Appendix A
Contents
1. A 2 2 Machine Bottle Runs Dry When a bottle on the machine runs dry air bubbles can get into the lines and it will not be able to detect the solvent even after the bottle is refilled In order to fix this the line will need to be backflushed After the solvent bottle has been refilled go to the tab in the Procise software called Manual Control and find the solvent bottle that needs to be backflushed in either the Flask Functions tab or Cartridge Functions tab For example in Figure A 3 the Flask Function Backflush S3 is highlighted Once it is highlighted click execute on the bottom of the screen and watch the bottom of the hose in the solvent bottle until bubbles can be seen coming out Then press All Off to shut off the backflush and the solvent should now be detected The run can now be restarted A 2 3 HPLC Bottle Runs Dry Sometimes the HPLC solvent bottles run dry and this is not as easily fixed as the machine bottles First the HPLC bottles need to be refilled Then the pump must be purged manually using the buttons on the front of the pumps Push the function key for purge then select the pump to be purged When the purge is complete push Manual on the front of the pump to turn the pumps on to free running The pumps should be allowed to run freely until bubbles are no longer coming from the end of the column as seen in the waste line If the machine is needed immediately the flow rate can be turned up to m2. Dip at front of Spectra Non Secure Mode A dip at the front of the spectra as seen in Figure A 4 causing difficulty in reading the amino acid sequence is a fairly common problem that indicates that the pump seals need to be changed In order to do this you must unscrew the grey metal screw going into the pump cylinder then unscrew the black ring around the top of the cylinder Remove the cylinder by using the purge function of the pumps to purge the pump you are working on and PUSH the cylinder out NEVER PULL the cylinder out it could damage the pumps which are nearly impossible to replace on this machine Once the cylinder has been pushed out fully and starts getting pulled back in push stop to keep it out Gently pull the cylinder off of the post One of the pump seals that needs to be changed is the one on the end of the post It is frequently hard to remove and 134 can be sliced off or yanked with pliers The second pump seal is the ring inside of the metal part at the top of the cylinder pull this off to expose the seal To put the cylinder back onto the post do NOT just push If it is not aligned correctly this can damage the inside of the cylinder which cannot be replaced Use the metal alignment tool in the spare parts drawer This part has two pieces that come together to form a tunnel that fits onto the end of the cylinder through which you can slide the cylinder post Before doing this take a pi
3. a faulty rachet cap assembly or the part that the bottle screws into on the machine In order to test this go to the Test Procedure tab on the Procise software and select Leak test Scroll down to the appropriate bottle and try a leaktest If it fails but the bottle is screwed in tightly this assembly should be replaced See page 9 19 in the User Manual for details on this procedure Once this 136 assembly has been replaced the seal should no longer leak and the solvent delivery should function as normal RW PROCISE Test Procedure aea Fie Edit Sequencer Help PROCISE Test Procedure 7 AS depleted DUM d MODEL INFORMATION ABOUT Select a Test Fl Startup Idle Init Sensor E Shutdown Cleanup Electrical Procedures Available Do not pause on error Event Log Cart Input Blk Test Cart Reagent Blk Test Bottle Change Cartridge Leak Test Procedure r Status Cartridge B Leak Test Cartridge C Leak Test Name Pres amp Temp Cartridae N eak Test Remaining Start Test Stop Test Step of J Function Number Manual Control Test Procedure Sequencer Status Sequence Methods Name Total Time Cycles amp Procedi Remaining Gradients Pun Pause Hold Jump Step Sequencer Status Idle WIPFEFPFEPF B CONNECTED Non Secure Mode Figure A 5 Leak Test Procise Software Screen A 2 7 Pressure Errors Clogged Lines Overpress
4. in water 100uL stock Na2PO4 1mL 196 Acetone 10mL Buffer premix 20uL TFA Machine Solvents R1 PITC in Heptane 2 5mL PITC in 50mL heptane R2B N methyl piperidine MeOH H20 R3 TFA neat purchased from Sigma R4A 25 TFA in Water DTT 12 5mL TFA in 37 5mL water with 0 01 DTT 32uL 1M stock R5 Acetonitrile with 0 000196 DTT S1 N heptane purchased from Sigma S2B Ethyl Acetate 450mL Ethyl Acetate with 225uL 1M DTT S3 N Butyl Chloride purchased from Sigma S4B 20 Acetonitrile in water 20 Acetonitrile in water Stocks Na2POA stock 2g in 15mL 3 5 THF in water 1M DTT stock 0 231g in 1 5mL Ethyl acetate or water A 1 3 Ordering Table A 2 Parts and Chemicals Commonly Ordered from Applied Biosystems Part Chemical Part Number Premix Buffer Concentrate 100mL 401446 R5B Acetonitrile n Acetylcysteine 40 mL 4340966 Seal Cylinder Head 200240 Seal 5mm Micro Pump 201399 Procise Cartridge Seals 50 pk 401611 PTH C18 Column 0 8mm x 250mm 401882 PTH Amino Acid Standards 4340968 R2C N Methylpiperidine in isopropanol 4310689 butanol water 9mm TFA Filters 401111 Table A 3 Parts and Chemicals Commonly Ordered from Sigma Aldrich Part Chemical Part Number Ethyl Acetate CHROMASOLV Plus 99 996 650528 4L Phenyl Isothiocyanate 78780 Heptane CHROMASOLV Plus 99 9 650536 4L 2L Chromasolve N Butyl Chloride for HPLC 34958 2L Trifluoroacetic Acid 9996 reagent plus T650
5. 125 Appendix A Use Care and Maintenance of Procise 494 CLC Edman Degradation Protein Sequencer 126 A 1 Regular Use A 1 1 Loading a Bead There are 4 cartridges on the Edman and each can be loaded with a bead The cartridge is unscrewed from the machine and the lid with the hose coming out of the top should ALWAYS be rested on the cartridge holder and not left to dangle This stretches out that hose and it will need to be replaced The cartridge top unscrews Inside there are two quartz sample holders and the bead fits in between them With gloves on use a finger on each side of the cartridge to hold the second quartz sample holder in place while you turn the cartridge upside down and let the top part slide out These quartz sample holders are VERY expensive and chip easily Do not drop them and be very careful when handling them Once the top sample holder is out there should be a used filter and cartridge seal that can be removed and thrown out A new cartridge seal is slid into the cartridge on top of the bottom sample holder that was left in place A new filter gets tapped into the groove in the top sample holder that was removed It can be gently tapped in place with tweezers The bead is placed onto this filter and the whole cartridge is then turned upside down with the bottom sample holder and cartridge seal held in place by a finger on each side of the cartridge and slid down on top of the top sample holder that has the bea
6. 8 1L A 1 4 Contacting AB 128 The number to call to place a service call with Applied Biosystems is 800 831 6844 press 4 then 1 Our serial number is 4CLO00092 Ask for service on the instrument and get a reference number for the call Type this onto the purchase order and have the purchasing department fax it to 650 554 2193 Include a description of the problem on the purchase order 129 A 1 5 Idle machine The machine should never be left completely idle or the column can dry out and will need to be replaced If the machine is not running press the Manual button on the front of the pumps It should turn the pumps on to 5uL min at 50 B and the bottom of that window will change to say free running This low flow of solvent keeps the solvents moving and the column wet A 1 6 Settings These are our common settings for the machine in the off chance that something is lost or rest and needs to be reprogrammed These are also the settings used for all of the amino acid sequencing in this thesis pum Tw PROCISE Sequence Methods E PROCISE SsveneeMetnods m r Stating Temperatures Injector Optimization cLC PTH Standards cLC Catig tt 5 D EZET Casto E H Mb RunGiadent cLC nto 5 PulsedLiquid cLC Full s pines EX ELO Table A 4 PulsedLiquid cLC Method PROCISE Pressure and Temperature P RO C SE Pressures and Temperatures f Pressure p
7. d on top Then the cartridge can be turned right side up the lid can be screwed back on and it can be placed back into its slot on the Edman The run is set up on the Procise software on the computer Each run should be set to use the PulsedLiquid cLC method unless a special one is required The cycles should be set to equal the number of desired amino acids plus 3 one warm up cycle one blank cycle and one standard cycle that run automatically All of the solvent bottles should be checked to ensure that the levels are appropriate for running the desired amount of cycles You can check the amount of each solvent required by clicking the button on the bottom of the screen 127 It will provide an amount in mL required to run the set cartridges and cycles A 1 2 Most of the solvents used are purchased from somewhere other than Applied Biosystems Solvents and mixed in house See Table A 1 for details When refilling solvents the bottle should be screwed on until you hear three Clicks Over tightening can damage the assembly such as the R1 bottle To loosen or tighten this bottle you must first push up hard on the rachet assembly to hold it in place while you screw or unscrew the bottle Table A 1 Solvent Compositions Letter Name Composition HPLC Solvents B2 Acetonitrile in Isopropanol 9 1 Acetonitrile Isopropanol A3 THF in water 3 596 THF
8. ece of Parafilm to cover the hole into the pump cylinder If one of the metal alignment pieces falls in it will be VERY difficult to remove Then while holding the metal pieces to the end of the cylinder align this with the post and apply steady but hard pressure to push the cylinder onto the post You will feel a click when it is on properly and it will not pull back off if you tug on it Then by tilting the cylinder back and forth pull off the metal alignment pieces then the Parafilm Use the purge function to pull this cylinder back down into the pump One that is in it will need twisted so that it aligns properly and slides into place There is a notch it must fit into Then the black ring can be screwed back on followed by the metal screw 135 Figure A 4 Dip at Front of Spectra Indicative of Bad Pump Seal A 2 5 Baseline Errors Stretched Out Amino Acid Standards If the amino acid standards look very stretched out and are not completing by the end of the HPLC run or if the DPTU peak is shifting from cycle to cycle there is most likely a leak in the pumps This is a fairly easy fix which requires looking at all of the screws around the pumps One of them will more than likely have a ring of salt around it Wash the salt off and tighten the screw turn It should fix the spectra problems immediately A 2 6 Pressure Errors Change Bottle Seals Occasionally when the machine has difficulty adding a solvent it is due to
9. ove this process along faster Everything should be done to minimize the times that these bottles are allowed to run dry so that the pumps are not pumping dry and the column does not dry out SN PROCISE Manual Control File Edit Sequencer Help PROCISE edem ABOUT Event Log Bottle Change Pres amp Temp Manual Control Test Procedure Sequence Methods Cycles amp Procedures i Gradients Functions Cartridge 133 AS Goose x MODEL INFORMATION oe Controls Additional Valves Continuous Run C Timed Run Elapsed Time Active Valves Cartridge Function Load 53 Cart sm loop Load S3 Cart Ig loop Vent S3 3 FlushS3 Del X1 Cart top Del X1 Cart bottom Del X1 Cart sensor Flask Function 451 Del R4 Flask 152 Load R4 Flask sm loop 153 Load R4 Flask Ig loop 154 Vent R4 155 Flush R4 156 Backtlush R4 157 Del R4 Waste Del X1 Waste 161 Del R5 Flask Load X1 Cart sm loop 152 Load R5 Flask sm loop Load X1 Cart Ig loop Vent X1 Regulators 4 Active Reaction Func 153 Load R5 Flask la loop 164 Vent R5 Regulators Active Conversion Func Valves Sensor Sequencer Status Idle Figure A 3 Procise Screen for Backflushing a Line A 2 4 Valves Sensor All Off Execute HH wconnecten Baseline Errors
10. si Set Actual Rl Regixe1 25 5 R2 Regisa2 08 08 R3 Registos fe 1 52 3 Regdso4 17 7 Cartridge Dry_Reguste5 35 B5 R4 54 X3FlskDry Reguao8 30 BO FlaskBubbe Regua 18 13 Loadinector Regulator 10 08 10 Main Tank B Figure A 1 Temperature and Pressure Settings 130 131 SW PROCISE Gradients File Edit Sequencer Help lt 3 MODEL INFORMATION Gradient Gradients Max Pressure i Prepare Pump cLC Full Gradient Min Pressure joo gt Full Gradient 0 Target Pressure Beso B2 28 0 psi psi Normal 1 cLC R User Defined Gradient Template Target Time a Data Collection Time Cum Volume A Cum Volume B elei3i3 3 a a A Delete How Sequencer Status Idle CONNECTED Non Secure Mode Figure A 2 Normal1 cLC Gradient A 2 Troubleshooting A 2 1 Computer Errors Freezing or Not Saving Spectra The computer needs to be restarted frequently more so when people use the computer for other uses Try to restart the computer once a week to prevent the loss of spectra and keep people from using the computer for any other purpose especially while the machine is running If a computer error occurs or the computer or sequencer freeze shut down the computer and both the Procise machine and the pumps Turn them both back on and they should sync up properly and continue running 132
11. ure errors on the Edman can sometimes be related to a clogged line Use the error message to try to find the line that might be clogged The line can either be replaced by new line from stocks in the spare parts drawers or flushed clear with methanol
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