PS/3 User Manual - Protein Technologies, Inc.
Contents
1. Catalog No Accessories Quantity PS3 300001 Reaction Vessel Small Scale 30 mL 1 ea PS3 300005 Reaction Vessel Large Scale 75 mL 1 ea SMP 220056 RV Cap 1 ea EA Reagent Vial Cap 8 Septa en PS3 SEP100 Vial Sepia Pkg of 100 PS3 SEP500 p Pkg of 500 Catalog No Replacement Parts Quantity SMP 300007 Bottle amber 4 L 1 ea SMP 300008 Bottle amber 1 L new style 1 ea SMP 260205 Cap SOLV and ACT DEP new style 1 ea SMP 260207 Cap ACT DEP old style 1 ea SMP 260076 Insert SOLV and ACT DEP new style 1 ea SMP 260077 Insert ACT DEP old style 1 ea PS3 270042 O Ring SOLV and ACT DEP new style 1 ea PS3 270041 O Ring ACT DEP old style 1 ea SMP RF 100 Bottle Filter Pkg of 100 SMP 260187 Filter Housing Bottle 1 ea PS3 260088 Part A Inline Filter Housing 1 ea PS3 260089 Part B Inline Filter Cap 1 ea PS3 010040 Filter Washer Set 30 Filters amp 10 Washers 1 ea PS3 270066 Membrane Valve Manifold new style 1 ea PS3 270010 Membrane Valve Manifold old style 1 ea SMP 350019 Quick Connect Male 1 ea PS3 350024 RV Fitting Upper Reducing Elbow Connector 1 ea PS3 350032 RV Fitting Lower Reducing Straight Connector 1 ea SMP 690015 SMC Valve new style manifold 1 ea PS3 870007 Tubing Pneutronics 0 078 ID Per Foot PS3 002 Teflon tubing 25 ft 1 ea PS3 310006 Waste Tank 1 ea Catalog No Tools Quantity SMP 680001 Hex Allen Wrench Set 1 ea SMP 680010 0 125 Lee Tool 1 ea2. 1W C Chan and P D White 2000 Fmoc Solid Phase Peptide Synthesis A Practical Approach 43 1 800 477 6834 4 3 2 Cleavage Procedure 1 1 Prepare the appropriate cleavage solution 2 Remove the RV from the PS3 and move it to the hood 3 Setup a 250 mL side arm flask with a coarse 15 mL fritted disc B chner funnel in a rubber stopper 4 Squirt DCM into the RV and pour the DCM resin slurry into the filter funnel NOTE If resin sticks to the sides of the RV after rinsing with DCM try DMF 5 Draw off the rinse solvent with a vacuum line Rinse the resin 2 3 more times with DCM 6 Empty the solvent s from the flask into a waste container 7 Cover the funnel with a piece of tissue paper and hold it in place with a rubber band 8 Move the covered funnel to a vacuum desiccator and dry under vacuum for 30 minutes to an hour 9 Remove from the desiccator and move the resin to a labeled appropriately sized vial with a well sealing cap Add approximately 1 mL of TFA cocktail Section 4 3 1 for every 20 mg of resin in the vial 10 Every 10 minutes gently shake each vial The resin will stick to the sides Try to wash it off the sides by swirling the mixture Mix for a total of 1 to 2 hours depending on the peptide and TFA cocktail mix 11 After mixing set up a side arm flask and B chner funnel Slowly pour the cleavage mixture into the funnel and collect the peptide solution in the side arm flask Rinse
3. Fmoc L Arg Pbf OH HBTU 2 an Leer Fmoc L Arg Pmc OH HBTU aa PSSHSNT Fmoc L Asn Trt OH HBTU nn PSSH5DB Fmoc L Asp OtBu OH HBTU gt a PSSHSCT Fmoc L Cys Trt OH HBTU 2 A Iesse Fmoc L Glu OtBu OH HBTU pe O PSSH5OT Fmoc L Gin Trt OH HBTU nn PSSHSG Fmoc L Gly OH HBTU pe u PSSHSHT Fmoc L His Trt OH HBTU ne d lu Fmoc L lle OH HBTU peut Fmoc L Leu OH HBTU RA alte PS3H5KBO Fmoc L Lys Boc OH HBTU 2 an kn Fmoc L Met OH HBTU or Fmoc L Phe OH HBTU nn Lesser Fmoc L Pro OH HBTU Ge d PSSH5SB Fmoc L Ser tBu OH HBTU 2 re psa meta Fmoc L Thr tBu OH HBTU pe O Iessen Fmoc L Trp OH HBTU nn PSSH5WRC Fmoc L Trp Boc OH HBTU pe kal E meva Fmoc L Tyr tBu OH HBTU n an Fmoc L Val OH HBTU Ry ler 51 1 800 477 6834 A 2 Bulk N Fmoc Protected Amino Acids Preweighed Catalog No B 25 A B 100 A B 25 RBF B 100 RBF B 25 RP B 100 RP B 25 NT B 100 NT B 25 DB B 100 DB B 25 CT B 100 CT B 25 EB B 100 EB B 25 QT B 100 QT B 25 G B 100 G B 25 HT B 100 HT B 25 1 B 100 I B 25 L B 100 L B 25 KBC B 100 KBC B 25 M B 100 M B 25 F B 100 F B 25 P B 100 P B 25 SB B 100 SB B 25 TB B 100 TB B 25 W B 100 W B 25 WBC B 100 WBC B 25 YB B 100 YB B 25 V B 100 V Description Fmoc L Ala OH Fmoc L Arg Pbf OH Fmoc L Arg Pmc OH Fmoc L Asn Trt OH Fmoc L Asp OtBu OH Fmoc L Cys Trt OH Fmoc L Glu OtBu OH Fmoc L Gln Trt OH Fmoc L Gly OH Fmoc L
4. SMP 680020 0 125 Lee Torque Tool 1 ea SMP 680017 Modified 14 Nut Driver 1 ea 54 www peptideinstruments com Appendix C Ninhydrin Test Fmoc solid phase peptide synthesis consists of two alternating steps 1 Deprotection of the N terminal amino group on the peptide 2 Coupling of the next protected amino acid to the peptide It is critical that both steps go to completion to produce a reasonable yield of the desired peptide For example 15 coupling steps that each go to 98 completion will yield approximately 74 of the desired peptide sequence If coupling efficiency drops to 95 each cycle only 46 of the desired peptide sequence will be recovered Deprotection results in a free amino group on the N terminus of the peptide Coupling the next activated amino acid replaces the free amine function with a protected amino acid The ninhydrin test or Kaiser test is a sensitive assay testing for the presence of free primary amine groups on a peptide and is used to determine whether deprotection and coupling steps have gone to completion Prepare the following solutions and store in small dropper bottles Solution A 1 Dissolve 2 5 g of ninhydrin in 50 mL of n butanol or ethanol 2 Warm the alcohol in a water bath or with a stir plate to help dissolve the ninhydrin Solution B 1 Melt 40 g of crystalline phenol in a water bath 2 Pour the liquefied phenol into 20 mL of n butanol or ethanol Solution C 1 Dissol
5. 1 ADVANGE CAROUSEL 2 GO TO CAROUSEL POSITION 3 INJECT POSITION 4 UP POSITION To start an operation press the number or use the arrow buttons to move to the operation then press ENTER Each operations are described below 1 ADVANCE CAROUSEL This will advance the carousel one position 2 GO TO CAROUSEL POSITION 4 __ Type in the position number of an amino acid vial and press RUN to move it under the injector Use two digits when entering in position number 3 INJECT POSITION The injector will inject the needles into the capped vial This operation is useful in determining if the carousel is aligned properly Always have a vial in place when using this operation to avoid damaging the synthesizer 4 UP POSITION The needles will return to home position WARNING Needles can cause serious damage Keep hands away from the injector needles Press EXIT to return to the MANUAL OPERATION screen 2 3 3 Reaction Vessel Control The REACTION VESSEL CONTROL screen allows the synthesizer to perform individual operations outside of a synthesis To open the REACTION VESSEL CONTROL screen from the MAIN MENU press 2 for MANUAL OPERATION then 3 for REACTION VESSEL CONTROL FILL MIX DISP REP RV 1 SOLV 05 0 00 00 10 1 RV2 SOLV 05 0 00 00 10 1 RV 3 SOLV 05 0 00 00 10 1 The screen shows the three reaction vessels with four columns 1 FILL Choose an action SOLV DEP AA or ACT by moving the cursor u
6. 1 2 Instrument Setup 1 2 1 Instrument Installation Procedure To install the PS3 you will need 1 Aflat sturdy surface 4 x 3 feet capable of supporting 150 Ibs to allow easy access for loading reagents operating the keypad and viewing the display The surface should be near a primary power outlet a fume hood and a nitrogen source Ambient temperature should be 50 100 F with relative humidity below 90 Do not place the instrument where it can be exposed to extreme temperatures e g near heating or cooling ducts near open windows or in direct sunlight 2 A grounded primary power source A 6 foot power cord is supplied with the PS3 Plug the instrument into the power outlet 3 Arelatively pure gt 99 9 and dry source of pressurized nitrogen The PS3 is designed to use two nitrogen tanks although one tank may also be used When one cylinder is depleted the instrument automatically switches to the second cylinder without interrupting the synthesizer Each standard size nitrogen cylinder should last for two weeks under continuous operation Two 10 foot tubing sets with inch FNPT fittings are supplied 10 www peptideinstruments com with the PS3 Attach these fittings to the pressure regulators on each nitrogen tank Connect the other end of each tube directly to the quick disconnect bulkhead fittings labelled N2 IN on the left side of the PS3 Insert each fitting until secured and turn on both nitr
7. Customize Delivery Volumes 35 37 User Programs 35 36 Depa stas 8 14 15 18 19 53 BOM dol 13 26 54 Ell keete 26 30 37 EET REE 15 Dispose 8 22 27 29 30 36 37 EMO rra 47 Double Coupling 19 21 35 56 Edit and Run 15 17 20 22 35 38 A Besten 31 47 49 Filter Ge ss 13 54 PINE des 33 34 47 54 Final Deprotection 35 38 Fmoc Chemistry 14 15 43 GROMMET since ceai OWE 9 11 HB TO ee 8 14 16 51 53 Injectar cc ia 12 24 25 34 61 AOE pp E RI RAA 48 Installation Amino Acid Vial 12 DOME aa 13 14 Instrument 10 Reaction Vessel 11 Manual Operation 17 23 26 36 37 39 Mix 22 23 26 27 29 30 36 38 Mx RV Vol 28 30 Ninhydrin Test 55 57 Nitrogen 8 10 11 14 23 24 28 29 35 41 42 48 49 Pause Synthesis 26 31 37 38 56 Printen EE 11 27 30 33 Programs ASSION Senter 17 21 33 37 38 o EE 17 21 36 PREG age 15 18 21 30 USER rc 17 23 30 35 36 Wash ae 23 28 GNOME 23 25 34 36 37 Eolia ee 47 Installation reerorrvvrnrrevrnnrerrn 11 Silanization 22 22121 57 58 Wash wana RR RI 28 Reagents 16 49 51 53 Rep srs 20 23 26 28 36 38 Replacement Parts 16 54 Run Button 25 26 34 36 38 39 56 57 Set Up nees dese ere 17 27 30 37 Shu
8. 00 00 10 1 Press the RUN button Place the vials for the peptide in the carousel in the synthesis order NIYDIAAQV See Section 1 2 3 Amino Acid Vial Installation Place an empty cartridge after the last amino acid cartridge Val Enter the start position carousel position of the Asn cartridge and the final position carousel position of the empty cartridge See Section 2 2 1 Set Amino Acid Vial Positions Press the ASSIGN button and assign the following programs See Section 2 2 3 Assign Programs a N program 7 b through V program 3 c Empty program 5 59 1 800 477 6834 8 Press EXIT to save the assignments 9 Before starting make sure that all the bottles are pressurized and primed See Section 2 4 Bottle Preparation 10 Press RUN 60 www peptideinstruments com Index AA Fill Step ne 20 26 36 Accessories 15 16 54 Act8 14 15 18 20 25 34 36 37 53 Bottle 13 26 34 54 Files 26 28 30 Amino Ac 16 Assign Amino Acid Positions 17 18 33 Button 17 21 57 Programs 17 21 33 37 38 Automatic Volume Incrementing Ee 29 BOG CHEMISUY A cc aie oda 15 Bottle EE sn NE 14 Installation owania 13 14 Bottle Preparation 17 26 34 Capping wasza nik w 19 20 35 37 Carouse POSION camara tintas 18 Carousel 12 13 17 24 25 35 49 EE ee eg 48 POSO Aust 30 31 39 57 A Waita 23 41 Gi 51505 9 ae 43 45
9. 5 to the empty vial 3 3 Stopping and Restarting a Synthesis The PS3 may be stopped at any point in a program Depending on the number of times the STOP button is pressed the synthesis will 1 PAUSE If the STOP button is pressed once the PS3 will go into a pause mode and will restart when the RUN button is pressed STOP If the STOP button is pressed twice the cursor will move to the AA column and the synthesis program will temporarily halt You can now exit the EDIT AND RUN menu to perform functions in other menu areas then return to resume the synthesis program precisely where it was temporarily stopped by pressing RUN This type of stop is useful when replacing reagents NOTE If STOP is pressed twice editing functions are limited to the RV line that is currently active AA and STOP positions cannot be modified but new programs can be assigned to AA positions in the current RV line with the exception of the current AA position 3 TERMINATE If the STOP button is pressed three times the current synthesis will halt and all memory will be cleared of the current step in 38 www peptideinstruments com process Make any changes necessary i e load vials assign new AA and STOP positions edit other RV lines or edit coupling deprotection programs To resume the synthesis at the same position where the synthesis was halted note which step was completed on the printer output and enter the corre
10. All steps with this reagent should be carried out in a fume hood with gloves glasses and protective clothing Have the following chemicals available Squirt bottle of 100 ethanol Squirt bottle of DCM Squirt bottle of DMF Squirt bottle of deionized water 5 v v solution of dimethyldichlorosilane in DCM Fw WDK NOTE Pre mixed silanizing reagents such as Sigmacote from Sigma Aldrich and Enviro Coat from EMD Biosciences may be used in place of 5 v v dimethyldichlorosilane in DCM Procedure for silanizing reaction vessels 1 Wash the reaction vessel with DMF then 100 ethanol to remove resin and other chemicals 2 Wash with hot soapy water 57 1 800 477 6834 3 Use a squirt bottle of water to force water through the top and bottom frits of the reaction vessel 4 Rinse with 100 ethanol Force ethanol through the top and bottom frits of the reaction vessel with a squirt bottle 5 Dry the reaction vessel and frits to force the ethanol out of the reaction vessel 6 Cap the top and bottom of the reaction vessel with a small micro centrifuge tube 0 5 mL 7 Fill the reaction vessel with 5 v v dimethyldichlorosilane in DCM 8 Screw the cap back on and place the reaction vessel on a rack in the hood for 1 hour to overnight 9 Empty the silanizing solution from the reaction vessel It can be poured back into the original container and reused several times 10 Rinse with DCM twice
11. REACTION VESSEL CONTROL 2 3 1 Wash Programs All wash programs deliver SOLV or solvent bottle 1 fluid To select wash programs press 1 or ENTER REACTION VESSEL 5 REAGENT MANIFOLD REACTION VESSEL 6 AA INJECTOR REACTION VESSEL 7 SYSTEM VENT MANIFOLD un 1 2 3 4 IMPORTANT Make sure that reaction vessels are actually connected to the instrument if reaction vessels are to be washed or DMF will spill onto the instrument There are seven sub selections to choose from for cleaning the PS3 1 REACTION VESSEL Washes the first reaction vessel with SOLV for the set time listed in UNIVERSAL TIMES under RV WASH Section 2 5 1 The default time is 05 seconds The SOLV is delivered from the top of the RV drains out the bottom then goes to waste Nitrogen is delivered to clear the lines after the SOLV is drained 23 1 800 477 6834 2 REACTION VESSEL Washes the second reaction vessel with SOLV for the set time listed in UNIVERSAL TIMES under RV WASH Section 2 5 1 The default time is 05 seconds The SOLV is delivered from the top of the RV drains out the bottom then goes to waste Nitrogen is delivered to clear the lines after the SOLV is drained 3 REACTION VESSEL Washes the third reaction vessel with SOLV for the set time listed in UNIVERSAL TIMES under RV WASH Section 2 5 1 The default time is 05 seconds The SOLV is delivered from the top of the RV drains out the bottom then goes to waste Nitrog
12. RVs but they must be in order An example i is RV 2 then RV 3 or RV 2 only The PS3 cannot synthesize on RV 1 and then RV 3 The carousel has room for 45 amino acid vials If more positions are required the instrument must be reloaded and restarted as a new synthesis 1 2 4 Bottle Installation Four sets of tubing emerge from the front of the instrument and are attached to bottle caps Each bottle tube is labelled as follows 1 SOLV two 4 L bottles Solvent is used to wash the resin between coupling and deprotection steps and rinse the amino acid vials after the activated amino acid has been transferred from the carousel to the reaction vessel NOTE Two SOLV bottles must be attached in order for the PS3 to run properly 2 DEP 1 L bottle Deprotectant removes the protecting group from the N terminus of the growing peptide chain in preparation for coupling with an activated amino acid 3 ACT 1 L bottle Activating solution promotes the formation of a carboxyl activated amino acid and neutralizes the deprotected a amine nitrogen on the peptide chain Each bottle position has a bottle filter to prevent particulates from entering the fluid system A Teflon encapsulated o ring in a Teflon insert establishes the 13 1 800 477 6834 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 To install a bottle 1 Vent
13. equal moles of HBTU and amino acid in pre packed vials 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 and kits for peptide synthesis on the PS3 See Appendix A 3 for listings 1 4 4 Replacement Parts Accessories Protein Technologies Inc supplies replacement parts for the PS3 as well as various accessories including bottles and waste containers A partial listing of replacement parts and accessories is located in Appendix A 4 For additional part and accessory information please call our support desk at 1 800 477 6834 www peptideinstruments com Chapter 2 Introduction to Software This chapter covers the function of each software screen Use the keypad below the display to make selections 2 1 Main Menu After the PS3 is powered up and the carousel has stopped at its home position the MAIN MENU will open 1 EDIT AND RUN 2 MANUAL OPERATION 3 BOTTLE PREPARATION 4 SET UP CAUTION Pressing the ASSIGN button in the MAIN MENU will delete the user programs 2 2 Edit and Run Open the EDIT AND RUN screen from the MAIN MENU by pressing 1 RV AA PRG REAG STAT TIMER REP STOP 1 01 01 1SOLV FILL 0 00 30 1 01 2 01 01 1SOLV FILL 0 00 30 1 01 3 01 01 1SOLV FILL 0 00 30 1 01 The EDIT AND RUN sc
14. of fluid NOTE The DISP time should typically be twice as long as the FILL time to drain properly 3 Press RUN Check the volume of fluid in the RV If necessary repeat with different times 36 www peptideinstruments com 4 Once you have determined the proper FILL and DISP times press EXIT twice and press 4 for SET UP and then press 1 for SET UNIVERSAL TIMES REAG WASH 07 SOLV FILL 07 CLEAR 10 RV WASH 05 DEP FILL 07 DISPOSE 14 VENT WASH 05 ACT FILL 3 MIX ON 01 AA WASH 05 MX RV VOL 35 MIX OFF 10 5 Change REAG WASH SOLV FILL and DEP FILL times to the new FILL time determined in MANUAL OPERATION Change DISPOSE to the new time for DISP determined in MANUAL OPERATION 6 Press EXIT twice to return to the MAIN MENU 3 2 3 Capping Free amino groups at the N terminus of the peptide can be permanently blocked by acetylation with acetic anhydride This prevents the free amino groups from participating in subsequent coupling steps and producing n 1 n 2 deletion peptides with internal amino acids missing Capping will sometimes simplify subsequent purification of the peptide but indiscriminate use can result in a very poor yield of the desired peptide If automatic capping is desired after a coupling add 2 mL of acetic anhydride to a clean empty vial and put it in the carousel following the amino acid to be coupled Assign program 6 to the vial When the synthesis reaches the capping vial it will add an aliq
15. screen e Chapter 3 Running a Synthesis explains the basic steps for setting up and running a synthesis e Chapter 4 Post Synthesis Procedures explains post synthesis cleaning shutdown and cleavage procedures e Chapter 5 Errors and Recovery describes PS3 recovery steps error messages and 1 2 About The Company Protein Technologies Inc PTI is a private company based in Tucson Arizona Founded in 1985 by researchers affiliated with the University of Arizona PTI has a long history of developing and manufacturing high quality peptide synthesizers Our instruments are used in major universities pharmaceutical companies and biotechnology companies worldwide We support our products with a dedicated field service team and are proud of our reputation for reliability We value the trust our customers and partners have placed in PTI Today we continue to grow and innovate to serve the needs of the solid phase synthesis market 1 800 477 6834 1 3 Common Abbreviations AA ACT Boc DEP C F min mmol umol MTBE Mtr Amino acid Activator tertButyloxycarbonyl Deprotection Solution Degrees Celcius Degrees Fahrenheit Dichloromethane Methylene Chloride Dimethylacetamide Dimethylformamide Dispose Drain Ethanedithiol 9 Fluorenylmethyloxycarbonyl Female National Pipe Thread 2 1H Benzotriazol 1 yl 1 1 3 3 Tetramethyluronium Hexafluorophosphate 1H Benzotriazolium 1 Bis Dimethylamino Methylene 5Chl
16. terminal amino acid is proline or B benzyl aspartate When these amino acids are fully deprotected the beads will be red and little or no color will be present in the solution When coupling is complete the beads will be white and the solution clear or slightly blue indicating a negative result If beads are blue following a coupling it is recommended to stop the synthesis and add another coupling double coupling to the synthesis to improve the quality of the peptide as follows 56 www peptideinstruments com 1 Press STOP three times to terminate the synthesis and exit the synthesis program 2 Place a new vial with the same amino acid the ninhydrin test was performed on in front of the next amino acid on the carousel 3 Enter the new carousel position in the AA column The STOP column should have the final amino acid carousel position entered 4 Press ASSIGN and change the program to 6 for the new AA position Press EXIT Press RUN to resume the synthesis 5 If necessary repeat the ninhydrin test after the coupling to verify it has a negative result Appendix D Silanizing the reaction vessels Peptide resin is hydrophobic and tends to adhere to glass If resin is not in contact with the fluid in the reaction vessel there may be incomplete reactions lt is recommended to silanize the glass reaction vessels before a synthesis to prevent resin from sticking to the sides CAUTION Dimethyldichlorosilane is very toxic
17. the bottles are pressurized bottles may leak causing damage to the instrument NOTE PTI suggests rinsing and clearing bottle lines as described in Section 4 2 Steps 4 6 once or twice a year as regular maintenance 4 2 Instrument Shutdown If the synthesizer is not to be used immediately follow the following shutdown routine 1 Place 3 empty RVs on PS3 2 Place an empty fully assembled amino acid vial in the carousel Move the vial under the injector using the AMINO ACID SYSTEM CONTROL screen Section 2 3 2 41 1 800 477 6834 CAUTION Needle can cause serious damage Keep hands away from the needle 3 Perform a SYSTEM wash Section 2 3 1 4 When complete clear bottle lines as follows a Vent all bottles Section 2 4 b Discard or store used chemicals c Place empty bottles on the instrument d Pressurize and prime empty bottles Section 2 4 e Vent all bottles Section 2 4 5 Rinse the bottle lines as follows a Place methanol or dichloromethane DCM in all three bottle positions NOTE If time is a concern DMF may be substituted for methanol and dichloromethane b Pressurize and prime all bottles Section 2 4 6 Clear the bottle lines by repeating step 4 7 Remove all RVs bottles and amino acid vials 8 Turn off the power on the PS3 CAUTION Bottles must be vented before turning off PS3 Otherwise bottles may leak and damage to the instrumen
18. the vial several times with a minimal amount of TFA cocktail and pour it through the filter funnel to rinse the resin 12 Optional To remove extra TFA cocktail pour the TFA cocktail from the side arm flask into a 250 mL round bottom flask and rinse the side arm flask with TFA to transfer all the peptide solution to the round bottom flask Attach the round bottom flask to a Rotovap with a water bath at 30 32 C Make sure the acid trap coldfinger is set up to trap the TFA Evaporate as much TFA as possible 44 www peptideinstruments com NOTE Make sure that you have a good acid trap coldfinger set up to trap the TFA It is an extremely corrosive acid Alternatively you can try directly precipitating the peptide after step 11 by adding the TFA peptide solution to ice cold ether or MTBE Best results are obtained when the volume of TFA used to cleave the resin and rinse the beaker and funnel is kept to a minimum 13 Precipitate the peptide by adding ice cold ether to the peptide solution 14 Pour the precipitate ether mixture into a fine 15 mL fritted disc B chner funnel in a 250 mL side arm flask Use a stainless steel spatula to scrape the peptide off the sides of the flask 15 Remove the ether with a vacuum line connected to the side arm 16 Add 10 mL of ether to the funnel and stir the precipitate with a stainless steel spatula 17 Repeat 2 3 more times to remove most of the anisole and thioanisole 18 Allow th
19. then the dissolved mixture is transferred to the RV The amino acid vial is then rinsed with an equal volume of SOLV which is also transferred to the RV before the mixing begins If the REP for an AA step is increased additional volumes of ACT will be delivered to the same AA vial and transferred to the RV prior to the SOLV rinse 3 2 2 Customize Delivery Volumes When synthesizing at larger scales it may be necessary to adjust fluid delivery volumes and drain times To determine the volume of fluid and drain time necessary for the synthesis add the amount of resin for the synthesis to an RV and adjust the fill and dispose times using the MANUAL OPERATION screen NOTE Each RV should be run at the same synthesis scale to ensure the proper volume of fluid is added to saturate the resin 1 From the MAIN MENU press 2 to open MANUAL OPERATION then press 3 to open REACTION VESSEL CONTROL Enter Fill Enter Drain Time Time FILL MIX DISP REP RV 1 SOLV 07 0 00 30 14 1 RV 2 SOLV 05 0 00 30 10 1 RV 3 SOLV 05 0 00 30 10 1 2 Move the cursor to the FILL column and press ENTER until SOLV is displayed Move the cursor to the two digit number to the right of SOLV and change the fill time in seconds then move the cursor to the DISP column to change the drain time in seconds Set the MIX time to 0 00 30 Always have the DISP time greater than the FILL time to drain all the fluid NOTE One second of fill time equals approximately 1 mL
20. vials for Fmoc chemistry on the PS3 See Section 1 4 3 3 The default programs 1 6 on the PS3 are appropriate for Fmoc chemistry 1 3 2 t Boc Chemistry To run t Boc chemistry on the PS3 1 Bottles should contain the following a SOLV High purity methylene chloride DCM b ACT 10 v v diisopropylethylamine in DCM c DEP 50 v v trifluoroacetic acid in DCM 2 Amino acid activator vials for the carousel should be packed with t Boc protected amino acids and an equal molar quantity of a carboxyl activating reagent such as HBTU HCTU or PyBOP 3 The fixed coupling deprotection programs should be modified to extend the deprotection step DEP MIX to 30 minutes Section 2 2 2 For example in the EDIT AND RUN menu press EDIT then 3 to modify program 3 Change 0 05 00 under TIMER to 0 15 00 Press EXIT and save with a new program number The modified program will conduct two 15 minute deprotection steps to remove the t Boc group before the next coupling step 1 4 Accessories 1 4 1 Reaction Vessels 4 e 4 Protein M 30 mL 75 mL Cat PS3 300001 1 ea Cat PS3 300005 1 ea 15 1 800 477 6834 1 4 2 Amino Acid Vials Vial Cat PS3 VX 010 Pkg of 10 Cat PS3 VX 100 Pkg of 100 1 4 3 Amino Acids amp Reagents for Peptide Synthesis Protein Technologies Inc supplies high quality pre tested N Fmoc protected amino acids preweighed in 1 0 mmol and 0 4 mmol quantities with
21. 34 1 Place RV top flush into top connector Tighten connector hand tight 2 Place RV bottom flush into bottom connector Tighten connector hand tight IMPORTANT Never use tools to tighten connectors To remove loosen connectors one at a time and remove the RV 1 2 3 Amino Acid Vial Installation The capped amino acid vial contains five parts a O C lt O Ring Screw Septa Washer Vial Cap To assemble the capped vial 1 2 3 1 Place the septa into the cap 2 Put the o ring over the washer and drop the washer into the cap on top of the septa 3 Screw the cap onto the vial tightly Place filled amino acid vials into the carousel in the order of the peptide sequence from the C terminus to the N terminus The first amino acid to couple should be placed in position 1 Check to make sure each vial fits under the injector port Repeat for up to three peptides Make sure the amino acids are in order for each peptide Do not skip carousel positions 12 www peptideinstruments com To load amino acid vials for a double coupling place two vials of the given amino acid in the sequence onto the carousel For example if a peptide with the sequence PEPTIDE is going to be synthesized and double coupling is desired for D the amino acid vials should be placed in the carousel as follows Position Amino Acid Vial ESTA EE paa D LETS D be NOTE The PS3 can use one two or three
22. 5 STAT Displays the operation status for the current step FILL Fill MIX Mix DISP Dispose Drain WASH Top Wash a0sp 6 TIMER Displays the time remaining for each of the operations in the STAT field The format is H MM SS NOTE The TIMER pauses during valve operations in all MIX steps This increases the actual MIX time in an amount proportional to the number of times the valves are activated during a MIX step The following table illustrates the effect of different combined MIX ON and MIX OFF times See Section 2 5 1 on the actual mix time OFF Time Factor actual Time min actua Timo min OFF Time s Actual Time min Actual Time min EEE 128 64 e 257 22 www peptideinstruments com NOTE To calculate the number of minutes to enter for a specific MIX time use the inverse of the multiplication factor For example to conduct a DEP that mixes for exactly 4 minutes with MIX ON 1 and MIX OFF 4 multiply 4 minutes by 0 752 1 1 33 and enter the product 3 minutes in a user defined program for the DEP MIX step 7 REP Displays the number of repetitions remaining for the current step in the synthesis 8 STOP Displays the last amino acid vial position for each RV 2 3 Manual Operation To open the MANUAL OPERATION screen from the MAIN MENU press 2 or use the down arrow button then press ENTER 1 WASH PROGRAMS 2 AMINO ACID SYSTEM CONTROL 3
23. 5 seconds The time should be changed depending on the synthesis scale Two RV WASH steps follow each step of a synthesis VENT WASH The VENT WASH or Vent Manifold Wash delivers SOLV through the vent manifold to waste The wash is followed by nitrogen to remove the residual fluid This operation is only available in WASH PROGRAMS under MANUAL OPERATION The default time is 5 seconds and it represents the SOLV flow time AA WASH The AA WASH or Amino Acid System Wash rinses the amino acid injection needles with SOLV after the AA step in a synthesis The SOLV is transferred from the amino acid vial to waste The default time is 5 seconds and it represents the SOLV flow time SOLV FILL The SOLV FILL or solvent fill delivers SOLV to the designated RV for a set time The default time is 5 seconds but may be adjusted from 2 to 40 seconds If the SOLV FILL time is less than the MX RV VOL the value will automatically increase by 1 second for every fifth cycle that is completed up to a maximum 40 second delivery DEP FILL The DEP FILL or deprotection fill delivers DEP to the designated RV for a set time The default time is 5 seconds but times may be adjusted from 2 to 40 seconds If the DEP FILL time is less than the MX RV VOL the value will automatically increase by 1 second for every fifth cycle that is completed up to a maximum 40 second delivery ACT FILL The ACT FILL delivers ACT to the designated amino acid vial for t
24. His Trt OH Fmoc L lle OH Fmoc L Leu OH Fmoc L Lys Boc OH Fmoc L Met OH Fmoc L Phe OH Fmoc L Pro OH Fmoc L Ser tBu OH Fmoc L Thr tBu OH Fmoc L Trp OH Fmoc L Trp Boc OH Fmoc L Tyr tBu OH Fmoc L Val OH Quantity 259 100g 259 100g 259 100g 259 100g 259 100 g 259 100 g 259 100 g 259 100 g 259 100g 259 100g 259 100g 259 100g 259 100g 259 100g 259 100g 259 100g 259 100 g 25g 100 g 25g 100 g 25g 100 g 25g 100 g 25g 100 g 52 www peptideinstruments com A 3 Reagents amp Kits Catalog No Reagents Quantity PS3 PPR L 20 Piperidine DMF DEP 0 9L PS3 MM L 0 4 N Methylmorpholine DMF ACT 0 9L B 100 HBTU 100 g B 500 HBTU HBTU 500 g B 1K HBTU 1 kg B 100 HCTU 100 g B 500 HCTU HCTU 500 g B 1K HCTU 1 kg p Pack of H5 1 0 mmol amino acids includes varies pe quantities of the 20 amino acids LES Pack of H1 0 4 mmol amino acids includes varies Roots quantities for the 20 amino acids DER Catalog No Start Up Kit Quantity Fmoc Amino Acid Start up Kit for the PS3 Contains 5 each of H1 amino acids 0 9 L Deprotectant 0 9 L PS3 STARTKIT o AM NMM 100 g HBTU 3 of 0 1 mmol scale Rink GR amide resin 1 of 0 1 mmol scale Fmoc Gly Wang resin 53 Appendix B Replacement Parts amp Accessories 1 800 477 6834
25. P 1 SOLV 0 00 30 6 2 ACT 0 00 30 1 3 AA 0 20 00 1 4 SOLV 0 00 30 3 19 Washes resin 1 800 477 6834 IMPORTANT During an AA step ACT solution is first delivered to the amino acid vial then the dissolved mixture is transferred to the RV The amino acid vial is then rinsed with an equal volume of SOLV which is also transferred to the RV before the mixing begins If different reaction times or repetitions are desired you may edit the programs as follows 1 Inthe EDIT AND RUN screen press the EDIT button A screen will open EDIT WHICH PROGRAM 1 2 3 4 5 CAP 6 2 Select the program to edit by pressing the number on the keypad followed by ENTER if selecting program 1 If single couplings will be used edit program 1 or 3 If double couplings will be used edit program 2 or 4 If a final deprotect will be performed edit program 5 If capping will be used edit program 6 3 The screen will display the first line of the program in the RV 1 line and the cursor will be in the TIMER column Program steps will RV AA PRG REAG STAT TIMER REP STOP be displayed in 1 01 01 1SOLV FILL 0 00 30 1 01 this line only 2 01 01 1SOLV FILL 0 00 30 1 01 3 01 01 1SOLV FILL 0 00 30 1 01 4 Use the left or right arrows to move between the TIMER and REP columns Use the up or down arrows to scroll through the steps Use the keypad to enter different times or repetitions NOTE Amino acid vials have a capacity of 5 mL 3 mL is
26. PS3 Technologies Inc USERN AQNU NE PS3 Peptide Synthesizer USER MANUAL Technologies Inc O 2006 Protein Technologies Inc 4675 S Coach Dr Tucson AZ 85714 USA All Rights Reserved Document 49030004 Rev 03 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 WARNING DO NOT ATTEMPT TO MOVE THE PS3 WHILE ANY OF THE SOLVENT OR WASTE CONTAINERS CONTAIN LIQUIDS www peptideinstruments com Contents COMMONS PARROT sde seedet v A 7 Pet OU INe MENA Lu 7 2 ABGUL The CP A an act Beinen 7 1 3 Common Ee 8 Chapter 1 General Information use aaa aa ala aaa 9 1 1 General System Rei EE 9 TAA tc Fosnes kuet 9 a A NARA 9 1 1 3 PS3 Right EE 10 1 2 Instrument Rai ee Ee AO aia Aga 10 1 2 1 Instrument Installation Procedure 224444444400Rnnnnnnn nennen 10 1 2 2 Reaction Vessel re ee 11 1 2 3 Amino Acid Vial Installation na 12 1 2 4 Bottle Installation ssrdsn na oa aaa ees tos a E RAE aaa ae 13 1 3 Fmoc and t Boc CHE EE 14 ESTEMOS GENE veden 14 LE TOC CHESS 15 o GE era cit Alo 15 1 4 1 Reaction NESS S d 15 1 4 2 Amino Acid Vials a eee aaa oda 16 1 4 3 Ami
27. action vessel number The first carousel position The program assigned to the first carousel position RON 30 www peptideinstruments com While a synthesis is running the printer prints each step after it completes If the synthesis is paused the message printed is PROCESS PAUSED If the synthesis is stopped the message printed is PROCESS STOPPED If the synthesis is terminated the universal times will reset and the message printed is PROCESS TERMINATED See Section 3 3 If there is an error during the synthesis the same message on the screen will print along with the reaction vessel number carousel position program number and program step 31 1 800 477 6834 32 www peptideinstruments com Chapter 3 Running a Synthesis 3 1 Basic Synthesis Checklist Check inline filters Clean or replace as necessary Section 3 1 1 Perform startup and instrument check Section 3 1 2 Place resin in RVs and install on instrument Section 1 2 2 Load AA vials into carousel Section 1 2 3 Load and pressurize all bottles Sections 1 2 4 amp 2 4 Assign start and stop positions for each RV Section 2 2 1 Assign programs for each amino acid Section 2 2 3 Turn on printer Section 2 5 2 Start synthesis Section 3 1 3 ESE eng 3 1 1 Check Inline Filters There are five inline filters on the PS3 One for each of the three reaction vessels and one for each of the two injection needles To Reaction Vess
28. and ethanol twice 11 Move the reaction vessel to an oven preset to 80 C for 30 minutes 12 Remove from the oven and cool Appendix E ACP Test Peptide Synthesis The sequence of the test peptide that is being made is H2N VQAAIDYING COOH Since the peptide has a C terminal acid group we will be using a Wang resin which has the first amino acid G already coupled to it The amino acid couplings are then affected in the C terminus to N terminus direction which means we have to load the carousel NIYDIAAQV We also need to modify a program in order to allow us to swell the resin before the first deprotection and coupling We will modify program 3 in order to obtain this swelling program See Section 2 2 2 Edit Programs Swell Program 1 Goto the EDIT AND RUN screen 58 5 6 www peptideinstruments com Press the EDIT button Select program 3 Change the time value for the first step SOLV from 0 00 30 to 0 10 00 Press the EXIT button Save as program 7 Synthesis set up 1 Transfer the 100 mmol of Fmoc Gly Wang resin from the supplied vial to an RV Place the RV on the instrument See Section 1 2 2 Reaction Vessel Installation If there is a lot of resin stuck to the sides of the RV you can rinse the resin down using DMF or DCM To drain the RV before the synthesis go to MANUAL OPERATIONS REACTION VESSEL CONTROL and select the following for the RV you are using FILL MIX DISP REP RV X SOLV 00 0
29. ct carousel position in the AA column The cursor will shift to the REAG column Press ENTER until the correct step in the synthesis program appears i e 1 SOLV 2 DEP 3 SOLV 4 ACT etc To start at the same substep within the step move the cursor to the STAT column with the right arrow key and press ENTER until the correct substep appears i e FILL MIX DISPOSE WASH All REPS assigned to this step will be repeated Once the correct AA position correct REAG and correct STAT appear on the display press RUN to restart the synthesis NOTE It may be easier in some instances to complete a step using the MANUAL OPERATION menu then proceed to the next step in a coupling deprotection program 39 1 800 477 6834 40 www peptideinstruments com Chapter 4 Post Synthesis Procedures 4 1 Post Synthesis Cleaning After a synthesis is complete 1 Remove the RVs from the PS3 and move them to a fume hood for cleavage Replace with empty RVs Remove used amino acid vials 2 Place an empty fully assembled amino acid vial in the carousel Move the vial under the injector using the AMINO ACID SYSTEM CONTROL screen Section 2 3 2 CAUTION Needle can cause serious damage Keep hands away from the needle 3 Perform an AA INJECTOR wash Section 2 3 1 4 Discard or store used chemicals 5 Empty the waste container NOTE The PS3 bottles must be vented when not in use If nitrogen pressure is lost while
30. e arrow keys to navigate the screen and the keyboard to enter new values The default values are below REAG WASH 05 SOLV FILL 05 CLEAR 10 RV WASH 05 DEP FILL 05 DISPOSE 10 VENT WASH 05 ACT FILL 3 MIX ON 01 AA WASH 05 MX RV VOL 35 MIX OFF 10 NOTE It is important to understand that the amount of reagent necessary to suspend and wash the resin during peptide synthesis increases as the scale or peptide chain length increases Because of this the PS3 was designed with an automatic volume incrementing feature in which the SOLV FILL DEP FILL and DISPOSE times in the UNIVERSAL TIMES menu are automatically increased by 1 second after every fifth coupling deprotection program is completed This feature is designed primarily for large scale 0 5 mmol syntheses to ensure that additional solvent is delivered to the RV to thoroughly suspend the resin as the resin mass increases during synthesis 27 1 800 477 6834 The UNIVERSAL TIMES are as follows 1 REAG WASH The REAG WASH or Reagent Manifold Wash delivers SOLV through the two reagent manifolds then to the waste The wash is followed by nitrogen to remove the residual fluid This operation is only available in WASH PROGRAMS under MANUAL OPERATION The default delivery time is 5 seconds and it represents the SOLV flow time RV WASH This function delivers SOLV to the top of the reaction vessel through the RV then drains the fluid to waste The default delivery time is
31. e carousel and clean encoder wheel on carousel shaft CAROUSEL NOT AT BOTTLE POSITION PRESS EXIT Carousel bumped out of position or sensor hole is blocked Restart the PS3 by turning off the power for 5 seconds then turning it on Remove carousel and clean encoder wheel on carousel shaft NO BOTTLE AT THIS POSITION PRESS EXIT Amino acid vial is not detected at the position Use AMINO ACID SYSTEM CONTROL to rotate the carousel If vial is present at that position restart the PS3 by turning the power off for 5 seconds then turning it on Verify carousel alignment Injector Errors Errors Cause Possible Action s INJECTOR UNABLE TO RETRACT PRESS EXIT Injection needles stuck In the AMINO ACID SYSTEM CONTROL screen press 4 UP POSITION to retract the injector INJECTOR UNABLE TO MOVE TO INJECT POSITION PRESS EXIT Injector is prevented from reaching the inject position Check the nitrogen tanks and verify they have nitrogen If nitrogen tanks need to be disconnected be sure to vent bottles first to avoid solvent leakage onto the instrument UNABLE TO MOVE CAROUSEL INJECTOR NOT UP PRESS EXIT Injector not retracting In the AMINO ACID SYSTEM CONTROL screen press 4 UP POSITION to retract the injector Restart the PS3 by turning off the power for 5 seconds then turning it on 48 System Pressure www peptideinstru
32. e peptide to air dry or lyophilize the peptide 4 3 3 Cleavage Procedure 2 1 Perform steps 1 12 from Cleavage Procedure 1 Section 4 3 2 2 Transfer the peptide solution to a centrifuge vial and add ice cold ether to precipitate the peptide 3 Centrifuge the vials then decant the ether Repeat the ether centrifuge process 3 times 4 Allow the peptides to air dry or lyophilize the peptides CAUTION Many peptides have powerful biological activities and can cause adverse physiological responses Peptides can be easily absorbed through mucous membranes such as nasal passages Therefore always wear an approved particle respirator when weighing handling or otherwise working with dry peptide powders 45 1 800 477 6834 46 Chapter 5 Errors and Recovery www peptideinstruments com The following tables list common errors their cause and possible corrective actions to take If the error still persists after all suggested actions have been tried please contact your PTI Technical Service representative at 1 800 477 6834 Reaction Vessel Error Cause Possible Action s UNABLE TO DISPOSE OF RV FLUID PRESS EXIT The fluid sensor in the dispose line does not detect an air fluid transition during a dispose step Check reagent bottle fluid level completely covers the filter frit at the bottom of the Teflon tubing Add fluid if necessary Tighten reagent bottle cap Check for defecti
33. els To Injection Needles d 5 a Injection Needle Filter Housing Housings To Waste gt To check inline filters for particulates 1 Turn off the PS3 2 Unscrew the fitting from the cap then unscrew the cap from the housing To PS3 33 1 800 477 6834 Washer J Filter Fitting If the filter is dirty remove the washer and filter from the cap and wash with ethanol to remove particulate matter Place a new or clean filter in the cap with the washer over the filter and screw the cap back into the PS3 housing Screw the fitting with the tube into the cap Do not use tools to tighten the fittings and cap Avoid over tightening To check for leaks 1 2 Turn on PS3 M From the MAIN MENU press 3 to select BOTTLE PREPARATION Pressurize and prime SOLV and ACT by making the appropriate selection s and pressing RUN Press EXIT to return to the MAIN MENU Press 2 then press 3 to open REACTION VESSEL CONTROL Under the FILL column press ENTER until SOLV is displayed in each row Press RUN for each row to deliver SOLV to all 3 RVs Check for leaks Under the FILL column in row 1 press ENTER until ACT is displayed Press RUN to deliver ACT to the empty amino acid vial Check for leaks IMPORTANT The filters for the injector needles should be cleaned or replaced after each synthesis Particles may cause valve malfunction and may damage the synthesizer 34 www peptideinstruments co
34. en is delivered to clear the lines after the SOLV is drained 4 VENT MANIFOLD SOLV is delivered to the vent manifold for the set time listed in UNIVERSAL TIMES under VENT WASH Section 2 5 1 The default time is 05 seconds Nitrogen is then delivered clearing the manifolds and sending the solvent to waste 5 REAGENT MANIFOLD SOLV is delivered to the two reagent manifolds for the set time listed in UNIVERSAL TIMES under REAG WASH Section 2 5 1 The default time is 05 seconds Nitrogen is then delivered clearing the manifolds and sending the solvent to waste 6 AA INJECTOR SOLV is delivered to the injector where it is injected into an amino acid vial The amount of solvent delivered is determined by the set time listed in UNIVERSAL TIMES for AA WASH Section 2 5 1 The default time is 05 seconds After the solvent is delivered to the vial it is transferred to waste NOTE An empty capped amino acid vial must be under the injector for the AA INJECTOR wash operation 7 SYSTEM The system wash performs all of the wash steps NOTE All RVs must be in place and an empty capped amino acid vial must be under the injector for the SYSTEM wash operation 2 3 2 Amino Acid System Control The AMINO ACID SYSTEM CONTROL screen allows the user to move the carousel and control the injector From the MAIN MENU press 2 for MANUAL OPERATION then 2 for AMINO ACID SYSTEM CONTROL 24 www peptideinstruments com
35. he set time The coupling solution is then transferred to the designated RV SOLV is delivered to the same amino acid vial for the same set time mixed and transferred to the same RV The default delivery time is 3 seconds for the ACT delivery and the SOLV delivery to the vial Do not increase the delivery time above 3 seconds or the coupling mix will bubble out the top of the vial If a larger volume is needed change the REP value in an AA step when editing a program to deliver another volume of ACT to the same AA vial and transfer it to the RV prior to the SOLV rinse 28 www peptideinstruments com CAUTION Do not increase the ACT FILL value above 3 seconds Allowing more than 3 seconds of fluid may cause the coupling mix to bubble out the top of the vial 8 MX RV VOL The MX RV VOL puts an upper limit on the volume delivered to the reaction vessels The recommended maximum time for the small RV 30 mL is 20 seconds and for the large RV 75 mL is 40 seconds The default time is 35 seconds SOLV FILL DEP FILL and DISPOSE times are automatically increased by 1 second after every fifth coupling deprotection program until the MX RV VOL limit is reached by either SOLV FILL or DEP FILL Automatic volume incrementing and the MX RV VOL limit can be overridden by entering a value in the SOLV FILL or DEP FILL times that is greater than the value in the MX RV VOL time CLEAR The CLEAR operation opens the nitrogen valve and pushe
36. m 3 1 2 Startup amp Instrument Check To startup the PS3 Peptide Synthesizer 1 Turn on the power switch located on the right side of the instrument The synthesizer will go through its self check by moving the carousel around to home or position 1 It will display the message FINDING CAROUSEL HOME POSITION PLEASE WAIT 2 After the carousel has stopped at the home position the screen will display 1 EDIT AND RUN 2 MANUAL OPERATION 3 BOTTLE PREPARATION 4 SET UP 3 Check the nitrogen supply and waste level The nitrogen pressure should be greater than 70 psi and there should be enough nitrogen in the tank for the synthesis The waste tank should be empty 3 1 3 Start A Synthesis After completing steps 1 8 in the synthesis checklist Section 3 1 to start a synthesis 1 Press 1 or ENTER in the MAIN MENU to select EDIT AND RUN 2 The cursor will be under the AA column for RV 1 Move the cursor to the desired RV row then press RUN to start 3 2 Additional Synthesis Options In addition to the basic synthesis setup the following synthesis procedures may be run Customize user programs Customize delivery volumes Capping Final deprotection PONS 35 1 800 477 6834 3 2 1 Customize User Programs Program times and repetitions may be edited as described in Section 2 2 2 When changing REP values it is important to remember that during an AA step ACT solution is first delivered to the amino acid vial
37. ments com Nitrogen pressure closes the valves holding the reagents in the bottles until needed If nitrogen pressure is lost while the bottles are pressurized the valves will open and DMF the primary solvent in the PS3 will move out through the valve blocks and injection needles onto the carousel DMF is a very effective paint remover and will damage the paint on the instrument In order to prevent this from happening a 10 minute emergency vent routine will go into effect when a sensor determines that there is insufficient pressure to operate the instrument Errors Cause Possible Action s NO SYSTEM PRESSURE CHECK If the pressure is below 70 psi TANKS increase the pressure EMERGENCY VENT IN Insufficient pressure Check the nitrogen tanks and verify PROGRESS they have nitrogen and they are on PRESS STOP THEN completely PRESS EXIT If the pressure is below 70 psi NO SYSTEM increase the pressure PRESSURE CHECK Check the nitrogen tanks and verify TANKS The user stopped the they have nitrogen and they are on EMERGENCY VENT emergency vent cycle completely FINISHED After fixing the system pressure PRESS EXIT press EXIT then RUN to restart synthesis 49 1 800 477 6834 50 Appendix Appendix A Reagents For Peptide Synthesis www peptideinstruments com A 1 PS3 Pre Packed N Fmoc Protected Amino Acids and HBTU Preweighed Catalog Description Quantity Leen Fmoc L Ala OH HBTU So a PSGH5RBF
38. nder the FILL column and pressing the ENTER button The cursor will scroll through the choices Press the right arrow key and use the numeric keypad to enter the number of seconds to deliver fluid 25 1 800 477 6834 a SOLV delivers solvent from bottle 1 SOLV to the RV b DEP delivers reagent from bottle 2 DEP to the RV c ACT delivers base from bottle 3 ACT to the selected amino acid vial d AA delivers the coupling solution from the amino acid vial to the RV The amino acid vial is rinsed with SOLV and the contents are transferred to the reaction vessel 2 MIX The MIX column displays the time in H MM SS Use the numeric keypad to enter the time 3 DISP The DISP column displays the number of seconds entered to drain the solvent from the reaction vessel The DISP number should typically be twice as large as the FILL delivery time Use the numeric keypad to enter the number of seconds to drain fluid 4 REP The REP column displays the number of repetitions entered for the FILL step Use the numeric keypad to enter the number of repetitions To start an RV select an RV row using the up and down arrow buttons then press the RUN button To pause a step press STOP then RUN to resume To cancel press STOP twice Press EXIT to return to the MANUAL OPERATION screen 2 4 Bottle Preparation The BOTTLE PREPARATION screen allows the user to pressurize prime and vent the solvent bottles From the MAIN MENU press 3 or
39. no Acids amp Reagents for Peptide Synthesis 16 1 4 4 Replacement Parts Accessories rrnnnrrrnnnnnnnnnnrrnnnnrrrnnnnnennnnvrnnnnnrnnn 16 Chapter 2 Introduction to Software uds A 17 SE Main Mea A AAA Een 17 2 2 EO SNAP es ee Ve co a o E de eta ah ed 17 2 2 1 Assign Amino Acid Positions ica 17 2 2 2 EGIl RE TE 18 223 ASSION Progra MS ati ae 21 2 24 Synthesis Slats une en 22 2 9 A Le sense 23 2 2 Wash Programs sr 23 2 3 2 Amino Acid System Control ata a eee d ema SEZ 24 2 3 3 Reaction Vessel Control 25 A tat a tit ta aa a 26 25 SOL LU Dica aaa eee 27 2 5 1 Set Universal TIMES seis nto tes canta eat ala cinta nd ta a no de da a ada eta 27 2 52 Piner Operations uran otel ee 30 Chapter 3 Running a Synthesis gudeverdenen 33 3 1 Basic Synthesis AAA tat ea 33 3 141 Check Inlin Finns inina A 33 3 1 2 Startup amp Instrument Check 35 3 1 3 SIDA STEG ERT 35 3 2 Additional Synthesis e UL EE 35 1 800 477 6834 3 2 1 Customize User PS e e eS 36 3 2 2 Customize Delivery Volumes usa a n i tn iat al 36 3 2 e et EE 37 GEI 38 3 3 Stopping and Restarting a Synthesis ee 38 Chapter 4 Post Synthesis Procedures nenea nenea 41 SEET 41 4 2 Instrument ShUIdoWN oi a 41 4 3 Manual Cleavage Procedures for Fmoc Chemistry 1111111112 43 4 3 1 Cleavage Cocktails au 43 4 3 2 Cleavage Procedure Tana 44 4 3 3 Cleavage Procedure 2 us o ee 45 Chapter 5 Errors and Recovery as
40. ogen tanks Adjust one regulator to 70 psi and the other to 80 psi The instrument will then use the 80 psi tank first before switching to the 70 psi tank Check for pressure leaks and tighten the fittings if needed IMPORTANT Securely fasten the cylinders with safety straps to prevent them from falling and do not move a cylinder or undo safety straps unless the metal cap is in place 4 An exhaust fume hood within 15 feet of the instrument or an elephant trunk exhaust line This will allow for safe disposal of waste products and their vapors Loosen the waste port fittings on the left side of the instrument and insert a length of 1 8 inch OD Teflon tubing into each port Secure the tubing by tightening each fitting Insert the tubing ends through the cap of the 5 gallon waste container It is best to place the waste container in a fume hood but if this is not possible a third tube is connected to the waste container cap This third tube may be directed to the fume hood from the waste container 5 Plug in the printer power cord into the right side of the PS3 along with the printer parallel cable into the printer parallel port if printer provided 1 2 2 Reaction Vessel Installation Three reaction vessels are provided with the PS3 Each vessel sits in a black rubber grommet in the black rack on the front of the instrument Elbow Connector ES by Sorew Cap Reaction Vessel Connector 11 1 800 477 68
41. olumn and type in the two digit program for the second amino acid position Continue until all amino acid positions have a selected program 4 Press the EXIT button to exit the assign programs section The cursor will return to the AA column 5 Repeat steps 1 4 to assign programs to all RVs that will be used in the synthesis 6 Press the EXIT button to return to the MAIN MENU To assign a double coupling program select from one of the two fixed double coupling programs 2 or 4 or select a modified version of those programs when assigning programs The double coupling programs automatically use the assigned amino acid position and the one next to it NOTE The double coupling program automatically overwrites any program assigned to the second amino acid position but for clarity it is suggested that the same program be assigned to the first and second coupling positions 21 1 800 477 6834 If a swelling program will be used Section 2 2 2 be sure to assign the swelling program to the first amino acid for each RV 2 2 4 Synthesis Status Once a synthesis is running the EDIT AND RUN screen will update its progress as follows 1 RV Displays the three reaction vessels available 2 AA Displays the current amino acid vial position for each RV 3 PRG Displays the program assigned to the current AA position 4 REAG Displays the current step number of the assigned program and the reagent for that step
42. olves AA HBTU with base 3 AA 0 20 00 1 AA coupling 4 SOLV 0 00 30 6 Washes resin 5 DEP 0 05 00 2 Deprotects N terminus 6 SOLV 0 00 30 8 Washes resin www peptideinstruments com Program 2 Double Coupling Coupling First COMMENTS Washes resin Dissolves AA HBTU with base AA coupling Dissolves AA HBTU with base AA coupling Washes resin Deprotects N terminus Washes resin COMMENTS Washes resin Deprotects N terminus Washes resin Dissolves AA HBTU with base AA coupling Washes resin COMMENTS Washes resin Deprotects N terminus Washes resin Dissolves AA HBTU with base AA coupling Dissolves AA HBTU with base AA coupling Washes resin COMMENTS Washes resin Deprotects N terminus Washes resin COMMENTS Washes resin Mixes Capping Reagent In Vial Caps resin STEP REAG TIMER REP 1 SOLV 0 00 30 3 2 ACT 0 00 30 1 3 AA 0 20 00 1 4 ACT 0 00 30 1 5 AA 0 20 00 1 6 SOLV 0 00 30 6 7 DEP 0 05 00 2 8 SOLV 0 00 30 3 Program 3 Single Coupling Deprotect First STEP REAG TIMER REP 1 SOLV 0 00 30 3 2 DEP 0 05 00 2 3 SOLV 0 00 30 6 4 ACT 0 00 30 1 5 AA 0 20 00 1 6 SOLV 0 00 30 3 Program 4 Double Coupling Deprotect First STEP REAG TIMER REP 1 SOLV 0 00 30 3 2 DEP 0 05 00 2 3 SOLV 0 00 30 6 4 ACT 0 00 30 1 5 AA 0 20 00 1 6 ACT 0 00 30 1 7 AA 0 20 00 1 8 SOLV 0 00 30 3 Program 5 Final Deprotect STEP REAG TIMER REP 1 SOLV 0 00 30 3 2 DEP 0 05 00 2 3 SOLV 0 00 30 6 Program 6 Capping STEP REAG TIMER RE
43. oro Hexafluorophosphate 1 3 Oxide Potassium Cyanide Liter Pounds Molarity moles L Minute Milliliter Millimoles Micromoles Methyl tert Butyl Ether 4 methoxy 2 3 6 Trimethylphenyl Sulfonyl Nitrogen N Methylmorpholine N Methyl 2 Pyrrolidone Outside Diameter tert Butyl Ester 2 2 5 7 8 Pentamethylchroman 6 Sulfonyl 2 2 4 6 7 Pentamethyldihydrobenzofuran 5 Sulfonyl Piperidine 1H Benzotriazole 1 yl Oxy Tris Pyrrolidinophosphonium Pounds Per Square Inch Reaction Vessel Reagent Repetitions Solvent Status tert Butyl Trifluoroacetic Acid Triisopropylsilane Trityl www peptideinstruments com Chapter 1 General Information 1 1 General System Description 1 1 1 PS3 Front RVs and a d Display Grommets gt 8 4 d and a JA ES o Nz Ports gt Keypad Needles and Injector Needle E Deliver Ep e 3 lt Power and Tubes a gt k yt ls em Printer Ports Inlet Ports dC MT TTT Carousel IPSS en Bottle Amino Acid Holder Vials 1 1 2 PS3 Left Side Nitrogen Inlets Delivery E Tubes A E Injection Needle gt Inline Filter Housings Waste pe Ports 1 800 477 6834 1 1 3 PS3 Right Side Pinter PRINTER Port PRINTER ON ONLY On Off Printer Switch T gt Power Port OF FOR 100V 120V INPUT USI 2 5A 250V JAG FUSE Power Port gt FOR 200V 240V INPUT LSA 250V JAG PUSE
44. reen has multiple functions It is used to Assign start and stop amino acid positions for each RV Edit programs Assign a program to each amino acid position Display synthesis status during a run EON 2 2 1 Assign Amino Acid Positions 1 From the MAIN MENU press 1 to open the EDIT AND RUN screen 17 1 800 477 6834 First amino Last amino acid acid vial position vial position RV AA PRG REAG STAT TIMER REP STOP 1 01 01 1SOLV FILL 0 00 30 1 15 2 16 01 1SOLV FILL 0 00 30 1 26 3 27 01 1SOLV FILL 0 00 30 1 32 2 Under the AA column enter the carousel position number that contains the first amino acid C terminus to be coupled to the resin for that RV Enter 2 digits such as 03 instead of 3 3 Use the right arrow button to move the cursor to the STOP column 4 Enter the carousel position number that contains the last amino acid N terminus to be coupled to the resin for that RV 5 Repeat steps 2 4 for each RV NOTE Do not skip amino acid positions Amino acid positions must be sequential from the stop position of an RV to the start position of the next RV 2 2 2 Edit Programs The PS3 can store fifteen programs The fixed programs 1 6 provided with the PS3 are listed below Programs 1 6 cannot be saved over or deleted however they can be edited and stored as programs 7 15 Program 1 Single Coupling Coupling First STEP REAG TIMER REP COMMENTS 1 SOLV 0 00 30 8 Washes resin 2 ACT 0 00 30 1 Diss
45. s any fluid into its designated destination such as reaction vessels amino acid vials or waste The default time is 10 seconds 10 DISPOSE The DISPOSE or reaction vessel dispose time drains the fluid from the reaction vessel to the waste after fills and mixes The DISPOSE time should be twice as long as the SOLV FILL and DEP FILL times to ensure fluid is out of the RV The default time is 10 seconds but may be adjusted from 2 to 99 seconds It is automatically increased by 1 second for every fifth cycle that is completed up to a maximum of 50 seconds DISPOSE times increment until the MX RV VOL limit is reached by either SOLV FILL or DEP FILL If 50 seconds is not sufficient to drain the RV DISPOSE may be set from 51 seconds up to 99 seconds without automatic volume incrementation MIX ON The MIX ON is the amount of time the nitrogen valve is open to allow nitrogen to enter the bottom of the reaction vessel and mix the resin and fluid The default time is 1 second Increasing the number of seconds will increase the mixing time but too much mixing may cause evaporation of solvent 12 MIX OFF The MIX OFF is the amount of time the nitrogen valve is closed during a mix No nitrogen enters the reaction vessel The default time is 10 seconds NOTE MIX ON and MIX OFF together control the amount of nitrogen bubbling during a mix If MIX ON is 1 and MIX OFF is 4 it will bubble 1 second every 5 seconds It is no
46. sisen dada O 47 A Na OO ROA OO 51 Appendix A Reagents For Peptide Gvnthesis 51 A 1 PS3 Pre Packed N Fmoc Protected Amino Acids and HBTU PRE WIG CG Leren eee 51 A 2 Bulk N Fmoc Protected Amino Acids Preweighed 52 A S Reagents EST 53 Appendix B Replacement Parts amp ACcessortes nenea 54 Appendix Ninhydrin TOS une SD ia 55 Appendix D Silanizing the reaction vessels AEN 57 Appendix E ACP Test Peptide Synthesis oococicicicicicicininincnconocononononos 58 Po A EE 61 vi www peptideinstruments com Introduction Thank you for purchasing your new PS3 peptide synthesizer from Protein Technologies Inc The PS3 is a fully automated microprocessor controlled instrument for synthesizing peptides using solid phase synthesis principles It is designed for users with minimal experience with peptide synthesis The PS3 allows for total flexibility in choosing synthesis scale coupling conditions deprotection conditions wash steps and reagent volumes Standard chemical protocols are included in the software and pre weighed reagents for these protocols are provided in convenient disposable containers 1 1 About The Manual In this manual Chapter 1 General Information describes the instrument layout basic installation procedures and PS3 accessories available for purchase from Protein Technologies Inc e Chapter 2 Introduction to Software explains the function of each software
47. t may occur 9 Turn off the nitrogen supply To move the PS3 1 Follow Instrument Shutdown Procedure above 2 Disconnect all cables and tubes from the PS3 3 Move the PS3 carefully 42 www peptideinstruments com 4 3 Manual Cleavage Procedures for Fmoc Chemistry CAUTION TFA is a corrosive acid and must be handled with care Safety glasses protective clothing and acid resistant gloves should be worn at all times All steps should be performed in a hood Have the following chemicals available Trifluoroacetic acid TFA Anisole Thioanisole Ethanedithiol EDT HPLC grade water Phenol Triisopropylsilane TIS Anhydrous ether or methyl tert butyl ether MTBE DCM 10 Squirt bottle of DMF 11 Squirt bottle of DCM 12 Squirt bottle of ethanol OBWNOJNAWNDU Have the following equipment available Rotovap with water bath Cold finger acid trap Vacuum pump acid resistant Centrifuge tubes minimum 15 mL oN gt 4 3 1 Cleavage Cocktails Different scavengers are used depending on the side chain protecting groups 1 A peptide containing Arg Mtr or unprotected Trp requires a cleavage solution of 81 5 TFA 5 thioanisole 5 phenol 5 water 2 5 EDT and 1 TIS 2 Peptides containing Cys Trt or Met requires a cleavage solution of 92 5 TFA 2 5 EDT 2 5 water and 2 5 TIS 3 Peptides with no Cys Trt or Met require a cleavage solution of 95 2 5 water and 2 5 TIS
48. t recommended or necessary to mix more frequently The default values in SET UNIVERSAL TIMES are for a 0 1 mmol scale synthesis in the 30 mL RV The default times may also be used for a 0 5 mmol scale synthesis of small 10 15 amino acids peptides with moderate 0 5 29 1 800 477 6834 mmol g resin substitution Syntheses of longer peptides at the 0 5 mmol scale should be done in the larger 75 mL RV with the following suggested times REAG WASH 05 SOLV FILL 15 CLEAR 10 RV WASH 05 DEP FILL 15 DISPOSE 30 VENT WASH 05 ACT FILL 05 MIX ON 01 AA WASH 05 MX RV VOL 40 MIX OFF 10 Press EXIT to return to the SET UP screen or press EXIT twice to return to the MAIN MENU 2 5 2 Printer Operations PRINTER OPERATIONS allow the user to turn the printer on off and print programs From the SET UP screen press 2 1 PRINTERON 2 PRINT USER PROGRAMS 3 PRINT FIXED PROGRAMS 4 PRINT ALL PROGRAMS The four printer options are 1 PRINTER ON Turns the printer on or off 2 PRINT USER PROGRAMS Prints programs 7 15 the programs created and saved by the user 3 PRINT FIXED PROGRAMS Prints programs 1 6 the fixed programs that cannot be saved over or deleted 4 PRINT ALL PROGRAMS Prints programs 1 15 Press EXIT to return to the SET UP screen or press EXIT twice to return to the main menu The printer prints the following information each time a synthesis starts The universal times The re
49. tdown eee 41 Solv 8 14 15 18 20 23 24 28 36 Bottle 13 26 34 54 Elli atte nc 26 30 37 38 el 20 26 28 36 SEP re 33 35 Stop Button 26 37 38 57 Column eee 18 57 SYNNESIS ar eee 38 1 800 477 6834 31 38 57 15 20 22 62 Universal Times23 24 27 29 31 37 47 Waste Container 11 41 North America Europe m 4675 S Coach Dr Banbury Oxfordshire Tucson AZ 85714 United Kingdom Phone 520 629 9626 Phone 44 0 7917 874456 Technologies Inc Toll Free 800 477 6834 Fax 520 629 9806 Website info peptideinstruments com www peptideinstruments com
50. the bottle position Section 2 4 2 Verify the o ring is properly installed in the bottle insert and the insert is in the cap Verify that the solution 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 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 unscrew the cap when the bottle position has been vented 1 3 Fmoc and t Boc Chemistries NOTE When switching between Fmoc and t Boc reagents on the PS3 be sure to perform steps 1 5 of the Instrument Shutdown procedure then repeat steps 4 and 5 two more times to fully clear reagents from the instrument Section 4 2 1 3 1 Fmoc Chemistry To run Fmoc chemistry on the PS3 1 Bottles should contain the following a SOLV High purity N N dimethylformamide DMF NOTE Dimethylacetamide DMA or N methyl 2 pyrrolidone NMP may also be used b ACT 0 4 M N methylmorpholine in DMF Cat 4 PS3 MM L c DEP 20 piperidine in DMF Cat 4 PS3 PPR L 2 Amino acid activator vials for the carousel should be packed with Fmoc protected amino acids and an equal molar quantity of a carboxyl activating reagent such as HBTU HCTU or PyBOP www peptideinstruments com NOTE PTI supplies bulk amino acids and pre packed
51. the default ACT delivery volume resulting in 6 mL total coupling solution being delivered to the RV during an AA step 3 mL ACT and 3 mL SOLV If a larger volume is needed the REP can be changed in the AA step to deliver another volume of ACT to the same AA vial and transfer it to the RV prior to the SOLV rinse 5 When finished making changes press the EXIT button A new screen will display STORE AS PROGRAM 7 8 9 10 11 12 13 14 or 15 OR ENTER 0 FOR NO CHANGE 20 www peptideinstruments com 6 Enter the number of a program file to save the changes There are 9 program files available for use 7 15 Press 0 if the program is not to be saved 7 The STORE AS PROGRAM screen will close and the EDIT AND RUN screen will open To swell the resin before a synthesis edit the first step of the program that will be used for the first coupling so that SOLV is mixed for 0 10 00 instead of 0 00 30 Be sure to assign the special swelling program to the first amino acid for each RV Section 2 2 3 2 2 3 Assign Programs Each amino acid position must have a program assigned to it To assign programs in the EDIT AND RUN screen 1 Use the arrow keys to move the cursor to an RV and press the ASSIGN button 2 The cursor will move to the PRG column Use the keypad to type in the two digit program for the first amino acid position 3 Press the down arrow button to change to the next amino acid position indicated in the AA c
52. uot of ACT reagent to the vial then transfer the mixed solution to the RV for a 20 minute capping procedure NOTE Program 4 may also be used if capping is desired following a single coupling Capping may also be done using the MANUAL OPERATION screen 1 Press STOP twice to pause the synthesis after the coupling solution is drained and the resin is washed Press EXIT to open the MAIN MENU 2 Press 2 for MANUAL OPERATION then 3 for REACTION VESSEL CONTROL 3 Press ENTER until SOLV appears under FILL Enter a FILL time that will completely suspend the resin during a MIX step 37 1 800 477 6834 Move the cursor to the MIX column and enter 0 20 00 for a 20 minute mix step Enter a DISP time that is twice as long as the FILL time Enter 1 for REP Press RUN to start the SOLV FILL and MIX Press STOP once as soon as the MIX step starts Remove the RV cap and add 2 mL of acetic anhydride Press RUN to resume the MIX step The solvent will be drained at the end of the 20 minute acetylation step After the operation is complete change the MIX time to 0 00 30 and REP to 6 Press RUN to wash the acetylated resin six times with SOLV Press EXIT twice after completing to return to the MAIN MENU Press 1 to open EDIT AND RUN and press RUN to restart the synthesis 3 2 4 Final Deprotection If final deprotection is desired place an empty amino acid vial after the last amino acid in a peptide Assign program
53. use the down arrow key to move to BOTTLE PREPARATION then press ENTER BOTTLE PREPARATION PRESSURE SOLV DEP ACT ALL PRIME SOLV DEP ACT ALL VENT SOLV DEP ACT ALL The bottles can be pressurized primed or vented separately or together For a given operation use the arrow keys to select the bottle or bottles in that row Press RUN to perform an operation NOTE All the bottles must be pressurized and primed before starting a synthesis 26 www peptideinstruments com The PS3 does not have a backflush option To remove solvent place an empty bottle in a vented position then pressurize and prime the bottle It may take two to three primes to remove small droplets Watch the fluid stream going to the waste to make sure the fluid is out of the line CAUTION Bottles are under pressure Use with caution Protein Technologies Inc recommends using safety coated bottles on the PS3 to prevent bodily damage if a bottle should break under pressure Press the EXIT button to return to the MAIN MENU 2 5 Set Up The SET UP screen allows the user to access the universal times and the printer To open from the MAIN MENU screen press 4 or use the down arrow key and press ENTER The SET UP screen will have 2 options 1 SET UNIVERSAL TIMES 2 PRINTER OPERATIONS 2 5 1 Set Universal Times Universal times allow the user to change fill drain and mix times for a synthesis To open press 1 from the SET UP screen Use th
54. ve 16 5 mg of potassium cyanide KCN in 25 mL of distilled water for a 0 001 M solution 2 Dilute 1 mL of 0 001 M KCN solution with 49 mL of pyridine Use redistilled or high purity pyridine Kaiser et al 1970 Analytical Biochemistry 34 595 598 55 1 800 477 6834 Have the following equipment and chemicals available in a hood PRE Squirt bottle of ethanol Small test tubes Paper towel Prepared solutions A B and C 100 C sand or water bath Small spatula or glass pipet Label two test tubes one is the control and the other is the sample Perform a ninhydrin test following each deprotection and coupling step as follows 1 Press STOP once to pause the PS3 after the DMF wash steps following the deprotection step Unscrew the cap on the reaction vessel and remove 10 15 mg of resin with a pipet or spatula Transfer the resin beads to the sample test tube and wash with 2 3 mL of ethanol twice Gently decant the ethanol onto a paper towel Add 3 drops of Solution A B and C to both test tubes The control will not have resin Mix gently Place the test tubes in a 100 C heated bath for 5 minutes Press RUN to resume the PS3 Compare the control to the sample Press STOP once to pause the PS3 a coupling step after the DMF wash steps following 10 Repeat steps 2 8 When deprotection is complete the beads and solution will appear an intense blue color indicating a positive result unless the
55. ve reagent bottle cap o ring Replace as necessary Tighten amino acid vial cap Check inline solvent filters Clean or replace as necessary Tighten RV screw cap Exchange RVs to check that RV frit is not plugged with resin fines Increase the DISPOSE time in the UNIVERSAL TIMES menu Increase the FILL times in the UNIVERSAL TIMES menu NOTE The PS3 automatically conducts a second dispose if the air fluid transition was not detected during the first dispose cycle DOUBLE DISPOSE is printed if this occurs during the last repetition of a step and the second dispose step is successful If the air fluid transition is not detected during the second dispose the UNABLE TO DISPOSE error message is displayed on the screen Universal Times Error Cause Possible Action s UNIVERSAL TIME ERROR ZERO TIME VALUES ARE NOT ALLOWED PRESS EXIT One of the universal times is set to 00 Check SET UNIVERSAL TIMES to correct the action times All actions must be greater than 00 47 Bottle Position 1 800 477 6834 Error Cause Possible Action s UNABLE TO MOVE CAROUSEL TO BOTTLE POSITION PRESS EXIT The carousel cannot find the next position Vials may be obstructing the carousel path There may also be a blockage at the sensor Remove the obstruction Restart the operation Restart the PS3 by turning off the power for 5 seconds then turning it on Remov
Download Pdf Manuals
Related Search