Agilent LC walk-up system using the Agilent 1200 Series LC Method Development Solution Agilent Easy Access software Manual
Contents
1. 40000000 20000000 MG mAUJ 0 J Educts 05 1 15 2 25 min A0 8000000 J 40000005 LEIC from product 2004 1 J 0 ji T T T J 1 1 5 2 25 min 150 600 4000000 EIC Educt 1004 a J Product 7 7 7 J 0 5 1 1 5 2 25 min 507 8000000 40000007 EIC from Educt 0 TT TT TT LE a T T7 07 T T T T T T 0 0 5 1 1 5 2 25 min 0 5 1 1 5 2 25 min Figure 9 UV chromatogram TIC and EIC of a reaction mixture Method Development Solution together References with the Agilent Easy Access software tailors the system even more to the needs of the chemists and allows them to become even more productive Different user groups can use up to eight columns with different selectivity or separation efficiencies always matching the properties of the com pounds and the analytical task without the need to change any hardware This provides optimum conditions for column scouting reaction monitoring and purity checks after preparative cleanup Different detectors can be attached to the system including UV and mass spectrometer The Easy Access software allows samples with different priorities to be run in a logical and convenient way Reporting can be done via e mail 1 A G Huesgen Agilent 1200 Series LC method development solution Automated method development with up to eight columns Agilent Technical Note 5989 9338EN 2008 2 W Duncan D McIntyre Increasing productivity with LC2. ass 20 RS Ske r 8 S jes Ss ays D Bises LR 0 i LL RE on er a 100 200 300 400 500 600 700 800 100 200 300 400 500 600 700 800 m z m z L J Figure 8 Mass spectra of submitted sample Group 3 submits a sample to control a reaction product The third group that has access to the LC MS system is submitting a sample that has been obtained from a reaction of two compounds The resulting chro matogram shows main peaks with very low resolution The LC MS analysis was used to give some information about the reaction products In Figure 9 the UV chromatogram is shown with three or four main peaks and four small peaks Also the MS scan mode total ion chromatogram and the resulting extracted ion chromatograms for the main peaks are shown The expected product with the mass 450 4 at RT 1 087 min was formed after 2 hours The reaction kinetic seems to be rather slow or the reaction parameters like temperature etc are not appropriate Therefore the yield of the expected product is low Conclusion Setting up an open access LC MS envi ronment with analytical and preparative LC MS systems increases the produc tivity of chemists so that they get the fastest access to the analytical results This allows the chemists to perform the next required step in their workflow without waiting for a remote analytical laboratory Adding multiple column and multiple solvent availability to such a system by using the Agilent 1200 Series LC
3. 5 min re equilibration at 5 B until 3 6 min Flow rate 0 8 mL min DAD and MS detection In addition to the UV chromatogram the MS signal in scan mode was used to control whether the expected prod uct was produced and also to get some rough ideas about the masses of the side products In Figure 8 the obtained mass spectra are combined The expected product was formed with in 1 5 hours with high yield Two side products were formed that have to undergo further structure evaluations 7 Educts Product Side products mAU 400 Reaction products after 0 5 h 200 4 o A oy 0 25 0 5 0 75 1 1 25 1 5 1 75 2 min mAU 200 4 Reaction products after 1h 100 4 a A A poo 0 25 0 5 0 75 1 1 25 1 5 1 75 2 min mAU 3005 Reaction products after 1 5h 200 4 100 5 0 lt lt 0 25 0 5 0 75 1 1 25 1 5 1 75 2 min LC Figure 7 Study of reaction kinetics di 2 1004 lt 80 4 3 Max 1 06906e 006 604 Product E 404 2012 011 L Sa 100 200 300 400 500 600 700 800 m z 1004 100 3 804 is Max 2 98957e 006 80 Max 4 431876 006 1 Educt 1 604e 60 Educt 2 4027 gt 40 ed 21 2 2 20 amp 04 se Frs se 0 mmo Heee 100 200 300 400 500 600 700 800 100 200 300 400 500 600 700 800 m z m z 10042 100 2 807 E Max 89880 8046 N Max 59272 604 Side product 1 ona 602 Side product 2 A s 4 N its MINS a BN lon wi Je 20187
4. 04 3S gt a S es 40 A st 0 6 0 8 1 1 2 1 4 1 6 1 8 min mAN Es CN phase 80 So Eu N 4 Sg 40 z e Be oe 0 6 0 8 1 1 2 1 4 1 6 1 8 min ques g 2 e Bonus RP C18 phase 4 o D ed Lo 407 RJZ S gt 6 0 6 0 8 1 1 2 1 4 1 6 1 8 min mAU D SB C18 phase 80 e al 2 Ls 40 2 Ss 3 8 53 A a yN 0 6 0 8 1 1 2 1 4 1 6 1 8 min ka Figure 4 Column scouting for optimum prep analysis conditions After purification Having purified the compound and evaporated the solvent the resulting fraction was again submitted by a co worker of group 1 and was analyzed on a 150 mm long column to see whether the fraction was clean that no thermal degradation occurred during solvent evaporation and if not to see as many impurities as possible The left part of Figure 5 shows the sam ple plates with positions filled with vials On the right side the done sample and the waiting samples are listed In this case the purity check sample will be the next sample injected At the top of this screen is the estimated time the system will be occupied by the remain ing runs The user can now estimate how long it will take to get the results After the analysis the user gets the results via e mail The fraction was pure enough for fur ther analytical evaluations Figure 6 Chromatographic conditions Column 2 1 mm id x 150 mm ZORBAX SB C18 1 8 pm Gradient 10 to 85 B in 8 min hold until 9 min at 85 B re equilibration with
5. 10 B until 11 min Flow rate 0 3 mL min DAD and MS detection Agilent Easy Access STATUS 1 run in queue est 8 mins to completion r Sample Queue Loc Input Notebook Method EI 1 P1 A 02 4 02 sam200Gexpoure ai Purity check on 100mm length 161008 P 4 JoblD User Group LoginDate 1 081016 134354 Amo Chemist 10 16 08 13 43 54 2 081016 130122 susan Chemist 10 16 08 13 01 22 3 081016 131458 Amo Chemist 10 16 08 13 14 58 4 081016 103754 Arno Chemist 10 16 08 10 37 54 4 gt Submit New Sample Figure 5 Monitoring screen of Agilent Easy Access software 3 gt f y a e So e So 300 L Figure 6 Purity check after preparative purification se N gt z o 3 5 Group 2 submits samples for reaction monitoring In the meantime a chemist from group 2 has submitted within 1 5 hours several samples to monitor a reaction for completion To get a result in time the samples were analyzed with high est priority Selecting the method Fast Kinetics sets these samples on first pri ority and analysis start immediately after the previous run has ended The results of the kinetic measurements are shown in Figure 7 Chromatographic conditions Column 2 1 mm id x 50 mm ZORBAX SB C18 Gradient 5 to 95 B in 1 5 min hold at 95 B until 2
6. Authors Angelika Gratzfeld Hiisgen Michael Frank Agilent Technologies Waldbronn Germany LC walk up system using the Agilent 1200 Series LC Method Development Solution and Agilent Easy Access software Test of reaction kinetics column scouting and impurity checks with one LC system and up to eight columns Application Note Drug Development Educts Product Side products mAU 400 Reaction products after 0 5 h 200 1 AA PR RTC LIER I 0 25 0 5 i 75 1 1 25 1 5 1 75 2 min mAU _ 200 4 Reaction products after 1h 100 0 A A TT oot 0 25 0 5 0 75 1 1 25 1 5 1 75 2 min mAU 4 3005 Reaction products after 1 5h 200 5 1005 0 SR oo 0 25 0 5 0 75 1 1 25 1 5 1 75 2 min In chemical synthesis laboratories different users are involved in the synthesis of new compounds Typically different projects are running for several groups or single users The needs and analytical tasks for liquid chromatography in combination with mass spectrometers are In time reaction monitoring Control of reaction products Column scouting before preparative LC e Purity analysis after preparative LC e Fast and easy access to a running LC system e Availability of columns of different selectivity e Predefined chromatographic methods e Fast reporting if sample has been analyzed Bo RE Agilent Technologies Introduction The main task of a chemical synthesis laboratory is to generate
7. MS Easy Access software Agilent Technical Note 5988 5525EN 2002 3 D Mcintyre Continuous Operation of a High Throughput LC MS System in a Walk Up Environment Agilent Technical Note 5989 6714EN 2007 www agilent com chem 1200mds Agilent Technologies Inc 2010 Published June 15 2010 Publication Number 5990 3358EN RE Agilent Technologies
8. ant the different projects often require different separation selectivity because of differ ent compound properties All these tasks can be performed on the Agilent 1200 Series LC Method Development Solution and Agilent Easy Access software This LC system can be used with up to eight columns of dif ferent length and internal diameter The superuser can define different analyti cal methods for the installed columns including appropriate equilibration methods The superuser can also define projects for different user groups or sin gle users Different permission levels can be set for users and groups Priorities can be set for example reac tion monitoring samples can have high est priority and are analyzed immediate ly After completion of the analysis reports can be sent via e mail to the appropriate user In this application we used the Agilent 1200 Series LC Method Development Solution with attached Agilent 6410 Single Quadrupole MS in combination with the Easy Access software for the following laboratory environment e Three user groups need access to the LC MS system e Four different projects are running All groups have the following analytical tasks Reaction monitoring e MS identification of the reaction product e Column scouting of reaction mixtures to find the most suitable column for preparative purification e Purity control after preparative LC Experimental Equipment The Agilent 1200 Se
9. ction valve installed on one pump channel which offers the possibility to have up to 15 different solvents The two clus tered column compartments offer four independent heating zones This allows setting columns at different tempera tures which is of advantage if for example higher temperatures are need ed for special applications Description of the Easy Access software The Agilent Easy Access software allows the users to walk up with their samples input sample information choose from the list of methods posi tion the samples as directed by the sys tem and then return to their labs and wait for an e mail containing the results 23 Depending on the permission level of the users they are allowed to change the selected method to a certain extent like injection volume or number of injections The system configuration and adminis tration is done by a superuser who is responsible for keeping the system run ning for all the other users Application example In the example presented in this appli cation note the system was set up with four short 50 x 2 1 mm columns of dif ferent stationary phases one 100 x 2 1 mm column one 150 x 2 1 mm column All columns were packed with 1 8 um particles Configuration and administration by the superuser A chromatographic method was set up for each column in the ChemStation software This has to be done by the superuser who is the only one who has access
10. ethod that should also be used for the analytical run This ensures that the column has seen the actual gradient 3 once already and that possible remain ing peaks on the column from the previ ous analysis are not falsifying the actu al chromatogram But any other prede fined method using the same column can be used to perform the equilibra tion The equilibration method will only be executed if the analytical method is changed Having made all needed entries the method setup in the Easy Access soft ware is finished In the next screen the superuser has to define the user groups and users who should have access to the system and to what extent they are allowed to modify methods and other parameters Having configured the system the user can now walk up and submit his sam ple Depending on his permissions he fills in the necessary information selects a method modifies the method puts one or several vials in the posi tions directed by the system and walks away Figure 3 Group 1 submits samples for prep purification work A chemist from group 1 has submitted a sample for column scouting He has selected four columns with different selectivity to find the best column for a preparative cleanup of the sample He puts the vial into the vial tray and the analysis can start using the selected columns with appropriate equilibration and run parameters At the end the user gets for each run a report and can decide whic
11. h column is the best Figure 4 Chromatographic conditions Columns 2 1 mm id x 50 mm 1 8 pm ZORBAX SB C18 ZORBAX SB CN ZORBAX Bonus RP C18 ZORBAX Eclipse PhenylHexyl 5 to 85 B in 0 8 min hold at 85 B to 1 5 min re equilibration at 5 B until 2 4 min at 0 8 mL min flow DAD and MS detection Gradient In this case the Agilent ZORBAX SB C18 column is the best for the prep analysis because resolution before and after the main peak is better than on the other three columns For the prep analysis the same column chemistry was chosen using a 21 2 mm id x 150 mm ZORBAX SB C18 5 um PrepHT870150 902 The preparative LC MS system is also operated as a walk up system controlled by the Agilent Easy Access software Agilent Easy Access Sample Data Input CHEMIST User Login if Sample Data Input il Sample Loading Description Methods 1 Colscout phenyl hexyl i gradient from 5 to 85 B 2 M column scoutingSB CN v mi gradient from 5 to 85 3 Columns couting Bonus RP Ai fast gradient from 5 to 85 B 4 M colurnnscouting SB C18 mi gradient from 5 to 85 ow Fast kinetic mi 5 to 85 Bin 3 min a curity check 100mm length fxd i Gradient from 10 to 85 B Back Load Samples Cancel amp Figure 3 User Arno submits a sample for column scouting y co mAU 2 PhenylHexyl phase 8
12. new com pounds like new drug compounds new pesticides or new dyes that fulfill cer tain prerequisites given by the govern ing company Typically several attempts to reach a desired structure are required and often several modifica tions of such a structure are desired as well The individual synthesis of com pounds involves reaction monitoring control of reaction products and purity check of compounds that were cleaned by preparative LC Traditionally many of these tasks have been done by thin layer chromatography however with rapid LC MS instrumentation results can be achieved even faster and most importantly with much more informa tion depth That is the reason why more and more companies make LCMS sys tems available to their chemists Recently walk up LC MS systems are used in these environments which pro vide easy access for inexperienced users analysis of the submitted sam ples with predefined columns and chro matographic methods and information about the results by for example e mail after completion of the run In many cases these users are unfamiliar with LC MS systems Therefore supe rusers take care of the LC MS system providing the chromatographic meth ods keeping the LC MS system run ning and in case of errors being able to restart the system Typically several projects are done in parallel Therefore different user groups or single users need access to a running LC system Most import
13. ries Rapid Resolution LC system comprised the following modules with firmware revisions A 06 10 or higher Agilent 1200 Series binary pump SL with degasser Agilent 1200 Series high performance autosampler SL Plus Two Agilent thermostatted Column Compartment SL Plus with installed valve drives Method development valve kit G4230B high pressure with method development capillary kit low disper sion for short columns Agilent 1200 Series diode array detector DAD SL Agilent 6410 single quadrupole MS with multimode source Several Agilent ZORBAX Rapid Resolution High Throughput RRHT 1 8 um columns Agilent ChemStation B04 01 with Agilent Easy Access software Results and discussion Description of the LC MS system The Agilent 1200 Series LC Method Development Solution consists of two or three clustered thermostatted col umn compartments SL Plus integrated into an Agilent 1200 Series standard LC or RRLC system One column compart ment contains the valve that is con nected to the pump and delivers the flow to the different columns The sec ond column compartment contains the valve that is connected to the detector and delivers the flow coming from the active column to the detector A maxi mum of eight columns up to 100 mm in length can be installed in two clustered column compartments using the low dispersion heat exchangers Figure 1 The system can be equipped with an additional clustered solvent sele
14. to the screen in Figure 2 The ChemStation methods are used in the method setup screen in the Easy Access software as CS method Figure 2 To facilitate the usage for the end user the naming of the easy access method was kept in a logical context with the usage in this case Figure 1 Clustered Agilent 1200 Series thermostatted column compartments SL plus with up to eight columns Agilent Easy Access Edit Existing Method Method Name fes kinetic Description E min Method priority 1 is highest 1 x Est runtime UY wavelength Channel A Sample Bw Reference Bw f fo peo ffm CS Method COLSCOUT BC13 M Ft 85 B in 3 min F Use Target Masses Time to equilibrate after change fi min CS Equil Method COLSCOUT SBC Method Type Injection volume Loop no column Minimum fi ul Chromatography Nominal E ul Maximum 40 pl LC MSD Method Parameters Method is in POSITIVE ion mode Default Target Masses l Update S Cancel Figure 2 Agilent Easy Access software screen to set up chromatographic parameters by the superuser fast kinetic Information about the gradient is given in the description sec tion Under CS EquilibrationMethod the equilibration of the column is deter mined In our case each column was equilibrated using the chromatographic m
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