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User Manual - Thermo Fisher Scientific

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1. STORAGE STORE IN ACCORDANCE WITH 29 CFR 1910 106 BONDING AND GROUNDING SUBSTANCES WITH LOW ELECTROCONDUCTIVITY WHICH MAY BE IGNITED BY ELECTROSTATIC SPARKS SHOULD BE STORED IN CONTAINERS WHICH MEET THE BONDING AND GROUNDING GUIDELINES SPECIFIED IN NFPA 77 1983 RECOMMENDED PRACTICE ON STATIC ELECTRICITY STORE AWAY FROM INCOMPATIBLE SUBSTANCES DISPOSAL DISPOSAL MUST BE IN ACCORDANCE WITH STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE 40 CFR 262 EPA HAZARDOUS WASTE NUMBER D001 100 POUND CERCLA SECTION 103 REPORTABLE QUANTITY CONDITIONS TO AVOID AVOID CONTACT WITH HEAT SPARKS FLAMES OR OTHER SOURCES OF IGNITION VAPORS 17 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B MAY BE EXPLOSIVE AND POISONOUS DO NOT ALLOW UNNECESSARY PERSONNEL IN AREA DO NOT OVERHEAT CONTAINERS CONTAINERS MAY VIOLENTLY RUPTURE AND TRAVEL A CONSIDERABLE DISTANCE IN HEAT OF FIRE SPILL AND LEAK PROCEDURES OCCUPATIONAL SPILL SHUT OFF IGNITION SOURCES STOP LEAK IF YOU CAN DO IT WITHOUT RISK USE WATER SPRAY TO REDUCE VAPORS FOR SMALL SPILLS TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS FOR LATER DISPOSAL FOR LARGER SPILLS DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL NO SMOKING FLAMES OR FLARES IN HAZARD AREA KEEP UNNECESSARY PEOPLE AWAY ISOLATE HAZARD AREA AND DENY ENTRY PROTECTIVE EQUIPMENT VENTILATION PROCESS ENCLOSURE VENTILATION RECOMMENDED TO MEET PUB
2. 001 001 Run Monitor HOLD PAUSE NEXT S jump S more MODULE a gt S 20 F 9 P F PAUSE T 1 Jump Step Menu la module JUMP Cvcle 10 Module jdacgfi Jump Module Menu le module JUMP MODULE g gt S 1 F 68 L gt WASTE T 6 module JUMP S 1 16 00 F68 L gt WASTE T 006 006 Run Monitor p HOLD PAUSE NEXT S jump S more Figure 2 12 How to jump out of module a after a powerfailure 6 When synthesis is interrupted at Step 16 in module g end the run a Inthe Run Monitor press the more soft key until the end run soft key appears Figure 2 13 b Press the end run soft key When the message Are you sure you want to end the run appears press the END RUN soft key 03 2002 Routine Operation 2 27 Applied Biosystems Run Monitor End Run Menu End of run report S 16 16 00 F 04 END LOOP T 001 001 HOLD PAUSE NEXT S jump S more S 16 16 C 10 15 M jdacgfi LOCK set int end run more l Are you sure you want to end the run cancel END RUN Run ended 14 15 Run began 11 50 Cvcles run 3 OK Figure 2 13 How to end the run after a powerfailure in module a The LCD displavs the time the run began and ended The autosampler jaws are closed on the AAC that contained the amino acid that was being activated when the powerfailure occurred In the steps that follow you will open the autosampler jaws to free the wheel and re arrange the AACs for the continuation of th
3. 2 11 5 16 peptide synthesis column see PSC i Peptide Synthesis Reagent A see kon ia 4 5 f 5 16 HBTU luer fittings 2 11 g 2 26 5 17 peptide resin 5 4 6 6 6 13 Lys 2 13 h 2 25 5 17 6 16 to 6 19 Phe 2 13 M I 5 17 PIP see piperidine i 5 17 piperidine 2 8 4 11 5 8 7 6 Man Menn 22195 224 20 44 illustrations 5 11 to 5 17 delivery 4 22 ibu 4 24 5 9 T 14 7 24 j 5 17 reagent bottle 7 15 7 17 mamienanee ee printout 5 9 plumbing schematic 5 11 Manual Control Menu 2 29 7 14 Material Safety Data Sh MsDs SS pea aterial Safety Data Sheet see ser defined 6 20 Delivery 2 30 McFerran N V 5 4 2 23 7 24 Module Editor Menu 5 9 Pressure 2 30 na PE 6 20 to 6 21 waste 2 30 2 33 enu Chart Recorder 4 4 module editor soft key ey eo cleavage 5 6 MONITOR DATA soft key Pave ras of motor assembly stand by consumption 2 30 End Run 2 28 i error message 7 20 power button 2 3 Function 2 29 7 14 MSDS 1 6 power switch 1 4 Jump Module 2 26 MTBE 3 4 powerfailure 2 25 to 2 29 Jump Step 6 18 7 18 Mutter M 5 5 error messsages 7 21 Main 2 15 2 24 2 29 4 8 AN mv see millivolt recovery 2 26 5 9 7 14 7 24 recovery during activation 2 26 Manual Control 2 29 7 14 N response 2 25 Module Editor 5 9 Next key l 2 4 pre activated compound 6 18 oe to 6 21 N methylpyrrolidone see NMP pre loaded PSC 2 11 Print Reports 2 24 7 24 NMP 2 7 pre programmed lines 2 5 5 9 03 2002 Index 3 Applied Biosystems pre programm
4. 2 4 2 5 2 5 2 5 2 6 2 8 2 9 03 2002 Applied Biosystems Select and Load the Columns for Your Synthesis Check the Run File Start the Automated Synthesis on Synergy Interpreting the conductivity trace Synthesis Interruptions Pressure Test Failures and Error Messages during syntheses Operator Interruptions to Synthesis Powerfailures Synergy Shutdown Procedure Return to Operation after Shutdown 3 Post Synthesis Procedures TFA Cleavage Procedure Two stage cleavage procedure Post cleavage recommendations Further reading Cleavage Reaction Procedure Required Reagents Equipment and Labware Recover the crude peptide from the cleavage solution Recovery by centrifugation Required Reagents Equipment and Labware Recovery by vacuum filtration Required Reagents Equipment and Labware Peptide Analysis and Purification Recommended Reading 4 Maintenance and Self Tests Maintenance of the Synergy Peptide Synthesizer Adjusting the Conductivity Trace Printout Changing Synergy Fuses Self Test Menus Bottle Self Test Leak Self Test Flow Tests Flow Test 1 DMF Delivery to Vessels and Peptide Synthesis Column Flow Test 2 Calibrate Tube 2 10 2 13 2 14 2 16 2 18 2 18 2 19 2 25 2 30 2 33 3 3 3 3 3 3 3 3 3 4 3 4 3 4 3 5 3 5 3 6 3 6 3 7 3 7 3 7 3 8 4 3 4 4 4 7 4 8 4 9 4 13 4 15 4 15 4 19 03 2002 Applied Biosystems Flow Tests 3 4 and 5 Flow Test 6 Pump AAC Flow Test 7 Pump Cell Fl
5. 35 THF tetrahydrofuran stabilized 200 mL 41 TFA trifluoracetic acid 49 Svnergv Waste Profile 55 03 2002 Material Safety Data Sheets and Synergy Waste Profile l 3 AR Applied ABI PART NUMBER 902035 Ad Biosystems OHS PART NRR O ev MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENCY RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION SUBSTANCE 0 4 M DIEA DMSO NMP REAGENT TRADE NAMES SYNONYMS ABI MSDS PART 902035 P N 401254 0 4 M N N DIISOPROPYLETHYLAMINE DIMETHYL SULFOXIDE N METHYLPYROLLIDONE CHEMICAL FAMILY MIXTURE CERCLA RATINGS SCALE 0 3 HEALTH 3 FIRE 2 REACTIVITY 0 PERSISTENCE 2 NFPA RATINGS SCALE 0 4 HEALTH 2 FIRE 2 REACTIVITY 0 COMPONENTS AND CONTAMINANTS COMPONENT PERCENT 49 2 DIMETHYL SULFOXIDE CAS 67 68 5 COMPONENT PERCENT 45 9 1 METHYL 2 PYRROLIDINONE CAS 872 50 4 COMPONENT PERCENT 4 9 N N DIISOPROPYLETHYLAMINE CAS 7087 68 5 EXPOSURE LIMITS NO OCCUPATIONAL EXPOSURE LIMITS ESTABLISHED BY OSHA ACGIH OR NIOSH 1 METHYL 2 PYRROLIDINONE 100 PPM 400 MG M3 DFG MAK TWA 100 PPM BASF CORP RECOMMENDED TWA PHYSICAL DATA DESCRIPTION LIQUID BOILING POINT NOT AVAILABLE SPECIFIC GRAVITY NOT AVAILABLE VAPOR PRESSURE NOT AVAILABLE SOLUBILITY IN WATER NOT AVAILABLE ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B FIRE AND EXPLOSION DA
6. Applied Biosystems deprotection 2 3 5 4 5 6 5 15 F Headers Menu 4 6 7 4 fade headers soft key 4 6 derivatives waste line 2 32 2 34 His 2 13 pene 5 4 Fields C G 5 7 HOBt 2 7 DIEA 2 8 4 11 5 7 7 6 Flow Test 1 4 15 hold prime 2 31 calibrate 7 25 Flow Test 2 4 19 HOLD soft key 2 4 2 31 2 32 calibration 4 30 Flow Test 3 4 22 diffusion rate TD Flow Test 4 4 22 I dimethyl sulfoxide see DMSO Flow Test 5 4 22 le 2 13 DMF 2 30 4 11 5 7 7 6 Flow Test 6 4 24 illustrated modules 5 11 to 5 17 basen LG low Test 4 26 IMPORTANT l 3 deliverv 4 15 4 22 Flow Test 8 4 27 incomplete coupling 6 6 Flow Test 3 4 22 Flow Test 9 4 30 instrument access 1 5 flow to PSC 5 16 internal gas pressure Fmoc 2 3 5 4 6 13 i DMSO A 2 amino acid non standard 6 16 MONOT l NE double coupling 6 10 to 6 12 deprot ti n 6 13 interrupt svnthesis 2 19 duct svstem l 9 FRA AU 1 8 procedure 2 20 i ion scavengers 5 6 E function ar 5 8 EG 2 10 edit manual activation 7 14 Run File 6 3 to 6 19 open jaws 2 28 7 18 J Edit amp Print Menu 6 4 powerfail pause 2 25 2 26 jaws 1 6 4 14 4 34 EDT 3 4 toggle 5 8 error messages 7 20 emergency Function Menu 2 29 7 14 open function 7 18 gas tank replacement 2 23 fuses 1 6 4 7 powerfailure 2 25 paper replacement 2 23 G sensor 4 34 powerfailure 2 25 fi Jump Module Menu 2 26 reagent Fn 2 22 8 OEP E Ja Jump Step Menu 6 18 7 18 synthesis interruptions 2 19 waste bottle relacement 2 23 838 tank 1 7 K end 6 3 empty Br
7. EDT 1 2 ethanedithiol HBTU 2 1 H benzotriazol 1 yl 1 1 3 3 tetra methyluronium hexafluorophosphate HOBt 1 Hydroxybenzotriazole MTBE methyl t butyl ether NH OH ammonium hydroxide NMP N methylpyrrolidone TFA trifluoroacetic acid THF tetrahydrofuran PIP piperidine 03 2002 Synergy Safety Guidelines 1 3 Applied Biosystems gt bl Synergy Safety Symbols The symbols shown here are affixed to the Synergy Peptide Synthesizer Read the explanations and make sure you understand what the symbols mean before you in teract with the instrument in any way Electrical Symbols This symbol indicates the on and off positions of a push push main power switch This protective grounding terminal must be connected to earth ground before any other electrical connections are made to the instrument This terminal either receives or delivers alternating current or voltage This terminal can receive or supply an alternating and a direct current or voltage This symbol indicates the presence of high voltage Other This symbol appears on the instrument to indicate that further information is avail able in this Safety supplement The information corresponding to these labels on Synergy is in the following section 1 4 Synergy Safety Guidelines 03 2002 Applied Biosystems Front panel to be removed only by trained personnel Synergy Safety Procedures General Laboratory Safety Your laboratory should have all equi
8. FIRST AID REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHEMICAL REMAINS AT LEAST 15 20 MINUTES IN CASE OF CHEMICAL BURNS COVER AREA WITH STERILE DRY DRESSING BANDAGE SECURELY BUT NOT TOO TIGHTLY GET MEDICAL ATTENTION IMMEDIATELY EYE CONTACT TRIFLUOROACETIC ACID CORROSIVE VAPORS MAY BE HIGHLY IRRITATING SEE INFORMATION ON ACIDIC CORROSIVES ACIDIC CORROSIVES ACUTE EXPOSURE DIRECT CONTACT MAY CAUSE PAIN LACRIMATION PHOTOPHOBIA AND BURNS IN MILD BURNS THE EPITHELIUM REGENERATES RAPIDLY AND THE EYE RECOVERS COMPLETELY IN SEVERE CASES THE EXTENT OF INJURY MAY NOT BE FULLY APPARENT FOR SEVERAL WEEKS ULTIMATELY THE WHOLE CORNEA MAY BECOME DEEPLY VASCULARIZED AND OPAQUE RESULTING IN BLINDNESS IN THE WORST CASES THE EYE MAY BE TOTALLY DESTROYED CHRONIC EXPOSURE EFFECTS DEPEND ON THE CONCENTRATION AND DURATION OF EXPOSURE REPEATED OR PROLONGED CONTACT MAY CAUSE CONJUNCTIVITIS OR EFFECTS AS IN ACUTE EXPOSURE FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OCCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS AT LEAST 15 20 MINUTES CONTINUE IRRIGATING WITH NORMAL SALINE UNTIL THE PH HAS RETURNED TO NORMAL 30 60 MINUTES COVER WITH STERILE BANDAGES GET MEDICAL ATTENTION IMMEDIATELY INGESTION TRIFLUOROACETIC ACID CORROSIVE SEE INFORMATION ON ACIDIC CORROSIVES ACIDIC C
9. Figure 7 6 shows a trace with an irregularitv during coupling due to either a prob lem with the Synergy pump system or with activator delivery To troubleshoot this irregularity first perform Flow Test 6 PUMP AAC If this flow test passes run Flow Tests 8 Calibrate HBTU and 9 Calibrate DIEA If Flow Test 6 does not pass run a Leak Test If the Leak Test fails at Step 26 check that the left transfer vessel is tightly screwed in place If the Leak Test continues to fail call Applied Biosystems Technical Support 7 8 Troubleshooting 03 2002 Applied Biosystems You may also perform a Circuit Test page 4 36 to scan the electrical circuitry If you cannot eliminate or determine the cause of your irregular trace call Applied Biosystems Technical Support Printer related malfunctions The printer paper may become mis aligned or jammed in the printer and generate aberrant conductivity traces If this happens during a synthesis go to the Setup amp Reports Menu and press the CANCEL PRINT soft key see How to replace or re align printer paper during synthesis on page 2 23 Check the printer paper and the ink cartridge on the printer Refer to the HP Person al Printer Thinkjet Owner s Manual for Troubleshooting recommendations If necessary you may re print the conductivity trace after the printer related prob lem has been corrected How to reprint the conductivity trace 1 Inthe Main Menu press the setup amp re
10. INTERRUPT L gt WASTE BEGIN LOOP GAS gt R XFER L gt WASTE END LOOP INTERRUPT GAS gt TVB DIEA gt L GAS gt L INTERRUPT L gt WASTE DMF gt R GAS gt R XFER L gt WASTE BEGIN LOOP GAS gt R XFER L gt WASTE END LOOP zi 3 D AOR HWWWNHADAWAONTIO HA W0WNOWON OH 0Q000NOAOLVLVNNONG UG IV 10 Test Printouts 03 2002 Applied Biosystems VENT TEST 7 e o u ANoOoahRWBDND 2 88 89 2 10 88 89 14 88 89 15 88 89 2 55 57 1 2 58 56 Function Function Name INTERRUPT PRS TEST PRS TEST INTERRUPT GAS gt TVB PRS TEST PRS TEST GAS gt L PRS TEST PRS TEST GAS gt R PRS TEST PRS TEST INTERRUPT FUNCTION VALVE WAIT INTERRUPT VALVE FUNCTION Time 30 10 10 30 10 10 30 10 10 30 10 10 18 60 18 10 03 2002 Test Printouts IV 11 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosvstems com www appliedbiosystems com AS Applied D Biosystems Applera Corporation is committed to providing the world s
11. R 1983 J Chromatogr 268 112 119 Tsarbopolous A 1989 Peptide Research 2 4 258 266 03 2002 Post Synthesis Procedures 3 9 Applied Biosystems 4 Maintenance and Self Tests Contents Maintenance of the Synergy Peptide Synthesizer 4 3 Adjusting the Conductivity Trace Printout 4 4 How to change the scales on the conductivity trace printout 4 4 How to change the chart speed 4 5 How to make the date time and cycle number appear on the printout 4 6 Changing Synergy Fuses 4 7 How to change fuses 4 7 Self Test Menus 4 8 Bottle Self Test 4 9 Bottle Changing Procedure 4 9 Priming Reagent Delivery Lines 4 11 Leak Self Test 4 13 How to perform a Leak Self Test 4 13 Flow Tests 4 15 Flow Test 1 DMF Delivery to Vessels and Peptide Synthesis Column 4 15 Adjusting the internal gas pressure to calibrate DMF delivery 4 18 Flow Test 2 Calibrate Tube 4 19 Flow Tests 3 4 and 5 4 22 How to monitor the internal gas pressure 4 23 Flow Test 6 Pump AAC 4 24 Flow Test 7 Pump Cell 4 26 Flow Test 8 Calibrate HBTU 4 27 Flow Test 9 Calibrate DIEA 4 30 Cycle Test 4 32 How to run the Cycle Test 4 32 Keyboard Self Test 4 34 Wheel Self Test 4 34 Circuit Self Test 4 36 03 2002 Maintenance and Self Tests 4 1 Applied Biosystems Maintenance of the Synergy Peptide Synthesizer Pressure Vent switch DMF Delivery port PSC position To Vent Reagent bottles Transfer vessels Figu
12. THF delivery is probably sufficient At the end of a synthesis THF delivery to the PSC washes the peptide resin with volatile solvent If the contents of the PSC after a complete synthesis are dry there is no need to increase THF delivery If THF delivery is inadequate and Flow Test 4 continues to fail call Applied Biosystems Technical Support If Flow Test 5 PIP fails If piperidine delivery is below the lower calibration mark check the bottle seal on the reagent bottle and prime the PIP reagent line page 4 11 If piperidine delivery is above the higher calibration mark check the internal gas pressure reading The current internal gas pressure should be 3 5 5 5 psi How to monitor the internal gas pressure The internal gas pressure is set to 3 5 5 5 psi during Synergy installation This pres sure is fairly stable and requires little adjustment during routine operation During a synthesis you can monitor the internal gas pressure in the Run Monitor S xx yy zz F F FUNCT NAME T xxx zzz _ HOLD PAUSE NEXT S jump S more 3ead pressure here La Current 345 10 ave 1234 change 7 pressure 5 3 psi more 03 2002 Maintenance and Self Tests 4 23 Applied Biosystems Press the more soft key until the Run Monitor displays the pressure reading on the bottom line The top line displays conductivity readings When Synergy is not running a synthesis use the following procedure to monito
13. WAIT PIP gt PSC B INTERRUPT PRS PIP GAS gt TVB GAS gt PSC BEGIN LOOP DMF gt TVB DMF gt PSC GAS gt TVB GAS gt PSC END LOOP THF gt PSC GAS gt TVB GAS gt PSC GAS gt TVB DMF gt BVB GAS gt BVB Time 999 Ow wo IV 6 Test Printouts 03 2002 Applied Biosystems Flow Test 6 PUMP AAC 2 e Oo u o NOOA ON 53 51 52 3 83 88 89 4 15 61 68 30 35 2 62 60 63 2 61 2 68 22 12 15 61 68 53 Function Function Name OPEN JAWS HOME AAC CLOSE JAWS BEGIN LOOP PRS AAC PRS TEST PRS TEST END LOOP GAS gt R XFER L gt WASTE DMF gt TVB DMF gt L INTERRUPT AAC PATH PUMP AAC PATH INTERRUPT XFER INTERRUPT L gt WASTE THF gt AAC GAS gt AAC GAS gt R XFER L gt WASTE OPEN JAWS Time 999 w oOo WOWNNHAHAN HHP WWWW HH 6 o awn ww 03 2002 Test Printouts IV 7 Applied Biosystems Flow Test7 PUMP CELL Function 72 e ke ANOOaBRWBNDY 3 84 88 89 4 31 34 2 30 35 45 14 30 35 64 3 60 61 4 65 2 68 3 36 15 61 68 4 61 68 30 33 13 20 23 10 13 11 Function Name BEGIN LOOP PRS PSC PRS TEST PRS TEST END LOOP DMF gt BVB DMF gt PSC B INTERRUPT DMF gt TVB DMF gt L HBTU gt L GAS gt L DMF gt TVB DMF gt L PSC PATH BEGIN LOOP PUMP XFER END LOOP PSC PATH INTERRUPT L gt WASTE BEGIN LOOP DMF gt R GAS gt R XFER L gt WASTE END LOOP XFER L gt WASTE DMF gt TVB DMF gt PSC GA
14. appears on the screen for 10 seconds After 10 seconds Synergy auto matically re starts itself In most instances you may continue using Synergy after an Exception Message without taking any remedial action If however an error message states that the function file was modified after an automatic re start re calibrate delivery of 03 2002 Troubleshooting 7 21 Applied Biosystems HBTU and DIEA before continuing a synthesis In these rare instances end the syn thesis that was in progress before the Exception Message appeared run Flow Tests 8 and 9 and re start synthesis Call Applied Biosystems Technical Support for addi tional assistance Table 7 5 Exception Messages Except 0 TRAPV Instruction Except 1 Privilege Violation Bus Error Trace Interrupt Address Error Line 1010 Emulator Illegal Instruction Line 1111 Emulator Divide by Zero Unexpected Interrupt CHK Instruction Spurious Interrupt Operator generated Error Messages Error messages that result from an operator s actions rarely occur during a synthesis that uses the standard pre programmed modules Instead they follow one of these operator actions Trying to use Manual Control during a synthesis Trying to run a Flow Test during a synthesis e Beginning a new synthesis when the printer is printing data Starting Flow Test 6 or the Wheel Test without an AAC in position one Starting a synthesis after an instrument re set without running Fl
15. 03 2002 Routine Operation 2 35 Applied Biosystems 3 Post Synthesis Procedures Contents TFA Cleavage Procedure 3 3 Two stage cleavage procedure 3 3 Post cleavage recommendations 3 3 Further reading 3 3 Cleavage Reaction Procedure 3 4 Required Reagents 3 4 Equipment and Labware 3 4 Recover the crude peptide from the cleavage solution 3 5 Recovery by centrifugation 3 5 Required Reagents 3 6 Equipment and Labware 3 6 Recovery by vacuum filtration 3 7 Required Reagents 3 7 Equipment and Labware 3 4 Peptide Analysis and Purification Recommended Reading 3 8 03 2002 Post Synthesis Procedures 3 1 Applied Biosystems TFA Cleavage Procedure WARNING POTENTIAL EXPOSURE TO HAZARDOUS CHEMICALS Chemicals used in these procedures can be hazardous and can cause injury illness or death Read the warnings prominently displayed on the bottle labels of all hazardous chemicals Wear protective eye glasses gloves and a laboratory coat when handling these chemicals Always handle hazardous materials in a working fume hood Cleavage is the process of chemically removing both the peptide from the resin and the side chain protecting groups from the peptide If you have no previous experi ence with cleaving a new peptide we suggest you cleave on a small scale at first with 10 20 mg of peptide resin and analyze the crude peptide with HPLC You may cleave the entire contents of a peptide synthesis column or 50 200 mg of peptide resin I
16. 14 Run File 2 14 6 4 edit 6 3 to 6 19 Run Monitor 2 15 2 19 2 27 2 31 7 19 7 24 S safetv carrier 1 7 2 8 2 9 7 6 scale trace printout 4 4 scavengers 5 6 Self Test Bottle 4 9 Circuit 4 36 Flow 4 15 to 4 31 Keyboard 4 34 Leak 4 13 Wheel 4 34 Self Test Menu 4 8 Ser 2 13 set an interruption procedure 2 20 set int soft key 2 20 Set Interrupt Menu 2 20 Set up amp Report Menu 4 4 4 24 set up amp report soft key 2 23 4 4 shutdown procedure 2 30 to 2 32 side chain 5 4 deprotection 5 6 soft key bottle 4 9 CANCEL PRINT 2 23 END RUN 2 27 7 11 headers 4 6 HOLD 2 4 2 31 2 32 leak 4 13 module editor 5 9 MONITOR DATA 2 24 PAUSE 2 4 PAUSE 2 18 prime 2 31 4 11 PRINT 4 15 print reports 2 23 run control 2 14 set int 2 20 set up amp report 2 23 4 4 soft keys 2 4 solid support 5 4 solid support see resin solid phase peptide synthesis 5 3 to 5 6 5 7 solvate 6 3 SPPS see solid phase peptide synthesis stand by power consumpion 2 30 4 Index 03 2002 Applied Biosystems start synthesis 2 14 vacuum filtration recovery startup checklist 2 14 3 7 to 3 8 step 5 8 Val 2 13 Stewart J M 5 23 valve switch circuits 4 36 on off 2 3 ventilation requirements Pressure Vent 2 3 2 30 1 8 to 1 11 synthesis voltage 1 4 interruptions 2 18 to 2 24 pressure test failure 7 13 W routine start 2 14 WARNING 1 3 termination 7 11 wash 5 17 synthesis column waste position 2 3 duct 1 9 syn
17. 1400 MG KG INTRAPERITONEAL RAT LD50 650 MG KG 14 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B INTRAPERITONEAL MOUSE LD50 1000 MG KG INTRAPERITONEAL RABBIT LD50 500 MG KG INTRAPERITONEAL CAT LD50 4000 MG KG INTRAPERITONEAL GUINEA PIG LDLO 3900 MG KG INTRAMUSCULAR MOUSE LD50 MUTAGENIC DATA RTECS REPRODUCTIVE EFFECTS DATA RTECS CARCINOGEN STATUS HUMAN LIMITED EVIDENCE ANIMAL INADEQUATE EVIDENCE IARC GROUP 2B AN EXCESS RISK FOR TESTICULAR GERM CELL TUMORS AND CANCER OF THE BUCCAL CAVITY OR PHARYNX AND A NONSIGNIFICANT EXCESS OF LUNG CANCER WERE OBSERVED IN OCCUPATIONALLY EXPOSED PERSONS LOCAL EFFECTS IRRITANT INHALATION SKIN EYES ACUTE TOXICITY LEVEL TOXIC BY INHALATION MODERATELY TOXIC BY INGESTION SLIGHTLY TOXIC BY DERMAL ABSORPTION TARGET EFFECTS HEPATOTOXIN POISONING MAY ALSO AFFECT THE SKIN KIDNEYS AND CARDIOVASCULAR SYSTEM AT INCREASED RISK FROM EXPOSURE PERSONS WITH SKIN KIDNEY OR LIVER DISORDERS ADDITIONAL DATA ALCOHOL MAY ENHANCE THE TOXIC EFFECTS HEALTH EFFECTS AND FIRST AID INHALATION N N DIMETHXLFORMAMIDE IRRITANT HEPATOTOXIN CARCINOGEN TOXIC 3500 PPM IMEDIATELY DANGEROUS TO LIFE OR HEALTH ACUTE EXPOSURE MAY CAUSE IRRITATION TO THE MUCOUS MEMBRANES FLUSHING OF THE FACE HEADACHE DIZZINESS LOSS OF APPETITE NAUSEA VOMITING COLICKY ABDOMINAL PAIN AND SPASMS CONSTIPATION DIARRHEA HYPERTENSION HEPATOMEGALY AND OTHER SIGNS OF LIVER INJURY ANIMALS EXPOSED TO 5
18. 2 14 Use the standard lines in the Run Start the svnthesis in the Run Control Menu as you would any other synthesis 03 2002 Advanced Operations 6 15 Applied Biosystems Module h Module h is a pre programmed module that can be used as an alternative to module a activation Use module h to add d forms of an amino acid or a non standard Fmoc amino acid such as ornithine or norleucine in the middle of a peptide or to add an organic acid such as acetic acid or biotin to the end of a peptide You can use module h to activate the compound on Synergy or to add pre activated com pounds How to use module h to activate non standard amino acids on Synergy 1 Assemble the amino acid columns for the synthesis and determine where the non standard residue will be added in the peptide Put a calibration column or a used AAC in the wheel position for the nonstandard residue IMPORTANT Do not leave any empty positions on the wheel between the AAC in position 1 and the N terminal AAC Place a calibration column or a used AAC in the wheel position for the non standard residue For example if you want to acetvlate the N terminal amino acid of a nonamer the acetyl group is added after the ninth amino acid in the sequence Assuming there are no double couples in the sequence the first nine cycles of the run can use the standard line of modules 2 In the Run Editor Menu enter the number of times the standard cycle line should be
19. 3 Remove the empty reagent bottles and the bottle seal for each bottle on the front of Synergy You may have to use tweezers to dislodge the bottle seal from inside the ratchet cap The DMF bottle seal is built into the DMF bottle cap assembly Always use new bottle seals Bottle seals should never be used more than once Note that there are two sizes of bottle seals 0o00 0 o o H8O an Cpa Bottle seal HBTU Piperidine Tetrahydrofuran Stabilized DIEA DMSO NMP Reagent line filter on kud HBTU deliverv line Figure 4 7 Reagent bottle positions on the front of the Synergy Peptide Svnthesizer 4 Open the full reagent bottle and check the rim of the bottle for chips With the exception of the DMF bottle place a bottle seal on the rim Note You must combine the contents of two reagent bottles to prepare the HBTU reagent Instructions for preparing the HBTU reagent are found on page 2 7 If vou are replacing one of the four bottles on the front of the instrument hold the bottle straight up with its reagent delivery line inside and turn it carefully into the threads of the ratchet cap on Svnergv Figure 4 7 The bottle should fit firmly in place Caution Do not overtighten the reagent bottles If you turn the bottle past the first point of resistance you may crack the bottle seal or damage the ratchet cap assembly When all the reagent bot
20. 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION CAS NUMBER 67654 71 1 SUBSTANCE 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE TRADE NAMES SYNONYMS ABI MSDS PART 901856 P N 401091 P N 401278 N N N N TETRAMETHYL O BENZOTRIAZOL 1 YL URONIUM HEXAFLUOROPHOSPHATE HBTU C11H16F6N5OP CHEMICAL FAMILY TRIAZOLE DERIVATIVE MOLECULAR FORMULA C11 H16 F6 N5 0 P MOLECULAR WEIGHT 379 25 CERCLA RATINGS SCALE 0 3 HEALTH U FIRE 1 REACTIVITY 0 PERSISTENCE 1 NFPA RATINGS SCALE 0 4 HEALTH U FIRE 1 REACTIVITY 0 COMPONENTS AND CONTAMINANTS COMPONENT 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 PERCENT 100 0 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE CAS 67654 71 1 OTHER CONTAMINANTS NONE EXPOSURE LIMITS NO OCCUPATIONAL EXPOSURE LIMITS ESTABLISHED BY OSHA ACGIH OR NIOSH PHYSICAL DATA DESCRIPTION ODORLESS WHITE TO OFF WHITE SOLID BOILING POINT DECOMPOSES MELTING POINT 428 464 F 220 240 C DECOMPOSES SPECIFIC GRAVITY NOT AVAILABLE SOLUBILITY IN WATER SOLUBLE 21 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B FIRE AND EXPLOSION DATA FIRE AND EXPLOSION HAZARD SLIGHT FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME DUST AIR MIXTURES MAY IGNITE OR EXPLODE FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR REGULAR FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPRAY FOG OR REGULAR FOAM 1990 EMERGENCY RESPO
21. A B C letters Lower case letter modules are pre programmed upper case letter modules are user defined To remove a module from a line in the Run Editor menu put the cursor under the module and press the Delete key To insert module in an existing line put the cursor under the module immediately to the right of the intended insertion When you press an alphanumeric key the letter appears to the left of the cursor When you have finished editing in the Run Editor Menu press the Menu key The message on the LCD now asks if you want to save your changes Press the cancel soft key if you want to continue editing Press the No soft key to get out of the Run Editor Menu without saving changes Press the Yes soft key if you want to save the edited run How to erase a line in the Run Editor 1 In the Run Editor Menu press the Next or Previous keys until the line you want to erase appears on the LCD Press the erase L soft kev to delete the line from the run Press the unerase L soft kev to put an erased line back in the run How to start the svnthesis after editing the run Follow the procedure Start the Automated Svnthesis on Synergy that begins on page 2 14 Start the synthesis in the Run Control Menu as you would any other syn thesis 03 2002 Advanced Operations 6 5 TOOT EO suoyniadQpaouvapy 9 9 Figure 6 4 Conductivity trace of a synthesis scaled to fit page with slow deprotections and
22. ACUTE EXPOSURE VAPORS MAY CAUSE IRRITATION REDNESS TEARING AND BLURRED VISION THE LIQUID MAY CAUSE SEVERE IRRITATION OR POSSIBLY BURNS CHRONIC EXPOSURE RATS EXPOSED TO 5000 PPM SHOWED MARKED EDEMA OR OPACITY OF THE CORNEA FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL SALINE OCCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY INGESTION TETRAHYDROFURAN ACUTE EXPOSURE MAY CAUSE GASTROINTESTINAL IRRITATION SORE THROAT ABDOMINAL PAIN NAUSEA VOMITING DIARRHEA AND DULLNESS ASPIRATION MAY OCCUR AND CAUSE SEVERE LUNG IRRITATION AND POSSIBLY DELAYED CHEMICAL PNEUMONITIS CHRONIC EXPOSURE REPEATED AND PROLONGED EXPOSURE HAVE BEEN REPORTED TO CAUSE LIVER AND KIDNEY DAMAGE FIRST AID IF THE PERSON IS CONSCIOUS AND NOT CONVULSING INDUCE EMESIS BY GIVING SYRUP OF IPECAC FOLLOWED BY WATER IF VOMITING OCCURS KEEP THE HEAD BELOW THE HIPS TO PREVENT ASPIRATION REPEAT IN 20 MINUTES IF NOT EFFECTIVE INITIALLY GIVE ACTIVATED CHARCOAL IN PATIENTS WITH DEPRESSED RESPIRATION OR IF EMESIS IS NOT PRODUCED PERFORM GASTRIC LAVAGE CAUTIOUSLY DREISBACH HANDBOOK OF POISONING 12TH ED TREAT SYMPTOMATICALLY AND SUPPORTIVELY GASTRIC LAVAGE SHOULD BE PERFORMED BY QUALIFIED MEDICAL PERSONNEL GET MEDICAL ATTENTION IMMEDIATELY ANTIDOTE NO SPECIFIC ANTIDOTE TREAT SYMPTOMATICALLY AND SUPPORTIVELY REACTIVITY REACTIVI
23. Advanced Operations describes how and when to modify the Run File IMPORTANT Edited Run lines remain part of the Run File until you return to the Run Editor and erase them If you have never used the Run Editor Menu to modify the Run File the Run File contains the pre programmed lines shown in Table 2 1 on page 2 5 03 2002 Routine Operation 2 13 Applied Biosystems If you have modified the Run File check that it contains the appropriate Run lines for the synthesis you are about to start Start the Automated Synthesis on Synergy When all the appropriate columns are in place and the amino acid column wheel is on the instrument you can begin the automated synthesis from the Run Control Menu Synthesis Startup Checklist 1 Check the external gas supply 2 Checks level of 5 reagent bottles 3 Check waste bottle empty full waste bottle 4 Check printer power paper supply and cable connections 5 Select and load PSC and AACs C terminal gt N terminal 6 Check the Run File 7 Check Run Control Menu settings How to start synthesis in the Run Control Menu 1 Press the run control soft key in the Main Menu to go to the Run Control Menu Figure 2 6 2 To direct Synergy to use the lines that are currently in the Run File the word YES should appear after Run Begin and after Run End on the Run Control Menu If you want the printer to generate a conductivity trace of the synthesis the word YES
24. Editor Menu and edit line 1 to read ja or h c c c g f i Adda second line with the standard cycle line j dac g f i for the cycles that follow 14 Press the Menu key to return to the Main Menu In the Main Menu press the run control soft key to go to the Run Control Menu Run Begin NO Run End YES Print YES Run Control Menu ___ VES NO START 15 In the Run Control Menu move the cursor to the space after Run Begin and press the No soft key Then press the START soft key to continue the synthesis 03 2002 Routine Operation 2 29 Applied Biosystems Synergy Shutdown Procedure If Synergy is used regularly there is no need to turn off the power or remove reagent bottles between syntheses Its stand by power consumption is equivalent to that of a 20 watt light bulb However if Synergy will not be used for a month or longer flush the reagent lines with DMF and nitrogen to prevent the lines from becoming blocked with reagent crystals To perform this procedure you need the four empty bottles that were shipped on Synergy the plastic cap for the Waste port and the loop that connected the Pressure and Delivery ports at the beginning of the Installation Procedure Pressure Vent switch Loop for Pressure and Delivery ports Waste Plug VENT pressure 6 AR Figure 2 15 Waste plug and loop for Pressure and Delivery ports WARNING CHEMICAL HAZARD Some chemical
25. IMPORTANT Avoid rotating the wheel hub as you remove and replace the amino acid column wheel during a synthesis interruption Examine both ends of the amino acid column AAC for irregularities The inner rings should be circular and concentric without irregularities or cracks If vou detect an irregularitv replace the AAC 7 14 Troubleshooting 03 2002 Applied Biosystems Concentric rings on ends of AAC E Figure 7 8 Check the ends of the AAC After the AAC has been replaced on the amino acid column wheel replace the wheel without rotating the wheel hub Manually activate Function 52 CLOSE JAWS before continuing the synthesis If after replacing the AAC the pressure test continues to fail end the synthesis see How to terminate a svnthesis on page 7 11 When synthesis has ended run the Leak Self Test see page 4 13 Check that the autosampler jaws close completely on the AAC If the jaws fail to close completely call Applied Biosystems Technical Support Pressure Test Failures during a Self Test Use Table 7 3 as a guide to the possible sources of leaks After you have performed the corrective actions described in this section Synergy automatically repeats the pressure test that failed If any pressure test fails repeatedly call Applied Biosystems Technical Support for assistance Pressure Test Failure after a Bottle Change Check bottle seals bottle cap threads of replaced reagent bottles Switc
26. It the liquid waste overflows into the external waste ventilation system during a synthesis IMMEDIATELY PRESS THE PAUSE SOFT KEY TO INTERRUPT SYNTHESIS DO NOT WAIT FOR MODULE c 1 If the waste bottle is almost full follow the procedure How to set an interruption to synthesis on page 2 20 and set the interruption to step 1 of Module c 2 When the PAUSE soft key appears follow the procedure How to replace the full waste bottle on page 2 9 3 When the empty waste bottle is in place press the PAUSE soft key to resume synthesis How to replace or re align printer paper during synthesis If the printer runs out of paper or if the paper is jammed in the printer you can re trieve the conductivity data from the buffer or memory for the run The buffer holds all the conductivitv data for one peptide synthesis only Each time a new synthesis begins old data is erased to clear the buffer Once the data is erased you cannot re trieve it or print it With the procedure described here you interrupt data printing first and then replace or re align the printer paper 1 Press the Menu key to return to the Main Menu 2 Inthe Main Menu press the set up amp report soft key then press the print reports soft key to go to the Print Reports Menu Figure 2 11 3 Press the CANCEL PRINT soft key to stop sending conductivity data to the printer 4 Replace or re align the printer paper Refer to the ThinkJet Owner s Manual for instru
27. Recommended disposal methods include high temperature incineration and solidification for secure chemical landfill disposal P N 902109 Rev B July 14 1993 56 VIII Special Protective Equipment RESPIRATORY PROTECTION An MSHA or NIOSH approved respirator for organic vapors is recommended A supplied air or SCBA respirator is recommended for high vapor concentration and emergencv situations VENTILATION Handle within a well ventilated area Minimize open exposure to air PROTECTIVE GLOVES Neoprene or latex rubber gloves are recommended EYE PROTECTION Safety glasses with side shields monogoggles or face shield OTHER PROTECTIVE EQUIPMENT AS necessary to prevent skin contact IX Special Precautions PRECAUTIONS TO BE TAKEN Handle as a flammable poisonous liquid Maintain adequate ventilation at all times Do not breathe vapor Do not get in eves on skin or on clothing Accidental contact should be washed immediatelv Keep awav from heat sparks and flame Keep containers tightiv closed Spill collection materials eve wash and safetv shower should be in area of use OTHER This waste solution has strong solvent properties and will attack manv forms of rubber plastics coating fabrics and finishes X Additional Information When not directiv attached to the instrument this waste material should be stored in a secure well ventilated location suitable for flammable materials Store away from l
28. acid column contains 75 umol amino acid IIl 2 Synergy Parts and Reagents 03 2002 Applied Biosystems Peptide Synthesis Columns PSCs Amino Acid Part Number Alanine Ala 401228 Arginine Arg Pmc 401229 Asparagine Asn Trt 401230 Aspartate Asp OtBu 401231 Cvsteine Cvs Trt 401232 Glutamine Gin Trt 401233 Glutamate Glu OtBu 401234 Glycine Gly 401235 Histidine His Trt 401236 Isoleucine lle 401237 Leucine Leu 401238 Lysine Lys Boc 401239 Methionine Met 401240 Phenylalanine Phe 401241 Proline Pro 401242 Serine Ser tBu 401243 Threonine Thr tBu 401244 Tryptophan Trp 401245 Tyrosine Tyr tBu 401246 Valine Val 401247 Amide 401248 Each PSC contains 25 umol of pre loaded or amide resin Fmoc MAP Resin Columns PSCs Fmoc MAP resin Part Number 4 branched MAP 401391 8 branched MAP 401392 03 2002 Synergy Parts and Reagents IIl 3 Applied Biosystems Appendix IV Test Printouts This appendix contains printouts of all the steps and functions that compose each of the flow tests and the Vent Test The Vent Test is performed only during Synergy Installation while the waste port is plugged to check for internal leaks in the venti lation system Contents Flow Test 1 DMF gt L R PSC IV 2 Flow Test 2 CALIBRATE COLUMN IV 3 Flow Test 3 DMF gt PSC IV 4 Flow Test 4 THF gt PSC IV 5 Flow Test 5 PIP gt PSC IV 6 Flow Test 6 PUMP AAC IV 7 Flow Test7 PUMP CELL IV 8 Flow Test 8 CALIBR
29. an incomplete coupling Editing a Run to extend coupling time The sequence of some peptides can result in slow reactions during a synthesis as a result of the interactions with in the peptide resin and their influence on reagent diffusion rates see The Changing Peptide Resin Structure on page 5 5 Although it is difficult to predict which sequences will be difficult conductivity monitoring can minimize their adverse effects Incomplete coupling Figure 6 4 shows a conductivity trace with a series of two slow deprotections With con ductivity monitoring the time Synergy allows for coupling is twice the time that was needed for deprotection However as Figure 6 4 demonstrates even with a long coupling time at F a slow incomplete washout of re agents G followed the coupling a strong indication that coupling was incomplete su ystsorg pauddy Applied Biosystems Run Editor Menu line cycle Deletion peptides After an incomplete coupling or deprotection the final crude product will contain a deletion peptide A deletion peptide has one or more amino acids missing in its sequence and can often be detected by analysis with HPLC high performance liquid chromatographv Extended coupling time If you know a particular sequence is difficult you can ex tend the coupling time in the Run Editor before starting the synthesis To extend coupling time add an extra c coupling module to the cycle line for that coupling For example
30. and cap Prime DMF V S 02 06 Check DMF bottle seal and cap Prime HBTU w S 02 10 Check HBTU bottle seal and cap Prime DIEA X S 02 10 Check DIEA bottle seal and cap Prime PIP y S 02 11 Check PIP bottle seal and cap Flow Test 1 k S 04 50 Check flow test calibration tube Flow Test 2 Calibrate S 04 22 Check flow test calibration tube Flow Test 3 DM F gt PSC m S 04 20 Check flow test calibration tube Flow Test 4 THF gt PSC n S 04 17 Check flow test calibration tube Flow Test 5 PIP gt PSC o S 04 29 Check flow test calibration tube Flow Test 6 Pump AAC p S 06 26 Check test fixture on wheel Flow Test 7 Pump Cell q S 04 38 Check flow test calibration tube Bottles on the front of the instrument have ratchet caps Use a mirror to check that the threads in the ratchet cap have not been stripped Replace the reagent bottle in its ratchet cap with the reagent deliverv line inside the bottle Hold the bottle straight up and carefullv turn it into the ratchet cap threads The bottle should fit firmly in place Do not overtighten the bottle If the ratchet cap appears to be damaged call Applied Biosvstems Technical Support Pressure Test Failure during a Leak Test Check HBTU DIEA THF bottle seals and caps DMF bottle lines Follow the preceding instructions for checking bottle seals and ratchet cap threads after a bottle change If a pressure test fails at Step 2 in the leak test you do not need to check the piperidine bottle Chec
31. and h the pre pro grammed lines allow a powerfailure of up to 60 minutes If power resumes within 60 minutes of the failure Svnergv will continue the svnthesis If the powerfailure lasts longer than 60 minutes Synergy will interrupt the synthesis when the controller encounters Function 9 P F Pause If a powerfailure occurs during module a or h Synergy tolerates an outage of only 2 minutes or less If power resumes within 2 minutes of the failure Synergy will continue the synthesis If the powerfailure lasts longer than 2 minutes Syner gywill interrupt the synthesis when the controller encounters Function 9 P F Pause After a powerfailure in any module except module a or h you can press the PAUSE soft key in the Run Monitor to continue synthesis with little risk of jeopardizing the condition of the peptide If a powerfailure occurs during module a or h and lasts longer than 2 minutes it may be more cost effective to end the synthesis and start over from the beginning with fresh AACs and PSC If you prefer to continue the syn 03 2002 Routine Operation 2 25 Applied Biosystems thesis after along powerfailure occurs in module a or h the following procedure de scribes how to recover from the powerfailure How to recover from a long powerfailure in module a or h In this procedure any activated amino acid that may be left in the instrument lines or transfer vessels after the powerfailure is first washed out to the waste bottl
32. finish without any need for user interaction with the instrument However when a pressure test fails or a mechanical error is detected Synergy temporarily in terrupts synthesis At other times you may have to interrupt synthesis to fix a problem that needs im mediate attention such as an empty reagent bottle a full waste bottle a power fail ure or a printer jam Pressure Test Failures and Error Messages during syntheses Synergy automatically interrupts a synthesis whenever it detects a leak or a mechan ical error If a pressure test fails during a synthesis the LCD displays the following message Pressure test failed Pressure test failed initial 5 3 final 2 1 diff 3 2 OK Press the OK soft key to return to the Run Monitor when a pressure test fails S xx yy zz F F FUNCT NAME T xxx zzz HOLD PAUSE NEXT S jump S more After a pressure test failure the Run Monitor displays an asterisk after the word PAUSE to indicate that Synergy has automatically suspended operation If you press the PAUSE soft key to resume operation without first finding the leak in the pressure system the instrument may continue to fail the pressure test Refer to What to do after a pressure test failure on page 7 12 for further instructions Mechanical failures that cause an interruption in the synthesis can occur when the autosampler jaws do not completely open or close or when the amino acid colum
33. gt R 5 to waste through the AAC five 6 61 XFER 7 times 7 66 L gt ACC gt WSTE 30 8 4 ENDLOOP 1 9 22 THFPAAC Dry AAC 10 10 GAS gt TVB 3 11 12 GAS gt AAC 120 12 3 BEGIN LOOP 2 Dry pumping vessels 13 15 GAS gt R 3 14 61 XFER 4 15 68 L gt WASTE 3 16 4 END LOOP 1 Module i incremental movement of amino acid column wheel Step Function Function Name Time Action Description 1 2 53 50 OPEN JAWS NEXT AAC 5 Openjawsand move to next 2 AAC 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 21 Applied Biosystems Module j jawsclose on AAC Step Function Function Name Time Action Description 1 3 BEGIN LOOP 999 2 53 OPEN JAWS 3 52 CLOSE JAWS Close jaws 4 10 GAS gt TVB and 5 83 PRS AAC 10 pressure test AAC 6 88 PRS TEST 30 7 69 PRSTESTj 1 8 4 END LOOP 1 Module h alternate activation Step Function Function Name Time Action Description 1 8 P F TIME 2 2 2 INTERRUPT 3 10 GAS gt TVB 5 4 45 HBTU gt L 15 5 14 GAS gt L 5 Measure activators 6 46 DIEA gt L 10 7 14 GAS gt L 5 8 1 WAIT 60 9 30 DMF gt TVB 10 31 DMF gt BVB 11 35 DMF gt L 12 9 P F PAUSE 1 Begin transfer to PSC 13 8 P F TIME 60 14 67 L gt PSC gt WASTE 15 Substitute time 5 22 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Recommended reading In addition to the articles cited in the text of Chapter 5 the following books provide inf
34. gt TVB GAS gt PSC GAS gt TVB GAS gt BVB Time 999 10 30 MOR aA 30 30 QNN O 03 2002 Test Printouts IV 3 Applied Biosystems Flow Test 3 DMF gt PSC 72 e ke ANOOoaBRWND 3 84 88 89 4 31 34 10 13 31 1 34 2 10 13 23 10 13 10 11 Function Function Name BEGIN LOOP PRS PSC PRS TEST PRS TEST END LOOP DMF gt BVB DMF gt PSC B GAS gt TVB GAS gt PSC DMF gt BVB WAIT DMF gt PSC B INTERRUPT GAS gt TVB GAS gt PSC THF gt PSC GAS gt TVB GAS gt PSC GAS gt TVB GAS gt BVB Time 999 10 30 Q MRD IV 4 Test Printouts 03 2002 Applied Biosystems Flow Test 4 THF gt PSC 2 e Oo u o NOOA ON 3 84 88 89 4 21 24 10 13 21 1 24 2 10 13 10 11 Function FunctionName BEGIN LOOP PRS PSC PRS TEST PRS TEST END LOOP THF gt BVB THF gt PSC B GAS gt TVB GAS gt PSC THF gt BVB WAIT THF gt PSC B INTERRUPT GAS gt TVB GAS gt PSC GAS gt TVB GAS gt BVB Time 999 10 30 MOR aA 30 30 30 o 03 2002 Test Printouts IV 5 Applied Biosystems Flow Test 5 PIP gt PSC Function 72 e ke ANOOoaBRWND 3 84 88 89 4 86 44 43 10 13 44 1 43 2 87 10 13 3 30 33 10 13 4 23 10 13 10 31 11 Function Name BEGIN LOOP PRS PSC PRS TEST PRS TEST END LOOP PRS PIP PIP gt WASTE PIP gt PSC B GAS gt TVB GAS gt PSC PIP gt WASTE
35. in Figure 6 4 there are four couplings preceding the incomplete cou pling at E Therefore you would add an extra c module to the second line in this case the fifth coupling in the Run Editor IMPORTANT Edited Run lines remain in the Run File until you return to the Run Editor and erase them How to extend coupling time L 1 gt C 1 Repeat 4M jdacgfi ADD L ERASE L a f PRINT L 2 gt C 5 Repeat 1M jdacgfi ADDL ERASE L avy PRINT L 3 gt C 6 Repeat 1 M de ADD L ERASE L a f PRINT Press Menu kev Save edit session changes cancel NO VES Figure 6 5 Adding an extra c module to the second cycle line fifth cycle in a run 1 In the Main Menu press the edit amp print soft key to go to the Edit amp Print Menu then press the run editor soft key The LCD displays the Run Editor Menu with the Begin line 03 2002 Advanced Operations 6 7 Applied Biosystems Press the Next key to go to the Run Editor Menu that displays the cycle line Figure 6 5 After the word Repeat in the top line of the LCD use the alpha numeric keys to enter the number of standard cycle lines that should precede the cycle line with extended coupling For the example shown in Figure 6 4 there are four cycles before the coupling that must be extended To have four standard cycles before the cycle with extended coupling enter 4 after the word Repe
36. jaws open and the amino acid column wheel moves counterclockwise one position Module j jaws close on AAC Autosampler jaws close on amino acid column and a pressure test checks the system for leaks 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 17 Applied Biosystems Annotated Module Printouts The following pages show the steps and functions in each pre programmed module with descriptions of the synthesis actions that occur in those steps In these module printouts a few functions have an asterisk after the time allowed for that function The asterisk indicates the time allowed for that function is variable and dependent on a condition For instance in the printout for module a shown here Step 3 Function 45 HBTU gt L has a substitute time of 15 seconds This value is dependent on the result of Flow Test 8 and may be different on your instrument The default value for all substitute times is 0 zero Module a extraction and activation Step Function Function Name Time Action Description 1 8 P F TIME 2 2 10 GAS 2 TVB 5 3 45 HBTU gt L 15 4 14 GAS gt L 5 Measure activators 5 46 DIEA gt L 10 6 14 GAS gt L 5 7 30 DMF gt TVB 3 8 35 DMF gt L 3 9 62 AAC PATH 1 10 3 BEGIN LOOP 12 11 60 PUMP 10 Cycle activators through 12 61 XFER 3 the AAC 13 4 END LOOP 1 14 60 PUMP 60 15 63 AAC PATH 1 16 61 XFER 7 17 30 DMF gt TVB 3 18 31 DMF gt BVB 3 19 35 DMF gt L 1 Begi
37. of lines Most interruptions should occur in one of these modules When the first Set Interrupt Menu appears the cursor is under the Cycle number Figure 2 9 2 20 Routine Operation 03 2002 Applied Biosystems S 02 08 C 01 01 M jdacgfi Run Monitor l LOCK set int end run more Press Menu key to return to Run The first Set Interrupt Cycle 1 Module jdacgfi Step Monitor Menu BEGIN END set Figure 2 9 How to set the interrupt Cycle number 4 Use the numeric keys to enter the Cycle number where the interruption should occur 5 Use the arrow key to move the cursor under the appropriate module letter Press the set soft key and the Set Interrupt Step Menu appears Figure 2 10 The top line in the Set Interrupt Step Menu displays the Module letter you have just designated The cursor appears after the letter S under step number 1 one Most interruptions should occur in Step one Cycle 1 Module jdacgfi Step The first Set Interrupt Menu BEGIN END set MODULE g gt S 1 F 68 L gt WASTE T 6 Set Interrupt Step Menu T SET The Set Interrupt Cvcle 1 Module jdacgfi Step Confirmation CLEAR Figure 2 10 How to set the interrupt Step number 6 With Step 1 selected in the Set Interrupt Step Menu press the SET soft key to complete the Set Interruption The top line of the next LCD should confirm the 03 2002 R
38. piperidine bottle 10 40 PIP 60 Add piperidine to DMF flow 11 5 Begin Loop 30 12 WAIT 30 Wait until the conductivity 13 CHECK COND 1 Change lt 1 in 30 seconds 14 End Loop 1 15 87 PRS PIP 1 16 41 PIP 300 Turn off piperidine delivery and 17 38 DMF gt PSC 1 wash PSC with DMF for 5 min Module e end synthesis Step Function f Function Name Time Action Description 1 30 DMF gt TVB 3 Wash PSC with DMF 5 minutes 2 33 DMF gt PSC 300 3 20 THF gt TVB 3 Wash PSC with THF 5 minutes 4 23 THF gt PSC 300 5 10 GAS gt TVB 3 Gas dry PSC contents 10 min 6 13 GAS gt PSC 600 7 10 GAS gt TVB 10 8 11 GAS gt BVB 10 5 20 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Module f DMF flow to PSC Stept Function f Function Name Time Action Description 1 3 BEGINLOOP 999 2 84 PRS PSC 10 3 88 PRS TEST 60 Pressure test PSC 4 89 PRS TEST 1 5 4 END LOOP 1 6 30 DMF gt TVB 3 7 37 DMF gt PSC 1 Turn on DMF delivery to PSC 8 5 Begin Loop 30 Wait until conductivity change 9 1 WAIT 30 lt 1 in 30 seconds 10 CHECK COND 1 11 End Loop 1 12 38 DMF gt PSC 1 Turn off DMF delivery to PSC Module g wash solvents through AAC and transfer vessels Step Function Function Name Time Action Description 1 52 CLOSE JAWS 6 Close jaws on AAC 2 68 L gt WASTE 5 Drain coupling solution to waste 3 3 BEGIN LOOP 5 4 36 DMF gt R 5 Wash pump with DMF and drain 5 15 GAS
39. repeated For our example you would enter 9 after Repeat 3 Press the ADD L soft key to add a line that will activate and couple the compound In our example the compound will be added to the N terminal amino acid 4 In cycle line 2 enter the modules j dh c g fi If the non standard residue is being added in the middle of the peptide you must press the ADD L soft key to add another cycle line in this case line 3 that will contain the standard activation module module a 5 Press the Next key to go to the End line Move the cursor under the d module and press the delete key Line Cycle Repeat Module List Beg 0 1 bf 1 1 8 jdacgfi 2 9 1 jdhegfi End 9 1 e Figure 6 14 Sample Run File for N acetvlating a 9 residue peptide 6 16 Advanced Operations 03 2002 Applied Biosystems 6 Continue with the procedure Making a Peptide on Synergy that begins on page 2 5 Start the synthesis in the Run Control Menu as you would any other synthesis When the synthesis reaches Step 1 of module h Synergy automatically pauses 7 When the interruption occurs during module h remove the left transfer vessel Figure 6 15 Figure 6 15 Removing the left transfer vessel 8 Dissolve 75 umol of the non standard residue in 300 uL solvent Most compounds will dissolve in DME If 75 umol of the compound does not dissolve in 300 uL DMF you may add up to 100 uL DMSO to encourage s
40. replace the vessel OK 8 Again remove the left pump vessel place it on a scale that is accurate to 1 mg and set the scale to zero 9 When prompted repeat Steps 5 and 6 a third time Test Passed OK 10 When this display appears the flow test has been successfully completed Press the OK soft key to complete this flow test If you see the message Test failed press the OK soft key and repeat the flow test If the flow test continues to fail call Applied Biosystems Technical Support 03 2002 Maintenance and Self Tests 4 31 Applied Biosystems Cycle Test During this test you load a peptide synthesis column and 3 amino acid columns on to Synergy The instrument then performs a pre determined set of modules to syn thesize LAGV a test peptide It takes approximately 3 hours to complete this test As synthesis progresses the printer produces a trace of the deprotection and cou pling reactions of the synthesis Compare the features of the trace from your synthe sis with the Trace Features described on page 2 16 How to run the Cycle Test 1 Before running the Cycle Test turn on the printer 2 To begin the Cycle Self Test press the cycle soft key in the Self Test Menu Figure 4 6 on page 4 8 Load the PSC V AACs l 6 2 A 3 L START 3 Place a V valine peptide synthesis column PSC in the synthesis column position Figure 4 16 How to place a PSC on t
41. soft key to release the hold and finish the prime When the HOLD soft key is active an asterisk appears after the soft key name As long as it is active the software does not progress to the next step and Synergy continues to perform the step being held The values that appear after the letter T count the number of seconds the step was held While the HOLD soft key is active during these prime procedures the contents of the bottle continue to flow out of the bottle through the valve block and into the waste bottle After all the reagent lines are flushed perform the next four steps to complete the instrument shutdown procedure 7 When all the reagent primes are completed switch the Pressure Vent switch to Vent 8 Shut off the external gas supply at the gas tank regulator 9 If you are moving Synergy after the shutdown remove the waste line at the Waste port Save the compression nut on the Waste line Use a 5 8 in wrench to screw the waste plug into the Waste port Figure 2 15 If you are not moving Synergy after the shutdown empty the waste bottle but leave the waste tubing connected at the Waste port 10 Press the power switch on the front of Synergy to turn it off 2 32 Routine Operation 03 2002 Applied Biosystems Return to Operation after Shutdown Use the following procedure to re start Synergy after you have used the shutdown procedure This procedure assumes that Synergy is still connect
42. the OK soft kev and continue the Wheel Test to the end Test Passed OK 5 This displav appears if the autosampler jaw and wheel are working 03 2002 Maintenance and Self Tests 4 35 Applied Biosystems Press the OK soft key to complete the Wheel Test If the message Test failed appears on the LCD press the OK soft key and repeat the wheel test If the Wheel Test continues to fail call Applied Biosystems Technical Support Circuit Self Test The Circuit Self Test rapidly checks the ROM read only memory card and the cir cuit connections to the valves To perform the Circuit Test press the circuit soft key in the Self Test Menu Figure 4 6 on page 4 8 The message Testing appears on the LCD If the Circuit Self Test passes press the OK soft key If the Circuit Self Test fails one of the following messages appears e ROM memory error e Valve continuity failure XXXXXXXXXXXXXXXXXXXX The zeros and ones that appear in place of the X s shown here are a code that in dicates which valves have caused the test failure Make a note of the number code and call Applied Biosystems Technical Support 4 36 Maintenance and Self Tests 03 2002 Applied Biosystems 5 Principles of Solid Phase Peptide Synthesis on Synergy Contents Solid Phase Peptide Synthesis 5 3 Amino acid derivatives 5 4 Deprotection 5 4 Activation 5 4 Coupling 5 4 Solid Support 5 4 The Changing Peptide Resin Structure 5 5 Con
43. the difference be tween two consecutive averages is lt 1conductivity unit Gas Oy KOSE DMF gt PSC i Flow through Deprotection 03 2002 Principles of Solid Phase Peptide Svnthesis on Svnergv 5 15 Applied Biosystems Module e end synthesis The PSC is washed with DMF for 5 minutes followed by THF for 5 minutes Gas dries the contents of the PSC for 10 minutes Function 23 THF deliverythrough the PSC THF gt PSC Naana Module f DMF flow to PSC DMF flows into PSC and conductivity readings are averaged every 30 seconds until the difference between two consecutive averages is lt 1 conductivity unit Function 37 Deliver DMF to the peptide synthesis column PSC DMF gt PSC Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Function 66 Deliver DMF to the amino acid column AAC and out to waste Module g wash solvents through AAC and transfer vessels Coupling solution flows into waste bottle Transfer vessels and AAC are washed five times with DMF DMF washes go to waste bottle AAC and transfer vessels are gas dried bi L gt AAC gt Waste Module h alternate activation Similar to module a this module also has a built in interruption that allows you to add non standard residues to the peptide Chapter 6 gives procedures that incorpo rate module h Module i incremental movement of amino acid column wheel Autosampler
44. the luer fittings push the luer fittings onto both column ends as tightly as possible Be careful not to twist the column Figure 2 4 Place the PSC in the peptide synthesis position 3 Place the AACs on the wheel If you used a pre loaded resin in the PSC the AAC for the second amino acid from the C terminal goes in position 1 Ifyou used an amide resin the AAC for the C terminal amino acid goes in position 1 Place the N terminal AAC on the wheel last after all the other amino acids in the sequence are in place Do not leave any positions empty between the AAC in position 1 and the N terminal AAC With the pre programmed lines you do not have to tell Synergy how many amino acids are in the peptide Instead when the sensors on the autosampler detect a posi tion on the wheel without an AAC they signal the controller to end the synthesis Therefore do not leave any empty positions on the wheel except after the N termi nal AAC IMPORTANT The order of the amino acid columns on the amino acid column wheel determines the sequence of amino acids in the peptide For example to synthesize Acyl Carrier Protein 65 74 shown on page 2 10 place the G Gly peptide synthesis column in the PSC position Place an N Asn amino acid column in position 1 on the amino acid column wheel Place an I Ile amino acid column in position 2 Place a Y Tyr AAC in position 3 Place an D Asp in position 4 place another I Ile
45. the next amino acid to be added to the growing chain by con verting its carboxyl group to an active ester a chemically reactive form Coupling Coupling occurs when the activated amino acid forms an amide bond CO NH with the a amino group of the deprotected amino acid in the peptide Solid Support The most widely used support for solid phase peptide synthesis is a lightly cross linked polystyrene resin in the form of tiny beads On Synergy the first step in every synthesis is the solvation of these beads with DME With solvation the beads swell to several times their dry volume and form a solvated gel phase 3 McFerran N V Walker B McGurk C D and Scott F C 1991 Conductance measurements in solid phase peptide synthesis Int J Pept Protein Res 37 382 387 5 4 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems The Changing Peptide Resin Structure s the peptide grows within the solvated gel matrix its sequence influences the composition conformation and behavior of the resin beads The conformation of the peptide resin may effect the chemical reactivity of the syn thesis through the for mation of inter and intra chain interactions 7797 Some peptide resin structures may bury the growing N terminus which decreases reactivity Inter chain inter actions may increase the effective crosslinking of the matrix causing the structure to collapse and reducing the diffus
46. these flow tests repeatediv fail call Applied Biosvstems Technical Support for assistance Flow Test 1 DMF Deliverv to Vessels and Peptide Svnthesis Column This flow test checks three deliveries of DMF first through the top valve block second through the bottom valve block and third through the svnthesis column During this flow test vou first tare the left transfer vessel and then when prompted weigh the vessel after each of three DMF deliveries and enter the values If the val ues vou enter do not meet specifications the flow test fails and vou must repeat the test IMPORTANT Alwavs tare the left transfer vessel at the beginning of Flow Test 1 especiallv if vou repeat the test 03 2002 Maintenance and Self Tests 4 15 Applied Biosystems 1 To begin Flow Test 1 press the flow soft key in the Self Test Menu Figure 4 6 Run Flow Test 1 DMF L R PSC PRINT START 2 Verify that a flow test calibration tube is in place in the peptide synthesis position Press the START soft key Place a flow test calibration tube in the peptide synthesis position for Flow Test 1 Figure 4 11 Remove the left transfer vessel Remove the left vessel and put on scale Zero scale then replace the vessel OK 3 Remove the left transfer vessel Figure 4 11 and place it on a scale that is accur
47. upper case letter name Use the alphanumeric keys to select an upper case module In the example shown in Figure 6 18 module a will be copied into module A Press the copy soft key to complete the copy The LCD now displays the new module You can press the Next and Previous keys to see the steps displayed one at a time You can also add erase and unerase bring back steps that were just erased in this menu 5 Press the Menu key and press Yes to save the editing session 6 After you save the editing session the Module Editor Menu appears Press the PRINT soft key in the Module Editor Menu to generate a printout of the selected module How to view the steps in a module 1 In the Run Editor Menu Press the a 7 soft key to toggle between lower case letters pre programmed modules and upper case letters user defined modules To review the steps in a pre programmed module go to the Module Editor Menu and press the view soft key To review the steps in a user defined module press the edit soft key 03 2002 Advanced Operations 6 21 Applied Biosystems 7 Troubleshooting This section describes most of the problems you could encounter while operating Synergy and how to respond to them As the operator you may become aware of a problem when one of the following events occurs e asynthesis generates an irregular conductivity trace e Flow Test fails e a pressure test fails e the LCD
48. with the deprotected L amino group Piperidine in combination with DMF deprotects the Fmoc protected a amino group on the peptide resin to prepare it for coupling THF tetrahydrofuran is the secondary solvent It is both volatile and miscible with DME At the end of synthesis THF deliveries follow after DMF washes to displace the primary solvent Pressurized gas delivery then removes THE drying the con tents of the peptide synthesis column Modules and Functions Modules are composed of a series of steps When all the steps in a module are per formed a defined action is accomplished Some modules are flow tests other mod ules perform routines within the larger process of peptide synthesis Each step ina module is defined by one of the 99 software functions Each function governs a par ticular action that is usually described in the function s name Some functions open or close Synergy s valves For example Function 10 GAS gt TVB opens valves 1 7 and 12 to direct pressurized gas through the valves in the top valve block Function 12 GAS gt AAC opens 5 valves to direct the flow of nitrogen gas to the amino acid column and out to waste Other functions contain directions for the Synergy controller For example when the controller reads Function 7 CHECK COND the controller determines the average of 30 conductivity readings The function takes no time to execute and appears only briefly on the LCD during a run It h
49. 0 PPM 590 MG M3 NIOSH RECOMMENDED TWA 250 PPM 737 MG M3 NIOSH RECOMMENDED STEL 200 PPM 590 MG M3 DFG MAK TWA 1000 PPM 2950 MG M3 DFG MAK 30 MINUTE PEAK AVERAGE VALUE 2 TIMES SHIFT MEASUREMENT METHOD CHARCOAL TUBE CARBON DISULFIDE GAS CHROMATOGRAPHY WITH FLAME IONIZATION DETECTION NIOSH VOL III 1609 1000 POUND CERCLA SECTION 103 REPORTABLE QUANTITY 41 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B PHXSICAL DATA DESCRIPTION COLORLESS VOLATILE MOBILE LIQUID WITH AN ETHER LIKE ODOR BOILING POINT 153 F 67 C MELTING POINT 162 F 108 C SPECIFIC GRAVITY 0 8892 VAPOR PRESSURE 145 MMHG 20 C EVAPORATION RATE N BUTYL ACETATE 1 14 5 SOLUBILITY IN WATER COMPLETE ODOR THRESHOLD 20 50 PPM VAPOR DENSITY 2 5 SOLVENT SOLUBILITY SOLUBLE IN BENZENE ALCOHOLS ETHERS KETONES ESTERS HYDROCARBONS VISCOSITY 0 5 CPS 20 C FIRE AND EXPLOSION DATA FIRE AND EXPLOSION HAZARD DANGEROUS FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME VAPOR AIR MIXTURES ARE EXPLOSIVE VAPORS ARE HEAVIER THAN AIR AND MAY TRAVEL A CONSIDERABLE DISTANCE TO A SOURCE OF IGNITION AND FLASH BACK FLASH POINT 6 F 14 C CC UPPER EXPLOSIVE LIMIT 11 8 LOWER EXPLOSIVE LIMIT 2 AUTOIGNITION TEMP 610 F 321 C FLAMMABILITY CLASS OSHA IB FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPR
50. 000 PPM FOR 6 HOURS SHOWED SIGNS OF LIVER LUNG AND KIDNEY INJURY THE LETHAL DOSE REPORTED IN MICE IS 9400 MG M3 2 HOURS CHRONIC EXPOSURE IN ADDITION TO THE EFFECTS DESCRIBED IN ACUTE INHALATION EFFECTS FROM OCCUPATIONAL EXPOSURE HAVE INCLUDED FATIGUE WEAKNESS NERVOUSNESS SLEEP DISORDERS VERTIGO FACIAL CONGESTION DIGESTIVE DISTURBANCES EPIGASTRIC PAIN CARDIOVASCULAR ABNORMALITIES NUMBNESS OF THE EXTREMITIES FUNCTIONAL NERVOUS SYSTEM DISORDERS AND INCREASED LEVELS OF SERUM AMYLASE SUGGESTING PANCREATITIS OTHER REPORTED EFFECTS HAVE INCLUDED SLOW COAGULATION TIMES A SIGNIFICANT REDUCTION IN PLATELET COUNT INCREASED CHROMOSOMAL ABERRATIONS AN EXCESS RISK FOR TESTICULAR GERM CELL TUMORS AND CANCERS OF THE BUCCAL CAVITY OR PHARYNX AND A NONSIGNIFICANT EXCESS OF LUNG CANCER PROLONGED EXPOSURE OF DOGS RESULTED IN POLYCYTHEMIA AND CARDIOVASCULAR EFFECTS WITH DECREASED PULSE RATE LOW BLOOD PRESSURE AND HEART DAMAGE REPEATED EXPOSURE OF RATS TO CONCENTRATIONS OF 100 TO 450 PPM FOR 4 MONTHS RESULTED IN NECROSIS OF THE LIVER AND MILD KIDNEY DAMAGE FETAL GROWTH RETARDATION BUT NO MALFORMATION WAS OBSERVED FOLLOWING EXPOSURE OF RATS FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED PERFORM ARTIFICIAL RESPIRATION KEEP PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY GET MEDICAL ATTENTION IMMEDIATELY SKIN CONTACT N N DIMETHXLFORMAMIDE IRRITANT ACUTE EXPOSURE MAY CAUSE IRRI
51. 1 3 Synergy Safety Symbols 1 4 Electrical Symbols 1 4 Other 1 4 Synergy Safety Procedures 1 5 General Laboratory Safety 1 5 Instrument Access 1 5 Fuses 1 6 Jaw Assembly 1 6 Hazardous Chemicals 1 6 Waste Bottle 1 6 Secondary Containment 1 7 Compressed Gas Cylinders 1 7 External Pneumatic Supply 1 7 Laboratory Ventilation Requirements 1 8 Fume Hood 1 8 Routine Maintenance Tasks 1 9 Duct System 1 9 Duct Work 1 9 Tubing 1 10 Canopy 1 10 Routine Maintenance Tasks 1 11 03 2002 Synergy Safety Guidelines l 1 Applied Biosystems Synergy User s Manual Conventions User Attention Words Throughout the Synergy Installation Guide and the Synergy User s Manual four kinds of information are set off from the regular text Each user attention word re quires a particular level of observation or action that is significant to user safety or proper instrument operation Note Used to call attention to information IMPORTANT _ Indicates information that is necessary for proper instrument operation Caution Damage to the instrument could result if you do not complv with this information WARNING Physical injury to the user or other persons could result if these precautions are not implemented Chemical abbreviations The following chemical abbreviations are used throughout the Synergy Installation Guide and the Synergy User s Manual DIEA N N diisopropylethylamine DMF N N dimethylformamide DMSO dimethyl sulfoxide
52. 11 123 25 C REFRACTIVE INDEX 1 4534 6 20 C CONVERSION FACTOR 1 PPM 3 48 MG M3 FIRE AND EXPLOSION DATA FIRE AND EXPLOSION HAZARD DANGEROUS FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME MODERATE EXPLOSION HAZARD WHEN EXPOSED TO HEAT OR FLAME VAPOR AIR MIXTURES ARE EXPLOSIVE VAPORS ARE HEAVIER THAN AIR AND MAY TRAVEL A CONSIDERABLE DISTANCE TO A SOURCE OF IGNITION AND FLASH BACK DUE TO LOW ELECTROCONDUCTIVITY OF THE SUBSTANCE FLOW OR AGITATION MAY GENERATE ELECTROSTATIC CHARGES RESULTING IN SPARKS WITH POSSIBLE IGNITION FLASH POINT 61 F 16 C CC FLAMMABILITY CLASS OSHA IB FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR REGULAR FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPRAY FOG OR REGULAR FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 ALCOHOL FOAM NFPA 325M FIRE HAZARD PROPERTIES OF FLAMMABLE LIQUIDS GASES AND VOLATILE SOLIDS 1991 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK DO NOT GET WATER INSIDE CONTAINER APPLY COOLING WATER TO SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT STAY AWAY FROM ENDS OF TANKS WITHDRAW IMMEDIATELY IN CASE OF RISING SOUND FROM VENTING SAFETY DEVICE OR ANY DISCOLORATION OF TANK DUE TO FIRE ISOLATE FOR 1 2 MILE IN ALL DIRECTIONS IF TANK RAIL CAR OR TANK TRUCK IS INVOLVED IN FIRE 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 GUIDE PAGE 29 EX
53. 2699 C2HF302 CHEMICAL FAMILY HALOGEN COMPOUND ALIPHATIC MOLECULAR FORMULA F3 C C 02 H MOLECULAR WEIGHT 114 03 CERCLA RATINGS SCALE 0 3 HEALTH 3 FIRE 0 REACTIVITY 1 PERSISTENCE 1 NFPA RATINGS SCALE 0 4 HEALTH 3 FIRE 0 REACTIVITY 1 COMPONENTS AND CONTAMINANTS COMPONENT TRIFLUOROACETIC ACID PERCENT 100 0 CAS 76 05 1 OTHER CONTAMINANTS NONE EXPOSURE LIMITS NO OCCUPATIONAL EXPOSURE LIMITS ESTABLISHED BY OSHA ACGIH OR NIOSH PHYSICAL DATA DESCRIPTION COLORLESS FUMING HYGROSCOPIC LIQUID WITH A STRONG PUNGENT ODOR RESEMBLING ACETIC ACID BOILING POINT 162 F 72 C MELTING POINT 5 F 15 C SPECIFIC GRAVITY 1 535 PH STRONGLY ACIDIC SOLUBILITY IN WATER VERY SOLUBLE VAPOR DENSITY 4 0 SOLVENT SOLUBILITY SOLUBLE IN ALCOHOL ETHER ACETONE CARBON TETRACHLORIDE HEXANE BENZENE 49 ABI PART NUMBER 901812 OHS PART NUMBER ABI24050 Rev B FIRE AND EXPLOSION DATA FIRE AND EXPLOSION HAZARD NEGLIGIBLE FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR REGULAR FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPRAY FOG OR REGULAR FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK APPLY COOLING WATER TO SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT STAY AWAY FROM ENDS OF TANKS 1990 EM
54. 3 After you have adjusted the internal gas pressure press the more soft key to return to the Run Monitor that displays the PAUSE soft key 4 Press the PAUSE soft key to continue Flow Test 1 As Flow Test 1 continues repeat the complete procedure to verify that all deliveries are within range If all delivery weights are within range Flow Test 1 passes If any delivery weights are not in range repeat the internal gas pressure adjustment procedure Flow Test 2 Calibrate Tube During this test two deliveries of DMF go into a flow test calibration tube placed in the peptide synthesis column position You need an indelible ink marker to mark the level of DMF in the column after each flow The marked flow test calibration tube is used to perform Flow Tests 3 4 and 5 1 Place a flow test calibration tube in the PSC position 2 To begin Flow Test 2 press the flow soft key in the Self Test Menu Figure 4 03 2002 Maintenance and Self Tests 4 19 Applied Biosystems 6 Then press the Next key or press the numeric key 2 to make Run Flow Test 2 appear on the LCD Run Flow Test 2 CALIBRATE TUBE PRINT START Press the START soft key In the next two minutes Synergy performs a brief pressure test and delivers DMF to the flow test calibration tube Mark the tube OK Make a thin horizontal mark on the flow test calibration tube to indicate the level of the DMF in the column F
55. 54500 UG KG INTRAVENOUS MOUSE LD50 63300 UG KG INTRAVENOUS DOG LD50 2472 MG KG INTRAPERITONEAL RAT LD50 3050 MG KG INTRAPERITONEAL MOUSE LD50 7 GM KG UNREPORTED ROUTE RAT LD50 MUTAGENIC DATA RTECS REPRODUCTIVE EFFECTS DATA RTECS CARCINOGEN STATUS NONE LOCAL EFFECTS IRRITANT INHALATION SKIN EYES ACUTE TOXICITY LEVEL MODERATELY TOXIC BY INGESTION SLIGHTLY TOXIC BY DERMAL ABSORPTION TARGET EFFECTS POISONING MAY AFFECT THE CENTRAL NERVOUS SYSTEM N N DIISOPROPYLETHYLAMINE CARCINOGEN STATUS NONE LOCAL EFFECTS IRRITANT INHALATION SKIN EYE ACUTE TOXICITY LEVEL NO DATA AVAILABLE TARGET EFFECTS NO DATA AVAILABLE HEALTH EFFECTS AND FIRST AID INHALATION DIMETHYL SULFOXIDE IRRITANT TOXIC ACUTE EXPOSURE VAPORS MAY CAUSE MODERATE IRRITATION OF THE RESPIRATORY TRACT WITH COUGHING HIGH CONCENTRATIONS MAY CAUSE SYSTEMIC EFFECTS SUCH AS NAUSEA VOMITING CHILLS CRAMPS HEADACHE DIZZINESS AND LETHARGY ALLERGIC RESPIRATORY REACTIONS MAY ALSO OCCUR THE LETHAL DOSE REPORTED IN RATS WAS 1600 MG M3 FOR 4 HOURS CHRONIC EXPOSURE ANIMALS SHOWED LIVER DAMAGE AND BRONCHOPNEUMONIA ON BEING SUBJECTED TO SPRAY FOR 5 MINUTES 10 TIMES OVER 15 DAYS BUT NO EVIDENCE OF TOXICITY ON EXPOSURE TO HEATED VAPOR FOR 30 MINUTES UNDER SIMILAR CONDITIONS RABBITS EXPOSED TO 25 50 ML HOUR OF MIST FOR 5 MONTHS DEVELOPED CHEMICAL PNEUMONIA CLOUDY SWELLING OF THE LIVER AND SIGNS OF RENAL TOXICITY 1 METHYL 2 PYRROLIDINONE M PYR
56. 84PRSPSC T 09 10 HOLD PAUSE NEXT S jump more l Press more two times Current 345 10 ave 1234 change 1 pressure 5 3 psi more Read pressure here 2 Press the more soft kev twice until the Run Monitor displavs the current pressure reading on the bottom line of the LCD If the pressure is too low use a flat edged screwdriver to adjust the internal pressure to 3 5 5 5 psi Insert the screwdriver blade into the small hole located behind the reagent bottles Figure 4 12 A quarter turn clockwise raises the pressure reading bv about 0 2 psi Turn the screw clockwise in one direction only and wait 30 seconds for the pressure reading to stabilize IMPORTANT Turn the internal gas adjustment screw in one direction only and then wait 30 seconds for the pressure to stabilize 4 18 Maintenance and Self Tests 03 2002 Applied Biosystems Insert screwdriver blade here to adjust the internal gas pressure Figure 4 12 Adjusting the internal gas pressure If the pressure is too high a Note the current internal gas pressure reading that appears after the word pressure on the LCD b Set the Pressure Vent switch to Vent to vent the excess pressure c Turn the pressure adjustment screw one quarter turn counterclockwise to lower the pressure by 0 2 psi d Set the Pressure Vent switch to Pressure and wait 30 seconds
57. 992 OCCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 11 08 91 REVISION DATE Julv 14 1993 12 IB Applied ABI PART NUMBER 901822 A l Biosystems OHS PART NUMBER n ev MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENCY RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION CAS NUMBER 68 12 2 RTECS NUMBER 102100000 SUBSTANCE N N DIMETHYLFORMAMIDE TRADE NAMES SYNONYMS ABI MSDS PART 901822 P N 400143 DIMETHYLFORMAMIDE N N DIMETHYLMETHANAMIDE FORMYLDIMETHYLAMINE DMF AMIDE DMFA DMF N FORMYLDIMETHYLAMINE PEPTIDE SYNTHESIS REAGENT 10 FORMAMIDE N N DIMETHYL STCC 4913157 UN 2265 C3H7NO CHEMICAL FAMILY AMIDE MOLECULAR FORMULA H C 0 N C H3 2 MOLECULAR WEIGHT 73 09 CERCLA RATINGS SCALE 0 3 HEALTH 3 FIRE 2 REACTIVITY 0 PERSISTENCE 0 NFPA RATINGS SCALE 0 4 HEALTH 1 FIRE 2 REACTIVITY 0 COMPONENTS AND CONTAMINANTS COMPONENT N N DIMETHYLFORMAMIDE PERCENT 100 0 CAS 68 12 2 OTHER CONTAMINANTS NONE EXPOSURE LIMITS N N DIMETHYLFORMAMIDE 10 PPM 30 MG M3 OSHA TWA SKIN 10 PPM 30 MG M3 ACGIH TWA SKIN 10 PPM 30 MG M3 NIOSH RECOMMENDED TWA SKIN 20 PPM 60 MG M3 DFG MAK TWA SKIN 40 PPM 120 MG M3 DFG MAK 30 MINUTE PEAK AVERAGE VALUE 4 TIMES SHIFT MEASUREMENT METHOD SILICA GEL TUBE METHANOL GAS CHROMATOGRAPHY WITH FLAME IONIZATION
58. ALATION RAT LC50 24000 MG M3 2 HOURS INHALATION MOUSE LCLO 1650 MG KG ORAL RAT LD50 2300 MG KG ORAL MOUSE LD50 2300 MG KG ORAL GUINEA PIG LD50 2900 MG KG INTRAPERITONEAL RAT LD50 1900 MG KG INTRAPERITONEAL MOUSE LD50 500 MG KG INTRAPERITONEAL GUINEA PIG LDLO MUTAGENIC DATA RTECS CARCINOGEN STATUS NONE LOCAL EFFECTS IRRITANT INHALATION EYE ACUTE TOXICITY LEVEL MODERATELY TOXIC BY INGESTION SLIGHTLY TOXIC BY INHALATION TARGET EFFECTS CENTRAL NERVOUS SYSTEM DEPRESSANT POISONING MAY AFFECT THE LIVER AND KIDNEYS AT INCREASED RISK FROM EXPOSURE PERSONS WITH IMPAIRED LIVER KIDNEY OR PULMONARY FUNCTION EYE OR SKIN DISORDERS ADDITIONAL DATA ALCOHOL MAY ENHANCE THE TOXIC EFFECTS HEALTH EFFECTS AND FIRST AID INHALATION TETRAHYDROFURAN IRRITANT NARCOTIC 20 000 PPM IMMEDIATELY DANGEROUS TO LIFE OR HEALTH ACUTE EXPOSURE TECHNICIANS WORKING WITH TETRAHYDROFURAN EXPERIENCED SEVERE OCCIPITAL HEADACHES AND DULLNESS OTHER EFFECTS REPORTED FROM HIGH VAPOR CONCENTRATIONS INCLUDE MUCOUS MEMBRANE IRRITATION WITH SORE THROAT AND COUGHING NAUSEA CENTRAL NERVOUS SYSTEM DEPRESSION WITH WEAKNESS FATIGUE UNCONSCIOUSNESS AND POSSIBLY ASPHYXIATION CONCENTRATIONS ABOVE 25 000 PPM PRODUCED ANESTHESIA WITH PROLONGED INDUCTION PROFUSE SALIVATION VOMITING MARKED FALL IN BLOOD PRESSURE POOR MUSCULAR RELAXATION AND STRONG RESPIRATORY STIMULATION IN EXPERIMENTAL ANIMALS CHRONIC EXPOSURE ANIMALS EXPOSED TO OVER 3000 PPM FOR 20 DAYS E
59. ALSO OCCUR THE LETHAL DOSE REPORTED IN RATS WAS 1600 MG M3 FOR 4 HOURS CHRONIC EXPOSURE ANIMALS SHOWED LIVER DAMAGE AND BRONCHOPNEUMONIA ON BEING SUBJECTED TO SPRAY FOR 5 MINUTES 10 TIMES OVER 15 DAYS BUT NO EVIDENCE OF TOXICITY ON EXPOSURE TO HEATED VAPOR FOR 30 MINUTES UNDER SIMILAR CONDITIONS RABBITS EXPOSED TO 25 50 ML HOUR OF MIST FOR 5 MONTHS DEVELOPED CHEMICAL PNEUMONIA CLOUDY SWELLING OF THE LIVER AND SIGNS OF RENAL TOXICITY 1 METHYL 2 PYRROLIDINONE M PYROL IRRITANT ACUTE EXPOSURE INHALATION OF VERY HIGH VAPOR CONCENTRATIONS MAY CAUSE MUCOUS MEMBRANE IRRITATION HEADACHE GIDDINESS MENTAL CONFUSION AND NAUSEA INHALATION OF 180 200 MG M3 FOR 2 HOURS AND A 6 HOUR EXPOSURE TO SATURATED VAPORS CAUSED NO DEATHS IN MICE AND RATS RESPECTIVELY CHRONIC EXPOSURE PROLONGED EXPOSURE TO VERY HIGH VAPOR CONCENTRATIONS MAY CAUSE HEADACHE GIDDINESS MENTAL CONFUSION AND NAUSEA INHALATION STUDIES IN LABORATORY ANIMALS FAILED TO SHOW ANY GROSS OR HISTOPATHOLOGICAL ABNORMALITIES WHEN EXPOSED TO CONCENTRATIONS OF 50 PPM 8 HOURS DAY FOR 20 DAYS OR 370 PPM 6 HOURS DAY FOR 10 DAYS 1 HYDROXYBENZOTRIAZOLE ACUTE EXPOSURE NO DATA AVAILABLE CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED PERFORM ARTIFICIAL RESPIRATION KEEP PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY GET MEDICAL ATTENTION IMMEDIATELY SKIN CONTACT DIMETHYL SULFOXIDE IR
60. AND ODOR IV Fire and Explosion Hazard Data FLASH POINT Closed Cup THF only Colorless liquid with a mild ammonia like odor 23 C 74 F FLAMMABLE LIMITS THF only 1 8 LEL 11 8 UEL FIRE EXTINGUISHING MEDIA Dry chemical alcohol foam carbon dioxide or Halon Percentages of the different waste components will vary depending on the length and sequence of the peptide synthesized and the version of software used P N 902109 Rev B July 14 1993 55 SPECIAL FIRE FIGHTING PROCEDURES Use self contained breathing apparatus and protective clothing to prevent skin and eye contact V Health Hazard Data EXPOSURE LIMITS See Section Ill For THF the STEL is 250 ppm and the IDLH level is 20 000 ppm EFFECTS OF ACUTE EXPOSURE SWALLOWING Harmful if swallowed Causes severe irritation of eyes nose and throat Higher concentrations may cause liver and kidney damage unconsciousness and death SKIN May cause severe irritation or burns Allergic skin sensitization may also occur INHALATION May cause irritation of eyes nose throat and lungs Higher concentrations may cause pulmonary edema unconsciousness and death EMERGENCY AND FIRST AID PROCEDURES SWALLOWING If conscious give large volumes of water immediatelv Get medical attention immediatelv SKIN Remove contaminated clothing Flush the contaminated area with water for at least 15 minutes and wash with mild soap or d
61. ARLIC LIKE TASTE AND ODOR TO THE BREATH AND SKIN LARGE AMOUNTS MAY CAUSE NAUSEA VOMITING CRAMPS DIARRHEA ANESTHESIA LETHARGY DROWSINESS HEADACHE CHILLS CHEST PAINS BURNING OR ACHING EYES AND TRANSIENT DISTURBANCES OF COLOR VISION AND PHOTOPHOBIA TRANSIENT HEMOLYSIS WITH HEMOGLOBINURIA HAS ALSO BEEN REPORTED ENHANCED IRRITATION EPIDERMAL VESICULATION HISTOLOGICAL EVIDENCE OF DERMAL DEATH AND PERIVASCULAR DERMAL INFILTRATES WERE NOTED AFTER OCCLUDED PATCH TESTING OCCASIONAL HYPERSENSITIVITY REACTIONS INCLUDING ANAPHYLAXIS HAVE BEEN REPORTED DUE TO ITS SOLVENT PROPERTIES DMSO FACILITATES THE ABSORPTION OF SUBSTANCES PRESENT ON THE SKIN WHICH MAY RESULT IN TOXIC EFFECTS CHRONIC EXPOSURE 9 MILLILITERS OF 90 DMSO WAS APPLIED TO THE ENTIRE TRUNK OF 20 MEN ONCE DAILY FOR 26 WEEKS THE EFFECTS NOTED WERE BAD BREATH TRANSIENT ERYTHEMA BURNING AND STINGING THE DERMATITIS ACCOMPANIED BY ONLY MODERATE INFLAMMATION REGRESSED AS TREATMENT CONTINUED DAILY CONTINUOUS APPLICATION WITH OCCLUSION PRODUCED HARDENING OF THE SKIN IN MOST SUBJECTS WITHIN 1 MONTH CRYSTALLINE LENS ALTERATIONS RESEMBLING JUVENILE NUCLEAR SCLEROSIS HAVE BEEN PRODUCED IN SOME ANIMAL SPECIES BUT NOT IN HUMANS NO LENS ABNORMALITIES WERE FOUND IN 25 PATIENTS TREATED DAILY WITH UP TO 30 ML APPLIED TOPICALLY FOR 19 MONTHS 1 METHYL 2 PYRROLIDINONE M PYROL IRRITANT ACUTE EXPOSURE CONTACT MAY CAUSE MILD IRRITATION CHRONIC EXPOSURE PROLONGED CONTACT HAS BEEN REPO
62. ATE HBTU IV 9 Flow Test 9 CALIBRATE DIEA IV 10 VENT TEST IV 11 03 2002 Test Printouts IV 1 Applied Biosystems Flow Test 1 DMF gt L R PSC 2 ae Oo u tr ANOOaBRWBNDY 3 84 88 89 4 68 61 68 2 30 1 35 10 14 2 68 61 68 31 1 36 11 15 61 15 61 2 68 31 34 68 61 68 30 1 39 11 15 61 15 61 2 68 61 68 10 13 11 Function Function Name BEGIN LOOP PRS PSC PRS TEST PRS TEST END LOOP L gt WASTE XFER L gt WASTE INTERRUPT DMF gt TVB WAIT DMF gt L GAS gt TVB GAS gt L INTERRUPT L gt WASTE XFER L gt WASTE DMF gt BVB WAIT DMF gt R GAS gt BVB GAS gt R XFER GAS gt R XFER INTERRUPT L gt WASTE DMF gt BVB DMF gt PSC B L gt WASTE XFER L gt WASTE DMF gt TVB WAIT DMF gt PSC gt R GAS gt BVB GAS gt R XFER GAS gt R XFER INTERRUPT L gt WASTE XFER L gt WASTE GAS gt TVB GAS gt PSC GAS gt BVB Time 999 oOo oOo WOWWWH WH WWOWWWWNHWWOWH WOWOW0 No oO G0OOWWWWO WOwWwwWwWh WH WwW Ww oO OOo IV 2 Test Printouts 03 2002 Applied Biosystems Flow Test 2 CALIBRATE COLUMN Function o e Oo u o NOOA ON 3 84 88 89 4 31 34 10 13 31 1 34 2 34 2 10 13 23 10 13 10 11 Function Name BEGIN LOOP PRS PSC PRS TEST PRS TEST END LOOP DMF gt BVB DMF gt PSC B GAS gt TVB GAS gt PSC DMF gt BVB WAIT DMF gt PSC B INTERRUPT DMF gt PSC B INTERRUPT GAS gt TVB GAS gt PSC THF gt PSC GAS
63. AY FOG OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 ALCOHOL FOAM NFPA 325M FIRE HAZARD PROPERTIES OF FLAMMABLE LIQUIDS GASES AND VOLATILE SOLIDS 1991 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK APPLY COOLING WATER TO SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT STAY AWAY FROM ENDS OF TANKS FOR MASSIVE FIRE IN CARGO AREA USE UNMANNED HOSE HOLDER OR MONITOR NOZZLES IF THIS IS IMPOSSIBLE WITHDRAW FROM AREA AND LET FIRE BURN WITHDRAW IMMEDIATELY IN CASE OF RISING SOUND FROM VENTING SAFETY DEVICE OR ANY DISCOLORATION OF TANK DUE TO FIRE ISOLATE FOR 1 2 MILE IN ALL DIRECTIONS IF TANK RAIL CAR OR TANK TRUCK IS INVOLVED IN FIRE 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 GUIDE PAGE 26 EXTINGUISH ONLY IF FLOW CAN BE STOPPED USE WATER IN FLOODING AMOUNTS AS FOG SOLID STREAMS MAY NOT BE EFFECTIVE COOL CONTAINERS WITH FLOODING QUANTITIES OF WATER APPLY FROM AS FAR A DISTANCE AS POSSIBLE AVOID BREATHING TOXIC VAPORS KEEP UPWIND EVACUATE TO A RADIUS OF 1500 FEET FOR UNCONTROLLABLE FIRES CONSIDER EVACUATION OF DOWNWIND AREA IF MATERIAL IS LEAKING 42 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B WATER MAY BE INEFFECTIVE NFPA 325M FIRE HAZARD PROPERTIES OF FLAMMABLE LIQUIDS GASES AND VOLATILE SOLIDS 1991 TOXICITY TETRAHYDROFURAN TOXICITY DATA 25000 PPM INHALATION HUMAN TCLO 21000 PPM 3 HOURS INH
64. BTU delivery 1 Gausepohl H Pieles U and Frank R W 1992 Schiff base analog formation during in situ activation by HBTU and TBTU In Peptides Chemistry and Biology Proceedings of the Twelfth American Peptide Symposium ed J A Smith and J E Rivier 523 524 ESCOM Leiden 03 2002 Troubleshooting 7 7 Applied Biosystems Malfunctioning instrument hardware Table 7 1 Occurrence of Irregular Trace Features and Recommended Flow Tests Irregular missing feature Corresponding modules Run Self Test Initial resin solvation b Begin f Flow Flow Test 3 DMF gt PSC 1 2 0r3 d Deprotection Flow Test 5 PIP gt PSC Flow Test 3 DM F gt PSC 4 a Activation Leak Test 5 c Coupling Flow Tests 8 Calibrate HBTU Flow Test 9 Calibrate DIEA 6 g Gurgle Flow Test 6 PUMP AAC 7 f Flow Flow Test 3 DM F gt PSC End of synthesis e End Flow Test 4 THF gt PSC See page 2 16 for a discussion of Trace Features Table 7 1 provides guidelines for choosing the appropriate Self Test to perform de pending on where irregularities occur in your conductivity trace see Flow Tests on page 4 15 and Leak Self Test on page 4 13 You may be able to pinpoint the source of the irregularity and eliminate it by performing the appropriate corrective actions Trace Feature 5 Deprotection Trace Feature 1 Se l FEJN Trace Feature 7 ae Figure 7 6 Trace with irregularity at coupling
65. CCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY INGESTION N N DIMETHXLFORMAMIDE ACUTE EXPOSURE MAY CAUSE HEADACHE DIZZINESS NAUSEA VOMITING DIARRHEA AND ABDOMINAL PAIN AND SPASMS AND OTHER SYSTEMIC EFFECTS AS DETAILED IN INHALATION ANIMALS EXPOSED TO A LETHAL DOSE EXHIBITED WEIGHT LOSS RESTLESSNESS AND IRRITABILITY PATHOLOGICAL EXAMINATION SHOWED DEPRESSED BONE MARROW ACTIVITY CHRONIC EXPOSURE REPEATED ORAL EXPOSURE IN RATS RESULTED IN LIVER INJURY THIS SUBSTANCE ADMINISTERED TO PREGNANT RABBITS PRODUCED AN INCREASED INCIDENCE OF FETAL INTERNAL HYDROCEPHALUS AT MATERNAL TOXIC DOSES ABORTION RETARDED FETAL GROWTH AND ADDITIONAL MALFORMATIONS WERE ALSO OBSERVED FIRST AID IF THE PERSON IS CONSCIOUS AND NOT CONVULSING INDUCE EMESIS BY GIVING SYRUP OF IPECAC FOLLOWED BY WATER IF VOMITING OCCURS KEEP THE HEAD BELOW THE HIPS TO PREVENT ASPIRATION REPEAT IN 20 MINUTES IF NOT EFFECTIVE INITIALLY GIVE ACTIVATED CHARCOAL IN PATIENTS WITH DEPRESSED RESPIRATION OR IF EMESIS IS NOT PRODUCED PERFORM GASTRIC LAVAGE CAUTIOUSLY DREISBACH HANDBOOK OF POISONING 12TH ED TREAT SYMPTOMATICALLY AND SUPPORTIVELY GASTRIC LAVAGE SHOULD BE PERFORMED BY QUALIFIED MEDICAL PERSONNEL GET MEDICAL ATTENTION IMMEDIATELY ANTIDOTE NO SPECIFIC ANTIDOTE TREAT SYMPTOMATICALLY AND SUPPORTIVELY REACTIVITY REACTIVITY STABLE UNDER NORMAL TEMPE
66. CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED INA PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND 18 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B OR OTHER POSITIVE PRESSURE MODE CLOTHING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOTHING AND EQUIPMENT TO PREVENT REPEATED OR PROLONGED SKIN CONTACT WITH THIS SUBSTANCE GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES TO PREVENT EYE CONTACT WITH THIS SUBSTANCE EMERGENCY EYE WASH WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE S EYES MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN EYE WASH FOUNTAIN WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE COPYRIGHT 1992 OCCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 07 31 91 REVISION DATE July 14 1993 19 20 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B AR Applied ABI PART NUMBER 902034 Ad Biosystems OHS PART R R ev MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENCY RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570
67. Chain Deprotection Once the synthesis is complete you must remove or cleave the peptide from the solid support and deprotect the side chain protecting groups on the peptide by treat ing the peptide resin with a mixture of trifluoroacetic acid TFA and ion scaven gers Methyl t butyl ether MTBE can then be added to precipitate the peptide out of the cleavage mixture This cleavage reaction procedure is detailed in Chapter 3 of this manual After cleavage the crude peptide can then be dissolved and lyophilized for storage 5 6 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems The Synergy Synthesis Process Synergy s peptide synthesis process incorporates traditional Fmoc chemistry with HBTU 2 1 H benzotriazol l yl 1 1 3 3 tetramethyluronium hexafluorophosphate activation 10 11 The three stages of SPPS deprotection activation and coupling are broken down into a series of pre programmed modules or instrument operations shown in Table 5 1 Table 5 1 Pre programmed Synergy Modules and Operations Module Operation Activation Begin synthesis Coupling Deprotection End synthesis Flow DMF delivery through PSC to waste Gurgle solvents wash AAC and transfer vessels 02 020 GU 2 TT a alternate activation for non standard residues i Increment moves the wheel to the next AAC position j Jaws close on AAC and perform a leak test Synthesis Columns and Reagen
68. DETECTION NIOSH VOL III 2004 PHYSICAL DATA DESCRIPTION COLORLESS TO VERY SLIGHTLY YELLOW HYGROSCOPIC MOBILE LIQUID WITH A FAINT AMINE LIKE ODOR BOILING POINT 307 F 153 C MELTING POINT 78 F 61 C SPECIFIC GRAVITY 0 9487 VISCOSITY 0 802 CP 6 25 C VOLATILITY 100 VAPOR PRESSURE 3 7 MMHG 13 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B EVAPORATION RATE N BUTYL ACETATE 1 lt 1 PH 6 7 0 5 M SOLUTION SOLUBILITY IN WATER SOLUBLE ODOR THRESHOLD 100 PPM VAPOR DENSITY 2 5 SOLVENT SOLUBILITY SOLUBLE IN ALCOHOL ACETONE BENZENE CARBON TETRACHLORIDE CHLOROFORM ETHER ESTERS CHLORINATED AND AROMATIC HYDROCARBONS AND OTHER ORGANIC SOLVENTS FIRE AND EXPLOSION DATA FIRE AND EXPLOSION HAZARD MODERATE FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME VAPORS ARE HEAVIER THAN AIR AND MAY TRAVEL A CONSIDERABLE DISTANCE TO A SOURCE OF IGNITION AND FLASH BACK VAPOR AIR MIXTURES ARE EXPLOSIVE ABOVE FLASH POINT FLASH POINT 136 F 58 C CC UPPER EXPLOSIVE LIMIT 15 2 212 F 100 C LOWER EXPLOSIVE LIMIT 2 2 212 F 100 C AUTOIGNITION TEMP 833 F 445 C FLAMMABILITY CLASS OSHA II FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPRAY FOG OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 ALCOHOL FOAM NFPA 325M FIRE HAZARD PROPERTIES OF FLAM
69. E CONTACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY STORAGE STORE IN ACCORDANCE WITH 29 CFR 1910 106 STORAGE TEMPERATURE AMBIENT TO 40 C SHELF LIFE UNKNOWN STORE AWAY FROM INCOMPATIBLE SUBSTANCES SPILL AND LEAK PROCEDURES OCCUPATIONAL SPILL SHUT OFF IGNITION SOURCES STOP LEAK IF YOU CAN DO IT WITHOUT RISK USE WATER SPRAY TO REDUCE VAPORS FOR SMALL SPILLS TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS FOR LATER DISPOSAL FOR LARGER SPILLS DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL NO SMOKING FLAMES OR FLARES IN HAZARD AREA KEEP UNNECESSARY PEOPLE AWAY ISOLATE HAZARD AREA AND DENY ENTRY PROTECTIVE EQUIPMENT VENTILATION PROVIDE LOCAL EXHAUST OR PROCESS ENCLOSURE VENTILATION TO MEET THE PUBLISHED EXPOSURE LIMITS VENTILATION EQUIPMENT MUST BE EXPLOSION PROOF RESPIRATOR THE FOLLOWING RESPIRATORS ARE RECOMMENDED BASED ON INFORMATION FOUND IN THE PHYSICAL DATA TOXICITY AND HEALTH EFFECTS SECTIONS THEY ARE RANKED IN ORDER FROM MINIMUM TO MAXIMUM RESPIRATORY PROTECTION THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST BE BASED ON THE SPECIFIC OPERATION MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND MUST BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION NIOSH MSHA ANY TYPE C SUPPLIED AIR RESPIRATOR WITH A
70. E PROLONGED AND RELAPSES ARE POSSIBLE IN SEVERE EXPOSURES DEATH DUE TO ANOXIA MAY OCCUR WITHIN A FEW HOURS AFTER ONSET OF PULMONARY EDEMA SYMPTOMS OR FOLLOWING A RELAPSE CHRONIC EXPOSURE DEPENDING ON THE CONCENTRATION AND DURATION OF EXPOSURE 50 ABI PART NUMBER 901812 OHS PART NUMBER ABI24050 Rev B REPEATED OR PROLONGED EXPOSURE MAY CAUSE EROSION OF THE TEETH INFLAMMATORY AND ULCERATIVE CHANGES IN THE MOUTH AND POSSIBLY JAW NECROSIS BRONCHIAL IRRITATION WITH COUGH AND FREQUENT ATTACKS OF BRONCHIAL PNEUMONIA MAY OCCUR GASTROINTESTINAL DISTURBANCES ARE ALSO POSSIBLE FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED GIVE ARTIFICIAL RESPIRATION MAINTAIN AIRWAY AND BLOOD PRESSURE AND ADMINISTER OXYGEN IF AVAILABLE KEEP AFFECTED PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY ADMINISTRATION OF OXYGEN SHOULD BE PERFORMED BY QUALIFIED PERSONNEL GET MEDICAL ATTENTION IMMEDIATELY SKIN CONTACT TRIFLUOROACETIC ACID CORROSIVE VAPORS MAY BE HIGHLY IRRITATING SEE INFORMATION ON ACIDIC CORROSIVES ACIDIC CORROSIVES ACUTE EXPOSURE DIRECT CONTACT MAY CAUSE SEVERE PAIN BURNS AND POSSIBLY BROWNISH OR YELLOWISH STAINS BURNS MAY BE DEEP WITH SHARP EDGES AND HEAL SLOWLY WITH SCAR TISSUE FORMATION CHRONIC EXPOSURE EFFECTS DEPEND ON THE CONCENTRATION AND DURATION OF EXPOSURE REPEATED OR PROLONGED CONTACT MAY RESULT IN DERMATITIS OR EFFECTS SIMILAR TO ACUTE EXPOSURE
71. EA N N Diisopropylethylamine can irritate skin eyes and mucous membranes Wear protective clothing and gloves when handling DIEA See the MSDS in the Synergy User s Manual 1 To begin Flow Test 9 press the flow soft key in the Self Test Menu Figure 4 6 on page 4 8 Then press the Next key or press the numeric key 9 to make Run Flow Test 9 appear on the LCD Run Flow Test 9 CALIBRATE DIEA PRINT START 2 Press the START soft key to begin Flow Test 9 Remove the left vessel and put on scale Zero scale then replace the vessel OK 3 When this display appears remove the left pump vessel on the front of Synergy Figure 4 15 on page 4 28 Place the vessel on a scale that is accurate to 1 mg and set the scale to zero 4 Replace the left transfer vessel on Synergy Press the OK soft key Remove the left vessel and weigh mg OK 5 When this display appears the left transfer vessel contains DIEA Without spilling the contents carefully remove and weigh the left vessel of DIEA Use the numeric keys to enter this value on the LCD Press the OK soft key 4 30 Maintenance and Self Tests 03 2002 Applied Biosystems Replace the vessel OK 6 Replace the left transfer vessel Press the OK soft key 7 When prompted repeat steps 5 and 6 The second DIEA weight should be greater than the first Remove the left vessel and put on scale Zero scale then
72. ERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 GUIDE PAGE 60 EXTINGUISH USING AGENTS INDICATED DO NOT USE WATER DIRECTLY ON MATERIAL IF LARGE AMOUNTS OF COMBUSTIBLE MATERIALS ARE INVOLVED USE WATER SPRAY OR FOG IN FLOODING AMOUNTS USE WATER SPRAY TO ABSORB CORROSIVE VAPORS COOL CONTAINERS WITH FLOODING AMOUNTS OF WATER FROM AS FAR A DISTANCE AS POSSIBLE AVOID BREATHING CORROSIVE VAPORS KEEP UPWIND TOXICITY TRIFLUOROACETIC ACID TOXICITY DATA 10 GM M3 INHALATION RAT LC50 13500 MG M3 INHALATION MOUSE LC50 1200 MG KG INTRAVENOUS MOUSE LD50 150 MG KG INTRAPERITONEAL MOUSE LDLO CARCINOGEN STATUS NONE LOCAL EFFECTS CORROSIVE INHALATION SKIN EYE INGESTION ACUTE TOXICITY LEVEL TOXIC BY INHALATION TARGET EFFECTS NO DATA AVAILABLE HEALTH EFFECTS AND FIRST AID INHALATION TRIFLUOROACETIC ACID CORROSIVE TOXIC THE LETHAL DOSE REPORTED IN RATS WAS 10 GM M3 SEE INFORMATION ON ACIDIC CORROSIVES ACIDIC CORROSIVES ACUTE EXPOSURE MAY CAUSE RESPIRATORY TRACT IRRITATION WITH COUGHING CHOKING AND POSSIBLY BURNS OF THE MUCOUS MEMBRANES OTHER INITIAL SYMPTOMS MAY INCLUDE DIZZINESS HEADACHE NAUSEA AND WEAKNESS IN SOME CASES PULMONARY EDEMA MAY DEVELOP EITHER IMMEDIATELY IN SEVERE CASES OR MORE LIKELY AFTER A LATENT PERIOD OF 5 72 HOURS THE SYMPTOMS MAY INCLUDE TIGHTNESS IN THE CHEST DYSPNEA FROTHY SPUTUM AND CYANOSIS PHYSICAL FINDINGS MAY INCLUDE HYPOTENSION WEAK RAPID PULSE AND MOIST RALES RECOVERY MAY B
73. ES OCCUPATIONAL SPILL SHUT OFF IGNITION SOURCES STOP LEAK IF YOU CAN DO IT WITHOUT RISK USE WATER SPRAY TO REDUCE VAPORS FOR SMALL SPILLS TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS FOR LATER DISPOSAL FOR LARGER SPILLS DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL NO SMOKING FLAMES OR FLARES IN HAZARD AREA KEEP UNNECESSARY PEOPLE AWAY ISOLATE HAZARD AREA AND DENY ENTRY 32 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B PROTECTIVE EQUIPMENT VENTILATION PROVIDE LOCAL EXHAUST OR PROCESS ENCLOSURE VENTILATION TO MEET THE PUBLISHED EXPOSURE LIMITS VENTILATION EQUIPMENT MUST BE EXPLOSION PROOF RESPIRATOR THE FOLLOWING RESPIRATORS ARE RECOMMENDED BASED ON INFORMATION FOUND IN THE PHYSICAL DATA TOXICITY AND HEALTH EFFECTS SECTIONS THEY ARE RANKED IN ORDER FROM MINIMUM TO MAXIMUM RESPIRATORY PROTECTION THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST BE BASED ON THE SPECIFIC OPERATION MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND MUST BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION NIOSH MSHA ANY TYPE C SUPPLIED AIR RESPIRATOR WITH A FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE OR WITH A FULL FACEPIECE HELMET OR HOOD OPERATED IN CONTINUOUS FLOW MODE ANY SELF CONTAINED BREATHING APPARATUS WITH A FULL
74. FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS ANY SELF CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE CLOTHING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOTHING AND EQUIPMENT TO PREVENT REPEATED OR PROLONGED SKIN CONTACT WITH THIS SUBSTANCE GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES AND A FACESHIELD TO PREVENT CONTACT WITH THIS SUBSTANCE EMERGENCY WASH FACILITIES WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE S EYES AND OR SKIN MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN EYE WASH FOUNTAIN AND QUICK DRENCH SHOWER WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE COPYRIGHT19920CCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 11 11 91 REVISION DATE 05 29 92 33 AR Applied ABI PART NUMBER 901837 Ad Biosystems OHS PART NUMBER ABI18940 Rev B MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENC
75. FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE OR WITH A FULL FACEPIECE HELMET OR HOOD OPERATED IN CONTINUOUS FLOW MODE ANY SELF CONTAINED BREATHING APPARATUS WITH A FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS ANY SELF CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE 11 ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B CLOTHING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOTHING AND EQUIPMENT TO PREVENT REPEATED OR PROLONGED SKIN CONTACT WITH THIS SUBSTANCE GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES AND A FACESHIELD TO PREVENT CONTACT WITH THIS SUBSTANCE EMERGENCY WASH FACILITIES WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE S EYES AND OR SKIN MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN EYE WASH FOUNTAIN AND QUICK DRENCH SHOWER WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE COPYRIGHT 1
76. HE BONDING AND GROUNDING GUIDELINES SPECIFIED IN NFPA 77 1983 RECOMMENDED PRACTICE ON STATIC ELECTRICITY KEEP IN A TIGHTLY CLOSED CONTAINER STORE IN A COOL DRY VENTILATED AREA STORE AWAY FROM INCOMPATIBLE SUBSTANCES STORE IN ACCORDANCE WITH 29 CFR 1910 106 THRESHOLD PLANNING QUANTITY TPQ THE SUPERFUND AMENDMENTS AND REAUTHORIZATION ACT SARA SECTION 302 REQUIRES THAT EACH FACILITY WHERE ANY EXTREMELY HAZARDOUS SUBSTANCE IS PRESENT IN A QUANTITY EQUAL TO OR GREATER THAN THE TPQ ESTABLISHED FOR THAT SUBSTANCE NOTIFY THE STATE EMERGENCY RESPONSE COMMISSION FOR THE STATE IN WHICH IT IS LOCATED SECTION 303 OF SARA REQUIRES THESE FACILITIES TO PARTICIPATE IN LOCAL EMERGENCY RESPONSE PLANNING 40 CFR 355 30 DISPOSAL DISPOSAL MUST BE IN ACCORDANCE WITH STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE 40 CFR 262 EPA HAZARDOUS WASTE NUMBERS D001 AND D003 100 POUND CERCLA SECTION 103 REPORTABLE QUANTITY CONDITIONS TO AVOID AVOID CONTACT WITH HEAT SPARKS FLAMES OR OTHER IGNITION SOURCES VAPORS MAY BE EXPLOSIVE MATERIAL IS CORROSIVE AVOID CONTACT WITH SKIN OR EYES DO NOT ALLOW CONTAMINATION OF WATER SOURCES SPILL AND LEAK PROCEDURES OCCUPATIONAL SPILL SHUT OFF IGNITION SOURCES DO NOT TOUCH SPILLED MATERIAL STOP LEAK IF YOU CAN DO IT WITHOUT RISK USE WATER SPRAY TO REDUCE VAPORS DO NOT GET WATER INSIDE CONTAINER FOR SMALL SPILLS TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS F
77. HING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOTHING AND EQUIPMENT TO PREVENT REPEATED OR PROLONGED SKIN CONTACT WITH THIS SUBSTANCE GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES TO PREVENT EYE CONTACT WITH THIS SUBSTANCE EMERGENCY EYE WASH WHERE THERE IS ANY POSSIBILITV THAT AN EMPLOVEE S EYES MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN BYE WASH FOUNTAIN WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE COPYRIGHT19920CCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 11 08 91 REVISION DATE 04 29 92 47 48 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B AR Applied ABI PART NUMBER 901812 Ad Biosystems OHS PART oe N ev MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENCY RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION CAS NUMBER 76 05 1 RTECS NUMBER AJ9625000 SUBSTANCE TRIFLUOROACETIC ACID TRADE NAMES SYNONYMS ABI MSDS PART 901812 P N 400003 P N 400137 P N 400445 2 2 2 TRIFLUOROACETIC ACID TRIFLUORACETIC ACID TRIFLUOROETHANOIC ACID PERFLUOROACETIC ACID ACETIC ACID TRIFLUORO PROTEIN SEQUENCER REAGENT R3 TFA PEPTIDE SYNTHESIS REAGENT 2 TFA SEPARATION PRODUCTS HPLC GRADE TFA UN
78. HING AND EQUIPMENT TO PREVENT REPEATED OR PROLONGED SKIN CONTACT WITH THIS SUBSTANCE 24 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES TO PREVENT EYE CONTACT WITH THIS SUBSTANCE EMERGENCY EYE WASH WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOVEE S EYES MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN BYE WASH FOUNTAIN WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE COPYRIGHT 1992 OCCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 09 06 91 REVISION DATE 02 28 92 25 AR Applied ABI PART NUMBER 902034 Ad Biosystems OHS PART R R ev MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENCY RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION SUBSTANCE HOBT 0 2 M DMSO NMP TRADE NAMES SYNONYMS ABI MSDS PART 902033 P N 401279 1 HYDROXYBENZOTRIAZOLE DIMETHYL SULFOXIDE N METHYLPYROLLIDONE CHEMICAL FAMILY MIXTURE CERCLA RATINGS SCALE 0 3 HEALTH 3 FIRE 2 REACTIVITY 0 PERSISTENCE 2 NFPA RATINGS SCALE 0 4 HEALTH 2 FIRE 2 REACTIVITY 0 COMPONENTS AND CONTAMINANTS COMPONENT PERCENT 50 22 DIMETHYL SULFOXIDE CAS 67 68 5 COMPONENT PERCENT 47 12 1 METHYL 2 PYRROLIDIN
79. HOWER WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE COPYRIGHT 1992 OCCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 07 31 91 REVISION DATE July 14 1993 54 ABI MODEL 432A Fast Moc Fmoc CHEMISTRY WASTE PROFILE EMERGENCV PHONE NUMBERS USA 415 570 6667 ext 3333 UK 0925 825650 I Identification he liquid waste for the 432A FastMoc Fmoc Chemistry is collected in a 1 gallon carbov This waste is a complex mixture of reagents which may have properties of greater hazard than the individual waste components by hemselves HANDLE THIS MATERIAL WITH EXTREME CAUTION DO NOT DISPOSE OF THIS WASTE IN SINKS OR DRAINSI THIS MATERIAL SHOULD BE DISPOSED OF AS A REGULATED HAZARDOUS WASTE II Approximate Composition MATERIAL PEL Units CAS NUMBER Dimethviformamide DMF 10 ppm 68 12 2 Tetrahydrofuran THF 200 ppm 109 99 9 Piperidine 110 89 4 N Methylpyrolidone NMP 872 50 4 Dimethyl sulfoxide DMSO 67 68 5 Diisopropylethylamine DIEA 7087 68 5 l Hvdroxvbenzotriazole HOBt 2592 95 2 Tetramethvlurea N A Diisopropylethylammonium hexafluoro phosphate DIEA PF N A Amino Acids Ill Physical Data BOILING POINT 760 mm N A N A Not available N A N A SPECIFIC GRAVITY H20 1 0 94 N A VOLATILITY vol 99 SOLUBILITY IN WATER Soluble APPEARANCE
80. IN ALL DIRECTIONS IF TANK RAIL CAR OR TANK TRUCK IS INVOLVED IN FIRE 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 GUIDE PAGE 26 EXTINGUISH ONLY IF FLOW CAN BE STOPPED USE WATER IN FLOODING AMOUNTS AS FOG SOLID STREAMS MAY NOT BE EFFECTIVE COOL CONTAINERS WITH FLOODING AMOUNTS OF WATER APPLY FROM AS FAR A DISTANCE AS POSSIBLE AVOID BREATHING VAPORS KEEP UPWIND TOXICITY DIMETHYL SULFOXIDE IRRITATION DATA 10 MG 24 HOURS OPEN SKIN RABBIT MILD 500 MG 24 HOURS SKIN RABBIT MILD 100 MG EYE RABBIT 500 MG 24 HOURS EYE RABBIT MILD TOXICITY DATA 1600 MG M3 4 HOURS INHALATION RAT LC50 38MKAJ 40 GM KG SKIN RAT LD50 50 GM KG SKIN MOUSE LD50 gt 11 GM KG SKIN DOG LD50 38MKAJ gt 11 GM KG SKIN MONKEY LD50 38MKAJ 14500 MG KG ORAL RAT LD50 7920 MG KG ORAL MOUSE LD50 gt 11 GM KG ORAL GUINEA PIG LDLO gt 10 GM KG ORAL DOG LD50 12 GM KG SUBCUTANEOUS RAT LD50 14 GM KG SUBCUTANEOUS MOUSE LD50 606 MG KG INTRAVENOUS MAN TDLO 5360 MG KG INTRAVENOUS RAT LD50 3100 MG KG INTRAVENOUS MOUSE LD50 2500 MG KG INTRAVENOUS DOG LD50 200 MG KG INTRAVENOUS CAT LDLO 8200 MG KG INTRAPERITONEAL RAT LD50 2500 MG KG INTRAPERITONEAL MOUSE LD50 gt 5500 MG KG INTRAPERITONEAL GUINEA PIG LDLO 1300 MG KG UNREPORTED RAT LD50 MUTAGENIC DATA RTECS REPRODUCTIVE EFFECTS DATA RTECS TUMORIGENIC DATA RTECS CARCINOGEN STATUS NONE LOCAL EFFECTS IRRITANT INHALATION SKIN EYE ACUTE TOXICITY LEVEL TOXIC BY INHALATION SLIGHTLY TOXIC BY INGE
81. INFLAMMATION OF CONJUNCTIVA AND TEMPORARY CORNEAL CLOUDING CHRONIC EXPOSURE REPEATED OR PROLONGED EXPOSURE TO IRRITANTS MAY CAUSE CONJUNCTIVITIS N N DIISOPROPYLETHYLAMINE IRRITANT ACUTE EXPOSURE MAY CAUSE SEVERE IRRITATION WITH POSSIBLE BURNS CHRONIC EXPOSURE REPEATED OR PROLONGED CONTACT WITH IRRITANTS MAY CAUSE CONJUNCTIVITIS FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL SALINE OCCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY INGESTION DIMETHYL SULFOXIDE ACUTE EXPOSURE INGESTION OF LARGE AMOUNTS MAY CAUSE NAUSEA VOMITING DIARRHEA ABDOMINAL PAIN LETHARGY AND DROWSINESS CHRONIC EXPOSURE REPEATED LARGE DOSES PRODUCED CRYSTALLINE LENS CHANGES RESEMBLING JUVENILE NUCLEAR SCLEROSIS IN SOME ANIMAL SPECIES BUT NOT IN HUMANS IN ANIMAL STUDIES REPEATED DOSES OF 1 5 GM KG RESULTED IN LIVER NECROSIS AND RENAL LESIONS REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS 1 METHYL 2 PYRROLIDINONE M PYROL ACUTE EXPOSURE INGESTION MAY CAUSE GASTROINTESTINAL DISTURBANCES CHRONIC EXPOSURE NINETY DAY FEEDING STUDIES IN LABORATORY ANIMALS AT CONCENTRATIONS UP TO 1 OF THEIR DIET FAILED TO DEMONSTRATE ANY TOXICOLOGICALLY RELEVANT EFFECT IT HAS BEEN DEMONSTRATED TO BE EMBRYOTOXIC TO RATS AND MICE IN VERY HIGH DOSES N N DIISOPROPYLETHYLAMINE ACUTE EXPOSURE MAY CAUSE SEVERE IRRITATION OF MOUTH THROAT AND
82. K soft key to continue S xx yy zz F Fit FUNCT NAME T xxx zzz HOLD PAUSE NEXT S jump S more The Run Monitor appears with an asterisk after the word PAUSE which means Synergy has automatically suspended operation The information in the top line identifies the Step and Function where the pressure failure occurred When a pressure failure occurs this line usually displays Function 89 PRS TEST If Synergy was running a Self Test when the pressure test failure occurred note the Step number and refer to Table 7 3 on page 7 16 for a description of the appropriate action If Synergy was running a synthesis when the pressure test failure occurred press the more soft key to determine which module was running S 02 16 C 00 01 M bf LOCK set int end run more The cursor appears under the module that was running when the pressure test failure occurred During a synthesis this would be either module b or module j Refer to Table 7 2 on page 7 13 for a description of the appropriate corrective action 7 12 Troubleshooting 03 2002 Applied Biosystems 2 Perform the appropriate corrective actions 3 Press the more soft key until the Run Monitor displays the PAUSE soft key 4 Press the PAUSE soft key to continue the synthesis or the Self Test Whenever a pressure failure occurs Synergy automatically repeats the pressure test after you press the PAUSE ke
83. LCD appears under text that you can modify Five soft keys under the LCD correspond to choices displayed on the bottom line of the LCD The HOLD and PAUSE soft keys are like toggle switches When you press either the HOLD or PAUSE soft key once an asterisk appears next to the key to indicate the key is operating or on Press the key again and the asterisk goes away to indicate the key is off Sixteen labeled keys to the right of the LCD let you interact with Synergy software controller by way of the LCD Use the alphanumeric keys to enter numbers on the LCD In some menus these keys can represent lower case letters a b c etc or upper case letters A B C etc Use the arrow keys lt gt to move the cursor on the LCD to the left or to the right e Use the Previous and Next keys to increase or decrease the value currently selected by the cursor e Use the Delete key to erase the entry that is currently selected by the cursor e Press the Menu key to see the preceding LCD display and to return to the Main Menu 2 4 Routine Operation 03 2002 Applied Biosystems Making a Peptide on Synergy With Synergy every step of solid phase peptide svnthesis has been incorporated into a series of pre programmed module lines Table 2 1 The conductivitv moni toring feature automaticallv optimizes deprotection and coupling to allow svnthesis of manv peptides without modifving these module lines See Ch
84. LENT OR EXPLOSIVE REACTION IODINE PENTAFLUORIDE POSSIBLE EXPLOSIVE REACTION METAL NITRATES FORMS AN EXTREMELY EXPLOSIVE MIXTURE METAL PERCHLORATES FORMS AN EXTREMELY EXPLOSIVE MIXTURE 31 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B NITRIC ACID POSSIBLE EXPLOSION HAZARD OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD PERCHLORIC ACID EXPLODES ON CONTACT PERIODIC ACID POSSIBLE EXPLOSIVE REACTION PHOSPHOROUS III OXIDE VIOLENT REACTION POTASSIUM VIOLENT REACTION POTASSIUM PERMANGANATE IGNITION ON CONTACT SILVER DIFLUORIDE VIOLENT REACTION SODIUM HYDRIDE POSSIBLE FIRE AND EXPLOSION AT ELEVATED TEMPERATURES SULFUR TRIOXIDE EXOTHERMIC REACTION 1 METHYL 2 PYRROLIDINONE M PYROL ACIDS INCOMPATIBLE OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD 1 HYDROXYBENZOTRIAZOLE OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD DECOMPOSITION THERMAL DECOMPOSITION MAY RELEASE TOXIC AND OR HAZARDOUS GASES POLYMERIZATION HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL TEMPERATURES AND PRESSURES STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANCE CONTACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY STORAGE STORE IN ACCORDANCE WITH 29 CFR 1910 106 STORAGE TEMPERATURE 4 C SHELF LIFE UNKNOWN STORE AWAY FROM INCOMPATIBLE SUBSTANCES SPILL AND LEAK PROCEDUR
85. LISHED EXPOSURE LIMITS VENTILATION EQUIPMENT MUST BE EXPLOSION PROOF RESPIRATOR THE FOLLOWING RESPIRATORS AND MAXIMUM USE CONCENTRATIONS ARE RECOMMENDATIONS BY THE U S DEPARTMENT OF HEALTH AND HUMAN SERVICES NIOSH POCKET GUIDE TO CHEMICAL HAZARDS NIOSH CRITERIA DOCUMENTS OR BY THE U S DEPARTMENT OF LABOR 29 CFR 1910 SUBPART Z THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION NIOSH MSHA N N DIMETHXLFORMAMIDE 100 PPM ANY SUPPLIED AIR RESPIRATOR ANY SELF CONTAINED BREATHING APPARATUS 250 PPM ANY SUPPLIED AIR RESPIRATOR OPERATED IN A CONTINUOUS FLOW MODE 500 PPM ANY SELF CONTAINED BREATHING APPARATUS WITH A FULL FACEPIECE ANY SUPPLIED AIR RESPIRATOR WITH A FULL FACEPIECE ANY SUPPLIED AIR RESPIRATOR THAT HAS A TIGHT FITTING FACEPIECE AND IS OPERATED IN A CONTINUOUS FLOW MODE 3500 PPM ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ESCAPE ANY AIR PURIFYING FULL FACEPIECE RESPIRATOR GAS MASK WITH A CHIN STYLE FRONT OR BACK MOUNTED ORGANIC VAPOR CANISTER ANY APPROPRIATE ESCAPE TYPE SELF CONTAINED BREATHING APPARATUS FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS ANY SELF
86. LLY RELEVANT EFFECT IT HAS BEEN DEMONSTRATED TO BE EMBRYOTOXIC TO RATS AND MICE IN VERY HIGH DOSES 1 HYDROXYBENZOTRIAZOLE ACUTE EXPOSURE NO DATA AVAILABLE CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID SEEK MEDICAL ATTENTION IMMEDIATELY TREAT SYMPTOMATICALLY AND SUPPORTIVELY MAINTAIN AIRWAY AND RESPIRATION IF VOMITING OCCURS KEEP HEAD BELOW HIPS TO PREVENT ASPIRATION IF A POISONOUS SUBSTANCE HAS BEEN INGESTED REMOVAL BY EMESIS OR GASTRIC LAVAGE WITH APPROPRIATE PRECAUTIONS TO PREVENT ASPIRATION IS GENERALLY RECOMMENDED PROVIDED THE PATIENT IS CONSCIOUS NOT CONVULSING AND THERE IS NO SIGN OF CORROSIVE INJURY ONLY QUALIFIED MEDICAL PERSONNEL SHOULD PERFORM GASTRIC LAVAGE IF A CORROSIVE SUBSTANCE HAS BEEN INGESTED DILUTION BY RINSING THE MOUTH AND GIVING WATER OR MILK TO DRINK IS GENERALLY RECOMMENDED IF PERFORATION HAS OCCURRED OR THE VICTIM IS UNCONSCIOUS THE VICTIM SHOULD NOT BE GIVEN ANYTHING TO DRINK REACTIVITY REACTIVITY STABLE UNDER NORMAL TEMPERATURES AND PRESSURES INCOMPATIBILITIES DIMETHYL SULFOXIDE ACID ANHYDRIDES POSSIBLE EXPLOSIVE REACTION ACID HALIDES POSSIBLE EXPLOSIVE REACTION ACYL HALIDES VIOLENT OR EXPLOSIVE REACTION ARYL HALIDES VIOLENT DECOMPOSITION REACTION BORON HYDRIDES MAY FORM EXPLOSIVE MIXTURE BORON HYDROBORATES MAY FORM EXPLOSIVE MIXTURE 4 4 BROMOBENZOYL ACETANILIDE MAY EXPLODE AT ELEVATED TEMPERATURES CARBONYL DIISOTHIOCYANATE EXPLOSIVE REACTION DINITROGEN TETRAOXIDE VIO
87. MABLE LIQUIDS GASES AND VOLATILE SOLIDS 1991 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK APPLY COOLING WATER TO SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT STAY AWAY FROM ENDS OF TANKS FOR MASSIVE FIRE IN CARGO AREA USE UNMANNED HOSE HOLDER OR MONITOR NOZZLES IF THIS IS IMPOSSIBLE WITHDRAW FROM AREA AND LET FIRE BURN WITHDRAW IMMEDIATELY IN CASE OF RISING SOUND FROM VENT ING SAFETY DEVICE OR ANY DISCOLORATION OF TANK DUE TO FIRE ISOLATE FOR 1 2 MILE IN ALL DIRECTIONS IF TANK RAIL CAR OR TANK TRUCK IS INVOLVED IN FIRE 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 GUIDE PAGE 26 EXTINGUISH ONLY IF FLOW CAN BE STOPPED USE WATER IN FLOODING AMOUNTS AS FOG SOLID STREAMS MAY NOT BE EFFECTIVE COOL CONTAINERS WITH FLOODING AMOUNTS OF WATER APPLY FROM AS FAR A DISTANCE AS POSSIBLE AVOID BREATHING VAPORS KEEP UPWIND TOXICITY N N DIMETHXLFORMAMIDE IRRITATION DATA 1003 24 HOURS SKIN HUMAN MILD 10 MG 24 HOURS OPEN SKIN RABBIT 20 MG OPEN EYE RABBIT 100 MG RINSED EYE RABBIT SEVERE TOXICITV DATA 9400 MG M3 2 HOURS INHALATION MOUSE LC50 4720 MG KG SKIN RABBIT LD50 2800 MG KG ORAL RAT LD50 3700 MG KG ORAL MOUSE LD50 3800 MG KG SUBCUTANEOUS RAT LD50 4500 MG KG SUBCUTANEOUS MOUSE LD50 2000 MG KG INTRAVENOUS RAT LD50 1800 MG KG INTRAVENOUS RABBIT LD50 2500 MG KG INTRAVENOUS MOUSE LD50 470 MG KG INTRAVENOUS DOG LD50 1050 MG KG INTRAVENOUS GUINEA PIG LD50
88. MICAL REMAINS AT LEAST 15 20 MINUTES IN CASE OF CHEMICAL BURNS COVER AREA WITH STERILE DRY DRESSING BANDAGE SECURELY BUT NOT TOO TIGHTLY GET MEDICAL ATTENTION IMMEDIATELY EYE CONTACT PIPERIDINE CORROSIVE ACUTE EXPOSURE MAY CAUSE IRRITATION AND BURNS WITH POSSIBLE PERMANENT DAMAGE SEVERE INJURY OF THE CORNEA OF RABBITS RATING A 9 ON A SCALE OF 1 TO 10 HAS BEEN REPORTED CHRONIC EXPOSURE PROLONGED CONTACT MAY CAUSE CONJUNCTIVITIS FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OCCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS AT LEAST 15 20 MINUTES CONTINUE IRRIGATING WITH NORMAL SALINE UNTIL THE PH HAS RETURNED TO NORMAL 30 60 MINUTES COVER WITH STERILE BANDAGES GET 37 ABI PART NUMBER 901837 OHS PART NUMBER ABI18940 Rev B MEDICAL ATTENTION IMMEDIATELY INGESTION PIPERIDINE CORROSIVE TOXIC ACUTE EXPOSURE MAY CAUSE SORE THROAT IRRITATION AND BURNS ABSORPTION MAY PRODUCE EFFECTS ON THE NERVOUS SYSTEM EFFECTS REPORTED IN ANIMALS INCLUDE WEAKNESS RESPIRATORY DISTRESS AND CONVULSIONS THE MEAN LETHAL DOSE REPORTED IN RATS WAS 400 MG KG CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID TREAT SYMPTOMATICALLY AND SUPPORTIVELY IF PERSON IS CONSCIOUS AND ABLE TO SWALLOW GIVE LARGE AMOUNTS OF WATER OR MILK TO DILUTE SUBSTANCE GET MEDICAL ATTENTION IMMEDIATELY GASTRIC LAVAGE PERFORMED BY QUALIFIED MEDICAL PERSONNEL MIGHT BE ADVISABLE IF THERE ARE NO SIGNS OF PERF
89. MMENDED BASED ON INFORMATION FOUND IN THE PHYSICAL DATA TOXICITY AND HEALTH EFFECTS SECTIONS THEY ARE RANKED IN ORDER FROM MINIMUM TO MAXIMUM RESPIRATORY PROTECTION THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST BE BASED ON THE SPECIFIC OPERATION MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND MUST BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION NIOSH MSHA ANY DUST AND MIST RESPIRATOR ANY AIR PURIFYING RESPIRATOR WITH A HIGH EFFICIENCY PARTICULATE FILTER ANY POWERED AIR PURIFYING RESPIRATOR WITH A DUST AND MIST FILTER ANY POWERED AIR PURIFYING RESPIRATOR WITH A HIGH EFFICIENCY PARTICULATE FILTER ANY TYPE C SUPPLIED AIR RESPIRATOR OPERATED IN THE PRESSURE DEMAND OR OTHER POSITIVE PRESSURE OR CONTINUOUS FLOW MODE ANY SELF CONTAINED BREATHING APPARATUS FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS ANY SELF CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE CLOTHING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOT
90. MPTOMATICALLY AND SUPPORTIVELY REACTIVITY REACTIVITY STABLE UNDER NORMAL TEMPERATURES AND PRESSURES INCOMPATIBILITIES 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE ACIDS STRONG DECOMPOSES WITH EVOLUTION OF GAS OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD DECOMPOSITION THERMAL DECOMPOSITION PRODUCTS MAY INCLUDE TOXIC AND CORROSIVE OXIDES OF PHOSPHOROUS AND HYDROGEN FLUORIDE POLYMERIZATION HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL TEMPERATURES AND PRESSURES STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANCE CONTACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY STORAGE STORAGE TEMPERATURE 4 C SHELF LIFE gt THREE YEARS STORE AWAY FROM INCOMPATIBLE SUBSTANCES 23 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B CONDITIONS TO AVOID MAY BURN BUT DOES NOT IGNITE READILY AVOID CONTACT WITH STRONG OXIDIZERS EXCESSIVE HEAT SPARKS OR OPEN FLAME SPILL AND LEAK PROCEDURES OCCUPATIONAL SPILL SWEEP UP AND PLACE IN SUITABLE CLEAN DRY CONTAINERS FOR RECLAMATION OR LATER DISPOSAL DO NOT FLUSH SPILLED MATERIAL INTO SEWER KEEP UNNECESSARY PEOPLE AWAY PROTECTIVE EQUIPMENT VENTILATION PROVIDE LOCAL EXHAUST VENTILATION VENTILATION EQUIPMENT MUST BE EXPLOSION PROOF RESPIRATOR THE FOLLOWING RESPIRATORS ARE RECO
91. NSE GUIDEBOOK DOT P 5800 5 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK DO NOT SCATTER SPILLED MATERIAL WITH HIGH PRESSURE WATER STREAMS DIKE FIRE CONTROL WATER FOR LATER DISPOSAL 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 GUIDE PAGE 31 USE AGENTS SUITABLE FOR TYPE OF SURROUNDING FIRE AVOID BREATHING HAZARDOUS VAPORS KEEP UPWIND TOXICITY 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE CARCINOGEN STATUS NONE ACUTE TOXICITY DATA NO DATA AVAILABLE TARGET EFFECTS NO DATA AVAILABLE AT INCREASED RISK FROM EXPOSURE PERSONS WITH ASTHMA BRONCHITIS OR OTHER RESPIRATORY DIFFICULTIES HEALTH EFFECTS AND FIRST AID INHALATION 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE ACUTE EXPOSURE MAY CAUSE BRONCHIAL IRRITATION WITH COUGHING AND OTHER SYMPTOMS OF BRONCHIAL DISTRESS CHRONIC EXPOSURE REPEATED OR PROLONGED EXPOSURE MAY CAUSE ALLERGIC RESPIRATORY SYSTEM SENSITIZATION FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED PERFORM ARTIFICIAL RESPIRATION KEEP PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY GET MEDICAL ATTENTION IMMEDIATELY SKIN CONTACT 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE ACUTE EXPOSURE MAY CAUSE IRRITATION CHRONIC EXPOSURE REPEATED OR PROLONGED EXPOSURE MAY CAUSE ALLERGIC SENSITIZATION FIRST AID REMOVE CONTAMINATED CL
92. OL IRRITANT ACUTE EXPOSURE INHALATION OF VERY HIGH VAPOR CONCENTRATIONS MAY CAUSE MUCOUS MEMBRANE IRRITATION HEADACHE GIDDINESS MENTAL CONFUSION AND NAUSEA INHALATION OF 180 200 MG M3 FOR 2 HOURS AND A 6 HOUR EXPOSURE TO SATURATED VAPORS CAUSED NO DEATHS IN MICE AND RATS RESPECTIVELY CHRONIC EXPOSURE PROLONGED EXPOSURE TO VERY HIGH VAPOR CONCENTRATIONS MAY CAUSE HEADACHE GIDDINESS MENTAL CONFUSION AND NAUSEA INHALATION STUDIES IN LABORATORY ANIMALS FAILED TO SHOW ANY GROSS OR HISTOPATHOLOGICAL ABNORMALITIES WHEN EXPOSED TO CONCENTRATIONS OF 50 PPM 8 HOURS DAY FOR 20 DAYS OR 370 PPM 6 HOURS DAY FOR 10 DAYS N N DIISOPROPYLETHYLAMINE IRRITANT ACUTE EXPOSURE MAY CAUSE IRRITATION TO THE UPPER RESPIRATORY TRACT SPASM INFLAMMATION AND EDEMA OF THE LARYNX AND BRONCHI CHEMICAL PNEUMONITIS AND PULMONARY EDEMA CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED PERFORM ARTIFICIAL RESPIRATION KEEP PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY GET MEDICAL ATTENTION IMMEDIATELY ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B SKIN CONTACT DIMETHYL SULFOXIDE IRRITANT ACUTE EXPOSURE MAY CAUSE IRRITATION WITH ERYTHEMA ITCHING SCALING A TRANSIENT BURNING SENSATION AND POSSIBLY BLISTERING IT CAN INITIATE THE IMMEDIATE RELEASE OF HISTAMINE WITH URTICARIAL WHEAL AND FLARE FORMATION ABSORPTION IS RAPID AND MAY CAUSE A G
93. ONE CAS 872 50 4 COMPONENT PERCENT 2 66 1 HYDROXYBENZOTRIAZOLE CAS 2592 95 2 EXPOSURE LIMITS NO OCCUPATIONAL EXPOSURE LIMITS ESTABLISHED BY OSHA ACGIH OR NIOSH 1 METHYL 2 PYRROLIDINONE 100 PPM 400 MG M3 DFG MAK TWA 100 PPM BASF CORP RECOMMENDED TWA PHYSICAL DATA DESCRIPTION LIQUID BOILING POINT NOT AVAILABLE SPECIFIC GRAVITY NOT AVAILABLE VAPOR PRESSURE NOT AVAILABLE SOLUBILITY IN WATER NOT AVAILABLE 27 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B FIRE AND EXPLOSION DATA FIRE AND EXPLOSION HAZARD MODERATE FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME VAPOR AIR MIXTURES ARE EXPLOSIVE ABOVE FLASH POINT FLASH POINT 190 F 88 C FLAMMABILITY CLASS OSHA IIIA FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPRAY FOG OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK APPLY COOLING WATER TO SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT STAY AWAY FROM ENDS OF TANKS FOR MASSIVE FIRE IN CARGO AREA USE UNMANNED HOSE HOLDER OR MONITOR NOZZLES IF THIS IS IMPOSSIBLE WITHDRAW FROM AREA AND LET FIRE BURN WITHDRAW IMMEDIATELY IN CASE OF RISING SOUND FROM VENTING SAFETY DEVICE OR ANY DISCOLORATION OF TANK DUE TO FIRE ISOLATE FOR 1 2 MILE
94. ONJUNCTIVITIS 1 METHYL 2 PYRROLIDINONE M PYROL IRRITANT ACUTE EXPOSURE EXPOSURE TO VAPORS MAY CAUSE IRRITATION CONTACT WITH THE LIQUID MAY CAUSE PAINFUL BURNING OR STINGING OF EYES AND LIDS WATERING OF THE EYES INFLAMMATION OF CONJUNCTIVA AND TEMPORARY CORNEAL CLOUDING CHRONIC EXPOSURE REPEATED OR PROLONGED EXPOSURE TO IRRITANTS MAY CAUSE CONJUNCTIVITIS 30 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B 1 HYDROXYBENZOTRIAZOLE ACUTE EXPOSURE NO DATA AVAILABLE CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL SALINE OCCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY INGESTION DIMETHYL SULFOXIDE ACUTE EXPOSURE INGESTION OF LARGE AMOUNTS MAY CAUSE NAUSEA VOMITING DIARRHEA ABDOMINAL PAIN LETHARGY AND DROWSINESS CHRONIC EXPOSURE REPEATED LARGE DOSES PRODUCED CRYSTALLINE LENS CHANGES RESEMBLING JUVENILE NUCLEAR SCLEROSIS IN SOME ANIMAL SPECIES BUT NOT IN HUMANS IN ANIMAL STUDIES REPEATED DOSES OF 1 5 GM KG RESULTED IN LIVER NECROSIS AND RENAL LESIONS REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS 1 METHYL 2 PYRROLIDINONE M PYROL ACUTE EXPOSURE INGESTION MAY CAUSE GASTROINTESTINAL DISTURBANCES CHRONIC EXPOSURE NINETY DAY FEEDING STUDIES IN LABORATORY ANIMALS AT CONCENTRATIONS UP TO 1 OF THEIR DIET FAILED TO DEMONSTRATE ANY TOXICOLOGICA
95. OR LATER DISPOSAL FOR LARGER SPILLS DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL NO SMOKING FLAMES OR FLARES IN HAZARD AREA KEEP UNNECESSARY PEOPLE AWAY ISOLATE HAZARD AREA AND DENY ENTRY REPORTABLE QUANTITY RQ 1 POUND THE SUPERFUND AMENDMENTS AND REAUTHORIZATION ACT SARA SECTION 304 REQUIRES THAT A RELEASE EQUAL TO OR GREATER THAN THE REPORTABLE QUANTITY FOR THIS SUBSTANCE BE IMMEDIATELY REPORTED TO THE LOCAL EMERGENCY PLANNING COMMITTEE AND THE STATE EMERGENCY RESPONSE COMMISSION 40 CFR 355 40 IF THE RELEASE OF THIS SUBSTANCE IS REPORTABLE UNDER CERCLA SECTION 103 THE NATIONAL RESPONSE CENTER MUST BE NOTIFIED IMMEDIATELY AT 800 424 8802 OR 202 426 2675 IN THE METROPOLITAN WASHINGTON D C AREA 40 CFR 302 6 PROTECTIVE EQUIPMENT 39 ABI PART NUMBER 901837 OHS PART NUMBER ABI18940 Rev B VENTILATION PROVIDE LOCAL EXHAUST OR PROCESS ENCLOSURE VENTILATION VENTILATION EQUIPMENT MUST BE EXPLOSION PROOF RESPIRATOR THE FOLLOWING RESPIRATORS ARE RECOMMENDED BASED ON INFORMATION FOUND IN THE PHYSICAL DATA TOXICITY AND HEALTH EFFECTS SECTIONS THEY ARE RANKED IN ORDER FROM MINIMUM TO MAXIMUM RESPIRATORY PROTECTION THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST BE BASED ON THE SPECIFIC OPERATION MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND MUST BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH AND THE MINE SAFE
96. ORATION FROM THE INGESTION OF A CORROSIVE SUBSTANCE IF VOMITING OCCURS KEEP HEAD BELOW HIPS TO HELP PREVENT ASPIRATION ANTIDOTE NO SPECIFIC ANTIDOTE TREAT SYMPTOMATICALLY AND SUPPORTIVELY REACTIVITY REACTIVITY WHEN DISSOLVED IN WATER THE SUBSTANCE IS A STRONG BASE IT REACTS VIOLENTLY WITH ACIDS AND IS CORROSIVE TOWARDS ALUMINUM ZINC AND OTHER METALS REACTS VIOLENTLY WITH OXIDANTS INCOMPATIBILITIES PIPERIDINE ACIDS INCOMPATABLE ACID ANHYDRIDES INCOMPATABLE ACID CHLORIDES INCOMPATABLE ALUMINUM CORROSIVE CARBON DIOXIDE INCOMPATABLE DICYANOFURAZAN INSTANTANEOUSLY EXPLOSIVE N NITROSOACETANILIDE THE DRY ANILIDE MAY EXPLODE ON CONTACT WITH A DROP OF PIPERIDINE 1 PERCHLORYLPIPERIDINE EXPLOSION ON CONTACT OXIDIZERS VIGOROUS REACTION ZINC CORROSIVE DECOMPOSITION THERMAL DECOMPOSITION PRODUCTS MAY INCLUDE TOXIC OXIDES OF CARBON AND NITROGEN POLYMERIZATION HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL TEMPERATURES AND PRESSURES STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANCE CONTACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY 38 ABI PART NUMBER 901837 OHS PART NUMBER ABI18940 Rev B STORAGE BONDING AND GROUNDING SUBSTANCES WITH LOW ELECTROCONDUCTIVITY WHICH MAY BE IGNITED BY ELECTROSTATIC SPARKS SHOULD BE STORED IN CONTAINERS WHICH MEET T
97. ORDANCE WITH STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE 40 CFR 262 EPA HAZARDOUS WASTE NUMBER D002 100 POUND CERCLA SECTION 103 REPORTABLE QUANTITY CONDITIONS TO AVOID MAY BURN BUT DOES NOT IGNITE READILY FLAMMABLE POISONOUS GASES MAY ACCUMULATE IN TANKS AND HOPPER CARS MAY IGNITE COMBUSTIBLES WOOD PAPER OIL ETC SPILL AND LEAK PROCEDURES OCCUPATIONAL SPILL DO NOT TOUCH SPILLED MATERIAL STOP LEAK IF YOU CAN DO IT WITHOUT RISK FOR SMALL SPILLS TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS FOR LATER DISPOSAL FOR SMALL DRY SPILLS WITH CLEAN SHOVEL PLACE MATERIAL INTO CLEAN DRY CONTAINER AND COVER MOVE CONTAINERS FROM SPILL AREA FOR LARGER SPILLS DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL KEEP UNNECESSARY PEOPLE AWAY ISOLATE HAZARD AREA AND DENY ENTRY PROTECTIVE EQUIPMENT VENTILATION PROVIDE LOCAL EXHAUST OR PROCESS ENCLOSURE VENTILATION SYSTEM RESPIRATOR THE FOLLOWING RESPIRATORS ARE RECOMMENDED BASED ON INFORMATION FOUND IN THE PHYSICAL DATA TOXICITY AND HEALTH EFFECTS SECTIONS THEY ARE RANKED IN ORDER FROM MINIMUM TO MAXIMUM RESPIRATORY PROTECTION THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST BE BASED ON THE SPECIFIC OPERATION MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND MUST BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRA
98. ORROSIVES ACUTE EXPOSURE MAY CAUSE CIRCUMORAL BURNS WITH DISCOLORATION AND CORROSION 51 ABI PART NUMBER 901812 OHS PART NUMBER ABI24050 Rev B OF THE MUCOUS MEMBRANES OF THE MOUTH THROAT AND ESOPHAGUS THERE MAY BE IMMEDIATE PAIN AND DIFFICULTY OR INABILITY TO SWALLOW OR SPEAK EPIGLOTTAL EDEMA MAY RESULT IN RESPIRATORY DISTRESS AND POSSIBLY ASPHYXIA MARKED THIRST NAUSEA VOMITING AND DIARRHEA MAY OCCUR DEPENDING ON THE AREA AND DEGREE OF CORROSION THE VOMITUS MAY CONTAIN FRESH OR DARK BLOOD AND LARGE SHREDS OF MUCOSA SHOCK MAY OCCUR WITH MARKED HYPOTENSION WEAK AND RAPID PULSE SHALLOW RESPIRATION AND CLAMMY SKIN CIRCULATORY COLLAPSE MAY DEVELOP AND IF UNCORRECTED LEAD TO RENAL FAILURE IN SEVERE CASES GASTRIC AND TO A LESSER DEGREE ESOPHAGEAL PERFORATION MAY OCCUR WITH PERITONITIS ACCOMPANIED BY FEVER AND ABDOMINAL RIGIDITY ESOPHAGEAL GASTRIC OR PYLORIC STRICTURE MAY OCCUR WITHIN A FEW WEEKS OR MAY BE DELAYED FOR MONTHS OR EVEN YEARS DEATH MAY RESULT WITHIN A SHORT TIME FROM ASPHYXIA CIRCULATORY COLLAPSE OR ASPIRATION OF EVEN MINUTE AMOUNTS IF DEATH IS DELAYED IT MAY BE DUE TO PERITONITIS SEVERE NEPHRITIS OR PNEUMONIA COMA AND CONVULSIONS SOMETIMES OCCUR TERMINALLY CHRONIC EXPOSURE DEPENDING ON THE CONCENTRATION REPEATED INGESTION MAY RESULT IN INFLAMMATORY AND ULCERATIVE CHANGES IN THE MUCOUS MEMBRANES OF THE MOUTH AND OTHER EFFECTS AS IN ACUTE INGESTION FIRST AID TREAT SYMPTOMATICALLY AND SUPPORTIVELY IF PER
99. OTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY 22 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B EYE CONTACT 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE ACUTE EXPOSURE MAY CAUSE IRRITATION TO THE EYES AND MUCOUS MEMBRANES OF THE EYELIDS CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL SALINE OCCASIONALLY LIFTING UPPER AND LOWER LIDS UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY INGESTION 2 1H BENZOTRIAZOL 1 YL 1 1 3 3 TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE ACUTE EXPOSURE NO DATA AVAILABLE CHRONIC EXPOSURE REPEATED OR PROLONGED INGESTION OF FLUORINE COMPOUNDS MAY CAUSE FLUOROSIS WITH SYMPTOMS INCLUDING OSTEOSCLEROTIC THICKENING AND CALCIFICATION OF THE LIGAMENTOUS ATTACHMENTS TO THE SKELETON AS WELL AS WEIGHT LOSS BRITTLENESS OF BONES REDUCED BONE MARROW SPACE ANEMIA WEAKNESS GENERAL ILL HEALTH STIFFNESS OF JOINTS AND DISCOLORATION OF DEVELOPING TEETH RARELY CENTRAL NERVOUS SYSTEM INVOLVEMENT MAY OCCUR FIRST AID TREAT SYMPTOMATICALLY AND SUPPORTIVELY GET MEDICAL ATTENTION IMMEDIATELY IF VOMITING OCCURS KEEP HEAD LOWER THAN HIPS TO PREVENT ASPIRATION ANTIDOTE NO SPECIFIC ANTIDOTE TREAT SY
100. Peptide Analysis and Purification Recommended Reading After cleavage the synthesized peptide may be sufficiently free of impurities for many applications The techniques most commonly used to analyze and purify syn thetic peptides incorporate high performance liquid chromatography HPLC espe cially RPC reverse phase chromatography Each synthetic peptide has its own unique sequence and structural characteristics that contribute to its solubility behavior These variables also influence the choice of HPLC techniques appropriate for analysis and purification Discussing all the possible variables and compatible techniques would be an ambitious project beyond the scope of this manual Instead we refer you to the following books and articles you may find useful Andrews P C 1988 Peptide Research 1 9 CRC Press 1984 Handbook of HPLC for the Separation of Amino Acids Peptides and Proteins Guo D Mant C T Taneja A K Parker J M R and Hodges R S 1986 J Chro matogr 359 499 Hoeger C Galyean R Boublik J McClintock R and Rivier J 1987 Bio Chromatography 2 134 3 8 Post Synthesis Procedures 03 2002 Applied Biosystems Mant Colin T and Hodges Robert S eds 1991 High Performance Liquid Chro matography of Peptides and Proteins Separation Analysis and Conformation Boca Raton Fla CRC Press Meek J L and Rossetti Z L 1981 J Chromatogr 211 15 Rivier J and McClintock
101. RATURES AND PRESSURES INCOMPATIBILITIES N N DIMETHYLFORMAMIDE ACID CHLORIDES INCOMPATIBLE ALKYL ALUMINUM VIOLENT REACTION 16 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B 2 5 BIS ENDO DICHLORO 7 THIABICYCLO 2 2 1 HEPTANE VIOLENT REACTION BOROHYDRIDE REACTS BRASS MAY BE ATTACKED CARBON TETRACHLORIDE VIOLENT REACTION ABOVE 65 C CHLOROFORMATES INCOMPATIBLE COPPER AND ALLOYS MAY BE ATTACKED DIISOCYANATOMETHANE VIOLENT POLYMERIZATION 2 5 DIMETHYLPYRROLE PHOSPHOROUS OXYCHLORIDE VIOLENT REACTION HALOGENS VIOLENT REACTION HALOGENATED HYDROCARBONS METALS DANGEROUS REACTION IRON VIOLENT REACTION LITHIUM AZIDE VIOLENT REACTION METHYLENE DIISOCYANTE MAY POLYMERIZE VIOLENTLY OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD PHOSPHOROUS TRIOXIDE VIOLENT REACTION PLASTICS RUBBER AND COATINGS MAY BE ATTACKED REDUCING AGENTS INCOMPATIBLE SODIUM AND COMPOUNDS VIOLENT REACTION 2 4 6 TRICHLORO 1 3 5 TRIAZINE VIGOROUS REACTION TRIETHYLALUMINUM MAY EXPLODE ON HEATING DECOMPOSITION THERMAL DECOMPOSITION PRODUCTS MAY INCLUDE TOXIC OXIDES OF CARBON AND NITROGEN POLYMERIZATION HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL TEMPERATURES AND PRESSURES STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANCE CONTACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY
102. RESSANT EFFECT ON SYMPATHETIC AND PARASYMPATHETIC GANGLIA HEALTH EFFECTS AND FIRST AID INHALATION PIPERIDINE CORROSIVE TOXIC ACUTE EXPOSURE MAY CAUSE IRRITATION AND BURNS OF THE NOSE THROAT AND MUCOUS MEMBRANES OF THE RESPIRATORY TRACT ABSORPTION MAY CAUSE EFFECTS ON THE NERVOUS SYSTEM ALSO SORE THROAT COUGH LABORED BREATHING DULLNESS AND PULMONARY EDEMA WHICH MAY BE DELAYED UP TO 2 DAYS MAY OCCUR THE REPORTED LETHAL DOSE IN MICE IS 6000 MG M3 2 HOURS CHRONIC EXPOSURE REPEATED OR PROLONGED EXPOSURE MAY CAUSE PULMONARY EDEMA REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED GIVE ARTIFICIAL RESPIRATION MAINTAIN AIRWAY AND BLOOD PRESSURE AND ADMINISTER OXYGEN IF AVAILABLE KEEP AFFECTED PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY ADMINISTRATION OF OXYGEN SHOULD BE PERFORMED BY QUALIFIED PERSONNEL GET MEDICAL ATTENTION IMMEDIATELY SKIN CONTACT PIPERIDINE CORROSIVE TOXIC ACUTE EXPOSURE MAY CAUSE SEVERE IRRITATION AND BURNS ABSORPTION MAY PRODUCE EFFECTS ON THE NERVOUS SYSTEM THE MEAN LETHAL DOSE APPLIED TO RABBIT SKIN WAS 320 MG KG NO EFFECTS FROM SKIN ABSORPTION WERE REPORTED CHRONIC EXPOSURE REPEATED OR PROLONGED CONTACT MAY CAUSE DERMATITIS FIRST AID REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHE
103. RITANT ACUTE EXPOSURE MAY CAUSE IRRITATION WITH ERYTHEMA ITCHING SCALING A TRANSIENT BURNING SENSATION AND POSSIBLY BLISTERING IT CAN INITIATE THE IMMEDIATE RELEASE OF HISTAMINE WITH URTICARIAL WHEAL AND FLARE FORMATION ABSORPTION IS RAPID AND MAY CAUSE A GARLIC LIKE TASTE AND 29 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B ODOR TO THE BREATH AND SKIN LARGE AMOUNTS MAY CAUSE NAUSEA VOMITING CRAMPS DIARRHEA ANESTHESIA LETHARGY DROWSINESS HEADACHE CHILLS CHEST PAINS BURNING OR ACHING EYES AND TRANSIENT DISTURBANCES OF COLOR VISION AND PHOTOPHOBIA TRANSIENT HEMOLYSIS WITH HEMOGLOBINURIA HAS ALSO BEEN REPORTED ENHANCED IRRITATION EPIDERMAL VESICULATION HISTOLOGICAL EVIDENCE OF DERMAL DEATH AND PERIVASCULAR DERMAL INFILTRATES WERE NOTED AFTER OCCLUDED PATCH TESTING OCCASIONAL HYPERSENSITIVITY REACTIONS INCLUDING ANAPHYLAXIS HAVE BEEN REPORTED DUE TO ITS SOLVENT PROPERTIES DMSO FACILITATES THE ABSORPTION OF SUBSTANCES PRESENT ON THE SKIN WHICH MAY RESULT IN TOXIC EFFECTS CHRONIC EXPOSURE 9 MILLILITERS OF 90 DMSO WAS APPLIED TO THE ENTIRE TRUNK OF 20 MEN ONCE DAILY FOR 26 WEEKS THE EFFECTS NOTED WERE BAD BREATH TRANSIENT ERYTHEMA BURNING AND STINGING THE DERMATITIS ACCOMPANIED BY ONLY MODERATE INFLAMMATION REGRESSED AS TREATMENT CONTINUED DAILY CONTINUOUS APPLICATION WITH OCCLUSION PRODUCED HARDENING OF THE SKIN IN MOST SUBJECTS WITHIN 1 MONTH CRYSTALLINE LENS ALTERATIONS RESEMBLING JUVENILE NUCLEAR
104. RS FOR SMALL SPILLS TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS FOR LATER DISPOSAL FOR LARGER SPILLS DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL NO SMOKING FLAMES OR FLARES IN HAZARD AREA KEEP UNNECESSARY PEOPLE AWAY ISOLATE HAZARD AREA AND DENY ENTRY REPORTABLE QUANTITY RQ 1000 POUNDS THE SUPERFUND AMENDMENTS AND REAUTHORIZATION ACT SARA SECTION 304 REQUIRES THAT A RELEASE EQUAL TO OR GREATER THAN THE REPORTABLE QUANTITY FOR THIS SUBSTANCE BE IMMEDIATELY REPORTED TO THE LOCAL EMERGENCY PLANNING COMMITTEE AND THE STATE EMERGENCY RESPONSE COMMISSION 40 CFR 355 40 IF THE RELEASE OF THIS SUBSTANCE IS REPORTABLE UNDER CERCLA SECTION 103 THE NATIONAL RESPONSE CENTER MUST BE NOTIFIED IMMEDIATELY AT 800 424 8802 OR 202 426 2675 IN THE METROPOLITAN WASHINGTON D C AREA 40 CFR 302 6 PROTECTIVE EQUIPMENT VENTILATION PROVIDE GENERAL DILUTION VENTILATION TO MEET PUBLISHED EXPOSURE LIMITS VENTILATION EQUIPMENT MUST BE EXPLOSION PROOF RESPIRATOR THE FOLLOWING RESPIRATORS AND MAXIMUM USE CONCENTRATIONS ARE RECOMMENDATIONS BY THE U S DEPARTMENT OF HEALTH AND HUMAN SERVICES NIOSH POCKET GUIDE TO CHEMICAL HAZARDS NIOSH CRITERIA DOCUMENTS OR BY THE U S DEPARTMENT OF LABOR 29 CFR 1910 SUBPART Z THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND IN THE WORK PLACE MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCC
105. RTED TO CAUSE SEVERE DERMATITIS WITH REDNESS CRACKING SWELLING BLISTERS AND EDEMA REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS N N DIISOPROPYLETHYLAMINE IRRITANT ACUTE EXPOSURE MAY CAUSE IRRITATION OR DERMATITIS AND POSSIBLY BURNS CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY EYE CONTACT DIMETHYL SULFOXIDE IRRITANT ACUTE EXPOSURE DIRECT CONTACT MAY CAUSE IRRITATION WITH REDNESS PAIN AND BLURRED VISION AQUEOUS SOLUTIONS CONTAINING 75 90 DMSO MAY CAUSE IRRITATION WITH TEMPORARY STINGING AND BURNING FIFTY PER CENT SOLUTIONS HAVE CAUSED A TRANSIENT BURNING SENSATION LOWER CONCENTRATIONS HAVE BEEN TOLERATED WELL WITHOUT INJURY TO THE EYE APPLICATION FULL STRENGTH INTO RABBIT EYES CAUSED PAIN MODERATE DISCHARGE CORNEAL EPITHELIUM INJURY AND DILATION OF CONJUNCTIVAL BLOOD VESSELS BUT NO HEMORRHAGING THE EYES RETURNED TO NORMAL IN 2 DAYS CHRONIC EXPOSURE REPEATED OR PROLONGED CONTACT WITH IRRITANTS MAY CAUSE CONJUNCTIVITIS ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B 1 METHYL 2 PYRROLIDINONE M PYROL IRRITANT ACUTE EXPOSURE EXPOSURE TO VAPORS MAY CAUSE IRRITATION CONTACT WITH THE LIQUID MAY CAUSE PAINFUL BURNING OR STINGING OF EYES AND LIDS WATERING OF THE EYES
106. S FORMS AN EXTREMELY EXPLOSIVE MIXTURE METAL PERCHLORATES FORMS AN EXTREMELY EXPLOSIVE MIXTURE NITRIC ACID POSSIBLE EXPLOSION HAZARD OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD PERCHLORIC ACID EXPLODES ON CONTACT PERIODIC ACID POSSIBLE EXPLOSIVE REACTION PHOSPHOROUS III OXIDE VIOLENT REACTION POTASSIUM VIOLENT REACTION POTASSIUM PERMANGANATE IGNITION ON CONTACT SILVER DIFLUORIDE VIOLENT REACTION SODIUM HYDRIDE POSSIBLE FIRE AND EXPLOSION AT ELEVATED TEMPERATURES SULFUR TRIOXIDE EXOTHERMIC REACTION 1 METHYL 2 PYRROLIDINONE M PYROL ACIDS INCOMPATIBLE OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD N N DIISOPROPYLETHYLAMINE COATINGS PLASTICS FINISHES RUBBER MAY BE ATTACKED OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD SEE AMINES AMINES ACROLEIN EXOTHERMIC POLYMERIZATION CALCIUM HYPOCHLORITE FORMATION OF EXPLOSIVE CHLOROAMINE MALEIC ANHYDRIDE EXPLOSIVE DECOMPOSITION NITROSYL PERCHLORATE EXPLOSIVE REACTION SODIUM HYPOCHLORITE FORMATION OF EXPLOSIVE CHLOROAMINE TRI ISO BUTYL ALUMINUM VIOLENT REACTION DECOMPOSITION THERMAL DECOMPOSITION MAY RELEASE TOXIC AND OR HAZARDOUS GASES POLYMERIZATION HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL TEMPERATURES AND PRESSURES 10 ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANC
107. S EYE RABBIT MILD TOXICITY DATA 1600 MG M3 4 HOURS INHALATION RAT LC50 38MKAJ 40 GM KG SKIN RAT LD50 50 GM KG SKIN MOUSE LD50 gt 11 GM KG SKIN DOG LD50 38MKAJ gt 11 GM KG SKIN MONKEY LD50 38MKAJ 14500 MG KG ORAL RAT LD50 7920 MG KG ORAL MOUSE LD50 gt 11 GM KG ORAL GUINEA PIG LDLO gt 10 GM KG ORAL DOG LD50 12 GM KG SUBCUTANEOUS RAT LD50 14 GM KG SUBCUTANEOUS MOUSE LD50 606 MG KG INTRAVENOUS MAN TDLO 5360 MG KG INTRAVENOUS RAT LD50 3100 MG KG INTRAVENOUS MOUSE LD50 2500 MG KG INTRAVENOUS DOG LD50 200 MG KG INTRAVENOUS CAT LDLO 8200 MG KG INTRAPERITONEAL RAT LD50 2500 MG KG INTRAPERITONEAL MOUSE LD50 gt 5500 MG KG INTRAPERITONEAL GUINEA PIG LDLO 1300 MG KG UNREPORTED RAT LD50 MUTAGENIC DATA RTECS REPRODUCTIVE EFFECTS DATA RTECS TUMORIGENIC DATA RTECS CARCINOGEN STATUS NONE LOCAL EFFECTS IRRITANT INHALATION SKIN EYE ACUTE TOXICITY LEVEL TOXIC BY INHALATION SLIGHTLY TOXIC BY INGESTION RELATIVELY NON TOXIC BY DERMAL ABSORPTION TARGET EFFECTS POISONING MAY AFFECT THE LIVER AND KIDNEYS ADDITIONAL DATA INTERACTIONS WITH MEDICATIONS HAVE BEEN REPORTED 1 METHYL 2 PYRROLIDINONE M PYROL IRRIITATION DATA 100 MG EYE RABBIT MODERATE TOXICITY DATA 8000 MG KG SKIN RABBIT LD50 3914 MG KG ORAL RAT LD50 4400 MG KG ORAL GUINEA PIG LD50 5130 MG KG ORAL MOUSE LD50 ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B 3500 MG KG ORAL RABBIT LD50 80500 UG KG INTRAVENOUS RAT LD50
108. S gt PSC THF gt TVB THF gt PSC GAS gt TVB GAS gt PSC GAS gt BVB Time 999 QD on ow 00 WWWHWWWWWODNH HHP HH Wan PR WO fe oO oO Oo WowWwownonaonoao IV 8 03 2002 Applied Biosystems Flow Test 8 CALIBRATE HBTU Step Function 1 10 2 3 3 15 4 61 5 68 6 4 7 2 8 10 9 45 10 14 11 2 12 45 13 14 14 2 15 68 16 3 17 15 18 61 19 68 20 4 21 2 22 10 23 45 24 14 25 2 26 68 27 36 28 15 29 61 30 68 31 3 32 15 33 61 34 68 35 4 Substitute time Function Name GAS gt TVB BEGIN LOOP GAS gt R XFER L gt WASTE END LOOP INTERRUPT GAS gt TVB HBTU gt L GAS gt L INTERRUPT HBTU gt L GAS gt L INTERRUPT L gt WASTE BEGIN LOOP GAS gt R XFER L gt WASTE END LOOP INTERRUPT GAS gt TVB HBTU gt L GAS gt L INTERRUPT L gt WASTE DMF gt R GAS gt R XFER L gt WASTE BEGIN LOOP GAS gt R XFER L gt WASTE END LOOP Time e RK 0QQMQNOANMOMRMNONUNOMTM 4000NAa i A 00MAM NOOLAFNNOI ma 03 2002 Test Printouts IV 9 Applied Biosystems Flow Test 9 CALIBRATE DIEA Step Function 1 10 2 3 3 15 4 61 5 68 6 4 7 2 8 10 9 46 10 14 11 2 12 46 13 14 14 2 15 68 16 3 17 15 18 61 19 68 20 4 21 2 22 10 23 46 24 14 25 2 26 68 27 36 28 15 29 61 30 68 31 3 32 15 33 61 34 68 35 4 Substitute time Function Name GAS gt TVB BEGIN LOOP GAS gt R XFER L gt WASTE END LOOP INTERRUPT GAS gt TVB DIEA gt L GAS gt L INTERRUPT DIEA gt L GAS gt L
109. SC the pressure test continues to fail end the synthesis see How to terminate a synthesis on page 7 11 When synthesis has ended run the Leak Self Test see page 4 13 Pressure Test Failure at module j Check low pressure gauge The autosampler jaws fail first when the input gas pressure drops below 60 psi Check AAC in jaws and action of jaws To inspect the amino acid column AAC first open the autosampler jaws by manually activating Function 53 OPEN JAWS 03 2002 Troubleshooting 7 13 Applied Biosystems How to manually activate a function 1 While the PAUSE soft key is active in the Run Monitor press the MAIN key to return to the Main Menu 2 Press the manual control soft key to go to the Manual Control Menu 3 Press the function soft key to go to the Function Menu Figure 7 7 4 Use the numeric keys to enter the number of the function you want to activate and then press the ON soft key To open the autosampler jaws activate Function 53 OPEN JAWS set up amp run edit amp manual self Main Menu report control print control test ON gt F V Manual Control Menu Press function valve ALL OFF Menu key to return to Main Menu Enter Function 53 OPEN JAWS Function Menu ON Figure 7 7 How to manually activate a function When the jaws are open carefully remove the amino acid column wheel without rotating the wheel hub
110. SCLEROSIS HAVE BEEN PRODUCED IN SOME ANIMAL SPECIES BUT NOT IN HUMANS NO LENS ABNORMALITIES WERE FOUND IN 25 PATIENTS TREATED DAILY WITH UP TO 30 ML APPLIED TOPICALLY FOR 19 MONTHS 1 METHYL 2 PYRROLIDINONE M PYROL IRRITANT ACUTE EXPOSURE CONTACT MAY CAUSE MILD IRRITATION CHRONIC EXPOSURE PROLONGED CONTACT HAS BEEN REPORTED TO CAUSE SEVERE DERMATITIS WITH REDNESS CRACKING SWELLING BLISTERS AND EDEMA REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS 1 HYDROXYBENZOTRIAZOLE ACUTE EXPOSURE NO DATA AVAILABLE CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY EYE CONTACT DIMETHYL SULFOXIDE IRRITANT ACUTE EXPOSURE DIRECT CONTACT MAY CAUSE IRRITATION WITH REDNESS PAIN AND BLURRED VISION AQUEOUS SOLUTIONS CONTAINING 75 90 DMSO MAY CAUSE IRRITATION WITH TEMPORARY STINGING AND BURNING FIFTY PER CENT SOLUTIONS HAVE CAUSED A TRANSIENT BURNING SENSATION LOWER CONCENTRATIONS HAVE BEEN TOLERATED WELL WITHOUT INJURY TO THE EYE APPLICATION FULL STRENGTH INTO RABBIT EYES CAUSED PAIN MODERATE DISCHARGE CORNEAL EPITHELIUM INJURY AND DILATION OF CONJUNCTIVAL BLOOD VESSELS BUT NO HEMORRHAGING THE EYES RETURNED TO NORMAL IN 2 DAYS CHRONIC EXPOSURE REPEATED OR PROLONGED CONTACT WITH IRRITANTS MAY CAUSE C
111. SON IS CONSCIOUS AND ABLE TO SWALLOW GIVE LARGE AMOUNTS OF WATER OR MILK TO DILUTE SUBSTANCE GET MEDICAL ATTENTION IMMEDIATELY GASTRIC LAVAGE PERFORMED BY QUALIFIED MEDICAL PERSONNEL MIGHT BE ADVISABLE IF THERE ARE NO SIGNS OF PERFORATION FROM THE INGESTION OF A CORROSIVE SUBSTANCE IF VOMITING OCCURS KEEP HEAD BELOW HIPS TO HELP PREVENT ASPIRATION ANTIDOTE NO SPECIFIC ANTIDOTE TREAT SYMPTOMATICALLY AND SUPPORTIVELY REACTIVITY REACTIVITY STABLE UNDER NORMAL TEMPERATURES AND PRESSURES INCOMPATIBILITIES TRIFLUOROACETIC ACID ACIDS REACTS TO FORM TOXIC GASES AROMATIC HYDROCARBONS HYDROGEN PEROXIDE EXPLOSION HAZARD BASES REACTS VIOLENTLY COMBUSTIBLE MATERIALS INCOMPATIBLE LITHIUM TETRAHYDROALUMINATE FIRE AND EXPLOSION HAZARD METALS ATTACKS DECOMPOSITION MAY RELEASE TOXIC AND HAZARDOUS HYDROGEN FLUORIDE FUMES UNDER THERMAL DECOMPOSITION OR BY REACTION WITH ACIDS POLYMERIZATION HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL TEMPERATURES AND PRESSURES STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANCE CONTACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY 52 ABI PART NUMBER 901812 OHS PART NUMBER ABI24050 Rev B STORAGE STORAGE TEMPERATURE 20 10 C SHELF LIFE gt 3 YEARS STORE AWAY FROM INCOMPATIBLE SUBSTANCES DISPOSAL DISPOSAL MUST BE IN ACC
112. STION RELATIVELY NON TOXIC BY DERMAL ABSORPTION TARGET EFFECTS POISONING MAY AFFECT THE LIVER AND KIDNEYS ADDITIONAL DATA INTERACTIONS WITH MEDICATIONS HAVE BEEN REPORTED 1 METHYL 2 PYRROLIDINONE M PYROL IRRTITATION DATA 100 MG EYE RABBIT MODERATE TOXICITY DATA 8000 MG KG SKIN RABBIT LD50 3914 MG KG ORAL RAT LD50 4400 MG KG ORAL GUINEA PIG LD50 5130 MG KG ORAL MOUSE LD50 3500 MG KG ORAL RABBIT LD50 80500 UG KG INTRAVENOUS RAT LD50 28 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B 54500 UG KG INTRAVENOUS MOUSE LD50 63300 UG KG INTRAVENOUS DOG LD50 2472 MG KG INTRAPERITONEAL RAT LD50 3050 MG KG INTRAPERITONEAL MOUSE LD50 7 GM KG UNREPORTED ROUTE RAT LD50 MUTAGENIC DATA RTECS REPRODUCTIVE EFFECTS DATA RTECS CARCINOGEN STATUS NONE LOCAL EFFECTS IRRITANT INHALATION SKIN EYES ACUTE TOXICITY LEVEL MODERATELY TOXIC BY INGESTION SLIGHTLY TOXIC BY DERMAL ABSORPTION TARGET EFFECTS POISONING MAY AFFECT THE CENTRAL NERVOUS SYSTEM 1 HYDROXYBENZOTRIAZOLE CARCINOGEN STATUS NONE ACUTE TOXICITY LEVEL NO DATA AVAILABLE TARGET EFFECTS NO DATA AVAILABLE HEALTH EFFECTS AND FIRST AID INHALATION DIMETHYL SULFOXIDE IRRITANT TOXIC ACUTE EXPOSURE VAPORS MAY CAUSE MODERATE IRRITATION OF THE RESPIRATORY TRACT WITH COUGHING HIGH CONCENTRATIONS MAY CAUSE SYSTEMIC EFFECTS SUCH AS NAUSEA VOMITING CHILLS CRAMPS HEADACHE DIZZINESS AND LETHARGY ALLERGIC RESPIRATORY REACTIONS MAY
113. STOMACH CHRONIC EXPOSURE NO DATA AVAILABLE FIRST AID SEEK MEDICAL ATTENTION IMMEDIATELY TREAT SYMPTOMATICALLY AND SUPPORTIVELY MAINTAIN AIRWAY AND RESPIRATION IF VOMITING OCCURS KEEP HEAD BELOW HIPS TO PREVENT ASPIRATION IF A POISONOUS SUBSTANCE HAS BEEN INGESTED REMOVAL BY EMESIS OR GASTRIC LAVAGE WITH APPROPRIATE PRECAUTIONS TO PREVENT ASPIRATION IS GENERALLY RECOMMENDED PROVIDED THE PATIENT IS CONSCIOUS NOT CONVULSING AND THERE IS NO SIGN OF CORROSIVE INJURY ONLY QUALIFIED MEDICAL PERSONNEL SHOULD PERFORM GASTRIC LAVAGE IF A CORROSIVE SUBSTANCE HAS BEEN INGESTED DILUTION BY RINSING THE MOUTH AND GIVING WATER OR MILK TO DRINK IS GENERALLY RECOMMENDED IF PERFORATION HAS OCCURRED OR THE VICTIM IS UNCONSCIOUS THE VICTIM SHOULD NOT BE GIVEN ANYTHING TO DRINK ABI PART NUMBER 902035 OHS PART NUMBER ABINF033 Rev B REACTIVITY REACTIVITY STABLE UNDER NORMAL TEMPERATURES AND PRESSURES INCOMPATIBILITIES DIMETHYL SULFOXIDE ACID ANHYDRIDES POSSIBLE EXPLOSIVE REACTION ACID HALIDES POSSIBLE EXPLOSIVE REACTION ACYL HALIDES VIOLENT OR EXPLOSIVE REACTION ARYL HALIDES VIOLENT DECOMPOSITION REACTION BORON HYDRIDES MAY FORM EXPLOSIVE MIXTURE BORON HYDROBORATES MAY FORM EXPLOSIVE MIXTURE 4 4 BROMOBENZOYL ACETANILIDE MAY EXPLODE AT ELEVATED TEMPERATURES CARBONYL DIISOTHIOCYANATE EXPLOSIVE REACTION DINITROGEN TETRAOXIDE VIOLENT OR EXPLOSIVE REACTION IODINE PENTAFLUORIDE POSSIBLE EXPLOSIVE REACTION METAL NITRATE
114. Sed gaseous waste ecurely attach the vent line from the waste bottle and avoid puncturing The vent line should be no longer than 15 ft 5 m Air flow indicator Minimum velocity is 80 ft min Maximum velocity is 125 ft min Figure 1 3 Venting Gaseous Waste Produced by Instrument to a Fume Hood Routine Maintenance Tasks e Periodically at least twice a year check and record face velocity If results fail to meet standards make ventilation adjustments as soon as possible to prevent employee exposure to hazardous fumes e Periodically at least once a year inspect and maintain exhaust system including fans and motors Duct System Gaseous waste is vented through a fume hood to a duct or is directly vented through the duct The following are important points about U S regulations and practices re lated to the duct Duct Work Should be constructed of materials compatible with the waste materials being generated 03 2002 Synergy Safety Guidelines 1 9 Applied Biosystems Should operate at all times including nights and weekends when vented waste bottle contents can escape to surroundings Should not come in contact with strong oxidizers bases or other chemicals that are incompatible with gaseous waste Should allow vapor or gas movement 1000 2000 fpm Tubing Do not let the open end of tubing face into oncoming air movement through duct or canopy Keep away from sources of pot
115. Svnergv Personal Peptide Svnthesizer User Manual Applied KS Biosystems O Copyright 2002 Applied Biosystems All Rights reserved For Research Use Only Not for use in diagnostic procedures Synergy is a trademark of Applied Biosystems Slo Blo is a registered trademark of Littlefuse Inc Speed Vac is a registered trademark of Savant Instruments Teflon is a trademark of E I duPont de Nemours amp Co ThinkJet is a trademark of Hewlett Packard Company Applied Biosystems Contents 1 Synergy Safety Guidelines Synergy User s Manual Conventions User Attention Words Chemical abbreviations Synergy Safety Symbols Electrical Symbols Other Synergy Safety Procedures General Laboratory Safety Instrument Access Fuses Jaw Assembly Hazardous Chemicals Waste Bottle Secondary Containment Compressed Gas Cylinders External Pneumatic Supply Laboratory Ventilation Requirements Fume Hood Routine Maintenance Tasks Duct System Duct Work Tubing Canopy Routine Maintenance Tasks 2 Routine Operation A Brief Description of Synergy The LCD and Keyboard Making a Peptide on Synergy Prepare Synergy for Synthesis 1 Check the external gas supply Check levels of 5 reagent bottles 2 3 Check the waste bottle and empty if necessary 4 Check the printer power paper supply and cable connections 1 3 1 3 1 3 1 4 1 4 1 5 1 5 1 5 1 6 1 6 1 6 1 6 1 7 1 7 1 7 1 8 1 8 1 9 1 9 1 9 1 10 1 10 1 11 2 3
116. TA FIRE AND EXPLOSION HAZARD MODERATE FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME VAPOR AIR MIXTURES ARE EXPLOSIVE ABOVE FLASH POINT FLASH POINT 160 F 71 C FLAMMABILITY CLASS OSHA IIIA FIREFIGHTING MEDIA DRY CHEMICAL CARBON DIOXIDE WATER SPRAY OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FOR LARGER FIRES USE WATER SPRAY FOG OR ALCOHOL RESISTANT FOAM 1990 EMERGENCY RESPONSE GUIDEBOOK DOT P 5800 5 FIREFIGHTING MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK APPLY COOLING WATER TO SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT STAY AWAY FROM ENDS OF TANKS FOR MASSIVE FIRE IN CARGO AREA USE UNMANNED HOSE HOLDER OR MONITOR NOZZLES IF THIS IS IMPOSSIBLE WITHDRAW FROM AREA AND LET FIRE BURN WITHDRAW IMMEDIATELY IN CASE OF RISING SOUND FROM VENTING SAFETY DEVICE OR ANY DISCOLORATION OF TANK DUE TO FIRE ISOLATE FOR 1 2 MILE IN ALL DIRECTIONS IF TANK RAIL CAR OR TANK TRUCK IS INVOLVED IN FIRE 1990 EMERGENCY RESPONSE GUIDE BOOK DOT P 5800 5 GUIDE PAGE 26 EXTINGUISH ONLY IF FLOW CAN BE STOPPED USE WATER IN FLOODING AMOUNTS AS FOG SOLID STREAMS MAY NOT BE EFFECTIVE COOL CONTAINERS WITH FLOODING AMOUNTS OF WATER APPLY FROM AS FAR A DISTANCE AS POSSIBLE AVOID BREATHING VAPORS KEEP UPWIND TOXICITY DIMETHYL SULFOXIDE IRRITATION DATA 10 MG 24 HOURS OPEN SKIN RABBIT MILD 500 MG 24 HOURS SKIN RABBIT MILD 100 MG EYE RABBIT 500 MG 24 HOUR
117. TACT THE DISTRICT DIRECTOR OF THE ENVIRONMENTAL PROTECTION AGENCY STORAGE STORE IN ACCORDANCE WITH 29 CFR 1910 106 BONDING AND GROUNDING SUBSTANCES WITH LOW ELECTROCONDUCTIVITY WHICH MAY BE IGNITED BY ELECTROSTATIC SPARKS SHOULD BE STORED IN CONTAINERS WHICH MEET THE BONDING AND GROUNDING GUIDELINES SPECIFIED IN NFPA 77 1983 RECOMMENDED PRACTICE ON STATIC ELECTRICITY STORE IN A COOL DRY WELL VENTILATED LOCATION STORE AWAY FROM HEAT OXIDIZING MATERIALS AND SUNLIGHT OUTSIDE OR DETACHED STORAGE IS PREFERRED INSIDE STORAGE SHOULD BE IN A STANDARD FLAMMABLE LIQUIDS STORAGE WAREHOUSE ROOM OR CABINET NFPA 49 HAZARDOUS CHEMICALS DATA 1991 STORAGE TEMPERATURE ROOM TEMPERATURE IN A WELL VENTILATED AREA SHELF LIFE INDEFINITE STORE AWAY FROM INCOMPATIBLE SUBSTANCES DISPOSAL DISPOSAL MUST BE IN ACCORDANCE WITH STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE 40CFR 262 EPA HAZARDOUS WASTE NUMBER U213 CONDITIONS TO AVOID AVOID CONTACT WITH HEAT SPARKS FLAMES OR OTHER SOURCES OF IGNITION VAPORS MAY BE EXPLOSIVE AND POISONOUS DO NOT ALLOW UNNECESSARY PERSONNEL IN AREA DO NOT OVERHEAT CONTAINERS CONTAINERS MAY VIOLENTLY RUPTURE AND TRAVEL A CONSIDERABLE DISTANCE IN HEAT OF FIRE 45 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B SPILL AND LEAK PROCEDURES OCCUPATIONAL SPILL SHUT OFF IGNITION SOURCES STOP LEAK IF YOU CAN DO IT WITHOUT RISK USE WATER SPRAY TO REDUCE VAPO
118. TATION REDNESS PAIN AND DESQUAMATION OF THE SKIN THERE ARE RARE INCONCLUSIVE REPORTS OF HUMAN SENSITIZATION EFFECTS OF ITCHING HYPEREMIA ABDOMINAL PAIN VOMITING AND AN INCREASED IN BLOOD PRESSURE WERE PRODUCED IN ONE CASE OF ACCIDENTIAL EXPOSURE THIS MATERIAL IS ABSORBED RAPIDLY THROUGH THE SKIN AND OTHER EFFECTS AS DETAILED IN INHALATION MAY OCCUR FETAL DEATHS HAVE OCCURED WHEN APPLIED 15 ABI PART NUMBER 901822 OHS PART NUMBER ABI07860 Rev B TO THE SKIN OF PREGNANT RATS CHRONIC EXPOSURE PROLONGED OR REPEATED EXPOSURE MAY CAUSE DERMATITIS AS A RESULT OF DEFATTING ACTION MATERNAL MORTALITY AND AN INCREASED RATE OF EMBRYONIC DEATH WERE OBSERVED IN A STUDY OF PREGNANT RABBITS RECEIVING REPEATED APPLICATIONS FIRST AID REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELV 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY EYE CONTACT N N DIMETHXLFORMAMIDE IRRITANT ACUTE EXPOSURE MAY CAUSE IRRITATION REDNESS PAIN TEARING AND BLURRED VISION A 50 SOLUTION IS SLIGHTLY IRRITATING TO RABBIT BYES A 75 100 SOLUTION CAUSES A MORE SEVERE REACTION WITH EDEMA OF THE CORNEAL EPITHELIUM MODERATE CONJUNCTIVITIS HAS ALSO BEEN NOTED CHRONIC EXPOSURE PROLONGED CONTACT WITH VAPORS OR LIQUID MAY CAUSE CONJUNCTIVITIS FIRST AID WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL SALINE O
119. TINGUISH ONLY IF FLOW CAN BE STOPPED COOL CONTAINERS WITH FLOODING AMOUNTS OF WATER FROM AS FAR A DISTANCE AS POSSIBLE DO NOT USE WATER DIRECTLY ON MATERIAL IF LARGE AMOUNTS OF COMBUSTIBLE MATERIALS ARE INVOLVED USE WATER SPRAY AND FOG IN FLOODING AMOUNTS USE WATER SPRAY TO ABSORB FLAMMABLE CORROSIVE VAPORS AVOID BREATHING CORROSIVE VAPORS KEEP UPWIND WATER MAY BE INEFFECTIVE NFPA 325M FIRE HAZARD PROPERTIES OF FLAMMABLE LIQUIDS GASES AND VOLATILE SOLIDS 1991 TOXICITY PIPERIDINE IRRITATION DATA 100 UG 24 HOURS OPEN SKIN RABBIT 5 MG 24 HOURS SKIN RABBIT SEVERE 250 UG 24 HOURS EYE RABBIT SEVERE TOXICITY DATA 4000 PPM 4 HOURS INHALATION RAT LCLO 6000 MG M3 2 HOURS INHALATION MOUSE LC50 6500 MG M3 INHALATION MAMMAL LD50 320 MG KG 36 ABI PART NUMBER 901837 OHS PART NUMBER ABI18940 Rev B SKIN RABBIT LD50 400 MG KG ORAL RAT LD50 145 MG KG ORAL RABBIT LD50 30 MG KG ORAL MOUSE LD50 22400 UG KG ORAL MAMMAL LD50 300 MG KG SUBCUTANEOUS RABBIT LDLO 460 MG KG SUBCUTANEOUS MOUSE LDLO 160 MG KG INTRAVENOUS RABBIT LDLO 50 MG KG INTRAPERITONEAL MOUSE LDLO MUTAGENIC DATA RTECS REPRODUCTIVE EFFECTS DATA RTECS CARCINOGEN STATUS NONE LOCAL EFFECTS CORROSIVE INHALATION SKIN EYE INGESTION ACUTE TOXICTY LEVEL TOXIC BY INHALATION DERMAL ABSORPTION INGESTION TARGET EFFECTS POISONING MAY AFFECT THE NERVOUS SYSTEM ADDITIONAL DATA INTRAVENOUS ADMINISTRATION HAD AN INITIAL STIMULANT AND SECONDARY DEP
120. TION NIOSH MSHA ANY TYPE C SUPPLIED AIR RESPIRATOR WITH A FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE OR WITH A FULL FACEPIECE HELMET OR HOOD OPERATED IN CONTINUOUS FLOW MODE ANY SELF CONTAINED BREATHING APPARATUS WITH A FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS 53 ABI PART NUMBER 901812 OHS PART NUMBER ABI24050 Rev B ANY SELF CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED INA PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE CLOTHING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOTHING AND EQUIPMENT TO PREVENT ANY POSSIBILITY OF SKIN CONTACT WITH THIS SUBSTANCE GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES AND A FACESHIELD TO PREVENT CONTACT WITH THIS SUBSTANCE EMERGENCY WASH FACILITIES WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE S EYES AND OR SKIN MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN EYE WASH FOUNTAIN AND QUICK DRENCH S
121. TY STABLE UNDER NORMAL TEMPERATURES AND PRESSURES IF PEROXIDATION IS INHIBITED IF UNINHIBITED MAY FORM EXPLOSIVE ORGANIC PEROXIDES WHEN EXPOSED TO AIR IF DISTILLED OR EVAPORATED TO DRYNESS AN EXPLOSION MAY OCCUR INCOMPATIBILITIES TETRAHYDROFURAN ACIDS REACTION AND POSSIBLE POLYMERIZATION BASES INCOMPATIBLE BORANE TETRAHYDROFURAN COMPLEX POSSIBLE EXPLOSION ON STORAGE BROMINE VIGOROUS REACTION HAFNIUM TETRACHLORIDE VIOLENT EXOTHERMIC REACTION LITHIUM ALUMINUM ALLOYS EXPLOSION LITHIUM ALUMINUM HYDRIDE LITHIUM TETRAHYDROALUMINATE POSSIBLE IGNITION METALLIC HYDRIDES POSSIBLE EXPLOSION WHEN HEATED OXIDIZERS STRONG FIRE AND EXPLOSION HAZARD PLASTICS RUBBER COATINGS SOME FORMS ATTACKED POTASSIUM DIOXIDE AND 2 AMINOPHENOL POSSIBLE EXPLOSION POTASSIUM HYDROXIDE POSSIBLE EXPLOSION IN PRESENCE OF PEROXIDE CONTAMINANTS 44 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B SODIUM HYDROXIDE POSSIBLE EXPLOSION IN PRESENCE OF PEROXIDE CONTAMINANTS SODIUM TETRAHYDROALUMINATE POSSIBLE VIOLENT EXPLOSION TITANIUM TETRACHLORIDE VIOLENT EXOTHERMIC REACTION ZIRCONIUM TETRACHLORIDE VIOLENT EXOTHERMIC REACTION DECOMPOSITION THERMAL DECOMPOSITION PRODUCTS MAY INCLUDE TOXIC OXIDES OF CARBON POLYMERIZATION MAY POLYMERIZE EXOTHERMICALLY IN CONTACT WITH SOME ACIDS STORAGE AND DISPOSAL OBSERVE ALL FEDERAL STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING OF THIS SUBSTANCE FOR ASSISTANCE CON
122. TY AND HEALTH ADMINISTRATION NIOSH MSHA ANY TYPE C SUPPLIED AIR RESPIRATOR WITH A FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE OR WITH A FULL FACEPIECE HELMET OR HOOD OPERATED IN CONTINUOUS FLOW MODE ANY SELF CONTAINED BREATHING APPARATUS WITH A FULL FACEPIECE OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS ANY SELF CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED INA PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE CLOTHING EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE IMPERVIOUS CLOTHING AND EQUIPMENT TO PREVENT ANY POSSIBILITY OF SKIN CONTACT WITH THIS SUBSTANCE GLOVES EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH THIS SUBSTANCE EYE PROTECTION EMPLOYEE MUST WEAR SPLASH PROOF OR DUST RESISTANT SAFETY GOGGLES AND A FACESHIELD TO PREVENT CONTACT WITH THIS SUBSTANCE EMERGENCY WASH FACILITIES WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE S EYES AND OR SKIN MAY BE EXPOSED TO THIS SUBSTANCE THE EMPLOYER SHOULD PROVIDE AN EYE WASH FOUNTAIN AND QUICK DRENCH SHOWER WITHIN THE IMMEDIATE WORK AREA FOR EMERGENC
123. The d module is not needed here because the Fmoc group has already been removed Extra c modules extend coupling time to compensate for the missing d module After editing Line 1 press the ADD L soft key Enter the standard cycle modules on Line 2 j d a c g f i By default a 1 one appears after the word Repeat Figure 6 3 shows the Run File for this example Line Cycle Repeat Module List Beg 0 1 bf 1 1 1 jacccgfi 2 2 1 jdacgfi End 2 1 de Figure 6 12 Run File for adding amino acids to peptide resin without Fmoc group Follow the procedure Start the Automated Svnthesis on Synergy that begins on page 2 14 Start the svnthesis in the Run Control Menu as vou would anv other svnthesis IMPORTANT Edited Run lines remain in the Run File until you return to the Run Editor and delete them 6 14 Advanced Operations 03 2002 Applied Biosystems How to add more amino acids to a dry peptide resin WITH an Fmoc group 1 Place the PSC that contains the peptide resin in the PSC position on Synergy 2 Place the appropriate AACs on the amino acid column wheel As with a routine svnthesis remember to put the AAC nearest the C terminal end in position 1 Linett Cycle Repeat Module List Beg 0 1 bf 1 1 1 jdacgfi End 1 1 de Figure 6 13 Run File for adding amino acids to peptide resin with an Fmoc group 3 Follow the procedure Start the Automated Synthesis on Synergy that begins on page
124. These columns are designed to be used one time only once a column has been opened its leakproof seal is broken Treat these used columns as a solid hazardous waste and dispose of them with other disposable equipment that has been in contact with chemicals Caution Re using PSCs or AACs can cause reagent leakage on the conductivity cell or the autosampler jaw seals and damage to Synergy Discard these columns after one use 2 12 Routine Operation 03 2002 Applied Biosystems Unused AACs and PSCs can be safely stored at room temperature for up to one year after the date of shipment WARNING SOLID HAZARDOUS WASTE Used AACs and PSCs have been exposed to DMF N N dimethyl formamide THF tetrehydrofuran and other hazardous chemicals Dispose of used columns in accordance with local state and federal regulations regarding solid hazardous waste See the Synergy Waste Profile in Chapter 1 of this User s Manual Table 2 3 Amino acid abbreviations AminoAcid 3 letter code 1 letter code alanine Ala A arginine Arg R asparagin Asn N aspartic acid Asp D cysteine Cys C glutamine Gin Q glutamic acid Glu E glycine Gly G histidine His H isoleucine lle leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr y valine Val V Check the Run File The three pre programmed lines in combination can perform most routine synthe ses Chapter 6
125. U reagent bottle position A can be used for six weeks e Verify that all the reagent bottles have been placed in the correct bottle positions 03 2002 Troubleshooting 7 5 Applied Biosystems O00 es om nn 7 I HBTU Piperidine Tetrahydrofuran Stabilized DIEA DMSO NMP ed Figure 7 4 Reagent bottle positions on the front of Synergy When all the reagent bottles are in place from left to right facing the front of the synthesizer the labeled reagent bottles should be HBTU 2 1H benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate in position A DIEA DMSO NMP 0 4M N N diisopropylethylamine dimethyl sulfoxide N methylpyrrolidone in position B Piperidine in position 1 and THF Tetrahydrofuran in position 2 DIEA Piperidine and THF Figure 7 4 The DMF N N dimethylformamide bottle sits on the right side of Synergy in a Safety carrier If you discover that one or more bottles are not in the correct position 1 Follow the first 6 steps of the Synergy Shutdown Procedure on page 2 30 but only for those bottle positions that are in the wrong bottle positions If the THF bottle was mistakenly placed in the piperidine bottle position and its contents contaminated with Piperidine discard the THF After you complete step 6 of the Synergy Shutdown Procedure replace the reagents in their correct positions Prime the reagent lines for the re position
126. UNCT NAME T xxx zzz HOLD PAUSE NEXT S jump S more Run Monitor v S 02 16 C 05 05 M jdacgfi LOCK set int end run more v Are you sure you want to end the run END RUN Menu cancel END RUN Run ended 14 15 Run began 17 50 End of run report Cycles run 3 OK 1 Inthe Run Monitor press the more soft key until the end run soft key appears 2 Press the end run soft key When the message Are you sure you want to end the run appears press the END RUN soft key The LCD displays the time the run began and ended 3 Ifthe jaws are closed when the synthesis ends go to the Manual Control Menu and activate Function 53 OPEN JAWS See How to manually activate a function on page 7 14 03 2002 Troubleshooting 7 11 Applied Biosystems Pressure System Leaks Synergy cannot operate properly with a leak in the pressure system It tests the pres sure system during module b and module j in a synthesis during the bottle change procedure the reagent line prime routines and some of the Self Tests What to do after a pressure test failure Pressure test failed initial 5 3 final 2 1 diff 3 2 OK If a leak is detected the LCD displays a message similar to the one shown here The value after the word diff is the difference between the initial pressure reading and the final reading Functions 88 and 89 direct the controller to take these two pressure readings 1 Press the O
127. UPATIONAL SAFETY AND HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION NIOSH MSHA TETRAHYDROFURAN 1000 PPM ANY POWERED AIR PURIFYING RESPIRATOR WITH ORGANIC VAPOR CARTRIDGE S ANY CHEMICAL CARTRIDGE RESPIRATOR WITH A FULL FACEPIECE AND ORGANIC VAPOR CARTRIDGE S 5000 PPM ANY SUPPLIED AIR RESPIRATOR OPERATED IN A CONTINUOUS FLOW MODE 10 000 PPM ANY AIR PURIFYING FULL FACEPIECE RESPIRATOR GAS MASK WITH A CHIN STVLE OR FRONT OR BACK MOUNTED ORGANIC VAPOR CANISTER ANY SELF CONTAINED BREATHING APPARATUS WITH A FULL FACEPIECE ANY SUPPLIED AIR RESPIRATOR WITH A FULL FACEPIECE 20 000 PPM ANY SUPPLIED AIR RESPIRATOR WITH A FULL FACEPIECE AND OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ESCAPE ANY AIR PURIFYING FULL FACEPIECE RESPIRATOR GAS MASK WITH A CHIN STYLE OR FRONT OR BACK MOUNTED ORGANIC VAPOR CANISTER ANY APPROPRIATE ESCAPE TYPE SELF CONTAINED BREATHING APPARATUS 46 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH CONDITIONS ANY SELF CONTAINED BREATHING APPARATUS THAT HAS A FULL FACEPIECE AND IS OPERATED IN A PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE ANY SUPPLIED AIR RESPIRATOR THAT HAS A FULL FACEPIECE AND IS OPERATED INA PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY SELF CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE CLOT
128. XHIBITED IRRITATION OF THE UPPER RESPIRATORY TRACT LIVER AND KIDNEY DAMAGE WAS OBSERVED BUT MAY HAVE BEEN DUE TO IMPURITIES OTHER REPORTED EFFECTS FROM REPEATED EXPOSURE INCLUDE DISCHARGE OR BLEEDING IN THE NASAL MUCOSA A CATALEPTOID POSTURE COMA AND CLONIC MUSCLE SPASMS LEVELS OF 5000 PPM FOR 91 DAYS PRODUCED ACANTHOSIS AND SUPPURATIVE INFLAMMATION OF THE FORESTOMACH MINIMAL CENTRILOBULAR HYPERTROPHY OF THE LIVER AND ATROPHY OF THE UTERUS AND DEGENERATION OF THE ADRENAL CORTEX FIRST AID REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY IF BREATHING HAS STOPPED PERFORM ARTIFICIAL RESPIRATION KEEP PERSON WARM AND AT REST TREAT SYMPTOMATICALLY AND SUPPORTIVELY GET MEDICAL ATTENTION IMMEDIATELY SKIN CONTACT TETRAHYDROFURAN ACUTE EXPOSURE LIQUID APPLIED TO THE SKIN OF 196 PERSONS WAS ESSENTIALLY NONIRRITATING HOWEVER AQUEOUS SOLUTIONS IN CONCENTRATIONS ABOVE 20 HAVE BEEN REPORTED TO CAUSE IRRITATION WHEN APPLIED TO RABBIT SKIN SKIN ABSORPTION MAY OCCUR CHRONIC EXPOSURE REPEATED OR PROLONGED EXPOSURE MAY CAUSE IRRITATION DEHYDRATION AND DEFATTING AND SUBSEQUENT DERMATITIS 43 ABI PART NUMBER 902034 OHS PART NUMBER ABI23010 Rev B FIRST AID REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY WASH AFFECTED AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO EVIDENCE OF CHEMICAL REMAINS APPROXIMATELY 15 20 MINUTES GET MEDICAL ATTENTION IMMEDIATELY EYE CONTACT TETRAHYDROFURAN IRRITANT
129. Y RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION CAS NUMBER 110 89 4 RTECS NUMBER TM3500000 SUBSTANCE PIPERIDINE TRADE NAMES SYNONYMS ABI MSDS PART 901837 P N 400629 HEXAZANE CYCLOPENTIMINE CYPENTIL AZACYCLOHEXANE PERHYDROPYRIDINE PENTAMETHYLENEIMINE HEXAHYDROPYRIDINE PEPTIDE SYNTHESIS REAGENT f 1 UN 2401 C5H11N CHEMICAL FAMILY PIPERIDINE MOLECULAR FORMULA C H2 5 N H MOL WT 85 15 CERCLA RATINGS SCALE 0 3 HEALTH 3 FIRE 3 REACTIVITY 3 PERSISTENCE 0 NFPA RATINGS SCALE 0 4 HEALTH 2 FIRE 3 REACTIVITY 3 COMPONENTS AND CONTAMINANTS COMPONENT PIPERIDINE PERCENT 100 CAS 110 89 4 OTHER CONTAMINANTS NONE EXPOSURE LIMITS NO OCCUPATIONAL EXPOSURE LIMITS ESTABLISHED BY OSHA ACGIH OR NIOSH PIPERIDINE 1 PPM AIHA RECOMMENDED TWA 1000 POUNDS SARA SECTION 302 THRESHOLD PLANNING QUANTITY 1 POUND SARA SECTION 304 REPORTABLE QUANTITY PHYSICAL DATA DESCRIPTION CLEAR COLORLESS LIQUID WITH A PEPPER LIKE OR FISHY ODOR AND A SOAPY FEEL BOILING POINT 223 F 106 C MELTING POINT 16 TO 19 F 9 TO 7 C SPECIFIC GRAVITY 0 862 VOLATILITY 100 VAPOR PRESSURE 40 MMHG 6 29 C PH STRONG BASE SOLUBILITY IN WATER 100 VAPOR DENSITY 3 0 SOLVENT SOLUBILITY SOLUBLE IN ACETONE ALCOHOL BENZENE CHLOROFORM AND 35 ABI PART NUMBER 901837 OHS PART NUMBER ABI18940 Rev B ETHER PKA
130. Y USE COPYRIGHT 1992 OCCUPATIONAL HEALTH SERVICES INC ALL RIGHTS RESERVED CREATION DATE 07 31 91 REVISION DATE 05 21 92 40 AR Applied ABI PART NUMBER 902034 Ad Biosystems OHS PART R R ev MATERIAL SAFETY DATA SHEET APPLIED BIOSYSTEMS 24 HOUR EMERGENCY RESPONSE 850 LINCOLN CENTRE DRIVE NUMBER 615 366 2000 FOSTER CITY CA 94404 415 570 6667 USA 0925 825650 UK SUBSTANCE IDENTIFICATION CAS NUMBER 109 99 9 RTECS NUMBER LU5950000 SUBSTANCE TETRAHYDROFURAN STABILIZED TRADE NAMES SYNONYMS ABI MSDS PART 902034 P N 401255 CYCLOTETRAMETHYLENE OXIDE FURANIDINE OXACYCLOPENTANE OXOLANE TETRAMETHYLENE OXIDE THF RCRA U213 TETRAHYDROFURAN FURAN TETRAHYDRO TETRAHYDROFURAN BHT STABILIZED BUTANE ALPHA DELTA OXIDE STCC 4908290 UN 2056 C4H80 CHEMICAL FAMILY FURAN DERIVATIVE MOLECULAR FORMULA C4 H8 0 MOLECULAR WEIGHT 72 10 CERCLA RATINGS SCALE 0 3 HEALTH 3 FIRE 3 REACTIVITY 1 PERSISTENCE 1 NFPA RATINGS SCALE 0 4 HEALTH 2 FIRE 3 REACTIVITY 1 CERCLA RATINGS SCALE 0 3 HEALTH 3FIRE 3REACTIVITY 1PERSISTENCE 1 NFPA RATINGS SCALE 0 4 HEALTH 2FIRE 3REACTIVITY 1 COMPONENTS AND CONTAMINANTS COMPONENT TETRAHYDROFURAN PERCENT gt 99 CASH 109 99 9 OTHER CONTAMINANTS MAY CONTAIN TRACES OF A PEROXIDATION INHIBITOR EXPOSURE LIMITS TETRAHXDROFURAN 200 PPM 590 MG M3 OSHA TWA 250 PPM 737 MG M3 OSHA STEL 200 PPM 590 MG M3 ACGIH TWA 250 PPM 737 MG M3 ACGIH STEL 20
131. ZARD Pressurized gas cylinders are explosive Attach pressurized gas cylinders firmly to a wall or bench by means of approved brackets chains or clamps Always cap the gas cylinder when not in use How to change the external gas tank 1 Switch the Pressure Vent switch on Synergy to Vent Figure 2 1 and wait one minute for the system to depressurize 2 Close both the main supply valve on top of the gas tank and the needle valve if one exists on the tank regulator 3 Remove the regulator from the empty tank and install it on a full replacement tank Use only pre purified compressed nitrogen gas on Synergy You do not need to disconnect either end of the tubing that connects the regulator to Synergy 4 Open the main tank supply valve Squirt soapy water around the connection between the regulator and the gas tank to check for leaks 5 Verify that the tank regulator setting returns to its original position 6 If the regulator has a needle valve open it completely 7 Switch the Pressure Vent switch to Pressure 2 Check levels of 5 reagent bottles WARNING POTENTIAL CHEMICAL HAZARD Regard all chemicals on the synthesizer including liquid in the lines as potentially hazardous Wear protective eyeglasses gloves and a labora tory coat when working with the chemicals used on the instrument If a spill occurs clean it up in accordance with instructions in the MSDSs or Waste Profile found in the Pre Installation Manual and in t
132. a conductivity trace or if the seven features do not look the same as those in Figure 7 1 it could indicate one of the following 1 A sequence dependent slow deprotection or incomplete coupling 2 One or more empty reagent bottles contaminated or outdated reagents incorrectly positioned bottles or a re used AAC 3 Malfunctioning instrument hardware 4 Malfunctioning printer 03 2002 Troubleshooting 7 3 TOOT EO 8unooysa1qnoL PL SIN Figure 7 2 Conductivity trace of a synthesis scaled to fit page with slow deprotection and incomplete coupling The conductivity trace in Figure 7 2 was generated from the synthesis of Ile Ile Ile Ile Ile Ile The first three deprotections Trace Feature 1 produced three sharp peaks A B C The fourth deprotection peak D is slight Iv broader and shorter and the fifth peak E is considerably broader and shorter Peaks D and E indicate slower diffusion rates in the peptide resin see page 5 5 for an explanation of diffusion rates and the corresponding coupling times Trace Features 5 and 6 are automatically increased to compensate F However the sluggish washout of reagents Trace Feature 7 at G indicates that coupling was incomplete At G the conductivitv did not return to the baseline in the maximum allotted time so the following deprotection H was abnormally high This particular coupling may have been improved by double coupling See page 6 10 for a description
133. agent bottles on the front of the instrument and replace them with full bottles of fresh reagents See Bottle Changing Procedure on page 4 9 Replace any cracked or damaged bottle seals Run the Bottle Prime Priming Reagent Delivery Lines on page 4 11 for all 5 reagent bottles If a pressure test failure occurs during a bottle prime check the bottle seal and cap for that bottle 2 34 Routine Operation 03 2002 Applied Biosystems 9 Use a marked flow test calibration tube to perform Flow Test 3 Flow Tests 3 4 and 5 on page 4 22 If Flow Test 3 passes go to step 10 If you do not have a marked flow test calibration tube perform Flow Tests 1 and 2 then perform Flow Test 3 If Flow Test 3 does not pass perform Flow Test 1 page 4 15 If Flow Test 1 does not pass check the internal gas pressure How to monitor the internal gas pressure on page 4 23 and if necessary adjust the internal pressure to 5 0 0 1 psi Repeat Flow Test 1 and then perform Flow Tests 2 through 9 If Flow Test 1 continues to fail call Applied Biosystems Technical Support 10 If you have not performed Flow Test 4 do so now See Flow Tests 3 4 and 5 on page 4 22 If Flow Test 4 does not pass call Applied Biosystems Technical Support 11 1f both Flow Tests 3 and 4 pass and you have not already done so perform Flow Test 8 page 4 27 and Flow Test 9 page 4 30 When Flow Tests 8 and 9 pass Synergy is ready to synthesize
134. al pressure the follow ing message appears on the LCD Pressure reading out of range OK During a synthesis this message could appear only in module b the beginning of synthesis when the PSC is tested for leaks or in module j the beginning of the pre programmed cycles when the AAC is tested for leaks If low pressure due to an empty gas tank is detected during module b you may change the gas tank as soon as Synergy interrupts synthesis or when the PAUSE soft key appears in the Run Monitor See How to change the external gas tank on page 2 6 for a procedure When the tank has been replaced press the PAUSE soft key to resume synthesis If low pressure due to an empty gas tank is detected during module j it may be dif ficult to determine if the synthesis has been affected Inadequate delivery of reagents may have already compromised activation or coupling in the previous cycle You 7 10 Troubleshooting 03 2002 Applied Biosystems may be able to detect irregularities in the conductivity trace It might be a waste of reagents amino acid columns and time to continue the synthesis If you have a conductivity trace of the svnthesis before the low pressure was detect ed and you are not sure whether to continue the synthesis call Applied Biosystems Technical Support How to terminate a synthesis Follow this procedure to prematurely end the synthesis S xx yy zz F Fit F
135. amount of waste generated is likely to exceed the space available in the waste bottle replace the waste bottle with an empty bottle Synergy generates hazardous organic waste Waste must be handled stored and dis posed of in accordance with federal state provincial and local hazardous waste reg ulations WARNING HAZARDOUS WASTE Waste produced by Synergy can be hazardous and can cause injury illness or death Handle all liquid solid and gaseous waste as potentially hazardous During transfer the waste container must be tightly sealed with the waste cap provided Read all applicable Material Data Safety Sheets and the Synergy Waste Profile Always wear gloves and handle hazardous waste in a fume hood that is connected in accordance with all installation requirements How to replace the full waste bottle Note Use this procedure when the instrument is not running a synthesis During a synthesis see How to remove and replace the full waste bottle during synthesis on page 2 23 1 Unscrew the waste cap assembly on the waste bottle You do not have to toggle the Pressure Vent switch 2 Tightly seal the waste bottle with the waste cap provided for transportation Remove the waste bottle from the safety carrier and safely dispose of its contents 3 Place an empty waste bottle in the safety carrier and screw the waste cap assembly onto the waste bottle 4 Check the printer power paper supply and cable connections Wh
136. ansfer vessel Screw the left vessel tightly in place 3 In the Run Monitor press the jump S soft key 4 Inthe Jump Step Menu enter 9 after S to jump to step 9 in module h This jump bypasses the steps that direct activator deliveries Caution If you do not perform steps 4 5 and 6 of this procedure activators may react with the peptide resin and partially cap the peptide essentially terminating the synthesis 6 18 Advanced Operations 03 2002 Applied Biosystems Jump from interrupt in module h to Step 9 Module h alternate activation Step Function f Function Name Time Action Description 1 8 P F TIME 2 2 2 INTERRUPT 1 3 10 GAS gt TVB 5 Measure activators 4 45 HBTU gt L 15 5 14 GAS gt L 5 6 46 DIEA gt L 10 7 14 GAS gt L 5 8 1 WAIT 60 9 30 DMF gt TVB 10 31 DMF gt BVB 11 35 DMF gt L 1 Begin transfer to PSC 12 9 P F PAUSE 1 13 P F TIME 60 14 67 L gt PSC gt WASTE 15 Substitute time Figure 6 17 Jumping over activator delivery steps in module h 5 Press the JUMP soft key to jump to the step 9 and return to the Run Monitor 6 Press the PAUSE soft key to continue synthesis 03 2002 Advanced Operations 6 19 Applied Biosystems The Module Editor Menu A module contains the steps needed to perform a particular process within a synthe sis All the pre programmed synthesis modules have lower case letter names such as a b c etc See Annotated Module Print
137. appear on the LCD as a result of either a mechanical failure or soft ware errors or after an operator error Mechanical Failure or Software Error Mechanical failures can occur during a synthesis or during some of the Self Tests Error messages that describe mechanical failures fall into one of these categories e Failure of the autosampler jaws or jaw sensors e Motor assembly or encoder failure e Powerfailure Table 7 4 Error Messages for Mechanical Failures Mechanical Failure Error Message Autosampler jaws or sensors Open jaw operation failed Close jar operation failed Jaw sensors failed or unplugged Home sensor failed Motor assembly or encoder Motor assembly failed Motor encoder failed Wheel failed to reach desired location The wheel belt is too loose Powerfailure There was a power failure while the synthesizer was running The power was off too long to resume the run A run control error occured before the power fail The run is paused The run cannot resume because the run file was modified The run cannot resume because the module executing was modified The run cannot resume because the function file was modified Autosampler jaw failure If any of the autosampler error messages listed in Table 7 4 appear on the LCD first check the gas tank and lower gas regulator gauge If one of these messages appear even with sufficient gas pressure call Applied Biosystems Technical Support Motor ass
138. apter 5 for a detailed explanation of automated peptide svnthesis on Svnergv Table 2 1 Pre programmed Synthesis Lines on Synergy Line Name Modules Action BEG bf initial resin solvation L cycle line 1 jdacgfi deprotection and coupling END de Fmoc deprotection and dry peptide resin This chapter describes the three stages of a peptide synthesis with these pre pro grammed module lines on Synergy 1 Preparing Synergy for synthesis page 2 5 2 Selecting and loading the columns for your synthesis page 2 10 3 Starting automated synthesis page 2 14 Prepare Synergy for Synthesis Synergy requires four visual checks to confirm the instrument is ready for operation 1 Check the external gas supply 2 Check levels of 5 reagent bottles 3 Check the waste bottle and empty it if necessary 4 Check the printer power connections and paper supply 1 Check the external gas supply Regulated pressure 60 70 psi Gas tank pressure gt 300 psi Pressurized gas controls reagent flow and the action of the autosampler jaws that close on the AACs during synthesis Without an adequate gas supply Synergy ceas es operation see Empty Gas Tank on page 7 10 To maintain adequate gas pres sure we recommend the nitrogen tank pressure high pressure gauge should not drop below 300 psi and the low pressure gauge should be set at 60 75 psi 03 2002 Routine Operation 2 5 Applied Biosystems WARNING GAS TANK EXPLOSION HA
139. as no effect on the valves Toggle functions come in pairs and turn a set of events or valves off and on Pairs of toggle functions include the following Function 86 PRS PIP and Function 87 PRS PIP turn on and off the valves that control pressure on the piperidine bottle Function 64 PSC PATH and Function 65 PSC PATH open and close valves to direct reagents through the peptide synthesis column and Function 3 BEGIN LOOP and Function 4 END LOOP repeat a series of functions until a pre deter mined condition is satisfied The following pages describe each module in the pre programmed synthesis cycles with a modified plumbing schematic that shows reagent flow for some of the func tions in these modules 5 8 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Table 5 2 Pre programmed Synthesis Lines on Synergy Line Name Modules BEG bf L cvcle line jdacgfi END de Xou mav print the steps of anv module from the Module Editor Menu See Anno tated Module Printouts on page 5 18 for step bv step descriptions of the svnthesis procedures in each module Table 5 3 summarizes the svnthesis procedures that oc cur during each module How to print modules set up amp run edit amp manual self Main Menu report control print control test run module function Press l Menu Edit amp Print Menu editor editor editor kev to return to Mai
140. ase peptide synthesis SPPS A unique system of gas pressurized valves reagent reservoirs and transfer vessels directs reagent flow through three stages of synthesis Deprotection of N terminal amino acid of peptide bound to resin support in the peptide synthesis column e Activation of amino acid in column on wheel Transfer of activated amino acid to the peptide synthesis column where coupling with N terminal amino acid occurs The Synergy software monitors the conductivity of the solution passing through the peptide synthesis column PSC and determines the reaction times of both deprotec tion and coupling With the printing option users may observe a real time graphic representation of each deprotection coupling cycle as synthesis progresses 03 2002 Routine Operation 2 3 Applied Biosystems Contrast adjustment Menu key Soft key The LCD and Keyboard With the liquid crystal display LCD and keyboard you can monitor each step of instrument operation and select software menus to perform instrument system tests or modify pre programmed lines GJEN e EY JET NE EE 1 Figure 2 2 The Synergy LCD and keyboard The LCD on the front of Synergy displays messages on two lines of 40 characters each Usually the top line displays information or directions The bottom line as signs names to the soft keys located directly under the LCD A cursor represented by a blinking horizontal line on the
141. at Press the ADD L soft key A new cycle line appears with L 2 gt C 5 in the top line and the cursor after the word Repeat the second LCD in Figure 6 5 Keep the default value 1 one after the word Repeat to indicate that this new line applies to only one cycle of deprotection and coupling Then move the cursor to the space after the M module Use the alpha numeric keys to enter jdaccgfi Line 2 now has two coupling modules in a row Since each c module is twice as long as the deprotection module that precedes it 2 c s extends coupling time to four times the deprotection time in that line The Synergy controller always repeats the last cycle line in any Run File until the jaw sensors detect an empty position on the amino acid column wheel If you do not want standard cycle lines to follow the extended coupling line go to step 8 of this procedure If you want standard cycle lines to follow the extended coupling line you must add a third line in the Run Editor Go on to step 6 Press the ADD L soft key to add a third cycle line A new line appears with L 3 gt C 6 on the top line and the cursor after the word Repeat Move the cursor to the space after the M and enter j d a c g f i If this is the last line cycle in the run you do not have to enter a number after Repeat By default the number 1 one appears after Repeat The proces
142. ate to 1 mg Set the scale to zero Replace the vessel on Synergy and press the OK soft key Remove the left vessel and weigh _mg OK Note This message appears 3 times during Flow Test 1 When it appears the left vessel contains DMF 4 16 Maintenance and Self Tests 03 2002 Applied Biosystems Carefully remove and weigh the left vessel and enter the weight on the LCD Do not discard the DMF Press the OK soft key Replace the vessel OK Replace the left vessel and press the OK soft key 6 When prompted repeat steps 4 and 5 two more times The values for the first two weights should be similar 460 740 mg because they both measure DMF deliveries through the valve blocks The third value results from DMF delivery through the PSC peptide synthesis column and should be 500 600 mg Test Passed OK When this display appears the values are within specifications Press the OK soft key to complete the flow test If any of the DMF delivery weights is out of range the LCD displays the message Value is out of range OK When this display appears press the OK soft key The flow test continues to ask for weights until all three deliveries have been weighed At the end of the test the LCD displays the message Test failed If Flow Test 1 fails repeat the test one more time Always re tare the left transfer vessel when prompted Weigh the DMF immedi
143. ately after each delivery to minimize weight loss due to evaporation or spillage During the repeat write down the value of the third DMF delivery weight If the repeat of Flow Test 1 fails adjust the internal gas pressure to calibrate the third DMF delivery 03 2002 Maintenance and Self Tests 4 17 Applied Biosystems If the flow test continues to fail e Check that the DMF reagent bottle is at least half full Occasionally a low volume of DMF in the reagent bottle may cause Flow Test 1 failure e Perform a Leak Test page 4 13 to check for internal system leaks e Adjust the internal pressure to calibrate the third DMF delivery Although the value of the third DMF delivery weight is acceptable in the range of 550 30 mg it is best to set the delivery as close to 550 as possible typically 550 10 mg The acceptable internal gas pressure range is 3 5 5 5 psi If the third DMF delivery weight was less than 520 mg increase the internal gas pressure If the third DMF delivery weight was greater than 580 mg decrease the internal pressure Adjusting the internal gas pressure to calibrate DMF delivery 1 Press the START soft key to begin Flow Test 1 Immediately press the PAUSE soft key to temporarily interrupt the flow test When you press the PAUSE soft key the current step number should be either 02 or 03 An asterisk appears PAUSE to indicate the flow test has been temporarily interrupted S 02 48 00 F
144. ceding cleavage reaction procedure WARNING CHEMICAL AND FIRE HAZARD MTBE is volatile and highly flammable Use only a spark free centrifuge Avoid prolonged centrifugation of MTBE Perform this procedure in a working fume hood 03 2002 Post Synthesis Procedures 3 5 Applied Biosystems Required Reagents MTBE glacial acetic NH4OH or acetonitrile distilled water Equipment and Labware disposable Pasteur type pipettes and a rubber bulb cotton balls or glass wool microliter pipetters 200 uL and 1000 uL 20 uL and 200 uL clinical centrifuge 1500 g 2000 g vortex mixer 1 Prepare a filter for step 6 of this procedure by pushing a small piece of cotton or glass wool into a disposable Pasteur type pipette as shown in Figure 3 2 es cotton B Figure 3 2 Prepare a filter from a disposable pipette 2 Centrifuge the tightly capped tube from the cleavage reaction for 2 3 minutes The peptide and spent resin should form a pellet in the bottom of the tube See Figure 3 3 MTBE scavengers and organic impurities Crude peptide Spent resin Figure 3 3 Peptide resin after MBTE wash and centrifugation 3 6 Post Synthesis Procedures 03 2002 Applied Biosystems 3 Aspirate or decant and discard the MTBE 4 Add 15 mL 2 mL MTBE to the tube tightly cap and vigorously vortex it to re suspend the peptide Repeat steps 2 4 at least three times 5 After the final MTBE wash decant or as
145. ctions 03 2002 Routine Operation 2 23 Applied Biosystems 5 Press the MONITOR DATA soft key to resume printing The printer will now start printing data from the beginning of the current synthesis It takes from 7 15 minutes per cycle to reprint data depending on the chart speed set in the Chart Recorder Menu See How to change the chart speed on page 4 5 Note Press the MONITOR DATA soft key to print the conductivity data for the most recent run if Synergy is not currently synthesizing a peptide Figure 2 11 set up amp run edit amp manual self Main Menu report control print control test pressure time amp print serial Budde Set up amp Report Menu L Menu settings date reports number more kev CANCEL MONITOR USER SYSTEM Print Reports Menu H PRINT DATA DATA DATA Figure 2 11 How to go to the Print Reports Menu 2 24 Routine Operation 03 2002 Applied Biosystems Powerfailures Whenever a powerfailure occurs the autosampler jaws open automatically When power returns the wheel automatically moves to home position so that position 1 on the wheel is between the jaws If you were running a synthesis before the powerfail ure when power returns the jaws close on the column in position I to check the wheel alignment If the wheel is aligned correctly the jaws open and the wheel re turns to the position it was in before the powerfailure occurred N
146. ctivity trace AC power input module s bottle seal 4 10 adjust printout 4 4 to 4 6 access 1 5 ti Bottle Self Test por enue 21 acetic acid glacial 3 6 3 7 Ne 7 34 irregularities 7 3 to 7 9 activation 2 3 5 4 5 12 peak height adjustment 4 4 alternate 22557 C contrast adjustment 2 4 6 16 to 6 19 calibrate copy activators 5 12 DIEA 4 30 module 6 20 Acyl Carrier Protein 65 74 2 11 HBTU 4 27 coupling 2 3 5 4 5 14 adjust CANCEL PRINT soft key 2 23 double 6 10 to 6 12 LCD contrast 24 canopv fume hood l 10 extended 6 6 to 6 9 peak heights 4 4 cap incomplete 6 6 7 4 printout 4 4 to 4 6 DMF bottle 7 16 C terminal amide 2 11 Ala 2 13 ratchet 4 10 7 16 C terminal amino acid 2 10 alphanumeric key 2 4 capped peptide 7 7 C terminus 5 3 amide l carboxyl group 5 4 cursor 2 4 a 2 10 Carpino L A 5 7 cycle 2 16 6 3 ST er l Caution 1 3 cycle line 2 5 6 3 derivatives 5 4 i fs 6 16 centrifugation recovery 3 3 cycle number 4 6 non standar ae 3 5 to 3 7 Cycle Test 4 32 to 4 33 Table of Abbreviations 2 13 4 Chart Recorder Menu 4 4 Cys 2 13 amino acid column wheel 2 3 i chart speed 4 5 annotated module printouts checklist D 5 18 to 5 22 start synthesis 2 14 data storage 7 24 Arg 2 13 2 25 Circuit Self Test 4 36 7 9 Delete key 2 4 Page NERI 3 5 cleavage 5 6 deletion peptide 6 7 arginine ee 2 25 sachon 3 4 to 3 5 delivery line filter 2 8 EE TFA procedure 3 3 to 3 8 Delivery port 2 30 arrow key 2 4 solaris 03 2002 Index 1
147. d column wheel and repeat Flow Test 6 If the pressure test in Flow Test 6 fails repeatedly call Applied Biosystems Technical Support for assistance How to jump over steps in a module 1 With the PAUSE soft key active in the Run Monitor press the jump S soft key to go to the Jump Step Menu The top line of the LCD in the Jump Step Menu displays the letter name of the module the Step number in the module the Function number and name at that step and the Time in seconds allotted to the function The cursor highlights the Step number Figure 7 9 7 18 Troubleshooting 03 2002 Applied Biosystems Run Monitor Jump Step Menu Run Monitor S 06 28 00 F 89 PRS TEST T 003 010 HOLD PAUSE NEXT S jump S more MODULE p gt S 6 F 89 PRS TEST T 10 Select step module JUMP MODULE p gt S 26 F 53 OPEN JAWS T 5 module JUMP v S 26 26 00 F 53 OPEN JAWS T 001 005 HOLD PAUSE NEXT jump S more Figure 7 9 How to Jump Steps in a Module 2 Use the numeric keys to enter the number of the step the controller should jump to In Flow Test 6 Pump AAC module p jump to Step 26 to open the jaws after a pressure test failure 3 Press the JUMP soft key to return to the Run Monitor 4 Press the PAUSE soft key to continue Flow Test 6 at Step 26 03 2002 Troubleshooting Applied Biosystems Error Messages Error messages
148. d gas cylinder is not properly secured it could fall over and explode which could cause physical injuries WARNING GAS TANK EXPLOSION HAZARD Pressurized gas cylinders are explosive Attach pressurized gas cylinders to a wall or bench by means of approved brackets or chains Always cap the gas cylinder when not in use External Pneumatic Supply Synergy requires 60 75 psi nitrogen input for operation 03 2002 Synergy Safety Guidelines 1 7 Applied Biosystems Laboratory Ventilation Requirements The information presented here reflects U S regulations and practices for venting all Applied Biosystems Inc instruments to a fume hood or to a duct WARNING POTENTIAL FOR EXPOSURE TO HAZARDOUS WASTES Do not operate a vented instrument unless it is connected in accordance with all the ventilation requirements presented here The wastes produced by certain chemicals in Applied Biosystems instruments are hazardous and can cause injury illness or death Always wear protective clothing when handling hazardous waste Mix and prepare hazardous materials beneath a fume hood Read the Waste Profile in Appendix 2 Fume Hood Always mix and prepare hazardous materials beneath a fume hood The vent line to the fume hood should be no longer than 15 feet 5 meters Be certain that the line is not punctured or otherwise damaged Following are important points about the fume hood It should operate at all times including nights and wee
149. da TION RR 5 5 ku i enz 2 5 6 3 glacial acetic 3 6 3 7 key End Run Menu 2 28 OG 2 13 alphanumeric 2 4 END RUN soft key 2 27 7 11 O 2 13 arrow 2 4 end synthesis 5 16 7 11 oly 2 13 Delete 2 4 error messages 2 18 Grant G A 5 23 Menu 2 4 mechanical failure 7 20 H Next 2 4 operator generated 7 22 Han G Y 5 7 Previous 2 4 exception message 7 21 HBTU 2 3 5 7 keyboard 2 4 extended coupling 6 6 to 6 9 AE 4 27 7 25 Self Test 4 34 external gas Tank 2 deliverv line filter 2 8 PR pA emergency replacement 2 23 preparation 27 L extraction 5 12 Readers 4 6 LAGV 4 32 2 Index 03 2002 Applied Biosystems LCD 2 4 Run Control 2 14 2 15 2 29 non standard amino acids 6 16 contrast 2 4 Run Editor 6 3 to 6 19 norleucine 6 16 Leak Self Test 4 13 Self Test 4 8 Note 1 3 leak soft key 4 13 Set Interrupt 2 20 leak test Set up amp Report 4 24 O external gas 2 34 Menu key 2 4 OPEN JAWS 7 18 external gas delivery 2 34 Merrifield R B 5 3 OPEN JAWS function 2 28 Leu 2 13 Met 2 13 operator interruptions 2 19 line 6 3 methyl r butyl ether see MTBE ornithine 6 16 begin 2 5 millivolt 4 5 P cycle 2 5 module 5 8 to 5 10 6 20 nd 2 5 5 12 P F PAUSE function 2 25 2 26 pre programmed 2 5 5 9 powerfail recovery 2 26 PAUSE soft key 2 4 liquid crystal display 2 4 b 5 13 PA USE soft key 2 18 liquid crystal display see LCD c 5 14 peptide l Live D H 55 aay 6 20 deletion 6 7 load synthesis columns d 5 15 recovery after cleavage 3 3 2 10 to
150. displays an error message Contents Irregular Conductivity Traces 7 3 Sequence dependent slow deprotection 7 5 Reagent bottles and reagent delivery 7 5 If you discover that one or more bottles are not in the correct position 7 6 Re used AACs 7 7 Malfunctioning instrument hardware 7 8 Printer related malfunctions 7 9 How to reprint the conductivity trace 7 9 Pressure System Leaks 7 12 Empty Gas Tank 7 10 How to terminate a synthesis 7 11 Pressure System Leaks 7 12 What to do after a pressure test failure 7 12 Pressure Test Failures during a synthesis 7 13 How to manually activate a function 7 14 Pressure Test Failures during a Self Test 7 15 Pressure Test Failure during a Leak Test 7 16 How to jump over steps in a module 7 18 Error Messages 7 20 Mechanical Failure or Software Error 7 20 Autosampler jaw failure 7 20 Motor assembly or encoder 7 20 Powerfailure 7 21 Exception Messages 7 21 Operator generated Error Messages 7 22 How to discontinue data printing 7 24 Incorrect use of Manual Control or incorrect module edit 7 25 03 2002 Troubleshooting 7 1 Applied Biosystems Irregular Conductivity Traces initial resin solvation _ ER Begin The conductivity trace shown in Figure 7 1 has seven characteristic Trace Features that you should see in every synthesis conductivity trace generated by Synergy See page 2 16 for a description of these seven Trace Features If all seven features do not appear on
151. ductivity Monitoring 5 5 Post synthesis Cleavage and Side Chain Deprotection 5 6 The Synergy Synthesis Process 5 7 Synthesis Columns and Reagents 5 7 Modules and Functions 5 8 How to print modules 5 9 The Synergy Pump 5 10 Illustrated Module Descriptions 5 11 Module a extraction and activation 5 12 Module b begin synthesis 5 13 Module c coupling 5 14 Module d deprotection 5 15 Module e end synthesis 5 16 Module f DMF flow to PSC 5 16 Module g wash solvents through AAC and transfer vessels 5 17 Module h alternate activation 5 17 Module i incremental movement of amino acid column wheel 5 17 Module j jaws close on AAC 5 17 Annotated Module Printouts 5 18 Recommended reading 5 23 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 1 Applied Biosystems Solid Phase Peptide Synthesis Solid phase peptide synthesis SPPS originated by Merrifield gt involves the suc cessive addition of amino acids to create a linear peptide chain The C terminus of the chain is covalently bound to a solid support or resin during synthesis Three chemical reactions are repeated for each amino acid that is added to the growing peptide chain deprotection activation and coupling ll Deprotection NH CH C OH protected amino acid Activation Po G 79 NH CH C NH CH G deprotected peptide activated amino acid Coupling amino acid side chain protecting group a amino protecti
152. during synthesis Run ended 13 58 Cycles run 3 Run began 10 54 OK 4 This display appears when the synthesis is completed Press the OK soft key If the printer is on this record of the synthesis appears at the end of the conductivity trace See Interpreting the conductivity trace on page 2 16 for a description of the features of a conductivity trace Note After the synthesis the AACs still contain white inert material but very little amino acid remains AACs contain only enough amino acid for one synthesis cycle They cannot be re used 5 Verify the sequence of amino acids in the peptide sequence before removing and discarding the AACs on the wheel Record the sequence on the conductivity trace printout or in your laboratory log book 03 2002 Routine Operation 2 15 Applied Biosystems Interpreting the conductivity trace Compare the trace your instrument has generated to the conductivity trace of LAGV shown in Figure 2 7 Note that there are actually two overlapping conductivity trac es that reflect two conductivity ranges Trace Features 1 4 and Trace Feature 7 are in the low conductivity range Trace Features 5 and 6 are in the high conductivity range Typically at the beginning of every trace you should observe a series of upward spikes as the resin in the PSC solvates before the DMF baseline is established Fol lowing resin solvation each cycle consists of a deprotection peak Trac
153. e Then you end the synthesis run re arrange the AACs on the wheel and continue the syn thesis Note If vou continue synthesis after a powerfailure that was longer than 2 minutes occurred in module a or h you have to replace the AAC for the amino acid that was being activated when the powerfailure occurred After a powerfailure in module a or h the synthesizer interrupts synthesis at Func tion 9 P F Pause The Run Monitor displays the PAUSE soft key 1 In the Run Monitor press the jump S soft key Figure 2 12 The Jump Step Menu displays the module step and function of the synthesis interruption This is not necessarily the same step and function where the powerfailure occurred In the Jump Step Menu press the module soft key to go to the Jump Module Menu Inthe Jump Module Menu use the arrow key to move the cursor under module g Press the JUMP soft key twice to return to the Run Monitor Set an interruption to occur at Step 16 of module g See Steps 3 7 of How to set an interruption to synthesis on page 2 20 In the Run Monitor press the PAUSE soft key to start running module g The first 9 steps of module g wash out the activated amino acid that was left in Synergy after the powerfailure The last 7 steps of module g gas dry the Synergy pump 2 26 Routine Operation 03 2002 Applied Biosystems S 20 22 00 F 9 P F Pause T
154. e Feature 1 followed by a coupling plateau Trace Features 5 and 6 Your trace should have one cycle for each AAC on the wheel At the end of the synthesis the trace should show one more deprotection followed by a drop below the DMF baseline when THF washed through the PSC Seven features of the trace your instrument has generated should closely match the trace shown in Figure 2 7 These seven Trace Features should be present in each complete cycle of deprotection and coupling 1 Beginning deprotection a single sharp peak 2 Deprotection complete a level horizontal line slightly higher than the DMF baseline 3 Peperidine washout a return to DMF baseline 4 Amino acid activation a level baseline consistent with point 4 5 Coupling begins a rapid rise followed by a steady high plateau 6 Pump AAC wash a level consistent with point 5 7 Coupling solution washout a drop that returns to DMF baseline For descriptions of irregular traces see Irregular Conductivity Traces on page 7 3 2 16 Routine Operation 03 2002 TOOT EO uonp 2dQaunnoy LIT Initial resin solvation Begin 1 Cycle one T Cycle two 1 Cycle three T End Figure 2 7 Sample Synergy conductivity trace scaled to fit page suasksorg pauddy Applied Biosystems Run Monitor Synthesis Interruptions If you are using the pre programmed lines the synthesis normally progresses from start to
155. e Maintenance Tasks e Periodically at least once a year inspect and maintain fans and motors e Visually inspect tubing periodically for breakage crimping corrosion if metal or other damage Replace the tubing as necessary Flush the vent line with pure dry pressurized gas to remove buildup of condensation or particulates Handle and dispose of all wastes as if they were hazardous WARNING POTENTIAL EXPOSURE TO HAZARDOUS WASTE Always wear a laboratory coat eye protection and gloves that resist waste chemicals when you work on waste bottles exhaust lines tubing and liquid traps 03 2002 Synergy Safety Guidelines 1 11 Applied Biosystems 2 Routine Operation This section of the manual describes how you can use Synergy to perform routine peptide syntheses Before you follow the procedures in this section your instrument should be installed and should have already synthesized the test peptide LAGV as described in the Synergy Installation Guide Contents A Brief Description of Synergy 2 3 The LCD and Keyboard 2 4 Making a Peptide on Synergy 2 5 Prepare Synergy for Synthesis 2 5 1 Check the external gas supply 2 5 How to change the external gas tank 2 6 2 Check levels of 5 reagent bottles 2 6 0 2 M HBTU Reagent Preparation 2 7 3 Check the waste bottle and empty if necessary 2 8 How to replace the full waste bottle 2 9 4 Check the printer power paper supply and cable connections 2 9 Select and Load the C
156. e out of the Chart Recorder Menu 4 6 Maintenance and Self Tests 03 2002 Applied Biosystems Changing Synergy Fuses Two 2 A 250 V SB Slo Blo or equivalent fuses protect Synergy s interior elec trical circuitry against power surges and excessive power consumption If the fuses blow soon after they have been replaced there could be a short circuit inside the in strument Call Applied Biosystems Technical Support if this happens The fuse holder is part of the AC power input module that includes the 3 prong pow er cord receptacle How to change fuses 1 2 Turn off the power on the front of Synergy and unplug the power cord To remove the fuse holder use a screwdriver blade to push the flexible plastic snap tab closure to the left When the tab is pushed far enough to the left the fuse holder pops out AC power input module A Push a ainst snap tab with screwdriver blade Figure 4 5 Locate the fuse holder on the rear of the Synergy Peptide Synthesizer 3 3 Pull out and remove the two blown fuses Insert two 2A 250 V Slo Blo or equivalent fuses into the two round openings in the fuse holder Push the fuse holder back in place into the AC power input module The fuse holder snaps firmly in place so that the back of the fuse holder is flush with the back instrument panel Plug in the power cord and press the power button on the front of Synergy to turn the power on 03 2002 Ma
157. e synthesis soft key to go to the Manual Control Menu 8 Press the function soft key to go to the Function Menu Figure 2 14 Use the numeric keys to enter Function 53 OPEN JAWS Press the ON soft key Press the Menu key to go to the Main Menu then press the manual control 2 28 Routine Operation 03 2002 Applied Biosystems set up amp run edit amp manual self Main Menu report control print control test Press Menu ON gt F V ket to Manual Control Menu L return to function valve ALL OFF Main l Menu Enter Function 53 OPEN JAW Function Menu ON Figure 2 14 How to manuallv activate the OPEN JAWS function 10 When the jaws open remove the amino acid column wheel Remove all the used AACs including the column that contained activated amino acid when the powerfailure occurred During the first 5 steps of this procedure the amino acid that was being activated when the powerfailure occurred was washed out of the Svnergv pump to prevent its coupling to the peptide 11 Replace the column that contained activated amino acid when the powerfailure occurred with a fresh unused AAC Place this AAC in position 1 on the wheel Place the rest of the unused AACs on the wheel in the positions after position 1 There should be no empty positions between position I and the N terminal AAC 12 Replace the wheel 13 Go to the Run
158. e until you re start data printing You can make these modifications at any time however the modifications do not go into effect until you begin data printing If Synergy is already printing data when you make changes either cancel printing see How to replace or re align printer pa per during synthesis on page 2 23 or wait till the end of the current data printout to restart printing and see how the printout has been modified How to change the scales on the conductivity trace printout 1 Inthe Main Menu press the setup amp report soft key to go to the Setup amp Report Menu set up amp run edit amp manual self Main Menu report control print control test pressure time amp print serial Press Set up amp Report Menu settings date reports number more Menu key set act chart Set up amp Report Menu L acts default recorder more Range gt Upper 0085 mv Lower 1700 mv Chart Recorder Menu L Chart Speed 01 mm min headers ok Figure 4 2 How to go to the Chart Recorder Menu 4 4 Maintenance and Self Tests 03 2002 Applied Biosystems 2 In the Setup amp Report Menu press the more soft key until the chart recorder soft key becomes available Figure 4 2 Press the chart recorder soft key to go to the Chart Recorder Menu The top line in the Chart Recorder Menu has entry fields you can use to modify the scales of the two conductivity trace lines The Upper
159. ean vacuum filtration flasks 125 mL Teflon or stainless steel spatula vortex 03 2002 Post Svnthesis Procedures 3 7 Applied Biosystems 1 Collect the peptide by filtering the contents of the graduated conical tube through a medium porositv fritted glass funnel mounted on a vacuum filtration flask 2 Turn the vacuum off and add 10 mL MTBE to the contents of the funnel Stir the contents with a spatula to resuspend the peptide and turn the vacuum on to filter again Repeat this wash step three times 3 Remove the filtration flask that now contains MTBE and impurities and discard the filtrate Place the fritted funnel on a clean filter flask 4 Add distilled water to the funnel to dissolve the peptide e If the peptide is basic it will probably dissolve in H O If the peptide does not dissolve well in water try adding a few drops of glacial acetic acid Do not let the concentration of glacial acetic exceed 15 v v because high concentrations of acetic acid do not lyophilize e If the peptide is acidic dropwise add NH OH to solubilize If the peptide is hydrophobic add acetonitrile to solubilize 5 When the peptide dissolves turn on the vacuum to complete the filtration Transfer the dissolved peptide from the filtration flask to a clean freeze drying flask Freeze the contents by placing the flask in either liquid nitrogen or a container of isopropanol and dry ice The peptide solution may now be lyophilized
160. ed bottles 7 6 Troubleshooting 03 2002 Applied Biosystems Re used AACs Note After a svnthesis the AACs still contain white inert material but very little amino acid remains AACs contain only enough amino acid for a single coupling They cannot be re used If you accidentally re use an AAC in any position reagent washout Trace Feature 7 does not return to baseline level for that cycle and all subsequent synthesis cycles even if the rest of the AACs on the wheel have never been used See page 2 16 for a discussion of Trace Features Incomplete reagent washout Ma U Ai il kr Nee sree res ht Used AAC 1 New AAC Figure 7 5 Conductivity trace of synthesis with re used AAC During deprotection the a amine on the peptide becomes accessible to a chemical reaction with an activated amino acid When activators flow through a used AAC very little if any activated amino acid is released The accessible sites on the peptide instead bind to HBTU to form tetramethyl guanidine In the process the peptide is partially capped essentially terminating the synthesis The incomplete washout re sults from the presence of basic sites on the resin that prevent rapid washout If incomplete reagent washout such as Figure 7 5 demonstrates is present from the beginning of synthesis it may indicate an excessive delivery of HBTU Run Flow Test 8 to calibrate H
161. ed modules 5 7 Pressure port 2 30 pressure system leaks 7 12 to 7 18 pressure test 7 25 failure 2 18 7 12 to 7 18 Pressure Vent switch 2 3 2 30 4 9 Previous key 2 4 prime 4 11 hold 2 31 prime soft key 2 31 4 11 print module 5 9 Print Reports Menu 2 24 7 24 print reports soft key 2 23 PRINT soft key 4 15 printer paper replacement 2 23 printer assembly 2 9 to 2 10 printout cycle number 4 6 printout adjustments 4 4 to 4 6 printout headers 4 6 Pro 2 13 procedure cleavage reaction 3 4 to 3 5 interrupt synthesis 2 20 restart 2 33 to 2 35 routine synthesis 2 14 shutdown 2 30 to 2 32 terminate synthesis 7 11 protecting group 5 4 PSC 2 10 2 12 4 32 5 7 amide 2 10 5 7 leaks 4 32 pre loaded 2 11 storage 2 13 pump 4 24 4 26 5 10 Pump AAC 4 24 Pump Cell 4 26 R ratchet cap 4 10 7 16 reagent bottle positions 2 8 4 10 DIEA 5 7 DMF 2 6 2 8 5 7 HBTU 2 7 5 7 piperidine 2 6 5 8 prime 4 11 to 4 12 THF 5 8 usage 2 7 washout 5 17 6 6 7 7 reagent bottle piperidine 7 15 7 17 reagent bottles emergency replacement 2 22 reagent usage Table 2 7 recovery centrifugation 3 3 3 5 to 3 7 vacuum filtration 3 3 3 7 to 3 8 regulator pressure 2 34 residues adding 6 13 resin 5 3 solvation 2 16 5 4 6 3 re start procedure 2 33 to 2 35 ROM card 4 36 run 6 3 Run Begin 2 14 Run Control Menu 2 14 2 15 2 29 run control soft key 2 14 Run Editor Menu 6 3 to 6 19 Run End 2
162. ed to the external pressurized gas tank Loop for Pressure and Delivery ports Waste plug 5 8 in Synergy power switch Figure 2 17 Synergy after shutdown 1 Turn on Synergy power switch 2 If you have disconnected the waste bottle line use a 5 8 in wrench to remove the plug from the Waste port Save the waste plug for future Synergy Shutdowns compression nut Figure 2 18 Attaching the waste bottle line at the waste port fitting 03 2002 Routine Operation 2 33 Applied Biosystems Place the plastic compression nut on the shorter tube from the waste bottle cap assembly Figure 2 18 Be sure the wide end of the ferrule goes on the tube first so that the narrow end of the ferrule is closest to the waste port fitting Slip the tube into the Waste port and screw the plastic compression nut finger tight over the Waste port fitting Attach the longer tube from the cap assembly to the external ventilation system described in the Pre Installation Manual and in the Laboratory Ventilation Requirements on page 1 8 of the Synergy Safety Guidelines Check that the waste line runs up to the external ventilation without any downward dips where waste can accumulate If necessary cut off excess line to allow proper flow 3 Verify that the Pressure Vent switch is in the Vent position Open the main supply valve on the gas tank and the regulator needle va
163. eel The wheel can hold up to 30 amino acid cartridges Note AACs and PSCs cannot be used interchangeably 1 Determine the sequence of the peptide to be synthesized The labels on the AACs and PSCs display the amino acid abbreviations designated by the International Union of Pure and Applied Chemistry IUPAC Table 2 3 on page 2 13 lists these amino acid abbreviations Peptide sequences are traditionally written with the N terminal amino acid on the left and the C terminal amino acid or amide on the right Synergy synthesizes the peptide from the C terminal position to the N terminal position A peptide sequence is written with N terminal amino acid on the left SS C terminal Val Gin Ala Ala Ile Asp T yr Ile Asn Gly t amino acid Sequence of Acyl Carrier Protein 65 74 Peptide built on C terminal amino acid in PSC 2 Place the PSC that corresponds to the C terminal amino acid in the peptide synthesis column position Table 2 4 PSCs that contain resin attached to amino acid are also called pre loaded resins If the peptide has an amide NH at the C terminal position place an amide resin PSC in the peptide synthesis column position Amide resin PSCs are labeled with the abbreviation AM 2 10 Routine Operation 03 2002 Applied Biosystems Caution Leaks in the peptide synthesis column position can damage Synergy To assure a leak proof connection between the peptide synthesis column and
164. em has fewer moving parts than a motor driven pump and so is more simple efficient and reliable Illustrated Module Descriptions Figure 5 2 is a simplified plumbing schematic of Synergy that shows the pump sys tem the conductivity cell the valve blocks and reagent bottles with the tubing that connects all these components Pages 5 10 through 5 15 contain illustrations of some of the functions to demonstrate how the pumping system and pressurized re agent deliveries perform parts of the synthesis process Caution The exterior panels on Synergy may be removed only by trained Applied Biosystems Service Personnel dlassasdsdufens Amino acid column wheel cee Peptide synthesis column Pressurized gas delivery line 4 Conductivity cell Amino acid column Figure 5 2 Simplified Synergy Plumbing Schematic 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 11 Applied Biosystems Module a extraction and activation Measured amounts of the activators HBTU and DIEA are cycled through the amino acid column AAC Activated amino acid begins transfer to the peptide synthesis column PSC Or Waste ass IL 7 Function 45 Deliver the activator HBTU to the left transfer vessel Measure HBTU Function 46 Deliver the activator DIEA to the left transfer vessel Neveeesereed De Bi Measure DIEA Function 60 PUMP Cvcle activators from left transfer ves
165. embly or encoder If any of the motor assembly or encoder error messages listed in Table 7 4 appear on the LCD call Applied Biosystems Technical Support 7 20 Troubleshooting 03 2002 Applied Biosystems Powerfailure There was a power failure while the synthesizer was running This message ap pears as soon as power returns but its appearance does not necessarily indicate a problem Press the OK soft key to acknowledge the message The power was off too long to resume the run If the powerfailure occurred during a synthesis Synergy allows a one hour loss of power during all modules except module a or j where it allows only a two minute outage This message appears when the length of the powerfailure exceeds these limits See Powerfailures on page 2 25 for a discussion of recommended responses to a powerfailure Arun control error occurred before the powerfailure The run is paused If this message appears after a powerfailure it means that just before the powerfailure an other error message was displayed Synergy does not remember what the error was because the powerfailure erased the error message Look at the Run Monitor to see what step function and module is currently displayed From this information you may be able to deduce what occurred just before the powerfailure If you don t know which error occurred just before the powerfailure visually inspect the wheel and jaws assembly and check the regulator and inter
166. en press the Next key or press the numeric key 6 to make Run Flow Test 6 appear on the LCD 4 24 Maintenance and Self Tests 03 2002 Applied Biosystems Run Flow Test 6 Pump AAC PRINT START Press the START soft key to begin the flow test A brief pressure test and a delivery of DMF to the left vessel follows Is there liquid in the left vessel only NO YES Look at both transfer vessels You should see liquid in the left transfer vessel If you see liquid in only the left vessel press the Yes soft key If there is no liquid in the left transfer vessel or if there is liquid in the right transfer vessel press the No soft key Watch the liquid as it drains out of the left vessel At the end of the liquid drain a few small drops may flow back into the vessel Is there liquid in the right vessel NO YES Look at both transfer vessels You should see liquid in the right transfer vessel and no more than a drop of liquid in the left transfer vessel If you see liquid in the right vessel press the Yes soft key If there is no liquid in the right transfer vessel press the No soft key Again watch the liquid as it drains out of the right vessel At the end of the liquid drain a few small drops may flow back into the vessel Is there liquid in the left vessel NO YES Again check that there is liquid in only the left vessel and no more than a dr
167. en properly connected to Synergy and turned on the HP ThinkJet Printer prints a conductivitv trace of each cycle of deprotection and coupling that occurs during a synthesis e Verify that the mini DIN terminal end of the RS 232 cable is plugged into the printer port of Synergy e Check the printer paper supply and replenish if necessary Turn on the printer power switch on the rear of the printer The red light labeled PWR on top of the printer appears when the printer is turned on e If the yellow light next to the blue button labeled Aa is blinking press the blue button to set the top line of the paper to the current line 03 2002 Routine Operation 2 9 Applied Biosystems N terminal amino acid e Refer to Adjusting the Conductivity Trace Printout on page 4 4 for instructions on setting the printer speed choosing the cycle number option and adjusting the scale of the conductivity traces on the printout Note The Computer port above the Printer port currently has no user related functions It may be used occasionally by trained Applied Biosystems Service personnel Select and Load the Columns for Your Synthesis After you have completed all four of the synthesis preparation steps listed on page 2 5 Synergy is ready to perform a synthesis Place one peptide synthesis column PSC in the peptide synthesis column position and the appropriate amino acid col umns AAC on the amino acid column wh
168. ential damage such as contact heat or flame Place the tubing end as far as possible into the duct canopy or hood Use polypropylene tubing of the shortest possible length and straightest possible run Tubing length should not exceed 15 feet 5 meters Eliminate low points that can trap residue or condensation Fasten tubing securely Use fasteners of polypropylene or teflon Do not use brass it corrodes Be careful not to puncture tubing Canopy Operate air exhaust at all times including nights and weekends when vented waste bottle contents can escape to surroundings WARNING POTENTIAL EXPOSURE TO HAZARDOUS GASEOUS WASTE Do not connect the waste vent to a ductless hood or to a system that purifies filters air and returns it to the room See the Synergy Waste Profile after the Safety Data tab in this User s Manual 1 10 Synergy Safety Guidelines 03 2002 Applied Biosystems Figure 1 4 shows a duct system and fume hood canopy with tubing connected to the instrument waste exhaust line Duct at negative pressure with X positive room pressure ER Fume hood canopy at negative pressure 1 Movement of Waste exhaust line gaseous waste leads up to N fume hood canopv Port to waste bottle with secondarv containment ify Fl ik Figure 1 4 Venting gaseous waste directly to duct Routin
169. ermined to be covered by this Warranty shall be performed by AB No agent employee or representative of AB other than an officer of the company has any authority to bind AB to any affirmation representation or Warranty con cerning the Instrument that is not contained in the User s Manual or this Warranty AB shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument AB makes no Warranty whatsoever in regard to products or parts furnished by third par ties This Warranty is limited to the original customer and is not transferable 03 2002 AB Limited Warranty Il 1 Applied Biosystems THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS EXPRESSLY IN LIEU OF ANY OTHER EX PRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABLITY OR FITNESS FOR A PARTICULAR PURPOSE KNOWN TO THE SELLER AND OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS Il 2 AB Limited Warrantv 03 2002 Applied Biosystems Appendix III Synergy Parts and Reagents The following listed parts for the Synergy Personal Peptide Synthesizer may be or dered by Synergy users and replaced without removing the instrument s exterior panels User Replaceable Synergy Parts Part Description Part Number amino acid column wheel 004185 calibration test fixtures 401383 waste cap as
170. esence of residual conductive material Figure 6 9 Conductivity trace of repeat synthesis of peptide in Figure 6 8 This synthesis has a double couple at the sites of difficult coupling B D Each coupling is over twice as long as its equivalent in the first trace and the reagent washouts at Cl and E show a more complete return to the DMF baseline The arrows point to the beginning of the second coupling of the double couple 03 2002 Advanced Operations 6 11 Applied Biosystems The rest of the AACs for the sequence follow on the wheel Leu in 6 Leu in 7 Thr in 8 and Gln in 9 Go to the Run Editor Menu cycle line 1 Use the alpha numeric keys to enter how many times the standard line should be repeated The standard cycle line can be applied to the first amino acid in the sequence Gly Line 1 one is used once as the top LCD in Figure 6 7 demonstrates Press the ADD L soft key to enter the double couple line After Repeat enter the number of consecutive double couples In this example there are two double couples in a row so a 2 appears after Repeat If you want a double couple at every amino acid position until the end of the sequence leave the Repeat value at one the default value The Synergy controller automatically repeats the last line in a run until the jaw sensors detect an empty position on the wheel After M on the LCD for the second line enter jdacgijacgfi 5 P
171. ess the prime soft key ve Run Flow Test 1 Prime THF Priming Menu l PRINT START S 03 04 00 F 20 THF gt BVB T 003 005 Run Monitor Amount of time HOLD PAUSE NEXT S jump S more the HOLD soft key is active S 03 04 00 F 20 THF gt BVB T 000 060 4 HOLD PAUSE NEXT S jump S more Figure 2 16 How to hold the THF prime at step 3 6 To begin a flow test press the START soft key Observe the step number in the left corner of the Run Monitor With all the reagent primes except the PIP piperidine prime press the HOLD soft key when the prime is in step 3 With the piperidine prime press the HOLD soft key when the prime is in step 4 When one prime is finished press the key labeled Next to proceed to the next prime During both the THF and DMF primes hold the prime at step 3 for one minute to flush nitrogen gas through the reagent line When the values after the T are 000 060 press the HOLD soft key to release the hold 03 2002 Routine Operation 2 31 Applied Biosystems During both the HBTU and DIEA primes hold the prime at step 3 until all DMF in the reagent bottles has been delivered then hold the prime for 60 more seconds to flush nitrogen through the reagent lines Press the HOLD soft key to release the hold During the PIP prime hold the prime at step 4 until all DMF is drained from the reagent bottle then hold the prime 60 seconds more Press the HOLD
172. est 1 5 7 8 and 9 Loops also bracket most of the pressure tests which means that if a pressure test fails Synergy repeats the pressure test until it passes If you follow the procedure How to jump over steps in a module on page 7 18 and try to jump past the beginning of a loop the error message Unmatched Begin Loop End Loop function appears If you must jump over a Begin Loop you must also jump over the End Loop function Incorrect use of Manual Control or incorrect module edit With the pre programmed modules most Synergy users will seldom if ever edit modules or use the Manual Control Menu in such a way that would cause an error message Advanced users should refer to Chapter 6 for more information on these procedures 03 2002 Troubleshooting 7 25 Applied Biosystems Index Numeri CS Asn 2 13 see also AAC PSC 1 2 ethanedithiol see EDT Asp 2 13 loading for synthesis l hvdroxvbenzotriazole see HOBt Atherton E 35 2 10 to 2 12 2 1H benzotriazol 1 yl 1 1 3 3 attention words 1 3 compression nut tetramethvluronium phosphate see autosampler jaws 4 14 waste line 2 32 2 34 HBTU conductivity 2 3 9 fluorenylmethylcarbonyl see Fmoc B cell 4 32 A Bayer E 5 5 flow test 4 26 in li 2 t 4 27 AAC 2410919996 Sii Oe oo ee begin modules 6 3 monitoring 5 15 storage 2 13 f k wis d 7 7 begin synthesis 5 13 conductivity data ped 5 4 biotin 6 16 buffer 2 23 Q amino grou oe p p 4 bottle positions 4 10 7 6 condu
173. est failure on page 7 12 Flow Test 8 Calibrate HBTU WARNING CHEMICAL HAZARD HBTU can cause allergic reactions and skin irritations in sensitive persons Wear protective clothing and handle HBTU solution in a properiv vented fume hood See the MSDS in the Svnergv User s Manual This flow test measures deliverv of HBTU Peptide Svnthesis Reagent A to the left transfer vessel To perform this flow test you tare the left transfer vessel and then weigh the contents of the left vessel after each of three reagent deliveries 03 2002 Maintenance and Self Tests 4 27 Applied Biosystems 1 To begin Flow Test 8 press the flow soft key in the Self Test Menu Figure 4 6 Then press the Next key or press the numeric key 8 to make Run Flow Test 8 appear on the LCD Run Flow Test 8 CALIBRATE HBTU PRINT START Press the START soft key to begin Flow Test 8 Remove left vessel and put on scale Zero scale then replace vessel OK Remove the left transfer vessel on the front of Synergy Figure 4 15 To tare a scale place the vessel on a scale that is accurate to 1 mg and set the scale to zero Figure 4 15 Remove the left transfer vessel Replace the left transfer vessel on Synergy Press the OK soft key Remove the left vessel and weigh mg OK When this displav appears the left
174. est fixture in position one 1 on the amino acid column wheel Figure 4 9 Figure 4 9 Placing a wheel test fixture in position one 3 Place the wheel on top of the hub on the right side of Synergy Slowly rotate the wheel until the protruding pin on the bottom of the wheel slips into the corresponding groove in the hub 03 2002 Maintenance and Self Tests 4 13 Applied Biosystems 4 Place a flow test calibration tube in the peptide synthesis position Figure 4 10 Figure 4 10 Placing a flow test calibration tube in the peptide synthesis position Twist and push the luer fittings on at both ends of the flow test calibration tube Do not unscrew the black plastic bushing attached to the top liner fitting Caution Leaks at the peptide synthesis column position can damage the conductivitv cell To assure a leak proof connection push the luer fittings onto both column ends with a slight twisting motion 5 Press the START soft key WARNING PERSONAL INJURY HAZARD The autosampler jaws are operated by a pneumatic valve under high pressure Keep your fingers away from the jaws during the Leak Self Test and any other time the jaws are in operation During the Leak Self Test eight different pressure tests are performed If a pressure leak is detected the LCD displavs the message Pressure test failed The second line of the LCD reports the pressure readings that were
175. etergent Get medical attention INHALATION Provide fresh air and rest If breathing is difficult provide oxvgen and get medical attention immediatelv Flush eves immediatelv with large amounts of water for at least 15 minutes Get medical attention VI Reactivitv Data STABILITV Stable INCOMPATIBILITY Contact with strong oxidizing agents or concentrated acids or bases may cause fire or explosion HAZARDOUS COMBUSTION OR DECOMPOSITION Burning may release toxic vapors and gases including hydrogen flouride carbon monoxide and oxides of nitrogen HAZARDOUS POLYMERIZATION Will not occur VII Spill or Leak Procedures STEPS TO BE TAKEN Avoid inhalation and skin contact Wear protective clothing Ventilate area of spill or leak Remove all ignition sources Small quantities may be collected with absorbant towels or pads and removed to a well ventilated area away from ignition sources Larger amounts 1 liter or more may be collected with an inert absorbant kitty litter or similar material or commercially available spill pillows designed for solvent collection This waste material must not be allowed to enter confined spaces such as a sewer because of the possibility of an explosion WASTE DISPOSAL METHOD This instrument waste solution should be disposed of as a regulated hazardous waste by a properly permitted hazardous waste management facility in accordance with federal state and local regulations
176. f HBTU is delivered into the DMF solution and a second reading is taken The first reading falls within the range of low conductivity readings the second reading falls within the range of high readings 1 To begin Flow Test 7 press the flow soft key in the Self Test Menu Figure 4 6 Then press the Next key or press the numeric key 7 until Run Flow Test 7 appears on the LCD Run Flow Test 7 Pump Cell PRINT START 2 Press the START key A two minute procedure precedes the first conductivity reading 4 26 Maintenance and Self Tests 03 2002 Applied Biosystems Conductivity 444 OK 3 Two conductivity measurements are taken during this test with a two minute time lapse between each reading When the conductivity value is within range the LCD displays the conductivity Press the OK key to continue the flow test after each display Test passed OK 4 When this displav appears press the OK soft kev to complete the flow test Value is out of rangel OK This message appears when a conductivitv value is not within specifications Press the OK key to see the value Press the OK key again to continue Flow Test 7 If the Test failed message appears at the end of Flow Test 7 repeat the flow test If Flow Test 7 continues to fail call Applied Biosvstems Technical Support If a pressure test failure occurs during this flow test read What to do after a pressure t
177. g procedure press the bottle soft key in the Self Test Menu Figure 4 6 Then press the prime soft key 03 2002 Maintenance and Self Tests 4 11 Applied Biosystems Run Flow Test 1 Prime THF PRINT START appears on the LCD Press the Next key or the Previous key until the appropriate priming flow test s an alternative you may use the alphanumeric keys to enter the number of the priming flow test you wish to run Table 4 1 Press the START soft key Test Passed OK after a pressure test failure on page 7 12 When this display appears no leaks have been detected and the priming procedure is completed Press the OK soft key Ifa pressure leak is detected during the bottle prime procedure see What to do 4 12 Maintenance and Self Tests 03 2002 Applied Biosystems Leak Self Test Use the Leak Self Test to check the entire internal pressure system for leaks You need the calibration test fixtures to perform the Leak Self Test Wheel test fixture Peptide synthesis column PSC with pre loaded resin or amide resin Flow test calibration test tube Figure 4 8 Calibration Test Fixtures Amino Acid and Peptide Synthesis Columns How to perform a Leak Self Test 1 To begin the Leak Self Test press the leak soft key in the Self Test Menu Figure 4 6 Put columns in position 1 and in the PSC PRINT START 2 Place a wheel t
178. gent bottles to prepare the HBTU 2 1 H benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate re agent Instructions for preparing the HBTU reagent follow The prepared reagent is good for six weeks 0 2 M HBTU Reagent Preparation WARNING CHEMICAL HAZARD HBTU can cause allergic reactions and skin irritations in sensitive persons Wear protective clothing and gloves and prepare the HBTU solution in a properly vented fume hood 1 Pour the contents 40 mL of the bottle labelled HOBt DMSO NMP 0 2 M 1 hydroxybenzotriazole Dimethyl sulfoxide N Methylpyrrolidone into the bottle labelled Peptide Synthesis Reagent A HBTU 8 mmoles 2 Tightly cap and invert the bottle to dissolve the HBTU 3 Write the date of preparation on the HBTU bottle label This prepared reagent can be used for up to six weeks 4 Locate the delivery line for the HBTU Press a reagent line filter onto the end of the delivery line One reagent line filter can be used for five consecutive bottles or approximately 200 mL of HBTU reagent 03 2002 Routine Operation 2 7 Applied Biosystems Bottle seal 5 Place a bottle seal on the rim of the HBTU bottle With the reagent delivery line inside hold the bottle straight up and turn it carefully into the threads of the ratchet cap on Synergy Figure 2 3 The bottle should fit firmly in place O00 zs ge om E Delivery line filter on HBTU deliver
179. gin a synthesis when printer printing data The buffer or memory for the printer can only hold data for one synthesis As soon as you start a new synthesis the old data is erased from the buffer If the printer is printing the old data the con troller cannot erase it to start a new synthesis When this error message appears you may let the printer finish printing or you may discontinue printing to start the new synthesis If you decide to discontinue printing to begin a new synthesis all the old data will be erased How to discontinue data printing 1 Press the setup amp report soft key in the Main Menu 2 Press the print reports soft key in the Setup amp Report Menu Figure 7 10 3 Press the CANCEL PRINT soft key in the Print Reports Menu Main Menu Set up amp Report Menu Print Reports Menu set up amp run edit amp manual self report control print control test pressure time amp print serial setting date reports number more CANCEL MONITOR USER SYSTEM PRINT DATA DATA DATA Figure 7 10 How to go to the Print Reports Menu Press Menu Key Start Flow Test 6 or the Wheel Self Test without an AAC in position one A wheel test fixture must be in position one of the amino acid column wheel to run ei ther one of these tests If the jaw sensor does not detect a column in position one dur ing one of these tests the Synergy controller automatically in
180. h the Pressure Vent switch to Vent and wait 30 seconds for the pressure to drop Remove any reagent bottles that have recently been replaced and check the bottle seals for cracks or warped surfaces Replace any defective bottle seals WARNING CHEMICAL HAZARD Reagent bottles are pressurized With the exception of piperidine always switch the Pressure Vent switch to Vent before unscrewing a reagent bottle Piperidine is the only reagent that is on its own pressure system separate from the other bottles You may remove the piperidine bottle without venting the pressure system for the other reagents When you open the piperidine bottle you hear a small hiss as residual pressure is released 03 2002 Troubleshooting 7 15 Applied Biosystems Table 7 3 Pressure Test Failures During a Self Test and Recommended Response When Failure Occured Module Step where failure occured Recommended Response Bottle Change t S 07 07 Check bottle seals bottle cap threads of replaced reagent bottles Leak Test S 02 29 Check HBTU DIEA THF bottle seals and caps DMF bottle lines transfer vessels S 05 29 Check Piperidine bottle seal and cap S 08 29 Call ABI Service S 11 29 Call ABI Service S 14 29 Call ABI Service S 19 29 Check wheel test fixture in jaws seals S 23 29 Check PSC and luer fittings S 26 29 Check left transfer vessel S 29 29 Check left and right transfer vessels Prime THF u S 02 04 Check THF bottle seal
181. h AAC and transfer vessels h alternate to module a for non standard amino acids Increment moves the wheel to the next AAC position Jaws close on AAC and pressure test How to determine the cycle and module Synergy is currently running 1 Press the more soft key in the Run Monitor to make the second Run Monitor appear The set int soft key should be visible S xx yy zz F F FUNCT NAME T xxx zzz HOLD PAUSE NEXT S jump S more Press l Menu Run Monitor key to return to S 02 16 C 00 01 M bf Main Menu LOCK set int end run more Figure 2 8 How to determine the cycle and module currently running 03 2002 Routine Operation 2 19 Applied Biosystems 2 Read the top line of the second Run Monitor display to determine which module the instrument is currently running The abbreviations in the top line indicate from left to right e S Step the current step number total number steps in this module e C Cycle the current cycle number total number of cycles in this synthesis The begin cycle is always cycle 0 zero With pre programmed lines the value for the total number of cycles increases with each complete cycle until the END line is run e M Module the cursor blinks under the module that is running For example in the second Run Monitor shown in Figure 2 8 the instrument is on the second step of 16 in Module b and in the begin cycle of the synthesis Once you know the modu
182. he Safety Supplement of the Synergy User s Manual Table 2 2 lists the 5 reagent bottles found on Synergy with average expected usage You can expect the values on this table associated with HBTU and DIEA to remain constant regardless of which amino acids go into your peptide synthesis However piperidine and DMF usage vary as the deprotection reactions vary in length 2 6 Routine Operation 03 2002 Applied Biosystems Table 2 2 Synergy Approximate Reagent Usage Reagent Bottle size ML mL cycle mm cvcle Cvcles bottles approx approx HBTU 40 0 4 0 5 100 DIEA 40 0 4 0 5 100 Piperidine 200 2 1 0 100 THF 200 2 1 0 100 DMF 4000 60 3 67 DMF mm cycle value applies to that part of the reagent bottle below the neck where the bottle sides do not taper You may find it helpful to place a vertical strip of white tape on the side of each re agent bottle to track reagent usage After each synthesis place a horizontal mark on the tape that corresponds with the current level of reagent in each bottle The values in the column labeled mm cycle in Table 2 2 reflect the average change in the re agent level measured in millimeters per cycle Use Table 2 2 and the number of amino acids in your next peptide to estimate how much reagent you will need to complete the synthesis If necessary replace reagent bottles before synthesis begins see Bottle Changing Procedure on page 4 9 You must combine the contents of two rea
183. he Synergy Peptide Synthesizer Twist and push the luer fittings on at both ends of the PSC Do not unscrew the black plastic bushing attached to the top luer fitting Caution Leaks at the peptide synthesis column position can damage the conductivity cell To assure a leak proof connection between the peptide synthesis column and the luer fittings push the luer fittings onto both column ends with a slight twisting motion 4 32 Maintenance and Self Tests 03 2002 Applied Biosystems Remove the amino acid column wheel and place the Amino Acid Column ACC labelled G in position 1 place the AAC labelled A in position 2 and place the AAC labelled L in position 3 Figure 4 17 Figure 4 17 Load the amino acid columns for the test peptide LAGV Replace the wheel Press the START key to begin the cycle test As the cycle test progresses check around the PSC luer fittings for leaks If you detect a leak at either end of the PSC push and twist the end into its luer fitting If a drop of liquid begins to form at the middle of the PSC wick it away with a tissue If the PSC continues to leak terminate the synthesis see How to terminate a synthesis on page 7 11 and restart the synthesis with a new PSC It takes approximately 3 hours to complete this cycle test During the test the pre programmed modules Table 2 1 on page 2 5 direct the flow of reagents monitor pressure and generate a conductivity trace
184. ight heat or potential ignition sources Contact the appropriate state hazardous waste regulatory agency for proper disposal procedures and list of registered service companies THIS WASTE MATERIAL IS HAZARDOUS AND SHOULD ONLY BE HANDLED BY PERSONS THOROUGHLY TRAINED IN HAZARDOUS MATERIALS HANDLING PROCEDURESI DATE ISSUED July 14 1993 P N 902109 Rev B July 14 1993 57 Applied Biosystems Appendix II AB Limited Warranty Applied Biosystems AB warrants to the customer that for a period ending on the earlier of one year from completion of installation or 13 months from the date of shipment to the customer the Warranty Period the AB Model 432A the Instru ment purchased by the customer will be free from defects in material and work manship and will perform at least in accordance with the minimum performance specifications for flow test the Mechanical Performance and synthesis of the test peptide LAGV the Chemical Performance as specified in the Install Guide This Warranty does not extend to any Instrument or part thereof that has been sub jected to misuse neglect or accident that has been modified or repaired by anyone other than AB or that has not been used in accordance with the instructions con tained in the Instrument User s Manual Nor does this Warranty cover any customer installable consumable parts for the Instrument that are listed in the Instrument Us er s Manual This War
185. ight of the trace is not effected by changing the chart speed nor does the time it takes to print data during a synthesis change when the chart speed changes 1 Inthe Chart Recorder Menu Figure 4 2 on page 4 4 press the arrow key to move the cursor from the top line to the bottom line of the LCD Place the cursor in the field after the words Chart Speed 2 Use the alpha numeric kevs to enter either 1 one or 2 mm min 3 Press the OK soft key to move out of the Chart Recorder Menu How to make the date time and cycle number appear on the printout Range gt Upper 0085 mv Lower 1700 mv Chart Recorder Menu Chart Speed 01 mm min headers ok Print the cycle headers NO Headers Menu NO YES OK Figure 4 4 How to go to the Headers Menu When you choose headers Synergy prints the date and time each cycle started with the cycle number If you use a pre loaded PSC one that contains an amino acid cy cle one corresponds to the deprotection of the C terminal amino acid and coupling of the second amino acid from the C terminal 1 Go to the Chart Recorder Menu Figure 4 2 on page 4 4 2 Press the headers soft key in the Chart Recorder Menu to go to the Headers Menu 3 Press the Yes soft key if you want headers on the printout press the No soft key if you don t want headers on the printout 4 Press the OK soft key to move out of the Headers Menu Press the OK soft key to mov
186. igure 4 13 The mark should be even with the bottom of the meniscus Press the OK soft key Figure 4 13 Marking the flow test calibration tube After the second DMF delivery the LCD directs you to make a second mark on the flow test calibration tube The second mark should be higher than the first mark and even with the bottom of the meniscus IMPORTANT Save this marked flow test calibration tube it will be used again in other flow tests 4 20 Maintenance and Self Tests 03 2002 Applied Biosystems Test Passed OK 6 When this display appears press the OK soft key to complete the flow test If a pressure test failure occurs during this flow test see What to do after a pressure test failure on page 7 12 03 2002 Maintenance and Self Tests 4 21 Applied Biosystems Flow Tests 3 4 and 5 e DMF gt PSC c THF PSC PIP PSC Each of these three flow tests delivers reagent to the flow test calibration tube that you marked in Flow Test 2 During each flow test observe the meniscus of the re agent delivered to the flow test calibration tube The meniscus should fall on one of or between the two marks on the tube 1 To begin one of these flow tests press the flow soft key in the Self Test Menu Figure 4 6 Then press the Next key the Previous key or the numeric key that corresponds to the flow test n
187. ine and END end Most of the time you will probably edit on the cycle line BEG gt C 0 Repeat 1 M bf ADD L ERASE L a P PRINT Press Nextor L gt C 1 Repeat 1 M jdacgfi Previous keys ADD L ERASE L a fy PRINT END gt C 1 Repeat 1M de ADD L ERASE L te PRINT Figure 6 2 Three parts of the Run Editor Menu How to print a Run File The Run File contains all the lines shown in the Run Editor Menu To print a Run File press the PRINT soft key in the Run Editor Menu Figure 6 3 shows a printout of a Run File with the standard lines When you first start up Synergy the Run Editor contains this Run File Line Cycle Repeat Module List Beg 0 1 bf 1 1 1 jdacgfi End 1 1 de Figure 6 3 Standard Run File with the standard lines How to edit a run 1 In the Main Menu press the edit amp print soft key In the Edit amp Print Menu press the run editor soft key Figure 6 1 6 4 Advanced Operations 03 2002 Applied Biosystems Press the Next or Previous keys to go to the line you want to edit If you want to add a new line to the run go to the line sub menu and press the ADD L soft key The new line follows the current line Use the arrow keys to move the cursor to the right or left Use the alphanumeric keys to enter letters or numbers Press the a J soft key to toggle between lower case a b c and upper case
188. intenance and Self Tests 4 7 Applied Biosystems Main Menu Self Test Menu Self Test Menus When you run a self test a set of events automatically occurs to test a specific part of Synergy s hardware As the test progresses the LCD may display instructions that you must follow to complete the test When the test is finished the LCD an nounces whether or not the test passed The following are the seven types of self tests bottle consists of two categories CHANGE and PRIME for changing reagent bot tles and priming reagent delivery lines leak checks for leaks in the pressure system flow consists of nine flow tests used to check reagent deliveries cycle runs a synthesis of the test peptide LAGV keyboard checks that all the keys for the LCD are working wheel tests the pressurized autosampler jaws and the amino acid column wheel circuit tests the electrical circuitry to the valves and the ROM card set up amp run edit amp manual self report control print control test Select a test bottle leak flow cycle more Select a test keyboard wheel circuit more Figure 4 6 The Self Test Menu Press the self test soft key in the Main menu to go to the Self Test Menus e Press the more soft key to toggle between the two Self Test Menus e Press the Main key to return to the Main Menu 4 8 Maintenance and Self Tests 03 2002 Applied Biosystems Bott
189. ion rates through the gel Currently we cannot predict when these sequence dependent phenomena will occur Conductivity Monitoring In each synthesis cycle the Fmoc group is first removed by treatment with piperi dine in DMF generating a carbamate salt which can be detected by conductance monitoring The rate at which this deprotection reaction proceeds serves as a valu able indicator of the peptide resin reactivity A rapid reaction gives rise to a sharp deptrotection spike a slow reaction produces a broader peak The conductivity cell on Synergy measures the conductive species every seconds Synergy s controller records these conductivity values and every thirty seconds av erages these thirty values As long as conductive species are being released at sig nificant levels the controller continues to extend deprotec tion The controller ends the deprotection reaction either when the difference between two consecutive 30 second value averages is equal to or less than I conductivity unit or when thirty 30 second averages have been taken Peptide chemists at Applied Biosystems have observed that when a peptideresin re quires extended deprotection times the coupling time for the next amino acid should also be extended For each deprotection Synergy controller remembers the depro tection time and adjusts the subsequent coupling module The resulting coupling A Bayer E and Goldhammer C 1992 Conformation dependent coupli
190. k that the Pressure line to the DMF bottle is screwed on tightly at the Pressure port fitting Check that the DMF bottle cap threads are not stripped Check that both transfer vessels are tightly screwed in place 7 16 Troubleshooting 03 2002 Applied Biosystems Check piperidine bottle seal and cap Follow the instructions for checking bottle seals and caps described under Pressure Test Failure after a Bottle Change on page 7 15 Call ABI Service Department If a pressure test failure occurs at Step 8 11 or 14 of the Leak Test it could indicate loose fittings or valve failures that can only be repaired by trained Applied Biosystems Service person nel Call Applied Biosystems Service to report these pressure failures Check wheel test fixture in jaws Do not use an AAC or PSC that has been opened to perform this leak test To examine or replace the column first press the PAUSE soft key to continue the Leak Test and open the jaws After replacing the wheel test fixture repeat the Leak Self Test from the beginning Make sure the wheel test fixture is upright in the wheel Check that the autosampler jaws close completely on the wheel test fixture Insufficient gas pressure can cause jaw failures If the jaws fail to close completely even with adequate gas pressure call Applied Biosystems Technical Support Check PSC and luer fittings Reagent leaks can occur around the peptide synthesis column PSC if it has not been pushed firm
191. kends when vented waste bottle contents can escape to surroundings It is to be located away from air currents generated by air conditioning ducts fans windows doors and moving equipment and persons It should exhibit a sign or label indicating where doors and windows must be positioned to give an average flow of 100 ft min linear face velocity The minimum velocity of flow at any point in the hood must be 80 ft min linear the maximum flow velocity must not exceed 125 ft min It must meet local state and federal health and safety requirements In addition see current fume hood standards established by the American Society of Heating Refrigeration Air Conditioning Engineers ASHRAE American Conference of Governmental Industrial Hygienists ACGIH and Occupational Safety and Health Agency OSHA It must be constructed of materials that are compatible with the waste materials chemicals being generated exhausted Figure 1 3 shows a fume hood configuration with movement of gaseous waste through the hood into a duct the duct is described in the following section 1 8 Synergy Safety Guidelines 03 2002 Applied Biosystems Building fresh air intake located away from gaseous waste release to prevent intake of waste into the building air Average face velocity of air flow should be 100 ft min linear To duct TT use waste is not released to the outside in the vicinity of fresh air intake Movement of
192. le Self Test Change and prime the bottle s CHANGE prime e Press the CHANGE soft key before you replace any empty reagent bottles Press the prime soft key to flush reagent delivery lines with reagent Bottle Changing Procedure Use this bottle change procedure to change one or more of the following four reagent bottles HBTU DIEA THF or DMF WARNING CHEMICAL HAZARD Reagent bottles are pressurized With the exception of piperidine always switch the Pressure Vent switch to Vent before unscrewing a reagent bottle on the Synergy Peptide Synthesizer Piperidine is the only reagent that is on its own pressure system separate from the other bottles You may open or replace the piperidine bottle without depressurizing the pressure system for the other reagents When you open the piperidine bottle you may hear a small hiss as pressure is released Do not open the piperidine bottle when Synergy is running module d 1 To begin the bottle changing procedure press the bottle soft key in the Self Test Menu Figure 4 6 Then press the CHANGE soft key Switch the Pressure Vent to Vent OK 2 Before removing any bottles set the Pressure Vent switch to Vent Press the OK soft key During the 30 second interval that follows the internal pressure drops to zero Replace empty reagent bottles with full reagent bottles OK 03 2002 Maintenance and Self Tests 4 9 Applied Biosystems
193. le couples at each site of difficult coupling How to program a double couple L 1 gt C 1 Repeat 1 M jdacgfi Run Editor Menu cycle line ADDL ERASEL ta j PRINT L2 C 2 Repeat 2 M jdacgijacgfi ADD L ERASE L au PRINT L 3 gt C 4 Repeat 1 M jdacgfi ADDL ERASEL a PRINT Fa key Save edit session changes cancel NO YES Figure 6 7 How to program two sequential double couples 1 Determine where the double couples should occur in the sequence Place the appropriate AACs on the amino acid column wheel You need two AACs of the same amino acid for each double couple For example assume in the sequence GIn Thr Leu Leu Asn Arg Gly Ile we will double couple at both Asn and Arg Synthesis on Synergy begins with the C terminal amino acid in this case Ile in the PSC Gly goes in position 1 6 10 Advanced Operations 03 2002 Applied Biosystems on the AAC wheel Arg and Asn both will be double coupled so Arg goes in position 2 and 3 Asn goes in position 4 and 5 1 x Z Z W ea Ba SENA Figure 6 8 Conductivity trace showing slow deprotections and incomplete couplings Coupling at B and D is followed by sluggish reagent washouts at C and E without a return to the DMF baseline A within the maximum allotted time an indication of incomplete coupling Note the deprotection peaks after C and E are abnormally high due to the pr
194. le that is running refer to Table 2 5 to determine the best place to set an interruption Table 2 5 Action Items and Response During Synthesis Action Item Response Replace empty reagent bottle set int at Module g j or i step 1 one Replace external gas tank set int at Module g j or i step 1 one Remove full waste bottle set int at Module c step 1 one Powerfailure Continue synthesis unless failure occurred during module a or h and lasted more than 2 minutes Replace printer paper Go to Setup amp Report Menu to cancel print Piperidine may be changed at any module except Module d and requires no system depressurization Change external gas tank when pressure drops below 300 psi It waste is backing up into external waste ventilation IMMEDIATELY PRESS THE PAUSE soft key and empty the waste bottle How to set an interruption to synthesis 1 Follow the directions on page 2 19 to determine what cycle and module Synergy is currently running 2 Consult Table 2 5 to determine where to interrupt synthesis Choose the first possible module in the current or next cycle For example if you need to change the HBTU bottle and Synergy is running Cycle 1 Module c set the interrupt to occur at Cycle 1 one Module g 3 Press the set int soft key on the Run Monitor to go to the first Set Interrupt Menu The first Set Interrupt Menu displays the modules that make up Line 1 in the pre programmed set
195. le valve May only toggle valve functions Turn off by using opposite function Function does not work in Manual Control Incorrect edit of module or function Function cannot execute requested action Step time too short to execute function Attempted to turn on more than 8 valves Attempted to operate unavailable valve Attempted to execute undefined function May not move wheel when jaw is closed May only toggle valve functions Missing module list or number of repeats Attempted to run module with no steps No function number found in module line Do not press the PAUSE soft key without first carefully assessing what module is running and the importance of interrupting the synthesis As discussed in Operator Interruptions to Synthesis on page 2 19 only interrupt synthesis at the beginning of either module g module j or module i Caution If liquid waste is backing up into the tubing that goes to external waste ventilation press the PAUSE soft key immediately and empty the waste bottle Run a Flow Test during a synthesis The Synergy controller will not let you run flow test simultaneously with a synthesis If you must run a flow test to check re agent delivery or the conductivity cell either wait until the synthesis is complete or end the synthesis before the END cycle To end the synthesis prematurely see How to terminate a synthesis on page 7 11 03 2002 Troubleshooting 7 23 Applied Biosystems Be
196. leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 03 2002 Part Number 902168 Rev C an Applera business
197. lied Biosystems Main Menu Editing Pre Programmed Runs You can synthesize most peptides with the pre programmed modules built into Syn ergy operating software Occasionally you may want to edit the lines in the Run File to perform the following tasks e Extend coupling time e Double couple one or more amino acids in a sequence Svnthesize a peptide that contains a non standard residue such as a d amino acid e Acetylate the N terminal amino acid e Add residues to an existing peptide resin In this chapter we describe how to use the Run Editor Menu to perform these tasks The Run Editor Menu A run contains all the module lines necessary to perform a complete peptide synthe sis A standard run consists of the following three lines begin b f to solvate the resin e cycle line j dac g f i to couple residues e end de to deprotect and dry the peptide resin Edit amp Print Menu Run Editor Menu begin set up amp run edit amp manual self report control print control test run module function editor editor editor BEG gt C 0 Repeat 1M bf ADDL ERASE L a f PRINT Figure 6 1 How to go to the Run Editor Menu 03 2002 Advanced Operations 6 3 Applied Biosystems Run Editor Menu begin Run Editor Menu line Run Editor Menu end The Run Editor Menu has three parts or sub menus BEG begin L cycle l
198. lve if one exists Turn on the external gas regulator Adjust the external regulator pressure to 55 psi a Perform an external gas delivery leak test by turning the regulator knob 3 5 complete turns counterclockwise As you turn the knob the lower pressure gauge needle immediately drops 1 5 psi and then stabilizes Note the reading on the lower gauge when it stops dropping Wait five minutes b If the lower gauge reading has remained stable after 5 minutes there are no leaks in the external gas delivery system Continue with step 5 c If the regulator needle moves down more than I psi in 5 minutes there is a leak in the external gas delivery line Fill a large syringe with soapy water and squirt the solution around the connections between the regulator Synergy and the gas delivery line to locate leaks Tighten any leaky connections Repeat the external gas delivery leak test that starts at step a If you cannot locate the source of the leak do not continue this procedure Call Applied Biosystems Technical Support for assistance 5 Adjust the external gas tank regulator pressure to 65 psi 6 With the Pressure Vent switch in the Vent position use a 1 4 in wrench to remove the loop attached at the Pressure and Delivery ports Attach the D delivery line from the DMF bottle cap to the Delivery port Figure 2 17 Attach the P pressure line from the DMF bottle cap to the Pressure port Remove the 4 empty re
199. ly into both of its luer fittings Look at Figure 4 10 on page 4 14 for an illustration of the PSC placement and make appropriate adjustments If adjusting the PSC does not eliminate this pressure failure examine the inner ring of the female luer fitting on either end of the PSC These rings should be concentric without irregularities or cracks Check left and right transfer vessels Check that the transfer vessels have been screwed tightly in place with two delivery lines inside each vessel Pressure Test Failure during a reagent prime Check bottle seals and cap See instructions on the preceding page for checking bottle seals and cap after a bottle change Pressure Test Failure during Flow Test 1 2 3 4 5 or 7 Check PSC Reagent leaks can occur around the flow test calibration tube if it has not been pushed firmly into both of its luer fittings Look at Figure 4 10 on page 4 14 for an illustration of the PSC placement and make appropriate adjustments 03 2002 Troubleshooting 7 17 Applied Biosystems Pressure Test Failure during Flow Test 6 Pump AAC e Check the wheel test fixture in the amino acid column wheel Do not use an AAC that has been opened to perform this leak test Before you can check the wheel test fixture you have to jump over most of the steps in Flow Test 6 to Step 26 Function 53 OPEN JAWS See the procedure How to jump over steps in a module Replace the column in position 1 one on the amino aci
200. mv millivolt values corresponds to the trace line for Trace Features 1 through 4 and Trace Feature 7 on the conductivity trace see Figure 2 7 on page 2 17 for examples of all the Trace Features The Lower mv value corresponds to the trace line for Trace Features 5 and 6 Lower mv trace Upper mv trace Se FG p 1 b Figure 4 3 The Upper and Lower conductivity trace lines 3 Use the arrow key to move the cursor to either the Upper or Lower mv value Use the alphanumeric keys to increase or decrease the value When you increase the value you increase the range of millivolts represented by the trace So if the conductivity trace runs off the page increase the range to confine the trace within the page limits When you decrease the value you decrease the range of millivolts represented by the trace Decrease the value when the peaks on the conductivity trace are too short 4 Press the Menu key to move out of the Chart Recorder Menu How to change the chart speed In addition to the conductivity trace lines every printout has evenly spaced back ground dots The space between each vertical row of dots represents 5 minutes of data The chart speed has two settings I mm min and 2 mm min A trace printed ata chart speed of I mm min is half the width and uses half the amount of paper than the same 03 2002 Maintenance and Self Tests 4 5 Applied Biosystems trace printed at 2 mm min The he
201. n Menu Select action for MODULE a 19 steps view copy a T PRINT Module Editor Menu W 1 1 Press the edit amp print soft key in the Main Menu 2 Press the module editor soft key in the Edit amp Print Menu In the Module Editor Menu the cursor appears under a module name in the top line of the LCD Pre programmed synthesis modules are named a b c d e f g h i and j User defined modules are given the upper case letter names A B C D E F G H I and J 3 Press the Previous or Next keys to change the module letter As an alternative you may press the letter key for the module you wish to print 4 When the top line of the LCD displays the desired module name press the PRINT soft key 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 9 Applied Biosystems Table 5 3 Stepwise Description of Procedures within each Pre programmed Module Module Steps Synthesis procedure a 1 8 Measure activators HBTU and DIEA 9 16 Cycle activators though AAC 17 22 Begin transfer of activated amino acid to PSC b 1 6 Pressure test PSC 7 9 Position first AAC in jaws 10 17 Solvate PSC resin and displace air from PSC c 1 10 Cvcle coupling solution through PSC for twice the time used to deprotect module d Step 5 9 d 1 6 Pressure test PSC 7 9 Pressurize PIP bottle and deliver DMF to PSC 10 PIP and DM F flow through PSC for 60 seconds 11 14 Monitor conductivitv
202. n wheel does not move properly If the optical sensors do not detect a column in the jaws synthesis ends When a mechanical failure interrupts synthesis the LCD displays an error message on the top line Make a note of this error message and refer to Error Messages on page 7 20 Press the OK soft key to go to the Run Monitor when an error message appears 2 18 Routine Operation 03 2002 Applied Biosystems Operator Interruptions to Synthesis IMPORTANT The procedures described in this section should be used only in emergencies and not on a routine basis Before each synthesis follow the steps described at the beginning of this chapter to prevent unnecessary interruptions to synthesis When a problem requires immediate attention you can interrupt synthesis in the Run Monitor However before you interrupt a synthesis you should determine what module or stage in the process the instrument is performing and how an interrup tion at that stage might affect the final product Then you can determine at what fu ture module you can program the instrument to interrupt synthesis without adverse affects on the final product Table 2 4 lists the module names and the operation as sociated with that module Table 2 4 Pre programmed Instrument Modules and Operations Module Operation a Activation b Begin synthesis c Coupling d Deprotection e End svnthesis f reagents Flow through valve blocks and PSC g Gurgle solvents was
203. n the instructions presented here we describe glassware and reagent volumes for both small scale and large scale cleavages Large scale recommendations are print ed in regular type with the corresponding small scale recommendations in bold face italics and in parentheses Two stage cleavage procedure This TFA cleavage procedure is described in two stages the cleavage reaction fol lowed by crude peptide recovery We describe two different recovery procedures centrifugation and vacuum filtration Choose either of these recovery methods de pending on the equipment you have access to and the applicable federal state and local laboratory regulations Post cleavage recommendations After cleavage you may freeze dry or lyophilize the peptide to remove water and volatile salts If you don t have access to a lyophilizer you may use a Speed Vac U centrifuge to remove liquid from the crude peptide in solution To enhance removal of scavengers you may dissolve the lyophilized peptide and re lyophilize Store the lyophilized peptide in a desiccator If your application requires further purification the lyophilized peptide can be re dissolved and then purified by reverse phase HPLC Further reading The information in this section is only an introduction to post synthesis procedures See page 3 8 for a list of references you can read for more information on post cleavage analysis and purification of synthetic peptides 03 2002 P
204. n transfer to PSC 20 P F PAUSE 1 21 P F TIME 60 22 67 L gt PSC gt WASTE 15 Substitute time 5 18 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Module b begin synthesis Step Function f Function Name Time Action Description 1 8 P F TIME 60 2 3 BEGIN LOOP 999 3 84 PRS PSC 10 Pressure test PSC 4 88 PRS TEST 60 5 89 PRS TEST 5 6 4 END LOOP 1 7 53 OPEN JAWS 5 8 51 HOME AAG 5 Position first AAC 9 52 CLOSE JAWS 5 10 3 BEGIN LOOP 3 11 31 DMF gt BVB 3 12 34 DMF gt PSC B 30 Solvate synthesis resin and 13 1 WAIT 30 displace air from PSC 14 30 DMF gt TVB 3 15 33 DMF gt PSC 30 16 1 WAIT 30 17 4 END LOOP 1 Module c coupling Step Function Function Name Time Action Description 1 64 PSC PATH 1 2 Begin loop 00 3 BEGIN LOOP 4 Cycle coupling solution through 4 60 PUMP 14 the PSC for twice the 5 65 PSC PATH deprotection time 6 61 XFER 2 7 64 PSC PATH 1 8 END LOOP 1 9 End loop 1 10 65 PSC PATH 1 Substitute time 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 19 Applied Biosystems Module d deprotection Step Function f Function Name Time Action Description 1 9 P F PAUSE 1 2 3 BEGIN LOOP 999 Pressure test PSC 3 84 PRS PSC 10 4 88 PRS TEST 60 5 89 PRS TEST 1 6 4 END LOOP 1 7 30 DMF gt TVB 60 8 37 DMF gt PSC 60 Flow DMF through the PSC 9 86 PRSPIP 1 Pressurize the
205. nal gas pressure readings before resuming synthesis The run file cannot resume because the run file was modified The run file can not resume because the module executing was modified Chapter 6 describes how to use the Run Editor Menu and the Module Editor Menu You may edit either of these menus during a synthesis without affecting the synthesis because the Synergy controller looks at the instructions in the Run File only at the beginning of a synthe sis Either of these messages appear after a powerfailure because three sequential events happened you started a synthesis you edited a run or a module during the synthesis and then a powerfailure occurred during the synthesis When the power returned the Synergy controller recognized the editions did not know what instruc tions to follow and so ended the synthesis The run cannot resume because the function file was modified When this error message appears Synergy no longer has values for the measured delivery of HBTU and DIBA As a result no activators will be delivered during module a This mes sage is most likely to occur after an Exception Message has caused an automatic re start Run Flow Test 8 and Flow Test 9 to calibrate delivery of HBTU and DIEA be fore performing a synthesis Exception Messages Software errors unexpected software events that confuse the controller rarely occur When one occurs Synergy emits a loud beep and an Exception Message Table 7 5
206. ng and deprotection Diagnosis and cure In Peptides Chemistrv and Biologv Proceedings of the Twelfth American Peptide Symposium ed J A Smith and J E Rivier 589 590 ESCOM Leiden 5 Mutter M Altmann K H Belloff D Florsheimer A Herbert J Huber M Klein B Strauch L Vorherr T and Gremlich H U 1985 The impact of secondary structure formation in peptide svnthesis In Peptides Structure and Function Proceedings of the Ninth American Peptide Svmposium ed C M Deber V J Hrubv and K D Kopple 397 405 Pierce Chemical Co Rockford Ill Kent S B H 1985 Difficult sequences in stepwise peptide synthesis Common molecular origins in solution and solid phase ibid 407 414 Live D H and Kent S B H 1983 Correlation of coupling rates with physicochemical properties of resin bound peptides in solid phase synthesis In Peptides Structure and Function Proceedings of the Seventh American Peptide Symposium ed V J Hruby and D H Rich 65 68 Pierce Chemical Co Rockford Ill Atherton E and Sheppard R C 1985 Detection of problem sequences in solid phase synthesis In Peptides Structure and Function ed C M Deber V J Hruby and K D Kopple 415 418 8 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 5 Applied Biosystems time is twice that of the deprotection to assure adequate coupling reaction for most syntheses Post synthesis Cleavage and Side
207. ng group activating group Figure 5 1 Schematic representation of steps in solid phase peptide synthesis 1 Merrifield R B 1963 Solid phase peptide synthesis The synthesis of a tetrapeptide J Am Chem Soc 85 2149 2 Merrifield R B Stewart J M and Jernberg N 1966 Instrument for automated synthesis of peptides Anal Chem 38 1905 1914 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 3 Applied Biosystems Amino acid derivatives The raw materials of SPPS are amino acid derivatives During synthesis chemical reactions can occur at three different locations on an amino acid at the carboxyl group COON at the a amino group NH2 and at some side chain functional groups R Amino acids are derivatized to prevent unwanted reactions at side chain groups and to control reactions at the a amino group Derivatives used on Synergy have Fmoc protecting groups attached to the cL amino groups The amino acids shown in Figure 5 1 also have side chain protecting groups though not all side chains have to be protected Deprotection During deprotection the Fmoc 9 fluorenylmethylcarbonyl protecting group is re moved to make the c amino group on the peptide resin accessible to a chemical re action with an activated amino acid As the Fmoc protecting group is removed a conductive piperidine carbamic acid salt is generated in the deprotection solution Activation Activation prepares
208. nisole and 50 uL 5 uL EDT Lastly add 900 uL 90 uL TFA 4 Cap the tube and agitate it on an orbital shaker for one hour at room temperature If an orbital shaker is not available briefly shake the tube by hand once every 15 minutes for one hour Note Increase the reaction time approximately one hour for each Arg Pmc in the peptide 5 Add 15 mL 2 mL MTBE to the contents of the conical tube Tightly cap the conical tube and vigorously vortex the contents to completely suspend the peptide in the MTBE solution Vortexing should completely break up any sticky clumps of precipitated material from the sides of the tube Recover the crude peptide from the cleavage solution Following step 5 of the preceding cleavage reaction procedure you must separate the crude peptide from impurities and scavengers in the ether solution Here we sug gest two methods for recovering centrifugation and filtration In both procedures impurities are first removed with multiple MTBE washes and then the peptide is dis solved in water or another solvent With the centrifugation procedure you need no additional labware In those labora tories where regulations forbid centrifugation of MTBE the filtration procedure may be used as an alternative To perform the filtration procedure you need a vac uum system and filtration glassware Recovery by centrifugation This procedure starts with the MTBE suspension of crude peptide from step 5 of the pre
209. of deprotection until change in conductivitv in 30 seconds lt 1 15 17 Depressurize PIP and wash PSC with DMF for 5 min e 1 2 Wash PSC with DMF 5 minutes 3 4 Wash PSC with THF 5 minutes 5 8 Gas dry PSC 10 minutes f 1 6 Pressure test PSC 7 Start delivery of DMF to PSC 8 11 Monitor conductivity until change in conductivitv in 30 seconds 2 1 12 Stop delivery of DMF to PSC g 1 2 Drain coupling solution to waste bottle 3 8 Wash pump with 5 DMF deliveries and drain DMF wash though AAC to waste bottle 9 11 Dry AAC 12 16 Dry transfer vessels i 1 2 Open jaws and move wheel to next position j 1 8 Close jaws and pressure test AAC h 2 Interrupt svnthesis for manual addition of non standard residues 3 10 Measure activators HBTU and DIEA 11 14 Begin transfer of activated amino acid to PSC The Synergy Pump The system of pneumatically operated left and right transfer vessels on Synergy acts as a pump that directs reagent flow throughout the activation and coupling steps of peptide synthesis Pressurized gas and a series of valves deliver reagents through the pump to mix with the contents of either the peptide synthesis column or the amino acid column Continuous delivery eliminates bubbles from the conductivity cell and enhances accurate conductivity monitoring in the peptide synthesis column see 5 10 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Conductivity Monitoring on page 5 5 This pumping syst
210. of double coupling See page 2 16 for a discussion of Trace Features suaystsorg pauddy Applied Biosystems Sequence dependent slow deprotection Sequence dependent slow deprotections arise as a result of the interactions within the peptide resin and their influence on reagent diffusion rates see The Changing Peptide Resin Structure on page 5 5 Although it is difficult to predict which se quences will reduce reagent diffusion rates conductivity monitoring can respond to and may at times eliminate their adverse effects on synthesis Figure 7 2 shows the conductivity trace of a sequence dependent slow deprotection Reagent bottles and reagent delivery e Carefully examine all the reagent bottles on Synergy to verify that they all contain sufficient amount of reagent initial resin solvation Le AL Figure 7 3 Conductivity trace without deprotection peak Trace Feature I Figure 7 3 shows a conductivity trace without Trace feature 1 a deprotection peak See page 2 16 for a discussion of Trace Features This shows what happens when there is no piperidine delivery due to an empty piperidine bottle on Synergy e Verify that reagents have not expired Applied Biosystems reagents can be safely used up to a year after their date of shipment to your facility We recommend you record on all Synergy reagent bottles the date of shipment After preparation 0 2 M HBTU Reagent Preparation on page 2 7 the HBT
211. of the synthesis You may leave the instrument unattended during this self test Run ended 13 58 Run began 10 54 Cvclesrun 3 OK 6 This displav appears when the svnthesis is completed Press the OK soft kev Compare the Trace Features of the printout from vour svnthesis of LAGV to the example shown in Figure 2 7 on page 2 17 If anv Trace Features are missing call Applied Biosvstems Technical Support For descriptions of irregular traces see Irregular Conductivitv Traces on page T 3 03 2002 Maintenance and Self Tests 4 33 Applied Biosystems Keyboard Self Test Use the keyboard self test to check all the keys associated with the LCD liquid crys tal display and the keyboard 1 To begin the keyboard test press the keyboard soft key in the Self Test Menu Figure 4 6 on page 4 8 Press all keys MENU to exit S1S2838485 lt gt 0123456789 PREV NEXT DEL 2 Press each of the 5 soft keys under the LCD As you press each key the program emits a beep and the name of the key SI S2 etc disappears from the LCD 3 Press the left and right arrow kevs and each of the numeric keys 0 9 4 Press the keys labelled Previous Next and Delete 5 Press the Menu key If the keyboard is working properly the next display reads Test Passed OK 6 Press the OK soft key If the Test failed message appears on the LCD call Applied Biosystems Technical Sup
212. olumns for Your Synthesis 2 10 Check the Run File 2 13 Start the Automated Synthesis on Synergy 2 14 How to start synthesis in the Run Control Menu 2 14 Interpreting the conductivity trace 2 16 Synthesis Interruptions 2 18 Pressure Test Failures and Error Messages during syntheses 2 18 Operator Interruptions to Synthesis 2 19 How to determine the cycle and module Synergy is currently running 2 19 How to set an interruption to synthesis 2 20 How to replace nearly empty reagent bottles during synthesis 2 22 How to replace an empty external gas tank during synthesis 2 23 How to remove and replace the full waste bottle during synthesis 2 23 How to replace or re align printer paper during synthesis 2 23 Powerfailures 2 25 How to recover from a long powerfailure in module a or h 2 26 Synergy Shutdown Procedure 2 30 Return to Operation after Shutdown 2 33 03 2002 Routine Operation 2 1 Applied Biosystems Synthesis column position LCD and keyboard Four reagent bottles A Brief Description of Synergy To Vent Pressure Vent switch Transfer vessels Amino acid column wheel Power on off button Figure 2 1 Synergy after installation The Applied Biosystems Synergy Peptide Synthesizer combines Fmoc 9 fluorenylmethylcarbonyl synthesis chemistry with HBTU 2 1 H benzo triazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate activation to per form solid ph
213. olvation Or add 75 umol of the compound to 300 uL of a 1 1 solution of NMP DMSO Caution Undissolved particulate matter may damage the valves on Synergy Do not put cloudy solutions or undissolved material in the left transfer vessel If the solution of pre activated compound in DMF is cloudy or contains undissolved material filter the solution A suitable filter may be prepared by pushing a small piece of cotton or glass wool into a disposable Pasteur pipette as shown in Figure 6 16 je cotton B Figure 6 16 Prepare a filter from a disposable pipette 03 2002 Advanced Operations 6 17 Applied Biosystems 9 Use a pipette to transfer the resulting solution to the left transfer vessel Screw the left vessel tightly in place 10 Press the PAUSE soft key to continue synthesis How to use module h to add a pre activated compound 1 Follow the previous procedure through step 7 2 Dissolve 75 umol of the pre activated compound in 1 5 mL DMF Caution Undissolved particulate matter may damage the valves on Synergy Do not put cloudy solutions or undissolved material in the left transfer vessel If the solution of pre activated compound in DMF is cloudy or contains undissolved material filter the solution A suitable filter may be prepared by pushing a small piece of cotton or glass wool into a disposable Pasteur pipette as shown in Figure 6 16 Use a pipette to transfer the resulting solution to the left tr
214. op of liquid in the right vessel If you see liquid in the left vessel press the Yes soft key If there is no liquid in the left transfer vessel press the No soft key 03 2002 Maintenance and Self Tests 4 25 Applied Biosystems Test Passed OK 7 When this display appears press the OK soft key to complete the flow test If you have pressed the No soft key at any time during this flow test the LCD displays the message Test failed Press the OK soft key If Flow Test 6 fails e Check that the left and right transfer vessels are screwed on tightly Check the two lines that go into each of the vessels for kinks or obstructions There should be sufficient space 1 mm between the open tips of these lines and the bottom of the vessels to allow unobstructed flow of liquid After performing these checks repeat Flow Test 6 e Ifthis flow test fails again run Flow Test 3 to check DMF delivery page 4 22 and run a Leak Self Test page 4 13 to check for pressure leaks e Ifa pressure test failure occurs during this flow test read What to do after a pressure test failure on page 7 12 Flow Test 7 Pump Cell The pump in the name of this flow test refers to the configuration of transfer ves sels that circulates liquids during synthesis During this flow test two conductivity readings are taken The first one measures conductivity of DMF delivered to the flow test calibration tube Next a small amount o
215. ormation on the development of solid phase peptide synthesis Fields G B and Noble R L 1990 Solid phase peptide synthesis utilizing 9 fluorenylmethoxycarbonyl amino acid Int J Peptide Protein Res 35 161 214 Grant G A ed 1992 Synthetic Peptides A User s Guide W H Freeman and Company New York Stewart J M and Young J D 1984 Solid phase Peptide Synthesis Second Edi tion Pierce Chemical Company Rockford Illinois 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 23 Applied Biosystems 6 Advanced Operations Contents Editing Pre Programmed Runs 6 3 The Run Editor Menu 6 3 How to print a Run File 6 4 How to edit a run 6 4 How to erase a line in the Run Editor 6 5 How to start the synthesis after editing the run 6 5 Editing a Run to extend coupling time 6 6 How to extend coupling time 6 7 Double Coupling 6 10 How to program a double couple 6 10 Adding residues to a peptide resin 6 13 Has the PSC been opened 6 13 Is the Fmoc group still on the peptide 6 13 How to add more amino acids to a dry peptide resin WITHOUT 6 14 the Fmoc group How to add more amino acids to a drv peptide resin WITH an 6 15 Fmoc group Module h 6 16 How to use module h to activate non standard amino acids on Svnergv 6 16 How to use module h to add a pre activated compound 6 18 The Module Editor Menu 6 20 How to copy a module 6 20 How to view the steps in a module 6 21 03 2002 Advanced Operations 6 1 App
216. ost Synthesis Procedures 3 3 Applied Biosystems Cleavage Reaction Procedure WARNING CHEMICAL HAZARD Trifluoroacetic acid is an extremely dangerous and corrosive liquid Always wear gloves a lab coat and eye protection when handling it Note Thioanisole and EDT produce an unpleasant offensive stench Store and handle these reagents in a fume hood To diminish odors dispose of pipette tips that come in contact with these chemicals in a container of bleach Required Reagents TFA Trifluoroacetic acid thioanisole EDT 1 2 ethanedithiol MTBE methyl t butyl ether Equipment and Labware pliers or a decrimping tool ABI P N 400414 polypropylene graduated 50 mL 15 mL conical tubes with screw caps orbital shaker with an attached tube rack 1 With one hand firmly grasp the bottom of the peptide synthesis column PSC that contains the peptide resin With the other hand use pliers or a decrimping tool to carefully pry off the column cap Figure 3 1 Figure 3 1 Removing the PSC cap with pliers 2 Place the entire contents of the peptide synthesis column approximately 50 200 mg peptide resin in a 50 mL conical tube For small scale place 3 4 Post Synthesis Procedures 03 2002 Applied Biosystems 10 20 mg peptide resin in a 15 mL conical tube Tap the conical tube on a bench top to insure that all the resin falls to the bottom of the tube 3 Working in a fume hood first add 50 uL 5 uL thioa
217. ote If the printer is running when a powerfailure occurs you must realign the top of the paper in the HP PaintJet when power returns Follow the instructions in the ThinkJet Owner s Manual When power first resumes after a powerfailure Synergy displays the message There was a flower failure while the synthesizer was running Press the OK soft key after this message and Synergy displays the Main Menu If a synthesis was running before the powerfailure occurred the synthesis continues until the controller encounters Function 9 P F PAUSE If the length of the power failure exceeds pre determined limits Synergy displays the message The power was off too long to resume the run When an interruption occurs during synthesis you have to decide whether or not to continue synthesis Your decision should be based on the following subjective cri teria how urgently you need the peptide how much you have already invested in time and energy how much more you are willing to invest to synthesize this peptide During modules a and h the activation modules amino acids are rapidly activated For optimal chemistrv results coupling should occur immediatelv after activation especiallv after activation of arginine The greater the amount of time that passes be fore activated amino acids come in contact with the deprotected amine the greater the chances that side chain reactions will occur If a powerfailure occurs in anv module except modules a
218. outine Operation 2 21 Applied Biosystems Cycle number Module and Step number of the interruption setting The Cycle number of the interruption appears after the word Cycle the cursor appears under the Module letter the Step number appears after the word Step To change the interruption setting press the CLEAR soft key and repeat this procedure starting on page 2 20 To return to the Run Monitor press the Menu key When synthesis is interrupted the PAUSE soft key appears on the Run Monitor How to replace nearly empty reagent bottles during synthesis IMPORTANT If during a synthesis you notice that a reagent bottle on Synergy is completely empty and the conductivity trace is unusual the synthesized peptide will probably be inferior It is more efficient to end the synthesis and start over with full reagent bottles and fresh columns 1 Ifthe HBTU DIEA THF or DMF bottles are almost empty follow How to set an interruption to synthesis on page 2 20 Set the interruption at step 1 of either Module g j or i When synthesis is interrupted the PAUSE soft key appears on the Run Monitor If the piperidine bottle is almost empty you do not need to depressurize the system to change the bottle A soft hiss of pressurized gas escapes when you unscrew the ratchet cap Do not change the piperidine bottle when Module d is running When Synergy interrupts synthesis switch the Pressure Vent switch to Vent Wait 30
219. outs on page 5 18 for printouts of all the pre programmed synthesis modules The pre programmed modules may be viewed or copied but they cannot be edited In the Module Editor Menu you may edit any module that has an upper case letter name such as A B C etc To edit one of the pre programmed modules you must first copy all its steps into a module with an upper case letter name Modules with upper case letter names are called user defined modules How to copy a module run module function Edit amp Print Menu editor editor editor Select action for MODULE a 19 steps Module Editor Menu view copy a f PRINT Copy MODULE a to MODULE A 19 steps copy MODULE A gt S 1 F 10 GAS gt TVB T 5 Press ADDS ERASE S Menu key Save edit session changes cancel NO YES Figure 6 18 How to copy a module 6 20 Advanced Operations 03 2002 Applied Biosystems In the Main Menu press the edit amp print soft key In the Edit amp Print Menu press the module editor soft key In the Module Editor menu Figure 6 18 use the alphanumeric keys to select the module you wish to copy The letter name of the module appears after the words Select action for MODULE Press the a P soft key to toggle between lower case letters and upper case letters Press the copy soft key to copy the selected module into a module with an
220. ow Test 8 Calibrate HBTU Flow Test 9 Calibrate DIEA Cycle Test Keyboard Self Test Wheel Self Test Circuit Self Test 4 22 4 24 4 26 4 27 4 30 4 32 4 34 4 34 4 36 5 Principles of Solid Phase Peptide Synthesis on Synergy Solid Phase Peptide Synthesis Amino acid derivatives Deprotection Activation Coupling Solid Support The Changing Peptide Resin Structure Conductivitv Monitoring Post svnthesis Cleavage and Side Chain Deprotection The Svnergv Svnthesis Process Svnthesis Columns and Reagents Modules and Functions The Svnergv Pump Illustrated Module Descriptions Module a extraction and activation Module b begin synthesis Module c coupling Module d deprotection Module e end synthesis Module f DMF flow to PSC Module g wash solvents through AAC and transfer vessels Module h alternate activation Module i incremental movement of amino acid column wheel Module j jaws close on AAC Annotated Module Printouts Recommended reading 5 3 5 4 5 4 5 4 5 4 5 4 5 5 5 5 5 6 5 7 5 7 5 8 5 10 5 11 5 12 5 13 5 14 5 15 5 16 5 16 5 17 5 17 5 17 5 17 5 18 5 23 03 2002 iii Applied Biosystems 6 Advanced Operations Editing Pre Programmed Runs The Run Editor Menu Editing a Run to extend coupling time Double Coupling Adding residues to a peptide resin Has the PSC been opened Is the Fmoc group still on the peptide Module h The Module Editor Menu 7 Troubleshooting Ir
221. ow Test 8 or Flow Test 9 Trying to jump into a software program loop Incorrectly using Manual Control Menu Incorrectly editing a module or a function Table 7 6 lists operator generated error messages and operator actions that may pre cede the appearance of the message Use Manual Control during a synthesis The Synergy controller will not let you use the Manual Control Menu while a synthesis is running unless you first press the PAUSE soft key in the Run Monitor IMPORTANT When you press the PAUSE soft key during a synthesis you interrupt the module that is currently operating Some module interruptions could interfere with the peptide synthesis and damage the final peptide product 7 22 Troubleshooting 03 2002 Applied Biosystems Table 7 6 Operator generated Error Messages Operator Action Error Message Use Manual Control during a synthesis Pause or end run to operate manually Run a Flow Test during a synthesis Cannot flow test when running Begin a synthesis when printer printing data The monitor file is in use Start Flow Test 6 without AAC Jaw closed with column missing Start Wheel Test without AAC No column in position 1 Start synthesis without running Flow Test 8 HBTU and DIEA need to be calibrated or Flow Test 9 Try to jump into loop Unmatched Begin Loop End Loop function Incorrect use of Manual Control Attempted to turn on more than 8 valves Attempted to operate unavailab
222. pirate the MTBE and add 5 mL I mL distilled water to the conical tube Gently vortex the tube to dissolve the crude peptide e Ifthe peptide is basic it will probably dissolve in H O If it doesn t dissolve well in water try adding a few drops of glacial acetic acid or acetonitrile Do not let the concentration of glacial acetic exceed 15 v v because high concentrations of acetic acid do not lyophilize e Ifthe peptide is acidic dropwise add NH OH to solubilize e If the peptide is hydrophobic add acetonitrile to solubilize 6 Separate the crude peptide solution from the spent resin by passing it through the filter Figure 3 2 and into a second 50 mL 15 mL polypropylene tube 7 Re use the pipette and the filter to repeat Steps 5 and 6 two more times to give a total of 15 mL 3 mL solution 8 Place a lint free tissue over the top of the tube and freeze the peptide solution by placing it in either liquid nitrogen or a container of isopropanol and dry ice The peptide solution may now be lyophilized Recovery by vacuum filtration This procedure starts with the MTBE suspension of crude peptide from Step 5 of the cleavage reaction procedure WARNING CHEMICAL AND FIRE HAZARD MTBE is volatile and highly flammable Perform this procedure in a working fume hood Required Reagents MTBE distilled water glacial acetic NH4OH or acetonitrile Equipment and Labware a medium porosity fritted glass funnel 15 mL 2 cl
223. pment required for the safety of individuals working with chemicals including fire extinguishers first aid equipment safety shower and eye wash fountain and spill cleanup equipment Instrument Access Side panel to be removed only by trained personnel Figure 1 1 Front view of Synergy Side panel to be removed only by trained personnel Rear panel to be removed only by trained personnel Figure 1 2 Rear view of Synergy Figure 1 1 and Figure 1 2 illustrate instrument accessibility 03 2002 Synergy Safety Guidelines 1 5 Applied Biosystems Fuses For continued protection against the risk of fire replace fuses only with listed and certified fuses of the type and rating specified on the rear of the instrument Jaw Assembly The jaw assembly is operated by a pneumatic valve under high pressure Keep fin gers away from the jaws during the turntable self test and whenever the turntable is in motion Hazardous Chemicals This instrument contains hazardous chemicals in bottles lines and valve blocks Do not operate or interact with the instrument until vou have read all the Material Safetv Data Sheets MSDSs for chemicals associated with this instrument Table 1 1 lists the chemicals used on Synergy Table 1 1 Chemicals for FastMoc Chemistry on Synergy Bottle Volume Chemical mL A 40 0 2 M 2 1H benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophospha
224. port Wheel Self Test Use the Wheel Self Test to test operation of the autosampler jaws the jaw sensors and the mechanism that turns the amino acid column wheel During this test the wheel rotates to position one where the jaws close down on a wheel test fixture WARNING PERSONAL INJURY HAZARD The autosampler jaw is operated by a pneumatic valve under high pressure Keep your fingers away from the jaws during this wheel test 4 34 Maintenance and Self Tests 03 2002 Applied Biosystems 1 To begin the Wheel Self Test press the wheel soft key in the Self Test Menu Put a test fixture in position one on the wheel START 2 Place a wheel test fixture in position 1 one on the amino acid column wheel Figure 4 18 IMPORTANT Place only one wheel test fixture on the wheel for this test Figure 4 18 Placing a wheel test fixture in position one on the wheel 3 Place the wheel on top of the hub on the right side of Synergy Slowly rotate the wheel until the protruding pin on the bottom of the wheel slips into the corresponding groove in the hub 4 Press the START soft key The wheel should now rotate so that the wheel test fixture in position one moves into the jaw assembly Next a series of tests check the motor and the jaw sensors If anv of these tests detect a mechanical failure an error message appears on the screen See Error Messages on page 7 20 for an explanation of error messages Press
225. port soft key 2 nthe Setup amp Report Menu press the print reports soft key 3 In the Print Reports Menu press the MONITOR DATA soft key 03 2002 Troubleshooting 7 9 Applied Biosystems Pressure Test Failures A pressure test failure can result from attempting to run Synergy with an empty gas tank an obstruction in the gas line or a leak in the pressurized gas system Two functions control pressure tests Function 88 PRS TEST and Function 89 PRS TEST When Function 88 is active the Synergy controller takes an initial pressure reading and turns off incoming gas for a fixed amount of time When the fixed amount of time has passed Function 89 directs the controller to take a second pressure reading and compare it to the initial reading If the difference between these two readings exceeds the pre determined acceptable values the pressure test fails Synergy is shipped with a pre set internal gas pressure range of 4 0 6 0 psi During installation the internal gas pressure is set to 5 0 0 1 psi Empty Gas Tank For optimal routine operation of Synergy the high pressure gauge on the external nitrogen tank should not drop below 300 psi and the low pressure gauge should be set at 60 75 psi IMPORTANT Always check the gas tank gauges before starting a synthesis If you attempt to run Synergy with insufficient external gas pressure the autosam pler jaws fail first When Synergy detects a drop in the intern
226. position 5 place A Ala in positions 6 and 7 place Q Gln in position 8 03 2002 Routine Operation 2 11 Applied Biosystems Position 9 The N terminal amino acid V Val goes on last in position 9 Figure 2 5 illustrates how when the AACs are in the correct order you can hold up the wheel and read the peptide sequence with the N terminal AAC on the left and the missing C terminal amino acid in the PSC if the PSC contains a pre loaded res in Position 1 Figure 2 5 AACSs in positions on the wheel to synthesize ACP 65 74 Note After a svnthesis the AACs still contain white inert material but very little amino acid remains AACs contain only enough amino acid for one synthesis cycle They cannot be re used few words about amino acid AAC and peptide synthesis PSC columns Applied Biosystems AACs and pre loaded PSCs both contain amino acid and poly meric material The amino acid in the AACs is associated temporarily to an inert polymer When the amino acid is removed from the AAC during synthesis the inert polymer stays in the column In the PSCs the amino acid is bound to a lightly cross linked polystyrene resin The bond between the amino acid and resin in the PSC is not broken until after synthesis is complete during the TFA cleavage procedure de scribed in Chapter 3 IMPORTANT AACs and PSCs cannot be used interchangeably AACs have a light blue label PSCs have a completely white label
227. r the internal gas pressure In the Main Menu press the set up amp report soft key Figure 4 14 In the Set up amp Report Menu press the pressure settings soft key The minimum and maximum pressure settings appear on the top line of the Pressure Settings display The current pressure appears on the bottom line of the LCD set up amp run edit amp manual self Main Menu report control print control test pressure time amp print serial Set up amp Report Menu L settings date reports number more Pressure Min 4 0 psi Max 6 0 psi Pressure Settings Current Pressure 5 3 psi ok Figure 4 14 How to monitor the current pressure Press Menu key to return to Main Menu To adjust the internal gas pressure see Step 2 of the procedure Adjusting the inter nal gas pressure to calibrate DMF delivery on page 4 18 Flow Test 6 Pump AAC This test first checks the flow of reagent from the left transfer vessel through the column on the amino acid column wheel to the right transfer vessel Then it checks the flow from the right transfer vessel back to the left transfer vessel The pump in the name of this flow test refers to the configuration of transfer vessels that circu lates liquids during synthesis 1 Place a wheel test fixture in position 1 one on the amino acid column wheel 2 To begin Flow Test 6 press the flow soft kev in the Self Test Menu Figure 4 6 Th
228. ranty does not apply to the Instrument s valves or reagent lines unless the customer uses only reagents and solvents supplied by AB or those of highest purity available that have been filtered to be free of particulates or those proven to be com pletely dissolved using AB cycles If the use of reagents or solvents supplied by AB or those of highest purity available that have been filtered to be free of particulates or those proven to be completely dissolved using AB cycles causes such hardware damage or contamination during the Warranty Period AB will return the Instrument to specified Mechanical Performance at AB s expense However AB does not guar antee specified Chemical Performance of the Instrument if solvents or reagents other than those supplied by AB are used If the Chemical Performance of the Instrument deteriorates due to solvents and or reagents other than those supplied by AB at the customer s request AB will return the Instrument to specified Chemical Performance and Mechanical Performance at the customer s expense If only reagents and sol vents supplied by AB are used thereafter the Chemical Performance and Mechani cal Performance will be covered in full for the remainder of the Warranty Period AB s obligation under this Warranty is limited to repairs or replacements that AB deems necessary to correct covered defects or failures of which AB is notified prior to expiration of the Warranty Period All repairs and replacements det
229. re 4 1 Synergy after installation printer not shown The Synergy Peptide Synthesizer requires little maintenance This chapter contains descriptions of some minor adjustments you can make and the Self Tests Most of the time you will probably use only two Self Tests the Bottle Self Test page 4 9 to change reagent bottles and Flow Test 8 Calibrate HBTU page 4 27 to calibrate HBTU delivery after changing the delivery line filter for that reagent Procedures in Chapter 7 Troubleshooting may recommend performing other Self Tests under certain conditions All operations described in this chapter can be performed without removing the in strument s exterior panels If a Self Test repeatedly fails there may be a hardware malfunction inside the synthesizer that can best be repaired by a trained Applied Biosystems Service representative If a Self Test repeatedly fails call Applied Bio systems Technical Support for assistance 03 2002 Maintenance and Self Tests 4 3 Applied Biosystems Adjusting the Conductivity Trace Printout In the Chart Recorder Menu you may make three different modifications to the ap pearance of the conductivity trace e Change the scales of the two overlapping trace lines e Change the chart speed Make the date time and cycle number appear before each cycle on the trace Note Although you may make these modifications while the printer is printing a trace the changes will not show up on the trac
230. regular Conductivity Traces Sequence dependent slow deprotection Reagent bottles and reagent delivery Re used AACs Malfunctioning instrument hardware Printer related malfunctions Pressure Test Failures Empty Gas Tank Pressure System Leaks Pressure Test Failures during a synthesis Pressure Test Failure at module b d or f Pressure Test Failure at module j Pressure Test Failures during a Self Test Pressure Test Failure after a Bottle Change Pressure Test Failure during a Leak Test Pressure Test Failure during a reagent prime Pressure Test Failure during Flow Test 1 2 3 4 5 or 7 Pressure Test Failure during Flow Test 6 Pump AAC Error Messages Mechanical Failure or Software Error Autosampler jaw failure Motor assembly or encoder Powerfailure Exception Messages Operator generated Error Messages Incorrect use of Manual Control or incorrect module edit 6 3 6 3 6 6 6 10 6 13 6 13 6 13 6 16 6 20 7 3 7 5 7 5 TT 7 8 7 9 7 10 7 10 7 12 7 13 7 13 7 13 7 15 7 15 7 16 7 17 7 17 7 18 7 20 7 20 7 20 7 20 7 21 7 21 7 22 7 25 03 2002 Applied Biosystems Index Appendix I Material Safety Data Sheets and Synergy Waste Profile Appendix II AB Limited Warranty Appendix III Synergy Parts and Reagents Appendix IV Test Printouts v 03 2002 Applied Biosystems I Synergy Safety Guidelines Contents Synergy User s Manual Conventions 1 3 User Attention Words 1 3 Chemical abbreviations
231. ress the ADD L soft key to start line 3 Enter jdac g fi In this example we want standard cycle lines for all the amino acid couplings after the two double couple lines We leave the default value 1 one after Repeat Figure 6 10 shows the Run File for this example Linett Cycle f Repeat Module List Beg 0 1 bf 1 1 1 jdacgfi 2 2 2 jdacgijacgfi 3 4 1 jdacgfi End 4 1 de Figure 6 10 Run File with a double couple in line 2 6 When vou have finished editing the run press the Menu kev and save the editing session Then continue with the procedure Making a Peptide on Synergy that begins on page 2 5 Start the synthesis in the Run Control Menu as you would any other synthesis IMPORTANT Edited Run lines remain in the Run File until you return to the Run Editor and delete them 6 12 Advanced Operations 03 2002 Applied Biosystems Adding residues to a peptide resin Before you can add more amino acids to a peptide resin you must answer two ques tions 1 Has the PSC been opened 2 Is the Fmoc group attached to the peptide Has the PSC been opened PSCs peptide synthesis columns are sealed to prevent leaks that could damage Synergy This seal is broken when the PSC is opened Caution Do not re use PSCs that have been opened Leaks from a PSC can damage Synergy If the PSC has been opened put 25 umol of the peptide resin in a new empty frit ted PSC Tightly press the top and bottom par
232. rofuran stabilized 200 mL 41 TFA trifluoracetic acid 49 Synergy Waste Profile 55 03 2002 Material Safety Data Sheets and Synergy Waste Profile l 1 Applied Biosystems Material Safety Data Sheets MSDSs and Synergy Waste Profile Acronyms Used in MSDSs AGGIH American Conference of Governmental Industrial Hygienists CASH Chemical Abstract Service Reference Number for Specific Pure Chemical CERCLA Comprehensive Environmental Response Compensation and Liability Act CFR Code Federal Regulations IDLH Immediate Danger to Life and Health LEL Lower Explosion Limit NFPA National Fire Protection Association NIOSH National Institute of Occupational Safety and Health OSHA Occupational Safety and Health Administration PEL Permissable Exposure Limit The federal OSHA limit usually expressed as a TWA for an 8 hour work shift PPM Parts Per Million RTECS Registry of Toxic Effects of Chemical Substances SCBA Self Contained Breathing Apparatus TLV Threshold Limit Value The ACGIH recommended TWA usually for an 8 hour work shift TWA Time Weighted Average UEL Upper Explosive Limit MSDS Table of Contents DIEA DMSO NMP 0 4M N N diisopropylethylamine dimethy1 sulfoxide N methylpyrrolidinone 40 mL 5 DME N N dimethylformamide 4 L 13 HBTU 8 mmoles 2 1 H benzotriazol 1 yl 1 1 3 3 tetramethyl uronium hexafluorophosphate 21 0 2M HOBt DMSO NMP 1 Hydroxybenzotrizole dimethyl sulfoxide N methylpyrrolidinone 40 mL 27 Piperidine 200 mL
233. s described by this last line will be repeated until the jaw sensors detect an empty position on the amino acid column wheel Press the Menu key and save the edit session see How to edit a run on page 6 4 Figure 6 3 shows the printed Run File for this example Follow the procedure Making a Peptide on Synergy on page 2 5 Start the synthesis in the Run Control Menu as you would any other synthesis 6 8 Advanced Operations 03 2002 Applied Biosystems Linett Beg End Cycle O O AG Repeat Module List bf jdacgfi jdaccgfi jdacgfi de Figure 6 6 Sample Run File with extended coupling time in the fifth cycle only IMPORTANT Edited Run lines remain in the Run File until you return to the Run Editor and delete them 03 2002 Advanced Operations 6 9 Applied Biosystems Double Coupling In most instances of incomplete coupling extended coupling time may sufficiently improve synthesis However in some cases especially if the amino acid being cou pled is an arginine you should edit the line to a double coupling With a double cou ple two consecutive AACs on the wheel are activated in one cycle This improves coupling by increasing the concentration of activated amino acid and extending cou pling time Figure 6 8 shows the conductivity trace of a synthesis with incomplete couplings Figure 6 9 shows the conductivity trace when the same peptide was synthesized with doub
234. s used with this instrument can be hazardous and cause injury illness or death Wear protective eye glasses gloves and a laboratory coat when working with these reagents To minimize exposure to and inhalation of these chemicals cap and store the reagent bottles according to instructions found in the MSDSs 1 Pour DMF into two empty 40 mL bottles and one empty 200 mL bottle so that the approximate depth of reagent in each bottle is I cm 2 Switch the Pressure Vent switch to Vent and wait 30 seconds for the internal pressure to drop 2 30 Routine Operation 03 2002 Applied Biosystems 3 Remove all the reagent bottles currently in position on the front of Synergy with the following additional steps e Disconnect the DMF bottle lines at the Pressure and Delivery ports Remove the DMF pressure line first then remove the DMF delivery line Replace these lines with the connecting loop that connected the Pressure and Delivery ports during Synergy shipment Figure 2 15 Remove the cap assembly from the DMF bottle cap and seal the bottle tightly e Replace the HBTU and DIEA bottles with the 40 mL bottles of DMF from step 1 Replace the piperidine bottle with the 200 mL bottle of DMF from step 1 Replace the THF bottle with an empty 200 mL bottle 4 Switch the Pressure Vent switch to Pressure 5 Inthe Main Menu press the self test soft key to go to the Self Test Menu Press the bottle soft key in the Self Test Menu Then pr
235. seconds for the internal pressure to drop to zero Remove all the empty reagent bottles at once and replace them with full bottles Refer to Step 4 of the Bottle Changing Procedure on page 4 9 fora thorough description of this process When the full reagent bottles have been placed on Synergy switch the Pressure Vent switch to Pressure and wait 60 seconds for the internal pressure to stabilize Press the PAUSE soft key to resume synthesis With this procedure you change reagent bottles without going through the standard bottle change procedure described in Chapter 4 The Synergy controller does not check the replaced reagent bottles for leaks until synthesis progresses to module j If a pressure test fails in module j after you have replaced reagent bottles it could indicate a bad bottle seal a crack in the rim of the bottle or a bottle has not been tightly screwed in place 2 22 Routine Operation 03 2002 Applied Biosystems How to replace an empty external gas tank during synthesis 1 Follow the procedure How to set an interruption to synthesis on page 2 20 Set the interruption to occur at step 1 of either Module g j or i 2 When the PAUSE soft key appears on the Run Monitor follow the procedure How to change the external gas tank on page 2 6 3 When the gas tank has been replaced press the PAUSE soft key to resume synthesis How to remove and replace the full waste bottle during synthesis Caution
236. sel through the AAC and to the right transfer vessel Lied i SD Pump through AAC 5 12 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Function 61 Transfer the activated amino acid from the right transfer vessel through the oneway valve to the left transfer vessel Module b begin synthesis PSC is pressurized to test for leaks and DMF is delivered to PSC to solvate the resin C OD ETTE Function 33 Deliver DMF to the peptide synthesis column PSC to solvate the resin and displace air L l DMFsPSC 03 2002 Principles of Solid Phase Peptide Svnthesis on Svnergv 5 13 Applied Biosystems Module c coupling Coupling solution is cycled through the PSC for twice the amount of time used to complete deprotection in module d Function 60 PUMP coupling solution from the left transfer vessel to the PSC and then to the right transfer vessel Pump through PSC Function 61 Transfer the coupling solution from the right transfer vessel through the oneway valve to the left transfer vessel 5 14 Principles of Solid Phase Peptide Synthesis on Synergy 03 2002 Applied Biosystems Function 37 DMF delivery through the PSC Function 40 Delivery of piperidine to the PSC Module d deprotection Piperidine and DMF flow into PSC and after one minute conductivity monitoring begins Conductivity readings are averaged every 30 seconds until
237. sembly 602851 waste port nut ferrule and gripper 230034 bottle cap assembly 1 gal bottle 601396 bottle seals 40 mL bottles 10 pkg 400612 bottle seals 200 mL bottles 10 pkg 400790 safety carrier 1 gal bottle 140041 RS232 printer cable 100390 Synergy Synthesis Reagents by Bottle Position Bottle Position Reagent Description Quantity Part Number A HBTU 8 mmol 401278 HOBt DMSO NMP 40 mL 401279 B DIEA DMSO NMP 40 mL 401254 Piperidine 200 mL 400629 2 THF stabilized 200 mL 401255 10 DMF 4L 400143 The Synergy 100 Cycle Reagent Kit P N 401466 contains enough liquid reagents for approximately 100 synthesis cycles and includes two 200 mL bottles piperidine two 200 mL bottles THF two 4 L bottles DMF one 8 mmol bottle HBTU one 40 mL bottle HOBt DMSO NMP and one 40 mL bottle DIEA DMSO NMP 03 2002 Synergy Parts and Reagents IIl 1 Applied Biosystems Amino acid columns AACs Amino Acid Part Number Alanine Ala 401208 Arginine Arg Pmc 401209 Asparagine Asn Trt 401210 Aspartate Asp OtBu 401211 Cysteine Cys Trt 401212 Glutamine Gin Trt 401213 Glutamate Glu OtBu 401214 Glycine Gly 401215 Histidine His Trt 401216 Isoleucine lle 401217 Leucine Leu 401218 Lvsine Lvs Boc 401219 Methionine Met 401220 Phenvlalanine Phe 401221 Proline Pro 401222 Serine Ser tBu 401223 Threonine Thr tBu 401224 Trvptophan Trp 401225 Tvrosine Tvr tBu 401226 Valine Val 401227 Each amino
238. should appear after the word Print on the Run Control Menu To change any entry on the Run Control Menu use the arrow keys to move the cursor to the right or left Press the YES or NO soft key to change the entry 3 Press the START soft key in the Run Control Menu to begin synthesis The Run Monitor appears as synthesis begins Figure 2 6 Synergy s controller checks for pressure leaks in the PSC fittings during the first few minutes of synthesis Visually check around the PSC luer fittings for leaks If you detect a leak at either end of the PSC push and twist the end into its luer fitting 2 14 Routine Operation 03 2002 Applied Biosystems set up amp run Main Menu report control print self edit amp manual control test Run Begin YES Run Control Menu Run End YES Print YES YES NO START S xx yy zz F Fi FUNCT NAME T xxx zzz Run Monitor HOLD PAUSE NEXT S jump S more Figure 2 6 How to go to the Run Control Menu Press Menu key to return to Main Menu Occasionally small droplets may form around the middle of the PSC Use a tissue to wick away small amounts of liquid If the PSC continues to leak terminate the synthesis see How to terminate a svnthesis on page 7 11 and restart the synthesis with a new PSC If no leaks are detected in these initial minutes of operation you may leave the instrument unattended
239. taken when the pressure test failed Refer to What to do after a pressure test failure on page 7 12 Test Passed OK 4 14 Maintenance and Self Tests 03 2002 Applied Biosystems If no pressure leaks are detected the LCD displays the message Test passed Press the OK soft key to complete the Leak Test Flow Tests Use flow tests to test and calibrate the flow of chemicals through the instrument Perform Flow Test 8 every time you change the delivery line filter on the line to the HBTU bottle Procedures in Chapter 7 Troubleshooting direct you to use certain flow test for specific conditions To perform these Flow Tests 1 8 and 9 you must have access to a scale that is accurate to 1 mg If you want a list of the steps in any flow test turn on the printer and press the PRINT soft key at the beginning of the flow test Table 4 2 Flow Tests Test Number Test Name Description 1 DMF L R PSC DMF to each transfer vessel and to the peptide synthesis column 2 CALIBRATE TUBE DMF to flow test calibration tube 3 DMF PSC DMF to flow test calibration tube 4 THF gt PSC THF to flow test calibration tube 5 PIP gt PSC Piperidine to flow test calibration tube 6 Pump AAC DMF to each transfer vessel and to the amino acid column Pump Oell DMF and HBTU conductivitv test CALIBRATE HBTU Calibrate HBTU deliverv to left transfer vessel 9 CALIBRATE DIEA Calibrate DIEA deliverv to left transfer vessel If anv of
240. te 1 hydroxybenzotriazole Dimethyl sulfoxide N methylpyrrolidone B 40 0 4 MN N Diisopropylethylamine Dimethyl sulfoxide N methylpyrrolidone 1 200 Piperidine 2 200 Tetrahydrofuran 10 4000 Dimethylformamide The MSDSs for hazardous chemicals supplied by Applied Biosystems are presented after the Safety Data tab of this User s Manual Waste Bottle This instrument produces hazardous liquid and gaseous waste Ensure that the waste container is correctly installed as shown on page 23 of the Installation Guide Do not dispose of hazardous waste until you have read the Synergy Waste profile found after the Safety Data tab of this User s Manual 1 6 Synergy Safety Guidelines 03 2002 Applied Biosystems WARNING HAZARDOUS WASTE The Waste Profile gives information on composition physical properties reactivity spill and leak cleanup health fire and explosion hazards waste disposal special protective equipment and precautions Dispose of hazardous waste in accord with all local state and federal regulations The Synergy Waste Profile can be found after the Safety Data tab of this User s Manual Secondary Containment WARNING HAZARDOUS WASTE Synergy generates hazardous chemical waste Place the external waste bottle in the safety carrier provided for all hazardous materials and waste Read the Synergy Waste Profile found after the Safety Data tab of this User s Manual Compressed Gas Cylinders If the compresse
241. terrupts the procedure To continue the test after this error message 1 Place a wheel test fixture in position one on the wheel 2 Press the OK soft key after the error message 3 Press the PAUSE soft key in the Run Monitor to continue the test 7 24 Troubleshooting 03 2002 Applied Biosystems Start synthesis without running Flow Test 8 or Flow Test 9 Flow Tests 8 and 9 calibrate the delivery of the activators HBTU and DIEA If these flow tests are not run by default no activator is delivered during synthesis When an Exception Mes sage appears on the LCD see page 7 21 it is automatically followed by a software re set that may erase the HBTU and DIEA calibration values If the calibration val ues are erased the message Function file was modified appears on the LCD You must re calibrate HBTU and DIEA after this message Try to jump into a program loop A program loop is a set of steps in a module that are repeated until a predefined condition has been satisfied A loop on Synergy is always bracketed by the function pair Function 3 BEGIN LOOP and Function 4 END LOOP or by the function pair Function 5 begin loop and Function 6 end loop These loops are found in a number of modules During synthesis for example one loop implements conductivity monitoring during module d and another loop repeat edly pumps activation reagents through the amino acid column during activation There are also loops in Flow T
242. thesis reagents 5 7 port 2 30 waste bottle 1 6 T emergencv replacement 2 23 test replacement 2 9 Cycle 4 32 to 4 33 waste line external gas leak 2 34 compression nut 2 32 2 34 Flow 4 15 to 4 31 ferrule 2 32 2 34 Leak 4 13 waste per cycle 2 8 tetrahydrofuran see THF waste profile 1 7 TFA 3 4 5 6 wheel cleavage 3 3 to 3 8 amino acid column 2 3 THF 2 8 2 22 4 11 5 8 7 6 error message 7 20 delivery 4 22 Self Test 4 34 Flow Test 4 4 22 prime 2 31 thioanisole 3 4 Thr 2 13 toggle functions 5 8 Trace Features 2 16 transfer vessels 2 3 trifluoroacetic acid see TFA Trp 2 13 Tyr 2 13 U Upper mv 4 5 user defined modules 6 20 V vacuum filtration 3 3 03 2002 Index Applied Biosystems Appendix I Material Safety Data Sheets and Synergy Waste Profile The MSDSs found in the following pages are for chemicals that may be purchased from Applied Biosystems For MSDSs not found in this section contact the manu facturer or supplier of the related chemicals Contents Material Safety Data Sheets MSDSs and Synergy Waste Profile 3 Acronyms Used in MSDSs 3 DIEA DMSO NMP 0 4M N N diisopropylethylamine dimethyl sulfoxide N methylpyrrolidinone 40 mL 5 DME N N dimethylformamide 4 L 13 HBTU 8 mmoles 2 1 H benzotriazol 1 yl 1 1 3 3 tetramethyl uronium hexafluorophosphate 21 0 2M HOBt DMSO NMP 1 Hydroxybenzotrizole dimethyl sulfoxide N methylpyrrolidinone 40 mL 27 Piperidine 200 mL 35 THF tetrahyd
243. tles are in place from left to right facing the front of the synthesizer the labeled reagent bottles should be HBTU 2 IH benzotriazol l vI 1 1 3 3 tetramethyluronium hexafluorophosphate in 4 10 Maintenance and Self Tests 03 2002 Applied Biosystems position A DIEA DMSO NMP 0 4M N N diisopropylethylamine dimethyl sulfoxide N methvipvrrolidone in position B Piperidine in position 1 and THF Tetrahydrofuran in position 2 DIEA Piperidine and THF Figure 4 7 The DMF N N dimethylformamide bottle sits on the right side of Synergy When all the reagent bottles are in place press the OK soft kev Switch the Pressure Vent to Pressure OK Switch the Pressure Vent switch to Pressure and press the OK soft key A three minute pressure test follows Test Passed OK When this display appears all the bottle connections are tight Press the OK soft key If this test fails one or more of the reagent bottles have not been properly attached to Synergy Read What to do after a pressure test failure on page 7 12 Priming Reagent Delivery Lines There are 5 priming flow tests one for each of the reagent bottles During each prim ing routine a pressure test checks for leaks Table 4 1 Priming Flow Tests Test Number Bottle Primed Oo A O N THF DMF HBTU DIEA Piperidine 1 2 Set the Pressure Vent switch to Pressure before beginning any prime To begin the primin
244. transfer vessel contains HBTU Without spilling the contents carefully remove the left vessel from the Synergy and weigh the HBTU delivered Use the alphanumeric keys to enter this value in milligrams on the LCD Press the OK soft key 4 28 Maintenance and Self Tests 03 2002 Applied Biosystems Replace the vessel OK 6 Replace the vessel on Synergy Press the OK soft key 7 When prompted repeat Steps 5 and 6 The second HBTU weight should be greater than the first Remove left vessel and put on scale Zero scale then replace vessel OK 8 Again remove the left transfer vessel Figure 4 15 place it on a scale that is accurate to l mg and set the scale to zero 9 When prompted repeat Steps 5 and 6 a third time Test Passed OK 10 When this display appears the flow test has been successfully completed Press the OK soft key to complete the flow test If you see the message Test failed press the OK soft key and repeat the flow test If the flow test continues to fail call Applied Biosystems Technical Support 03 2002 Maintenance and Self Tests 4 29 Applied Biosystems Flow Test 9 Calibrate DIEA This flow test measures delivery of DIEA Peptide Synthesis Reagent B to the left vessel As with Flow Test 8 you tare the left transfer vessel and then weigh the con tents of the left vessel after each reagent delivery WARNING CHEMICAL HAZARD DI
245. ts Synergy peptide synthesis columns PSCs each contain 25 umol of either pre loaded resin or amide resin Synergy amino acid columns AACs each contain 75 umol amino acid enough for a single coupling Each of the five bottled reagents on Synergy have specific applications in the pep tide synthesis process DMF N N dimethylformamide is the primary solvent used in almost all stages of synthesis on Synergy It solvates the peptide resin at the beginning of synthesis acts as a solvent for the piperidine deprotection reaction is a cosolvent in activation with NMP N methylpyrrolidone and DMSO dimethyl sulfoxide and washes the valve blocks and peptide synthesis columns after each coupling HBTU reagent in combination with the DIEA N N diisopropylethylamine re agent are the activators These reagents rapidly convert the carboxyl group to an ac 9 Carpino L A and Han G Y 1972 The 9 fluorenylmethoxycarbonyl amino protecting group J Org Chem 37 3404 3409 10 Fields C G Lloyd D H Macdonald R L Otteson K M and Noble R L 1991 HBTU activation for automated Fmoc solid phase peptide synthesis Peptide Res 4 95 101 A Knorr R Trzeciak A Bannwarth W and Gillesen D 1989 New coupling reagents in peptide chemistry Tetrahedron Lett 30 15 1927 1930 03 2002 Principles of Solid Phase Peptide Synthesis on Synergy 5 7 Applied Biosystems tive ester a chemically reactive form that reacts
246. ts of the PSC together During the be gin line Synergy runs a pressure test to check for a leak in the PSC If the PSC has not been opened use it as is Is the Fmoc group still on the peptide If you used the standard End line d e to synthesize the peptide resin the Fmoc group has been removed from the peptide If you do not use the End line at all when you synthesize the peptide the peptide resin is still solvated and the contents of the PSC are still wet with DMF at the end of the last cycle Do not remove the PSC from Synergy without first running module e WARNING CHEMICAL HAZARD DMF is flammable and an irritant to skin and eyes Avoid all skin contact with and inhalation of DMF To keep the Fmoc group on the peptide resin remove the d module from the END line in the Run Editor The End line now contains only module e Line Cycle Repeat Module List Beg 0 1 bf 1 1 1 jdacgfi End 1 1 e Figure 6 11 Run File with End modified to keep the Fmoc group on the peptide resin 03 2002 Advanced Operations 6 13 Applied Biosystems How to add more amino acids to a dry peptide resin WITHOUT the Fmoc group 1 2 Place the PSC that contains the peptide in the PSC position on Svnergv Place the appropriate AACs on the amino acid column wheel As with a routine synthesis remember to put the AAC nearest the C terminal end in position 1 Go to the Run Editor Menu and edit Line 1 to read jacccg fi
247. umber 2 When the appropriate flow test appears on the LCD press the START soft key to begin the flow test Is there liquid between the marks NO YES 3 Ifthe reagent meniscus is on one of or between the two calibration marks on the flow test calibration tube press the YES soft key If the reagent meniscus does not fall on one of or between these two marks press the NO soft key After 2 more minutes the LCD reports if the test was successful Test passed OK 4 Press the OK soft key to complete the flow test Test failed OK If anv of these three flow tests fails e Check that each of the three reagent bottles contains fluid and that the ends of the reagent lines in the bottles extend to the bottom of the bottles 4 22 Maintenance and Self Tests 03 2002 Applied Biosystems Prime the reagent line for the reagent s that fail See Priming Reagent Delivery Lines on page 4 11 Check that the flow test calibration tube fits tightly in the luer fittings and re run Flow Test 2 If a pressure test failure occurs during one of these flow tests see What to do after a pressure test failure on page 7 12 If Flow Test 3 DMF fails Perform Flow Test 1 then repeat Flow Test 3 If Flow Test 3 continues to fail call Applied Biosystems Technical Support If Flow Test 4 THF fails If the THF level is near the marks in the flow test calibration tube during Flow Test 4
248. y line HBTU Piperidine Tetrahydrofuran Stabilized DIEA DMSO NMP i Figure 2 3 Reagent bottle positions on the front of Synergy 6 Run Flow Test 8 to check HBTU delivery each time you replace the filter on the end of the HBTU delivery line One delivery line filter can be used for five HBTU bottles or about 200 mL reagent Reagent bottle positions When all the reagent bottles are in place from left to right facing the front of the synthesizer the labeled reagent bottles should be HBTU in position A DIEA DMSO NMP 0 4M N N diisopropylethylamine dimethyl sul foxide N methylpyrrolidone in position B Piperidine in position 1 and THF Tet rahydrofuran in position 2 Figure 2 3 DMF N dimethylformamide sits in a safety carrier on the side of the instrument next to the waste bottle 3 Check the waste bottle and empty if necessary Waste generated per cycle approximately 65 mL The one gallon approx 4 L waste bottle accommodates approximately 62 synthe sis cycles Reagent usage is influenced by the conductivity produced during synthe sis so the waste generated varies according to the characteristics of the peptide sequence Difficult sequences require longer deprotection and washing times and so generate more waste 2 8 Routine Operation 03 2002 Applied Biosystems the RS 232 cable Multiply the number of amino acids in your synthesis by 65 mL to estimate how much waste will be generated If the
249. y to continue operation If a pressure test repeatedly fails there could be a leak inside Synergy that only a trained Applied Biosystems Service representative can repair Call Applied Biosystems Technical Support for assistance Pressure Test Failures during a synthesis Table 7 2 Pressure Test Failures During a Synthesis and Response When Failure Occurred Module Recommended Response Beginning synthesis b Check placement of PSC in luer fittings Check Pressure Vent switch During synthesis j Check low pressure gauge Check position of AAC in jaws and action of jaws Pressure Test Failure at module b d or f Check Pressure Vent switch Set the Pressure Vent switch to Pressure during a synthesis The message Pressure out of range appears on the LCD when this switch is set to Vent during a synthesis Check placement of PSC in luer fittings Reagent leaks can occur around the peptide synthesis column PSC if it has not been pushed firmly into both of its luer fittings see Figure 4 9 on page 4 13 for an illustration of the PSC placement and make appropriate adjustments If adjusting the PSC placement does not eliminate this pressure failure examine the rings of the luer fittings on either end of the PSC These rings should be concentric without irregularities or cracks Also check for fluid where the body meets the end of the PSC caps If you see such leaks stop the synthesis and discard the PSC If after replacing the P

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