Shimadzu LCsolution Chromatography Data System Operation Manual Read the instruction manual thoroughly before you use the product. Keep this instruction manual for future reference. @) sHimapzu Shimadzu Corporation ents Division Analytical & Measuring Instru Kyoto, Japan 223.0814Copyright Shimadzu Corporation 2004. All rights are reserved, including those to reproduce this publication or parts there of in any form, without the exprese written permission of Shimadzu Corporation. Information in this publication is subject to change without notice and docs aot represent a commitment on the patt of the vendor Any errors or omissions which may have oseutred in this publication will be corrected as soon as possible, but not nesessar~ ily immediately upon detection. Shimadzu Corporation makes no claims or warranty concerning the influence of the application of this publicstion on the ‘operation of the machine. [Note that Shimadzu does not have any obligation concerning the eflécts resulting ftom the application ofthe contents ofthis ‘mama Shimadzu Corporation will continue to supply original Shimadzu maintenance parts associated witha given product for a period up to 7 yeas fiom the cessstion of production ofthat product Please be informed that afte this period of time, Shimadzi Corporation cannot guarantee supply of such original mainte- nance parts. However, following diseontinuation of a product, the period of availabilty of maintenance parte which have ‘been produced on a aubcouttsct basis is up to the disertion ofthe eoncemed sub-contracting company. Microsoft® Windows, Windows NT® and MS-Excel® are registered trademarks of Misrosoft Corporation. ‘Microsoft® Windows" 2000, Windows? XP and MS-Word are products of Microsoft Corporation. Adobe®, Acrobat® and Distiller? are registerd trademarks of Adobe Systems Incorporated. Acrobat Reader™ and Acrobat PDFWriter™ are products af Adobe Systems IncorporatedIntroduction ‘Thank you very much for purchasing the LCsolution software for Shimadzu liquid chromatography workstations (hereafter called “LCselation’ [LCsalution allows you to control the liquid chromatograpa (hereafter called “LC*) from your personal compute, acquire chee= smatograms and other different kinds of date, and zeanalyze the acquired data under different parameters on your personal com+ ter ‘This manual has been put together in order to familiarize you with the basic knowledge required to operate LCsolution. Be sure to read it throughly before using this software. After reading the manual, keep i in a safe place so that it cam be accessed whenever necessary. ‘The "Administration Manual” for LCsolution for Shimadzu liquid chromatography workstations is attached as an on-line man- val ‘The “Administration manual” is tached asa separate volume ‘The Administration manual covers the information on the features useful for eystem administration such asthe support feae tures for GLP/GMP or FDA 21CFR Past, a set of regulations for eletronic records and electronic signature, For more infor :mation on the funstions of LCsoluion, refer to this on-line manual. ‘This manual assumes thatthe reader is knowledgeable of basi operations of Windows®, For the operation of Windows®, refer tothe instruction manual that comes with that product. ‘Thie manual sometimes explains commonly for LabSolutions series. And some explanations may use the drawings come from sister products like GCsolution, i it does not cause misunderstanding in the sange of explanationsUsing the instruction manual Hi Kinds of instruction manuals ‘The LCsolution package contains the following information that describes the operational procedures and functions ‘Name Media Description (persion mana for | Printed Document “Explains the operational procedures for data acquisition ard asaya Lesotution Leseluton Tastllation anual for | Print Document Explains the eperatonal procedures for sstllation of LCsolunon Lessisna “Admunisrien manval for | Paated Documeat ‘Explains te operational procedures aad base idea of system admiast- LCssisnon ton and data management wsing LCsohion Online help Lsottion program Provides detaled infomation oa parameters and sering ranges, This sccemble fom the Help ment in [Csoltion (For urna the on-line help refer to secon “18 11 Teng Help" inthe Operon manual) Operation manual for | CD-ROM dik for installa | Provides the operation volume ofthe isuction manual ab & PDF Sle 50 ECsoluton @DF version) | wow that st can be viewed oa your gersoaal computes Its accessible fom the Help mens in LCsoloton. (For sing his PDF, cefer to section °13.72 Using the Online Mara” in the Operation mana) Tnstaliason manuel for | CD-ROM disk for installa. | Provides the installation vohume ofthe istration manval 6a PDF fileso Lsolstion PDF version) | som that it can be viewed on your personal compter Tis yer inked 1 the sdvanced se up cr maitensnco procedures ithe Operation Administ ‘non mama) “Admunisuaton manval for | CD-ROM disk for iasala- | Explains the operational procedures and basic idea of system admunisea. Lesotstion PDF version) | sion ‘non and data management using LCsokion. This manual i provided asa PDF files that itcan be refered to online whenever operation ested 0 system adminiovaion are neaded. The goncal table of eoatens $9 acl se, saclay all the satvction manele (PDF version). Ie allows you's tse cach instvsion mana va the sper || Legends for instruction manual ‘This manual ses the following legends: ‘Legend Meaning ® Gives you tis <> Shows a window or view mame, ¢ =. 2 372 Checking data ycapayne Window . $2 Data Files Handled in the Window . 521 Imponing/Exporting Methods 522 lenpore Export Repor Formats $3 Setting Peak Integration Parameters S31 Serang Peak Ineprtion Parameters 532 Detectsg Peaks Based on the Peak Iteration Paramcters 533 Semag atearation Tame Program Examples 5.34 Manual peak integration feature (manipulation) 535 Seta Manual Pak lntegaicn Examples S4 Setting Peak Identification Parameters S41 Sutin Peak Weatscaton Parameters 542 Perfomnine Pak leeniaton SS Setting Quantitative Parameters .....essceeseeeeeeeeeeseeeseeeneeneenseeeees 5S: ‘Seng Quantitative Pasmatrs 55 Setiag Compound Tables 56 Seving in Exporting to) Method File 56 SG Operations in the Window ....6.cccssecseesceessesseeeee 87 8.7 Operations in the Window . 6.2 Using the Multi Chromatogram for Data Analysis 63 Extracting a Chromatogram/Spectrum in Different Situations 631 Exwactiag fom View 632 Exuacting fom View 64 Registering Extracted Chromatograms/Spectrums .....eecceeeceeseeeeeeeeeen es 65 G41 Ropisoring Fxractad Cheomatonms ie Muli Chyomstoztam Table 65 642 Registering Exracted Spectrams in Spectrum Table 66 6S Calculating Peak Purity ....6eececeeeeeeeeeeeeeeeeereenerteenteeneeeees 651 Setting Paty Parameters 6 652 Displaying Calculated Peak Party 6 6.6 Identifying Compounds by Their Spectrum . 0 651 Setting Library Search Parameters 70 652 Searching with UV Library n 67 Editing the Spectrum Library 68 Using Spectrums to Identify Peaks ll (HN LabSotutions/4.Csotution Operation jtttttttneeeem69 Performing Other Operations 691 Saving in Exporting to) a Method File 692 Exportng Chromatogram Spectrum, 693 Upiating Snapshot 6.94 Performing Arhmene Operations oa Chromatograms Spectrum 5 15 15 18 7 Batch Processing 7.1 Performing Continuous Data Acquisition . 711 Displaying a Batch Teble= Window ro Creme Batch Tables 742 Creating Bach Tabs Using the Wizard 713° Performing Batch Processing 7.2 Performing Continuons Postrun Analysis . 721 Displaying a Window to Open Batch Fes 722 Selecta Data Files Used to Cente Batch Table 723 Perfogaiag Batch Processing 7.3 Batch Processing Parameters . 731 Sema the Basket Callransa ype 732 Speatving Statup Shutdown conkers 733° Semag the Beckeround Data File 74 Performing Additional Analysis - Barch Queue Function ..... n 8 80 st SL 2 4 4 86 8 Browser Functions $7 se 8.1 Browsing the Quantitative Cale. Results at a Time S11 Displaying the Quanttanve Cale Result Quant Browser 812 Diaplayng the Necenay Information 82 Making a Postrun Analysts of Multiple Data 821 Changing the Data Processing Condstions via “Quant Browsex> 822 Starsically Celeulating the Quantitative Caution Values 83 Listing Multiple Data ....e.sceeseeeeeeeeeeeeeeereenees 831 Perfonine Operations with window 1337 Using the 2.1.1 LCsolut n Applications Installing this software into the PC, the [LCsolution] icon appears on the sereen (desk top), ‘Double-cticking thie ‘eon tas LCsolution Sy oe ‘Agent optionally ava tle) ie nealled CCidking on this kay allows yov to select {[Opearion) eon ed Clicking this icon starts an application that is used to enter instrument and device settings, to control instruments and devices, and to perform a single ‘or batch nas (This is in Online mode.) Clicking this icon tarts an application that is used to read acquired analy sis dota and to perform calibration and data processing Lc Postun) Clicking this icon starts an application that is sed to, during analysis, edit ‘a method file or bate fie that isnot sed for analysis (This is in Offline mode.) Clicking this igon starts an application that is used 10 browse multipule data or perform quantitative calculation for mubtipule data files in one the Note: The [LC Analyse #] and [LC Analysis Editor #] scons ae enabled fir the sumiber of HPLC that ae curently ensod to your pe sonal computer Note: The [LC Analysis #) andthe [LC Analysis Edo *] scons aro piven tho snstument nam restored on “System Configueation” a9 “ool Tips” with focusing by mouse cursor. Note: When another prostans i stares flsreen mode, the seen program icon will baceme isle bebind st Reduce of mi mize the curent program window t see tor pinto [LabSolatons) -[LCsouton] inthe [Start] mens to selec the program. {GE Labsolutions/LCsolution Operation jee‘This document does not cover the operations functions available from the [Administration] tab, but inchudes the following programs: Double-click on tis conf] pemrmermer “LCsolation Launcher is mesure LCsolution aluiluicustio) “Enabled when CLASS. Agent ( eal i ie)is Chdkng on tis key iti £ oo aulows yoo to sclst | Uo (Adminseasion) wb PCa us Lone ‘eure jm cna fincas Hl atk: ‘Used to register users or periodically change passwords. Also used to Bll cS Alejo w wa ig ops yw Oe wi Hl) Sees Log Browse] For farther dtails of th icons included nthe [Administration] tab, refer to [Adminizraton Volume], 2.2 Using he S)stam Administrae on Tole Ser dvatabe kame eT LabsotutionsACsotution Operation la_Affee confirming that the HPLC unit has been tuned on and initialized, click the [LC Analysis 1] icoa to call up the program. The following application will sat. Note: Tue <1 Analysis Eder secon ome the a:talenlss functions found inthe scree, The “LC Postrus> sere tems ae alo basicaly the ame as those used inthe window is available on both and programs Stores batch table used to perform series of analyses or postrun analyses, ‘The seme batch file ean be used on both and programs eT LabsotutionsACsotution Operation la39> 3 Performing an Analysis . I Introduction This chapter will serve ae a ttorial that will help you understand the basie LCsolution operations by providing you with the opportunity to use the LCeolution to perform actual analysis 3.1.1 Procedure for operating LCsolution in this example Description of table File extensions and description of files Method Analisi Location of application curve formation: if ‘" = QAIQC stings, calibration Batch File Batch able, batch seins Rel Data File Chromatogr, peak table, enscation/quent- tran excosion) | chapter terse analysis ests, report format (dma sepor) method (cory (omy 525 ¢ (Created automaticaly) = pain NZ Note: Tosh ch ie i a mtd Batch Table sd seport formu (Je) foe each data Planning and executing batch analysis" (le). After te _ NY Te Data Analysis Te Calibration Carve ‘Checking renal Checking results (calibration curve) flee) coun the saferuation on th Jean and ler that have been specified on each sow of the batch por is op) and on the Le eb) ead table (hen ts LabSolutions/LCsolution OperationNote: 1F loa i opened by solecing TT. Fn prepare the enlys sem Conte tha the units for he nai sytem (pump, tonapler, columa oven a etc) are somes tothe ste onl, andthe um then On. Aer the power has bee ured On, the wis wile inkalized. Chek tha 0 enor message appears on any of the ui, ses, fer oth incruction marae forth 2. sia the window will appear Note: Before staring up LCsoloion, be sure to check tar the analysis system is active Ifthe analysis system i acuated afer the LCso- ‘in has ben started, he LCsolution will ot eeogniz the sytem. 3. The dialog box will pen. Enter your user ID and password to log in. Jnpsolnitiois ‘Gicking on the right button sill play the user ID fst, ‘Afr lacing = UserID eick [OXY “iGsafution (Note: fhe initial state, you cat login by entering your user ID: Admin (with no password). (Note: To ster serup a password for an “Admun" level use, oto ad user(s). se the program (Gl —_LabSolutions/LCsolution Operation3.2.2 Setting up the system configuration ‘When the LCsolution is first connected to the analysis system or when the system configuration is changed, the following sertings will be required. If here is no change in the system configuration, skip this chapter and proceed to “3.2.3 Checkong analysis system A. Click on {System Configuration] a the top page ofthe Assistant Bar displayed onthe left side ofthe sereen you have logaed io ®D —_trany page other than the top page ([Real Time)) of the Assistant Baris displayed, click on the (Top) ison to display the (Rel Time] Assistant Ba 2. in the and elect sytem and communication prt, Then add th sytem controller tothe unit that are wed for analysis. Now itis posible to select the stings ofeach unit inthe system (automatic? manual stings) Click [System Configuration] = Spence: S108 Fe Pne dtc = B Peet: 1c 10 Bow By pasar Sk - 1 ame DOr: 100%» 1 “Fi Fence £3 ata: S008 Siem Check, G sacoe stomatal lode the nected ut the we of Mules, Used for Analysis) Use| 0 delete unused units from tree. & scott] tem | cs ok | tea] | window will appear. (Administration Manual] “5.2.1 Performing System Checks”) Click on the [Run] button, The system check will commenes, “Whon the system chook ashed, the evaluation rote we peed Aon chasing of Seavcoeee ‘it Sener system Check Resutes> | cater (Eilon EE (Note: To check Lamp Energy” and Wavelength Accuracy” ofthe ight seuce forthe detector, set [Check Level] t0"Advanced” Otber- wise, selet “Normal”. 1th patna logs (operation log, maitenance log, ad eer log) the outgu of varios comection coei- cent, eae sequited, seth ouput epion [Fort] 1 “Advanced” (See naxt pase) 3. During the system check, the screen indicates the progress ofthe system check, 4, Ait the system check has been completed (the progress bar has reached 100%), click om the [View Results] button to isplay the results. Check to see that no item that has failed the test. The results ofthe system check will be saved a8 a file that is automatically named (inches the analysis date) inthe Log Syscheck fer, which is located in the LCsolu- tion folder, Note: If the results of te system check show a flute refer to the instruction manual foreach nit and ake the necessary remedial actoes,isioding the replacement of consumables and the calibration ofthe detector. Te check the ference value forte epstena clack, open the Properiss window for etch system uit in accordance with °2.22 Sergi the ston conaurerion” ick en the (System Check] button, an then change the reference vals 11 [| LabSotutions/LCsotution Operation jie3.2.4 _ Setting instrument parameters Set the analysis conditions instrument parameters) inthe window. A. check that “Ready” is displayed in the status field. (Lf this field shows, eg., “Not Connected”, the system environment ‘needs tobe setup properly in accordance with “3.2.2 Setting up the system configuration”. 2. The View. A dialog box will be opened allowing you to save the settings (nethod). Give a name tothe method fle and then save it. The parameters you have set willbe transfered to the insta sent, Ifthe system controller is activated during communication (it can be made active by clicking om the (Instrument On/Off] button), each system unit will start operating withthe set parameters. ‘The CBM-20,20A8it will immediately reflect in each unit the parameters that have been downloaded from the PC. ‘To operate the equipment, use the following instrument contol tool buttons: Instrument On O# [Used to activate or deactivate the contolle IF the contollertemsins acuvated, the ‘astument wll star the specified operation when the issumest controle parameters are downoaded For the CBM this button cued to conta the pup and oven Controller's LCD OwOtt Uses turn ON/OFF the LCD of the contre (Controller Lock: Unlock [Used to lock unlock the buts located ou the main unt ofthe controll I the locked state, the burns can ely be conrlled by the LCsottion. Use this option to prevent a smalfnction Purp Ov 08 Used to start tp the pump. When this option is tumed On, the pump wil tart operat ing in sceondance with the set porsmcters Purge Avtosampler ‘Used to sat pursing the Autosampler. To specify the purge time forthe Auosamples, use the Autosampler] tab ofthe Iastumeet Paramstas> View n Advanced] mode. Rime Autosampler ‘Used to rinse the Autormpler. When ths option is slated, the sampling nedle will ‘be cleaned Oven On Of ‘had ose ON OFF the oven Zeros Detector Resetsthe signal intensity ofthe detector to zero immediately Note: To display ide he [lnstoment Conn] 0 bars, goto the [View] mens 4, you change any analysie condition, save the last settings ae method file, Click on [fllocated on the toolbar to over verte the settings inthe method file that was saved when the parameters were downloaded. Note: The "Method File" contain all he infomation on the dats analysis parameters, inching the snatical condition forthe configu ‘sion of the analysis system an instroment parameters, chromstogrm integration parameters, sad information cn quamative cal culation aed calibration curves. Once an appropriate method Slee eeatd, st nay be speciid inthe batch table or pareaetene say be Loaded to data ile to rable anlpsis under the carne aalieparaeters ‘The method file saved here will be used for subsequent analysis. I is, therefore, recommended that the file be saved in a folder that ea be found easily. D once project (Retr FF 5 7.1) folder is selected using [Select Project(Foldes)] on the [File] memo, the folder selected asthe efalt cas be specified a the destination for saving the fe (Gl —_LabSolutions/LCsolution Operation3.2.5 Monitoring the analysis system ‘The status information is displayed in real sime on the ~Chromatogram™ View (displayed by seleoting “LC Analysis> - ‘ View [instrament Monitor] bar Displays the values for the parameters for each instrament. ‘Used to monitor the signal intensity ofthe detec tor, pressure, oven'reom detestor temperatures. ‘The View allows you to specify the axial types and ranges ofthe graph displayed. (Click withthe right ‘mouse button on the graph and select [Display Sertings]. The following window will appear) “Display Settings ( View) Note: As forthe unit for stars items, the signal intensity ofthe detector isin the wnt specified on the View, temperatuce is in degrees C, Gow rate issn mL/min, and pressure i in the uit specified in the window forthe system con- olla. @ ‘The [lnstroment Monitoe] bar alos yout exter values and directs to he conta he sstrment ‘LabSolutions/LCsolution Operation. 3 Optimizing Single-Run Analysis, Analy- sis Parameters and Creating Reports 3.3.1. Making a single-run analysis fhe postions af chromatogram peaks and the optimal conditions fr peal detection are wlknown, you need to measure in advance the chromatogram of the sample and use that chromatogram to set the data analysis parameters. The procedure for actually measuring the chromatogram ofthe sample is described below: A. Prive analysis, confirm that “Ready” is displayed inthe View and thatthe baveline is stable enough 2. tw the Data Acquistion window, lick on the [Sinale Start] icon[M)] on the Assistant Bar or RL onthe Toolbar), The “Single Run> window will appea. In this window, specify the already created method file, the name of data file created after analysis, the vial forthe injected sample, andthe injetion volume. ‘Single Run> LT |) Spon the metodo ‘eset |) ati a = Ener the name of the dts le tobe crested Sangeto Melodie: — aioe Ener the vial number fr the sample injected ‘with the Avtosample If [Ener the tay momber for the sample injected ‘wih the Autosampler Note: The destinanon folder will be usually the ane (reference project) displayed by shies, display t sing Ll on the Tolbar ‘When the data file is opened, the chromatogram will be displayed LC Data Analysie> - View ee LabsotutionsCsotution Operation lH 163. Dislay the ltegetion Parameters window Select [Data Analysis Parameters] menu (displayed by rightelicking on the chromatogram), or lice “4 ‘on the Toolbar and click onthe [Intepraton] tb. Inthis window, adjust the integration parameters to determine the optimal integration parameters ‘Specifvine the minimal width of peaks 1 be detected wil allow the removing othe noise peaks. ‘When to leva is about 14 the wide val, nc is detected ae a peak. ‘This parameter determines the sat and end pons ofthe peak The position where the absolute value {or the slope ofthe chromatogra is equal 19 ths valve i uied as tae sar a ead pois ofthe peak detected ‘This parameters used to adj how the baseline ie awn when multiple piaks are overlapping and sust be separated, detected No pak with» “a genera, the chromatogram peak becomes ‘lus smaller than this ‘broader a the reterion time increases This param sting will be detected. ‘eri used to automatically change the Wid and Select ether area oe Slope values with ne so that tnoad peak ean bs height mode using the se a gy ton ine As (EDEL se [Cateulated by] option elases the Width value ss auromatically ad brctl below {(dovbled) andthe Slope value halve. ‘Data Analysis Parameters> * - [Integration] tab 4, after entering the appropriate values forthe integration parameters, click on the [Apply] button, The resulting effeet ean. te checked in the View: Find the optimal integration parameters by changing the integration paramc- ters in various ways. @ The following paragraph describes a simple procedure for setting the integration parameters. Determine the Wisth and Slope val- ue (for Wiki and 1000 for Slope inthis example, though they are diffrent depending on the tage peaks). Double thoge valoer nd cle the condition ofthe peaks detctad. (dhe Width value is ineresed, small asses wil ot be detected as peaks. Ifthe ‘Slope value increased ld slopes in the basline will no be detected as peaks.) Repet the above steps, and ado the integra: tion parameter in use at the ane when uaceceeary peak become undetectable 17 [| LabSotutions/LCsotution Operation jee5. We th appropriate integain resin stil on the [0] baton Note: 1th dosed inoaraton resi isnot obsined, use tho “ateeaton Tis Progrm> window, which allows you to perfor integra: ‘ion with 2 high degree of feedom. To star the program, click on the (Time Program] button pictured onthe [Integra- ‘on eb inthe previous section ‘The utegration Time Propran> window allows you t specify aay integration comsnaad at an arbitrary time. For the details of each commsnd, us the [Help] menu ee LabsotutionsCsotntion Operation la 1s3.3.3 Setting column performance parameters ‘To caleulate the column performance, set the following parameters, @ The LCsotution supports the calculation methods for USP, JP, JP2, EP, BP, DAB, EMG, EMG (50%), area, and beight, as well as ser defined eaeulaton equations Refer 1 [Adnaviration Marual} “5.11 Performing Colin Performance Caleulatons" ‘With the datafile opened in the window, click on the View (or 4 ‘onthe Toolbar and then click on the [Performance] tab In this window, set the column performance parameters. “Data Analysis Parameter=> [Performance] tab Select column performance ealeulaton method, and enter dead volume time and column length, For dead volume time, you may select either the retention time ofthe fist detected peak or any abitraty time (entered valve). ‘After the settings are complete, click on the [OK] button. The settings for column performance are now complete. ‘The column performance is caloulated for each peak detected (o identified), The calculation result can be checked in the “Compound Table” or the “Peak Table” in window, [Wizard] (on the Assistant Bar) can be selected. Click on the [Wizard] icon. The window wil be digplaed 2. Check that the integration paramcters are properly sc, and then click on the [Next] but= ton, (The integration parameters may be changed. For information on the integration parameter setings, refer tothe pre vvious section.) 3. eat, ers Sz ‘776 esses 9112059 Bama) e7a6 7 ear ‘Register the retention time forthe peak to be identified. fan arbiteary peak is ticked, the retention ime for that peak will be registered inthe “Compound Table” ‘The peaks that have been detected within the allowable retention time range around the retention time registered here ‘will be use in the quantitative ealeslations for analysis and postman, (The allowable range for retention time will be et oapage 3/3) (Check the peak (cetenton time) to be registered in the “Compound Table”, and then click onthe [Next] button. eee Labsotutions-Csotution Operation ja 204, “Cosgound Table Wizard 3% ‘Specidy quantitative calculation method and a calibration curve type. Set the necessary parameters such as Quantitative Method, Caleulated by, Curve Fit Type, and Units. For “Caleulated by”. specify whether the data used for quantitative calelation i peaks area or peak height. For “# of CCalib. Levels, enter the expected number of calibration curve levele, For example, to perform L-point calibration, enter 1; to perform 3-point calibration, enter 3, Note: Wheathe Group Type parameter is set, the following guamitative analysis of group canbe made + Group eaibraton- A calitration curve wl be created fom the total ares of peaks specifi for the same group wen guatite- the mlysinismade * Cons. ration: The concentration obtained by totaling the quantitatively calelated concentrations of peaks speciied forthe same group wll be outa, 5. “Compound Table Wizard 4% Specify a method for identifying peaks, If Window is selected ffom the Window/Band option, the peaks detected in the range of {specified retention time = (catered value) %} will be identified. If Band is selected, the peaks detected within the range of {specified retention time _= (entered value) min) will ke identified. If [Peak Selection] is, for example, set to “All Peaks”, multiple peaks included in the specified allowable range of retention time will be identified as a single peak: (using the total area or total height). 21 N__LabSotutions/1Csotution Operation ieeeAfter completing the settings forthe identification method, click on the [Next] button. [Note:The setine for Retention Tine Update is vali inthe continuous analysis (batch processing) mode If “None” is selected here, the peak inthe same position willbe detected among a series of all analtical dara, 1F "Replace is selected hee, the specified tein time for data during sly willbe replaced with retention time of the paak detected inthe previous analy, f“Averags” ia selected ere, the intermediate point willbe assumed 35 the retention time unlike the complete replacement as inthe case where “Replace” has ben selected This faction i effective for sample type “Standard” ia burch processing 6. Finally, make the advanced settings inthe “Compound Table”. Enter the component name of the peak which will be ‘dene, the peak type, the concentations of the standard substances, te ‘To make quantitative analysis by the extemal standard method, set ll Type parameters to “Target”. For the internal stan ard method, specify only the Type of the internal standard substance as “ISTD” and enter the same aumber in the [ISTD group] of the intemal standard peak and the peaks that are calculated using that peak. For Cone. 1, 2, and 3, enterthe level concentration, level? concentration, and level-3 concentration of each compound included inthe standard sample. (The “Level” ofthe sample tobe analyzed will be specified foreach sample in the Batch Table. “3.5.1 Creating Batch Table”) In addition, the quantitative analysis, identification method, and calibration curve type may be changed for each com- ‘pound, For the cell marked with “Default” the method specified in up to is applied. Entering a value in this cell allows you to male the settings foreach identified peaks ee Labsotutions.Csotntion Operation ja 22“The settings in the window are wed forthe following analyses: Quantitative substances: A Para-hydroxybenzole methyl B Parachydroxybenzoie ethyl © Para-hydroxybenzoic propyl (internal standard substance) Samples analyzed: Standard sample level 1: Each concentration of A, 8, and Cis 1 ug/ml ‘Standard sample level 2: Each concentration of A and 8 is 2 ugim! and the concentration of C is 1 ugim. ‘Standard sample level 3: Each concentration of A and 8 is 4 ugim! land the concentration of C is 1 ugin Unknown sample: ‘Sample used to quantitatively determine the concentrations of A and &: the concentration of Cis 1 ugiml. After finishing the settings in the “Compound Table”, click on the [Finish] button, Note: Ifthe quntitive snalvsis of aroup i specified, [Group] will be edde inthe “Compound Tab” Taclude the substances dat xe tobe calculated asthe same group, inthe same [Groups]. After closing the Compound Table Wizard window, ene these ‘uns foreach group oa the [GroupParam's] tab ofthe View: 7. After finishing the settings in the , lick on the [View] button located in the upper right cor ner of the “Compound Table” to establish the edited “Compound Table” Ifyou want to cancel the edited “Compound, Table” select [Table Bait] - [Cancel Edit] fom the menu before clicking on the [View] button [Now the identification and quantitative analysis parameters have been set. ‘You are ready to create calibration curve and perform quantitative calculations. To actually do so, carry out batch anal- yas (batoh postnan; “5 Caroyiig Out Batel Analyse"). Note: The seine: you have made up to thie pint cannot be uee forthe actual analysis unless they ate saved inthe mthod file (oe the ext section), 25 [NN LabSolutions/1.Csotution Operation eee3.3.5 Applying settings to original method Save the analytical parameters set in the “LC Data Analysis> window as a method file. Ifyou save the data file after setting the data analysis parameters (integration, column performance, identification, quanttae tive parameters, ete.) in the window, no change willbe reflected in the method file, The reason is that the parameters that have been set modified inthe window appears 25 [NNN LabSolutions/1.Csotution Operation ieee3. Wea he rpeeditm said out the Properties» window fr tha tm wl be dplayed Now psy fomat fo he cteomatopan ep ter- Siee the dfs salve isused bere lick cathe [OK] buton to clove the window. The dota file can be loaded to the report file by dragging and dropping that file icon from . Drag and drop file leon to-read in. @ Note: Altemaivels, youmay wart t lick onthe [Fl] tb ofthe “Properties window and specify she desired daa fi sing [Browse] “The speed ie will be loaded 10th sport Te tus box ie uckee, he foumat wal be “Fix the ile robe cepored. when (he Sie ie saved ara eport format 4, The report format con be checked in the workspace (or more previsely in the window that will be described later) In the window the report inthe specitied ‘eemat wil be automaticaly recorded and primed ou afer analysis For specifing« port format in the Batch tbl, refer to “3.5.1 Crating Batch Table” Note: Any ox and specified age lo mny be pst toa report Ifthey art be used use [Text] YA oe [Pinte] i place those report items inthe report format. 27 [MMMM LabSolutions/1Csotution Operation ie. 5 Carrying Out Batch Analysis 3.5.1. Creating Batch Table Set the schedule used to cary out batch analysis using the ereated method file. TL. openthe tr"? Back Proven wl dpa 2. Enter the batch analysis schedule in the , ‘Sanplo 1D Sample Type | Analysis Ty Method File | Data File | Levelt | ti for TStanded} IT aT Mate. iem Star 7 zo 1Standad I OT Mashed em Ste 2 a 1Standad ITT Methed em std 3 fa os Unknown IT OT Meth. em Sangle a 5 Unknown IT OT pa y 3 rain items entered ‘Tray Vial &: Enter the tray and vil numbers for the sample to be analyzed. Injection volume: Enter the volume to be injected in unite of ul, Sample type: Select sample type. For example, seleet “Standard” for a sample used to create a calibration curve, or “Uaknovn" fora sample used for quantitative analysis. For the first standard sample for cresting a calibration curve, enable the [Initialize Calibration Curve] option. “Analysis type Specify whether or not analytical processing will be performed. IT stands for Integration: QT for Quantitative caleulation ‘Method file: Specify the method file used for analysis Data file ‘Name the data obtained ffom analysis. Ifyou do not enter the full-path name ofthe file, data ‘willbe crested in the reference (specified) project folder. Level # Enter the levels of standard samples and QA/QC sample, Repost output Ifthe check box is ticked, the analysis result report will be generated and printed out automati- cally. [Report format fle Ifthe “Report Output” is ticked on, the analytical result report willbe created in the report for= mat specified here. In addition, the intemal standard volume, sample volume, and dilution ratio may be specified. (these are valid for sample ‘pes other than Standard ) This Table also allows you to specify Auto Purge, Baseline Cheek, and Actions based on the evale uustion ofthe analysis rest Note: Iethe necessany dit isnot dspaved forthe stems inthe Table, click with the ih mows horton wth te cesar position onthe ‘Batch Table lac [Table Se] om the duplayad menu and specify whether th mput tema tobe displayed or hidden In the Batch Table, enter a datafile name, method file name, and semple type on each row. For a standard sample, specify Standatd for the sample type and a level males. (For a sample to be analyzed quantitatively, specify Unknown forthe same ple ype. Entering a level number is optional) eee — Labsorutions.Csolntion Operation — ja 28‘The specified batch table should be named as a batch fle and saved. Click aem ‘Toolbar to save the bath file ‘The contents ofthe batch table will be saved. Note: This sections described the procedure forthe stings required to star the collection of data f you carry ou batch poston for exiting data, fellow the same procedure si thi section using the bate table in the window 3.5.2 Carrying out Batch Analy: ‘Canying out the analysis of a created batch table. A. Dispiay the desied batch table (bate file) by selecting - , Click on the [Batch Start] icon D>] onthe [Batch] Assistant Bar. The batch analysis willbe catied out stating with the fist ow. lsc he Bote Rets isn [HI] ote (tn on [a] on te 2. To paucetesume or stop batch analys paw P ys [Batch] page of the Assistant Bar @ Wienacht ow progmsng abi top ad che soe tetae eon canted ru D Tre same butons ac als avaiable onthe Toolbar, (Bah Star] [Pause Rest] [Sep] Note: These buttons are iden unless the “Batch Table> is active, they are hide, cick om any prt ofthe Batch Table 3. tycasanet check sce whether the doa wner atch anal wl be propery interted sing he specie metho, he “Snap” Suction is wea This ition loads he data under sls and plays it terpocly. This aloes yout chee he interation eu the data ring data ein, ‘To activate the “Snapshot”, click on the [Snapshot] icon on the [Acquisition] page of the Assistant Bar during analse when the - “LC Data Analyse window 4, The progress of the betch analysis can be checked on the

‘After the batch analysis has been completed, check the log messages to ensure that no error has oceurred during analysis, Ifthe batch analysis was not successful, also eeferto this log Message, ‘SubMessage Date Stat Batch Processing (Data Acquistion) Ti aalFiqeehBatcht eb: 1 arene, No peak hasbeen detcted Detector A (C \Data\Pioiecx\ St kel er72002 [PDA [C\Data\Pijech Sc Led) anr2002 29 [NNN LabSolutions/1.Csotution Operation ieee. 6 Creating a Summary Report ‘A summary report ie a simple report that catalogs the chromatograms for multiple analysis results and peak tables, ‘ek Sephiee Sagi REDOD SA Sept Sagh Some 32057 aoe ames 300516 gee EE 2B | E 2 @D The summary report is classified imo two pes: + Concession type: Displays al of the concentration, area an heat rents + Compound type Displays the posk information foreach compound, using the concent and column pexforanee “The summary report can be ereated in almost the same manner as in "3.4 Creating Report Forme The following deseribes the prosedure for creating the “Summary Report” in postran batch analysis, Te onthe Report sete repetie San (Lot ontario et a 2. Display the Postrun ‘The LCsolution is designed based on the basic concept of managing data, methods, batch files, and other related files by placing all of them in a single “Project Folder”. D he project folder may be rely created and duplicated using LCsolution’s A. Using the window, open the datafile (with the fle name entered inthe batch table) created by bate snalysis. If you double-click on the whitespace ofa file same cell in the batch table, the coresgonding data fle or sethod fie ea be opened. Similanly the data acquired through a single analysis run can be loaded to the window by double- licking on the icon forthe corresponding data file onthe « [Data File] tab. ‘The View, the View. Note: Clicking with he right noue buon on cach view wil diplay the pop-up mem related othe diplayseings, wich allows you {0 nile he enlargement of he chomaoyam, ea the tas frmation, display the peak tp comment, change the sale change the backround cole, check the cliraton cas equation a display and deh "Compound Tale items 53 [NN LabSolutions/1Csotution Operation ie2. Tock te infomation on each aiatonpoit ofthe eairaton eure (pes pofi it integration result pak ar, ct) diplay the LC Calteaon Cuve> window and led he etd fle which was sed ine anal ‘The View allows you to check the details of the calibration curve. The View allows you to check the information on each peak. Note: To reanalyze the dat under different analytical conditions or by a diffrent quantitative analysis method cany out the following procedure o make tach posta ealysix 1. Use the window to load a method ile, edt the data analysis parameters, and thea save the metho file Altematvely, you may load the datafile onthe LC Data Analjis> window, eit the data analysis parameter forthe mtd ia ‘he data ile, and then Select (Apply to Method] (save the method in the datafile as an independent method fie) Teaeeteenmaanemn a onthe toolbar to the Data Analves Parasctera> wader: (Canaan Pama 2 tothe Poston “Batch Tele> specify the method Sle saved instep 1s the method ie cel aad then eaery cr batch posta nals ee Labsotutions.Csotution Operation la 343.7.3. Checking data with CLASS-Agent 1 the optional software for data management, “CLASS-Agent” is st up oa the PC on which the analysis was cused out, the analytical results data can be registered automaticaly inthe Agent database “The following describes how to check the registered data with CLASS-Agent. A. start CLASS-Agent Manager. Double-click on the Database icon luton Lavacher, ‘onthe [Administration] tab of the window “Tine bor, sano informanion, and information set in View sn “Display Setings>, Propetios> TacseaseeDeciensee iplay arenvanon in “Allows maxaaizing of the clnmment Parametss> View di psy ‘Sends the set inirument paremitrs to the analy ‘Use opine to change display rave ‘Use scroll bar to] | Upper aren is called View arn, lower area called lestrumeat Paraeter> Viewares, played in tab format Perform sestrimeat parameter serinas to | Data Acquisition se for data acquisition *% (pasetme Cnecky putton |/EE 1 gs me) FA] tasrument conto bar — The [Instrument Control] bar allows you to perform operations before snd after the analysis, uch as startup and shutdown. If ‘you eck on the [Baseline Check] button, the noise and drift will be evaluated relative tothe baseline on the specified chan nl in accordance with the settings fo the method Note: No baseline chek can be made on the chal for which “Max Pl” i selected in - [Muli Chor] tb @D Analysis may be copistered in the window during the baseline check @D ie yos tick the check box for the baseline check on the row for the , the baseline can be checked before the specified analysis is stared. *% [zeros Detector] button jnnae a foi [Rea Tne tsteument Conta bar (Zen baeciaa) ‘The [Real Time Instrument Control] bar allows you to turn On/O# the pump and oven individually. Clicking on the [Zeros Detector] button will send a command tothe LC to calibrate the zero Level ofthe baseline for detector signals 39 [M__LabSolutions/1.Csolution Operation jt“ 9% (Data Anatysivsnapshor] toon AY (acct sana d) ‘During data acquisition, use the [Snapshot] icon to view current acquistion, a temporary datafile is ereated with chremato- ‘grams obtained fiom the stat of analysis to licked points, and these chromatograms are read into - , which started simultaneously. Using this feature, the peak area and ather data can be checked during analysis. D The data sead can be saved wider another name, f accessary. ‘Othenvise, this icon becomes the [Data Analysis] ion, which allows you to load the datafile obtained ftom the most recent (previows) data acquisition tothe - Views become for read only. Therefore, you ‘can no longer change the [Stop Time]. ‘To change the Stop Time during data acquisition, use the “Change Analysis Time> window: Click [Azguaioa) (Change Anaysis Time] ac =Data Acquisition menu Change Analysis Time> TF yeu click this baron whea mati ple detector are used, the setings fo fl the detectors aze changed 19 the Tages analysis ue (shortest axa {is tine if [Set to Minimem Vale] hack box i eke). (Note: Ifthe set analysis ime is shores than the acral analysis ane, data acquiston wil tp a tat ime anda dara file wil be created. Note: This cetins will not be eflecie in the method ut wil elf inthe operation los to ence traceability eee Labsotutions-Csotntion Operation la 4243 . 3 Changing the Data Sampling Interval ‘The data sampling interval bared upon the peak shapes of the chromatogram is designate here. Setting a smaller date sampling interval enables more accurate data processing of very sharp peak in the chromatogram, However, ifan excessively small sampling rate is designated, the datafile size will become unnecessarily large, wasting ¢ys- 4.3.1 Parameters Related to Data Sampling ‘The parameters related to the chromatogram data sampling interval are as follows: *S¢ - [Properties] of each detector - [Base Period] Enter the basic sampling interval unit forthe chromatogram. Initial Value | 100 meee Options | 20/40/60/80/100/200/500/1000 mace Note: The baseline cannot he desigaated es than 100 msc. in the following cass: + when the Dual modes selected in Detector “Properties” inthe «System Configuration window on the SPD-I0A serie lira ole visite ultraviolet sectophotometic detector: + the CRD optional ust connected wo the CDD-1OArp/p (eectrocondutvty detector. *% ofeach detector Options | Base ate xn (a= 1 to 10) [msec] the ssping interval is desanated on froqueucy, te above deftle values and options ate dplaved as averse uamibers of ane Note: sampling itercal becomes shorter, the data Bile capscity comes larg When the paak width ble, decrose the ee) Ae Sampling setting vale when n Hz) or increase it en in msec). *% {Integration} tab - [Width] “This parameter is used to perform chromatogram peak integration, To detect a peak, set the balftheight width value. Recom- | The 1/4 to 1/2 ofthe actual detection peak width ie the standard. {GE Labsolutions/LCsolution Operation jeeClick [System Configuration) < - “Detector Properties” 4.3.3 Setting the Detector [Sampling Rate] “Instrument Parameters> View - [Data Acquisition] tab ee LabsotutionsCsotution Operation la as.4 Setting Up HPLC Start/Stop Actions ‘The LC can be started ata designated time or automatically stopped after analysis has been finished. ‘Rilo: warming up wit the atnmnent param fer sotingsscluded i th specified shathod ‘Use Pumping Peo" to specify the warmup ‘inte op to the star ofthe anal cessed the batch queve “When the Startup dilog is opended fom he Instrument Cental bs, the Pumping Pesod inthis inlog wil not be displayed. eth ase, Warman ups continued ul th data acquisition i executed the speciied method. Afer (Cool oven Time} has elapsed, each insir- ent ie turned OF, @ The Batch Table alo has the same settings. When registering analysis in the batch queue, enter startup time, In the LCsolution soars, a singl-run analysis is handled like a batch table of single rw and cegisered inthe bach queue Note: The burch queue processing is executed asthe eepstre order. Therefore. the shutdown sein may not be putin aormally Ifthe ‘sarup setting is made when analyses are repistred in the batch quoue the startup witing mode becomes active aftr those anals~ ses have been complete, Ifthe ime specified in Start Date and Tine is exceeded 2 this pot, the ssp wll occu immediasly 45 NNN LnbSolutions/1.Csotution Operation iii. 5 Registering Column Information To change the column based on the analysis purposesappliation, frst attach a column to the HPLC, and then register that ‘column’ information inthe “System Configuration window. w. ii Ege Usp Eh Spare: S080 Syanerc 8 = ® * * * a a 2 a a ii a Window For convenience, the name window. Duoplay Setings> window appesrs, uo aah” lick menu a View are - (See Online Help) =Propartise> (Soe Online Help) TacrexeeDecressee Swiehes emneen [View] aad [Eda} modes. Repeo- ‘cessing i automaticaly per- ‘Use spina change display aio Upper area is View area, omerieft is «Calibre thon Curve> View ara, and lower Fight is View aes, orth abe and sample information are in the Calibration Curve> and Compound Table> View area. Note: One «LC Postrun> - window plus the View displayed using “LC Postrus> - window glve tae “Composed Table> View daplayed sing window is displayed by selecting the [Meth] - [Data Analysis Parameters] menu, its elated pop-up sen or oor bon Note: When parmeters related to data procensing have been set before data acquision,tning - Window ‘Data files are handled used in the “Data Analysis window: LCsolution stores a variety of information such as method and system configurnton inthe datafile, Also, wen performing postrun analysis frm the window, o¢ ‘wlien applying the method file to perform postrun analysis of other data files individually, impoit the data processing param ters (excluding the instrument parameters) tothe datafile metbod. The data file is reanalyzed by using the imported method. ‘The Method import can be performed using [File] - [Load Method] 5.2.2 _ Import/Export Report Formats a sede ie Cnet oo ete ne YON ene sin iuitia anise uttowr nase “Here, when the report format is saved as is after editing, the report format stored inthe datafile will be updated. Then, save the data file, andthe updated eport format will be saved inthe data fle ‘To perform report format importiexport to or from the datafile, use [Fle] - [Open Report Format File] or [Save Report For- mat File As). eee — Labsorutions.Csolntion Operation — fa 4s. 3 Setting Peak Integration Parameters 5.3.1 Setting Peak Integration Parameters ‘Drag ad crop fle con tw cpen dat ‘SEE TebeGst em = tick [Dura Analysis. (Click [Amayze] icon on [LCPDA Data] Assistant Bor or he (sa/6n wa [Frcs sl] >) = ls [py enaapan or = (hiking oa the Apply baton allows ‘yoo to reflect the changes an the rests wathoot exiting the cent window Repeat the enterand: Time pomam st up with ‘hangs operation so that the neces iathod fle i saved. You can sary peak canbe detected, dit and se for postrun anys, PAE 5.3.2 Detecting Peaks Based on the Peak Integration Parameters ‘Affer editing the parameters on cach tab of the View area's peak detection point mark, baseline dis- play, te (tick onthe (0K) baton BREE] . The window wil be closed just when th postua star. 49 (ll LabSolutions/LCsolution Operation‘5 Nent Step - Data Processing 5.3 Setting Peak Integration Parameters 5.3.3 Setting Integration Time Program Examples Integration time programs allow you t improve processing tht integration parameters alone cannot handle propery *% operational procedure for setting up integration time program (Peak Integration Of/0n) 1) Click the [tntegration OfFto On]* button + If you place the mouse pointer ver click a com mand button, the ewidance rmesnse for the button ‘ppenss i the boom et oF the screen Proceed. your ‘operation fllowine the mee ain aan OT ) Following the guid- ance, specify the starting and ending points of the estion by clicking on the chromatog 3) When the ending point is clicked, a sereen opens for your confirmation. Afer confirming the starting and ending times of the section, cick the [Simulate] button’, * Processing is done using your saciid integration ine program pro Crea cing chromatogram information such as basclise and area on the fs ER — on screen for 903 to check the rogram bebsioe 4) The specified processing command is shown on the program table* 4 i.) Check the processing renut and lick z the [OK] buiton to close the Integra- q tion Time Program oe Integration is done and the result is coz shown on the screen. et a + Yoo cals ett mam Tose oe LabSolutions/LCsolution Operation3 5.3.4 Manual peak integration feature (manipulation) By using the manual peak integration feature (manipulation), you can move the starting or ending point ofa specifie peak or remove unwanted peaks, @ —_Youcan we the manual ieroraion fearon tho “Data Analysis and , click the [Manual Peak Integration Bar] displayed by right-clicking on the or sereen, you ean perform manual integration on the data currently open. Processing commands for manual integration are saved inthe target data, Therefore, they will not be saved in a method file ‘even if the method is exported in the screen. Also, manual integration processing commands will not be ‘overwritten by any method file even if postrun analysis is run forthe data using another method file in or , 1 [ME LabSolutions/1.Csolution Operation jee5.3.5 Setting Manual Peak Integration Examples * operational procedure for setting up manual peak integratton (Shifting peak detection points) ee 1) Click the [Move BL] button |A guidance message will appear in the bottom-lef ‘comer area of the screen, 2) Click the detection point you wish to move, ‘The baveline for the peal tars moving along with the movre movement +3) Checking the baseline, clic atthe point to which you swish to move the peak detection point. 44) The peak detection point moves to that point and the resultant baseline s displayed Ifyou have mistakenly moved the point to an undesirable pace, you can cance the move by clicking the [BEV tation ‘LabSolutions/LCsolution Operation (ls. 4 Setting Peak Identification Parameters Peak identification refers to the data processing used to identify a particular acquired peak among the compound peaks in the analysis sample. Usually, peak identifieation and quantitative calculation are performed simultaneously, 5.4.1 Setting Peak Identification Parameters When performing peak identification and quantitative esleulation, frst enter each substance’s type, standard retention time, ‘concentration, ete. in a compound table. Then, when performing data analysis, the acquired chromatogram peak values and ‘compound table's set values are compared, Ifthe peak retention time falls within the identification allowance width the sub- stance is identified as the target substance ‘This section describes the time allowance used to cansder whether the peak retention time isin the standard retention time range, when perfomning peak identifiation based on the compound table's set values, This section also explains how to select an identification method from absolute and relative etention time. = 2 = (Sasa a co {Data Analyse) Drag and drop fe icon to open data (Click [Amatyzs) con oa (LCPDA Dat] Assis Bar or here cater percent value. To use [Band] method, eter band ‘vid in mainstes = Select action to wie of feding bask to compound tobe retention time of peak actualy identified during data acquis Selec aston to we rolipe peaks enter the identification lowes wth Note: For more information abou identification parameters, eer t Online Help 53 [M__LabSolutions/1.Csolution Operation je