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Prelude User Manual - Protein Technologies, Inc.

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1. r Peptide Programming Sequence PTI PTI PTI Standard standard Taran X3DDEDDDDD21 1 1 1 13 Standard Current Cycle Maar oaa mmm Sequence c g Original Modifies g Molecular NoAA o H Weight lde oa A BPEPTI DE g s paje PTHPEPTID E g vs os PHIPEPTIDE ae Dell g mol 1112 8 BTANDARD el ole e 878 ETANDARD ins da e 878 Nev Close Cancel Save j BTANDARD am va Print The Dynamic Sequence Programming buttons are located in the Synthesis Editor to the right of the peptide sequences and are as follows 1 Ins Opens the Cycle Selection window which allows the user to choose the type of cycle to insert Idle No AA or an amino acid See below Inserts the new cycle to the right of the selected cycle in a sequence NOTE Make all cycle inserts prior to assigning programs When a No AA or new amino acid cycle is inserted all program assignments return to the default program 2 Del Deletes the selected cycle in a sequence NOTE Make all cycle deletions prior to assigning programs When an Idle or a No AA cycle is deleted all program assignments return to the default program CAUTION This feature can delete amino acids in the original sequence file Use with caution If an amino acid is deleted by mistake use the Undo button or reload the original sequence file to start over 3 Undo U
2. www peptideinstruments com Catalog No Start Up Kits Quantity Fmoc Amino Acid Start up Kit for the Prelude Contains 27 amino acids PPS STARTKIT 0 9 L Deprotectant 0 9 L 0 4M NMM 1 ea 100 g HBTU 200 mg Fmoc Ala Wang resin 200 mg Fmoc Gly Wang resin Fmoc Amino Acid Start up Kit for the Prelude Contains 27 amino acids EESTI ES 100 g HBTU 200 mg Fmoc Ala Wang resin 200 mg Fmoc Gly Wang resin 1 ea B Reagent Shelf Life amp Handling CAUTION This instrument contains solvents and chemicals that should be handled carefully Many are easily absorbed through the skin and can cause adverse health effects Wear safety glasses protective clothing and rubber gloves at all times Follow MSDS handling guidelines provided with the individual reagents Respirators and adsorbent should be available in the event of a spill B 1 Reagent Shelf Life Proper handling and storage of peptide synthesis reagents is important for the successful performance of your instrument Please review the table on the following pages to be certain that your reagents are properly stored Be sure to rotate your stock of reagents using a first in first out method so that their shelf life is not exceeded before use Reagent Temperature Shelf Life Amino Acids Solid except Fmoc Trp Boc OH 20 25 C Stable Fmoc Trp Boc OH Solid 20 C Stable Amino Acids in DMF solution ex
3. 1 800 477 6834 allows the user to set a new start cycle start a synthesis and monitor a running synthesis To open the RV status screen click on the shortcut button M or click on the Operations menu select RV Operations and then select RV Status SUser File Operations Tools Reports View Help Bottle Preparations gt Ife R Y LIONS D Load Synthesis Calculations AA Calculations Solv Reag Manual Operations The Reaction Vessel Operations screen will open with the RV Status tab active Reaction Vessel Operations RY Status Load Synthesis Calculations AA Calculations Solv Reag Reaction Vessel Status Set Start Cycle PTI _ PTPEPTID123 PTIPEPTIDE123 PHPEPTID128 FTHPEPTID123 PHPEPTID128 PHPEPTID123 m Action Filing Timed Fill 8 DMF Top Wash oco om ae ww N o c amp w N For Help press F1 ep RVB Last Archive MASTER USER 20060309 Comm Waste Vac CHE On the left side of the RV Status screen is the Set Start Cycle button the Colored Status Box and the Error Reporting Box 61 www peptideinstruments com The Colored Status Screen displays various messages on a colored background depending on the status of the synthesis as follows 1 Synthesis is Loaded Ready is displayed on a dark green background 2 Synthesis i
4. CAUTION Fluid may not immediately stop when the process is paused 8 Cancel Click the Cancel button to cancel an active process CAUTION Fluid may not immediately stop when the process is cancelled Cancelling an action may result in solution left in the lines and valve blocks which could lead to cross contamination Always prime either Solvent 1 or Solvent 2 following a cancel to clear the valve blocks and lines 9 Close Click the Close button or click the X in the upper right corner to close the Bottle Preparations screen 2 5 1 2 Special Bottles Special Bottles allows the user to prevent solvent reagent bottles from being manipulated in the Bottle Preparations screen and set individual amino acid bottles to perform a Single Shot gelivery To open the Special Bottles screen click on the Operations menu and select Special Bottles File Operations Tools Reports View Help Bottle Preparations gt Bottle Preparations RV Operations b Special Bottles Cleaning gt Solvent Calibration This will open the Bottle Preparations screen with the Special Bottles tab active 54 No Prime 1 E DMF ZE DCH 3 Dep 4 Cap 5 7 Base B Acti Y Act2 8 M TFA Solvent Reagent 1 800 477 6834 m One Shot Delivery Amino Acid 1T Ala 2 Cys 3 Asp 47 Glu 5 J Phe B Gy 7T His 8 lle 9 Lys Bottle 8 has a special feature called No Prime it will preven
5. Following the cleavage remove collection vials and work up the cleaved peptide 103 www peptideinstruments com 8 Perform a Collect Back Flush Section 2 5 3 5 9 Replace Solv 8 bottle with an empty bottle Perform a Cleave Bottle Solvent Back Flush Section 2 5 3 3 10 Discard rinse solution 4 2 Dynamic Sequence Programming The optional Dynamic Sequence Programming feature consists of advanced operations in the Synthesis Editor that may be activated by purchasing a license from PTI To puchase a license contact PTI customer service at 1 800 477 6834 Catalog No Description Quantity Dynamic Sequence eens Programming License Dynamic Sequence Programming allows the user to 1 Insert or delete an Idle cycle from the sequence An Idle cycle makes the RV inactive during that cycle 2 Insert or delete a No AA cycle from the sequence A No AA cycle performs all program steps in a cycle except for amino acid addition 3 Insert or delete an amino acid from a sequence If used incorrectly these operations can easily ruin a synthesis Therefore once activated Dynamic Sequence Programming may only be performed when a supervisor is logged in 104 1 800 477 6834 Synthesis Editor PTI Program Sequence Synthesis Synthesis Name PTI Comments Program Sets D Standard 2 Program2 X ul Program v al Program3 SS GE Solvent Reagent Standard Amino Acid Standard
6. 1 800 477 6834 Index About SUser 99 100 Accessories 27 29 126 Activator 10 15 43 67 69 Amino Acid 10 28 121 123 Bottle 9 14 21 28 36 51 53 66 73 95 116 126 Installation 24 25 Seal Replacement 14 76 113 118 Systema ccce tens 13 14 116 Degradation Testing 125 ee ue EEN 35 37 95 File 32 35 37 45 46 48 49 52 66 90 101 File Manager 35 37 Shelf EIS ante ed 124 Solubility Testing 125 Archive All Data 86 88 Back Flush Collect 13 15 72 79 81 101 103 113 114 Nitrogen 53 75 76 103 117 Cleave Bottle 72 76 78 79 103 114 Solvent 53 75 76 103 Cleave Bottle 15 72 76 78 101 103 106 113 114 Bottle Amino Acid9 13 14 20 21 28 36 51 53 66 73 95 116 126 Installation 24 25 Seal Replacement 14 76 113 118 i i RE 15 Filter 13 16 25 76 101 102 117 Replacement 13 113 117 Position Flush 15 72 74 76 114 119 PreSSUl eegene uge NN 17 Single Shot 110 126 Solvent Reagent 9 14 39 51 54 55 69 73 95 126 Installation 25 26 Seal Replacement 16 113 118 127 ee E 114 Bottle Preparations 13 14 16 24 25 31 40 50 56 73 85 90 117 11
7. Technologies Inc Prelude Peptide Synthesizer USER MANUAL The World s Leading Manufacturer of Peptide Synthesizers Prelude Peptide Synthesizer USER MANUAL Technologies Inc 2010 Protein Technologies Inc 4675 S Coach Dr Tucson AZ 85714 USA All Rights Reserved Document 9030002 Rev 06 gt EPP WARNING ALL REACTION VESSELS MUST BE IN PLACE AT ALL TIMES WARNING COLLECTION VIALS MUST BE IN PLACE AT ALL TIMES WARNING SAFETY SHIELD DOORS MUST BE CLOSED WHILE A SYNTHESIS OR CLEAVAGE IS RUNNING WARNING SYNTHESIS WILL HALT IF WASTE CONTAINER IS FULL WARNING DO NOT ATTEMPT TO MOVE THE PRELUDE WHILE ANY OF THE SOLVENT OR WASTE CONTAINERS CONTAIN LIQUIDS WARNING THIS INSTRUMENT CONTAINS SOLVENTS AND CHEMICALS THAT SHOULD BE HANDLED CAREFULLY MANY ARE EASILY ABSORBED THROUGH THE SKIN AND CAN CAUSE ADVERSE HEALTH EFFECTS WEAR SAFETY GLASSES PROTECTIVE CLOTHING AND RUBBER GLOVES AT ALL TIMES FOLLOW MSDS HANDLING GUIDELINES PROVIDED WITH THE INDIVIDUAL REAGENTS RESPIRATORS AND ABSORBENT SHOULD BE AVAILABLE IN THE EVENT OF A SPILL 1 800 477 6834 Contents EE eich eae ip aue oe e C Hee thi a PR Et OP a UE ER Ee pte ete V NITRO Elle GE 9 US ABOUT The E Al MR Tc 9 2 About The Company en See AE ee 9 ES OmOob Abbreviations EE 10 Chapter 1 General Inte mma nie EE 11 1 1 General System D8seniplions aut er 11 SR S elt EE 11 1 1 2 Solvent Feedthrough Panel NEE 12 121 83 UNE le
8. x RY Status Load Synthesis Calculations AA Calculations Solv Reaa m Select Synthesis E P RV 1 PTT EI RY 2 PTT E RV 3 PTT Sequence PTIPEPTIDE123 Termination COOH Weight 1057 182 Modified 8 28 2006 3 58 25 PM Comment EI RY 4 PTT RV 5 PTT EI RY 6 PTT E Program Template From Cycle 1 PTI Modified 8 30 2006 4 50 17 PM Comment E Standard Program standardCleave zl C Now AtEnd Delay 08 30 2006 09 00 00 Load PTI For Help press F1 B ave Last Archive MASTER_USER_20060309 Gm Ga Gap GD 7 The Select Synthesis section displays all synthesis files that share the default solvent reagent and amino acid files specified in the Settings screen To view syntheses using different solvent reagent or amino acid files the default files must first be changed in the Settings screen Section 2 6 3 Click on to view more details Click on to hide details The first level of detail shows the names of the sequences assigned to each reaction vessel The second level of 64 1 800 477 6834 detail shows the sequence termination molecular weight date and time the synthesis file was modified and any comments in the file Below the sequences assigned to each reaction vessel is the Program Template It lists the starting cycle for each program used in the synthesis The Cleave section assigns a cleavage program to a synthesis After selecting a synthesis turn
9. 1 New The New button creates a new file The Sequence File Manager will open Enter a new name or use the default and click OK or click the Cancel button to return to the Sequence Editor without creating a new file 2 Close The Close button is not active until the sequence is saved After saving the sequence click the Close button to exit the Sequence Editor Screen 3 Cancel The Cancel button removes any changes made since the last Save A screen will appear reading Abandon All Changes The Yes button will permanently remove the changes the No button will leave the changes 4 Save Once a change is made to the sequence the Save button becomes active Click Save to save the changes The Save and Cancel buttons will deactivate once the sequence is saved 5 Save As To copy the file on the screen click the Save As button The Sequence File Manager will appear with a default name for the copy Change the file name or accept the default and click OK The Cancel button will close the screen and return to the Sequence Editor 6 Print To print the open sequence file click the Print button It will go automatically to the printer NOTE To view the sequence before printing click the Close button and preview the file using the Print button in the Sequence File Manager Section 2 3 46 1 800 477 6834 2 4 3 3 Synthesis Editor The Synthesis Editor is used to create synthesis files Synthesis fi
10. The Yes button will permanently remove the changes the No button will leave the changes 4 Save Once a change is made to the file the Save button becomes active Click Save to save the changes The Save and Cancel buttons will deactivate once the file is saved 5 Save As To copy the file on the screen click the Save As button The Amino Acid File Manager will appear with a default name for the copy Change the file name or accept the default and click OK The Cancel button will close the screen and return to the Amino Acid Editor 6 Print To print the open amino acid file click the Print button It will go automatically to the printer NOTE To view the program before printing click the Close button and preview the file using the Print button in the Amino Acid File Manager Section 2 3 NOTE The Prelude software comes with a standard amino acid file that can be copied opened or viewed by selecting the file Standard in the Amino Acid File Manager See Section 2 3 The Standard file contains the names and molecular weight values of 20 standard amino acids 37 www peptideinstruments com 2 4 2 Solvent Reagent Editor The Solvent Reagent Editor allows the user to assign abbreviations and operation names to each solvent reagent bottle and create a good laboratory practice record of the volumes and concentrations of solvents or reagents consumed as well as the date opened the lot number and the sou
11. indicates the position is pressurized and N indicates the position is vented Amino acids 1 9 10 18 and 19 27 are pressurized together in groups of nine The Primed columns indicate the prime status Y indicates the position is primed while N indicates the position is not primed The Status Bar near the bottom of the screen displays the status of the current operation The buttons are 1 Select All Clear All The Select All Clear All buttons simplify the bottle preparations process Click on Select All to select all bottle positions in the Solvents or Amino Acids sections When one or more positions are 52 1 800 477 6834 selected the Select All button will be replaced by the Clear All button Click on Clear All to deselect all bottle positions in the Solvents or Amino Acids sections 2 Pressurize Click on the Pressurize button to pressurize all selected bottles When pressurization is complete the Pressurized column will display a Y next to the selected bottles Allow a minute for the bottle s to equilibrate NOTE Because amino acid bottle positions 1 9 10 18 and 19 27 share a common pressure manifold when one bottle in the column is selected and pressurized all bottles sharing the same valve will be pressurized 3 Prime Click on the Prime button to prime all selected bottles When priming is complete the Primed column will display a Y next to the selected bottles 4 Vent
12. the No button will leave the changes Save Once a change is made to the synthesis the Save button will become active Click Save to save the changes The Save and Cancel buttons deactivate once the synthesis is saved Save As To copy the file on the screen click the Save As button The Synthesis File Manager will appear with a default name for the copy Change the file name or accept the default and click OK The Cancel button will close the screen and return to the Synthesis Editor 49 www peptideinstruments com 6 Print To print the open synthesis file click the Print button It will go automatically to the printer NOTE To view the synthesis before printing click the Close button and print the file using the Print button in the Synthesis File Manager Section 2 3 2 4 4 Exit The Exit function closes the Prelude SUser software To exit the user software click on the File menu and select Exit 2 5 Operations Menu 2 5 1 Bottle Preparations The Bottle Preparations screen automatically opens when the Prelude software opens To open the Bottle Preparations screen click on the shortcut button or click on the Operations menu and select Bottle Preparations OE y File Operations Tools Reports View Help Ee EIER Bottle Preparations RV Operations gt Special Bottles Cleaning gt Solvent Calibration Then select from one of the following three screens 1 Bottle Preparations 2 Sp
13. the amino acid solution The software calculates this value based on the Amino Acid Concentration mM and the Suggest Volume mL values The Print button prints the calculated values from the Calculations AA and Calculations Solv Reag screens NOTE Use the Print button only after modifying both the Calculations AA and the Calculations Solv Reag screens to avoid having to print twice 2 5 2 4 Calculations Solv Reag The Calculations Solv Reag screen is located under the Calculations Solv Reag tab in the Reaction Vessel Operations window It aids the user in preparing chemicals for a synthesis by calculating resin weights solvent reagent minimum volumes and the amount of activator necessary for the coupling solutions in a given synthesis To open the Calculations Solv Reag screen click on the Operations menu select RV Operations and then select Calculations Solv Reag Ce File Operations Tools Reports View Help Bottle Preparations gt In I RV Operations RV Status Cleaning gt Load Synthesis Calculations AA Calculations 5 Manual Operations The Reaction Vessel Operations screen will open with the Calculations Solv Reag tab active 67 www peptideinstruments com Reaction Vessel Operations x RV Status Load Synthesis Calculations AA Calculations Solv Reag Synthesis Name Prelude QA r Peptides Sequence Termination Sub mm
14. 4 1 Amino Acid Editor 35 2 4 2 Splvarib heagehtEdiEOl sese Seo Eee EE M Pei e Be coo tue E Ee EE SES o res 38 2 4 93 DVINIMOSIS ue ie hier hr ER I ways 40 22 3 1 P ele nn Ee te coo ea 41 2 4 3 2 Seqlence Edif e rito essen ee en en 44 www peptideinstruments com 24 33 Synthesis Editor cas ciis cata Denen De 47 ZIEL EX erp mE 50 2 5 Operations Mehl 50 2 5 1 ln EE 50 2 5 1 1 Bottle Preparall ns use RE E E Rod 51 2 5 12 Special Bottles en ee 54 2 5 1 3 Solvent Calibration E 56 25 2 RV Operations m ee ae nee ee een EANA En 60 PA 2 HRV Ee 60 2 5 2 2 Load Synthesis nenne een 64 2 5 2 3 Calculations ARE Ares ek ee 65 2 5 2 4 Calculations Solv Reag nu oe UE run 67 2 3 2 3 Manual e 70 2 5 9 GIO AMING xxi mens 72 2 5 3 1 EE 72 2 5 3 2 Botle POSITION TEE 74 2 5 3 3 Cleave Bottle Solvent Back Flush eeessseessss 76 2 535 Oullect Back Flush een een 78 2 5 9 5 RINSE All Blocks na et ae ere e e 79 259 T olea BIOOKS Susanne 81 2 9 3 9 M aSLU FIO eod rra tue ie ERR EE RR ERR EAE 82 2 6 Tools MENU M 84 26 1 DatabaSe xs ud ea a ne ae ae EE 84 2 6 1 1 Rebuild Errors Table An een 84 2 OT ATODIVOUATTDIBES rennen 84 2 52 LOgIH 99 OU ux cs eua heo e eMe LU UU E UC er 86 2 6 3 SENGS RR 87 RE e ee 90 2 9 9 ODE AMON Times cueste cote ti ernennen ee 90 2 5 6 DDSratlofis cite Do eo b e Di bL tne bate D te eed 90 2 mepoHs Ee EE 90 NIE P cs 90 SEO 95 BBW LOW d ip
15. A Phone 1 520 629 9626 Technologies Inc Toll Free 800 477 6834 Fax 1 520 629 9806
16. Click on the Vent button to vent all selected bottles When complete the Pressurized column will display an N next to the vented positions Allow a minute for the pressure to return to 1 atm 14 7 psi before opening the bottle 5 Nitrogen Back Flush Click the Nitrogen Back Flush button to back flush selected bottles with nitrogen gas A SUser warning window will open CHEN x eo Warning This procedure back flushes Nitrogen into selected bottle s This procedure may contaminate amino acids reagents solvents Click OK to proceed or click Cancel to return to the Bottle Preparations screen without back flushing If the bottle is not already vented the bottle will vent prior to back flushing 6 Solvent Back Flush Click the Solvent Back Flush button to back flush selected bottles with Solvent 1 DMF A SUser warning window will open SuUser U xl Warning This procedure back flushes Solvent into selected bottle s This procedure may contaminate amino acids reagents solvents 53 www peptideinstruments com Click OK to proceed or click Cancel to return to the Bottle Preparations screen without back flushing If the bottle is not vented the bottle will vent prior to back flush with Solvent 1 7 Pause Resume Click the Pause button to pause an active process When the action is paused the Pause button will be replaced by the Resume button Click the Resume button to continue the paused process
17. E Content i Contents Index Search File Manager Files of every type Amino Acid Solv Reag Program Sequence Synthesis are manipulated using the File Manager As an example the Amino Acid File Manager is shown below El e perations Menu El e Cleaning Amino Acid File Manager e Setting Up a Synthesis A File Manager will list all files of that type in a box to the left of the screen with functional buttons to the right To select a file click on its name to highlight it When a file is highlighted all buttons to the right will become activated The function of each button is as follows New The New button creates a new file 1 Click on the New button This will open a new window X x SE Ce DH The Contents tab organizes the help files into directories A purple book icon indicates directories while a sheet of paper with a question mark icon indicates individual help files Click on a file name or icon in the Contents list to display the corresponding help file in the window to the right Type in a keyword in the box at the top of the Index tab to find it and highlight it in the alphabetical list below Click the Display button at the bottom of the Index tab to choose from the list of help files containing the keyword Click the Display button in the new window to display the help file in the window to the right Type in a keyword in the box at the top of the Search tab and click the List Topi
18. File Apes ene Standard peration Volume ul Mix Time HMG Drain Reps Comment O 00 00 00 pm L The Program Name box lists the name of the currently open program file To open a different file select a different file from the pull down menu The Select Solvent Reagent File box lists the name of the solvent reagent file whose values determine what operations are available under the Operation column pull down menu see below To use a different solvent reagent file select a different file from the pull down menu In addition to the operations defined by the selected Solvent Reagent File the following operations may be selected 1 Cleave and Collect Delivers Solvent 8 mixes every 2 minutes for the specified time and drains to collection vial Rinses for 30 seconds with specified volume of solvent 8 for specified number of reps Cleave Mix Performs a mix every 2 minutes for the specified time Collect Drains RV contents to collection vial Rinses with specified volume of solvent 8 for specified mix time Repeats rinse for specified number of reps Drain Dry Drains RV contents and flushes RV with nitrogen for the specified mix time E Mail Notification Sends an email describing the instrument status to the address in the Settings screen See Section 4 3 Mix Mixes RV contents for the specified time 42 1 800 477 6834 7 Waste C
19. Filter Frit 3 16 Tube Pkg of 100 PPS AA SEP 54 Seal AA Bottle Pkg of 54 PPS CLV SEP 012 Seal Collection Vial Top Pkg of 12 CLV 050 030 Pkg of 30 CLV 050 090 Vial 50 mL Collection Pkg of 90 CLV 050 180 Pkg of 180 Catalog No Replacement Parts Quantity 1020023 Assembly Waste Cable 1 ea 0100096 Assembly Waste Tank 1 ea 3010001 Safety Coated Bottle 1 L Amber 1 ea 3010002 Safety Coated Bottle 4L Amber 1 ea 2700042 O Ring for 1 and 4 L Bottles 1 ea 2600187 Bottle Filter Housing 1 8 Tube 1 ea 2600262 Bottle Filter Housing 3 16 Tube 1 ea 3500041 Ferrule 1 16 Tube Blue 1 ea 3500043 Ferrule 1 16 Tube Clear 1 ea 124 1 800 477 6834 Catalog No Replacement Parts Quantity 3500004 Ferrule 1 8 Tube Yellow 1 ea 3500071 Ferrule 3 16 Tube Blue 1 ea 3500040 Fitting Nut 1 16 Tube PP 1 ea 3500061 Fitting Nut 1 8 Tube PP 1 ea 3500072 Fitting Nut 1 8 Tube PPS 1 ea 3500070 Fitting Nut 3 16 Tube PPS 1 ea 3510002 Fitting Nut 1 16 Tube Headless PPS 1 ea 3510001 Fitting Nut 1 8 Tube Headless PPS 1 ea 4830003 Fuse 4A 250VAC AGC Type 1 ea 4830001 Fuse 5A 250VAC AGC Type Fast Acting 1 ea 4830002 Fuse 2A 250VAC AGC Type 1 ea Catalog No Tools Quantity 1010030 Assembly Flow Meter amp Tubing 1 ea 6800023 Tool 3 16 Tube Fitting Nut 1 ea 6800027 Tool 1 8 Tube Headless Fitting Nut 1 ea 125 www peptideinstruments com 126
20. N Fmoc Protected Amino Acids Preweighed n 119 A 2 Bulk N Fmoc Protected Amino Acids Preweighed 120 A S Reagents amp OS une nete atte mousse nu meu 121 B Reagent Shelf Life amp Handling see 122 B Reagent Shelf Dt 122 B 2 Amino Acid Solubility Testing ANEN 123 B 3 Amino Acid Degradation Testing NENNEN 123 C Accessories Replacement Parts amp Tools 124 EE 127 vii www peptideinstruments com viii 1 800 477 6834 Introduction Thank you for purchasing your new Prelude peptide synthesizer from Protein Technologies Inc The Prelude is a fully automated peptide synthesizer with six reaction vessels that operate simultaneously Twenty seven amino acid bottles and eight solvent reagent bottles allow for maximum flexibility Extra features include automated cleavage Dynamic Sequence Programming E mail Notification Single Shot deliveries and Wash After Error 1 1 About The Manual In this manual e Chapter 1 General Information describes the instrument layout basic installation procedures and Prelude accessories available for purchase from Protein Technologies Inc e Chapter 2 Introduction to Software explains the basics of using the software e Chapter 3 Basic Synthesis Operations describes the basic procedures for setting up a synthesis on the Prelude e Chapter 4 Advanced Synthesis Operations amp Optional Features describes advanced features and options on the Pre
21. Preparations and select Solvent Calibration SS File Operations Tools Reports View Help ee Bottle Preparations RV Operations gt Special Bottles Cleaning IM Solvent Calibration This will open the Bottle Preparations screen with the Solvent Calibration tab active 56 1 800 477 6834 A Bottle Preparations x Bottle Preparations Special Bottles Solvent Calibration Select solvent for calibration Set Target Delivery Volume to volume being used during synthesis Set Number of Deliveries to increase precision reading collect volume Select In Place RVs for testing Press Run to start test deliveries Note Delivery timeouts and Prime errors may result from incorrect Actual Volume entries Restore Default ele er SA Restore default values and retry if calibration deliveries fail Solvent Bottle Test RV Actual Volume uL OS se so po wm soi wire Wie Wi 0 Copy To All RVs Target Delivery Volume uL 1000 Rv2 0 Number of Deliveries Expected Collect Volume ul Run Close The Solvent Calibration screen has the following buttons 1 2 6 Run Starts running a calibration Close Closes the window Cancel Cancels a running calibration Refactor Calculates new calibration factors based on Expected Collect Volume uL and Actual Volume uL values Save Factors Saves new calibration factors Click the Save Factors button when the Actual Volume
22. RVs to clean RV lines Section 2 5 3 8 Perform Rinse All Blocks to clear waste valve Section 2 5 3 6 RV sensor may require service contact Technical Service Dept NOT PRIMED Sensor does not sense fluid Go to Bottle Preparations screen and prime bottle Section 2 5 1 1 Try FILL ERROR Possible Action s NO PRESSURE The bottle required by the program is not pressurized Check nitrogen supply pressure gauge Go to Bottle Preparations screen to pressurize Section 2 5 1 1 TIME OUT Operation was not performed in the allotted operation time Press Start to continue with RV operations RV NOT IN PLACE An RV is removed or not in place when an operation is initiated Replace RV Section 1 2 3 and press Start COLLECTION VIAL NOT IN PLACE A collection vial is removed or not in place when a cleave or collect operation is initiated Replace the required collection vial Section 1 2 4 and press Start 117 www peptideinstruments com 6 2 Critical Errors No Operations Allowed The following table lists critical errors on the Prelude their cause and possible corrective actions to take The following errors will cause the Prelude to pause all operations immediately and vent all bottles If the problem persists after the suggested actions are taken please contact your PTI Technical Service representative at 1 800 477 6834 Error Cause Possible Acti
23. Remove all chemicals and resins from the Prelude before the System Clean because they will be contaminated with Solvent 2 2 5 3 2 Bottle Position Flush Bottle Position Flush flushes selected amino acid and or solvent reagent bottle lines with Solvent 1 DMF or nitrogen Bottle Position Flush should be performed when changing reagents or to clear a clog caused by amino acid precipitate in the line Bottle Position Flush is performed as part of a System Clean but it can also be performed alone as follows 1 Replace amino acid and or solvent reagent bottles to be flushed with empty bottles 2 Click on the Operations menu select Cleaning and then select Bottle Position Flush 74 3 4 5 6 1 800 477 6834 File Operations Tools Reports View Help g Bottle Preparations gt Qg RV Operations ls Cleaning d System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Mitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash Ris This will open the Bottle Preparations screen Bottle Preparations Special Bottles Solvent Calibration r Solvents __ Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed 1 ror EH 1 E Ala EHE 10 TF Leu un is T E ia 2 Moim EH 2 E gs DR nn Tue lU 2 ity FT 3 Fide ENS MH 3 E Aen PR 12 E Aen EN 2 2r Am ER 4 To m i 4 E Gl EHE 2 E Be EN
24. SMP 10 WBC Fmoc L Trp Boc OH 10 mmol SMP 20 WBC 20 mmol SMP 05 YB 5 mmol SMP 10 YB Fmoc L Tyr tBu OH 10 mmol SMP 20 YB 20 mmol SMP 05 V 5 mmol SMP 10 V Fmoc L Val OH 10 mmol SMP 20 V 20 mmol A 2 Bulk N Fmoc Protected Amino Acids Preweighed Catalog No Amino Acid Quantity B 25 A 25g B 100 A Fmoc L Ala OH 100g B 25 RP 25g B 100 RP Fmoc L Arg Pmc OH 100g B 25 RBF 25g B 100 RBF Fmoc L Arg Pbf OH 100g B 25 NT 25g B 100 NT Fmoc L Asn Trt OH 100g B 25 DB 25g B 100 DB Fmoc L Asp OtBu OH 100g B 25 CT 25g B 100 CT Fmoc L Cys Trt OH 100g B 25 EB 25g B 100 EB Fmoc L Glu OtBu OH 100g B 25 QT 25g B 100 QT Fmoc L Glin Trt OH 100g B 25 G 25g B 100 G Fmoc L Gly OH 100 g 120 Bulk N Fmoc Protected Amino Acids Preweighed cont 1 800 477 6834 Catalog No Amino Acid Quantity Send Fmoc L His Trt OH ids E Fmoc L Ile OH oe e Fmoc L Leu OH ee EE Fmoc L Lys Boc OH ws EUM Fmoc L Met OH 1005 uus Fmoc L Phe OH Ped Pd Fmoc L Pro OH 1005 BE Fmoc L Ser tBu OH 1009 EE Fmoc L Thr tBu OH ae ren Fmoc L Trp OH 0 ee Fmoc L Trp Boc OH E ie Fmoc L Tyr tBu OH d P D Fmoc L Val OH e A 3 Reagents amp Kits Catalog No Reagents Quantity PS3 PPR L 20 Piperidine DMF Dep 0 9L PS3 MM L 0 4 N Methylmorpholine DMF Act 0 9L B 100 HBTU 100g B 500 HBTU HBTU 500 g B 1K HBTU 1 kg B 100 HCTU 100 g B 500 HCTU HCTU 500 g B 1K HCTU 1 kg 121
25. Synthesis es 47 49 E Mail Notification 42 109 110 EIFOL ois ttes 18 56 62 72 119 120 Critical enseyi snis 16 18 120 Email On ESPERE 110 Recovery ae 119 Reporting Box 61 62 Wash After 91 111 E STOD tense tea ua ctas aM Idi 63 File Manager 31 35 Amino Acida ainsi 35 37 Program 31 32 37 40 41 43 44 Sequence 31 44 46 Solvent Reagent 38 40 Synthesis 31 47 49 50 File Menu 35 38 41 44 47 50 Flow Control e 18 101 Nitrogen Push 18 Gauge Bottle Pressure 17 102 119 Nitrogen Pressure 17 102 119 Nitrogen Tank 115 Vacuum u 18 101 102 120 Valve Pressure 17 102 GLP Dal es ee 36 39 Help Merit e 97 99 Help TOPICS et eoe 97 99 Installation Amino Acid Bottle 24 25 Collection Vial 23 24 Instrument ue ose e tee 20 Ee 21 Reaction Vessel 21 22 Solvent Reagent Bottle 25 26 Waste Container 26 Job Reports 88 94 96 Load Synthesis 60 64 65 101 128 Logln LogOut 88 89 Maintenance Cleaning and 113 COMP ULSI eec oio 113 114 ei PE 86 Manual Operations 31 60 63 70 72 85 Eeer 42 OlGaVD ioca P ADS 42 Flow Con
26. and fluid may remain in the lines 1 1 9 Ventilation System The Prelude has two 4 inch vent holes one on each side of the instrument It comes equipped with an adjustable angle adaptor for one hole and a vent cap for the other The adaptor has a tube fitting for attaching the waste container vent line The Prelude should be connected to lab ventilation with a 4 inch 10 cm duct supplied by the user The ducting should be made of a chemically resistant material PVC or urethane but no rubber A minimum flow of 50 cubic feet min CFM must be maintained at the instrument 1 1 10 Nitrogen System The nitrogen inlet is located on the utility panel A minimum of 70 psi must be supplied for the instrument to operate The lack of nitrogen is a critical error and the instrument will pause all operations vent all bottles and display an error message No operations will be allowed until the supply is restored The high pressure nitrogen is diverted into three regulators 1 Valve Pressure Used to seal the valve membranes Should be set to 30 40 psi User should not adjust 2 Nitrogen Pressure Used for mixing and delivering fluid Should be set to 5 psi User should not adjust 3 Bottle Pressure Used to pressurize the bottles Should be set to 9 psi Regulator is accessible through the side access panel CAUTION Timed delivery volumes from solvent bottles 1 4 and 8 are dependent on the Bottle Pressure setting Any
27. below Current Cycle displays the current cycle position Assign Sequence The box above the peptide sequence column lists the programs assigned to a given cycle of the synthesis Use the arrow keys on the keyboard or the arrow buttons on the screen to move to different amino acids in the sequence Then click on a program button D 1 2 or 3 in the Program Sets section to assign a program to the cycle If more than four programs are needed additional programs may be assigned using the numbered buttons To assign an additional program select a new program using the pull down menu next to one of the numbered buttons Programs previously assigned with that number will be represented by an X This process m ay be repeated to assign additional programs Only the latest program selection will be represented by the number All others will be represented with an X The buttons are 1 New The New button creates a new file The Synthesis File Manager will open Enter a new name or use the default and click OK or click the Cancel button to return to the Synthesis Editor without creating a new file Close The Close button is not active until the synthesis is saved After saving the synthesis click the Close button to exit the Synthesis Editor screen Cancel The Cancel button removes any changes made since the last Save A screen will appear reading Abandon All Changes The Yes button will permanently remove the changes
28. each of these screens will be reviewed in each subsection below 2 4 3 1 Program Editor Two programs are recommended to run a synthesis on the Prelude 1 Swelling Program Synthesis program with extended initial wash times to swell the resin during the first step 2 Synthesis Program If automated cleavage is desired a Cleavage Program is also necessary NOTE The Prelude software has standard swelling synthesis and cleavage program files called Standardsw Standard and StandardCleave respectively that can be opened and viewed by selecting a file and opening it using the Program File Manager See Section 2 3 These files are the standard programs installed by Protein Technologies Inc and are used for the synthesis of PTI s test peptides To open the Program Editor click on the File menu select Synthesis and select Program File Operation Tools Reports View Help Amino Acid A d Solvent Reagent DN Exit Sequence Synthesis The Program File Manager will open as a new screen To create a new program file click the New button enter a name for the new file and click OK Alternatively select an existing program file from the list and click the Open button The program file will open in the Synthesis Editor under the Program tab 41 www peptideinstruments com E Synthesis Editor NewName E x Program Sequence Synthesis Program Name Select Solvent Reagent
29. feeding one corner into the manifold using a DULL instrument or fingernail to prevent cutting or tearing the surface of the seal 3 The seal can then be turned and pushed into the manifold in small increments 4 The metal backing disk floats and can be pressed upward to allow entry of the seal 5 Replace filter housing and frit 5 2 7 Solvent Bottle Seal Replacement The solvent bottle seal consists of an encapsulated o ring seated in a bottle cap insert The o ring can be damaged if not handled properly and should be replaced if a nitrogen leak is noted Extra caution should be taken not to damage the insert when replacing the o ring To remove the o ring simply lift the o ring off the insert with your fingertip The protective gloves will assist in preventing damage to the inserts by cushioning against fingernail damage IMPORTANT Never use sharp or pointed objects to remove the o rings from the inserts Even small nicks may cause a nitrogen leak Never use a razor blade or knife to cut off the o rings CAUTION Always wear protective clothing safety glasses and gloves when working on bottle seals 116 1 800 477 6834 Chapter 6 Errors amp Recovery IMPORTANT It is important to manually drain all reaction vessels prior to resuming after an error When a synthesis is resumed following an error it will start at the beginning of the step not where it left off as in a regular pause Thus if react
30. immediately and cancels the synthesis CAUTION There may be fluid left in the reaction vessels and or lines after using the E Stop Use the Manual button to open the Manual Operations screen and run a Drain Dry and a DMF wash step if necessary to clean the synthesizer after an emergency stop and avoid contaminating the next fluid delivery NOTE The synthesis must be reloaded to resume after an E Stop Reload the synthesis using the Load Synthesis screen then select the starting cycle and step using the Set Start Cycle button on the RV Status screen The synthesis will start at the beginning of the step 5 Manual The manual button will open the Manual Operations screen See Section 2 5 2 5 63 www peptideinstruments com 2 5 2 2 Load Synthesis The Load Synthesis screen is located under the Load Synthesis tab in the Reaction Vessel Operations window It allows the user to view the details of a synthesis file load a synthesis file onto the RV Status screen and assign a cleavage program to a synthesis To open the Load Synthesis screen click on RV Operations under the Operations menu and select Load Synthesis File Operations Tools Reports View Help Bottle Preparations gt Inn RY Operations RV Status Cleaning Loa sis Calculations AA Calculations Solv Reag Manual Operations The Reaction Vessel Operations window opens with the Load Synthesis tab active Reaction Vessel Operations E
31. in the lower box The buttons are as follows 1 Set Start Cycle By default the synthesis will start at cycle 1 step 1 To change the starting cycle and step of the synthesis click on the Set Start Cycle button This will open a new window 62 1 800 477 6834 Cycle 1 Step 1 i X 1 Standard v Cycle 1 to 6 OK D DMF Top Wash Step 1 to 6 Cancel Use the upper pull down menu to select a starting cycle Use the lower pull down menu to select a starting step Click the OK button to accept the changes or click the Cancel button to return to the RV Status screen without changing the start settings After clicking OK a SUser screen will appear with the message This will set the synthesis to Cycle lt gt Step gt Are you sure Click the Yes button to continue or No to cancel 2 Start Click the Start button to start the synthesis 3 Pause Click the Pause button to pause the synthesis The synthesis will stop after the current step is complete to allow the manifold blocks to be washed This eliminates the problem of residual fluid in the lines contaminating the next fluid delivery Click the Start button to resume the synthesis 4 Cancel E Stop Click the Cancel button during a pause or before the synthesis is started to delete the synthesis from the RV Status screen While a synthesis is running the Cancel button is replaced with an E Stop button The E Stop button ends the synthesis
32. is highlighted the Print button becomes active b Click on the Print button This will open the Report Preview window with a preview of how the document will look when it is printed Use the magnifying glass at the top of the screen to view the document at different magnifications and the left and right arrows to navigate between pages x wf 18 dal all ai Program Report Name Standard ID 48 Last Mod ified 5 15 2006 9 56 02 AM Solvent Reagent File Standard Step Operation Solvent Volume MixTime Repeats Drain On l Deprotection 20 Piperidine DMF 1000 000250 2 Tme 2 DMF Top Wash Dinethylfomamide 1000 000030 6 Tme 3 AA Building Block Reagent 1000 000000 1 Fake Cancel c To print the file click the Print button or click on the printer icon at the top ofthe screen Alternatively click on Cancel to return to the File Manager screen without printing the file d Click on the X in the upper right corner of the window or click Cancel to close the Report Preview window 6 Delete deletes an existing file a Click on the desired file name to highlight it Once a file is highlighted the Delete button becomes active 34 1 800 477 6834 b Click on the Delete button This will open a new window with the message You are about to delete 1 file Are you sure c Click OK to delete the file or click Cancel to return to the File Manager screen without deleting the file 7 Clo
33. ready for editing 2 Open opens an existing file a Click on the desired file name to highlight it Once a file is highlighted the Open button becomes active b Click on the Open button c The File Editor for that file type will open with the file ready for viewing or editing 3 Rename assigns a new name to an existing file a Click on the desired file name to highlight it Once a file is highlighted the Rename button becomes active b Click on the Rename button This will open a new window Program File Manager x Rename File lt CopyOfStandard gt as seem Cancel c Enter the new name and click OK to rename the file or click Cancel to return to the File Manager without renaming the file 4 Save As creates acopy of an existing file a Click on the desired file name to highlight it Once a file is highlighted the Save As button becomes active b Click on the Save As button This will open a new window with the name CopyOfX where X is the name of the original file displayed as the default name for the copy Program File Manager x Save File Standard as Cancel 33 www peptideinstruments com c You may choose to keep this name or enter a different name Click OK to create a copy of the file or click Cancel to return to the File Manager screen without copying the file 5 Print prints a file a Click on the desired file name to highlight it Once a file
34. step move the cursor to a step and click the Insert button A new step will be inserted above that step 2 Add To add a step to the bottom of the program click the Add button 3 Delete To delete a step move the cursor to that step and click the Delete button 4 New The New button creates a new file The Program File Manager will open Enter a new name or use the default and click OK or click the Cancel button to return to the Program Editor without creating a new file 43 www peptideinstruments com 5 Close The Close button is not active until the program is saved After saving the program click the Close button to exit the Program Editor Screen 6 Cancel The Cancel button removes any changes made since the last Save A screen will appear reading Abandon All Changes The Yes button will permanently remove the changes while the No button will leave the changes 7 Save Once a change is made to the program the Save button becomes active Click Save to save the changes The Save and Cancel buttons will deactivate once the program is saved 8 Save As To copy the file on the screen click the Save As button The Program File Manager will appear with a default name for the copy Change the file name or accept the default and click OK The Cancel button will close the screen and return to the Program Editor 9 Print To print the open program file click the Print button It will go automatically t
35. the database as needed following a software upgrade If you encounter an error message pertaining to this function please contact PTI customer service at 1 800 477 6834 2 6 1 2 Archive All Data The Archive All Data function archives all synthesis data and compacts the database It is located in the Tools menu under Database The selection becomes active when a supervisor logs in Section 2 6 2 This function makes a back up copy of all data from syntheses run since the last Archive All Data was performed The Archive All Data command archives all the synthesis log data contained in the current database file into an archive file named after the database file and the archive date Name_YYYYMMDD mob It is recommended to archive data every 3 6 months depending on the number of syntheses run This makes the system more responsive In addition data should be archived prior to deleting old program sequence and synthesis files as part of regular file maintenance NOTE Halt all other instrument operations before running an Archive All Data To Archive All Data 84 1 800 477 6834 1 Click on the Tools menu and select Logln Enter the supervisor password and click OK 2 Click on the Tools menu select Database and select Archive All Data File Operations Tools Reports View Help a Pn Ki Database Rebuild Errors Table LogOut Archive All Data Settings Diagnostics Operation Times Operations 3 The following war
36. times are used to prevent reagents from sitting in the valve system for extended periods of time prior to being rinsed out RV Size 10 mL or 40 mL Adjusts calibration curve Wash After Error wash volume and maximum delivery volume based on RV size The maximum delivery volume is 10 000 pL for the 10 mL RV and 20 000 uL for the 40 mL RV The System Settings are as follows 1 Default Operation Times The contents of this file control the times associated with operations on the instrument The default setting is Standard The Prelude software must be restarted for changes to this setting to take effect Report File This is the file whose contents are displayed when the Reporter window is opened The default setting is MASTER USER mdb To select a different file click the Browse button 89 www peptideinstruments com NOTE System Settings are not accessible under supervisor logins and should not be changed by the user Improper changes can cause the instrument to malfunction Only PTI factory engineers should modify System Settings The function of the E Mail Options section is described in detail in Section 4 3 To save changes click the OK button and restart the Prelude software by clicking on the X in the upper right corner of the main screen then double clicking on the Prelude user software icon on the desktop To cancel changes click the Cancel button 2 6 4 Diagnostics The Diagnostic
37. 0 RP 20 mmol SMP 05 RBF 5 mmol SMP 10 RBF Fmoc L Arg Pbf OH 10 mmol SMP 20 RBF 20 mmol SMP 05 NT 5 mmol SMP 10 NT Fmoc L Asn Trt OH 10 mmol SMP 20 NT 20 mmol SMP 05 DB 5 mmol SMP 10 DB Fmoc L Asp OtBu OH 10 mmol SMP 20 DB 20 mmol SMP 05 CT 5 mmol SMP 10 CT Fmoc L Cys Trt OH 10 mmol SMP 20 CT 20 mmol SMP 05 EB 5 mmol SMP 10 EB Fmoc L Glu OtBu OH 10 mmol SMP 20 EB 20 mmol SMP 05 QT 5 mmol SMP 10 QT Fmoc L Glin Trt OH 10 mmol SMP 20 QT 20 mmol SMP 05 G 5 mmol SMP 10 G Fmoc L Gly OH 10 mmol SMP 20 G 20 mmol SMP 05 HT 5 mmol SMP 10 HT Fmoc L His Trt OH 10 mmol SMP 20 HT 20 mmol SMP 05 I 5 mmol SMP 10 I Fmoc L lle OH 10 mmol SMP 20 I 20 mmol SMP 05 L 5 mmol SMP 10 L Fmoc L Leu OH 10 mmol SMP 20 L 20 mmol SMP 05 KBC 5 mmol SMP 10 KBC Fmoc L Lys Boc OH 10 mmol SMP 20 KBC 20 mmol 119 Prelude Bottles Containing N Fmoc Protected Amino Acids Preweighed www peptideinstruments com cont Catalog No Amino Acid Quantity SMP 05 M 5 mmol SMP 10 M Fmoc L Met OH 10 mmol SMP 20 M 20 mmol SMP 05 F 5 mmol SMP 10 F Fmoc L Phe OH 10 mmol SMP 20 F 20 mmol SMP 05 P 5 mmol SMP 10 P Fmoc L Pro OH 10 mmol SMP 20 P 20 mmol SMP 05 SB 5 mmol SMP 10 SB Fmoc L Ser tBu OH 10 mmol SMP 20 SB 20 mmol SMP 05 TB 5 mmol SMP 10 TB Fmoc L Thr tBu OH 10 mmol SMP 20 TB 20 mmol SMP 05 W 5 mmol SMP 10 W Fmoc L Trp OH 10 mmol SMP 20 W 20 mmol SMP 05 WBC 5 mmol
38. 01 102 Synthesis Checklist 101 EGO era 47 49 File 32 47 48 50 64 86 93 95 101 114 File Manager 31 47 49 50 Load 60 64 65 101 System PARIS aa ee 20 Amino Acid Bottle 13 14 116 Clean 15 72 74 76 78 84 103 113 114 Cleavage Collection 13 eu TEE 18 EO nico cera ERC 120 Nitrogen 17 115 120 Reaction Vessel 13 Setting Sa EM 91 Solvent Reagent Bottle 14 115 VACUUM EE 18 102 Ventilation 17 19 NEE 16 Tools Menu 85 86 88 89 USB Porta sn 18 20 88 www peptideinstruments com Utility Panel 11 12 17 18 20 26 ATTEN 97 102 Wash Vacuum Alter EON rennen 91 111 Gauge icit 18 101 102 120 RVs 15 72 84 85 101 103 113 Die sis eoe re 12 18 114 119 ln le ee 18 120 Waste System sa E 18 102 CONST ut D prin 43 Valve Container 16 17 26 27 29 103 Gei 15 18 Installation c e 26 Nitrogen Tank 115 Level Sensor 16 17 20 26 102 Pressure noian 17 51 120 E re BEE 17 102 POF au 12 26 Replacement 113 114 ON ee ae 12 NV ASICs nn n D RE 21 119 Syste EG 16 Ventilation System 17 19 Venta 17 20 130 info peptideinstruments com www peptideinstruments com Worldwide 4675 South Coach Drive Tucson AZ 85714 U S
39. 1 2 1 Laboratory Requirements In order to install and run the Prelude a laboratory must be able to supply the following 1 Ventilation System The Prelude has a 4 inch vent equipped with an adjustable angle adaptor The Prelude should be connected to a lab ventilation system with a 4 inch 10 cm duct supplied by the user The ducting should be made of a chemically resistant material PVC or urethane but no rubber A minimum flow of 50 cubic feet min CFM must be maintained at the instrument 2 Nitrogen Supply A relatively pure 299 996 and dry source of pressurized nitrogen is recommended The system uses nitrogen for solution transfers and agitation mixing Alternative gases can be utilized if desired e g Argon The user must supply all necessary regulators and nitrogen tanks One male and one female 1 4 NPT fittings are provided with the unit to connect it to the tank IMPORTANT Securely fasten cylinders with safety straps to prevent them from falling and do not move a cylinder or undo safety straps unless the safety cap is in place 3 Secondary container for waste recommended The supplied 5 gallon waste carboy should be placed in a secondary container that is resistant to the harsh chemicals in the waste The 19 www peptideinstruments com capacity of the secondary container should be enough to contain a 5 gallon spill Memory Stick optional Files may be transferred from the Prelude compute
40. 22732 FR 5 Bae ER MI 5 Ere IN Mm 14 Toh IN D 232F 53 N Mm 6 Tick DN NU 6 E Gy MM mp E Ae pH Mat 224 ENG 7 Tse EN fi 7 THis PR 16E Se DR 32045 ENG 8 irs Ni PN 8 Eis BH i7 T Fi war Aa Ey 9 TT Lys BH 18 five pu et Ag pn Clear All Select All Back Flushing Back Flush DCM bottle Eressunze Prime Ment Nitrogen Back Flush Pause Cancel Close If a Solvent Back Flush will be performed Solvent 1 DMF must be pressurized and primed first A Nitrogen Back Flush operation does not require Solvent 1 DMF to be pressurized and primed To pressurize and prime Solvent 1 DMF check the box next to Solvent 1 DMF by clicking in it Click the Pressurize button When complete select Solvent 1 again Click the Prime button Check the box es next to the bottle s that will be flushed To back flush bottle s with Solvent 1 DMF click on the Solvent Back Flush button To back flush bottle s with nitrogen click on the Nitrogen Back Flush button 75 www peptideinstruments com NOTE When changing reagents it is suggested to perform a Nitrogen Back Flush to flush reagent back into the bottle Replace the bottle with an empty bottle and perform Solvent Back Flush to flush residual reagent from the line Wipe excess fluid off the bottle tubing and load the new reagent bottle When trying to loosen a clog remove bottle filter and use a Solvent Back Flush NOTE Different flushing solvents may be used by placing them in t
41. 4 Program Operation Names Names used to describe program operations These operation names are displayed in programs on the synthesis log and in the Manual Operations screen a Bottom Delivery Solvent or reagent is delivered through the bottom of the RV b Top Wash Solvent is delivered through the top of the RV NOTE Only Solvents 1 amp 2 can be delivered through the top of the RV 5 GLP Data a Vol mL Volume used for synthesis in mL b Conc mM Concentration of reagent solution used for synthesis mM c Opened Date Date and time solution was opened d Lot Number Lot number e Source Number Source information catalog number company etc By default a new file contains the standard solvents and reagents Double click in a cell to modify the value The buttons are 1 New The New button creates a new file The Solvent Reagent File Manager will open Enter a new name or use the default and click OK or click the Cancel button to return to the Solvent Reagent Editor without creating a new file 2 Close The Close button is not active until the file is saved After saving the file click the Close button to exit the Solvent Reagent Editor screen 39 www peptideinstruments com 3 Cancel The Cancel button removes any changes made since the last Save A screen will appear reading Abandon All Changes The Yes button will permanently remove the changes the
42. 9 Bottle Pressure Gauge 102 119 Bottom Delivery 39 Calculations AA 60 65 67 70 125 Solv Reag 60 67 70 Calibration 41 51 56 57 59 102 113 Circuit Breakers 12 20 Cleaning 13 15 72 85 103 114 119 and Maintenance 113 Operations 15 Clear All Blocks 72 82 84 114 Cleavage 65 101 103 Automated 41 105 Collection System 13 Program 41 64 66 101 105 Solution 23 76 78 114 Cleave and Collect 16 42 51 Bottle na nee 15 Back Flush Nitrogen 72 76 78 79 103 114 Solvent 15 72 76 78 101 103 106 113 114 Back Flush 13 15 72 79 81 101 103 106 113 114 Cleave and Collection Vial 11 23 24 28 42 126 Installation nities tien Gen oder 23 COMM Me D 88 97 Computer Maintenance 113 114 SOY SOM een wert ee potete 18 Data Archiver 86 87 Database File 86 88 91 93 114 Deprotectant 14 15 124 DIAQHOSICS EN NN NEN 92 www peptideinstruments com De UE 42 43 71 72 85 96 Dynamic Sequence Programming 48 106 108 Editor Amino Acid 35 37 95 Program ei 40 43 44 Sequence sss 44 46 Solvent Reagent 38 40 95
43. Amino Acids amp Reagents for Peptide Synthesis Protein Technologies Inc supplies high quality pre tested N Fmoc protected amino acids preweighed in 5 mmol 10 mmol and 20 mmol quantities in synthesizer ready bottles see Appendix A 1 for listings as well as bulk N Fmoc protected amino acids preweighed in 25 g and 100 g quantities See Appendix A 2 for listings We recommend using our amino acids for all of your synthesis needs Protein Technologies Inc also supplies reagents for peptide synthesis on the Prelude See Appendix A 3 for listings 28 1 800 477 6834 1 3 5 Replacement Parts amp Additional Accessories Protein Technologies Inc supplies a full line of replacement parts for the Prelude as well as various accessories including solvent reagent bottles and waste containers A partial listing of replacement parts and accessories is located in Appendix C For additional part and accessory information please call our customer support desk at 1 800 477 6834 29 www peptideinstruments com 30 1 800 477 6834 Chapter 2 Introduction to Software This Chapter covers the components of each screen in the software The Prelude software was designed to mimic the Windows format to ease customer learning however there are some differences The main difference is the File Manager screen which is explained in Section 2 3 2 1 Main Menu The Main Menu is located at the top of the main SUser window and contains
44. Bottles Solvent Calibration Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed r Solvents T Ala Leu Trp Cys Met E Tw Asp Asn A21 T Glu Pro A22 Phe Gin A23 Gl Ag 424 His Ser A25 Ile Thr A26 Lys Val IN 427 Clear All Select All Pressurizing bottles done _Bressuize Prime Vent Nitrogen Back Flush Solvent Back Flush Pause Cancel Close 1 DMF 2 v DCM 3 M Dep 4 Cap 5 M Base 6 7 8 Act2 TFA oo oa OC 4 t nn The Solvents section lists the 8 solvent reagent positions while the Amino Acids section lists the 27 amino acid positions Select a position by clicking in the box to the right of a position number This will place a check mark in the box Click again to uncheck the position NOTE The Bottle Preparations screen is set to display the abbreviations from the amino acid and solvent reagent files entitled Standard To change the labels next to each bottle position change the abbreviation for that position in the amino acid or solvent reagent file entitled Standard To set the Bottle Preparations screen to display abbreviations from a different amino acid or solvent reagent file the default file name must be changed in the Settings screen See Section 2 6 3 The Pressurized columns indicate the pressurization status Y
45. Department if alternative solvents are desired Under no circumstances should TFA be used in the amino acid manifold system destruction of the seals will occur See Section 5 2 6 Amino Acid Bottle Seal Replacement for replacement procedures 1 1 7 Solvent Reagent Bottle System The eight solvent and reagent bottles are located outside the unit in a bottle container and are attached to the Prelude via the solvent feedthrough panel These glass bottles are pressurized with nitrogen to accomplish solution transfer For the safety of the user safety coated glass bottles should always be used The solvent and reagent bottles are controlled using the Bottle Preparations screen Section 2 5 1 1 CAUTION Safety coated glass bottles are supplied by PTI with each instrument and should always be used with this instrument Using regular glass bottles may result in serious bodily injury Bottle positions 1 4 are intended for solvents and volumes are measured out by timed deliveries These positions are appropriate for the primary and secondary wash solvents deprotectant and capping reagent Bottle positions 5 7 are intended for reagents and can precisely measure volumes in 150 500 and 1000 uL aliquots using a metering loop These positions are appropriate for coupling solutions Bottle position 8 is specifically intended for the delivery of cleavage solution Volumes for bottle 8 are measured by timed deliveries and upon draining from th
46. MMAN nee ee 95 Swellnd ini 41 Reaction Vessel 10 11 27 126 Installation 21 22 Operations 31 60 61 64 65 67 119 Syste M ee 13 Reagent sis eicere 28 123 Shelf Life amp Handling 124 Rebuild Errors Table 85 Replacement Parts 29 126 Reporter Window 91 92 96 Reports Ele iu oin E P eee Dus 91 MOD at oe reuera 88 94 96 Meriti ae 88 92 Preview 34 96 Rinse All Blocks 15 72 81 83 114 119 RV Statlis acest 16 60 65 70 Safety Shield 11 19 Sequence ECOL ceres re ire 44 46 File 32 37 40 44 46 68 86 94 101 114 File Manager 31 44 46 Set Start Cycle 61 62 SONIS ciscus a 42 52 87 92 Shortcut Buttons nen 31 Shutdown 103 114 120 Single Shot Gei sis Dec ease 110 126 RI E 54 56 110 Solvent Back Flush 53 75 76 103 Cleave Bottle 15 72 76 78 101 103 106 113 114 Calibration 41 51 56 59 102 113 Feedthrough Panel 11 12 14 20 26 Solvent Reagent Bottle 9 39 51 54 55 69 73 95 Installation onis 25 26 Seal Replacement 113 118 SVSIBTIBa ee ero ere 14 115 Editor oic c e s 38 40 95 File 32 38 40 42 48 52 69 101 File Manager 38 40 Special Bottles 16 41 51 54 56 114 Sf g o Me NN NN 1
47. No button will leave the changes 4 Save Once a change is made to the file the Save button becomes active Click Save to save the changes The Save and Cancel buttons will deactivate once the file is saved 5 Save As To copy the file on the screen click the Save As button The Solvent Reagent File Manager will appear with a default name for the copy Change the file name or accept the default and click OK The Cancel button will close the screen and return to the Solvent Reagent Editor 6 Print To print the open solvent reagent file click the Print button It will go automatically to the printer NOTE To view the program before printing click the Close button and preview the file using the Print button in the Solvent Reagent File Manager Section 2 3 NOTE The Prelude software comes with a standard solvent reagent file that can be copied opened or viewed by selecting the file Standard using the Solvent Reagent File Manager See Section 2 3 The Standard file contains standard solvents and reagents for Fmoc chemistry 2 4 3 Synthesis Editor To open the Synthesis Editor screen click on the File menu and select Synthesis File Operations Tools Reports View Help Amino Acid A n Solvent Reagent Synthesis Program Sequence Synthesis 40 1 800 477 6834 Then select from one of the following three screens 1 Program 2 Sequence 3 Synthesis The function of
48. RVs AND COLLECT VIALS MUST BE IN PLACE 3 This procedure may contaminate amino acids reagents solvents System cleaning can take more than 1 hour to complete Press OK to start and Cancel to cancel Cancel 5 During the System Clean the Bottle Preparations screen will open 73 www peptideinstruments com Bottle Preparations E E x Bottle Preparations Special Bottes Solvent Calibration r Amino Acids Solvents 1 Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed 1 FE is N F Leu H IER ipft 2 Tale hi 7 Met ER 20 Tw 3 E Asp n E Asn H A AEA d Glu ER E Pro HM 2 AT A 5 Phe d E Gin H 23 AT A23 6 7 8 9 F Gy N E Ata H 24 aT 524 His N MT Ser H 25 aT 25 E je N pF Thr N 26 Af Ae E Ly N T Val N a7 AT A27 o c co OC e w N VSEE EN il m Pos ei Priming DCM System Clean Pause Cancel The status of the operation will be displayed in the status bar Click the Pause button to pause the operation Click Resume to resume a paused operation Click the Cancel button to cancel the operation Click the Close button to close the Bottle Preparations screen 6 After the cleaning operation is complete remove the bottles and collection vials dispose of the rinse solution and replace the bottles and collection vials with clean ones for the next synthesis NOTE The System Clean takes over 1 hour to complete CAUTION
49. adjustments made to this regulator will require reverification of Solvent Calibration Section 2 5 1 3 Each pressure is displayed on pressure gauges on the front of the instrument The intensity of the mixing and fluid deliveries are controlled using the following two flow controls 17 www peptideinstruments com 1 Mix Flow Control Controls the nitrogen flow during a mix Control is located on the front of the instrument 2 Nitrogen Push Flow Control Controls the nitrogen flow during fluid delivery Control can be accessed through the side access panel CAUTION The Nitrogen Push Flow Control is factory set Adjusting the Nitrogen Push Flow Control will affect the way the instrument delivers fluid to the reaction vessels If it is adjusted too low it may cause the instrument to fail in its delivery and error out If you feel an adjustment is needed please contact a PTI customer service representative for proper instructions prior to adjusting this control IMPORTANT Adjusting the mixing or delivery flows too high can cause resin to stick to the top of the reaction vessels and possible reagent loss This can lead to incomplete reactions 1 1 11 Vacuum System Vacuum is supplied by a vacuum pump located inside the instrument Alternatively the instrument can be hooked up to an external laboratory supply through the vacuum inlet located on the utility panel see Section 1 1 3 The vacuum is display
50. als and work up peptides or cleave peptide from resin if on instrument cleavage was not performed 2 Perform a Wash RVs Section 2 5 3 8 3 Perform a Bottle Position Flush Section 2 5 3 2 on all bottles used in the synthesis First perform a Nitrogen Back Flush to flush reagent back into the bottles Replace used bottles with empty bottles and perform a Solvent Back Flush to flush residual reagent from the lines 4 If a cleavage was performed do a Collect Back Flush Section 2 5 3 5 and a Cleave Bottle Nitrogen Back Flush Section 2 5 3 4 Then replace Solv 8 bottle with an empty bottle and perform a Cleave Bottle Solvent Back Flush Section 2 5 3 3 5 Discard store or reuse used chemicals 6 Empty the waste container 7 If the instrument will not be used immediately shutdown the instrument Section 3 4 3 4 Instrument Shutdown It is not necessary to shutdown the Prelude following each synthesis Instrument shutdown is only necessary if the instrument needs to be moved or if the instrument will not be in use for an extended period To shutdown the Prelude 1 Perform a System Clean Section 2 5 3 1 then Nitrogen Back Flush all bottles using the Bottle Preparations screen Section 2 5 1 1 2 Empty all amino acid and solvent reagent bottles of fluid 3 Empty the waste container 4 Close the SUser software 5 Shutdown the computer by selecting Shutdown from the Start menu 101 www peptideinstrumen
51. base or an archived database file click on the button This will open the Open window Look in 3 Archive D ex E3 2 MASTER_USER_20060227 mdb 2 MASTER_USER_20060309 mdb 4 USER2_20060202 mdb 4 USER3_20060206 mdb Filename USER1 20060201 mdb Files of type Access Database Files mdb DI Cancel 91 www peptideinstruments com Go to C PTI Prelude Data Archive and select an archive file Click Open The columns of the job table are labelled as follows 1 Number Job number 2 Synthesis Name of the Synthesis file 3 Date Date and time synthesis was run To select a job file click on and highlight the job number listed in the Number column In the Include In Report section select the information that will be displayed in the job report Select from the following 1 Job Summary Displays the facility machine job number date and synthesis name as well as a table summarizing the following a RV RV number b Name Name of sequence file c Sequence Amino acid sequence of peptide d MW g mol Molecular weight in g mol of peptide e CONH2 Terminal group on C terminus of peptide Yes for CONH2 No for COOH 2 Job Details Displays the cycle job number program and amino acid added to each RV in the cycle as well as a table summarizing every software operation executed for each cycle The columns are a Step Program step b Ac
52. cept Fmoc Cys Trt OH 20 25 C 7 14 Days Fmoc Cys Trt OH in DMF solution 20 25 C 1 Day N N Dimethylformamide 20 25 C Stable Methylene Chloride 20 25 C Stable 2096 Piperidine DMF v v 20 25 C Stable Acetic Anhydride 20 25 C Stable 0 1M HBTU 0 4M 4 Methylmorpholine in DMF 20 25 C 5 7 Days Trifluoroacetic Acid 20 25 C Stable TFA Cocktail 20 25 C 1 Day 122 1 800 477 6834 B 2 Amino Acid Solubility Testing Supplies Required e Sealable vials or bottles 2 5 mL volume e Amino acids e Solubilizing solvent i e DMF or NMP Protocol Determine the concentration of amino acid to be used on the Prelude instrument In the Prelude software open the Calculations AA screen Section 2 5 2 3 Calculate the amount of amino acid to dissolve in 2 5 mL of solvent to create a solution at that concentration Prepare the solution and mix or sonicate until powder is fully solubilized Seal vial and place on a shelf with similar temperature and light conditions as would be experienced on the instrument Examine the vials daily for discoloration or precipitation and record the data Amino acids should not be left on the instrument longer than the time it takes for discoloration or precipitation to occur NOTE This test should use the same batch of solvent that will be used on the instrument B 3 Amino Acid Degradation Testing Supplies Required e Amino acid vials used for solubility study e HPLC gradien
53. ck the Cancel button to cancel the operation Click the Close button to close the Bottle Preparations screen 77 www peptideinstruments com 5 After the cleaning operation is complete empty solvent bottle 8 of any rinse fluid and replace with a clean solvent bottle for the next cleavage 2 5 3 5 Collect Back Flush The Collect Back Flush cleaning procedure flushes Solvent 2 DCM through the cleave system and collection lines into the collection vials to remove TFA solution and any residual peptide from the system Collect Back Flush should be performed after every collection to prevent contamination of the next synthesis product Collect Back Flush is performed as part of a System Clean but it can also be performed alone To perform a Collect Back Flush 1 Install empty collection vials to receive the rinse solvent 2 Pressurize and prime Solvent 2 DCM using the Bottle Preparations screen See Section 2 5 1 1 3 Click on the Operations menu select Cleaning and then select Collect Back Flush File Operations Tools Reports View Help g Bottle Preparations gt t RV Operations via Cleaning System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Mitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash Bus 4 The following warning window will open Verify the empty collection vials are in place and click OK Click the Cancel button to cancel the Co
54. cording to the database file name and archive date Name YYYYMMDD mdab NOTE t is also recommended to copy the current mdb database file to an external location as a backup Save to a memory stick inserted in one of the USB ports then transfer to another computer where the file may be saved to the hard drive or other external drive To print a job report from an archived file see Jobs Section 2 7 1 2 6 2 Logln LogOut The Logln LogOut feature prevents unauthorized users from accessing certain functions under the Tools menu The Archive All Data Section 2 6 1 2 function and the Settings Section 2 6 3 screen are only accessible with a supervisor password while the remaining selections under the Tools menu and Comm Log under the Reports menu may only be accessed with a PTI 86 1 800 477 6834 technician or factory password For general use access to the Tools menu is not required to run the instrument To LogIn 1 Click on the Tools menu and select Logln File Operations Tools Reports View Help up m Settings Diagnostics Operation Times Operations 2 The following window will open Enter Password for Diagnostics access 3 Enter the appropriate password and click OK Click Cancel to cancel the login To LogOut Click on the Tools menu and select LogOut File Operations Tools Reports View Help Si Pri emm Settings Diagnostics Operation Times Operations 2 6 3 Setting
55. cs button to display a list of the help files containing the keyword Click the Display button at the bottom of the Search tab to display the help file in the window to the right The icons are as follows 1 Hide Show Click the Hide icon to hide the Contents Index Search tabs Click the Show icon to show the tabs 96 1 800 477 6834 2 Back Click the Back button to return to the previously viewed help file 3 Print Click the Print button to print the currently open help file If the file has been selected from the Contents tab the following window will open Print Topics x You can print the selected topic or all the topics in the selected heading What would you like to do Print the selected topic Print the selected heading and all subtopics i Cancel This window gives the user the option to print the individual help file or all the help files under the selected heading Select an option and then click OK to print the file from the Print screen Click Cancel to return to the Help Topics window without printing 4 Options Click the Options icon to select from the following a b Hide Show Tabs Hides Shows the Contents Index Search tabs Back Returns to the previously viewed help file Forward If the Back button was pressed returns user to the previously viewed help file Home Returns the user to the Help Topics Home page Stop Stops the process of opening the help fil
56. e Refresh Restarts the process of opening the help file Internet Options Allows the user to set the Internet options Print Prints the currently open help file Search Highlight On Off Highlights the search term in the Help file s 97 www peptideinstruments com 2 9 2 About SUser To open the About SUser information box click on the Help menu and select About SUser File Operations Tools Reports View Help Help Topics KA Pi You Be iw Fe About SUser This will open the About SUser information box About SUser x Version 1 1 0 89 Compiled 9 8 2006 8 41 21 Location C PTI SSPP SUser exe Instrument Virtual 1 1 0 20 Sep 5 2006 Prelude Copyright C 2003 2004 Version numbers for Prelude User and Instrument software will be displayed 98 1 800 477 6834 Chapter 3 Basic Synthesis Operations 3 1 Synthesis Checklist Steps for running a synthesis without cleavage NC or with cleavage C are shown in the table below See Section 4 1 for more information about automated cleavage A zZ 9 Startup amp Instrument Check Turn on the Prelude Peptide Synthesizer Section 3 2 Check nitrogen supply and gauges Sections 3 2 amp 1 1 10 Check vacuum gauge Sections 3 2 amp 1 1 11 Check waste level Sections 3 2 1 1 8 amp 1 2 7 Change RV upper and lower o rings if necessary every 2 weeks Section 1 2 3 Change bo
57. e F Gly E His F Leu WEN Uff F Met i 20 F Ty Asn i 21 2r 22 FE Pe i 22 aT 22 E Gin i 23 AT 423 E Arg i 24 AT 424 I Ser Z ef As F lle pF Thr i 26 AT 426 E es E va i 2 p 427 Select All Select All E DMF E DCM Dep F cap Base E Acti Act F TFA p on mo c Qg N 333933333 Washing Top Wash Pressurize Vent Nitrogen Back Flush Solvent Back Flush Pause Cancel The status of the operation will be displayed in the status bar Click the Pause button to pause the operation Click Resume to resume a paused 83 www peptideinstruments com operation Click the Cancel button to cancel the operation Click the Close button to close the Bottle Preparations screen CAUTION If the procedure is cancelled there may be fluid left in the RVs lines or blocks Repeat the operation or perform a Drain operation using the Manual Operations screen See Section 2 5 2 5 to remove any residual fluid 5 After the cleaning operation is complete remove the reaction vessels and replace with clean resin filled reaction vessels for the next synthesis 2 6 Tools Menu 2 6 1 Database Synthesis data and other information are saved in a database The two functions available to help maintain this database are described in the following sections 2 6 1 1 Rebuild Errors Table The Rebuild Errors Table function is available for technician use only It is used to reconfigure
58. e reaction vessel are transferred to the collection vials Throughout the Prelude software program each bottle position is referred to by an abbreviated title These assignments may be changed in the Solvent Reagent Editor Section 2 4 2 The titles standard abbreviations bottle volumes and typical solution composition of each bottle position for Fmoc chemistry are as follows SOLV 1 or DMF Two daisy chained 4 L safety coated glass bottles for the primary wash solvent typically reagent grade DMF DMA or NMP may also be used Because this solvent is quite stable the 4 L containers 14 1 800 477 6834 may be installed and left in place throughout several sets of syntheses This solvent is utilized in the automated cleaning operations for the valve fluid system Therefore SOLV 1 must be in place for normal operation of the instrument This solvent position is also utilized during the Bottle Position Flush Section 2 5 3 2 Rinse All Blocks Section 2 5 3 6 and Wash RVs Section 2 5 3 8 cleaning operations SOLV 2 or DCM 1 L safety coated glass bottle for a secondary wash solvent such as DCM to wash the peptide resin in preparation for automated cleavage This bottle position is utilized during the System Clean Section 2 5 3 1 Cleave Bottle Solvent Back Flush Section 2 5 3 3 and Collect Back Flush Section 2 5 3 5 cleaning operations DCM may be installed and left in place for several sets of syntheses SOLV 3 or D
59. each RV The Prelude software calculates this amount based on the values in the Sub mmol g and Scale umol columns 68 1 800 477 6834 7 Yield mg Displays the theoretical amount of peptide in mg expected from the synthesis The Prelude software calculates this amount based on the value in the Scale umol column The Solvents Reagents section calculates the minimum volume of solvent or reagent needed for each of the 8 solvent reagent bottle positions The columns are labelled as follows 1 Pos Displays the bottle position number 2 Description Displays the full name of the solvent or reagent based on the solvent reagent file associated with the selected synthesis 3 Calc Vol mL Displays the calculated minimum volume in mL necessary for the selected synthesis 4 Suggest Vol mL Displays the suggested volume in mL to use for the synthesis The user may change the value in this column The Activators section calculates the volume and or weight of various activators needed for the activator solutions The columns are 1 Description Displays the name of the activator as entered in the solvent reagent file associated with the selected synthesis 2 MW g mol Displays the molecular weight in g mol of the activator as entered in the solvent reagent file associated with the selected synthesis 3 Density g mL Displays the density in g mL of the activator as entered in the solve
60. ecial Bottles 3 Solvent Calibration The function of each of these screens will be reviewed in each subsection below 50 1 800 477 6834 2 5 1 1 Bottle Preparations The Bottle Preparations screen is located under the Bottle Preparations tab in the Bottle Preparations window It allows the user to pressurize prime vent and back flush the 8 solvent reagent bottles and the 27 amino acid bottles Each solvent reagent bottle has a separate pressure valve while amino acid bottles 1 9 10 18 and 19 27 share common pressure manifolds Each solvent and amino acid bottle is primed separately so it is not necessary to keep solutions in unused bottles during a synthesis Position 8 is the only position available for the Cleave and Collect operation while positions 1 and 2 are used specifically for the primary and secondary wash solvents that are also used in certain cleaning operations The Bottle Preparations screen opens when the Prelude software is opened To open the Bottle Preparations screen click on the shortcut button or click on the Operations menu and select Bottle Preparations File Operations Tools Reports View Help i Bottle Preparations A Bottle Preparations RY Operations gt Special Bottles Cleaning gt Solvent Calibration This will open the Bottle Preparations screen with the Bottle Preparations tab active 51 www peptideinstruments com A Bottle Preparations E E x Bottle Preparations Special
61. ect a synthesis The Cleave Program box displays the current cleavage program Use the pull down menu to select a cleavage program or leave the box blank When a cleavage program is displayed calculated values will correspond to both the selected synthesis and the selected cleavage program The Amino Acid Concentration mM box allows the user to input a solution concentration in mM When the ReCalc button is pressed amino acid weights will be calculated based on this value and the Suggest Volume mL value explained below The Amino Acids section displays a table of the 27 amino acid bottle positions The columns are labelled as follows 1 Pos Amino acid bottle position 2 Description Displays the 1 letter abbreviation and full name of the amino acid or other chemical based on the amino acid file associated with the selected synthesis 3 Res Displays the number of times the amino acid solution will be delivered during the synthesis 66 1 800 477 6834 4 Calc Volume mL Displays the calculated minimum volume in mL necessary for the synthesis CAUTION It is not recommended to use less than the minimum volumes Reagent bottles may run out of fluid during the synthesis 5 Suggest Volume mL Displays the suggested volume in mL to place in the bottle at the start of the synthesis The user may change the value in this box 6 Weight mg Displays the amount of dry amino acid in mg needed for
62. ective clothing safety glasses and gloves when working on the filter assemblies Replacement Procedure 1 From the Bottle Preparations screen Section 2 5 1 1 select the positions that need replacement filters 2 Press the Nitrogen Back Flush button to blow out the reagent from the lines and filter housing 3 When the operation is complete remove bottle s and wipe exterior reagent off the filter assembly 4 Unscrew the filter assembly from the tube and remove filter frit from filter housing 5 Clean and rinse housing with desired solvent and dry 6 Install new frit by pressing housing over frit 7 Screw filter assembly back onto tubing IMPORTANT When installing the filter assembly onto the tubing be sure the tube is threaded into the filter housing as far as it will go to prevent nitrogen bubbles from being introduced when the reagent level goes below the top of the filter housing 115 www peptideinstruments com NOTE To expedite the replacement procedure it is best to have extra filter assemblies The clean filter assembly can be used and the dirty filter can then be cleaned while the instrument is running 5 2 6 Amino Acid Bottle Seal Replacement The amino acid bottle seal should be replaced annually or as needed 1 To remove use forceps or tweezers to grab the seal and pull it out of the manifold 2 To replace remove filter housing then put new seal over tube and start by
63. ed on the vacuum gauge on the front of the instrument The normal operating range is 10 22 in Hg When the vacuum drops to 10 in Hg the vacuum pump will turn on The vacuum is diverted directly to the valve blocks and is used to lift the valve membranes to allow fluid flow from different locations The lack of vacuum is a critical error and the instrument will pause all operations vent all bottles and display an error message This occurs when the vacuum pump fails to bring the vacuum gt 10 in Hg No operations are allowed until the vacuum is restored 1 1 12 Computer System The Prelude has an internal computer that operates the Prelude instrument and user software A monitor keyboard mouse and USB printer are supplied with the Prelude They are connected to the Prelude s internal computer via the utility panel A second USB port allows data to be transferred from the computer via a user supplied memory stick 18 1 800 477 6834 1 1 13 Safety Shield Safety doors are installed for the protection of the user The doors of the safety shield MUST be CLOSED when the Prelude is running Before opening the doors all of the reaction vessels must be in a non operational state i e paused or completed and drained of all fluids IMPORTANT Minimum safety equipment to be used at all times are NIOSH MSHA approved respirator face shield chemically resistant gloves and other protective clothing 1 2 Instrument Setup
64. ed to a fitting on the 4 vent duct The waste container is a 5 gallon carboy fitted with a waste level sensor to prevent overfilling If the waste container is full all operations in the instrument will stop automatically and all the bottles will vent No operations will be allowed until the container is emptied and reconnected IMPORTANT The waste container being full is a critical error on the Prelude To prevent overfilling the instrument will automatically pause all operations and vent all bottles To resume operations first empty and reconnect the waste container then re pressurize and prime all the bottles using the Bottle Preparations screen Section 2 5 1 1 Go to the RV Status screen Section 2 5 2 1 and press the Start button to continue the operations on the paused reaction vessels The waste level sensor is wired in a normally closed NC configuration so if the switch is disconnected it is the same as if the container is full This logic prevents waste from being delivered when the container is not connected The connectors are resistant to the aggressive waste solutions Do not attempt to 16 1 800 477 6834 disassemble the switch connector assembly lt is not a field service item and damage may occur CAUTION Be sure to backflush bottles before removing the waste level sensor connector If the sensor connector is removed from the waste container while any bottles are primed the bottles will vent
65. ee en Dc dC E 95 2 9 emm 95 2 9 1 Help Keele 95 29 2 ADOUESUSDE cci sures EE E Et er En t d 98 Chapter 3 Basic Synthesis Operations ENEE 99 Sob Synthesis Checklists ttita an hara an teen 99 3 2 Startup amp Instrument Check rennen res 100 3 3 Post Synthesis Procedures oo OS en 101 3 4 EST HE 101 Chapter 4 Advanced Synthesis Operations amp Optional Features 103 4 1 Automated Bleavage oat a aM tede cos lets eco ae ec each hee teh eg 103 4 2 Dynamic Sequence Programming EEN 104 e e Meute EE 107 vi 1 800 477 6834 4 4 Single Shot Delivery seen 108 4 5 Wash Aller ENO s s eitis net INNO I totas ee 109 Chapter 5 Cleaning amp Maintenance un 111 5 1 Cleaning amp Maintenance Schedule unn 111 5 2 DCT ANOS sosocas ee RU WORT OE ERE 112 5 2 d lee pe AON S ee 112 5 2 2 Computer Maintenance seen en 112 5 2 3 Solv 8 Valve E En 112 5 2 4 Nitrogen Leak Check eoi oio EP dit qe D Un 113 5 2 5 Bottle Filter Replacement sseeeeeee 115 5 2 6 Amino Acid Bottle Seal Replacement sssnnnsneeeeseeeenerrnneeeseerrerrnnee 116 5 2 7 Solvent Bottle Seal Replacement kA 116 Chapter 6 Errors amp PIG DO VB V eec eater uc opa demas edlen Ee 117 6 1 COMMON EIFOIS ans 117 6 2 Critical Errors No Operations Allowed sese 118 B 3SOINEFERIOlS EE 118 EE 119 A Reagents for Peptide Gvnthesies AAA 119 A 1 Prelude Bottles Containing
66. ely select the Copy To All RV s feature Click the Run button A SUser window will open with the message WARNING Calibration can take 15 minutes to complete Make sure RVs and Collect Vials are in place Press OK to start and Cancel to cancel CEN 0x 1 o WARNING Calibration can take 15 minutes to complete K Make sure RVs and Collect Vials are in place Press OK to start and Cancel to cancel Cancel Click OK to continue or Cancel to return to the Solvent Calibration screen without starting the calibration After the calibration is complete measure the volumes in the collection vials s using a calibrated collection vial or a graduated cylinder and enter the values in microliters in tne Actual Volume uL column next to the appropriate RV J Bottle Preparations E o X Bottle Preparations Special Bottles Solvent Calibration Measure volume in collect tube and enter in appropriate Actual Volume field hen done press Refactor button to calculate new factors Re Run to confirm then press Save Note Delivery timeouts and Prime errors may result from incorrect Actual Volume entries Restore Default pasen xA i Restore default values and retry if calibration deliveries fail r Solvent Bottle Test RV Actual Volume uL Q5 da Sg m sa Cao tone d nece Target Delivery Volume ul Number of Deliveries Expected Collect Volume ul Cancel Refactor Click the Refactor button t
67. enzotriazolium 1 bis dimethylamino methylene 5chloro hexafluorophosphate 1 3 oxide In Hg Inches of Mercury M Molarity moles liter uL Microliters mL Milliliters MW Molecular Weight N2 Nitrogen NMM N Methylmorpholine NMP N Methyl 2 Pyrrolidone NPT National Pipe Thread Pip Piperidine Pro Protected Psi amp Psig pound s per square inch gauge PVC Polyvinylchloride Reag Reagent Rep Repetition Res Residues RV Reaction Vessel Solv Solvent TFA Trifluoroacetic Acid THF Tetrahydrofuran Vac Vacuum Vol Volume 1 800 477 6834 Chapter 1 General Information 1 1 General System Description 1 1 1 Prelude Front Reaction Vessels Collection Vials Section 1 1 4 Section 1 1 5 4 Vent also on opposite side Safety Shield H Section 1 1 9 Section 1 1 13 Pressure Gauges Section 1 1 10 Bottle Pressure Regulator amp N2 Push Flow Control Access Panel Section 1 1 10 Amino Acid Bottles Section 1 1 6 Utility Panel Section 1 1 3 Solvent Feedthrough Panel Section 1 1 2 N2 Mixing Flow Control Vacuum Gauge Section 1 1 10 Section 1 1 11 11 www peptideinstruments com 1 1 2 Solvent Feedthrough Panel Fluid Delivery Pressure Delivery Ports Ports Solvent Bottle Ports Waste 2 Outlet Fluid Bottle Vent Waste 1 Outlet Vent 1 1 3 Utility Panel Main Power Switch Circui
68. ep 1 L safety coated glass bottle for deprotectant to remove the N terminal Fmoc protecting group The standard composition is 2096 v v piperidine in DMF This solution is also quite stable and may be installed and used for several sets of syntheses Other reagents may be loaded for alternate chemistries SOLV 4 or Cap 1 L safety coated glass bottle for capping solution to permanently block any unreacted amino groups following a coupling reaction or to acetylate the N terminus of a completed peptide Typical compositions include 1 1 3 acetic anhydride pyridine or DIPEA DMF Other reagents may be loaded for alternate chemistries SOLV 5 or Base 1 L safety coated glass bottle for base if separation of base and coupling reagent is desired The standard base composition is 0 4 M NMM in DMF Bottle 6 or 7 should then contain a coupling reagent such as 0 1 M HBTU in DMF Alternatively base and coupling reagent may be combined into a single activator solution as described below SOLV 6 or Act1 1 L safety coated glass bottle for activator solution to form the activated Fmoc amino acid for the coupling reaction The standard composition is 0 1 M HBTU in 0 4 M NMM in DMF Other reagents may be loaded for alternate chemistries NOTE The activator solution must be equimolar with the amino acid solutions The activator solution should be prepared fresh for each synthesis SOLV 7 or Act2 1 L safety coated glass bottle for additional reage
69. er seat 2 Raise the cam lever into the horizontal position to lock the collection vial in place 23 www peptideinstruments com To remove collection vial 1 Hold the collection vial with one hand while lowering the cam lever to the vertical position with the other hand 2 Tilt the vial from the bottom and remove from the instrument 1 2 5 Amino Acid Bottle Installation To install an amino acid bottle first make sure the bottle position is vented and if necessary back flushed with nitrogen See Bottle Preparations screen Section 2 5 1 1 then 24 1 800 477 6834 1 Make sure the metal slide is pushed all the way in Insert the bottle filter and tube into the bottle and push the amino acid bottle upward 2 The metal slide is spring loaded and will pop out when the bottle is in place NOTE Check that the bottle filter is resting against the lower rear of the bottle This will ensure that all of the reagent in the bottle will be used To release the bottle make sure the bottle position is vented Hold the amino acid bottle with one hand while pushing in the metal slide with the other Carefully slide the bottle off the tubing and filter CAUTION Failure to hold the bottle while releasing will cause the bottle to fall and spill which may result in personal injury loss of reagent and or damage to the instrument 1 2 6 Solvent Reagent Bottle Installation To install a
70. eration of the Prelude and should be maintained like any other component Overloading of the PTI directory can occur if the program sequence and synthesis files are not removed regularly Files that are no longer required should be removed quarterly Database files should be archived Section 2 6 1 2 every quarter or semi annually depending on the number of syntheses performed and backups of important files programs job files should be performed on a regular basis Lost or truncated clusters can occur if power failures or accidental shutdowns occur while the instrument is operating Perform a Scandisk check or a Disk Cleanup routine to fix disk problems Use regular Microsoft Windows conventions to maintain the computer and operating system 5 2 3 Solv 8 Valve Replacement Due to the extremely aggressive nature of cleavage solutions annual replacement of the Solv 8 valve on the cleavage block is recommended as preventative maintenance Please contact your PTI Technical Service representative at 1 800 477 6834 to perform this replacement NOTE The lifetime of the valve can be increased by using the No Prime feature in the Special Bottles screen Section 2 5 1 2 and by flushing the cleavage system by performing a Collect Back Flush Section 2 5 3 5 and Cleave Bottle Solvent Back Flush Section 2 5 3 3 after every cleavage 112 1 800 477 6834 5 2 4 Nitrogen Leak Check It is recommended to routinely check the sealing o
71. f all the reagent supply bottles NOTE For all of the following tests use only one nitrogen supply Do not allow the nitrogen tank gauge to fall below 75 psi during the test or the bottle positions that are pressurized for the test will be automatically vented Test A Regulator amp QC Test 1 Remove the nitrogen quick connect i e no nitrogen line connected to unit 2 Turn off the nitrogen tank valve 3 Watch nitrogen tank gauge on tank for drop in pressure within 15 minutes then turn on nitrogen tank valve 4 f the gauge on the nitrogen tank regulator drops there is a leak If this is the case check the tank regulator and tank fitting for leaks Also check the tank regulator outlet fitting and gauges 5 If the gauge does not drop there is no leak If there are no leaks reconnect the nitrogen quick connect Test B Internal Nitrogen System Test 1 Connect a nitrogen flow meter between the nitrogen tank and the nitrogen inlet to the unit 2 f the flow is greater than 25 cc min there is a leak in the internal nitrogen system Call the PTI Technical Service Department at 1 800 477 6834 3 If the flow is less than 25 cc min proceed to Test C Test C Solvent System Test 1 Connect the nitrogen flow meter and pressurize all solvent bottles in the Bottle Preparations Section 2 5 1 1 screen 2 Allow the system to stabilize for 10 15 minutes 3 Check the nitrogen flow meter to see if there is any fl
72. greater than 25 cc min call your local Technical Service Department representative at 1 800 477 6834 If the flow is less than 25 cc min proceed to the next manifold Repeat steps 2 4 for all three amino acid manifolds If the flow is less than 25 cc min the system check is complete 114 1 800 477 6834 5 2 5 Bottle Filter Replacement The bottle filter should be replaced on a regular basis the frequency depends upon the quality and concentration of the reagents utilized Always replace filters for reagents that show any precipitation If a specific reagent cannot be delivered replacement of the bottle filter should be the first solution The bottle filter consists of a filter housing and a frit The frit fits into the housing via a press fit On the other side of the housing is a partially threaded entrance for the tube To thread the filter housing onto a bottle tube gently twist the housing clockwise while pushing it onto the tube Be certain to thread the assembly completely onto the tubing or bubbles may be introduced between the top of the housing and the tubing The filter assemblies are easily removed by gently twisting counterclockwise while pulling down To remove the filter frit either press the frit out with a swab etc from the top or lift the frit out with a spatula etc To replace the frit put the new frit on a clean flat surface and press the filter housing firmly over the frit CAUTION Always wear prot
73. he Solvent 1 bottle CAUTION Under no circumstances should TFA be used in the amino acid manifold system destruction of the bottle seals will occur See Section 5 2 6 Amino Acid Bottle Seal Replacement for replacement procedures 7 After the cleaning operation is complete empty the flushed bottles of any rinse fluid and replace with clean bottles for the next synthesis 2 5 3 3 Cleave Bottle Solvent Back Flush The Cleave Bottle Solvent Back Flush cleaning procedure flushes solvent bottle 8 with Solvent 2 DCM to remove TFA solution from the line Cleave Bottle Solvent Back Flush is performed as part of a System Clean but it can also be performed alone NOTE When changing cleavage solutions it is suggested to perform a Cleave Bottle Nitrogen Back Flush Section 2 5 3 4 to flush reagent back into the bottle Replace the bottle with an empty bottle and perform a Cleave Bottle Solvent Back Flush to clear residual reagent from the line Wipe excess fluid off the bottle tubing and load the new reagent bottle To perform a Cleave Bottle Solvent Back Flush 1 Pressurize and prime Solvent 2 DCM using the Bottle Preparations screen See Section 2 5 1 1 2 Click on the Operations menu select Cleaning and then select Cleave Bottle Solvent Back Flush 76 1 800 477 6834 ra A File Operations Tools Reports View Help g Bottle Preparations gt d RY Operations le Cleaning 2 System C
74. hesis Procedures Cleave peptides from resin Section 3 3 Remove collection vials and work up peptides Section 3 3 Perform Wash RVs Sections 3 3 amp 2 5 3 8 Perform Bottle Position Flush on used bottles Sections 3 3 amp 2 5 3 2 Perform Collect Back Flush Section 2 5 3 5 Perform Cleave Bottle Solvent Back Flush Section 2 5 3 3 Perform System Clean if necessary every 2 weeks Section 2 5 3 1 Empty the waste container Section 3 3 amp 1 2 7 99 www peptideinstruments com 3 2 Startup amp Instrument Check To startup the Prelude Peptide Synthesizer turn on the On Off switch located on the utility panel on the side of the instrument Turn on the monitor and printer The SUser software will startup automatically Before starting a synthesis perform the following checks 1 Check the nitrogen supply amp gauges Make sure there is enough nitrogen in the tank for the synthesis and that the tank is on Check the nitrogen status in the lower right corner of the screen N2 should be displayed on a green background If N2 is displayed on a red background nitrogen is not getting to the instrument Check the tank and lines Check the pressure gauges The Valve Pressure Gauge should read 25 35 psi and the setting should not be adjusted The Nitrogen Pressure Gauge should read 5 psi and the setting should not be adjusted The Bottle Pressure Gauge should read 9 psi See Sectio
75. hose files for the synthesis Only sequences with amino acid and solvent reagent files matching those of the first sequence chosen will be allowed in the synthesis To run a sequence with a different amino acid or solvent reagent file a different synthesis file must be created 2 Length The length of the peptide is displayed in the Length column 3 Peptide Sequence The sequence is displayed to the right of the Length column and is right justified with the N terminus to the left and the C terminus to the right The residues involved in the current cycle are highlighted 4 Dynamic Sequence Programming The function of the Ins Del and Undo buttons next to the text Sequence Modifiers No AA Idle are part of this optional advanced feature described in detail in Section 4 2 5 COOH CONH2 Click under COOH or CONH2 to choose an acid or amino C terminus respectively for the peptide When COOH is chosen the sequence will shift one character to the right and the first character will not be included in the synthesis 48 6 1 800 477 6834 Molecular Weight The molecular weight of the peptide with the assigned COOH or CONH2 C terminus is displayed The molecular weight is calculated using the deprotected molecular weight values in the amino acid file assigned to the sequence Current Cycle The box to the left of Current Cycle displays the name of the program assigned to the current cycle The box
76. ion vessels 1 3 were filled prior to the error it will start filling at reaction vessel 1 and reaction vessels 1 3 will have been filled twice 6 1 Common Errors The following table lists common errors their cause and possible corrective actions to take If the error still persists after all suggested actions have been taken please contact your PTI Technical Service representative Error Cause Possible Action s FILL ERROR RV sensor did not sense fluid during a Fill operation Check for fluid covering the filter in delivery bottle If many RVs are pausing frequently on the same solvent reagent check clean or replace bottle supply filter at the source Check that the bottle pressure gauge is set to 9 psi Check bottle seal for improper fit damage missing parts that may cause a nitrogen leak Check for nitrogen leaks using external nitrogen flow meter Section 5 2 4 Check for plugs or precipitates and perform Bottle Position Flush cleaning operation if required Section 2 5 3 2 Check waste line for plugs If only one RV is pausing frequently on numerous solvents check RV for clogged frits and clean or replace RV if required Perform Wash RVs to clean the RV lines Section 2 5 3 8 RV sensor may require service contact Technical Service Dept CLEAR ERROR The RV fluid sensor senses fluid after the Clear operation Check adjust nitrogen pressure gauge to 5 psi See Section 1 1 10 Perform Wash
77. ions 44 Calculations Solv Reag Manual Operations Alternatively open the Manual Operations screen by clicking the Manual button on the RV Status screen See Section 2 5 2 1 70 1 800 477 6834 Manual Operations 8 lt r Select Active Reaction Vessels Iv AVI Iv Rv2 M Rev3 IT Ava TD pp AVE Clear All vs r Operation jaa Building Block x Volume uL Mix time Drain Reps fi 000 00 01 00 Iv fi 5 elect Amino Acid C Ala C Leu C Tp C Cys C Met C Ty C Asp C Asn C A21 C Glu Pro C A22 C Phe C Gin C A23 C Gly C Arg C A24 C His C Ser C A25 C lle C Thr C A26 C Lys C Val C A27 Close Clear Cancel Actio Check Reps Sensor Fill Rinse and Clear Sensor Fill Rinsing Block 4 Clearing Block 4 Step Done Done Select one or more RVs for an operation in the Select Active Reaction Vessels section Click on the Select All RVs Clear All RVs button to select or deselect all RVs respectively The Clear All RVs button replaces the Select All RVs button when one or more RVs are selected Use the pull down menu in the Operation section to select an operation Input the delivery volume in microliters in the Volume uL box Volumes will be rounded up to volumes that can be delivered in increments of 150 500 or 1000 uL up to a maximum volume of 10 000 uL for 10 mL RV s and up to a maximum volume of 20 000 uL for 40 mL RV s The RV size should be selected in the Se
78. ions DIN Cleaning System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs 2 The following warning window will open Click OK to proceed or click the Cancel button to cancel the Clear All Blocks operation Suse MUN Q WARNING Clearing can take 20 minutes to complete press OK to start and Cancel to cancel 3 During the Clear All Blocks operation the Bottle Preparations screen See Section 2 5 1 1 will open 81 www peptideinstruments com Bottle Preparations x Bottle Preparations Special Bottles Solvent Calibration Solvents p Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed Leu Met a m ee ey nen Sn s os oO Om MN oo syon e wn The status of the operation will be displayed in the status bar Click the Pause button to pause the operation Click Resume to resume a paused operation Click the Cancel button to cancel the operation Click the Close button to close the Bottle Preparations screen CAUTION f the procedure is cancelled there may be fluid left in the lines or blocks Repeat a Clear All Blocks to remove any residual fluid 2 5 3 8 Wash RVs Wash RVs rinses the reaction vessels and lines by delivering Solvent 1 DMF from the
79. lean Bottle Positi Cleave Bott vent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs 3 The following warning window will open Click OK to continue or Cancel to cancel the Cleave Bottle Solvent Back Flush operation LM 09 Warning This procedure back flushes Solvent into selected bottle s This procedure may contaminate amino acids reagents solvents E Cancel 4 During the Cleave Bottle Solvent Back Flush the Bottle Preparations screen will open Bottle Preparations E AXI Bottle Preparations Special Bottles Solvent Calibration Solvents 34 r Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed Tom PS E Ala F Leu IEL lt E DEM Ja las Met 2 Ty F Dep Asp I Asn 2 Araz Cap E Glu E Pro 22 AT 422 F Base Phe F Gin 23 aT 423 E Acti E Gly F Ag 24 AT 424 E Act F His F Se 25 ef Ap E TFA E je E Th 26 AT 25 E Jee F va 2 AT 527 Clear All Select All Back Flushing Back Flush TFA bottle 333333333 ii ceo o a o t MM sl oc eh M O d nae won 383855333 333333332 Eressurze Ment Nitrogen Back Flush Solvent Back Flush The status of the operation will be displayed in the status bar Click the Pause button to pause the operation Click Resume to resume a paused operation Cli
80. les are used to assign sequences to reaction vessels choose an acid or amino N terminus for each peptide and assign programs to each cycle of the synthesis A program assigned to a given cycle is run on all active reaction vessels in parallel unless Dynamic Sequence Programming is used Section 4 2 To open the Synthesis Editor click on the File menu select Synthesis and then select Synthesis File Operation Tools Reports View Help RI Synthesis Program Sequence Synthesis Amino Acid Solvent Reagent The Synthesis File Manager will open as a new screen To create a new synthesis file click the New button enter a name for the new file and click OK Alternatively select an existing synthesis file from the list and click the Open button The synthesis file will open in the Synthesis Editor under the Synthesis tab I Synthesis Editor PTI x Program Sequence Synthesis Program Sets Synthesis Name stna Posem W Solvent Reagent pmo s D Standard m Program2 x tnaa 1 Program H Program3 D Amino Acid Standard Comments r Peptide Programming Program4 Current Cycle Undo M A e gt en c Original ifiers 9 N Molecular DDDDBGDD21DD bog Lu PrrPBPTIDE123 2 is paje c 172 PTIPBPTIDE123 ins Da c 1072 PTIPBPTIDE123 _ins Daf c Standard BTANDARD wl palco e 88758 Standard QTANDARD i C m Sta
81. llect Back Flush operation suser x WARNING COLLECT VIALS MUST BE IN PLACE Press OK to start and Cancel to cancel E Cancel 5 During the Collect Back Flush the Bottle Preparations screen will open 78 1 800 477 6834 3 Bottle Preparations a x Bottle Preparations Special Bottles Solvent Calibration r Solvents 34 r Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed 1 E As i F Leu N Ig let 2 F Gs i I Met N 20 F Tyr 3 sp Asn N a aT An 4 E Su E Pio N 22 AT A 5 Pe i Gin i 23 AT 423 6 7 8 3 oo ug o o qc m Di d Vi Ti E Gy i E Arg H 24 Af 424 E His E Ser H 25 aT 425 E Thr N 26 AT A F Lys Val NE 2 AT amp 27 su n Ep w w N t tes ds Roe Eon n pn i n d Collect Back Flush Rinse tue Ss The status of the operation will be displayed in the status bar Click the Pause button to pause the operation Click Resume to resume a paused operation Click the Cancel button to cancel the operation Click the Close button to close the Bottle Preparations screen CAUTION f the procedure is cancelled there may be fluid left in the instrument lines or blocks Repeat the Collect Back Flush procedure to clear the lines 6 After the cleaning operation is complete remove the collection vials and discard the rinse solution Place clean empty collection vials
82. lude e Chapter 5 Cleaning amp Maintenance explains the cleaning procedures for the Prelude and its maintenance schedule e Chapter 6 Errors amp Recovery describes common instrument errors and how to recover from them I 2 About The Company Protein Technologies Inc PTI is built on the belief that our products and services are of the highest possible quality PTI s products are supported by a dedicated field service team and we are proud of our reputation for reliability Founded in 1985 by researchers affiliated with the University of Arizona PTI launched its first peptide synthesizer in 1990 Since then PTI has manufactured and sold the world s finest solid phase synthesizers Today we are growing and innovating to serve the needs of the solid phase synthesis market If you have any questions concerning your PTI synthesizer please feel free to contact us Tel 520 629 9626 Toll Free 800 477 6834 Email info peptideinstruments com www peptideinstruments com www peptideinstruments com 1 3 Common Abbreviations AA Amino Acid Act Activator or Action Conc Concentration DCM Methylene Chloride or Dichloromethane Dep Deprotection Solution or Deprotected DIPEA Diisopropylethylamine DMA Dimethylacetamide DMF Dimethylformamide Fmoc 9 Fluorenylmethyloxycarbonyl GLP Good Laboratory Practice HBTU 2 1H Benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate HCTU 1H B
83. ments com The amino acid bottles have a special feature called a Single Shot delivery One Shot Delivery section which will deliver all of the amino acid in a bottle to a specific reaction vessel To activate this feature check the box next to an amino acid Selected amino acid bottles will display a picture of a syringe next to their position in the Bottle Preparations screen see figure above When the Sequence reaches the amino acid in the Synthesis the Prelude will deliver all of the amino acid solution in the selected bottle to the reaction vessel NOTE If a bottle is set to perform more than one Single Shot delivery from the same bottle position during the same synthesis pause the synthesis after the first delivery then vent refill and repressurize the bottle before resuming Otherwise the instrument will error when it reaches the second delivery 2 5 1 3 Solvent Calibration The Solvent Calibration screen is located under the Solvent Calibration tab in the Bottle Preparations window It allows the user to calibrate the volume deliveries of the timed solvent reagent bottles 1 4 and 8 NOTE Calibration accuracy is dependent on the Bottle Pressure Section 1 1 10 If the pressure reading on the Bottle Pressure gauge changes by more than psi in either direction following a calibration the calibration should be checked To open the Solvent Calibration screen click on the Operations menu select Bottle
84. n 1 1 10 for more information 2 Check the vacuum gauge The vacuum gauge is located on the front of the instrument and should read 17 22 in Hg Also check the vacuum status in the lower right corner of the screen Vac should be displayed on a green background If Vac is displayed on a red background there is a problem with the vacuum system If this occurs call your local PTI Service Technician at 1 800 477 6834 Meanwhile the Prelude may be run with an external vacuum source See Section 1 1 11 for more information 3 Check the waste level Check the waste status in the lower right corner of the screen Waste should be displayed on a green background If Waste is displayed on a red background the waste is full or the waste level sensor is disconnected Empty the waste if necessary and make sure the waste level sensor is connected properly See Sections 1 1 8 and 1 2 7 for more information 4 Change bottle filters if necessary Bottle filters should be changed in the event of a clogged filter or change of reagent See Section 5 2 5 for instructions 5 Select RV size 10 mL or 40 mL in the Settings screen Section 2 6 3 6 Calibrate solvent delivery volumes if necessary If consumption volumes are not matching calculated volumes for solvents 1 4 and 8 it may be necessary to perform a solvent calibration See Section 2 5 1 3 for instructions 100 1 800 477 6834 3 3 Post Synthesis Procedures 1 Remove oollection vi
85. ndard BTANDARD ler RE NeW EE Cancel Save Save As Pint The Synthesis Name box lists the name of the currently open synthesis file To open a different file select a different file from the pull down menu 47 www peptideinstruments com Enter synthesis comments in the Comments box The Program Sets section defines programs that can be assigned to different cycles of the synthesis Use the pull down menus to assign programs to each button D 1 2 and 3 D stands for the default program which is initially assigned to every cycle in the synthesis If more than four programs are needed additional programs may be assigned using buttons 1 2 and 3 See Assign Sequence 8 in Peptide Programming section below for more information about programming assignments The Solvent Reagent box displays the solvent reagent file assigned to the synthesis This file is determined based on the first sequence entered The Amino Acid box lists the current amino acid file assigned to the synthesis This file is determined based on the first sequence entered The Peptide Programming section has the following functionalities 1 Sequence Assign a sequence to each RV using the pull down menus in this column The first row corresponds to RV1 the second row corresponds to RV2 and so on down to RV6 NOTE The amino acid and solvent reagent files assigned to the first sequence chosen will define t
86. ndoes the most recent cycle modification The Cycle Selection window allows the user to choose the type of cycle that will be inserted when the Ins button is clicked 105 www peptideinstruments com Cycle Selection x E mj Em mu Em mu EN mu Em 9 Bi mj Em mu B i mu En mun Carce AI The buttons are as follows 1 Idle Cycle Inserts an Idle cycle represented by a hyphen to the right of the selected cycle in the sequence 2 No AA Inserts a No AA cycle represented by an asterisk to the right of the selected cycle in the sequence 3 Amino Acid Inserts an amino acid cycle to the right of the selected cycle in the sequence 4 Cancel Cancels the insert operation When an advanced operation is assigned to a sequence H is displayed to the right of the sequence to alert the user The following examples demonstrate possible uses of Dynamic Sequence Programming 106 1 800 477 6834 Example 1 Run different programs during cycle 1 Program Assignment D RV 1 Sequence A A RV 2 Sequence Bh e RV 1 will couple A with program 1 while RV 2 sits idle When program 1 is complete RV 2 will couple C with program 2 while RV 1 sits idle Both RV s will then couple their next amino acid with the default program D Example 2 Use different activators in cycle 1 Program Assignment DX32 1 RV 1 Sequence A A RV 2 Sequence ET Program 1 Depro
87. ning window will open Click Yes to continue or click No to cancel the operation AN This will archive then compact and repair the database This application must be shut down to perform the operation Any syntheses currently in process will be lost Do you wish to shut down this app and perform this operation D Data Archiver Version 1 0 0 40 Pata MASTER_USER mdb Success Pracess File o 5 When the archiving process is complete the word Success will be displayed 6 To archive a database file other than the current database file specified under Settings Section 2 6 3 click the Select File button This will open a new window 85 www peptideinstruments com open o Look in ES Data EI ex E3 Archive 4 MASTER_USER mdb UserEventLog mdb iUJSER1 mdb USER2 mdb 7 USERS mdb File name USERT mdb Files of type Access Database Files mdb DI Cancel 7 Select a user database file from the Data folder and click Open This will return the user to the Data Archiver window where the Process File button will have become active 8 Click the Process File button to archive the file 9 Click the Close button or click on the X in the upper right corner to close the window 10 Double click on the SUser icon to restart the SUser software Archived data is located in the C PTI Prelude Data Archive folder and is located in a single file named ac
88. nt SOLV 8 or CLEAV 1 L safety coated glass bottle of cleavage reagent for cleavage of the peptide from the resin after synthesis is complete This position is specifically designed to handle the aggressive TFA cleavage 15 www peptideinstruments com solution It can only be accessed through the Cleave and Collect operation NOTE The cleavage solution should be prepared fresh for each synthesis NOTE The No Prime feature must be selected for the SOLV 8 bottle in the Special Bottles screen Section 2 5 1 2 when in use However the No Prime feature should be deselected for the SOLV 8 bottle during Solvent Calibration Section 2 5 1 3 Each solvent position has a bottle filter to prevent particulates from entering the fluid system For replacement procedures see Section 5 2 5 An encapsulated o ring in the bottle cap insert establishes the bottle seals and is inert to the reagents Damage to the insert or o rings will result in nitrogen leakage and potential loss of reagent volatiles like TFA DCM For bottle seal replacement procedures see Section 5 2 7 Custom bottle configurations and assemblies can be arranged through technical service 1 1 8 Waste System The only exit for the closed fluid flow paths of the instrument is through the waste system Waste exits the Prelude to the waste container through three ports on the Solvent Feedthrough Panel The waste container is vented through a fourth tube attach
89. nt 8 bottles Section 1 2 6 and loctite fittings to solvent feedthrough panel connectors Install 27 empty amino acid bottles Section 1 2 5 Unpack and setup monitor keyboard mouse printer and connect to utility panel Section 1 1 3 Attach power cord to power cord receptacle Section 1 1 3 and plug in Attach nitrogen supply lines Turn on main power switch and the three circuit breakers Turn on computer monitor and printer Verify all systems alarms are OK 4 icons on lower right corner of main screen are green Perform Nitrogen Leak Check see Section 5 2 4 20 1 800 477 6834 Back flush Solvent 1 to all 27 amino acid lines and verify liquid delivery to each amino acid bottle using the Bottle Preparations screen Section 2 5 1 1 Use the Manual Operations screen Section 2 5 2 5 to deliver 1000 uL of Solvent 1 to all RV s Verify liquid delivery and drain into waste check for leaks on all waste valve fittings Pressurize amino acid bottles using the Bottle Preparations screen Section 2 5 1 1 and check for leaks 1 2 3 RV amp O Ring Installation To install reaction vessel o rings 1 2 3 4 e e gt o M em Unscrew the cap from the lower RV seat on the instrument Place a reaction vessel bottom o ring in the center of the lower RV seat Screw the cap on over the o ring until tight Slide a reaction vessel top o ring into the groove on the uppe
90. nt reagent file associated with the selected synthesis 4 Conc mM Displays the concentration of the activator solution in mM The user may change the value in this column 5 Actual Volume mL Displays the total volume of activator solution The user may change the value in this column 6 Weight mg Displays the weight in mg of activator needed The software calculates this value based on the entry in the MW g mol column 7 Volume mL Displays the volume in mL of activator needed The software calculates this value based on the entries in the MW g mol and 69 www peptideinstruments com Density g mL columns If there is no entry in the Density g mL column the value will be displayed as 0 000 The Print button prints the calculated values from the Calculations AA and Calculations Solv Reag screens NOTE Use the Print button only after modifying both the Calculations AA and the Calculations Solv Reag screens to avoid having to print twice 2 5 2 5 Manual Operations The Manual Operations screen allows the user to perform individual operations on the instrument outside of a synthesis To open the Manual Operations screen click on the shortcut button or click on the Operations menu select RV Operations and then select Manual Operations File Operations Tools Reports View Help Bottle Preparations n a RV Operations RV Status Cleaning Load Synthesis Calculat
91. o calculate new calibration factors Repeat steps 1 7 until the Actual Volume uL and Expected Collect Volume uL values match Then click the Save Factors button Click Close to exit the Solvent Calibration screen 59 www peptideinstruments com CAUTION After calibration is complete the fluid system may be contaminated Perform a Rinse All Blocks followed by a Clear All Blocks to clean the instrument Sections 2 5 3 6 amp 2 5 3 7 2 5 2 RV Operations The Reaction Vessel Operations screen automatically opens when the Prelude software is opened To open the Reaction Vessel Operations screen click on the shortcut button M or click on the Operations menu select RV Operations and select a sub section File Operations Tools Reports View Help g Bottle Preparations Inn RY Operations RV Status Cleaning Load Synthesis Calculations AA Calculations Solv Reag Manual Operations This will open the Reaction Vessel Operations screen with the sub section tab active The Reaction Vessel Operations screen has 5 sub sections RV Status Load Synthesis Calculations AA Calculations Solv Reag Manual Operations ae co mc The function of each of these sub sections will be reviewed in each subsection below 2 5 2 1 RV Status The RV status screen is located under the RV Status tab in the Reaction Vessel Operations window Once a synthesis has been loaded this screen 60
92. o the printer NOTE To view the program before printing click the Close button and preview the file using the Print button in the Program File Manager Section 2 3 2 4 3 2 Sequence Editor To open the Sequence Editor click on the File menu select Synthesis and select Sequence File Operation Tools Reports View Help Amino Acid ER Solven Program Sequence Synthesis The Sequence File Manager will open as a new screen To create a new sequence file click the New button enter a name for the new file and click OK Alternatively select an existing sequence file from the list and click the Open button The sequence file will open in the Synthesis Editor under the Sequence tab 44 1 800 477 6834 imi Synthesis Editor PTI Program Sequence Synthesis Sequence Name Select Amino Acid File Amino Acids 4 a NE Standard Description EJ KAN E i Technologies Inc E KA Sequence Definition COOH 11057 182 Current Position Length ME re er EES EES EES 13 n3 CONH2 1056 175 PTIPEPTIDE123 ES 38 7 ca P Tree D Tee PE pE pen PE New Close Cancel Save Save s Print The Sequence Name box displays the name of the currently open sequence file To open a different file select a different file from the pull down menu The Select Amino Acid File box displays the name of the amino acid file wh
93. ol g Scale umol Resin ma Yield mg ber oa neen KKRCSMWIIPTDDEZA IQAAIDYING RCSMWIIPTDFGA QAAIDHL1G KKRCSMWIIPT234A AAID567G Solvents Reagents SSC Activators Actual Voli J Pos Description Calc vol mL Rn Description MW g mol Density g mL Conc mM mL le Weight g Volume mL 1 Pimethyfformamide 224 3000 m 101 556 0 920 2 PicHloramethane o Jeu 379300 vo 3 P Piperidine OMF u 321 100 0 000 4 Pimethyfformamidez pmo IT a 000 6 7 8 ban NMM O 1M DTI rao Pinethyfformamides 100 cca 0 v The Synthesis Name box displays the current synthesis Use the pull down menu in the Calculations AA screen Section 2 5 2 3 to select a different synthesis The Peptides section calculates the resin amount needed for each reaction vessel It displays a table with columns labelled as follows 1 Sequence File Displays the name of the sequence file in each RV Row 1 corresponds to RV1 row 2 to RV2 and so on down to RV6 2 Sequence Displays the amino acid sequence of the peptide 3 Termination Displays the termination group on the C terminus COOH or CONH2 4 Sub mmol g Displays the substitution on the resin in mmol g The user may change the value in this column 5 Scale umol Displays the synthesis scale in umol The user may change the value in this column 6 Resin mg Displays the amount of resin in mg to weigh out into
94. ollect Drains RV contents to collection vial The columns are 1 Operation Use the pull down menu to select an operation for each step 2 Volume uL Enter a volume in microliters The Prelude delivers in 150 500 and or 1000 uL increments Entries will be rounded up to the nearest value possible with a minimum volume of 150 uL and a maximum volume of 10 000 uL for the 10 mL RV The maximum volume increases to 20 000 uL for the 40 mL RV The size of the RV must be specified in the Settings screen Section 2 6 3 for these volumes to take effect 3 Mix Time H M S Enter the mix time in Hours Minutes Seconds The maximum mix time is 99 59 59 4 Drain Click to check or uncheck the box When checked the reaction vessel will be drained at the end of the program step When unchecked the reaction vessel will not be drained at the end of the program step NOTE t is important to leave Drain unchecked when delivering amino acid so the amino acid remains in the reaction vessel for the activator delivery CAUTION When Drain is unchecked multiple deliveries may be made to the same RV without draining Be careful not to exceed the RV s maximum capacity as this may force resin into the showerhead causing clogs or contamination 5 Reps Enter the number of times to repeat the step The maximum number of repetitions is 9 6 Comment Record comments for the step The buttons are 1 Insert To insert a
95. on s NO NITROGEN The nitrogen supply switch in the pneumatic inlet assembly senses lt 65 psig from the nitrogen supply system Check nitrogen tanks and regulators Check quick connect fittings for proper fit and or leaks NO VACUUM Vacuum supply switch senses 10 in Hg vacuum after pump stops running Attach external vacuum gauge Check tube fittings on vacuum pump head for leaks and tightness WASTE FULL Waste level sensor indicates the tank is full or not connected to the instrument Empty waste tank and reconnect Section 1 2 7 Check reconnect waste tank connector No communication NEE ee Turn Prelude power off for 30 seconds then back on instrument software 6 3 Other Errors The following errors indicate an internal computer problem NO MODULE NO COMMAND STRING MODULE NOT IN PLACE SYSTEM ERROR 1 SYSTEM ERROR 2 SYSTEM ERROR 3 SYSTEM ERROR 4 If the error persists shutdown then restart the instrument If this does not fix the problem contact your PTI Technical Service representative at 1 800 477 6834 118 Appendix 1 800 477 6834 A Reagents for Peptide Synthesis A 1 Prelude Bottles Containing N Fmoc Protected Amino Acids Preweighed Catalog No Amino Acid Quantity SMP 05 A 5 mmol SMP 10 A Fmoc L Ala OH 10 mmol SMP 20 A 20 mmol SMP 05 RP 5 mmol SMP 10 RP Fmoc L Arg Pmc OH 10 mmol SMP 2
96. on the instrument for the next collection 2 5 3 6 Rinse All Blocks Rinse All Blocks rinses each block with Solvent 1 DMF then flushes each block with nitrogen to remove residual fluid It does so without venting the bottles to prevent contamination of the amino acids solvents and reagents Rinse All Blocks is performed using Solvent 2 as part of a System Clean but it can also be performed alone using Solvent 1 DMF 1 To perform a Rinse All Blocks operation pressurize and prime Solvent 1 using the Bottle Preparations screen See Section 2 5 1 1 2 Click on the Operations menu select Cleaning and then select Rinse All Blocks 79 www peptideinstruments com File Operations Tools Reports View Help g Bottle Preparations gt Dr RV Operations y E Cleaning 2 System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash Dys 3 The following warning window will open Click OK to proceed or click the Cancel button to cancel the Rinse All Blocks operation WARNING Rinse and clear can take 15 minutes to complete press OK to start and Cancel to cancel Cancel 4 During the Rinse All Blocks operation the Bottle Preparations screen will open Bottle Preparations ES xl Bottle Preparations Special Bottles Solvent Calibration Solvents _ r Amino Acids Press
97. on the cleave option by clicking in the On box When the box is checked the functions in the Cleave section become active Select a cleavage program using the pull down menu in the Program box Set the cleavage start time by selecting one of three options 1 Now To start the cleavage immediately click the circle next to Now If this is selected the loaded synthesis will be ignored and only the cleavage program will be performed 2 At End To start the cleavage at the end of the synthesis click the circle next to At End 3 Delay To delay the start of the cleavage program click the circle next to Delay then enter a date and time to start the cleavage program There is no need to delete or backspace in the date box Type over the date and time to change the numbers The Load button loads the selected synthesis and cleavage onto the RV Status screen and opens the Calculations AA screen 2 5 2 3 Calculations AA The Calculations AA screen is located under the Calculations AA tab in the Reaction Vessel Operations window It calculates solution volumes and the amount of dry amino acid necessary for a given concentration To open the Calculations AA screen click on the Operations menu select RV Operations and then select Calculations AA Se File Operations Tools Reports Yiew Help Bottle Preparations gt In nl RV Operations RV Status Cleaning Load Synthesis Calculations AA Calculations Sol
98. or detailed information on the Collect Back Flush 1 1 6 Amino Acid Bottle System 27 amino acid bottle positions are located on the front of the Prelude Amino acid bottles are available in 10 120 and 400 mL capacities and can be connected or detached quickly and easily Pressurizing the bottles with nitrogen accomplishes solution transfer and this and other operations are controlled using the Bottle Preparations screen Section 2 5 1 1 Each amino acid bottle has a bottle filter to prevent particulates from entering the fluid system These filters should be changed on a regular basis depending on the quality and concentration of reagent utilized See Section 5 2 5 Bottle Filter Replacement for instructions CAUTION DO NOT install or remove amino acid bottles when they are pressurized 13 www peptideinstruments com IMPORTANT Each row of nine amino acid bottles is connected to a separate pressurization manifold All nine amino acid bottles in each row are vented or pressurized together All amino acid positions in a row must have bottles in place for that row to pressurize Empty bottles should be placed in any unused positions IMPORTANT The amino acid manifold seals are not affected by DMF or NMP Recent studies have shown that THF and DCM can also be utilized without destruction of the seals if extra caution is used to prevent the liquid from contacting the seals Contact PTI s Technical Service
99. ose values will determine what amino acids or other chemicals are available for inclusion in the sequence To use a different amino acid file select a different file from the pull down menu The Amino Acids box to the right of the screen contains buttons for each of the 27 bottle positions on the instrument The buttons are labeled with the abbreviations and key codes of the currently open amino acid file Enter a sequence by clicking the buttons with the mouse or using the keyboard Enter comments in the Description box The Sequence Definition box contains the following boxes 1 Current Position lists the current position of the cursor within the sequence 2 Length lists the total number of characters in the sequence 3 Molecular Weight COOH displays the molecular weight of the deprotected peptide with an acid N terminus 4 Molecular Weight CONH2 displays the molecular weight of the deprotected peptide with an amino N terminus 45 www peptideinstruments com NOTE The peptide molecular weights are calculated from the amino acid molecular weight values entered in the amino acid file 5 Sequence Box large white box where the sequence is entered Click in the box to place the cursor Enter a sequence by clicking on the buttons to the right of the screen or by using the keyboard NOTE Entered characters must match those available in the current amino acid file The remaining buttons are as follows
100. ow 4 Ifthe flow is greater than 25 cc min there is a leak in one of the bottles If the flow is below 25 cc min proceed to Test D 113 www peptideinstruments com To identify the leaky bottle start at the Solvent 8 position or 7 if no Solvent 8 and vent the bottle Check nitrogen flow meter for flow If the flow is greater than 25 cc min move up one bottle and vent Solvent 7 or 6 Check nitrogen flow meter for flow If the flow is still greater than 25 cc min continue to the next bottle s until the flow is below 25 cc min 10 When the flow is below 25 cc min the last vented bottle has a leak 11 Check the bottle cap insert and o ring 12 Check the supply tubing for cracks or leaks 13 Pressurize the bottle and re test If the flow is still above 25 cc min call your PTI Technical Service Department representative at 1 800 477 6834 If the flow is below 25 cc min proceed to Test D Test D Amino Acid System Test 1 2 Make sure all 27 amino acid bottles are in place Pressurize the first amino acid manifold and let the system stabilize for 5 10 minutes Check nitrogen flow meter If the flow is greater than 25 cc min vent the system and examine the amino acid bottle seals for solids cracking tears or other damage that would interfere with sealing Pressurize the amino acid manifold again let stabilize and check the nitrogen flow meter If the flow is still
101. print the reports from the Report Preview window and click the Close button or click on the X in the upper right corner to close the window 2 Print The Print button will automatically send the reports to the printer 94 1 800 477 6834 3 Print to File Click the Print to File button to save the job report A Save As window will open and allow you to save the report as a text file or other file type 4 Close Click the Close button to exit the Reporter window 2 7 2 Comm Log The Comm Log screen is available for technician use only It is used to view and troubleshoot communications between the user software and the instrument computer 2 8 View Menu The View menu displays the names of all open windows The active top window is indicated by a checkmark next to its name Click on the View menu and select an open window to go to it quickly File Operations Tools Reports View Help Era pmAn E m Si ea v R 1 Reaction Vessel Operations e Reaction Vessel Operations v 2 Bottle Preparations imi Bottle Preparations EE SSS SSS 2 9 Help Menu 2 9 1 Help Topics To open the Help Topics window click on the Help menu and select Help Topics File Operations Tools Reports View Help Help Topics Bashy WE About SUser 95 www peptideinstruments com On the left side of the Help Topics window are the Contents Index and Search tabs igi xi He m D Hide B ack Print S Options
102. r RV seat Test for leaks by installing an empty reaction vessel below and performing a DMF Top Wash using the Manual Operations screen Section 2 5 2 5 21 www peptideinstruments com To install reaction vessel 1 Lower the cam lever to the vertical position Push the RV firmly into the lower seat first CAUTION Make sure the RV bottom is pressed through the bottom o ring in the lower RV seat or the RV may leak A slight twisting may help 2 Line up RV with the upper seat and raise the cam lever up into a horizontal position to lock the RV in place To remove reaction vessel 22 1 800 477 6834 1 Hold the RV with one hand while lowering the cam lever into the vertical position 2 Pull the top of the RV away from the instrument then pull the RV up out of the lower seat to remove 1 2 4 Collection Vial Installation IMPORTANT It is not recommended to have cold ether in the collection vial when the cleavage solution is collected The vial may overfill during the collection of the product causing both loss of the product and potential damage to the instrument from the TFA solution Rather collect cleavage solution remove collection vial from instrument then precipitate peptide with cold ether lt 0 C To install collection vial 1 Lower the cam lever to the vertical position Place the uncapped vial into the upper seat first then line up with the low
103. r to an external computer using the USB port and a memory stick 1 2 2 Instrument Installation Procedure IMPORTANT Installataion of the Prelude should be performed by trained personnel only Improper installation may result in damage to the instrument or operators Uncrate main unit lift off pallet use caution gt 100 Ibs Remove all materials from crate check off list 2 4 L safety coated glass bottles 7 1 L safety coated glass bottles 1 5 gallon waste container package of reaction vessels package of collection tubes package of amino acid bottles 1 vent connector 1 vent cap 1 bottle container optional bottle cap and tubing assemblies waste lines hardware Remove top panel and side panel where vent will be installed Install adjustable vent adaptor and bend flaps out to fasten Place vent cap in other side panel vent hole Attach waste level sensor cable connector 4 pin to utility panel Section 1 1 3 Place waste tank in user provided secondary container and dress cleanly Attach the other end of the connector to the waste level sensor on the waste container by lining up the red dots then pushing down Attach the three shorter 1 4 waste lines from waste tank to waste fittings on solvent feedthrough panel Section 1 1 2 Attach the long 1 4 vent line from waste tank to vent duct adaptor fitting Install RV s and collection tubes into positions Sections 1 2 3 amp 1 2 4 Install Solvent 1 through Solve
104. rce number To open the Solvent Reagent Editor click on the File menu and select Solvent Reagent File Operation Tools Reports View Help Amino Acid The Solvent Reagent File Manager will open as a new screen To create a new solvent reagent file click the New button enter a name for the new file and click OK Alternatively select an existing solvent reagent file from the list and click the Open button The solvent reagent file will open in the Solvent Reagent Editor 4 Solvent Reagent Editor Standard Solvent File Name Standard Program Operation Names GLP Data Pos Name Abbrv Bottom Delivery Top Wash Vol mL Conc mM Opened Date Lot Number Source Number Of 8 Tracoctal TFA leave and Collect 1 0 0 Jos gaz Time Calibrated Delivery X Sensor Delivery Cancel Save Savehs Print x The Solvent File Name box displays the name of the currently open solvent reagent file To open a different file select a different file from the pull down menu To the left of each row is an O or an X O indicates volumes for that bottle position are measured by timed deliveries while X indicates volumes for that bottle position are measured by sensor controlled deliveries using a metered loop 38 1 800 477 6834 The columns are 1 Pos Solvent reagent bottle position 1 8 2 Name Name of solvent or reagent 3 Abbrv Abbreviation for solvent or reagent
105. s The Settings window allows a logged in customer or technician Section 2 6 2 to set the system defaults Click on the Tools menu and select Settings 87 www peptideinstruments com File Operations Tools Reports View Help m Pm Ki Database D Logout Settings Diagnostics Operation Times Operations This will open the Settings window Settings BE x r Operation Settings Facility Protein Technologies Inc Machine Name Pe Current Amino Acid File Standard Current Solvent File standard e Operator DB DataiMASTER_USER mdb Wash After Error IV Enabled Solvent 1 Reps 1 Force Block Rinse Before Mixing Enabled RV Size 10 mL or 40 mL 40mL r System Settings Default Operation Times standard D Report File Data MASTER_USER XML r E Mail Options Email Address info peptideinstruments com On Error IV Cycle Progress On Notified Step Indicates that the system must be re started for changes in these settings to take effect cm The Operation Settings are as follows 1 Facility Enter name of Facility 2 Machine Name Enter name for Prelude computer 3 Current Amino Acid File Values from this file are displayed in the Bottle Preparations screen in the Name column The default setting is Standard Select a different amino acid file using the pull down menu 88 1 800 477 6834 The Prelude software m
106. s Running Cycle step program operation repetition and current action are displayed on a light green background 3 Synthesis is Paused Cycle step program operation repetition and Pause are displayed on a yellow background 4 Synthesis is in Error Cycle step program operation repetition and action are displayed on a flashing red and yellow background IMPORTANT It is important to manually drain all reaction vessels prior to resuming after an error When a synthesis is resumed following an error it will start at the beginning of the step not where it left off as in a regular pause Thus if reaction vessels 1 3 were filled prior to the error it will start filling at reaction vessel 1 and reaction vessels 1 3 will have been filled twice 5 Synthesis is Complete Done is displayed on a white background When there is an error the details of the error are reported in the Error Reporting Box located in the lower left corner In the center of the screen the synthesis name is displayed at the top over an arrow that indicates the direction of the synthesis Below the synthesis name peptide sequences are displayed in numbered rows corresponding to the 6 reaction vessels The white box at the lower center of the screen displays the status of the current operation On the right side of the screen the name of the currently running program is displayed in the upper box while the steps of the program are displayed
107. s positions 1 9 Bank 2 displays positions 10 18 and Bank 3 displays positions 19 27 The columns are labeled as follows 1 Pos Amino acid bottle position 1 27 2 Name Name of amino acid or other chemical 3 Abbrv Three letter abbreviation for amino acid or other chemical 4 Key One letter abbreviation 5 Dep MW g mol Molecular Weight without protecting groups 6 Pro MW g mol Molecular Weight with protecting groups 7 GLP Data a Vol mL Volume used for synthesis in mL b Conc mM Concentration of amino acid solution used for synthesis mM c Opened Date Date and time solution was opened 36 1 800 477 6834 d Lot Number Lot number e Source Number Source information catalog number company etc By default a new file contains the names and molecular weight values of 20 standard amino acids Double click in a cell to modify its value The function of each button is 1 New The New button creates a new file The Amino Acid File Manager will open Enter a new name or use the default and click OK or click the Cancel button to return to the Amino Acid Editor without creating a new file 2 Close The Close button is not active until the file is saved After saving the file click the Close button to exit the Amino Acid Editor screen 3 Cancel The Cancel button removes any changes made since the last Save A screen will appear reading Abandon All Changes
108. s screen is available for technician and factory use only It is used to troubleshoot and repair the instrument by allowing the user to control individual valves 2 6 5 Operation Times The Operation Times screen is available for factory use only It is used to edit the control times for individual software operations 2 6 6 Operations The Operations screen is available for factory use only It is used to edit the software operations available for use on the instrument 2 7 Reports Menu 2 7 1 Jobs When a synthesis is run all information related to that synthesis is stored in a job file Job files are assigned a job number and are stored in the default database file defined under Settings Section 2 6 3 Jobs may be previewed on screen or sent to a printer To access and print a job file click on the Reports menu and select Jobs 90 1 800 477 6834 File Operations Tools Reports View Help E m ds pi Gomm Log The Reporter window will open fi Reporter Ver 1 0 0 93 ES UserJArchive DataBase Include In Report Data MaSTER_USER mdb ona IV Job summary IV Job Details Date PTI 3 9 2006 11 J_ Job Amino Acids Standard 3 9 2006 11 Job Solvents Reagents Job Program Summary Preview Print Print to File The User Archive DataBase section displays the current database file while the job files from the database are listed in the table To select a job file from a different data
109. se closes the File Manager screen Click the Close button or the X in the top right corner of the screen to exit the File Manager 2 4 File Menu 2 4 1 Amino Acid Editor The Amino Acid Editor allows the user to describe the contents of each amino acid bottle and create a good laboratory practice record of the volumes and concentrations of amino acids consumed as well as the date opened the lot number and the source number To open the Amino Acid Editor click on the File menu and select Amino Acid File Operation Tools Reports View Help Solvent Reagent Synthesis gt Exit The Amino Acid File Manager will open as anew screen To create anew amino acid file click tne New button enter a name for the new file and click OK Alternatively select an existing amino acid file from the list and click the Open button The amino acid file will open in the Amino Acid Editor 35 www peptideinstruments com 1 Amino Acid Editor Standard d x Amino Acid File Name Standard x Bank 1 C Bank2 C Bank3 GLP Data Pos Name Abbrv Key Dep MW g mol Pro MW g mol vol mL Conc mM Opened Date Lot Number Source Number Cancel Save Save s Print The Amino Acid File Name box displays the name of the currently open amino acid file To open a different file select a different file from the pull down menu Bank 1 Bank 2 and Bank 3 selections determine which amino acid positions are displayed Bank 1 display
110. solution 5 Job Program Summary Displays the job number name of the synthesis ID synthesis file number and the date and time the synthesis file was last modified A table lists the programs run at each cycle The columns are a Starting Cycle The first cycle the program was run at b Program Run The name of the program run at that cycle and subsequent cycles until the next Starting Cycle listing Job Program Details Displays the job number name of the synthesis ID synthesis file number and the date and time the synthesis file was last modified A table lists the individual steps of all programs run during the synthesis The columns are a Step Program step b Operation Operation executed during the program step c Solvent Name of the solvent reagent amino acid delivered during the operation d Mix Time Nitrogen mixing time in HH MM SS e Repeats Number of times the step was repeated f Drain On Displays a checked box if the RVs were drained following the step and an unchecked box if they were not The buttons are 1 Preview Clicking the Preview button will open the job reports selected in the Include In Report section in a Report Preview window Use the magnifying glass at the top of the Report Preview window to view the document at different magnifications and the left and right arrows to navigate between pages Click on the printer icon at the top of the window to
111. solvent reagent bottle 1 Make sure the solvent reagent bottle position is vented See Bottle Preparations screen Section 2 5 1 1 2 Verify the o ring is properly installed on the cap insert and that the insert is in the cap Also verify that the fluid line has a bottle filter with frit attached 25 www peptideinstruments com 3 Place the bottle in the bottle container Insert the line so that it is straight and at the bottom of the bottle the tubing can be molded by gentle bending Do not kink or the tubing integrity will be compromised 4 Attach the cap and tighten to a firm hand tight To remove the bottle make sure the bottle position is vented and unscrew the cap while carefully guiding the tubing and filter out of the bottle 1 2 7 Waste Container Installation To install the waste container Waste Level Cable Connector Wade Waste C Level ap Sensor 1 Place the waste container into a secondary containment vessel if available 2 Align both red dots to properly insert the waste level cable connector into the waste level sensor located at the top of the waste container Plug the other end of the waste level cable connector into the waste level sensor port located on the utility panel 3 Connect the three shorter 1 4 waste lines to the waste fittings on the solvent feedthrough panel and insert them into the waste tank cap 4 Connect the longer 1 4 vent line to the exhaust vent adaptor fi
112. t Action c Pos Position of RV bottle or other code depending on the operation d Rep Repetition e Operation Operation 92 f g h 1 800 477 6834 Description Detailed description of operation OT Operation Type i M Manual operation ii A Automated operation ii R Restart iv D Done Date Time Date and time of operation 3 Job Amino Acids Displays the job number and a table of the information entered in the Amino Acid Editor Section 2 4 1 The columns are a b C g Position Amino acid bottle position Amino Acid Full name of amino acid Source Number Source information for amino acid Opened Date and time bottle was opened Lot Number Lot number of amino acid Concentration mM Concentration in mM of amino acid solution Volume mL Volume in mL of amino acid solution 4 Job Solvents Reagents Displays the job number and a table of the information entered in the Solvent Reagent Editor Section 2 4 2 The columns are a b C Position Solvent reagent bottle position Solvent Full name of solvent reagent Source Number Source information for solvent reagent Opened Date and time bottle was opened Lot Number Lot number of solvent reagent 93 www peptideinstruments com f Concentration mM Concentration in mM of reagent solution g Volume mL Volume in mL of solvent reagent
113. t Breakers Power Cord e Power Supply Receptacle e Computer e Vacuum Pump USB Ports Waste Level Sensor Port Video Port Vacuum Mouse amp Nitrogen Keyboard Inlets Ports Network Port 12 1 800 477 6834 1 1 4 Reaction Vessel System The Prelude reaction vessel system is designed around a simple and reliable quick release mechanism Cam levers allow the operator to remove and install the six reaction vessels quickly and easily An In Place detection sensor verifies that an RV is present prior to solution transfer operations NOTE All active RV s must be in place at all times for the instrument to function 1 1 5 Cleavage Collection System The collection system for the Prelude accepts 50 mL polypropylene vials The system is made of materials resistant to the aggressive reagents associated with cleavage solutions A positive seal ensures cleavage solution vapors are vented An In Place detection sensor verifies that a vial is present at all times NOTE All six collection vials must be in place at all times for the instrument to function CAUTION The collection tubing is not rinsed automatically since it exits into the collection vials Therefore it is important to perform a cleaning process after each collection operation is completed and the collection vials have been replaced The cleaning operation is denoted as a Collect Back Flush under Cleaning in the Operations menu See Section 2 5 3 5 f
114. t EE 12 1 1 4 Reaction Vessel SVSIGITI 0 denos rep iene a 13 1 1 5 Cleavage Collection System ENNEN 13 1 1 6 Amino Acid ee E 13 1 1 7 Solvent Reagent Bottle System ENEE 14 11 8 Waste System minin Me hU eM D ee 16 1 1 9 Ventilation System een 17 US Bi La Be tee Systen anna nt 17 RIA Vacuum System uenis Cete ue Certus uu ie canner ec E ER E CA 18 1 1 12 Computer System escis oi e t ee Di as D co Ex donus 18 EN 83ey SHIBIO nie hte Fere Tace ere Fe ec fe MeL en 19 Tee IBS DIRIGE SEID AEE A 19 1 2 1 Laboratory Requirements 111 19 1 2 2 Instrument Installation Procedure seeeeeeeeeeseseeeeeesss 20 1 23 BV amp O Ring Installation ae essen 21 1 2 4 Collection Vial Ins tallatiori ss 22 2222er 23 1 2 5 Amino Acid Bottle Installation u 4444444444HHnnnnnnnnnn nennen 24 1 2 6 Solvent Reagent Bottle Installation ernennen 25 1 2 7 Waste Container Installation uuu44444444444BHnnnnnnnnnn nennen 26 EE eegen 27 1 3 1 Reaction Vessels amp O RIIngs 22 22 ea 27 1 3 2 Amino Acid Ra EE 28 aes ro Collection Vals een tere a a E 28 1 3 4 Amino Acids amp Reagents for Peptide Synthesis 28 1 3 5 Replacement Parts amp Additional Accessories 29 Chapter 2 Introduction to SOTIWaLre nn 31 E Be BEE Mec LM LUN D DN E 31 2 2 SNORCUFDUNONS e es 31 23 Elle Manager sauna 32 24 Fle NIBH Sdn hai D n x I SE 35 2
115. t system with 214 nm detection water 0 1 TFA and acetonitrile 0 1 TFA mobile phases and a reverse phase column e Optional autosampler Protocol Inject a small amount of each amino acid over a two week period to determine if there is any degradation of the amino acid For example 10 pL of the amino acid solution diluted to 1 mM at the following time periods initial 3 days 7 days 10 days and 14 days This data will provide you with the stability of the amino acid in the primary solvent Gradient is dependent on column Typically a 5 to 75 gradient over 20 minutes is used 123 www peptideinstruments com C Accessories Replacement Parts amp Tools Catalog No Accessories Quantity PPS R10 030 Pkg of 30 PPS R10 090 Reaction Vessel 10 mL PP Pkg of 90 PPS R10 180 Pkf of 180 PPS R45 030 Pkg of 30 PPS R45 090 Reaction Vessel 45 mL PP Pkg of 90 PPS R45 180 Pkg of 180 PPS GRV40 1 Reaction Vessel 40 mL Glass 1 ea P EINE E Ud O Ring Reaction Vessel Bottom PI SE EE O Ring Reaction Vessel Top Ee 2 Ls PPS ORING BK 06 O Ring Premium Reaction Vessel Bottom Pkg of 6 PPS ORING TK 06 O Ring Premium Reaction Vessel Top Pkg of 6 Neid Bottle 10 mL Single Shot AA UM MP VX 2 Pkg of 20 ee oe EE Pkg of 100 AAR 400 I 1 ea Mee BOE A00 MAA Pkg of 10 SMP RF 100 Bottle Filter Frit 1 8 Tube Pkg of 100 SON 270060 Bottle
116. t the user from F a Bottle Preparations E Bottle Preparations Special Bottles Solvent Calibration x manipulating the bottle using the Bottle Preparations screen by making the position inactive see figure below This feature keeps the TFA bottle vented and unprimed except during a delivery for safety reasons and to minimize the release of fumes This feature must be selected for the TFA bottle during use since the Cleave Collect operation includes pressurizing and priming the bottle prior to the delivery and venting and back flushing the TFA bottle following a delivery This feature must be deselected for the TFA bottle during Solvent Calibration Section 2 5 1 3 E Bottle Preparations E Bottle Preparations Special Bottles Solvent Calibration r Solvents 1 DMF 2 M DCM 3 M Dep 4 Cap D M Base 6 i 7 8 Pressurized Primed m Amino Acids Hla Cys Asp N Glu Phe Gy IN Clear All His Ile Lys 0 cO So C 4 w N Pressurized Primed Leu Met Asn Pro Gln Ag Ser Th Val Select All Pressurized Primed Pressurized Primed 19 Tp 20 T Ty 21 aT Az 22 AT A22 23 423 24 AT A24 25 AT A25 26 AT A26 27 AT A27 _Bressuize Prime Vent Nitrogen Back Flush Solvent Back Flush Pause Cancel Close Pressurizing bottles done 55 Xj www peptideinstru
117. tection DMF wash amino acid addition no drain Program 2 Activator addition from bottle 5 no drain Program 3 Activator addition from bottle 6 no drain Program X Coupling mix DMF wash RV s 1 amp 2 run program 1 RV 2 sits idle while RV 1 runs program 2 When program 2 is complete RV 1 sits idle while RV 2 runs program 3 Finally both RV s run program X to complete the cycle Both RV s will then couple their next amino acid with the default program D Example 3 Couple different modifiers at the end of the synthesis Program Assignment 2 1 D D RV 1 Sequence 1 A A RV 2 Sequence 2 AA At the end of the sequence poly A RV 1 couples a modifier from amino acid bottle 1 to peptide 1 using program 1 while RV 2 sits idle When program 1 is complete RV 1 sits idle while RV 2 couples a modifier from amino acid bottle 2 to peptide 2 using program 2 4 3 E Mail Notification The optional E Mail Notification feature allows the Prelude to send emails to a specified email address under the following conditions 107 www peptideinstruments com 1 At the beginning of each synthesis cycle 2 Ata specific step of a synthesis program 3 When a user error occurs This feature may be activated by purchasing a license from PTI To purchase a license contact PTI customer service at 1 800 477 6834 Catalog No Description Quantity PPS TXT EML RPT E Mail Notifica
118. the following menus File Operation Tools Reports View Help a Im ra pa ap in 2 2 Shortcut buttons Six shortcut buttons are located below the main menu They open the following screens ra Program File Manager Pi Sequence File Manager SCH Synthesis File Manager ea Bottle Preparations Screen y Manual Operations Screen i My Reaction Vessel Operations Screen 31 www peptideinstruments com 2 3 File Manager The File Manager is the main interface screen for manipulating the following file types Amino Acid Solvent Reagent Program Sequence Synthesis mp ol As an example the Program File Manager is shown below Program File Manager 1 bx Open Rename Save As Print Delete Close The File Manager lists all files of that type in the box to the left of the screen with function buttons to the right To select a file click on its name to highlight it When a file is highlighted all buttons to the right activate The function of each button is 1 New creates a new file a Click on the New button This will open a new window Program File Manager x New File Name Cancel 32 1 800 477 6834 b Enter a name for the new file or accept the default name and click OK to create a new file or click Cancel to return to the File Manager screen without creating a new file c After clicking OK the File Editor for that file type will open with the new file
119. the Solvent box Solvent 1 or 2 and the number of repetitions in the Reps box 1 9 109 www peptideinstruments com 110 1 800 477 6834 Chapter 5 Cleaning amp Maintenance 5 1 Cleaning amp Maintenance Schedule e Wash RVs Section 2 5 3 8 e Bottle Position Flush used bottles Section 2 5 3 2 Every e Collect Back Flush Section 2 5 3 5 Only After Synthesis Cleave e Cleave Bottle Solvent Back Flush Section 2 5 3 3 Only After Cleave Every Two e System Clean Section 2 5 3 1 Weeks e Replace RV upper and lower o rings Section 1 2 3 Quarterly or e Computer Maintenance Section 5 2 2 Semi Annually e Solvent Calibration Section 2 5 1 3 e Solv 8 Valve Replacement Section 5 2 3 Ainnueny e Amino Acid Bottle Seal Replacement Section 5 2 6 e Nitrogen Leak Check Section 5 2 4 As Needed e Bottle Filter Replacement Section 5 2 5 e Amino Acid Bottle Seal Replacement Section 5 2 6 e Solvent Bottle Seal Replacement Section 5 2 7 111 www peptideinstruments com 5 2 Operations 5 2 1 Cleaning Operations The following cleaning operations are available on the Prelude and are covered in Section 2 5 3 System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs Qo OD OT qe OF rorem 5 2 2 Computer Maintenance The computer is an important component in the op
120. the box a second time will deselect the RV The Copy To All RVs selection runs the calibration in RV 1 only then copies the resulting calibration factors to all 6 RVs CAUTION Using the Copy To All RVs feature significantly shortens the calibration time but may result in a delivery volume variation of up to 10 which is sufficient for most solvents The greatest accuracy is obtained when all 6 RVs are selected during a calibration This is recommended for reagent deliveries that require greater accuracy After running a calibration the Actual Volume uL column becomes active Measure the actual volumes delivered to the collection vial s and enter them in the Actual Volume uL column CAUTION Make sure RVs and collection vials are installed on the instrument for the selected RV positions before running a calibration To calibrate a bottle 1 Select a solvent bottle in the Solvent Bottle section by clicking in the appropriate circle 2 Enter the volume in microliters that will be delivered to an RV from the selected bottle during a synthesis in the Target Delivery Volume uL box 3 Enter the number of times the volume will be delivered to the selected RV s in the Number of Deliveries box 4 Select the RV positions to be tested by clicking in the box to the left of the desired RV s in the Test RV In Place column Click in the box a second 58 1 800 477 6834 time to deselect an RV Alternativ
121. the running operation as well as errors 2 5 3 Cleaning The cleaning operations are System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs Qo ED en PO e Each operation is decribed in the sections below 2 5 3 1 System Clean System Clean rinses the entire fluid system with Solvent 2 and should be performed every two weeks Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks and Wash RVs are all performed as part of a System Clean 72 1 800 477 6834 1 To perform a System Clean place empty amino acid and solvent reagent bottles in all positions Place empty RVs and collection vials in position 2 Place 1 L of methanol or other cleaning solvent in the Solvent 2 bottle 3 Click on the Operations menu select Cleaning and then select System Clean File Operations Tools Reports View Help g Bottle Preparations gt In RY Operations Bottle P Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RV s 4 The following warning window will open Verify all bottles RVs and collection vials are in place then click OK to start the procedure or Cancel to exit the cleaning procedure suser 0 9 WARNING ALL BOTTLES
122. tion License 1 To use 1 Login as a supervisor Section 2 6 2 2 Open the Settings screen Section 2 6 3 and go to the E Mail Options section 3 Enter an email address in the Email Address box 4 Select one of the following options a On Error sends email when the Prelude goes into error b Cycle Progress sends email at the beginning of each synthesis cycle c On Notified Step sends email when the operation E mail Notification is performed as part of a synthesis Program 4 4 Single Shot Delivery The Single Shot delivery feature is an advanced feature that allows the entire contents of an amino acid bottle to be delivered to a specified reaction vessel A 10 mL amino acid bottle is available for use with this feature Catalog No Description Quantity AAR SSI e TM 1 ea AAR SSX Bottle 10 mL Single Shot AA Pkg of 10 The functions of this feature are described in detail in Section 2 5 1 2 Special Bottles 108 1 800 477 6834 4 5 Wash After Error The Wash After Error feature can be enabled using the Settings screen Section 2 6 3 When enabled the Prelude will wash all six RVs with Solvent 1 or 2 for up to 9 times if an error occurs This keeps your reaction safe from unwanted side reactions and their resulting impurities To enable this safety feature check the Enabled box in the Settings screen to the right of Wash After Error Enter a wash solvent in
123. top of all 6 reaction vessels through the vessels to waste After draining the solvent nitrogen is delivered from the top to dry the RVs Wash RVs should be performed every time a reaction vessel is used to remove residual reagent from the RVs delivery lines and block Wash RVs is performed as part of a System Clean but it can also be performed alone using Solvent 1 DMF 1 To perform a Wash RVs pressurize and prime Solvent 1 DMF using the Bottle Preparations screen See Section 2 5 1 1 2 Click on the Operations menu select Cleaning and then select Wash RVs 82 1 800 477 6834 File Operations Tools Reports View Help Bottle Preparations gt tu RV Operations ht System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks 3 The following warning window will open Verify all 6 reaction vessels are in place and click OK or click the Cancel button to cancel the Wash RVs operation SUser B xj 2 WARNING ALL Rs MUST BE IN PLACE Press OK to start and Cancel to cancel Cancel 4 During the Wash RVs operation the Bottle Preparations screen will open be Ei Bottle Preparations Bottle Preparations Special Bottles Solvent Calibration r Solvents r mino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed Fs E F Cys Asp E Gu FT Ph
124. trol 11 18 101 TME sushi 43 71 96 Molecular Weight 10 36 37 45 49 65 69 94 Nitrogen Back Flush 53 75 76 103 117 Cleave Bottle 72 78 79 103 114 Flow Meter 115 116 119 E iso ee 102 Ile 12 17 115 Leak Check 20 113 115 Pressure 17 Pressure Gauge 102 119 Push Flow Control 11 18 SPAMS ss teca eosdem cn 102 SUD Vai oes 19 101 102 115 Syste Miao cientes 17 115 120 Tank Gauge 115 No Prime 16 55 101 105 114 Operations eene 92 114 Cleaning 15 51 72 114 Manual 31 60 63 70 72 85 Menu 13 40 50 51 54 60 84 PEOGFOITI s suo cs 39 42 43 62 Reaction Vessel31 60 65 67 119 Synthesis doe Sete 101 Bi 92 Operator DB 90 O Ring Bottle Insert 16 25 116 118 126 Installation Reaction Vessel Bottom21 22 126 Reaction Vessel Top 21 126 Pressure en TEEN 17 Gauge eun 11 17 102 1 800 477 6834 E ue Le WEE 51 53 NIG CN Rete REDE NO DNNEN 17 NAIVE sis dus er atau 17 51 Pressurize 16 17 21 51 53 75 76 79 81 84 101 115 116 119 Prime 16 51 54 75 76 79 81 84 119 Print IER anne 96 Program Cleavage 41 64 66 101 105 Details sense 96 EE 40 43 44 a E 32 41 42 44 86 114 File Manager 31 32 37 40 41 43 44 Operations 39 42 43 62 SEIS en 48 49 SU
125. ts com 6 Turn off the instrument 7 Disconnect the nitrogen tank 102 1 800 477 6834 Chapter 4 Advanced Synthesis Operations amp Optional Features In addition to its basic synthesis operations the Prelude has the following advanced synthesis operations GI equa D Automated Cleavage Dynamic Sequence Programming E Mail Notification Single Shot Delivery Wash After Error Some of these features are optional but can be purchased by contacting PTI customer service at 1 800 477 6834 The functions of each feature are reviewed in the subsections below 4 1 Automated Cleavage The optional automated cleavage feature performs cleavage and collection on the instrument To perform an automated cleavage 1 Select No Prime for the Solv 8 bottle using the Special Bottles screen Section 2 5 1 2 Create a cleavage program using the Program Editor Section 2 4 3 1 Set the cleavage to occur after a synthesis or alone using the Load Synthesis screen Section 2 5 2 2 Calculate necessary solvent volumes for cleavage by selecting the cleavage program in the Calculations AA Section 2 5 2 3 screen and viewing the calculated solvent volumes in the Calculations Solv Reag Section 2 5 2 4 screen Prepare cleavage cocktail in the Solv 8 bottle then pressurize and prime the bottle using the Bottle Preparations screen Section 2 5 1 1 Run synthesis and or cleavage using the RV Status screen Section 2 5 2 1
126. tting and insert the other end into the waste tank cap 5 Screw the cap onto the waste tank To empty a full container 1 Carefully disconnect the waste level sensor connector by grasping the knurled area of the fitting firmly and pulling directly up Unscrew the cap 26 1 800 477 6834 2 Empty the waste container and place it back into the secondary containment vessel 3 Screw the cap back on and reconnect the waste level sensor being careful to line up the red dots before applying pressure Do not force 1 3 Accessories 1 3 1 Reaction Vessels amp O Rings Technologies be 40 mL Glass 10 mL Disposable 45 mL Disposable Cat PPS R10 030 Pkg of 30 Cat PPS R45 030 Pkg of 30 Cat PPS GRV40 1 1 ea Cat PPS R10 090 Pkg of 90 Cat PPS R45 090 Pkg of 90 Cat PPS R10 180 Pkg of 180 Cat PPS R45 180 Pkg of 180 Reaction Vessel O Rings Bottom Premium Cat PPS ORING BK 06 Pkg of 6 Top Premium Cat PPS ORING TK 06 Pkg of 6 27 www peptideinstruments com 1 3 2 Amino Acid Bottles gt 10 mL Single Shot 120 mL 400 mL Cat AAR SSI 1 ea Cat SMP VX 20 Pkg of 20 Cat AAR 400 l 1 ea Cat AAR SSX Pkg of 10 Cati SMP VX 100 Pkg of 100 Cat AAR 400 X Pkg of 10 1 3 3 Collection Vials 5 il ECH 50 mL IEN Cat CLV 050 030 Pkg of 30 Ir 3 Cat CLV 050 090 Pkg of 90 E Cat CLV 050 180 Pkg of 180 1i 11 7 1 3 4
127. ttings screen Section 2 6 3 Input the mix time in Hours Minutes Seconds in the Mix time box Use the Drain box to select whether the selected RVs will be drained at the end of the operation If the Drain box is checked the selected RVs will drain at the end of the operation If the Drain box is unchecked the RVs will not drain at the end of the operation Input the number of times the operation will be repeated up to a maximum of 9 in the Reps box CAUTION When Drain is unchecked multiple deliveries may be made to the same RV without draining Be careful not exceed the RV s maximum capacity as this may force resin into the showerhead causing clogs or contamination 71 www peptideinstruments com When the operation AA Building Block is selected the Select Amino Acid section becomes active Click in the circle next to the desired amino acid position to select an amino acid for delivery The buttons are as follows 1 Start Starts the manual operation 2 Close Closes the Manual Operations screen 3 Clear Resets the Manual Operations screen to default values 4 Cancel Action Cancels the running operation CAUTION If an operation is cancelled during a delivery or drain residual fluid in the lines may contaminate the next operation Perform a Drain operation to remove fluid from the lines prior to running a new operation The Status Box at the bottom of the screen displays the actions of
128. ttle filters if necessary i e change of reagent Sections 3 2 amp 5 2 5 Select RV size in Settings screen Section 2 6 3 Calibrate solvent delivery volumes if necessary Sections 3 2 amp 2 5 1 3 2 Z Software Setup Create amino acid file Section 2 4 1 Create solvent reagent file Section 2 4 2 eje one oo oo oo Create swelling and synthesis program file s Section 2 4 3 1 Create cleavage program file s Section 2 4 3 1 Create sequence file s Section 2 4 3 2 Create synthesis file Section 2 4 3 3 e e e oo oo e Ole eoo eoe oe CO Set default amino acid and solvent reagent files in Settings screen Section 2 6 3 Load synthesis Section 2 5 2 2 Load synthesis amp cleavage program Section 2 5 2 2 Calculate amino acid solvent reagent resin amounts needed Sections 2 5 2 3 amp 2 5 2 4 Instrument Setup Prepare amino acids solvents reagents and load bottles on instrument Sections 1 2 5 1 2 6 Add resins to RVs and install on instrument Section 1 2 3 Install collection vials on instrument Section 1 2 4 Select No Prime for Solv 8 bottle Section 2 5 1 2 Pressurize and prime all bottles needed for the synthesis Section 2 5 1 1 Run Synthesis Click on Start in RV Status screen to run synthesis Section 2 5 2 1 Adjust nitrogen mix flow control if necessary Section 1 1 10 Olele Olelelele Post Synt
129. uL matches the Expected Collect Volume uL Restore Default Restores default calibration factors to selected solvent The Status Box located at the top of the screen displays instructions for how to perform a calibration While a calibration is running it displays the current actions of the instrument The Solvent Bottle section is where you select a solvent bottle for calibration To select a bottle click in the circle next to the appropriate label S1 S8 stand for Solvent 1 Solvent 8 bottom deliveries while Top1 amp Top2 stand for the Solvent 1 and Solvent 2 top wash deliveries respectively Only one solvent bottle may be selected at a time 57 www peptideinstruments com The Target Delivery Volume uL box is where you enter the calibration delivery volume For best results this volume should be the same as the volume in microliters that will be delivered by the selected bottle during a synthesis The Number of Deliveries box is where you enter the number of times the volume will be delivered to an RV s during the calibration The higher this value the more accurate the calibration The software calculates the theoretical volume that should be in the collection vial at the end of the calibration and displays it in the Expected Collect Volume uL box The Test RV In Place column allows you to select the RV positions to be calibrated Check the box to the left of the desired RV s to select an RV Clicking
130. uri i Pressurized Primed T Ala F Cys Asp E Glu E Phe E Gly E His F ile F Lys Select All Select All Rinse All Blocks Pressure Prime ve Nircgen Beck Flush Solvent Back Flush Pause Cancel Close DMF DEM F Dep Cap Base E Acti cd ach uj Ll 23 AT 423 24 AT 224 25 AT A 26 AT A 2 AT 527 E Act2 pF TFA on nao fF tQ Ww 443333333 BEE EME NE ENE E The status of the operation will be displayed in the status bar Click the Pause button to pause the operation Click Resume to resume a paused operation Click the Cancel button to cancel the operation Click the Close button to close the Bottle Preparations screen 80 1 800 477 6834 CAUTION f the procedure is cancelled there may be fluid left in the lines or blocks Perform a Clear All Blocks operation See Section 2 5 3 7 to remove any residual fluid 2 5 3 7 Clear All Blocks The Clear All Blocks function flushes each block with nitrogen gas to remove residual fluid without venting the bottles and contaminating the amino acids solvents and reagents Clear All Blocks is performed as part of a System Clean but it can also be performed alone as follows 1 To perform a Clear All Blocks operation click on the Operations menu select Cleaning and select Clear All Blocks File Operations Tools Reports View Help Bottle Preparations gt dg RV Operat
131. ust be restarted for changes to this setting to take effect Current Solvent File Values from this file are displayed in the Bottle Preparations screen in the Name column The default setting is Standard Select a different solvent reagent file using the pull down menu The Prelude must be restarted for changes to this setting to take effect Operator DB This database file will store all system data bottle settings synthesis definitions and the synthesis log file data of any syntheses run on the instrument while it is selected Individual operators may have their own database files for their own syntheses The default setting is MASTER USER mdb The Prelude software must be restarted for changes to this setting to take effect To select a different file click the Browse button To create a new database file make a copy of the MASTER USER mdb file preferably after an archive and rename it using Windows Explorer Wash After Error When enabled the Prelude will wash all six RVs with the entered solvent for the entered repetitions if an error occurs Click in the Enabled box to enable this safety feature A check mark indicates the feature is enabled Enter a wash solvent in the Solvent box Solvents 1 2 and the number of repetitions in the Reps box 1 9 Force Block Rinse Before Mixing When enabled the Prelude will perform a block rinse before mixing rather than after draining This is useful when long mix
132. v Reag Manual Operations or load a synthesis in the Load Synthesis screen and the Calculations AA screen will open automatically 65 www peptideinstruments com 3 Reaction Vessel Operations E x RV Status l Load Synthesis Calculations AA Calculations Solv Reag Synthesis Name Ste Del Cleave Program e Amino Acid Concentration mM 100 ReCalc Amino Acids R Calc Suggest Weight eS Volume Volume mg mb mL 1 3 GG i2 22 685 lanine 2 E E 6f 16 337 ysteine Trt 3 P 8 GI 22 905 partic Acid OtBu cere Ey Im ES lutamic Acid OtBu 5 i H 2 12 494 spartic Acid 6 E 3 ED i 468 lutamic Acid 7 F ia 2 12 551 yrosine tBu 2 8 d 8 20 30 1060 isoleucine 9 f E 12 22 1031 ysine Boc Pos Description R Calc Suggest Weight eS Volume Volume mg mb mL Eee iate gt m Re PDCDTDT me Fe PEA a aes Si Ee PT TT LI 18 kk FD Pos Description R Calc Suggest Weight eS Volume Volume mg ml mL 19 3 amp 16 843 ryptophan Boc 20 f ls 2 12 551 yrosine tBu 2 ff p il on 656 sparagine Trt 2 22 Er E ms e spartic Acid 23 E E ET 1 2 494 spartic Acid 24 mE E 1 11 468 lutamic Acid 25 f D 2 12 551 yrosine tBu 3 26 E D EN 2 424 soleucine3 Pos Description 27 E E 1 11 656 sparaaine Trt Print The Synthesis Name box displays the current synthesis Use the pull down menu to sel

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