Agilent Technologies Automated method development using Agilent 1100 Series HPLC systems Agilent ChemStation ILS ChromSmart MD software Application Note
Contents
1. Automated method development using ee Agilent 1100 Series HPLC systems me A Agilent ChemStation and ILS ChromSmart o e MD software Application Jeffrey D DeCicco Douglas R Myers _ Ralf Honsberg o a a e missal QD Method ils m 5 InSmart from Intelligent Labors host situations a Abstract The development of an HPLC method can be a time consuming and knowledge intensive process Experienced chromatographers often spend a good portion of their effort on routine tasks rather than perform ing value added research and development The ChromSmart MD soft ware from Intelligent Laboratory Solution ILS automates these routine tasks and the experimental method development process ChromSmart uses real time expert system technology to plan execute and evaluate methods This Application Note demonstrates the use of ChromSmart with Agilent 1100 Series HPLC systems and Agilent ChemStation soft ware for automating the experimental method development process oh Agilent Technologies ChromSmart MD software ChromSmart integrates HPLC instruments sample descriptors experiment constraints and method objectives to develop separation methods ChromSmart MD emu lates the actions and decisions of analysts by automating the time consuming repetitive tasks of method planning development execution and interpretation ChromSmart MD does these tasks unattended and online The analysts b2. including control of column switching systems e Automated equilibration with detection and removal of retained materials e Total area tracking to stop the method early if the sample has eluted through the column e Automated column washing also known as a zero injection gradient or ZIG for new columns or columns with possible retained material Interfacing with Agilent 1100 Series HPLC and Agilent ChemStation ChromSmart integrates and con trols Agilent 1100 Series 1100 HPLC whereby method control and data acquisition are provided through the Agilent ChemStation software The communication between ChromSmart and ChemStation occurs through a real time software bridge This bridge resides on the same computer as the ChemStation online software ChromSmart can reside on the same computer or on any computer on the local area network LAN Figure 1 shows a typical configuration of equipment Figure 2 shows how to connect to ChromSmart from the ChemSta tion s Method and Run Control menu ChromSmart does not interface directly with the Agilent 1100 Series modules Instead ChromSmart collects data in real time from the ChemStation ChromSmart evaluates this data in real time and instructs ChemStation to perform tasks automatically For example ChromSmart instructs ChemStation to perform a column equilibration with a given column and mobile phase During equilibra tion the UV signal is monitored in real time When a s
3. e scouting gradi ents one for each solvent If the scouting gradient suggests an iso cratic separation is possible then the plan is changed dynamically during the method development process to try running an isocratic method ChromSmart was present ed with the sample and allowed to operate in unattended mode At the conclusion of the method develop ment session ChromSmart has performed 25 methods in about 6 hours This included the following e 3 scouting gradients e 9 isocratic experiments e 3 zero injection gradients ZIG to wash the column e 6 column equilibrations The final result was an isocratic method that met the desired user requirements of resolution and retention The final method had a mobile phase composition of 81 water 19 acetonitrile on a C8 column Figure 3 shows the chro matogram obtained using the final method Notice that the method was stopped automatically because ChromSmart detected that sample had completely passed through the column Eee Method stopped at 18 861 minutes Time minutes Sample ALLTECH1095 Method Date 05 13 2002 03 12 Method Type isocrat injection Method Number 23 Column Waters CS pas 1 Solvent 812 H2O 10 0 ACN Figure 3 Final method found by ChromSmart for the reversed phase sample Browsing method development results in ChromSmart Several aspects of a method develop ment or sample analysis session can be browsed Thi
4. egins the method development process by specifying analysis conditions for the initial experiment as well as criteria that the final method must fulfill in terms of chromatographic perfor mance such as retention time and resolution ChromSmart MD analyses the results of the initial experiment determines the next logical step required to drive the method towards fulfillment of the criteria and then executes this step while monitoring the results ChromSmart MD bases the deci sions about which experimental parameters to vary on expert interpretation of the results With intelligent analysis performed by ChromSmart there are no lengthy systematic searches covering redundant or irrelevant parameter spaces Instead navigating the search space for method planning is done in a more directed manner substantially increasing the efficien cy of the method development process ChromSmart provides the following capabilities for increasing laboratory productivity and through put e Automated development of HPLC separation methods using the analysts expert knowledge e Automated identification of separation conditions including gradient isocratic reversed phase normal phase e Handling of a wide range of samples including ionic non ionic and chiral non chiral e Post run analyses method results interpretation and recommendations for subsequent runs e Automated search through the analysts selectivity variable preferences
5. hromSmart optimized the mobile phase screening process for a reversed phase separation Mini mal configuration was required to start the optimization process The desired mobile phases and columns to optimize were specified as well as operating preferences method criteria and optimization prefer ences Compounds Concentration 1 Uracil 0 015 mg mL 2 Phenol 0 7 mg mL 3 Benzaldehyde 0 05 mg mL 4 N N Diethyl M Toluamide 1 0 mg mL 5 Toluene 4 0 mg mL 6 Ethyl Benzene 4 0 mg mL Table 1 Sample compounds ChromSmart Server ChromSmart Client pa on other PC _ optional on Agilent posteo Figure 1 Typical ChromSmart MD configuration amp Instrument 1 online Method 4 Run Control Eile RunControl Instrument Method Sequence View Abort Help fage Method and Run Control 3 ils m E DEF E S N i 1 E ChromS mart Connection Dia aa Host fiisdev1 Socket fi 150 Stack Name 1100 single es ee MiA N my u nSmart from Intelligent Labor hast situations Figure 2 Connecting to ChromSmart from ChemStation The sample presented to the sys tem contained the six compounds shown in table 1 For this sample a method of 42 water 58 acetonitrile was known to provide adequate separation results on a C18 column at a flow rate 1 0 mL min with a detection wavelength of 245 nm To run ChromSmart the analyst only has to know the sepa
6. ions only Monitor Equilibration Method 1 y gt Meihad feel 2 Anglo Chromatogr Baseline Stabie m O o yl o Fite Test Mode Stop Mathod arar Chrematogram 2 Monitor Matnog Figure 5 Equilibration decision tree reversed phase chromatography is the Rule of 3 There is approxi mately a three fold increase in k for a 10 decrease in B strong solvent This rule would follow an analysis that analyzes the current experimental results and deter mines that a decrease in Bis Jo Malos Not Seas Y o A Fhag Tamperatura Has Minin Time Elapsed JO 107 Tonlinae O Mathes Dora No Stable Baseline 2 High Pressure a arty Mernon Is Stospes Continia mary Mitos Ext Loop Is stoppen required that is the peaks are bunched left or an increase in selectivity is required These rules of thumb are abundant in the field of chromatographic analysis Often analysts are implicit ly making decisions based upon these rules of thumb To simplify matters rules of thumb are separat ed into two categories in method and post method In method refers to decisions made and actions taken during an actual experiment Such rules of thumb are concerned with equipment and method moni toring Post method refers to deci sions made after an experiment is run and may look back at a history of results Many analysts may perform thi
7. ration type that is reversed phase and the desired selectivity variables columns and solvents In this example the analyst entered the following information into ChromSmart e Reversed phase chromatography as appropriate separation approach e Mobile phase selectivity variables in following order 1 Ethanol EtOH 2 Methanol MeOH 3 Acetonitrile ACN e C8 column as stationary phase selectivity variable more columns could be chosen if an automatic column switcher is available on the HPLC system The following method and optimization preferences where chosen e Perform automatic column washes before sample is injected for the first time or if retained material is suspected on the column e Perform automatic column equili bration with minimum equilibra tion time of 5 minutes and maxi mum equilibration of 60 minutes e Optimize to find best isocratic method with a minimum resolu tion of 1 0 and retention range between 1 0 and 10 0 k e Perform initial scouting gradients between 5 and 80 strong solvent in 20 minutes Once these preferences were entered in the appropriate dialogs ChromSmart automatically gener ates an initial plan of experiments and starts the method development process This initial plan is based on the optimization and selectivity preferences The preferences chosen were isocratic optimization with a single selectivity variable type organic phase The initial plan consisted of thre
8. s analysis implicitly by simply observing the chromatogram It is possible to summarize such rules by simply taking a step by step approach to the decision making process The first step is to summarize the information that is relevant In this case the UV signal level and the method run time are important The second step is to summarize the relevant rules A flow sheet is a useful tool to explore depict and test the infor mation and rules Once the infor mation and rules are summarized it is possible to capture and execute them as ChromSmart decision trees Within ChromSmart decision trees are essentially automated flow sheets Figure 5 shows a portion of the in method automatic equilibration decision tree References 1 The Rule of 3 can be found in the reference L R Snyder J J Kirk land J L Glajch Practical HPLC Method Development Second Edi tion Wiley New York 1997 Links Further information about ChromS mart MD software can be found on the Web at www chromsmart com Jeffrey D DeCicco Ph D is Marketing Manager and Douglas R Myers Ph D is President at Intelligent Laboratory Solutions Inc Naperville IL USA Ralf Honsberg is Product Manager for Data Systems at Agilent Technologies GmbH Waldbronn Germany www agilent com chem nds The information in this publication is subject to change without notice Copyright O 2003 Agilent Technologies All Rights Reserved Reprod
9. s includes a summary of all injections a slide show of chromatograms session configuration the execution plan formal reports and an audit trail of the decisions made by Chrom Smart There are two views to browse quickly the results of a session The first is a spreadsheet summary and the second is a slide show of chromatograms The results can be queried further to show only methods that involved sample injections that is remove ZIGs and equilibrations Figure 4 shows this view From the spreadsheet summary the analyst can review the chro matogram the decision making audit trail a peak report and over all rating of the method The val ues in green represent good method conditions that met the users requirements A more visual approach to review the results is with the slide show option This option allows the analyst to review the results and notes in a step by step fashion Using decision trees in ChromSmart ChromSmart uses automated rules of thumb to analyze chromato graphic data make recommenda tions and execute those recom mendations Rules of thumb can range from heuristics to the analysis of model predictions A good example of a rule of thumb for Years OB Pos 1 fe scones os ajena Tz NEON fis eon E os We MEAT o EN CA E CA IO E En ajen enen waes co res1 memo asa Pam ose EC EA ZAC EI O vir ws avs ome Figure 4 Summary of method that involved sample inject
10. table baseline is detected ChromSmart instructs ChemStation to stop equilibration and initiate a sample injection ChromSmart collects the real time UV spectrum temperature and flow measurements In addition the integrated peak and spectra are collected and analyzed ChromSmart takes advantage of the advanced control and data analysis capabili ties of ChemStation This includes peak integration and spectral analysis capabilities For example ChromSmart can request automati cally that ChemStation perform spectral peak purity analysis or spectral comparisons for a set of peaks ChromSmart also provides advanced equipment monitoring The objective is to ensure equip ment wellness and avoid operating conditions that lead to a shutdown For example back pressure is monitored in real time If the rate of change causes the high pressure limit to be exceeded ChromSmart decreases flow or changes the mobile phase concentration In addition ChromSmart ensures that columns are protected from mobile compositions that may damage the packing The Agilent 1100 Series HPLC systems and ChemStation software are very flexible and can incorporate equipment such as automatic column switching valves ChromSmart takes advantage of these switching valves and can perform automatically smart column screening using multiple columns during the method development process Experimental case study The following example illustrates how C
11. uction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Published March 1 2003 Publication Number 5988 8963EN SEE Agilent Technologies
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