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Peptide Synthesizer USER MANUAL

1. z Bi Synthesis Editor QC ES Program Sequence Synthesis Synthesis N il Solvent Reagent esis Name a f olvent Reager C D ec y 2 acena a oc 1 acsw y 3 y Comments Amino Acid fc Peptide Programming QCsw Current Cycle Tee Program Heat M J K 4 J 1 l gt J UN Sequence E e Original O N Molecular No Weight Sequence Length 2 DDDDDDDDHA ide p n hat e acacei 9 11 7 51132Y3HB6 g vs Del 192 c QCGLHRH 10 GHWSYGLRPH ins Dell e 11282 c QCACP2H x 1 T 6CCKEYKUG Bi ins Dal o 192 sofe acGLHRH 10 GHWSYGLRP ins Det 11282 li acacrs V QAAIDYING H ns Dal 1082 il QC G LHRH 10 GHWSYGLRPS rs dal e 11222 50C _ Cancel _Saveas Pm ie 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 a
2. File Operations Tools Reports View Help Settings Diagnostics Operation Times Operations 2 The following window will open Enter Password for Diagnostics access x Cancel 3 Enter the appropriate password and click OK Click Cancel to cancel the login To LogOut Click on the Tools menu and select LogOut 91 File Operations Tools Reports View Help ES PT oi er Settings Diagnostics Operation Times Operations 2 6 3 Settings 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 File Operations Tools Reports View Help CAE LogOut Settings Diagnostics Operation Times Operations This will open the Settings window 92 Operation Settings Facility Protein Technologies Inc Machine Name Prelude Current Amino Acid File standard bd Current Solvent File Standard y Operator DB Data MASTER_USER mdb Browse Wash After Error V Enabled Solvent 1 Reps 1 Force Block Rinse Before Mixing Enabled RY Size 10 mL or 40 mL 40 mL r System Settings Default Operation Times standard y Report File Data MASTER_USER XML Browse E Mail Options Email Address info peptideinstruments com On Error IV Cycle Progress Tl On Notified Step Indicates that the system must be re started for changes in these setti
3. 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 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 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 99 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 SUser File Operations Tools Reports View Help Oy a Pi rail 0 w 1 Reaction vessel Operations jel Reaction esse Operations Y 2 Bottle Preparations E E Bottle Preparations 2 9 Help Menu 2 9 1 Help Topics To ope
4. 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 Concentration mM Concentration in mM of reagent solution Volume mL Volume in mL of solvent reagent 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 98 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 6 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
5. If the flow is still greater than 25 cc min continue to the next bottle s until the flow is below 25 cc min When the flow is below 25 cc min the last vented bottle has a leak Check the bottle cap insert and o ring Check the supply tubing for cracks or leaks 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 iL 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 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 5 2 4 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
6. ap es H N N 3 H OH HN Narr 0 NHsww NHunn o wn vN o H H HO 0 ln 0 3 Diketopiperazine Formation Diketopiperazine formation is an example of cyclative cleavage In peptide synthesis peptides containing a C terminal proline attached to the solid support via a Wang linker can undergo diketopiperazine formation during the addition of the next amino acid This resulting cyclized product is cleaved from the resin resulting in lower overall yields for the synthesis Heat can significantly accelerate this side reaction in peptides containing C terminal prolines attached to the Wang linker resulting in very low to negligible yields It is highly recommended to turn the heat off during the addition of the next amino acid after a C terminal proline or to use Pro 2 chlorotrityl resin R Oop o Y R yAn hia o N HN A wo R Ry 4 Gamma Lactam Formation Gamma lactam formation occurs when the activated ester of the incoming Arg amino acid reacts with its own side chain and forms a ring This cyclized product is unable to couple to the growing peptide chain resulting in arginine deletion sequences Heat accelerates this side reaction creating higher levels of arginine deletion in sequences prepared with heat The best way to prevent this side reaction may be to turn the heat off during the arginine coupling step Double coupling methods have been reported to aid in the synthesis however they include a significant amount of time
7. K A O DO Date and time 8 28 2015 13 38 09 1000 qe on gt Step 1 Cyde 3 AA F Date and time 800 p nnn nnn nnn nn nnn enn nen nnn nen nnn nent n enna teen nent ence enn cate sansa santas eaten sansa neces 8 28 2015 13 38 09 Step 1 Cyde 4 AA N n MN re tions wen nnanAnk AO N Date and time 8 28 2015 13 38 09 w0 FM ee i Step 1 Cyde 5 AA E Date and time o el E rece 8 28 2015 13 38 09 Sten 1 Cuda A Abra 1 7 7 7 7 rd z 7 Time min T 2 2 lila 1 4 Step ij2 a2 2 s e Cyde RFlinle lk r n le la AA Page 1of 1 Individual Graph Refresh Back Forward Open Print a The buttons are as follows E Individual Graph Allows the user to change between graphs of a general UV synthesis or an individual repetition of deprotection Refresh Redraw the graph Back Move back through pages of the same graph or between data files Forward Move forward through pages of the same graph or between data files Open Shows an Open dialog where can be selected the UV file to see the graph Print Prints an image of the current graph and also the text information is at the right side OK Closes the dialog 76 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 Eo NI E S p N oe Each operation is d
8. 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 44 7 1 800 477 6834 Waste Collect Drains RV contents to collection vial The columns are La 2 Operation Use the pull down menu to select an operation for each step Volume uL Enter a volume in microliters The Prelude X 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 Mix Time H M S Enter the mix time in Hours Minutes Seconds The maximum mix time is 99 59 59 N2 Click to check or uncheck the box When checked the mix will occur with N2 bubbling Shake Click to check or uncheck the box When c
9. 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 105 3 3 Post Synthesis Procedures 1 Remove collection vials 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 lfa 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 lf 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 X 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 X 1 Perform a System Clean Section 2 5 3 1 then
10. mixing right diagram This process occurs once every 10 seconds during a monitored mix E 1 3 Advantages of the IntelliSynth UV Monitoring System By taking a measurement every 10 seconds during a mix it is possible to determine when the reaction has stopped progressing This means unlike other UV monitoring systems on the market the IntelliSynth system can actually determine the shortest deprotection time required for a step rather than just the number of repeats E 2 UV Graph Screen The UV Graph screen displays the UV absorbance graphs for individual deprotection reactions as well as overall syntheses The UV Graph screen can be accessed by selecting the UV Graph button at the bottom right of the RV Status screen or Manual Operations screen UV Graphs generate when UV Monitored steps are included in syntheses which are run in RV Automated Operations There are two graph types that can be displayed on the UV Graph screen 1 UV Summary Graph 2 UV Individual Graph 139 The buttons at the bottom of the screen are as follows Individual Graph Summary Graph When viewing a Summary graph selecting Individual Graph displays UV monitoring data for the Individual Mixes of the latest synthesis When viewing an Individual graph selecting Summary Graph displays UV monitoring data for latest synthesis Refresh Refreshes data for current screen Back Scrolls the graph to the left when full data set does not fit on scree
11. 10 EB Fmoc L Glu OtBu OH 10 mmol SMP 20 EB 20 mmol SMP 05 QT 5 mmol SMP 10 QT Fmoc L Gln Trt OH 10 mmol SMP 20 QT 20 mmol SMP 05 G 5 mmol SMP 10 G Fmoc 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 5 mmol SMP 10 1 Fmoc L lle OH 10 mmol SMP 20 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 SMP 05 M 5 mmol SMP 10 M Fmoc L Met OH 10 mmol SMP 20 M 20 mmol 124 Catalog No Amino Acid Quantity 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 WBC 5 mmol 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 FLA 5 A FLA 25 A FLA 100 A FLA 1KG A FLA 5 RBF FLA 25 RBF FLA 100 RBF FLA 1KG RBF FLA 5 NT FLA 25 NT FLA 100 NT FLA 1KG NT FLA 5 DB FLA 25 DB FLA 100 DB FLA 1KG DB FLA 5 CT FLA 25 CT FLA 100 CT FLA 1KG CT Description Quantity Fmoc L Ala OH Fmoc L
12. 119 Prime 17 51 52 53 54 77 78 79 81 84 119 Ermita ando lo 96 Program Cleavage 40 64 65 66 101 105 DetallS tar naan 96 Ed 39 42 43 Ela ee 31 40 41 43 86 File Manager 30 31 36 39 40 43 Operations 38 41 42 62 SEIS rr eee rere aca te 48 49 SUMMA osc ete eee toca ae ace 95 SS WETILMG mee eea E 40 Reaction Vessel 10 11 27 126 Installation anio 23 Operations 30 60 61 64 65 67 119 Sytem a eee e 13 Reagent aee E ence 29 Shelf Life amp Handling 125 Reagens o eee a 121 124 Rebuild Errors Table 85 Replacement Parts 29 126 Reporter Window 91 92 96 Reports ara 91 a 88 94 95 96 Meno aan 88 92 e AR 33 96 Rinse All Blocks 15 74 81 82 83 119 RV Status 17 60 61 63 65 70 Safety Shield 11 20 Sequence Edito 44 45 46 File 31 36 39 44 46 68 86 94 101 File Manager 30 44 45 46 Sel Stan Cycle idas 61 62 149 Settings41 52 87 88 89 90 91 92 ShoriculDBUtOnS A 30 SAMUI ONNIN cd 103 120 Single Shot DOM a ETAT 28 110 127 Delivery eenen 54 56 110 Solvent Back FUS Monsano 53 77 103 Cleave Bottle 15 74 78 79 101 103 106 113 Calibration 40 50 56 57 58 59 102 113 Feedthrough Panel 11 12 14 22 26 Solvent Reagent Bottle 38 51 54 55 69 74 95 Installation
13. 25 minutes at room temperature as part of the first coupling making it impossible to conclude whether the improved incorporation of Arg was due to the reaction being performed at room temperature before the heat was turned on or actually double coupling at the higher temperatures 135 NHR 5 Phosphate Group Cleavage Heat can cleave phosphate groups during the deprotection reaction Therefore when synthesizing peptides containing phosphate groups it is important to perform all deprotection steps after the phosphate group has been incorporated with the heat turned off 136 Appendix E Intellisynth UV Monitoring And UV Extend System The Prelude X Peptide Synthesizer can be field upgraded in your facility to a unit with UV monitoring PPX UV OPT With the UV monitoring and extend control system it is possible to monitor the extent of the deprotection reaction and use that data to control deprotection times and repeats and extend coupling times There are 3 main UV Monitoring operations on the Symphony X 1 Basic Monitoring Measures UV absorption of the reaction solution over time to determine the extent of the deprotection reaction but does not interfere with the synthesis 2 UV Extend Operations Measures the UV absorption of the reaction solution to determine the extent of the deprotection reaction and uses that data to control the deprotection reaction times and repetitions 3 UV Extend Operations and C
14. 93 Deprotectamt o eere e 15 Diagnostics eee vee cer ter see 92 DIM ias 41 42 72 85 96 Dynamic Sequence Programming 48 106 108 Editor AMINO AC ota 34 36 95 Program ricardo 39 42 43 SEQUENCE cocooocccccccccnnnnns 44 45 46 Solvent Reagent 37 38 39 95 SynthesiS 47 48 49 50 E Mail Notification 41 109 110 EMOS oee 18 56 62 72 119 120 Gritical crates ce eats 17 18 120 Email OG toe eee eee 110 FIECOVENY eaea A ian tnacitieteatnen 119 Reporting BOXG 2ceet 61 62 Wash After ecese 91 111 ESO E Acc 63 File Manager 30 31 32 33 34 AMINO ACI canada 34 36 Program 30 31 36 39 40 43 Sequence 0 30 44 45 46 Solvent Reagent 37 38 39 SynthesiS 30 47 49 50 File Menu 34 37 40 44 47 50 Flow Control Mixto a tatoo 18 101 Nitrogen PUSI 2 2 ce scccee eenean 18 Gauge Bottle Pressure 18 102 119 Nitrogen Pressure 18 102 119 Vacuum teca 18 101 102 120 Valve Pressure occccccnnn 18 102 GLPData an aa ace 35 38 Help Menu ceee en 97 100 Help TOPICS eae 97 99 Induction Heating 127 Infrared Heating Side Reactions cccccnnonn 131 Installation Amino Acid Bottle 24 25 Collection Vial ostias 24 ak iW lag tele meneteeerer ererepererererercrecrrs 21 P eee cece renee eee cee ene cece 23 Reaction Vessel eee 23 Solven
15. Arg Pbf O Fmoc L Asn Trt OH Fmoc L Asp OtBu O Fmoc L Cys Trt OH 125 Catalog No Description Quantity FLA 5 EB 59 FLA 25 EB 259 FLA 100 EB Fmoc L Glu OtBu O 100 9 FLA 1KG EB 1 kg FLA 5 QT 59 FLA 25 QT 259 FLA 100 QT Fmoc L Gln Trt OH 100g 1 kg FLA 5 HT FLA 25 HT FLA 100 HT Fmoc L His Trt OH FLA 1KG QT FLA 5 G 5g FLA 25 G 25g FLA 100 G poe eer 1009 FLA 1KG G 1 kg 59 259 FLA 1KG HT FLA 5 I 5g FLA 25 25g FLA 100 Eee er 1009 FLA 1KG I 1 kg 59 259 FLA 5 L FLA 25 L FLA 100 L Fmoc L Leu OH 100g FLA 1KG L 1 kg FLA 5 KBC 5g FLA 25 KBC 259 FLA 100 KBC Fmoc L Lys Boc OH 100 9 FLA 1KG KBC 1 kg FLA 5 M 59 FLA 25 M 25g FLA 100 M Fmoc L Met OH 100g FLA 1KG M 1 kg FLA 5 F 5g FLA 25 F 25g FLA 100 F Fmoc L Phe OH 100g FLA 1KG F 1 kg FLA 5 P 5g FLA 25 P 25g FLA 100 P OR 1009 FLA 1KG P 1 kg FLA 5 SB 59 FLA 25 SB 25g FLA 100 SB Fmoc L Ser tBu OH 100g FLA 1KG SB 1 kg FLA 5 TB 5g FLA 25 TB 25g FLA 100 TB Fmoc L Thr tBu OH 100g FLA 1KG TB 1 kg FLA 5 WBC 59 FLA 25 WBC 25g FLA 100 WBC A lt Oln 1009 FLA 1KG WBC 1 kg 126 Catalog No Description Quantity FLA 5 YB 59 FLA 25 YB 259 FLA 100 YB Fmoc L Tyr tBu OH 100g FLA 1KG YB 1 kg FLA 5 V 59 FLA 25 V 25g FLA 100 V Fmoc L Val OH 1009 FLA 1KG V 1kg A 3 Reagents amp Kits Please see www ptipep com for all of your peptide synthesis reagent needs Protein Technologies Inc offers a wid
16. Calculations Solv Reag Section 2 5 2 4 screen Prepare cleavage cocktail in the Solv 8 bottle Run synthesis and or cleavage using the RV Status screen Section 25 2 1 Following the cleavage remove collection vials and work up the cleaved peptide 108 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 BRS RNIN ISS AS Programming License i 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 Not Applicable in Prelude X mode 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 109
17. Cleave M on Program StandardCleave y C Now AtEnd Delay 08 30 2006 09 00 00 Load PTI For Help press F1 BB Ave Last Archive MASTER_USER_20060309 Gm AE GD GD 4 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 67 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 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
18. Individual Read will be added to the repeat If the absorbance changes less than the threshold for a set number of reads the repeat will end The repeat will end automatically if it has run for a maximum amount of time The number of repeats can increase depending on the observed UV absorbance in a Synthesis Graph After a minimum number of repeats is completed the instrument checks if the absorbance is above a set absorbance threshold If the absorbance is above the threshold another repeat is added and if the absorbance is below the threshold the operation will end The operation will end automatically if it has run for a maximum number of repetitions 143 A repeat will end when either condition is met A a a 3 3 10000 4 g Rep Fi Fi E E 2009 Sep 26 04 42 m 1 Loa a OD 3 yO 1 The number of consecutive 2 The Maximum Repetition absorbance readings that fall Time is reached within the Reaction Read Delta value of each other is equal to the Reaction Read Count The operation will stop repeating when any condition is met Le ee O ee O A 25000 25001 23000 xi SIB A gt y u 20000 EH a a ee PPP sE a Absorbance 3 15000 Threshold 15000 ai 13000 SE ee nee 10000 x09 SE Z s a O Mor sooo E o 5000 SI g 3 E 0 0 pi Time Time Time s Step 2 Step 2 Step 2 Cycle 1 Cycle 1 Cycle 1 144 1 The peak height i
19. PAOA BEE ote t A T PT E 88 264 Rebuild Ernrors Table ena 88 261 2 AS a e E R eg oe et Aree 89 216 2 OG kooo Ut e areata A e E A AeeAna ey 91 216 3 SEntingS aeaee e E toe pred E E E E 92 O aA DNE E MOE ET E E E E E EE T 95 216 5 Operation NIMES nores aaea ee oneness 95 2 6 6 Operations ae Aces cee cere ee ere E er Ro 95 ZF GD OUTS CME 0 ote Some hee cee o ae toca A ae ceo 95 BET AOS os Beck sot ate seco ce teen i doo eee e 95 PAT geod SF 011064 OEA 100 eas ola TENN UA teak eats tle T 100 ZO ClO eae erecetecceestte cece ce cecece e e EEEREN 100 2 9 CIDA OPICS i e ten tee semen tae E bomen Ota ence tte hee tee eee ence eR ee 100 NS USC A aca 103 Chapter 3 Basic Synthesis Operations se erae eae ea 104 a GICCKIIGL sage E A Seen ae Penstue fear caeeaetarize 104 3 2 Slat ec Instrument CNeck o 105 33 Post Synthesis PIO ed 106 A o REA RE EAE 106 Chapter 4 Advanced Synthesis Operations amp Optional Features 108 viii 1 800 477 6834 41 Automated Cleavage oo 108 4 2 Dynamic Sequence Programming oooooocccccccccnnnnncononcccnnnncnonnnnnnnnnncnnnnnnnnnns 109 AE MN e ac 112 AS also Di arrears career eer ee ere a 113 EEES O A em mA 114 Chapter 5 Cleaning amp Maintenance eco eee cca 116 5 1 Cleaning amp Maintenance Schedule ccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 116 A A neni rap ane ane cee nian re ee e ace aa 117 52 A Cleaning Operations eos e 117 5 2 2 Computer MMalimteManCer a near ensue ence
20. T RV2 Number of Deliveries iv AVS M RV4 Expected Collect Volume uL Tr AVS M AVE Run Close Cancel Refactor The Solvent Calibration screen has the following buttons 1 Run Starts running a calibration 2 Close Closes the window 3 Cancel Cancels a running calibration 4 Refactor Calculates new calibration factors based on Expected Collect Volume uL and Actual Volume uL values 5 Save Factors Saves new calibration factors Click the Save Factors button when the Actual Volume uL matches the Expected Collect Volume uL 6 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 60 1 800 477 6834 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 synthes
21. The waste container is vented through a fourth tube attached 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 19 1 800 477 6834 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 X 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 disassemble the switch connector assembly Itis 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 contai
22. This procedure may contaminate amino acids reagents solvents 3 Cancel 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 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 delivery To open the Special Bottles screen click on the Operations menu and select Special Bottles File Operations Tools Reports View Help Ed Bottle Preparations RY Operations gt Speci
23. 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 File Operations Tools Reports Yiew Help Bottle Preparations gt lan id RY Operations RV Status Cleaning Load Synthesis Calculations 44 Calculations Solv Reag Manual Operations or load a synthesis in the Load Synthesis screen and the Calculations AA screen will open automatically 68 1 800 477 6834 b e Reaction Yessel Operations x RY Status Load Synthesis Calculations AA Calculations Solv Reag Synthesis Name SiN mer Cleave Program X Amino Acid Concentration mM 100 ReCalc Amin
24. 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 85 File Operations Tools Reports View Help a Bottle Preparations gt lg RV Operations gt EL Cleaning ia 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 Ris 2 The following warning window will open Click OK to proceed or click the Cancel button to cancel the Clear All Blocks operation suse ee 2 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 Solvents F DHF F DCM F Dep F Cap F Base F Acti F Act2 M TFA o sn on euw N 4 Bottle Preparations Bottle Preparations Special Bottles Solvent Calibration m Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed F Ala F Leu PRE F Tp F Cys E T Het PA 20 F Ty F Asp E F Asa MT 212T 221 F Gu EN F Pro ME 2241 422 I Phe nn T Gin HN 23241 423 M Gy ENE
25. 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 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 xi Rename File lt CopyOfStandard gt as 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 a copy 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 3 x Save File lt Standard gt as Cancel 35 1 800 477 6834 c You may choose to keep this name or enter a different name Click OK to create a copy of
26. 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 X Data Archive folder and is located in a single file named according to the database file name and archive date Name_YYYYMMDD madb 90 NOTE Itis 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 Login LogOut The LogIn 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 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 Login
27. 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 1 4 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 Preparations and select Solvent Calibration File Operations Tools Reports View Help a SEEN Bottle Preparations RY Operations gt Special Bottles Cleaning Solvent Calibration This will open the Bottle Preparations screen with the Solvent Calibration tab active 59 1 800 477 6834 A Bottle Preparations xj 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 F s for testing Press Run to start test deliveries Note Delivery timeouts and Prime errors may result from incorrect Actual Volume entries Restore Default egal ieee samy j Restore default values and retry if calibration deliveries fail Test RY Actual Volume uL In Place FP RVI T Copy To All RVs p Solvent Bottle Ob er er AAA 150 C Topl Top2 Target Delivery Volume uL 1000
28. 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 121 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 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 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
29. 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 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 A xl Warning This procedure back flushes Nitrogen into selected bottle s This procedure may contaminate amino acids reagents solvents E Cancel 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 56 1 800 477 6834 o x Warning This procedure back flushes Solvent into selected bottle s
30. 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 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 Program Heat box shows a red dot icon if the program has the Heat option enabled and a gray icon if it is not The boxes below the icon show the temperature assigned to each Reaction Vessel These are informative only and cannot be edited from t
31. 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 3 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 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 11 TIME OUT Operation was not performed in the allotted time Press Start to continue with RV o
32. 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 Notification 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 X 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 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 113 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 X will wash all s
33. 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 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 A x uja A gt Q 4 Program Report Name Standard ID 48 Last Modified 5 15 2006 9 56 02 AM Sok ent Reagent File Standard Step Operation Solvent Volume MixTime Repeats Drain On l1 Deprotection 20 Piperidine DMF 1000 000230 2 Tme 2 DMFTop Wash Direthylfommamide 1000 000030 6 Tme 3 AA Buildirg 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 of the 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 36 1 800 477 6834 b Click on the Delete button This will open a ne
34. uncompromised speed yield reagent savings and flexibility creating the most complete peptide synthesis solution available Heating conditions in each of the 6 reaction vessels can be independently set to enable flexible customized reactions Real time UV monitoring with available Single Shot amino acid delivery ensures that reactions are completed efficiently and no excess reagent is wasted Combined with automatic cleavage and flexible pre activation options the Prelude X enables researchers to synthesize routine and difficult peptides with unparalleled speed and efficiency 1 1 About The Manual In this manual e Chapter 1 General Information describes the instrument layout basic installation procedures and Prelude X 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 X e Chapter 4 Advanced Synthesis Operations amp Optional Features describes advanced features and options on the Prelude X e Chapter 5 Cleaning amp Maintenance explains the cleaning procedures for the Prelude X 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 ser
35. 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 File Operations Tools Reports View Help a Bottle Preparations gt Qg RV Operations gt jane Cleaning System Clean Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitragen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs 3 This will open the Bottle Preparations screen 79 Y3 Bottle Preparations ay Solvents Pressurized Primed 1 Pom Mi E 2 Foo m 3 EL N 4 Fcp N 5 F Bae NI HI 6 Fic wW OP 7 T Ac N FRE 8 Tin Mi En Bottle Preparations Special Battles Solvent Calibration Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed ks 10 fa i 19 F Tp FR 2 6 HE qx FA 20 Ffy A S E de La aa 42 ii a fa te E 224 Az HE Sc E N T 14 5 N WM 2341742 N FEE amp M iw 6 E Mi 24241 42 iia re Bl mo E Ml 254142 E eaS Pen i 26 AT 4
36. 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 solvent 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 72 1 800 477 6834 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 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 Opera
37. 117 O EE cece eeres 117 5 2 4 Bottle Filter Replacement erene e E AEE E 119 5 2 5 Amino Acid Bottle Seal Replacement 2 ccceeeeeeeeeeeeeeeeeeees 120 5 2 6 Solvent Bottle Seal Replacement oooooocccccccccccccoononcccnonononnnnnnanananonoss 121 Chapter Gr o A cee eect ae eet cee eet aac eet cae REEERE ea seer 122 KCNN EMMONS ee ra eRe he te NRL ne RBs 122 6 2 Critical Errors No Operations Allowed ccccceeeeeeeeeeeeeeeeeeeeeeeeeeees 123 A aantet neeotemecenetgniatansobaneTet abner chan bem E EE ET NEEE 123 Appendix A Reagents For Peptide Synthesis ooooooooccccccccccconocccccccccnnnnnnnannns 124 A 1 Prelude X Pre Packed N Fmoc Protected Amino Acids Preweighed 124 A 2 Bulk N Fmoc Protected Amino Acids Preweighed cccccccccncccccnnnos 125 ASS ETEO IEE e nece 127 Appendix B Reagent Shelf Life amp Handling oooococonnncccconccccccconnnoncnanano 128 Balneagent Shellie x sonata aetna Aa a E A A E AE E AE ance 128 B 2 Amino Aid Solubility Festing meee meeeere nese econ etecereee teres rene 128 B 3 Amino Acid Degradation Testi c cccccesccecccec ceteeeseeeeesecccctceeceeesesesscccceeee 129 Appendix C o 129 Appendix D Induction Heating SyStemM coooonnnnnncccccnnncccnnnncnonncccnncnnnnnnnnananos 130 DEN EEI OAE E E E E E nn teas naee de 130 D 2 Advantages of the Induction Heating System ooonninccccccccnnncccccncnanannnnos 131 BRA COPIA A E E E A 131 BACH SNO POO
38. 2 FRE E Ni 18 fa N 2 al 427 E AAA Back Flushing Back Flush DCM bottle rose teva Close 4 lfa 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 5 Check the box es next to the bottle s that will be flushed 6 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 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 the 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 5 Amino Acid Bottle Seal Replacement for replacement procedures 7 After the cleanin
39. 34 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 y Og Solvent Reagent RI Program Exit Sequence Synthesis 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 5 Ef synthesis Editor PTI Program Sequence Synthesis Sequence Name Amino Acid File _ Amino Acids CV Single Shot PTI X Standard NA Desoto E Ee Sequence Definition coon 87 mee Current Position Length Molecular Weight g mol a Temperature PTIPEPTIDE123 E n ae amp se DEN a Bas will be applied if Heat is On in the Program Cancel Save 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 whose 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 contains buttons for each of the 27 b
40. 6 hours 21 min 51 sec PC ACPS_ModRvs frQaaIDviNe foon 0 47 20 42 55 22 71 6 hours 21 min 51 sec REGIHRH EnwaYeLRPE fronre 0 41 20 48 78 22 57 6 hours 21 min 51 sec rSolvents Reagents ona Activators ua Vas Pos Description Calc Yol mL no Description MW g mol Density g mL Conc mM mL Weight g Volume mL 1 Pimethylformamide 1078 2000 NMM 101 556 0 920 400 100 4 06 4 42 2 pi hk thi 38 E A 4 14 0 00 ichloromethane 1000 HCTU 413 700 0 000 100 100 3 pow Piperidine DMF 114 200 HBTU 379 300 0 000 0 0 0 00 0 00 4 pi thylfo ide 57 Ps 4 h 0 00 0 00 imethylformamide 100 yBop 442 300 0 000 0 0 5 pamnmom f 7 100 occ 206 300 ooo f of o 0 05 0 00 Ine 6 p 4MNMMIO 1M 57 3 EA z 7 Pimethyformamide 57 100 8 fra Cocktail 0 0 Print 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 th
41. 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 Appendix C Accessories 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 180 129 Reaction Vessel 10 mL Coated Glass PPX FGRV10 6 required for heat Pkg of 6 Reaction Vessel 40 mL Coated Glass PPX FGRV40 6 required for heat Pkg of 6 AAR SSI TM 1 ea AAR SSX Bottle 10 mL Single Shot AA Pkg of 10 SMP VX 20 Pkg of 20 SMP VX 100 UU as Pkg of 100 AAR 400 1 ea AAR 400 X SU GU o Pkg of 10 CLV 050 030 Pkg of 30 CLV 050 090 Vial 50 mL Collection Pkg of 90 CLV 050 180 Pkg of 180 Appendix D Induction Heating System The Prelude X Peptide Synthesizer can be field upgraded in your facility to a unit with induction heating and shaking Cat PPX IHEAT OPT amp PPX SHAKE OPT D 1 History of Heat in SPPS Heat has been used to aid in the syntheses of difficult peptides for the last 30 years Believed to be first used in 1984 by Janda and colleagues heating methods range from a simple oil bath to specially designed heated reaction blocks to infrared and induction heating to
42. 77 113 lead 15 16 Filter 14 16 25 26 77 101 102 Replacemert 14 113 Position Flush 15 74 76 77 119 PRESS UNO aro 18 Single Shot 28 110 127 Solvent Reagent14 38 51 54 55 69 74 95 mstallationm e 26 Seal Replacement 16 113 147 Bottle Preparations 14 17 24 26 30 39 50 51 52 53 54 55 56 75 76 77 78 79 80 81 82 83 84 85 90 119 Bottle Pressure Gauge 102 119 Bottom Delivery oooooccccccccono 38 Calculations DA A 60 65 67 70 126 SolV Rea g a 60 67 70 Calibration 40 50 56 57 59 102 113 Circuit Breakers ccicscsscasaaooonanns 12 22 Cleaning 13 15 74 75 77 78 79 81 82 83 84 85 103 119 and Maintenance 113 Operations arc eases erences eee ee 15 Clear All Blocks 74 82 83 84 Cleavage eioten 65 101 103 AUTOMATE ee ea 40 105 Collection System 13 Program 40 64 65 66 101 105 Solutio m a a camera 24 78 Cleave and Collee 16 41 51 DO Sr 15 16 Back Flush Nitrogen n se 74 78 103 Solvent 15 74 78 79 101 103 106 113 Collect Back Flush 13 15 74 79 80 81 101 103 106 113 Cleave ang cscicsccasnocacas 16 41 51 Collection Vial 11 29 41 127 COMITE sedante cansada 88 97 Computer Meaintenales nan 113 System eae e tects cee 20 Data Archiver ceee eee 86 87 Database File 86 87 88 91 92
43. 9 585 700 0 O 10 06 2015 08 50 52 3 Aspartic Acid OtBu Asp D 133 105 411 500 0 O 10 06 2015 08 50 52 4 Glutamic Acid OtBu Glu E 147 132 425 500 0 O 10 06 2015 08 50 52 5 Phenylalanine Phe F 165 194 387 400 0 O 10 06 2015 08 50 52 6 Glycine Gly G 75 068 297 300 0 O 10 06 2015 08 50 52 7 Histidine Trt His H 155 158 619 730 0 0 10 06 2015 08 50 52 8 Isoleucine lle I 131 176 353 400 0 O 10 06 2015 08 50 52 9 Lysine Boc Lys K 146 191 468 600 0 O 10 06 2015 08 50 52 new cose _saveas 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 displays positions 1 8 Bank 2 displays positions 9 17 Bank 3 displays positions 19 27 and CV Single Shot displays positions 28 33 The columns are labeled as follows 1 Pos Amino acid bottle position 1 33 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 w
44. Bottle System ea aa aaae e AA E AA ee 17 1 1 7 Solvent Reagent Bottle System osisscccsnie toe sesc tonne eeoteiesdoosnie cecusieetees 17 A A A E anna Wena eee oh eet eer 19 LTO Ventilation System araeir ent en eee ene tere eterna 20 ANO ii 20 Er Vacuum Syster e a E atom ieee seat oie ae beeen 21 1112 Right Gauge Ranell ene meee ee con aconbens EEE ea 22 e Gae aea e EE 22 Isi Emergency Stop BUOM enee neeaae e A aen 23 11 15 A ier ee a eaen eaae 23 O 23 AMstument SetUp ma aaa eaa coins aos ios 23 1623 Laboratory REQUIES MENS Iso isa 23 1 2 2 Instrument Installation Procedure c cceccgeecdec2 ceecece eeeeeee ceecceeceeerececuce 24 12 3 RV amp O Ringilnstallation eiee ee e E anera 26 122 4 Collect Viallnstallation n eee e veer eres E eee 27 1 2 5 Amino Acid Bottle Installation a meee eea Aa Raae 27 1 2 6 Solvent Reagent Bottle Installation connnnnccnnnnnccccnccnnnnnnnnnnn o 29 1 2 7 Waste Container lingtallatiOtcicc 2 ccccccteeeeece lt a reetceeteest ac eeaet ese tecetazctece 29 ME BIACCOSSONES en acres E EN ee en ee Ae Rar ATEN TW 30 181 Reaction Vessels amp OSOS ias 30 ca lo Bottles ae ane er shar Sen etreaten cine Tea iene EAE EE E 31 E A a 32 1 3 4 Amino Acids amp Reagents for Peptide Synthesis oooooncccccccccccccccccccnnn 32 1 3 5 Replacement Parts 8 Additional Accessories sseeeeeeeeeeeees 32 Chapter 2 InthOGucon to Software mieren ee e ee Ee aE E Eeee aee 33 ZV Wan CIN a A E AAE 33
45. 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 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 If a CV Single Shot is desired then click in circle next to CVSS 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 5 UV Graphs Shows the UV Graph dialog 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 the running operation as well as errors The UV Graph screen shows the user the UV Summary and Individual graphs of a synthesis To open it click on the UV Graphs button in the Manual Operation and RV Status screens 75 1 800 477 6834 m UV Graph Step 1 Cyde 1 AA F a Date and time UV Summary 1002 8 25 2015 14 47 30 Step 1 Cyde 2 AA
46. Clean and rinse housing with desired solvent and dry 6 Install new frit by pressing housing over frit Ye 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 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 5 Amino Acid Bottle Seal Replacement 120 The amino acid bottle seal should be replaced annually or as needed ill 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 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 Reinstall filter housing and frit see 5 2 5 5 2 6 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
47. DYING OH dissolving the final valine in DMSO so its coupling occurred in a 1 1 mixture of DMF DMSO virtually eliminated the valine deletion peak from the HPLC of the crude peptide 4 Try a more efficient activator If HCTU was insufficient to do the job try HATU In rare cases using an activator that operates via a different mechanism i e PyBOP or PyClock can improve results for specific sequences i e C peptide H EAEDLQVGQV ELGGGPGAGS LQPLALEGSL G OH PTI offers these high efficiency coupling reagents and others in our chemical catalog 5 Try increasing the temperature See below The following four items must be in balance in order to maximize coupling efficiency 1 Activator Efficiency 2 Reaction Time 3 Temperature 4 Sequence Difficulty Increasing each of the first three items alone can increase the coupling efficiency However increasing all three for an easy sequence may actually allow side reactions to occur resulting in a lower purity peptide Therefore when adding heat it is important to balance it with the other factors In general heat should be used with lower efficiency activators for short amounts of time i e DIC HOBt or HBTU for 1 5 minutes at 75 C If a coupling is extremely difficult as in the case of Aib or N methylated amino acids it may be necessary to use a higher efficiency activator like HATU and multiple couplings along with elevated temperatures D 3 4 Deprotection Reaction Typically
48. E Cleave Mix 9 Auto E q 0Q gt z_ oK 7 Collect 4 GHIKLMNPORSTVWY123456 4 8 DCM Top Wash Manual 9 Deprotection 5 GHIKLMNPORSTVWY123456 5 10 DMF Top Wash 6 GHIKLMNPORSTVWY123456 6 Action Filling Sensor Fill UY Graph Temps AVI RY2 RY3 Ava RAYS RYE 0 0 0 0 0 0 On the left side of the RV Status screen is the Set Start Cycle button the Colored Status Box and the Error Reporting Box The Colored Status Screen displays various messages on a colored background depending on the status of the synthesis as follows 64 1 800 477 6834 1 Synthesis is Loaded Ready is displayed on a dark green background 2 Synthesis is 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 bee
49. F Ara i 24 AT 42 N N N N N N N N I His ME TT Ser Mi 2521425 Select All F ile on TF Th MI war 426 F Lys FRE Ya fm 2 aT 427 0 00 nt oD oOo amp WwW NY Pressurize Prime 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 86 CAUTION If 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 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 File Operations Tools Reports View Help E Bottle Preparations gt E RY Operation
50. Molecular amp Sequence Length DD DDDDDDDDDDD DDD gt O H Wagh G PTIPEPTIDEN235 10562 7c GAdmanufact y 2 I MNPOQRSTVWY 128456 s Da 22867 c PTI aj PTIPEPTIDEM235 hs Da 10562 ae GAdmanufact j 2 I MNPORSTVWY 128456 ns Det 7286 7 c aa PTIPEPTIDEM235 ns Da o 10562 70fC QAtmenu act Y MNPORSTVWY 126 456 ns Del 77867 i No Heat Cancel Save 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 50 1 800 477 6834 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 Prog
51. NH Select All Select A Collect Back Flush Rinse Pressurize Prime Ven Nitrogen Back Flush Solyent Back Flush Pause cnca 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 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 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 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 File Operations Tools Reports Yiew Help E Bottle Preparatio
52. 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 106 6 Turn off the instrument 7 Disconnect the nitrogen tank 107 Chapter 4 Advanced Synthesis Operations amp Optional Features In addition to its basic synthesis operations the Prelude X has the following advanced synthesis operations IS 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 de 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
53. O A E A AA EEA arise 132 D 3 2 LOW Resin eee e E E ee ee 132 D3 COUDIMG KEACUO IN stein dem erates E E aR Ocean 132 DS Denrotectlom Reaction ca coda 133 D 3 5 Pseudoprolines Hmb amp Dmb Amino Acids and Dipeptides 134 D 4 Side Reactions Accelerated by Heat ccceeeesessseseeeeeeeeeeeeeeeeeeeeees 134 Appendix E Intellisynth UV Monitoring And UV Extend System 2 6 137 Es blew WN Monto MO WOrkS osito crsaetatetta a came nee cada 138 A A aia 138 E 1 2 The IntelliSynth UV Monitoring System ooooococccccccccccconccccccconononnnannns 139 E 1 3 Advantages of the IntelliSynth UV Monitoring System 245 139 ESA A obio 139 E O E cerere ae 140 p A O A a 141 E S Basic Mo Me a ee OMC cata 142 1 800 477 6834 E E 142 A Programe eee E E cee ae eeguaen ema cee eee a ae 142 E 4 Deprotection with UV Extend OperatiONS cccccoonnnnccccncnnnonicnnananncnnos 143 os Ma ie Oise cios one eee 143 E 4 2 Wntingia Program iia it Aenea nee en Re en ne ence as 145 E 5 Deprotection and Coupling with UV Extend Operations 0000 145 ENS THOVGIVICW aia ancora dios 146 E 5 2 o A a a 146 aero erect 147 1 800 477 6834 Xi 1 800 477 6834 Introduction Thank you for purchasing your new Prelude X peptide synthesizer from Protein Technologies Inc Building upon the strength of the original Prelude platform the Prelude X adds heating oscillation mixing and UV monitoring to deliver
54. 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 X 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 Yiew Help Amino Acid ry 90 Solvent Reagent Synthesis Program Sequence Synthesis 42 1 800 477 6834 Then select from one of the following three screens 1 Program 2 Sequence 3 Synthesis The function of each of these screens will be reviewed in
55. PEPA SINOGECUUI OUT GINS dotaciones E ane ar oeta eke eae eee anaes aa 33 2 OHI UM ANAC GT trees etek aca o 34 ae aS O OS 37 24 V Amino Acid Editon meae e Deh ee tere caee tat oe see cece tetas 37 242 Solvent Reagent Edit 40 vii 1 800 477 6834 A VIM SIS E ILO cece wena ge cere caterer E eee ete 42 2 ALS AUP FOOKan Editon acc cceace cams e E E ano ages 43 24 32 Seguenc ccrscteccer cen cee Base E cena econ ace eeeeages 47 A Roe 50 A AO nun AD ERRE ARDE OO anes 53 O allOme MOM Uess cee ce ecetecececte e ce ceed ne verk cet EL eNe Eae 53 a Preparations seere erea tau toe pecea nee eens enor na eats aes 53 Zoos Bottle Preparatoria omnia 54 PANS feller are od 57 251 9 Solvent Calibration rota ccuce decena enetace fant cee reaerans face 59 Die FAN OCT AUIOMS o2tcectcececiuae care cestcaseetsceececececs ert vacecerererevest sess see ctcereci ccs 63 25 O Ge CaN ee era 63 25 2 2 OAC Synthesis anan m ee Marinade ese A anew AE 67 25 23 Calculations AA e E E AE EE 68 2524A Calculations SOMA 2222 2 nackte eat eee eE eeaeee eaea 70 215 2 5 Manual Operations m reaa e ee e aee A 73 A E T 77 253 System elean e ee E AEE E AE EE AAE 77 25 3 2 BotleRostiomRI Sh aaee ce eee eee 79 2 5 3 3 Cleave Bottle Solvent Back Flush ooooooocccccccccccccconoccccccconnnnnnnnnnn 81 253 5 Collect BackoRlUshitwes Anti e A Ren E casa Ae 82 2 5 3 6 Rinse All Blocks mee E 84 215 3 M Cleats ABIKS eoe E AE E EE 85 OKO Ma RVS a AA AT 87 2 6 mools O 88
56. Prelude X Technologies Inc USERN E Prelude X Peptide Synthesizer USER MANUAL O 2015 Protein Technologies Inc 4675 S Coach Dr Tucson AZ 85714 USA All Rights Reserved Document 9030024 Rev 01 gt gt P 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 INSTRUMENT 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 COM a apn aaa on AREA E R DOS IEUN vii A E osa 12 Pr A A o 12 EE oa 12 13 COMMON ADDIGVIATIONS eater ate tte heats 13 Chapter General limonmation anios es oie hen eran nen acota icon Ont 14 1i General System DES eo acen 14 o et 14 Tele Solvent Feedthrough Panel ect 15 e ANG a A A T E A A E A EN 15 11 4 Reaction Vessel System reece cee e eee E Ee aE EE eai 16 12125 Cleavage Colection System s s tos 16 Tales Amino Acidi
57. 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 Sar pe Prt Restore default values and retry if calibration deliveries fail Solvent Bottle Test RY Actual Volume uL Es C52 C53 Css Cose Cm C Topz MPlece Jo SPFA Target Delivery Volume ful 1000 Ava WM Pv3 Number of Deliveries 3 Expected Collect Volume uL 3000 PAV 4 MF RAYS F RAYS Close Cancel Refactor Click the Refactor button to 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 62 1 800 477 6834 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 X software is opened To open the Reaction Vessel Operations screen click on the shortcut button or click on the Operations menu select RV Operations and select a sub section File Operations Tools Reports view Help iz Bottle Preparations gt la a RY Operations Cleaning Load Synthesis Calculations 44 Calculations S
58. This button should only be used in the event of an emergency and is not a viable way to pause an operation during a synthesis Pressing the Emergency Stop Button can result in a loss of reagents and or the current synthesis 1 1 15 Computer System The Prelude X has an internal computer that operates the Prelude X instrument and user software A monitor keyboard and mouse are supplied with the Prelude X They are connected to the Prelude X s internal computer via the utility panel Extra USB ports allow data to be transferred from the computer via a user supplied memory stick 1 1 16 Safety Shield Safety doors are installed for the protection of the user The doors in front of the reaction vessels MUST be CLOSED when the Prelude X 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 Opening the doors while a synthesis is running will result in an error and the synthesis will be paused 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 1 2 1 Laboratory Requirements In order to install and run the Prelude X a laboratory must be able to supply the following 1 Ventilation System The Prelude X has a 4 inch vent equipped with an adjustable angle adaptor The Prelude X should be c
59. V PV UVD Reps Deprotection 3000 0 00 30 X X RV Xtend Fb 1 DME Tep i sonar on o es al lees 3 Wash AA Building An RV Block 1000 0 00 00 X 1 Activator 1 1000 0 00 00 X RV 1 Base 1000 0 10 00 X X RV Use Fb 1 Top Delivery 3000 0 00 30 X X RV 1 AA Building QM RV Block 1000 0 00 00 xX 1 Activator 1 1000 0 00 00 X RV 1 Base 1000 0 10 00 X X RV Use Fb 1 Top Delivery 3000 0 00 30 X X RV 3 a4 Oo 146 Index About SUSOT 0 cccceeeeeeeeeeeee 100 Accessories 27 29 126 127 Activator 10 16 42 67 69 Alpha Keyboard 135 136 137 139 140 142 143 Amino ACIO o 10 29 122 Bottle 22 28 35 51 53 66 74 95 127 Installation 24 25 Seal Replacement 14 77 113 e sean eee cere aes 14 Degradation Testing 126 EGO ligarse E S 34 36 95 File 31 34 35 36 44 45 48 49 52 66 90 101 File Managet 00000 34 36 PRED ACKE scaner 121 Sie lillie aen eters ES 125 Solubility Testing 125 126 Archive All Data s cscenstecdasen 86 88 Back Flush Collect 13 15 74 79 80 81 101 103 113 NITROGEN eta 53 77 103 Cleave Bottle 74 78 103 SOIVEMILG aa 53 77 103 Cleave Bottle 15 74 78 79 101 103 106 113 Bottle Amino Acid 14 22 28 35 51 53 66 74 95 127 Installation ee 24 25 Seal Replacement 14
60. 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 the following menus SUser File Operations Tools Reports View Help ES Pr YS PA e En 2 2 Shortcut buttons Six shortcut buttons are located below the main menu They open the following screens A Program File Manager Sequence File Manager Mal Synthesis File Manager on Bottle Preparations Screen y Manual Operations Screen EL Reaction Vessel Operations Screen 33 1 800 477 6834 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 ci lee As an example the Program File Manager is shown below Program File Manager lx 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 34 1 800 477 6834 b Enter a name for the new file or accept the default name and click OK to
61. al Bottles Cleaning gt Solvent Calibration This will open the Bottle Preparations screen with the Special Bottles tab active 57 1 800 477 6834 43 Bottle Preparations xi Bottle Preparations Special Bottles Solvent Calibration m No Prime 1 r One Shot Delivery Solvent Reagent Amino Acid 1 DHF 1T Ala 10 T Leu 19 T Tip 2 F DCM 2T Cys 11 T Met 20 T Tyr 3 F Dep 3 T Asp 12 T Asn 21 M A21 4 F Cap 4 F Glu 13 T Pro 22 Y A22 5 Base 5 7 Phe 14 Gin 23 M 423 6 F Acti 6 7 Gy 15 7 Arg 24 IV 424 Z F Act2 7T His 16 J Ser 25 M 525 8 M TFA 8 lle 17 T Th 26 V A26 3 7 Lys 18 T Val 27 Mw Ae Bottle 8 has a special feature called No Prime it will prevent the user from manipulating the bottle using the Bottle Preparations screen by making the position inactive 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 Bottle Preparations Special Bottles Solvent Calibration Solvents Amino Acids Pressurized Primed Primed Primed Prime
62. as opened 38 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 Close The Close button is not active until the file is saved After saving the file click the Close button to exitthe Amino Acid 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 the No button will leave the changes 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 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 Print To print the open amino acid file click the Print button It will go automatical
63. d 1 for MM MM 1 Aa WW 10 Eeu WN 19 Emr N 2 moo mM N 2 Cys M 1 Me UN 2 ty MN 3 Dep Mu i 3 Asp UN 12 Asn UN 21 4 a21 4 icp MN N 4 Gu WW 13 Pro UN 2 gt Maz 5 Mbase MM N 5 ElPhe IN 14 Gn ON 23 4 F A23 6 Ati Mi N 6 Fay M 15 Ha WW 24 4 EJA24 7 Maz N 7 His UN 16 Hse N 25 2 EA 8 Emra E N 8 le oN 17 Th 26 2 PAG 9 bs M 18 va N 27 4 A27 Pressurized Select Al MIN Select Al Pressurizing bottles done pesanz i tose Ba Back Rush Pause Close 58 1 800 477 6834 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 X 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
64. d 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 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 to 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 46 1 800 477 68
65. day Below is a brief history of heating methods used in SPPS 1985 Tam synthesized TGFa using an oil bath 1986 Barlos synthesized leucine enkephalin using an oil bath 1991 Foutch synthesized AT III and other peptides using a recirculating oil bath 1992 Wang synthesized ACP 65 74 using a domestic microwave 2002 Erdelyi and Gogoll synthesized TVI and others using a microwave synthesizer 2012 PTI introduces the first peptide synthesizer to use infrared IR heating and synthesizes ACP 65 74 Aib enkephalin and others on the Tribute IR peptide synthesizer 2013 PTI introduces the Symphony X Multiplex Peptide Synthesizer available with infrared IR heating 2015 PTI introduces the Prelude X the first peptide synthesizer available with induction heating 130 D 2 Advantages of the Induction Heating System PTI s induction heating system offers extremely fast time to temperature as well as accurate temperature sensing without overshooting or overcorrecting Vortex mixing is used to ensure even temperature profiles With independent control and a compact design the use of induction heating allows parallel independent heating of all six reaction vessels Instruments from Protein Technologies Inc are the only rapid heating systems to come with the patented PTI fluidics system giving you maximum up time minimum solvent usage and worry free operation that lasts for years D 3 Recommended Use PTI r
66. 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 all RVs are present prior to executing a synthesis The Prelude X is also available with an induction heating and shaking option Induction heating makes it possible to assign unique heating temperatures to each reaction vessel position CAUTION All RV positions that are being heated must use an induction compatible reaction vessel Failure to use the correct RV will cause the heater to time out and result in a system error See Section 1 3 1 for a listing of available reaction vessels and accessories NOTE All RV positions including unused positions must have an RV present for the instrument to function 1 1 5 Cleavage Collection System The collection system for the Prelude X 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 col
67. dual 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 Itis used to reconfigure 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 88 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 mdb 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 maint
68. e 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 X Front Emergency Stop Right Gauge Panel Reaction Vessels Section 1 1 14 Section 1 1 12 Section 1 1 4 A ZN E ad 4 Vent also on Safety Shield nae pea me opposite side Section 1 1 16 Section 1 1 9 Mix Flow Control Section 1 1 13 Bottle Pressure Regulator amp N2 Push Flow Control Access Panel Section 1 1 10 Left Gauge Panel Section 1 1 13 A Collection Vials d Section 1 1 5 Utility Panel Section 1 1 3 Solvent Feedthrough Panel Section 1 1 2 Amino Acid Bottle Positions Section 1 1 6 14 1 800 477 6834 Fluid Delivery 1 1 2 Solvent Feedthrough Panel Ports Pressure Delivery Ports Bottle Vent Waste 1 Outlet Waste 2 Outlet Vent Fluid 1 1 3 Utility Panel Heater Power Switch Heater Power Receptacle USB Ports Network Port Power Supply Fan 3 Waste Level Sensor and Video Ports Nitrogen Inlet Main Power Switch Power Cord Receptacle 15 1 800 477 6834 1 1 4 Reaction Vessel System The Prelude X reaction vessel system is
69. e also used in certain cleaning operations The Bottle Preparations screen opens when the Prelude X 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 Bottle Preparations gt Bottle Preparations RV Operations gt Special Bottles Cleaning gt Solvent Calibration This will open the Bottle Preparations screen with the Bottle Preparations tab active 54 1 800 477 6834 Bottle Preparations Bottle Preparations Special Bottles Solvent Calibration Solvents Amino Acids Pressurized Primed Primed Primed Primed 1 Em a i 1 fa EN 10 le IN 19 Em M 2 DoM i i 2 Cys IN 11 Met N 20 Ty N 3 Der ia 3 Asp IN 12 Asn IN 21 azi IN 4 Cap N N 4 Gu N 13 Pro N 22 A22 N 5 Base N N 5 Phe N 14 Gin N 23 A23 N 6 Act1 N N 6 Gly N 15 Arg N 24 A24 N 7 Maz PEN N 7 His iN 16 Ose N 2 Dazs N 8 TFA N N 8 lle N 7 Thr N 26 A26 N 9 ys IW 18 Fiva IN 27 a27 IN Pressurized Select Al Select All Step Done Dep bottle 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 abbreviat
70. e selection of high quality coupling reagents resins and solvents as well as pseudoprolines and other specialty reagents Catalog No Start Up Kits Quantity Fmoc Amino Acid Start up Kit for the Prelude X Contains 30 x 10 mL disposable RVs 30 x 45 mL disposable RVs 6 x 10 mL coated glass RVs 6 x 40 mL coated glass RVs 0 9 L of 20 Piperidine DMF PPX STARTKIT 0 9 L of 0 4M NMM 0 1 mmol scale Rink amide resin 1 ea 0 1 mmol scale Fmoc Gly Wang resin twenty 5 mmol and twenty 20 mmol prepacked AA bottles one of each amino acid 100 g HCTU Assorted 5 mmol prepacked AA bottles for running test peptides Fmoc Amino Acid Start up Kit for the Prelude X Contains 30 x 10 mL disposable RVs 30 x 45 mL disposable RVs 6 x 10 mL coated glass RVs 6 x 40 PPX STARTKIT ML coated glass RVs 0 1 mmol scale Rink amide resin 0 1 mmol scale Fmoc Gly Wang resin twenty 5 mmol and twenty 20 mmol prepacked AA bottles one of each amino acid 100 g HCTU Assorted 5 mmol prepacked AA bottles for running test peptides 1 ea Catalog No Cleavage Kits Quantity PTI Universal Cleavage Kit Suitable for cleaving i peptides containing all 20 standard amino acids CEE Contains 95 mL TFA 2 mL water 2 mL anisole 1 mL EDT Makes 100 mL 1 ea Reagent K Cleavage Kit Suitable for cleaving if peptides containing all 20 standard amino acids ER Contains 82 5 mL TFA 5 mL thioanisole 5 mL water 5 g phenol and 2 5
71. e the bottles Should be set to 9 psi Regulator is accessible through the side access panel Displayed on Left Gauge Panel See Section 1 1 1 CAUTION Timed delivery volumes from solvent bottles 1 4 and 8 are dependent on the Bottle Pressure setting Any adjustments made to this regulator will require reverification of Solvent Calibration Section 2 5 1 3 The intensity of the mixing and fluid deliveries are controlled using the following two flow controls 1 Mix Flow Control Controls the nitrogen flow during a mix Located on Left Gauge Panel See Section 1 1 13 Counter clockwise to increase clockwise to decrease 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 ins
72. each subsection below 2 4 3 1 Program Editor Two programs are recommended to run a synthesis on the Prelude X 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 X 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 ly g Solvent Reagent RI 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 43 1 800 477 6834 El Synthesis Editor QA4manufacture Program Sequence Synt
73. ecommends synthesizing most peptides at room temperature first The majority of peptides produced worldwide are successfully synthesized at room temperature using conventional methods When a peptide does not come out well in the first try it is possible to further optimize the synthesis using more efficient activators using lower loaded resins or more hydrophilic solid supports adding pseudoproline dipeptides or Dmb or Hmb dipeptides or adding heat See Sections D 3 2 D 3 5 In the same way that it does not make sense to use pseudoprolines or more expensive hydrophilic solid supports for every synthesis it is also not recommended to use heat to synthesize all peptides In the case of the former it is a needless expense in the case of the latter it is to prevent the acceleration of unwanted side reactions Heat is best used selectively ideally just for the specific cycles in which it is needed Difficult cycles can be identified using the UV monitoring feature on the Prelude X and protocols can be modified accordingly Heat works by accelerating reactions In certain cases the use of heat promotes various side reactions and the best results may be obtained by removing the heat altogether Heating during the coupling of cysteine or histidine can produce unacceptable levels of racemization and heating during the coupling of arginine residues may promote gamma lactam formation During the synthesis of phosphopeptides the heater must be turn
74. ed off during all deprotection reactions once the phosphate group has been incorporated or it will cleave the phosphate group Care must also be used when synthesizing peptides containing aspartic acid as heat can accelerate aspartimide formation during the deprotection step Finally heating during the coupling of amino acids to C terminal proline residues may accelerate diketopiperazine formation See Section D 4 131 D 3 1 Starting Protocol PTI recommends synthesizing all unknown peptide sequences at room temperature then adding enhancements as needed from there All reagents listed below are available in our Chemical Catalog An example starting protocol is Resin Rink amide MBHA resin or preloaded Wang polystyrene resin loading 0 5 mmol g Deprotection 2 x 5 minutes 20 piperidine DMF Coupling 2 x 10 minutes 100 mM AA 100 mM HCTU 200 mM NMM in DMF 5x excess Washing 6 x 30 seconds DMF Cleavage 95 2 2 1 TFA water anisole EDT for 2 hours Most peptides using the standard 20 amino acids can be made using the above protocol Additional tools may be used for difficult sequences PTI recommends using PTI peptide predictor software and or UV monitoring to identify difficult cycles Difficult sequences are caused by aggregation or steric hindrance Aggregation occurs when hydrophobic side chains clump together causing a single peptide chain to fold in on itself or neighboring peptide chains to interact with one another obscur
75. eee 26 Seal Replacement 113 System ee 14 Editor A renee 37 38 39 95 File 31 37 39 41 48 52 69 101 File Managev 37 38 39 Special Bottles 16 40 50 54 56 StatUP r ae e a E ca 101 102 Synthesis eE E E cearnsatenas 101 EMO aa 47 49 50 File Manager 30 47 49 50 Loadi Or a Nera noe 60 64 65 101 System Alarms e a a A 22 Amino Acid Bottle 14 Clean 15 74 75 76 78 79 81 82 84 103 113 Cleavage Collection 13 CGOmMpUtEr enee e aa 20 Eion E e E A A N 120 Nitrogen aea 17 120 REACTION VESSEl ronnen 13 A Ni 91 Solvent Reagent Bottle 14 Vacuum a area 18 102 Menta iO aera 17 20 Waste ins 16 Tools Menu 85 86 88 89 View Menu ooccocconccncccnccnnconccnncnncos 97 VSB ROn ron rra 20 21 88 Wash Utility Panel 11 12 17 20 22 26 Ateneo eaaaeeca arcane 91 111 102 RVs 15 74 84 85 101 103 113 Vacuum 119 GAUGE te 18 101 102 120 Waste o 12 A isonet cece tnartat 42 A ceases 18 120 Container 16 17 26 27 29 103 SM a oe 18 102 Installation tes 26 Valve Level Sensor 16 17 22 26 27 Bloc 15 18 102 120 PRESS US teu 18 51 Otis aaa e 12 26 Gage ci 18 102 Cmtlet AA A ana 12 MaS E ae 22 119 A 16 Ventilation System 17 20 MEN me 17 22 150 For more information Email info ptipep com Website www ptipep com Technologi
76. enance NOTE Halt all other instrument operations before running an Archive All Data To Archive All Data 1 Click on the Tools menu and select Login 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 Cai Rebuild Errors Table i E LogOut Archive All Data Settings Diagnostics Operation Times Operations 3 The following warning window will open Click Yes to continue or click No to cancel the operation suser 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 4 The SUser software will exit and the Data Archiver window will open 89 Aa Data Archiver Version 1 0 0 40 PatalMASTER_USER mdb Success 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 ES Look in a Data gt e aea Archive 4 MASTER_USER mdb 4 UserEventLog mdb USER 1 mdb 7 USER2 mdb 7 JUSER3 mdb File name USER mdb Files of type Access Database Files mdb y Cancel 7 Select a user
77. ens 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 sus n U File Operations Tools Reports View Help Bottle Preparations gt la a RY Operations RY Status Cleaning Load Synthesis Calculations AA Calculations Solw Reag Manual Operations The Reaction Vessel Operations screen will open with the Calculations Solv Reag tab active 70 1 800 477 6834 RY Status Load Synthesis Calculations AA Calculations Solv Reag Synthesis Name Re m Peptides Sequence Termination Sub mmol g Scale umol Resin mg Yield mg Time SET 7 51132734G foon 0 47 20 42 55 21 26 6 hours 21 min 51 sec REGUERA fWSYGLRPF frome 0 41 20 48 78 22 57 6 hours 21 min 51 sec PeAcr2_ModRv3 6CCKEYKMG foon 0 47 20 42 55 21 26 6 hours 21 min 51 sec PC GLHRH faHWSYGLRPF fonz 0 41 20 48 78 22 57
78. ent Bottle Installation To install a 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 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 acta Waste T 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 29 1 800 477 6834 4 Connect the longer 1 4 ve
79. ent Check Turn on the Prelude X 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 bottle filters if necessary i e change of reagent Sections 3 2 amp 5 2 4 Select RV size in Settings screen Section 2 6 3 Calibrate solvent delivery volumes if necessary Sections 3 2 amp 2 5 1 3 Software Setup Create amino acid file Section 2 4 1 Create solvent reagent file Section 2 4 2 oe oe pn oejo o joejoejoe oe e 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 olojojo o o o Ololo ojololoje o lO Set default amino acid and solvent reagent files in Settings screen Section 2 6 3 Load synthesis Section 2 5 2 2 e 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 Y NC C 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 Instal
80. es Inc
81. escribed in the sections below 2 5 3 1 System Clean System Clean flushes the entire fluid system with Solvent 2 This operation should be executed every two weeks in order to prevent precipitate from building up A system clean performs the 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 operations 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 Premium Wash solvent in the Solvent 2 bottle Please contact PTI to order Premium Wash Solvent 3 Click on the Operations menu select Cleaning and then select System Clean File Operations Tools Reports Yiew Help E Bottle Preparations gt Qg RY Operations pR 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 77 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 A gt WARNING ALL BOTTLES RYs AND COLLECT VIALS MUST BE IN PLACE 7 This procedure may contaminate amino acids reagents solvents System cleaning can take more than 1 hour to complete Press OK to start and Cance
82. f 20 Cat AAR 400 1 1 ea Cat SMP VX 100 Pkg of 100 Cat AAR 400 X Pkg of 10 31 1 800 477 6834 1 3 3 Collection Vials 5 A 50 mL Em Cat CLV 050 030 Pkg of 30 Tal Cat CLV 050 090 Pkg of 90 Ar Cat CLV 050 180 Pkg of 180 5 10 1 5 1 3 4 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 X See Appendix A 3 for listings 1 3 5 Replacement Parts amp Additional Accessories Protein Technologies Inc supplies a full line of replacement parts for the Prelude X 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 32 1 800 477 6834 Chapter 2 Introduction to Software This Chapter covers the components of each screen in the software The Prelude X software was designed to mimic the
83. g operation is complete empty the flushed bottles of any rinse fluid and replace with clean bottles for the next synthesis 80 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 3 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 File Operations Tools Reports View Help E Bottle Preparations gt tog RY Operations gt E System clean 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 Te hhh Warning This procedure back flushes So
84. h for each synthesis SOLV 7 or Act2 1 L safety coated glass bottle for additional reagent 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 solution ltcan 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 4 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 6 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 X to the waste container through three ports on the Solvent Feedthrough Panel
85. he vertical position Pull the RV gently up and out of the lower seat A slight twisting motion may help release the RV from the bottom seat 2 When installing an RV gently insert the RV into the bottom seat Be sure to line the RV up with the upper seat Hold the RV with one hand while carefully lowering the cam lever to the horizontal position to lock the RV in place CAUTION Make sure the RV bottom is pressed through the bottom o ring in the lower RV seat or the RV may leak Be cautious when lowering the RV cam lever It can snap closed and break the RV if it is not guided down To install reaction vessel o rings 2 3 4 E o gt ps i 26 1 800 477 6834 1 Unscrew the cap from the lower RV seat on the instrument 2 Place a reaction vessel bottom o ring in the center of the lower RV seat 3 Screw the cap on over the o ring until tight 4 Slide a reaction vessel top o ring into the groove on the upper RV seat 5 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 1 2 4 Collect Vial Installation The collection vials install into a threaded port on the machine Install by turning the vial clockwise by hand until the top of the vial is seated firmly against the seal inside the threaded port To remove turn the vial counterclockwise IMPORTANT It is not reco
86. he delivery of cleavage solution Volumes for bottle 8 are measured by timed deliveries and upon draining from the reaction vessel are transferred to the collection vials Throughout the Prelude X 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 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
87. hecked during the mix the RVs will shake RPM Enter a value for RPM Heat Click to check or uncheck the box When checked the RV will be heated when the mix occurs 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 RV PV UVD Use the pull down menu to select an UV mode during a Deprotection operation The options are None Basic Xtend Xtend Fb To apply an extended time of the feedback of a UV deprotection in an AA Building Block Base Activator 1 or Activator 2 operation select Use Fb NOTE It 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 45 1 800 477 6834 10 Reps Enter the number of times to repeat the step The maximum number of repetitions is 9 11 Comment Record comments for the step The buttons are 1 Insert To insert a 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 Ad
88. hen 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 DE RV 1 Sequence A BA RV 2 Sequence Ce 16 Program 1 Deprotection 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 DD RV 1 Sequence TERN RV 2 Sequence 2 A A 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 X to send emails to a specified email address under the following conditions 112 1 At the beginning of each
89. hesis Program Name Select Solvent Reagent File QA4manufacture Zi Standard Z Operation Volume ul Mix Time H M S N2 Shake RPM Heat Drain RV PV UVD Reps Comment 1 AA Building Block x 3650 00 00 01 O M 120 M M rv None x 1 Manufacturing Quality Control 2 Activator 1 y 4000 00 00 01 fa 0 O M av None x 1 3 Base y 4000 00 00 01 la 0 O M RW None x 1 4 Cleave and Collect y 2000 00 00 00 la 17 TA 1 A 5 Mix a 0 00 00 01 Mm 00O Oa 1 6 leave Mic y 0 00 00 01 mM O Em 1 insert 7 Colect x 0 00 00 00 mil 0 0O Mme 1 8 DCM Top Wash y 300 00 00 01 OH 0 Mm 1 a 3 Deprotection x 4000 00 00 01 Ef 0 O M Rw Basie y 1 a 10 DMF Top Wash y 1000 00 00 01 a 0 O ma av 1 mu E Delete m 1 uu Cancel Save Save As 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
90. his screen The buttons are E 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 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 52 1 800 477 6834 5 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 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 X SUse
91. ick 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 file 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 102 2 9 2 About SUser To open the About SUser information box click on the Help menu and select About SUser ECT File Operations Tools Reports View Help Help Topics FA Pr you PA Y i 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 X User and Instrument software will be displayed 103 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 NI Z O Startup amp Instrum
92. ide the instrument The vacuum is displayed 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 21 1 800 477 6834 vacuum pump fails to bring the vacuum gt 10 in Hg No operations are allowed until the vacuum is restored 1 1 12 Right Gauge Panel The right gauge panel contains 1 Vacuum Displays the vacuum in mm Hg See Section 1 1 11 2 Nitrogen Pressure Displays the nitrogen pressure in psi See Section 1 1 10 1 1 13 Left Gauge Panel The left gauge panel contains 1 Valve Pressure Displays the valve pressure in psi The valve pressure should be at 30 40 psi 2 Bottle Pressure Displays the solvent bottle pressure in psi The bottle pressure should be at 9 psi 3 N2 Mix Flow Control Flow knob controls the nitrogen flow during a mix Counter clockwise to increase clockwise to decrease 22 1 800 477 6834 1 1 14 Emergency Stop Button Press down to stop the Prelude X in the event of an emergency All actions will cease and all bottles will be vented Simply twist clockwise to release
93. ils Number Synthesis Date 41 PTI 3 9 2006 11 Job Amino Acids 42 Standard 3 9 2006 11 I Job Solvents Reagents Tl Job Program Summary Recsevcesseccsessectnceeseonsecsecesscecssesees Preview Print Print to File 4 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 database or an archived database file click on the button This will open the Open window Look in Gachive o de ex En lal MASTER_USER_20060227 mdb Fy MASTER_USER_20060309 mdb 21 USERZ_20060202 mdb 21 UsER3_20060206 mdb File name JUSER1_20060201 mdb Files of type Access Database Files mdb y Cancel 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 96 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 A
94. ing the reactive group at the end of the growing chains The presence of highly sterically hindered side chains can also prevent the facile formation of bonds at the end of the growing peptide chain as well The sections below list different strategies you can use to improve the outcome of difficult cycles D 3 2 Low Loaded Resin Using a low loaded resin lt 0 4 mmol g can significantly improve purities presumably by eliminating interchain aggregation D 3 3 Coupling reaction 1 Try increasing the amino acid excess and or concentration Performing the coupling reaction at a higher concentration can significantly improve the coupling efficiency This can be accomplished simply by increasing the amino acid excess from 5x to 10x or by decreasing the overall coupling reaction volume assuming the resin can be sufficiently covered for good mixing 2 Try increasing the reaction times or the number of couplings For very sterically hindered amino acids such as Aib it was found that 4 x 90 minute couplings with HATU were necessary to incorporate them at 132 high efficiency within the sequence VQ Aib Aib IDYING OH 89 final peptide crude purity although the other amino acids were able to be coupled for just 2 x 1 minute each 3 Try dissolving the difficult amino acid in DMSO Dissolving hydrophobic sterically hindered amino acids in DMSO has been found to improve the coupling efficiency in some cases In the synthesis of ACP VQAAI
95. ino acid into its other enantiomeric form Heat can increase the amount of racemization during the coupling reaction especially for histidine and cysteine residues Lowering the temperature to 50 C or below during the coupling of these residues and the use of coupling methods that do not rely on the presence of base can help to minimize this side reaction 2 Aspartimide Formation Aspartic acid and the nitrogen of an adjacent residue in a peptide sequence may form an aspartimide in the presence of an acid and or a base Once formed the aspartimide can reopen into various forms In Fmoc chemistry the aspartimide can form piperidides when exposed to piperidine in subsequent deprotection steps Heat can accelerate this side reaction It is especially prevalent in peptide sequences containing Asp Gly Asp Ala or Asp Ser To minimize this side reaction in aspartimide prone sequences HOBt can be added to the piperidine deprotection solution However HOBt can interfere with UV monitoring If UV monitoring is being used concurrently with heated protocols a better solution would be to use an alternative deprotection reagent such as piperazine or to simply turn the heat off during the deprotection steps after aspartic acid has been incorporated in the sequence Finally replacing the amino acid immediately preceding the Asp with the Fmoc Hmb protected version can also help minimize aspartimide formation 134 o Ri LA mro N 0 7 Ah AS o
96. ions 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 indicates the position is pressurized and N indicates the position is vented Amino acids 1 27 are pressurized together 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 55 1 800 477 6834 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 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
97. is 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 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 b
98. is 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 each RV The Prelude X software calculates this amount based on the values in the Sub mmol g and Scale umol columns 71 1 800 477 6834 7 Yield mg Displays the theoretical amount of peptide in mg expected from the synthesis The Prelude X software calculates this amount based on the value in the Scale umol column 8 Time Displays the estimated time for the synthesis in hours min sec 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
99. ix 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 the Solvent box Solvent 1 or 2 and the number of repetitions in the Reps box 1 9 114 115 Chapter 5 Cleaning amp Maintenance 5 1 Cleaning 8 Maintenance Schedule e Wash RVs Section 2 5 3 8 Ever e Bottle Position Flush used bottles Section 2 5 3 2 Sieci e Collect Back Flush Section 2 5 3 5 Only After 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 Computer Maintenance Section 5 2 2 Quarterly or Semi e Solvent Calibration Section 2 5 1 3 Annually e Amino Acid Bottle Seal Replacement Section 5 2 5 Annual e Replacement of RV Top Upper valve block membrane y must be performed by PTI service personnel e Calibration of UV and IR components Nitrogen Leak Check Section 5 2 3 As Needed Bottle Filter Replacement Section 5 2 4 Amino Acid Bottle Seal Replacement Section 5 2 5 Solvent Bottle Seal Replacement Section 5 2 6 116 5 2 Operations 5 2 1 Cleaning Operations The following cleaning operations are available on the Prelude X and are covered in Section 2 5 3 System Clean Bottle Position Flush Cleave Bo
100. l 30 16 50 26 Solv 1 MIX 2014Jul 30 18 19 28 Solv 1 MIX 2014Jul 30 19 11 53 Solv 1 MIX 2014Jul 30 20 13 41 Solv 1 MIX 2014 Jul 30 16 05 59 Solv 1 MIX 20 14Jul 30 17 345 Ss BA i 2014Jul 30 21 15 21 Solv 1 MIX 20 14 Jul 30 22 07 52 Solv 1 MIX 2014Jul 30 15 21 26 Time aa a a ao aaa aaa ae Step ri 2 2 2 Cycle 1 2 3 4 5 9 AA 1 1 1 1 1 1 1 Individual Cycle E 2 2 Individual Read Graph An Individual Read Graph displays UV data for Individual Repeats of UV Monitored steps A peak is recorded once every 10 seconds when a UV reading is taken In an Individual Read graph each peak represents an individual absorbance reading Individual readings are shown for each UV Monitored step Below each cycle are the following labels 1 Step Program step number 2 Cycle Cycle number 3 AA One letter code of the AA coupled that cycle 141 Individual Reading EN nN a g i 8 Individual Repeat E 3 Basic Monitoring Mode E 3 1 Overview Basic monitoring takes absorbance readings every 10 seconds during the UV Monitored step but it does not interfere with the synthesis You can get two types of graphs from this data 1 UV Summary Graph 2 UV Individual Graph E 3 2 Writing a Program To use basic monitoring during a deprotection step select Basic in the UVD drop down menu for that step NOTE The recommended minimum vol
101. l 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 amp 1 1 13 2 Z O Olele Olelelele Post Synthesis Procedures e 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 7 104 3 2 Startup amp Instrument Check To startup the Prelude X Peptide Synthesizer turn on the On Off switch located on the utility panel on the side of the instrument and also the On Off switch for the heaters if installed 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
102. l to cancel Cancel 5 During the System Clean the Bottle Preparations screen will open i53 Bottle Preparations x Bottle Preparations Special Bottles Solvent Calibration Solvents Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed 13 Fin IE a an 2 aT Az 22 0 422 222417423 24 AT 524 Mon N EN F ptm NE F Dep F Cap TT Base Y 241 425 26 AT 426 27 AU 427 0 1002400 oon none w N a a Aa Pla la eet at 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 7 If Premium Wash Solvent is used for the System Clean it is recommended to perform a second System Clean with DMF or NMP in the Solvent 2 bottle in order to remove the Premium Wash Solvent from the lines Any remaining Premium Wash Solvent in the system can adversely affect chemistry 78 NOTE The System Clean takes over 1 hour to complete CAUTION Remove all chemicals and resins from the Prelude X before the System Clean because they
103. lection 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 for detailed information on the Collect Back Flush 16 1 800 477 6834 1 1 6 Amino Acid Bottle System 27 amino acid bottle positions are located on the front of the Prelude X 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 4 Bottle Filter Replacement for instructions CAUTION DO NOT install or remove amino acid bottles when they are pressurized IMPORTANT All 27 amino acid bottles are vented or pressurized together For this reason all amino acid positions must have bottles in place for the pressurization to occur 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 i
104. 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 Solvent 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 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 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 3 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 25a 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 25 1 800 477 6834 1 2 3 RV 8 O Ring Installation To install or remove reaction vessel Upper RV Seat moves with lever Bottom RV Seat 1 To remove an RV apply slight downward pressure to the RV with one hand while lifting the cam lever with the other until the lever locks into t
105. ll 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 Undoes 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 110 Cycle Selection PE Idle Cycle No y Boa E A 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 Not Applicable in Prelude X mode Buttons will be greyed out 4 Cancel Cancels the insert operation When an advanced operation is assigned to a sequence EA is displayed to the right of the sequence to alert the user The following examples demonstrate possible uses of Dynamic Sequence Programming 111 Example 1 Run different programs during cycle 1 Program Assignment DEZI RV 1 Sequence A A RV 2 Sequence Cier RV 1 will couple A with program 1 while RV 2 sits idle W
106. lose button and preview the file using the Print button in the Sequence File Manager Section 2 3 49 1 800 477 6834 2 4 3 3 Synthesis Editor The Synthesis Editor is used to create synthesis files Synthesis files 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 Amino Acid y lg Solvent Reagent ve Program Sequence Synthesis Synthesis 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 Pel synthesis Editor PTI Se Program Sequence Synthesis Program Sets Synthesis Name Solvent Reagent pr D Standard y Bl gS Gade Comments 1 zj a zj Amino Acid Standard Peptide Programming Standard Current Cycle Und Pogan A at M 4 4 4 gt 1 i Sequence gt Original Modifiers y
107. lower right corner of the screen N2 should be displayed ona 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 Section 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 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 4 for instructions 5 Select RV size 10 mL or 40 mL in the Settings screen
108. lvent 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 X will perform a block rinse before mixing rather than after draining This is useful when long mix 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 uL for the 10 mL RV and 20 000 uL for the 40 mL RV The System Settings are as follows de Default Operation Times The contents of this file control the times associated with operations on the instrument The default setting is Standard The Prelude X 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 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 modif
109. lvent into selected bottle s This procedure may contaminate amino acids reagents solvents Cancel 81 4 During the Cleave Bottle Solvent Back Flush the Bottle Preparations screen will open k a Bottle Preparations xj Bottle Preparations Special Bottles Solvent Calibration Solvents Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed FRE Ex IN N N N E PA oN DO e w N BG ay al Se Ee ee ono Y oo 2 WwW NY EA EV ER ERN SE bay ta tal ta are a Mata a 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 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 Fl
110. ly 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 X 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 39 1 800 477 6834 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 source number To open the Solvent Reagent Editor click on the File menu and select Solvent Reagent File Operation Tools Reports Yiew 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 Ls Solvent Reagent Editor Standard Solvent File Name Standard g Program Operation Names GLP Data Pos Name Abbry Bottom Delivery Top Wa
111. mL EDT Makes 100 mL 1 ea 127 Appendix B Reagent Shelf Life 8 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 except 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 20 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 B 2 Amin
112. may be installed and left in place for several sets of syntheses SOLV 3 or Dep 1 L safety coated glass bottle for deprotectant to remove the N terminal Fmoc protecting group The standard composition is 20 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 1 800 477 6834 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 fres
113. ment or operators e Uncrate main unit lift off pallet use caution gt 200 lbs e Remove all materials from crate Check off list 1 Prelude X user manual 2 4L safety coated glass bottles 1L safety coated glass bottles 5 gal waste container 10 ml reaction vessel assemblies Package of collection tubes 2 7 1 12 2 7 Package of amino acid bottles 1 1 1 1 1 Waste duct assembly Nitrogen tubing assembly Flow meter assembly Bottle tray Bottle cap and tubing assembly 4 4 2 24 1 800 477 6834 1 Waste tubing assembly 1 Waste cable assembly 1 Power cord 1 Monitor 1 USB Keyboard 1 USB Mouse 1 Mouse pad 1 Plastic fitting 1 4 FPT x 1 4 tube 1 Peptide Predictor software 1 AA replacement filters 100 pack 1 Solvent bottle filter 100 pack 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
114. mino 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 Act Action c Pos Position of RV bottle or other code depending on the operation d Rep Repetition e Operation Operation f Description Detailed description of operation g OT Operation Type i M Manual operation ii A Automated operation 97 h iii 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 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 f g
115. mmended 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 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 27 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 28 1 800 477 6834 1 2 6 Solvent Reag
116. n Summary Graph or toggles to previous data screen Individual Graph Forward Scrolls the graph to the right when full data set does not fit on screen Summary Graph or toggles to next data screen Individual Graph 5 Open Use to view UV monitoring data from another synthesis 6 7 OK Use to return to the RV operations or Manual operations screen Print Prints graphs from currently selected synthesis UV Graph data updates after a UV Monitored step is complete giving access to view the Summary Graph and Individual Graph data of that step E 2 1 Summary Graph A UV Summary Graph displays a summary of the UV absorbance data for a total synthesis In a Summary Graph each peak represents an individual repeat in a UV Monitored step where the lighter portion represents the minimum absorbance measured during that repeat and the darker portion represents the maximum absorbance measured during that repeat Below each cycle are the following labels IS Time Time for UV Monitored step to complete in minutes Step Program step number for monitored step Cycle Cycle number AA One letter code of the AA coupled that cycle 140 Synthesis View UV Absorption UVLogFiles UVLog_RV0O1_DATE_073014_TIME_152227_UVGraphPagel bmp Individual 50000 Repeat Maximum Absorbance 40000 Minimum 30000 Absorbance 20000 Solv 1 MIX Solv 1 MIX 10000 Ma B E E 2 B fE o o EE 2014jJu
117. n 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 in the lower box The Shake box allows the user to change between Shake modes Auto will keep the set RPM and Manual will set the RPM with the knob on the instrument When a synthesis is running the RPM box shows the current RPM The current temperature of each RV will be shown in the bottom labels The UV Graph dialog will be shown clicking in the UV Graph button 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 65 1 800 477 6834 Cycle 1 Step 1 x 1 Standard v Cycle 1 to 6 OK fi DMF Top Wash y Step 1 to 6 Cancel Use the upper pull d
118. n the Help Topics window click on the Help menu and select Help Topics File Operations Tools Reports View Help m AEC Help Topics FA Pm Yee GA AA About SUser On the left side of the Help Topics window are the Contents Index and Search tabs 100 o amp Br Hide Back Print Options Contents Index Search E Y General 2 Main Menu 2 Shortcut Buttons f File Manager 5 e File Menu 5 o Dperations Menu a Cleaning F Setting Up a Synthesis File Manager Files of every type Amino Acid SolvfReag Program Sequence Synthesis are manipulated using the File Manager As an example the Amino Acid File Manager s shown below Amino Acid File Manager Standard 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 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
119. ner while any bottles are primed the bottles will vent and fluid may remain in the lines 1 1 9 Ventilation System The Prelude X 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 cover for the other The adaptor has a tube fitting for attaching the waste container vent line The Prelude X 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 Section 1 1 3 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 20 1 800 477 6834 1 Valve Pressure Used to seal the valve membranes Should be set to 30 40 psi User should not adjust Displayed on Left Gauge Panel See Section 1 1 1 2 Nitrogen Pressure Used for mixing and delivering fluid Should be set to 5 psi User should not adjust Displayed on Right Gauge Panel See Section 1 1 1 3 Bottle Pressure Used to pressuriz
120. ngs to cme take effect The Operation Settings are as follows La a Facility Enter name of Facility Machine Name Enter name for Prelude X computer 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 The Prelude X software must 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 X 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 93 may have their own database files for their own syntheses The default setting is MASTER_USER mdb The Prelude X 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 X will wash all six RVs with the entered so
121. ns gt E a RV Operations gt E y 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 RYs 3 The following warning window will open Click OK to proceed or click the Cancel button to cancel the Rinse All Blocks operation Te hl gt WARNING Rinse and clear can take 15 minutes to complete press OK to start and Cancel to cancel 84 4 During the Rinse All Blocks operation the Bottle Preparations screen will open norte preparan uu z Bottle Preparations Special Bottles Solvent Calibration Amino Acids Solvents Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed DO on a w N Mp a A a ns oak 1 2 3 4 5 6 7 8 9 E e a Sida Si at Se n Osea 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 If 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
122. nt line to the exhaust vent adaptor fitting 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 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 roteir 10 mL Induction Compatible 40 mL Induction Compatible 10 mL Glass Cat PPX FGRV10 1 1 ea Cat PPX FGRV40 1 1 ea Cat TPS GRV10 1 1 ea Cat PPX FGRV10 1 Pkg of 6 Cat PPX FGRV40 1 Pkg of 6 Cat TPS GRV10 10 Pkg of 10 30 Technologies inc 40 mL Glass Cat TPS GRV40 1 1 ea Cat TPS GRV40 10 Pkg of 10 1 800 477 6834 10 mL Disposable 45 mL Disposable Cat PPS R10 030 Pkg of 30 Cat PPS R45 030 Pkg of 30 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 1 3 2 Amino Acid Bottles Protem 10 mL Single Shot Cat AAR SSI 1 ea Cat AAR SSX Pkg of 10 120 mL 400 mL Cat SMP VX 20 Pkg o
123. o 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 128 Determine the concentration of amino acid to be used on the Prelude X instrument In the RV Operations menu of the Prelude X software open the Calculations AA screen 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 ona 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 gradient 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 uL of the amino acid solution diluted to 1 mM at the following time periods initial 3 days
124. o Acids Calc Suggest Weight Volume volume mg ml mi 1 R E 12 22 685 lanine 2 E E 6 16 937 ysteine Trt 3 P E 12 22 905 spartic Acid OtBu 4 E E 1 11 468 lutamic Acid OtBu 5 f E 2 12 494 spartic Acid 6 E E 1 11 468 lutamic Acid 7 F E 2f 12 551 yrosine tBu 2 Pos Description Res Calc Suggest Weight Volume Volume mg mt mL 10 a 2f 12 424 soleucine2 1 3 E 6 16 594 ethionine 12 fi E 1 11 656 sparaaine Trt 13 3 El 6 16 540 roline 14 E El 6 16 977 lutamine Tre 15 El 6 16 1038 rainine PDF 16 E 3 6 16 613 jerine tBu pl Pos Description Res Re Cale Suggest Weight 5 Volume Volume mg mL mi 19 7 El 6 1 843 tryptophan Boc 20 f E Bd iz 551 yrosine tBu 2 f E 1 11 656 sparagine Trt 2 22 a E E p Es spartic Acid a PRA spartic Acid a LN ES ECO lutamic Acid 25 F E By 12 551 yrosine tBu 3 Pos Description fe ai 30 1060 Jim El e Bl El al 2 747 zaf H a3 hreonine tBu soleucine3 18 F aline g soleucine f E Al 22 1031 27 F ysine Boc sparaqine Trt Print The Synthesis Name box displays the current synthesis Use the pull down menu to select 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 syn
125. olv 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 Ea S p A ae 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 63 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 or click on the Operations menu select RV Operations and then select RV Status RETA File Operations Tools Reports View Help E Bottle Preparations gt lan l RY Operations Load Synthesis Calculations 44 Calculations Solv Reag Manual Operations The Reaction Vessel Operations screen will open with the RV Status tab active RV Status Load Synthesis Calculations AA Calculations Solv Reag Reaction Vessel Status p 2 GAdmanutacture Program QA4manufacture Shake Program 1 GHIKLMNPORSTVWY123456 MM 8 Buia Bok RPM 2 Activator 1 KLMNPORSTVUY Base 2 jen s 123456 z 4 Cleave and Collect g FAR e i MIK 3 GHIKLMNPORSTVWY123456 3
126. onnected 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 23 1 800 477 6834 2 Nitrogen Supply A relatively pure gt 99 9 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 capacity of the secondary container should be enough to contain a 5 gallon spill 4 Memory Stick optional Files may be transferred from the Prelude X computer to an external computer using the USB port and a memory stick 1 2 2 Instrument Installation Procedure IMPORTANT Installation of the Prelude X should be performed by trained personnel only Improper installation may result in damage to the instru
127. ons 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 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 48 1 800 477 6834 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 C
128. ottle positions on the instrument The buttons are labeled with the abbreviations and key codes of the 47 1 800 477 6834 currently open amino acid file Enter a sequence by clicking the buttons with the mouse or using the keyboard The CV Single Shot box contains a button to add the key of the CV single shot amino acid Each CV Single Shot position is tied to a specific RV For example CV1 can only deliver to RV1 The Temperature box to the right of the screen allows a temperature to be set for each position in the sequence Each position can have a unique temperature or a single temperature can be used across all positions Entering a 0 in the Temperature box will leave the position unheated 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 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 butt
129. oupling Feedback Measures the extent of the deprotection reaction and uses that data to control the deprotection reaction times and repetitions and extend the coupling times based on the deprotection reaction time 137 E 1 How UV Monitoring Works E 1 1 Chemistry During the deprotection reaction piperidine removes the Fmoc group and forms a piperidine dibenzofulvene adduct with the byproduct See below Fmoc Amino Acid Resin o R AMAS Lo H 0 Piperidine O F Dibenzofulvene E Y gt Intermediate O Je H H R iia Piperidine Piperidine N Deprotected Amino l Acid Resin UV Monitoring 1301 nm Conductivity Monitoring Dibenzofulvene Piperidine Adduct Piperidine Carbamate Salt The IntelliSynth UV Monitoring System monitors the absorbance of this adduct at 301 nm during the deprotection reaction 138 E 1 2 The IntelliSynth UV Monitoring System 1 UV es I 1 UV o l I Light O Detector I I Light O Detector I bee gs iets ee AA The IntelliSynth UV Monitoring System consists of a light source and detector encased in a 1 3 8 x 1 3 8 x 1 3 5 cm x 3 5 cm x 4 5 cm housing which measures the absorbance of the fluid in the tubing directly below the reaction vessel During a mix part of the fluid is pushed down into the section of tubing exposed to the light source and detector and a measurement is taken left diagram The fluid is then pushed back up into the reaction vessel to resume
130. own 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 lt 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 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 res
131. perations 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 RV DOORS OPEN RV door sensor senses an open door Check the RV doors are closed RV door sensor may require service contact Service Dept 122 6 2 Critical Errors No Operations Allowed The following table lists critical errors on the Prelude X their cause and possible corrective actions to take The following errors will cause the Prelude X 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 Action 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 lt 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 no
132. r 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 X software opens 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 Bottle Preparations E Bottle Preparations gt RY Operations gt Special Bottles Cleaning gt Solvent Calibration Then select from one of the following three screens 1 Bottle Preparations 2 Special Bottles 3 Solvent Calibration 53 1 800 477 6834 The function of each of these screens will be reviewed in each subsection below 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 ar
133. ramming 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 those 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 51 1 800 477 6834 6 Molecular Weight The molecular weight
134. reagents that show any precipitation If a specific reagent cannot be delivered replacement of the bottle filter should be the first solution 119 The bottle filter consists of a filter housing and a frit The frit is press fit into the housing 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 or dental pick 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 protective 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
135. rogen tank valve Watch nitrogen tank gauge on tank for drop in pressure within 15 minutes then turn on nitrogen tank valve If 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 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 le 3 Connect a nitrogen flow meter between the nitrogen tank and the nitrogen inlet to the unit If 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 If the flow is less than 25 cc min proceed to Test C Test C Solvent System Test il Connect the nitrogen flow meter and pressurize all solvent bottles in the Bottle Preparations Section 2 5 1 1 screen Allow the system to stabilize for 10 15 minutes Check the nitrogen flow meter to see if there is any flow If the 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 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 118 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
136. s 2 The AUIS ey 3 The maximum below the Absorbance SMS number of repetitions Individual Repeats Threshold Da is reached Absorbance Threshold is equal to the Repeat Threshold Count E 4 2 Writing a Program To use UV Extend operations during a deprotection step select Xtend in the UVD drop down menu for that step NOTE The recommended minimum volumes for UV operations are 3000uL for 10mL RVs and 5000uL for 45mL RVs Example Program An example program using UV Extend Operations is shown below These programs will produce a UV graph of the deprotection reactions for the synthesis They will control the deprotection reaction times and repetitions The coupling reaction is unaffected Example UV Deprotect Extend Synthesis Program n Situ Ste Operation Volume bas p p Time N2 Shake RPM Heat Drain RV PV UVD Reps 1 Deprotection 3000 0 00 30 X Xx RV Xtend 1 DMF Top ra Xx RV 2 Wash 3000 0 00 30 X 3 AA Building RAY RV 3 Block 1000 0 00 00 X 1 4 Activator 1 1000 0 00 00 X RV 1 5 Base 1000 0 10 00 X X RV 1 6 Top Delivery 3000 0 00 30 X X RV 1 AA Building O RV 7 Block 1000 0 00 00 x 1 8 Activator 1 1000 0 00 00 X RV 1 9 Base 1000 0 10 00 X X RV 1 10 Top Delivery 3000 0 00 30 X X RV 3 E 5 Deprotection and Coupling with UV Extend Operations 145 E 5 1 Overview This mode is the same as the Deprotection wi
137. s pR 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 US gt WARNING ALL R s MUST BE IN PLACE 5 Press OK to start and Cancel to cancel Cancel 4 During the Wash RVs operation the Bottle Preparations screen will open 87 A Bottle Preparations xl Bottle Preparations Special Bottles Solvent Calibration Solvents Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed 1 g la lla la la ial a A z 223 Z 1 2 3 4 5 6 7 8 Di De SY Oo ch amp ww y ala lea ala la ik iad ads ie la le la EE in al W 7 R a l A2 i Select All Washing Top Wash 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 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 resi
138. s used to prevent the liquid from contacting the seals Contact PTI s Technical Service 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 5 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 X 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 1 800 477 6834 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 t
139. scribe 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 8 2 can be delivered through the top of the RV Se 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 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 41 1 800 477 6834 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
140. sh Vol mL Conc mM Opened Date Lot Number Source Number Ol 1 Dimethylformamide DMF ome Wash ome Top Wash 4000 o 06 20 2006 09 43 23 A107495 100 20 300 of 2 Dichloromethane DCM DCM Wash DCM Top Wash 500 O 06 20 2006 09 43 23 P857463 404 50 600 of 3 20 Piperidine DMF Dep Deprotection 500 o 06 20 2006 09 43 23 012584 PS3 PPR L Ol 4 Acetic Anhydride cap capping soo o 06 20 2006 09 43 23 x 5 0 4M NMM DMF Base Base 250 400 06 20 2006 09 43 23 053809 PS3 MM L x 6 HBTU DMF Acti activator 1 250 100 06 20 2006 09 43 23 322158 B 1K HBTU x7 Activator 2 Act2 Activator 2 500 O 06 20 2006 09 43 23 O 8 TFA Cocktail tra leave and Collect toc 0 fosjzojz0 094323 O Time Calibrated Delivery X Sensor Delivery Ney Close Cancel Save Save s Print 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 40 1 800 477 6834 The columns are i 2 Pos Solvent reagent bottle position 1 8 Name Name of solvent or reagent Abbrv Abbreviation for solvent or reagent Program Operation Names Names used to de
141. t Reagent Bottle 26 Waste Containe e 26 Job Reports 88 94 95 96 Load Synthesis 60 64 65 101 OG MROQW UL ects eeee 88 92 Maintenance Cleaning and 113 COMPLE rnea 113 File eee ancien 86 Manual Operations 30 60 63 70 72 85 M A recente 41 Cleave a R 41 Flow Control cea 11 18 101 Morado 42 72 96 Molecular Weight 10 35 36 45 49 65 69 94 Nitrogen Back FIUSH cscs1 c 0 53 77 78 103 Cleave Botile 74 103 Flow Meten ane i E TEE nenenerens 119 Gage eoe e eee et oon 102 aE rra 12 17 Leak Gheck T 22 113 PES UI acid 18 Pressure Gauge 102 119 Push Flow Control 11 18 SLAMS hates hase E E 102 SOD Vecee eeeaencceces cee 21 101 102 System ee e ea 17 120 NoPE 16 55 101 105 Operations res aaae ee eaae a E 92 Cleaning acid 15 51 74 Manual 30 60 63 70 72 85 Menu 13 39 50 51 54 60 61 65 67 70 74 76 78 79 81 82 84 Program ereere 38 41 42 62 Reaction Vessel 30 60 61 64 65 67 119 Synthesis e e eee 101 MMES e e a 92 Operator DB aa dosis 90 O Ring Borens e daa 16 26 Installation eee 23 Reaction Vessel Bottom 23 24 Reaction Vessel TOp occooo 24 Pressure Bote tre erie Precare mater a 18 19 Galgo era 11 102 Manto tasa 51 59 Nidos ccoo 18 MANO Vat 18 19 51 Pressurize 17 18 22 51 52 53 77 78 79 81 84 101
142. t connected to the instrument Empty waste tank and reconnect Section 1 2 8 Check reconnect waste tank connector NOT COMMUNICATING No communication between the user and instrument software Turn Prelude X power off for 30 seconds then back on E STOP ENGAGED The E stop button is pressed Disengage E stop button THERMAL CUT OFF A thermal cut off has occurred Ensure that Induction Compatible RVs are being used in all heated positions 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 123 Appendix A Reagents For Peptide Synthesis A 1 Prelude X Pre Packed 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 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
143. temperatures to each RV which RV1 starting at the left Checking the N2 box activates N2 bubbling during the mix Select a mix mode in Mix Actions checking the box of Shake and introducing a RPM for automatic shake checking Heat and setting the temperatures at the left side corresponding to each RV and checking the N2 box for N2 bubbles The actual temperatures are shown in the Actual boxes and the actual RPM is also shown in the RPM box when a manual operation is running If a Deprotection step is selected the UV Mode box becomes active Select a Basic or Xtend UV deprotection 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 74 1 800 477 6834 volume of 20 000 uL for 40 mL RV s The RV size should be selected in the Settings 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
144. th UV Extend Operations however the UV data from the deprotection step is also used to extend the coupling time in the cycle The deprotection time of the first repeat during deprotection with UV Extend is multiplied by the Coupling Multiplier default setting of 2 to obtain the coupling time as long as this value falls below the Maximum Reaction Time E 5 2 Writing a Program To use UV Extend and Feedback control operations during a deprotection and coupling step select Xtend Fb in the UVD drop down menu for the deprotection step and Use Fb from the UVD drop down menu during the coupling step NOTE The recommended minimum volumes for UV operations are 3000uL for 10mL RVs and 5000uL for 45mL RVs NOTE If Use Fb is included in a program in order for its reaction time to calculate correctly it must be included in a program after an Xtend Fb step 7 N ojo N O OI A Example Program An example program for the Deprotection and Coupling with UV Extend Operations is shown below These programs will produce a UV graph of the deprotection reactions for the synthesis They will control the deprotection reaction times and repetitions and extend the coupling time based on the total deprotection reaction time does not include wash time between repeats Example UV Synthesis Extend Program in situ Step Operation Volume Mix Time N2 Shake RPM Heat Drain R
145. 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 Topics 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 2 Back Click the Back button to return to the previously viewed help file 101 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 l 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 C Print the selected heading and all subtopics E 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 Cl
146. thesis 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 69 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 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 scre
147. tions screen click on the shortcut button or click on the Operations menu select RV Operations and then select Manual Operations File Operations Tools Reports Yiew Help Bottle Preparations gt la a RY Operations gt RY Status Cleaning Load Synthesis Calculations 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 73 1 800 477 6834 Lal Manual Operations Select Active Reaction Vessels Mix Actions YIRAY1 VJRV2 VIRV3 V RV4 V RV5 V RY6 Shake Clear All RVs 2 S A M RPM Temperatures Heat N2 Set Y Y 70 80 80 80 60 70 Actual UV Mode 0 0 0 0 0 0 None Operation Volume uL Op time Drain Reps AA Building Block x 150 00 0200 7 1 Select Amino Acid Ala Cys Asp Glu Phe Gly His lle Lys Leu Met Asn Pro Gin Arg Ser Thr Val Tip Tyr A421 A22 423 A24 A25 A26 A27 CvSs la Clear j UV Graphs Close 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 Select a mix mode in Mix Actions Checking the A box under Shake and entering a value in the RPM box sets a specific shake RPM Checking the Heat box allows the user to assign
148. ttle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs go N o E5 Go ND 5 2 2 Computer Maintenance The computer is an important component in the operation of the Prelude X 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 Nitrogen Leak Check It is recommended to routinely check the sealing of 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 117 Test A Regulator 8 QC Test 1 Remove the nitrogen quick connect i e no nitrogen line connected to unit Turn off the nit
149. ume 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 66 1 800 477 6834 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 A Bottle Preparations gt lon RY Operations RY Status Cleaning Calculations Solv Reag Manual Operations The Reaction Vessel Operations window opens with the Load Synthesis tab active 3 Reaction Vessel Operations Ry Status Load Synthesis Calculations AA Calculations Solv Reag Select Synthesis B P fA RY 1 PTT fH RY 2 PTT RY 3 PTI Sequence PTIPEPTIDE123 Termination COOH Weight 1057 182 Modified 8 28 2006 3 58 25 PM Comment fl RY 4 PTT fA RY 5 PTT fy RY 6 PTT E Program Template From Cycle 1 PTI Modified 8 30 2006 4 50 17 PM Comment E Standard
150. umes for UV operations are 3000uL for 10mL RVs and 5000uL for 45mL RVs Example Program An example program for synthesis using in situ couplings and basic UV monitoring is shown below These programs will produce a UV graph of the deprotection reactions for the synthesis without interfering with the synthesis 142 Step 1 ojoj N Oyj ine Operation Volume ple P Time N2 Shake RPM Heat Drain RV PV UVD Reps Deprotection 3000 0 02 30 X X RV Basic 2 DME Top 3000 0 00 30 Xx ay 3 Wash AA Building aya RV Block 1000 0 00 00 x 1 Activator 1 1000 0 00 00 X RV 1 Base 1000 0 10 00 X X RV 1 Top Delivery 3000 0 00 30 X X RV 1 AA Building Ry RV Block 1000 0 00 00 X 1 Activator 1 1000 0 00 00 X RV 1 Base 1000 0 10 00 X X RV 1 Top Delivery 3000 0 00 30 X X RV 3 Exam le UV Monitored Synthesis Program E 4 Deprotection with UV Extend Operations E 4 1 Overview This mode is the same as the Basic Monitoring mode however the data is used to extend the deprotection time and number of repetitions without affecting any other steps in the cycle The UV Extend Operations alter the deprotection reaction time in two ways ika The time of an Individual Repeat can lengthen depending on the observed UV absorbance in an Individual Read Graph If the absorbance changes from one Individual Reading to another above a set threshold another
151. ush 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 82 File Operations Tools Reports View Help E Bottle Preparations gt E RY Operations gt Bottle Position Flush Cleave Bottle Solvent Back Flush Cleave Bottle Nitrogen Back Flush Collect Back Flush Rinse All Blocks Clear All Blocks Wash RVs 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 Collect Back Flush operation pu 2 WARNING COLLECT VIALS MUST BE IN PLACE Press OK to start and Cancel to cancel Cancel 5 During the Collect Back Flush the Bottle Preparations screen will open t33 Bottle Preparations xj Bottle Preparations Special Bottles Solvent Calibration Solvents Amino Acids Pressurized Primed Pressurized Primed Pressurized Primed Pressurized Primed F DMF EA ii ia Mite ii Ei 19 Ftp F pew I Cys NE 7 Met Pi 20 OF Ty FT Dep E Asp NE F Asn i Ara I Cap F Glu PRT F Fro 2 va 522 I Base T Phe ia F Gin i 8 2a Ae TE Acti Pr Gly in F Ara FRE 24 AT 424 F His FRE F Se M 25 41 425 Fe aa F Thr fy 2 AT 426 Lys al FT Val ME 22417427 FT amp ct2 TF TFA DD 10022002 oon nn ks Ww
152. vices 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 1 800 477 6834 Tel 520 629 9626 Toll Free 800 477 6834 Email info 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 Benzotriazolium 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 Piperidin
153. w 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 Close 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 a new screen To create a new amino acid file click the 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 37 1 800 477 6834 Pel Amino Acid Editor Standard E Amino Acid File Name Standard Bank 1 Bank 2 Bank 3 CV Single Shot SS SS 3 GLP Data Pos Name Abbrv Key Dep MW g mol Pro MW g mol Vol mL Conc mM Opened Date Lot Number Source Number 1 Alanine Ala A 89 095 311 380 0 O 10 06 2015 08 50 52 2 Cysteine Trt Cys c 121 15
154. when a difficult sequence presents itself it is the coupling reaction that is causing the problem Occasionally however it is possible to improve results by optimizing the deprotection reaction Try adding 2 1 8 diazabicyclo 5 4 0 undec 7 ene DBU to 20 piperidine DMF Prolonged exposure to DBU can promote aspartimide formation in Asp containing sequences so in this case reaction times should be decreased to 2 x 30 seconds or at most 2 x 1 minutes 133 D 3 5 Pseudoprolines Hmb amp Dmb Amino Acids and Dipeptides The addition of a proline can break up aggregation up to 6 residues down the growing peptide chain Pseudoproline dipeptides and Hmb and Dmb amino acids and dipeptides are rigid structures that can break up aggregation in a similar way Strategic placement of such building blocks in a difficult peptide sequence can significantly improve its synthesis as in the synthesis of h amylin 1 37 H KCNTATCATORLANFLVHSSNNFGAILSSTNVGSNTY NH gt pseudoproline dipeptides underlined Pseudoproline dipeptides were necessary to obtain the peptide with a usable purity and yield One limitation is pseudoproline dipeptides must contain a serine or threonine and can therefore only be used in sequences containing those amino acids Hmb and Dmb amino acids are less restricted but are not available for all 20 standard amino acids D 4 Side Reactions Accelerated by Heat 1 Racemization Racemization is the partial conversion of a chiral am
155. y 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 X software by clicking on the X in the upper right corner of the main screen then double clicking on the Prelude X user software icon on the desktop To cancel changes click the Cancel button 94 2 6 4 Diagnostics The Diagnostics 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 Bian MECA The Reporter window will open 95 Reporter Yer 1 0 0 93 x User Archive DataBase Include In Report Pata MAsTER_USER mdb ade Y Job Summary IV Job Deta
156. y 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 61 1 800 477 6834 time to deselect an RV Alternatively 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 US 2 WARNING Calibration can take 15 minutes to complete Make sure RYs 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 the Actual Volume uL column next to the appropriate RV x Bottle Preparations Special Bottles Solvent Calibration Measure volume in collect tube and enter in appropriate Actual Volume field hen done press

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