spectrum is derived from the selected spectr
98. n the new r cluster and the i cluster is calculated as follows pan GEOG 4 4 n p n q This algorithm is a generalization of Average Linkage Median Algorithm D p q is the spectral distance between p and q Dr i ARAIA 4 5 Centroid Algorithm n is the total number of reference spectra D r i is calculated according to the following equation p WP D UZU DIU np tntg D P 4 6 n n Ward s Algorithm The previous algorithms merge the two groups which are most similar Ward s Algorithm however tries to find as homogeneous groups as possible This means that only two groups are merged which show the smallest growth in heterogeneity factor H Instead of determining the spectral distance the Ward s Algorithm determines the growth of heterogeneity H This method can 30 OPUS IDENT Bruker Optik GmbH Performing a Cluster Analysis especially be used for the cluster analysis of bacteria spectra i e these clusters correlate extremely well with microbiological affinity n i is the number of spectra merged in the i cluster H r i is calculated according to the following equation H r i D r i a 4 7 If you want to test different cluster algorithms you do not need to calculate the spectrum to spectrum distance matrix again because the clustering does not have any effect on this kind of matrix 4 2 Performing a Cluster Analysis To perform a cluster analysis the following steps are req
99. o perform a cluster analysis For further details on this subject refer to chapter 7 12 Bruker Optik GmbH OPUS IDENT 37 Cluster Analysis 38 OPUS IDENT Bruker Optik GmbH Setting up Conformity Test Conformity Test The conformity test is a an easy method to test the deviations of measured NIR spectra within certain limits To set these limits you need samples which belong to at least one batch or one production cycle of the final product as reference spectra These reference spectra vary within the accepted range of specifications The NIR spectra of these samples reflect the different sample variations and form a confidence band in the spectral range To pass the conformity test the spectrum of a new sample has to be within this confidence band on each wavelength The conformity test is mainly used for the quality control of defined products for which a quantitative calibration would be too time consuming or even impossible First you have to calculate the average and the standard deviation o of the absorbance values for each wave lengthi The mean value plus minus the standard deviation determine the confidence band within the spectral range and define which amount of variations on each spectral wavelength is acceptable for the particular product Second you have to check whether the spectrum of a sample to be tested is within the defined confidence band in the spectral range The difference between this sample and
100. ons Add Spectra for New Group Add Spectra to Sel Group Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentTutoria Ix Set Sub Libraries bO Assign Classes GATA Diclofenac potassiu Diclofen Acetylcystein Acetylcy Klucel HF Klucel H Cellulose microcryst Cellulos Corn starch Corn sta Trimethoprim Trimetho Cyanocobalamin Eudragit RS PO Cyanocob Eudragit 1 2 3 4 5 6 7 8 9 Syloid silica Syloid s C Progra Syloid_silica 01 Syloid silica C Progra Syloid silica 02 Syloid 244 FP 10791 Figure 62 Setup Identity Test Method Missing reference spectra Missing reference spectra may be due to file renaming or as exemplified in figure 62 to a different data path In this case store the missing reference spectra into the right path and load the IDENT method again This is important as you cannot perform any calculation using an IDENT method with missing spectra If you want to know the total number of spectra included in a particular group click on the Threshold tab The Spectra column includes the total number of spectra per group Load Method Reference Spectra Parameters Threshold Validate Store Method m Maximum hit SDev 0 25 Set M Confidence Level Set 939 99 Setup Identity Test Method C Program OPUS6_0 Dat
101. oups are called classes or clusters The clustering can be displayed in a dendrogram Figure 18 shows a simplified dendrogram including 5 spectra Cluster Analysis C Program OPUS6_0 IDENT Study_5 CLA Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Dendrogram Be Window E GO OO a a E E D D a oo Figure 18 Cluster Analysis Dendrogram 4 1 Theory The spectral distance indicates the degree of spectral similarity Two spectra with a spectral distance of 0 are entirely identical within the frequency ranges tested The higher the difference between two spectra the higher the spectral distance Bruker Optik GmbH OPUS IDENT 25 Cluster Analysis The hierarchical cluster algorithms perform the following tasks First the spectral distances between all spectra are calculated The two spectra with the highest similarity i e spectra with the smallest spectral distance are merged into a cluster The distances between this cluster and all other spectra are calculated Several methods Single Linkage Complete Linkage are available to calculate the distances The two spectra spectrum spectrum or spectrum cluster with the smallest distance are merged again into a new cluster The distances between this new cluster and all other spectra spectra cluster are calculated The two spectra spectrum spectrum or spectrum cluster or cluster c
102. r Analysis C Program OPUS6_0 IDENT Factor_scoreCoeff CLa Figure 103 Cluster Analysis Store Method tab This dialog box allows to store the cluster analysis method created Click on the Store Method button to open the standard Save File dialog box The method file has the extension CLA 120 OPUS IDENT Bruker Optik GmbH Index Numerics 3D Files 114 A A Given Oblong 114 A Given Triangle 114 Abs Threshold 68 Add Average Spectra 6 Add Region 12 86 Add Spectra 6 Artificial Spectrum 29 Average Linkage 27 29 30 113 Average Spectrum 51 C Calculate Distances 33 108 Can Be Confused With 69 74 Centroid Algorithm 113 Centroid Technique 30 Class Test 70 Class Test NOT OK 71 Class Test NOT PERFORMED 71 Class Test OK 71 Classes 25 Assign 9 83 Clear Selected Regions 87 Cluster Analysis 25 105 Performing 31 Clusters 25 Complete Linkage 30 113 Composing 88 Confidence Band 44 Confidence Level 67 Conformity Data Block 49 Conformity Index 39 46 Maximal 46 Conformity Index Limit 43 45 Conformity Test 39 Performing 48 Report 49 Setup 39 D Data Preprocessing 10 61 107 Data Processing 84 Dendrogram 25 36 109 Derivative 85 Detailed Report 94 Diagnosis 110 E Eigen Vectors 23 Eigenvalues 23 54 Euclidian Distances 28 Expected Reference 21 F Factor Spectrum 57 Factorization 23 28 53 88 Original Spectra 88 Factorization Method 58 File List 114 Fixed Algorithm 6
103. r this is actually correct or not can be tested by a validation For details see chapter 6 7 3 Abs Threshold This option allows you to define the threshold for each reference spec trum This threshold however will only take effect if the group consists of several spectra If you define the threshold make sure to consider the spectral differences of the original spectra 6 5 Identity Test The identity test routine generates a hit list which is stored in the IDENT report sorted by ascending spectral distances Provided the expected reference has been selected automatically from sample name or has been defined by the user the following three categories of identity test results are possible Identical Not Identical or Can Be Confused With see below If a group consists of only one spectrum the identity test result will be Identity Not Checked If no expected reference has been defined the identity test results will be Identified As Not Identified No Unique Identification Possible Identical to in case of expected reference The first hit must be the expected reference The Hit Quality of the first substance i e the spectral distance between a test spectrum and the first reference spectrum in the report has to be smaller than the threshold of the expected reference and the spectral distances between the query spectrum and all other average spectra are always larger than the corresponding thresholds Identified As in ca
104. raries IP2 IP3 etc it may be better to use the Factorization method and limit the spectral region which of course causes the spectral noise to be insufficiently smoothed but the spectrum shows many significant details Note If you update library data new spectra have to be re calculated on each library level Click on the Parameters tab This tab defines the spectral regions which have to be considered for identification You also have to select the preprocessing method from the drop down list as well as the IDENT method algorithm Vector normalization is frequently selected as data preprocessing method Sometimes however you get even better results if you select First and 2nd Derivative For further details refer to chapter 7 Select Vector Normalization from the Preprocessing drop down list The Always use lowest IP level check box is only enabled on the first library level as this is a global setting for the entire library If you activate the check box the IDENT analysis will be performed on the lowest IP level available This will also be done even if an IDENTICAL TO result has been achieved at any higher IP level 10 OPUS IDENT Bruker Optik GmbH Setting Parameters Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentTutorial ident_ Demodata IPI 4 1ormalization 10502 4 4099 9 EO Standard B E Figure 8 Setup Identity Test Method Parameters tab If you want
105. re you can sort the reference spectra by ID sample or group name and sub library in ascending or descending order Double click on the respective column Note that if you sort the spectra e g by sample name the ID number in the first column however will keep the original order see figure 61 x Load Method Reference Spectra Parameters Threshold Validate Store Method EN Options Set Sub Libraries 3 Add Spectra for New Group Add Spectra to Sel Group Assign Classes o Pan rename I Sartre Growpniame zar 2 Acetylcystein Acetylcystei A eee de Biotin Biotin 1 Lactose 80 mesh Lactose 80 m 6l Maisst rke_109526_0 Maisst rke_1 3 Nifedipin high_10866 Nifedipin hi 5l Syloid silica Syloid silic Figure 61 Setup Identity Test Method Reference spectra sorted in descending order 7 2 2 Missing Reference Spectra If you load an IDENT method it may occur that certain spectrum files listed in the particular method are missing in the data path To be able to localize the missing spectra the group name which the missing spectra belong to will be highlighted in red If you click on the sign of the particular group the name of the missing spectra will also be highlighted in red Bruker Optik GmbH OPUS IDENT 79 Reference Section Load Method Reference Spectra Parameters Threshold Validate Store Method Opti
106. re 77 Validate pop up menu Bruker Optik GmbH OPUS IDENT 93 Reference Section Two validation options are available e Validate this library This option validates the main and sub library you are currently working with e Validate this library and all sub libraries below This option validates the main and sub library you are currently working with and all additional sub libraries belonging to the current library The progress of the validation process is indicated by the status bar 7 5 1 Validation Report Validation reports will not be overwritten and the report file name is always the same defined for the main or sub library Groups assembled in common classes are not considered to overlap Nevertheless the IDENT reports include the groups assigned to classes You can select between the following reports which are based on single spectra except for the selectivity report and histogram e Summary Report The Summary Report outlines all important information on the current IDENT method e g path and file name sub library names date time operator name and comments It includes all the groups which can be confused with other groups e Result Report The Result Report outlines all important information on the current IDENT method e g path and file name sub library names date time operator name and comments It gives additional information on which groups should be assigned to a new common sub library e D
107. rence Test Display Cl Spectra CI limit Print Save Max Conformity Index i 3 0 Sum limit Figure 32 Setup Conformity Test Graph tab The green data points A represent the reference spectra and the blue ones B represent the test spectra The CI Limit is indicated by the red line C which can be moved by using the CI limit slider on the left side To display the reference and test spectra separately select either Reference or Test from the upper drop down list In most cases it is recommended to select the Reference Test option to be able to directly compare the scattering of reference vs test spectra If you position the cursor on one specific data point a text frame pops up indicating the exact spectrum identity see figure 33 144 00 1 7658 i No 144 Sprungantwort 1143 0 140 160 180 200 220 Figure 33 Spectra plot with spectrum description You can also select single data points only Move the cursor to the respective data point section press the left mouse button and draw the mouse over this section If you leave the mouse button only the data points selected will be displayed in the plot To undo this just right click into the plot Bruker Optik GmbH OPUS IDENT 45 Conformity Test You can select between the fo
108. rivative 0 Smoothing Points for Internal Derivative 1 Reduction Factor 1 _Hit No Hit Quality SampleName FieName Threshold 1 0 964332 000001 Sample L Leucin Av of 11 An000007 100 15 463419 2 6 198979 000004 Sample DL Alanin Av of 11 An000004 100 17 56358 3 8 598950 000005 Sample L Alanin Av of 11 An000005 100 16 701758 4 8 967188 000015 Sample L Methionin Av of 11 An000015 100 17 573791 5 9 232257 000003 Sample L Isoleucin Av of 11 An000003 100 16 73790 G 9 693203 000014 Sample DL Methionin Av of11 An000014 100 18 17575 7 11 084043 000002 Sample DL Isoleucin le of11 An000002 100 8 758205 8 11 231035 000006 Sample DL Tryptophan Av of11 An000006 100 15 760675 3 11 261703 000007 Sample L Tryptophan Av of11 An0 00007 100 21 082051 10 12836980 000008 Sample Glucose H20frei Av of 11 An000008 100 18 293217 Figure 40 Ident Report 6 1 2 Factorization The Factorization method represents spectra as linear combinations of so called factor spectra loadings a To hy aa he Tay ha eza 6 3 The a vector shows the a spectrum and the factor spectra are denoted f fo f3 etc T indicates the coefficients scores required to reconstruct the original a spectrum To calculate the spectral distance D between the two spectra a and b the T coefficients are used in the Factorization method D D Gat 6 4 Bruker Optik GmbH OPUS IDENT 53 IDENT Theory The summation is performed for a certain num
109. rization or Factorization orig specs algorithm the additional Set Factors button will be displayed Click on the Set Factors button to open the following dialog Factor Spectra x Eigenvalues of factor spectra 103 9 0 000487084 10 0 000276177 98 11 0 000235058 94 12 0 000125255 99 13 005 85 No of used factor spectra 5 I Use residuals Cancel Figure 72 Factor Spectra 88 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Parameters Example Selecting the optimum number of factors is not easy OPUS facilitates this procedure by highlighting those factors which consists of the most non overlapping score coefficient clusters as shown in figure 72 The following graph is a simplified depiction of such non overlapping score coefficient clusters of two groups A and B A B Mra nm a ool m mmm oo EI SE E OE Score coefficients The factor selection field in figure 72 has to be interpreted as follows 5 0 002231 74 6 0 00169531 120 10 7 0 00112543 118 3 8 0 000652048 103 9 0 000487084 113 10 0 000276177 98 11 0 000235058 94 9 dc Figure 73 Factor spectra highlighted A Eigen values B Number of separated score coefficient clusters it is always started with the factor which includes the most groups separated C Number of separated score coefficient clusters additional to the factors selected before In case of factor 6 e g there are 120 clu
110. rogra Cellulose_microcrys Microcrist Cellulose RC 581_ 109200 01 M EZA EZA EZA EZA EZA EZE EZA EZA EZA EO pel EEK OPO rdi an ei DI barr GE 19 C Progra Cellulose_microcrys Microcrist Cellulose RC 581_109200_03_K an ba LLET asi MM Anh m zz eee tee err ezi ME err erte ie Hi le e e DZ Od ANNAN ND I Figure 89 Cluster Analysis Reference Spectra tab This dialog box is the same as the Reference Spectra dialog box of the Setup Identity Test Method command and has been described in section 7 2 106 OPUS IDENT Bruker Optik GmbH Cluster Analysis Parameters 7 10 Cluster Analysis Parameters Cluster Analysis New E x Load Method Reference Spectra 3d File Filelist Parameters Report Store Method m Preprocessing Vector normalization Regions Interactive Region Selection Clear Selected Regions 11995 Method m Making distance matrix Standard v Calculate Distances Figure 90 Cluster Analysis Parameters tab 7 10 1 Preprocessing The cluster analysis uses the same data preprocessing methods as described in section 7 3 7 10 2 Regions The Regions table allows to limit the data to one or several spectral regions to be considered for the cluster analysis The frequency limits for the spectral regions can either be entered manually or selected interactively Bruker Op
111. rs For k 1 and 1 n the result is identical to the Whole Matrix option result e A Given Oblong The distances are determined for those spectra between the ku and 1 position rows and between the k and l position columns of the list This corresponds to a rectangle within the matrix The value specified in the Width for Classes of Distances field determines the number of classes The default value corresponds to a division of 20 classes i e the range from 0 up to the maximum distance is divided into 10 equal areas You can change this value The maximum number of classes is 20 If you enter an invalid number the value will automatically be corrected The Window button opens the dendrogram diagnosis or histogram within a Report window You can have the report printed out using the print options from the OPUS Print menu 7 12 3D File Filelist You can also set up a cluster analysis using either a 3D file or a file list When working with a 3D file you first have to load such a file OPUS automatically opens the 3D display indicated by the 22a tap 114 OPUS IDENT Bruker Optik GmbH 3D File Filelist Rotation E ay Inclination E 4 n x 1893 85 E SEE x EE 70 2 1350 00 Z 2727 00 Index 1350 pitta Gi Z ClusterList N m bei o 1000 0 es ZG 10015 20 25C3003500 X VVavenumber cm 1 E g 515 5 p w o o c c 5 1 0 5 2 3 EE 0 5 Kl o Al n A AU y JA
112. s Parameters 2 404 4 dead aqcgs dante Sees een aoe Hae 107 LAQA Preprocessing 4 d a Karea Kei i elea Bae goai 107 GK REINS agia EO ei arraia age ween ge ges ae agitz a aes 107 PIO Method orea IS grils SY Jet caters adres tte fata Na daar 108 7 10 4 Calculate Distances era age elas Ea die SEO eia 108 7 11 Cluster Analysis Report AAA 109 PALA OE Tar alee ead Lesa Weg rira atara EE 111 PAL ARDURE sentana bic deay edit daure edarra Git edehiet pais 112 7 12 SD Fil Filelisto n Gowan Gus a aa RA gb arg ei e 114 ebe MORI EAEN EO 119 7 13 Cluster Analysis Store Method 0 cece cece ee eee eee 120 About OPUS IDENT This manual consists of two parts The first part describes how to create a user defined reference library and generate an IDENT method Apart from this the IDENT analysis and the resulting report files are explained in detail as well as the theory of the IDENT software test routines The cluster analysis and conformity test are described in a separate chapter The reference section refers to all IDENT functions and supports you if you have questions about the IDENT functionalities or problems while using IDENT Introduction OPUS IDENT in the following referred to as IDENT is a software package designed to identify substances by their IR spectra An unknown spectrum in the following called test spectrum is directly compared to reference spectra of a library IDENT identifies those reference spectra whi
113. s E Reports AB Method file Compare Spectra from date x Ranges time Description IDENTICAL TO 000001 Sample L Leucin Av of 11 Hit quality with expected reference Threshold for expected reference reshold calculation Algorithm Vector normalized spectra Order of Derivative Smoothing points No of used factor sp 15 hits of 15 gt Ranges Class Name Class Test OK Using residuals Order of Internal Derivative Smoothing Points for Internal Derivative Reduction Factor Hit No HitQualty Sample Name Group _ Threshold 0 010556 000001 Sample L Leucin Av of 11 depends on each ref spectrum 0 023866 C MOPUSSIDENTSexamplesiSTANDARD FAA 1 PE 0 047006 000003 Sample L Isoleucin Av of 11 000003 0 023866 3 0 095210 000002 Sample DL Isoleucin Ay of 11 4 0 104591 000015 Sample L Methionin Av of 11 5 0 146645 000004 Sample DL Alanin Av of 11 6 0 158939 000005 Sample L Alanin Av of 11 i 0 172990 000014 Sample DL Methionin Ay of 11 8 0 266797 000006 Sample DL Tryptophan Ay of 11 g 0 288849 000007 Sample L Tryptophan Av of 11 10 0 668990 000011 Sample Xplit Av of 11 11 0 671159 000010 Sample Fructose Av of 11 12 0 690747 000012 Sample Sorbit Av of 11 13 0 750100 000013 Sample Mannit Av of 11 14 0 763355 000008 Sample Glucose H20frei Av of 11 15 0 848422 000009 Sample Glucose H20 Av of 11 d Figure
114. s case the report indicates the corresponding average spectrum sample name and threshold confidence region specified for this substance The file names of the original spectra which have not been identified and the spectral distances between these original spectra and the average spectrum Hit are listed under the Original Spectra Outside Confidence Region definition Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata Id Load Method Reference Spectra Parameters Threshold Validate Store Method Selectivity ag a2 4 4 5 Detailed report br Ident_Demo Y02 bd Set Print 0 085380 0 110454 a Raspherry_aroma 35 sugar 0 079495 0 110454 Raspherry_aroma 36 sugar 0 080934 0 110454 Raspherry_aroma 37 sugar 0 066776 0 110454 Raspherry_aroma 36 sugar 0 089263 0 110454 Raspherry_aroma 39 sugar 0 083548 0 110454 HEH HE HE HE HE HE HE HE HE HE HE HE HE E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E A E E E E E E E E E E E E E E E A EE HE HE HE HE HE HE HE OE HE HE HE HE HE E E E E E E E E E E E E E d E E E E E E E E E E E E E E E E E E E E E E E E E A E E E E E E E E E E E E E E E A HHI Summary Original Spectra which can be confused with other references 198 of La E pn o I Original Spectra which can be uniquely identified 526 of 726 o be AG b D 3 da aroen be Figure 57 Validation Report Spectra ident
115. s the method However the spectra have an additional numeric file extension starting with 0 for the first factor spectrum These spectra can be loaded into OPUS like any other spectrum 54 OPUS IDENT Bruker Optik GmbH Algorithms 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 Figure 42 Reference spectrum and first factor spectrum 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 Figure 43 Second third and forth factor spectrum The spectra see figure 42 and 43 show the signal to noise ratio of a certain factor spectrum The factor spectrum displayed in figure 44 mainly consists of noise It is not recommended to use this spectrum to calculate spectral distances Bruker Optik GmbH OPUS IDENT 55 IDENT Theory You can easily check the factor spectra orthogonality by multiplying two factor spectra using the OPUS Spectrum Calculator This is followed by an integration across the whole frequency range of the result spectrum The integration result will be 0 or approximately 0 due to the finite computing accuracy 0 000000050 o 5 GO Q 0 000000050 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 Figure 44 Factor spectrum with excessive noise 6 2 Factorization Theory Assuming that s reference spectra consist of d data points each The reference spectra are represented by d d d3 column vectors
116. se Isosorbide dinitrate Isosorbide dinitrat Lactose 80 mesh Lactose 80mesh Lactose 200 mesh Lactose 200mesh Nifedipin high Nifedipin high Nifedipin slow Nifedipin slow Nitrofurantoin powd Nitrofurantoin po Pharmacoat 606 Pharmacoat Raspberry aroma Raspberry aroma Sugar crystalline Sugar Figure 5 Setup Identity Test Method Reference Spectra tab In both cases a dialog box opens where you can select the spectra to be used for the IDENT method Having loaded spectra they will be assigned to the respective group and are given a consecutive JD number The Sample Name as well as the Group Name are automatically read from the file parameters To see the list of group specific spectra click on the sign in the first column figure 5 Click on the sign to close the list You can also remove entries from the file list Select one or more entries by clicking on the left mouse button and holding down the Shift or CTR key Use the DEL key on the PC keyboard to delete the entry ies Before you can delete a group or spectrum a menu pops up and asks you whether you really want to delete the spectra selected If you want to create new sub libraries click on the Set Sub Libraries button These sub libraries are an integral part of the main IDENT method and are indicated in green in the Sub Library column figure 5 The following dialog opens Bruker Optik GmbH OPUS
117. se of no reference defined All threshold values are taken into account In case of Identified As the Hit Quality of the first substance i e the spectral distance between a test spectrum and the first reference spectrum in the report has to be smaller than the corresponding threshold But the Hit Quality of all other substances has to be larger than the corresponding threshold 68 OPUS IDENT Bruker Optik GmbH Identity Test Not Identical to in case of expected reference The spectral distance between the test spectrum and expected reference spectrum is larger than the threshold value Not Identified in case of no reference defined In this case the Hit Quality of all reference substances has to be larger than the threshold Can be Confused with in case of expected reference This result indicates that the spectral distance of the test spectrum to at least one other average spectrum is smaller than the confidence region If e g the Hit Quality of 4 substances is smaller than the corresponding threshold and one of these substances is identified to be the expected reference No Unique Identification Possible in case of no reference defined The Hit Quality of more than one substance is smaller than the corresponding threshold Code numbers are assigned to the individual test results Therefore the results can be easily evaluated in an OPUS macro Can be confused with lt N gt Other Hits in case of expected
118. sters separated with 10 of these 120 clusters not being separated by factor 5 In case of factor 7 there are 118 clusters separated with 3 of these 118 clusters not being separated by factor 5 nor factor 6 Select the factor spectra from the list which are used to calculate the spectral distance It is not advisable to accept this value without performing a validation first We recommend to perform several validations of the IDENT library using different numbers of factor spectra Select the optimum number of factor spectra according to the result obtained It is not necessary to use a consecutive sequence of factor spectra You can select factor 2 3 and 5 Delete factor 1 if you do not want to get any baseline information If you want to calculate spectral distances by using spectral residuals activate the Use Residuals check box Bruker Optik GmbH OPUS IDENT 89 Reference Section 7 4 Setup Identity Test Method Threshold Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentTutoria x Load Method Reference Spectra Parameters Threshold validate Store Method i de Maximum hit SDev Confidence Level 0 25 99 99 Set Set Outlier TE 0 017578 Cellulose PH101 99 9988 0 0061317 3 Cellulose PH102 99 9968 0 0038525 4 Cellulose PH112 99 8456 0 0040854 5 Corn starch 99 9999 0 018445 6 Diclofenac potassi F 99 9849 0 011674 Diclofenac sodium 99
119. ta Extended Demodata IdentT utorial Ident_Demodata IP1 IP3 class_en FAA Load Ident Method Change m Output options b Show results immediately J Print results automatically Identity Test Cancel Help Figure 84 Identity Test Select File s tab Select a test spectrum and drag and drop the spectrum absorption block from the OPUS browser window into the File s for identity test selection field To load or modify an IDENT method click on the Load Ident Method button and select the desired method from the dialog that opens If an identity test method has already been loaded e g if you have created one prior to starting the analysis the path and method name will be indicated in the Loaded Identity Test Method field The Show results immediately check box in the Output options group field is activated by default and the identity test results will be shown in a special evaluation result display see figure 85 and 86 Bruker Optik GmbH OPUS IDENT 101 Reference Section Result of IDENT Evaluation Sample C OPUS Daten_Ident_Kurs Cellulose microcryst Cellulose microcryst 2 Method File C OPUS Daten_Ident_Kurs DemoMethod FAA Date and Time 2002 08 27 15 29 27 UTC 1 Hit No Sample Name Hit Qual Threshold Group 1 Cellulose microcryst n 0 00823 0 01046 Cellulos 2 Starch from rice n 0 14093 0 01013 Starch f01 3 Starch from corn n 0 17524 0 00905 Starch f 4 Starch soluble n 0 19326
120. the average of the reference samples is calculated on each wave lengthi This absolute deviation is now weighted by the corresponding standard deviation oon the respective wavelength which results in a relative deviation referred to as Conformity Index CI JE EEE E Asample Oreference i The maximum of all CI values is derived as test result 5 1 Setting up Conformity Test Select the Setup Conformity Test command in the Evaluate menu On the Load Method tab you can load an already existing method file generated for a conformity test Bruker Optik GmbH OPUS IDENT 39 Conformity Test Setup Conformity Test General information Reference spectra 41 Test spectra 220 Aa ae Frequency ranges 1 Selected datapoints 631 Preprocessing First derivative C Program OPUS6_0 Konformitatstest Mixing_TU_Munich Mixing_TU_Mur ict E Load Method Reference Spectra Test Spectra Parameters Validate Graph Report Store Method Load Method Figure 26 Setup Conformity Test Load Method tab The General information group field includes information on the kind of data block data points selected as well as the data preprocessing type It is distinguished between reference and test spectra Reference spectra have been created by a specific method whereas test spectra can be tested for their conformity with this specific method for validation purposes To create a new method click on the
121. tik GmbH OPUS IDENT 107 Reference Section 7 10 3 Method Select an algorithm to identify the spectrum see section 6 1 and 7 3 If you select the Factorization method you have to specify the number of factor spectra to be used to calculate the spectral distances in the Factor Spectra dialog box This dialog box opens automatically after clicking on the Calculate Distances button In contrast to the identity test the Use Residuals option is not available during cluster analysis Spectral residuals are not taken into account when calculating spectral distances The calculation of factor spectra is not necessary during cluster analysis as test spectra will not be analyzed To determine the spectrum to spectrum distance see section 6 1 2 only Z covariance matrix and L Eigen vectors will be calculated Click on the OK button to return to the Parameters dialog box Factor Spectra x Eigenvalues of factor spectra 10 0 00219666 16 0 00058 17 0 000485223 18 0 000476858 191 0 000443329 E No of used factor spectra fis E Use residuals Cancel Figure 91 Cluster Analysis Factor Spectra 7 10 4 Calculate Distances Click on the Calculate Distances button to start the calculation of the spectrum to spectrum distance If the calculation has been finished it is recommended to store the method before you generate a dendrogram 108 OPUS IDENT Bruker Optik GmbH Cluster Analysis Report 7 1
122. tion on the data preprocessing method the algorithm used for the identity test and how many sub libraries are part of the reference library For further details on how to rename libraries refer to chapter 1 To print the library structure use the drop down list to select the respective printing option and click on the Print button For further details see chapter 1 Setup Identity Test Method Reference Spectra Setup Identity Test Method C Program OPUS6_0 Data Extended Demodata IdentTutori x Load Method Reference Spectra Parameters Threshold Validate Store Method Set Sub Libraries bO Assign Classes Pe ets A aea Ee Sub ea Acetylcystein Acetylcystein Cellulose microcryst Cellulose PH101 Cellulose microcryst Cellulose PH102 Cellulose microcryst Cellulose PH112 Corn starch Corn starch Diclofenac potassiu Diclofenac potassi Diclofenac sodium Diclofenac sodium EO wN GO ON db LA bd e Eudragit RS PO Eudragit Glucose dried Glucose Isosorbide dinitrate Isosorbide dinitrat Lactose 80 mesh Lactose 80mesh Lactose 200 mesh Lactose 200mesh Nifedipin high Nifedipin high Nifedipin slow Nifedipin slow Nitrofurantoin powd Nitrofurantoin po Pharmacoat 606 Pharmacoat Raspberry aroma Raspberry aroma Sugar crystalline Sug
123. tra Parameters Validate Graph Report Store Method r Preprocessing Smoothing points 21 GO Interactive Region Selection Conformity Test parameters Conformity Index limit g I Use signed Conformity Index values m View spectra Display Preprocessed Spectra Figure 30 Setup Conformity Test Parameter tab Frequently the Vector normalization is selected as preprocessing method Sometimes better results can be obtained by using the First or 2nd derivative method In both cases you have to additionally define the amount of Smoothing points you can select between 5 9 and 25 The optimal number of smoothing points however has to be evaluated empirically The Conformity Index Limit parameter records spectra between an upper and lower limit see graph below The best possible scaling factor is between 3 and 4 which is of course not mandatory The evaluation basis for the self adapting conformity test is the reference spectra scaling indicated by means of the standard deviation Upper CI limit gd be Lower CI limit Bruker Optik GmbH OPUS IDENT 43 Conformity Test Activate the Use signed Conformity Index Values check box if you want to use non absolute index values Click on the Display Preprocessed Spectra button to have the respective spectra and confidence band displayed The confidence band is displayed in red and is calculated as follows Average value CI limit
124. tra in an identity test you have to enter the number of average spectra into the Number of Classes field Define which part of the matrix you want to include into the histogram Whole Matrix In this case all distances will be used With the matrix being symmetrical and diagonal elements being 0 only a triangular matrix without diagonal elements is used The matrix size is n n 1 2 Only Pairs for repro tests The data record is divided into pairs and the distances between the first and second spectra first pair third and fourth spectra second pair etc are calculated The number of distances being considered is n 2 This value can be used to determine the reproduction level of measurements which have been repeated twice Only Triplets for repro tests Same as above but this time the data record is divided in triplets The number of distances used for statistics is n 3 3 3 This value can be used to determine the reproduction level of measurements which have been repeated three times Bruker Optik GmbH OPUS IDENT 113 Reference Section e Only Reference i e the last column This option only considers distances between the last spectrum indicated in the list and all other spectra The number of distances is n 1 e A Given Triangle The distances are calculated for those spectra between amp and rows of the list This results in a triangle within the matrix You have to enter the k and paramete
125. u can perform an analysis using this IDENT method and the analysis results will be displayed in the form of a report To perform an identity test the following steps are required 1 Measuring at least 1 reference spectrum per substance 2 Incorporating the reference spectra into a library the spectra need to belong to batches of one single substance which have been identified by means of conventional analytics 3 Defining a suitable spectral range for identification 4 Selecting a data preprocessing method 5 Generating an IDENT method 6 Measuring test spectra 7 Analyzing test spectra This chapter assumes theoretical basic knowledge and therefore only briefly outlines the methods involved The theory of the identity test method is explained in chapter 6 If you generate an IDENT method a main spectra library IP1 will be created All sub libraries e g IP2 IP3 IP4 etc which are related to this reference library will be stored in the same method file which uses the extension SEA The data structure of the complete library is displayed in a kind of browser window and OPUS ensures a common internal validation of the entire library For further details see chapter 7 1 1 Loading Existing Method Start the IDENT software from the OPUS Evaluate menu Select the Setup Identity Test Method command Bruker Optik GmbH OPUS IDENT 3 Setting Up an Identity Test Method If you want to load an existing method clic
126. uired e Measuring at least 1 spectrum per substance e Incorporating the spectra into the list e Defining a suitable spectral range for identification e Selecting a data preprocessing method e Calculating spectral distances e Defining a cluster algorithm e Generating a dendrogram histogram or diagnosis Several tabs of the Cluster Analysis dialog box are identical to the Setup Identity Test Method dialog and will only be briefly explained For details see the respective sections in chapter 1 Click on the Cluster Analysis command from the Evaluate menu On the Load Method tab you can load an already existing method file generated for cluster analysis This tab is identical to the Load Method tab of the Setup Identity Test Method command As you will generate a new Cluster Analysis method click on the Reference Spectra tab The following dialog opens Bruker Optik GmbH OPUS IDENT 31 Cluster Analysis Cluster Analysis New Load Method Reference Spectra 3d File Filelist Parameters Report Store Method Add Spectra Change Path led e ar eza C Progra Cellulose_microcrys Cellulose microcrystalline PH 101 C Progra Cellulose_microcrys Microcist Cellulose PH 101_108349_02 KK C Progra Cellulose_microcrys Microcrist Cellulose PH 101_ 108349 02 M C Progra Cellulose_microcrys Microcrist Cellulose PH 101 108349 05 K C Progra Cellulose_microcrys Microcist Cellulos
127. well as combinations of both methods e Vector Normalization The Vector Normalization data preprocessing normalizes a spectrum i e the average y value is calculated first and subsequently subtracted from the spectrum Then the sum of squares of all y values is calculated and the spectrum is divided by the square root of this sum This method is used in case of different optical thickness to compare the samples with each other The form of the different spectra will be preserved which facilitates the interpreting of spectra However the result extremely depends on the spectral region selected i e specific differences of one region are distributed to all data points 84 OPUS IDENT Bruker Optik GmbH Setup Identity Test Method Parameters e First Derivative Calculates the first derivative of the spectrum by interpolation Steep edges of a peak become more important compared to flat structures This method is mainly used to preprocess pronounced but small features which are overlaid by a high and broad background In case of this method the window size selected is very important The smaller the window size the more spectral details are shown with the spectral to noise ratio being apparently higher e 2nd Derivative This method is similar to First Derivative but it allows to evaluate extremely flat structures If you use one of the derivative methods an additional selection field will be displayed to define the amount o
128. which form D data matrix d x s dimension D d d d d 6 5 When exchanging the rows for the columns in this matrix you obtain DT transposed matrix of s xd dimension Multiply DT transposed matrix by D original matrix to obtain Z covariance matrix ZED D 6 6 A diagonalization and orthogonal transformation of Z produce Eigen vectors and Eigen values of Z NED e E 6 7 56 OPUS IDENT Bruker Optik GmbH Factorization Theory The column vectors of L matrix s x s dimension are l l 13 Eigen vectors of Z matrix These Eigen vectors are orthonormal i e the following equations are valid for the scalar product of two Eigen vectors l l 0 E 6 8 l 6 9 A matrix s x s dimension contains A ka A3 A Eigen values of Z matrix as the main diagonal all other matrix elements are 0 This means Z 1 kl 6 10 D data matrix is factorized by L Eigen vector matrix using multiplication F D L 6 11 F matrix has the same dimensions as D data matrix d x s and includes the vectors of f f fz factor spectra as columns Multiplying E transposed matrix by F yields F F UD BI x D L L D D L L Z L A 6 12 The elements of FTF quadratic matrix s x s dimension are the scalar products which can be created in pairs together with factor spectra The 6 12 equation causes the factor spectra to be orthogonal to each other if 0 PES 6 13 Jef Ai 6 14 The vector norm of a factor

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