Automated method development usingAgilent 1100 Series HPLC systems,Agilent ChemStation andChromSword® software

Application

AbstractThe development of an HPLC method is invariably a repetitive and

time-consuming process of method planning, development, execution

and interpretation. Analysts are often forced to spend a good portion of

their effort on these routine tasks rather than concentrating on more

valuable work in the laboratory. Today, many tools are available to help

automate the method development process. This Application Note

demonstrates the use of ChromSword Auto software with Agilent 1100

Series HPLC systems and Agilent ChemStation software.

Sergey GalushkoRalf Honsberg

• to improve existing methods • to optimize separation only tar-

get compounds• to find conditions for separation

of main components, excipientsand impurities

Analysts need only to specify theirobjectives, for example, find iso-cratic or gradient method condi-tion, separate only target com-pounds or unknown compoundssuch as impurities, and so on, andthen press the GO button.

Configuring ChromSword forautomated method develop-ment

Before ChromSword can be usedfor automated method developmentwith an Agilent 1100 Series HPLCsystem and the Agilent ChemSta-tion software (revision A.09.01 orhigher), it’s necessary to configureChromSword correctly using theHPLCAdmin program, see figure 1

There are several parameters,which need to be configured:• Current Instrument — specifies

the number of instruments con-nected to ChemStation. A newinstrument can be added bypressing the + (plus) button. The– (minus) button deletes the lastinstrument from the list. The Cur-rent Instrument dialog box allowsthe analyst to choose the currentinstrument, for which the otherparameters can then be specified.

• Basic Directory — selects a direc-tory where ChemStation willstore the data (for example,C:\cstest2\) during method devel-opment. All data and method fileswill be stored in this directory.

For normal phase chromatography:• composition of the mobile phase

(isocratic, linear and multi-stepgradients)

• temperature

For ion-exchange chromatography:• buffer concentration• temperature

One approach to develop or opti-mize reversed-phase methods is touse virtual chromatography basedon structural formula and column/solvent type. The ChromSwordsoftware contains a database ofproperties with more than 60 dif-ferent commercial reversed-phasecolumns that allows to simulateretention behavior of compoundsin isocratic and gradient mode. When ChromSword and ChemSta-tion software work togetheronline HPLC method developmentfor reversed-phase chromatographycan be performed in unattendedmode and can optimize:• composition of the mobile phase

(isocratic, linear and multi-stepgradients)

• column temperature

If the Agilent 1100 Series system is equipped with a quaternarypump and a column thermostatwith a column selection valve,ChromSword and ChemStationcan perform fully automaticallyHPLC optimization with differentcolumns and up to three differentorganic modifiers.ChromSword has different func-tions in a HPLC laboratory: • to develop completely new

methods

Introduction

ChromSword is a software packthat supports rapid developmentof separation methods for liquidchromatography, whereby a mini-mum number of experiments arerequired. ChromSword can be usedfor optimizing separations in re-versed-phase, normal-phase and ion-exchange liquid chromatography.

ChromSword can be used offlineas standalone software for com-puter-assisted HPLC methoddevelopment or, in combinationwith Agilent 1100 Series HPLC sys-tems and Agilent ChemStationsoftware, it creates a powerful,specialized method developmentsystem capable of developing newmethods or improving existingmethods fully automatically. The following types of optimiza-tion and computer simulation canbe performed using the softwarein offline mode.

For reversed-phase chromatography:• composition of the mobile phase

(isocratic, linear and multi-stepgradients)

• pH value, pK determination • temperature• 2-dimensional optimization: con-

centration isocratic/temperature,gradient profile/temperature,concentration/pH value, temperature/pH value, ternarysolvent mixtures using one column or column combinations,(column combinations/concen-tration, gradient profile, temperature, pH value)

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The subdirectories correspond-ing to each instrument will becreated in this directory. Forexample, if the basic directory is C:\Test\ and 2 instrumentswere specified, the directoriesC:\cstest2\Instr1\ andC:\cstest2\Instr2\ will be createdby the administration program,see figure 2.

• Default Project — specifies projects and choose the defaultproject. Projects can be addedby the + (plus) and deleted bythe – (minus) buttons. Each project is the name of the sub-directory where the results andmethods will be stored. Thissubdirectory may appear in anyinstrument directory. For exam-ple, if the project ADrugs isselected for instrument 1, thedata and methods will be storedin the C:\cstest2\Instr1\ADrugsdirectory. Removing a projectwith the Delete button will onlymake the project unavailable forfurther usage, however all dataand methods created remain ondisk.

• Template Method — ChromSworduses a ChemStation method as a template for generating newmethods during the methoddevelopment process. Any validexisting ChemStation methodcan be used as a template. Theadministration program willautomatically copy the templatemethod to the destination direc-tory (into the TPL directory withthe name Template.m. Multipleinstruments require separatetemplates, because their configu-rations may be different. TheSave&Exit function in the Filemenu is used to save the

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1 Start the ChemStation in offlineor online mode.

2 Load any valid method for theAgilent 1100 Series HPLC systemthat was used before with theinstrument. Choose the SaveMethod as function from theMethod menu and save themethod it as Template.m in thecorresponding directory<BasicDirectory>\Instr<n>\tpl\.

Tips and tricks for operation ofChromSword with ChemStation

• The Sequence subdirectory fieldin the Sequence parameters window of the ChemStation has to be empty (loading thesequence DEF_LC.S sets the correct default parameters).

• ChromSword resets the datapaths of the ChemStation to theproject directory. If samples are

Figure 2 ChromSword data path

Figure 1 HPLCAdmin program used to configure the data-system connection for ChromSword

changes and close the HPLCAdministration program.

It is also possible to select a tem-plate method. For this a methodhas to be saved with the name Tem-plate.m in the TPL subdirectory ofthe ChromSword instrument direc-tory. To prepare a template methodthe following steps are needed.

run without ChromSword, thedata might be saved in thewrong directory. Loading amethod, sequence and data filefrom the default path (for exam-ple, \HPCHEM\1\DATA) resetsthe path to the correct directory.

• During the automated develop-ment process ChromSword cre-ates new sequence files. Theseshould not be used for creating anormal sequence in the Chem-Station. As a solution theDEF_LC.S or any other knownsequence should be loadedbefore doing analysis with theChemStation only.

• ChromSword revision 2.1 cur-rently supports the followingAgilent 1100 Series HPLC mod-ules: binary pump, quaternarypump, standard autosampler,column compartment includingthe built-in column selectionvalve, variable wavelength detec-tor, multi-wavelength detectorand diode array detector.

Automatic method optimization

This section gives a short descrip-tion of how to set up a simplemethod development experiment.The following Agilent 1100 SeriesHPLC system was used for allexperiments: quaternary pump,degasser, standard autosampler,column compartment with built-incolumn selection valve and diodearray detector.

Preparing a method developmentexperiment Configure the ChromSword soft-ware as described in the previoussection. The following proceduredescribes how to start the auto-matic experiment.

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1. Start ChromSword.2. Select Compounds tab

(normally default).3. Fill in compound names

(vial number in the autosamplerand compound number are thesame).

4. Double-click on the compoundname to activate the chemicalformula program and draw themolecular structure for eachcompound, if it is available(alternatively formulas can beloaded from .sdf or .mol files).

5. Select the First Guess and find a column and an organic solventaccording to the availablecolumns in your laboratory.

1. Prepare all standards (one compound per vial) plusone vial with a mixture of allstandards. The concentrationused is typically 1 mg/ml.

2. Prepare the Agilent 1100 SeriesHPLC system according to theproposed parameters such asmobile phase and column.

3. Select the Auto-Wizard functionand follow dialogs.

4. Make sure the Agilent 1100Series HPLC system is set upproperly, for example, no air inthe system, lamp(s) on for 0.5 h,oven temperature ready, allstandard vials in the predefinedposition, solvent bottles allfilled to maximum (you can alsouse 2-L bottles, if necessary).

5. Select Go from the Auto-wizard.If no structural formulas are available, fill in the list of com-pound names (step 3), skip steps 4through 6, and then prepare theAgilent 1100 Series HPLC system.You can use phosphate buffers asaqueous component (solvent A)but use low concentrations (<20 mM) to avoid plugging apump through precipitation.

Reviewing results and generating areportAfter the experiment is finishedsuccessfully the following stepsare needed to get a report print-out, with results tables and chro-matograms.

6. Start the Rptview program.7. Select the appropriate .txt file

under for example C:\Programfiles\Merck\ChromSword2.1\(name ofexperiment)<counter>.txt

8. Now the results can bereviewed, a report can beprinted, copied and saved inMicrosoft® Word format.

Using created data sets for furtheroptimizationThe data (sets) created by theautomatic experiment can be usedfor further optimization using thecalculation procedures availablein ChromSword. To take advan-tage of this feature the followingsteps should be followed (Goal:Further optimization of resolutionbased on the already availableautomatically processed data bychanging % B):

9. Open appropriate .smp file inthe ChromSword main screen,for example C:\Programfiles\Merck\ChromSword2.1\(name ofexperiment)<counter>.smp.

10. Double-click on Concentrationunder Available Data.

11. Select, for example, Gradientand move line to see separa-tion at different % B concentra-tions.

How to run a method develop-ment experimentFigure 3 show the screen thatappears after starting ChromSword.Here the names and the sequenceof compounds are entered firstfollowed by the molecular struc-ture of the molecules (if avail-able), see figure 4. A chemical editor program ChromDraw isincluded in the ChromSwordpackage and also offers informa-tion about sum formula and mass under the CHEM menu. The drawn molecular structurecan also be saved so that it can be used in other applications.

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Figure 3Start screen for setting up an experiment

Figure 4Drawing a molecular structure using ChromDraw

If names and structures areentered, the First Guess functionof the main screen can be selectedto get a proposal for column,mobile phase, type and concen-tration of an organic solvent, see figure 5.

Having selected the column andthe mobile phase, the Agilent 1100Series HPLC system should be setup and equilibrated as describedabove. During equilibration of thesystem the Auto Wizard dialogfrom the main screen can be fol-lowed and conditions for theexperiments can be selected andsaved, see figure 6.In the first dialog, it is determinedwhether one or several columnsshould be tested, and whether onemobile phase set or anothermobile phase set should be used.In the third dialog, it is determinedwhether standards are available.In the last screen, flow, tempera-ture, injection volume, columnparameters, elution test parame-ters, and so on are selected. Forthe elution test a high concentra-tion of an organic modifier shouldbe used that provides low or noretention of all the components of the mixture to be separated(the washing concentration).After all parameters have been set and if the Agilent 1100 SeriesHPLC system is equilibrated theGO button can be selected and theautomated experiment starts.

ReportingThe history of a method develop-ment project that ChromSword hasperformed is saved automatically ina history file. After the optimization

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Figure 5First proposal from the software based on the molecules and the molecular structure

Figure 6Auto Wizard screens

experiment is finished the reportviewer (RptView) can be activatedand the appropriate .txt file can beselected, see figure 7.

Report Viewer is a ChromSwordtool to navigate quickly within theresults of a method developmentproject.After the appropriate .txt file hasbeen loaded the results and reportappears, see figure 8. This reportcan now be printed, copied andsaved using the icons at the bottomof the screen.

Further optimization using a existingdata setThe automatically created data setcan be used for further optimiza-tion, for example, for increasing theresolution because more peaks areexpected in the real life sample. Inthis case the .smp file is reopenedand data set listed in Available Datalisted can be selected by a double-click, see figure 1.Here is an example where Concen-tration can be used for further opti-mizing the gradient and for improv-ing the resolution between threepeaks. After the data set has beenselected, the screen shown in figure9 appears.

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Figure 7Report viewer start screen

Figure 8Example of a ChromSword report

Figure 9Screen for using available data set for further optimization

Selecting the Model as nextscreen, see figure 10, the influenceof a different organic phase con-centration on the separation of thethree peaks can be simulatedusing the vertical cursor in the leftdiagram. To simulate the influenceof different gradients on the sepa-ration the Gradient page can beopened. In this screen completechromatograms can be displayedand the peaks are shown as blackbars. By adding more Nodes evenvery complicated gradients can besimulated, predicting the influenceon the resolution, see figure 11. Inthe diagram the Nodes appear ascrosses. By clicking on a crossand moving it, the gradient profilecan be altered. The Peaks moveaccordingly.An alternative and a rapid optionto optimize linear and multi-seg-ment gradients is to choose theOptimize page tab. In this case theprogram optimizes a gradient pro-file automatically and displaysparameters of the optimizingprocess.

Example experiments

Several experiments were done totest the performance and usabilityof the ChromSword software revision 2.1.• Analysis of a mixture of hydro-

cortisone, phenobarbital andphenacetin

• Analysis of a mixture of betablocking drugs: Pindolol, Timo-lol, Metoprolol and Propranolol

• Analysis of antiasthmatic drugs

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Figure 10Influence of a different organic phase concentration on the separation

Figure 11 Optimizing the gradient profile for better resolution using the available data set

Experiment 1: Analysis of amixture of hydrocortisone,phenobarbital and phenacetin

The chemical structures of allcompounds were entered and the ChromSword software (First Guess function) proposed touse a C-18 stationary phase andmethanol or acetonitrile as mobilephase. Based on this the followingsetup was selected using themethod development wizard.• Column: 3 x 150 mm ZORBAX

SB C-18 phase• Mobile phases: water-methanol

for the first experiment andwater-acetonitrile for the sec-ond experiment

• Flow rate: 0.8 mL/min• Detection wavelength: 220 nm• Column temperature: 30°C• Injection volume: 5 µL• Zero time: 0.80 min• Equilibration time: 8.00 min• Dwell time: 1.38 min

The Elution test conditions wereset to:• Concentration of % B: 80%• Run time: 3 min• Equilibration time: 1 minWhen all conditions were set upthe experiments were started andafter approximately 9 h all experi-ments were finished, giving thefollowing results.

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Results with water-methanol asmobile phaseThe best conditions for an isocrat-ic and a gradient runs were evalu-ated, see figure 12.The best isocratic conditions withnear-baseline separation of allpeaks were at 48% methanol. Thebest linear gradient conditionsinvolved a gradient from 10 %B to47 %B in 3.2 min. Shortest runtimes were obtained starting with46% Acetonitrile, holding it until0.4 min and increasing the per-centage of Acetonitrile then to55% at 0.5 min.

The isocratic method elutes all peaksearlier than the linear gradientmethod. For real-life samples itmight be of advantage to rinse thecolumn after peak elution with ahigher percentage of organic phase.This can help to avoid problems withmatrix peaks, which might co-elutewith peaks of interest after severalisocratic runs.

Results with water-acetonitrile asmobile phaseAfter completion of the runs usingwater and methanol as mobile phase,the organic phase was switched to

Fast gradient run: 46% for 0.4min to 55%B at 0.5minLinear gradient run: from 10% to 47%B in 3.2minIsocratic run: 48% B

Linear gradient run

Isocratic run

Fast gradient run

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Figure 12Analysis of phenobarbital (1), phenacetin (2) and hydrocortisone (3) using water-methanol asmobile phase

acetonitrile for the following runs.The best conditions for an isocrat-ic and gradient analysis of themixture were also evaluated. Fig-ure 13 shows both results, inwhich the isocratic run also pro-vides near-baseline separation forall peaks and shorter retentiontimes.

For high throughput analysis theisocratic method is best suited,whereas for real-life sample thegradient run with water andmethanol as mobile phase mightbe the better choice. Finally acomparison was made betweenthe method developed by an expe-rienced user and the methods pro-posed by ChromSword, see figure14. In this case the user did notneed much time to develop themethod. This was mainly based onthe fact that column and mobilephase were given as prerequisites.

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Figure 13Analysis of phenobarbital, phenacetin and hydrocortisone using water-acetonitrile as mobilephase

ChromSword isocratic method

User gradient method

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Figure 14Comparison between ChromSword and method developed by an experienced user

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Isocratic run

Linear gradient run: from 8% to 28%B in 18.2minIsocratic run: 31% B

Gradient method developed by user in 0.5 h:at 0 min 20% acetonitrileat 1 min 20% acetonitrileat 5 min 80% acetonitrileIsocratic method developed by ChromSword in 4.5 h: 31% acetonitrileLinear gradient method developed byChromSword in 4.5h: 10 to 47% methanol in 3.2min

All evaluated methods are applica-ble depending on the needs of thereal-life samples to be analyzed.The benefit of the ChromSwordmethod development software isthat reasonable results weredeveloped overnight, without anyfurther user interaction. The eval-uated methods can be used imme-diately or they can be optimizedfurther with real-life samples

Experiment 2: Analysis of beta-blocking drugs Pindolol,Timolol, Metoprolol and Propranolol

The chemical structures of thebeta-blocking drugs were enteredand ChromSword (First Guessfunction) made a proposal for col-umn and mobile phases. In thiscase no reasonable results wereobtained. This was mainly due tothe fact that software is not ableto recommend a modifier, if this isneeded.From former experiments it wasknown that the analysis of beta-blocking drugs can be done on aC-8 stationary phase using water-acetonitrile as mobile phase. Bothsolvents must contain trifluoro-acetic acid (TFA) as modifier forgood separation, see figure 15.

These experiments were doneusing 0.3 x 150 mm capillarycolumns. However, transfer to acolumn with an internal diameterof 4.6 mm was required. It has tobe considered that the limit ofdetection is much lower on a cap-illary column and that the injectedconcentration should be adjustedfor the 4.6-mm id column. Thisensures reasonable peak heightsfor method development as shownin figure 16.

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3 capillary columns were tested to find optimum conditions In total 52 runs were needed to find:best columnbest of mobile phasebest modifierbest gradientbest oven temperature

3. Zorbax SB C-8

2. Zorbax Extend C-18

1. Hypersil ODS C-18

Figure 15Method development for beta-blocking drugs Pindolol (1), Timolol (2), Metropolol (3), Propranolol(4) on three different capillary columns

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1 µl from vial 78 (level 8 from calibration) on 0.3 x 150 mm Zorbax SB C-8

30 to 90 times more sensitivethan 4.6 mm id column

3 µl from vial 78 on 4.6 x 150 mm Zorbax Eclipse C-8

Figure 16Method transfer from a 0.3-mm id column to a 4.6-mm id column

The method transfer requiredabout six runs and the followingconditions were evaluated:• Column: 4.6 x 150 mm ZORBAX

Eclipse C-8 phase • Mobile phase: water-acetonitrile • Flow rate 1.5 mL/min• Detection wavelength: 220 nm• Column temperature: 20 °C• Injection volume: 10 µL• Zero time: 0.87 min• Equilibration time: 10.10 min• Dwell time: 0.80 minThe elution test conditions wereset to:• Concentration of %B: 60 %• Run time: 5 min• Equilibration time: 1 min

These evaluated conditions wereused to start the method develop-ment of the ChromSword softwarefor further optimization of chro-matographic parameters. Figure17 shows the results that wereobtained within about 9.3 h.

Experiment 3: Applicationdevelopment of antiasthmaticDrugs using ChromSword

To test the application develop-ment of antiasthmatic drugs withChromSword already known sepa-ration conditions have been usedas reference. The reference chro-matogram is shown in figure 18.

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Start Gradient: 10 to 80 %B in 20 min developed by user within 1h, knowing the column and the modifier

4.6 x 150 mm Zorbax Eclipse C-8Flow rate 1.5 ml/min

Figure 17The benefit of the ChromSword software in this case is that the user found the starting conditionsand that the software further optimizes the gradient.

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Figure 18Reference chromatogram: Theobromine (1), Theophyline (2), Enprophylline (3), Caffeine (4)

Water: Acetonitrile 92:8 v/vFlow: 1 mL/minTemperature: 50 °CUV Detection: 270 nm standard cellColumn: ODS Hypersil, 5 µm, 125 x 4 mm

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In contrast to this application,Carbamazepine should have beenseparated in addition to Theo-bromine, Theophyline and Caf-feine, and because of the differentcolumn id the flow rate had to beadjusted to achieve identical lin-ear flow velocity. It was expectedto obtain a solution preferably forboth isocratic and gradient elutionconditions. Further, the influenceof temperature change between 40 °C and 60 °C should be investi-gated based on the obtained opti-mization conditions.

Optimization of isocratic and gradientelution conditions• System components:

Agilent 1100 Series system• Sample:

Theobromine, Theophyline, Carbamazepine, Caffeine

• Starting conditions:Acetonitrile-Water mobile phase

• Flow rate: 0.8 mL/min• Injection volume: 1 µL• Zero time t(0): 0.7 min• Gradient dwell time: 1.38 min• Column temperature: 50°C• UV detection: 270 nm• Column: ZORBAX SB C18, 5 µm,

150 x 3 mm

All analytes had been prepared as individual standards and posi-tioned in the sampler at positions 1through 4. Position 5 was the mix-ture.Elution Test Conditions:• Starting concentration: 40 % ACN• Test time and run time: 3 min• Equilibration time between each

individual run: 8 min • ChromSword method development

program: Isocratic or Gradient

ResultsChromSword delivered final resultsfor both isocratic and gradient elution conditions. Figures 19 and 20 show the final optimized chromatograms as obtained by the method development software for bothconditions.

Optimization of best temperature conditions based on both proposalsTo optimize further the separationconditions the temperature hadbeen taken into account. Here, thegoal was to find out the influenceof temperature to further reduceseparation time and at the sametime to monitor its impact on peak symmetry. ChromSwordenables to use the data from previous experiments and add further ChemStation data that hadbeen prepared in two additionalruns controlled by the ChemSta-tion only. These two additionalruns used the ChromSword gradient condition alreadyobtained except that the tempera-ture set point was changed from50°C to 40°C and 60°C.

Figure 18Optimized chromatogram under isocratic conditions based on ZORBAX StableBond column

Acetonitrile: Water 20:80 v/vFlow: 0.8 mL/minTemperature: 50°CUV Detection: 270 nm standard cellTime required for automatic optimization: 9 h overnight

The new data (based on retentiontime and peak width) were addedto the ChromSword table calledConcentration and Temperature.Having entered all additionalinformation, ChromSword wasused offline to find out new sepa-ration conditions that would givebest symmetry at shortest run time.The chromatograms in figure 21show that temperature has no sig-nificant impact on the total sepa-ration. Conditions used at 40°C donot differ dramatically whereas60°C does not yield a significanteduction in retention times.These results offered the opportu-nity to look for different decision-making criteria to identify theoverall best separation conditions.Eventually the separation condi-tions at 60°C were found to beoptimal, see figure 22, because ityielded the best signal-to-noiseratio in addition to shortest analy-sis time. When temperatureimpacts column longevity, lowertemperature conditions should beused. In this case high tempera-ture was not an issue, because ofthe excellent stability of ZORBAXcolumns at high temperatures.

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Figure 20Optimized chromatogram gradient conditions

Gradient Profile:(Flow 0.8 mL/min, Solvent A: Water, Solvent B: Acetonitrile)Step # Time (min) % A % B1 0.0 90 102 0.3 90 103 0.7 83 174 1.4 83 175 1.8 55 456 6.4 55 457 11.0 55 45

Figure 21Chromatograms at different temperatures

Conclusion

The chromatographic methoddevelopment softwareChromSword can be used forunattended optimization ofreversed-phase, normal-phase andion-exchange separations. Duringthe procedure the composition ofthe mobile phase, its pH and thecolumn temperature can be opti-mized. This can be done eitheronline in combination with Agilent1100 Series HPLC systems andAgilent ChemStation software, oralternatively offline. When used inoffline mode ChromSword per-forms virtual chromatographyusing the structural formulas ofthe compounds investigated andthe software's database of morethan 60 reversed-phase columnsand several mobile phases. Inonline mode real experiments areperformed whereby the separationis optimized using the resultingchromatographic parameters. As afirst step the structures of the dif-ferent substances have to bedetermined. The ChromSwordsoftware then automatically calcu-lates a first guess for column,mobile phase and temperature.

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Figure 22Final chromatogram at 60°

Different optimization experi-ments follow and finally a reportis generated, showing all theresults and including the mostsuccessful conditions in terms ofseparation and analysis time. Theconstructed data set can then beused for further optimizationeither virtually of by real experi-ments. The ChromSword software canachieve fast and unattended opti-mization of separations in combi-nation with complete control ofthe important chromatographicparameters and offers the oppor-tunity to simulate chromatogramsfor further optimization, making itan invaluable tool for both chro-matographic beginners andexperts.

Contacts and Links

For more information about ChromSword, contact Sergey Galushko by e-Mail atgalushko@t-online.de

ChromSword is distributed inEurope by Agilent TechnologiesSales & Services GmbH & Co KGProject Services OrganizationEurope pso_europe@agilent.com <mailto:Pso_europe@agilent.com> and in the US and Canada by IRISTechnologies, L.L.C., Lawrence,KS (www.iristechnologies.net )

Acetonitrile-Water 10 to 84 % Acetonitrile in 4.1 minFlow: 0.8 mL/minTemperature: 60°C UV Detection: 270 nm standard cell

Microsoft is a U.S. registered trademark ofMicrosoft Corp.; ChromSword is a U.S. regis-tered trademark of Merck KGaA

The information in this publication is subject to change without notice.

Copyright © 2003-2004 Agilent TechnologiesAll Rights Reserved. Reproduction, adaptationor translation without prior written permissionis prohibited, except as allowed under thecopyright laws.

Published March 15, 2004Publication number 5988-8927EN

www.agilent.com/chem/nds

Sergey Galushko is owner of

Dr. Galushko Software Entwick-

lung, Mühltal Germany

Ralf Honsberg is Requirement

Analyst for Data Systems, Angeli-

ka Gratzfeld-Huesgen is Applica-

tion Chemist and Bernd Hoff-

mann is Research and Develop-

ment Chemist, all at Agilent

Technologies GmbH, Waldbronn,

Germany.