Agilent RapidFire High-throughput MS System Troubleshooting Guide
Contents
1. Otherwise call Agilent as the valve on your system may need to be replaced Use a spare adapter 10 32 F to 6 32 M to redirect a thick OD 1 16 tubing into any valve port of your choosing can be very helpful in flushing grooves with both types of solvents in both directions 16 Agilent RapidFire High throughput MS Troubleshooting Guide Symptom 6 Pump 1 is overpressuring Pump 1 P1 can overpressure in any of the following three scenarios which correspond to the three valve position combinations during a RapidFire cycle e P1 overpressures with the valves positions as in State 1 Aspirate all 3 valves in their Inject blue positions See Table 1 below for troubleshooting steps for this scenario e Pl overpressures with the valves positions as in State 2 Wash Load V1 in Load green V2 and V3 in Inject blue In this case follow the same steps outlined in Table 1 valves configuration as in State 1 but be aware that the clog could be in the V1P2 V1P5 beige loop e P1 overpressures with the valves positions as in State 3 Elute all 3 valves in their Load green positions See Table 2 on page 19 for troubleshooting steps for this scenario Table 1 Scenario 1 Valve Positions as in State 1 Aspirate Actions Supporting actions Comments or Results 1 You may need to change the cartridge 2 You may need to replace the OD 1 32 red tubing between V2P1 and the botto2. f All three nanovalves should thereafter show green disks next the Home section of the Valve Configuration utility If they do not call Agilent for assistance g With all valves homed and in Inject blue position if the back pressure of P1 is still high then the clog is further up the lines coming from P1 5 Disconnect the pieces of tubing a Disconnect the tubing in the following Ifyou deduce that the clog is that lead step by step to P1 order within a valve at a port or ina Start with V3P1 V1P6 groove see Symptom 5 A Then V4P2 V3P6 valve port or groove is Then finally P1 V4P1 clogged on page 15 b Evaluate the results to determine where the clog is located 6 You may need to replace the OD If the back pressure of P1 is low with no 1 32 red tubing between the top cartridge in the column holder but becomes of the column changer and V2P4 high as soon as any cartridge gets inserted then the problem lies downstream of the column on the aqueous flow path Disconnect the column changer V2P4 red tubing at V2P4 If P1 still overpressures then this tubing is at fault Replace it 7 Be aware that clogs can occur inside the beige adapters 10 32 to 6 32 threads 18 Agilent RapidFire High throughput MS Troubleshooting Guide Table 2 Scenario 3 Valve Positions as in State 3 Elute Actions 1 You may need to rehome the a valves b c d Supporting actions Turn off the pumps Push the E STOP butto
3. Agilent RapidFire High throughput MS System Troubleshooting Guide Before you begin Understand and interpret the flowpath diagrams 2 Symptom 1 The RapidFire system does not seem to be sipping 9 Symptom 2 The sip sensor does not detect liquid sample 12 Symptom 3 A pump is experiencing Underpressure 13 Symptom 4 The mass spectrometer signal is verylow 14 Symptom 5 A valve port or groove is clogged 15 Symptom 6 Pump 1 is overpressuring 17 Symptom 7 Pump 2 is overpressuring 20 Symptom 8 Pump 3 is overpressuring 21 Symptom 9 The sipper guide needle crashed into the plate 22 How to use this guide Use the actions given in this guide to troubleshoot problems that may arise while using the Agilent RapidFire High throughput MS System If the problem persists after you perform the appropriate troubleshooting routine please contact Agilent for technical support phe Agilent Technologies Before you begin Understand and interpret the flowpath diagrams To debug most problems that you may encounter on your RapidFire high throughput mass spectrometry system you need to understand the following flow diagrams for the instrument which are shown in this section Figure 1 State 1 Aspirate on page 3 Figure 2 State 2 Wash Load on page 4 Figure 3 State 3 Elute on page 5 Figure 4 State 4 Re equilibrate on page 6 Figure 5 Flush the sipper tube on page 7 Figure 6 Physical colors of
4. e High throughput MS Troubleshooting Guide Actions Supporting actions Comments or Results 4 Assess aspiration proficiency by a measuring aspiration times 5 If sipping still appears to be inefficient after you ve taken b actions 1 4 above test for a flow constriction within V1 c With the vacuum on and with all three If the first action Step 4a results in valves in Inject blue position hold an poor sluggish aspiration while the eppendorf tube of water under the sipper second action Step 4b reveals tube efficient swift aspiration then Measure how long it takes to sip1mL The obstruction is likely to be in of water the sipper tube itself at the The normal range is 25 45 seconds V1P3 port or in the V1P2 V1P3 Disconnect the beige loop at V1P2 and groove stick the free end into the same little tube Change and flush the sipper tube of water as described in Symptom 5 A Is aspiration taking place valve port or groove is Is 1 mL of water sucked up in 35 clogged on page 15 seconds or less Manually actuate V4 to its Load green position in the Valve Tuner window Ascertain that all three nanovalves V1 V2 and V3 are in the Inject blue position as show below All Valves V1 V2 V3 V4 e eee Don t Change inject ml E Load O O O O we ollollollo Turn on pump 1 P1 using the commands of the main window Set the flow rate to 1 5 mL min Watch the backpressure of P1 If
5. es Q1 and Q3 14 Agilent RapidFire High throughput MS Troubleshooting Guide Symptom 5 A valve port or groove is clogged This section assumes that previous troubleshooting points to a clog in the V1P2 V1P3 groove between the sipper tube and the injection loop The affected valve port or groove may be different on your system Actions Supporting actions Comments or Results 1 Flush the groove in both directions a Manually actuate V4 to its Load green with aqueous solvent position in Valve Tuner b Ascertain that the three nanovalves V1 V2 and V3 are in the Inject blue position c Disconnect the sipper tube at V1P3 d Turn on P1 only using the command of When you try to flush the the Pump Settings section Keep its flow V1P2 V1P3 groove liquid will rate at 1 5 mL min at first drip out of V1P3 The presumed clog must have an exit path Pump Settings Pump 1 Active O 1 5 mL min 1 bars e Ifthe back pressure of P1 remains low then ramp up its flow rate incrementally 1 5 then 3 then 5 mL min f Ifthe clog fails to disappear now flushin Remove one end of the loop from the other direction V1P2 and connect it instead into g Turn on P1 only If the back pressure of P1 V1P3 Leave V1P2 open remains low then ramp up its flow rate Don t hesitate to remove the incrementally 1 5 then 3 then 5 center post if it is in the way The mL min This may force the clogging set screw that holds it in place is
6. hroughput MS Troubleshooting Guide 7 Figure 6 Physical colors of tubing flush the sipper tube Legend Port colors forall 3 valves pump2 wi vacuum sipsensor sipper w2 pump 3 cartridge Agilent RapidFire High throughput MS Troubleshooting Guide Symptom 1 The RapidFire system does not seem to be sipping Actions Supporting actions Comments or Results 1 Check that the vacuum is on e 2 Check that the sipper tube is going deep enough in the wells to aspirate the sample See the figure on the left below Verify the integrity of the connections to the sidearm waste flasks Check that the vacuum pressure displayed on the RapidFire UI is in the range of 60 to 70 kPa Present liquid such as water from an eppendorf tube to the sipper tube and verify that the liquid is aspirated slowly but noticeably The value sets the distance in millimeters that the tip of the sipper tube is above the plate bottom If necessary adjust the Sip Height onthe main RapidFire tab See the figure on the right below Make the value smaller to lower the position of the sipper tube in the wells RapidFire Mass Spectrometry RF MS Fie Toos Help Plateset Run Controls 3 Flush the sipper tube e gt wm m O Plates Sequences Sipper Setting Sip Height men 2 Click Flush Now on the main tab of the RF MS data acquisition software Agilent RapidFir
7. is clogged Replace it 4 You need to change the MS probe Gently replace the ESI or APCI injection probe of your MS Agilent RapidFire High throughput MS Troubleshooting Guide 21 Symptom 9 The sipper guide needle crashed into the plate Actions Supporting actions Comments or Results 1 Press the E STOP button and clear up the platform Repower the stages by lifting the E STOP Unscrew the damaged pink sipper guide needle luer lock as well as the beige PEEK sipper tube that runs through it a 1 Screw in a new sipper guide needle and from the bottom slide in a new sipper tube Make sure the sipper tube bottoms out in V1P3 and is tightly held in place Re home the sipper tube for both 96 and 384 well types of plates The tubing is most likely severely kinked With the help of tweezers if necessary cap the sipper tube with a red ferrule and screw it into V1P3 Pull on the sipper tube to make sure that it doesn t fall out Click the Home button under the Sipper Configuration Wizard and follow all of the calibration steps Agilent RapidFire High throughput MS Troubleshooting Guide Agilent RapidFire High throughput MS Troubleshooting Guide 23 www agilent com In this book This guide has troubleshooting information for common problems that may arise while using the Agilent RapidFire High throughput MS System Agilent Technologies Inc 2011 Printed in USA Revisio
8. m of the column changer 3 You may need to replace the V1P1 V2P6 OD 1 32 red tubing a b Turn off P1 Select the Load position from the System Tools gt Column Changer menu then click Go To Take out the cartridge and insert a new one Select the position of the new column from the System Tools gt Column Changer menu then click Go To If the back pressure of P1 returns to normal continue normal operation With the column changer disconnected if P1 is still overpressuring then the clog is upstream of the SPE cartridge on the aqueous flow path Disconnect the red tubing at V2P1 If pressure becomes low the red tubing is clogged Replace it Disconnect the V1P1 V2P6 red tubing at Valve 1 If pressure becomes low the red tubing is clogged Replace it For State 1 Aspirate flow diagram see Figure 1 on page 3 Agilent RapidFire High throughput MS Troubleshooting Guide 17 Table 1 Scenario 1 Valve Positions as in State 1 Aspirate Actions Supporting actions Comments or Results 4 You may need to rehome the a Turn off the pumps valves if with the V1P1 V2P6 red b Push the E STOP button tubing disconnected at V1 P1 is c Manually rotate the valve couplings in the still overpressuring counter clockwise direction or towards the Inject position as far as possible d Release the E STOP button e In RF MS under the Valve Tuner window click on the Find button for all three valves
9. material out of the groove accessible from the back of the h Ifthe clog fails to disappear flush the RapidFire platform on the groove with organic fluid as described in pyramidal bracket between V1 Step 2 and V3 Agilent RapidFire High throughput MS Troubleshooting Guide 15 Actions Supporting actions Comments or Results 2 Flush the groove in both directions with organic solvent 3 If you think that the clog has been removed from your RapidFire High throughput MS System then resume your experiments Tip Instead of feeding aqueous solvent through the OD 1 16 blue tubing normally connected to P1 s front panel fill this fluidic path up with organic solvent a Disconnect the OD 1 16 blue tubing from P1 s outlet and plug it instead into P3 s outlet after having temporarily removed P3 s beige tubing b With the sipper tube disconnected and the loop in its usual location between V1P5 and V1P2 Turn on P3 only using the commands of the Pump Settings section Keep its flow rate at 1 5 mL min at first c Ifthe back pressure of P3 remains low ramp up its flow rate incrementally 1 5 then 3 then 5 mL min d Ifthe clog fails to disappear now link the loop to V1P3 leaving V1P2 open for a backflush of the groove e Turn on P3 again and ramp up its flow rate if possible Doing this uses P3 to try and dissolve the V1P2 V1P3 clog This may force the clogging material out of the groove
10. n Manually rotate the valve couplings in the counter clockwise direction or towards the Inject position as far as possible Release the E STOP button e In RF MS under the Valve Tuner window 2 Disconnect the green waste line at a V3P5 3 Disconnect V4P2 V3P6 at V3 if the back pressure of P1 remains high click on the Find button for all three valves All three nanovalves should thereafter show green disks next the Home section of the Valve Configuration utility If they do not call Agilent for assistance With all valves homed and in Load green position if the back pressure of P1 is still high then the clog is between P1 and waste If the back pressure of P1 comes down then the flow is being obstucted by one of the following the adapter at V3P5 or the OD 1 16 green tubing itself Replace the culprit If P1 still overpressures then move up the lines and disconnect V4P2 V3P6 at V4 If P1 becomes low then you can assume that the V3P6 V3P5 groove is clogged Comments or Results For State 3 Elute flow diagram see Figure 3 on page 5 See Symptom 5 A valve port or groove is clogged on page 15 Agilent RapidFire High throughput MS Troubleshooting Guide 19 20 Symptom 7 Pump 2 is overpressuring Actions Follow essentially the same troubleshooting instructions as desribed in Symptom 6 Pump 1 is overpressuring on page 17 but focus your examination on the organic flow path downst
11. n A September 2011 G9314 90002 Ee Agilent Technologies
12. of liquid under the sipper value sipping air RapidFire High throughput MS tube and record the value displayed on System as shown below the sip sensor module c Compare the values recorded above 12 Agilent RapidFire High throughput MS Troubleshooting Guide Symptom 3 A pump is experiencing Underpressure Actions Supporting actions Comments or Results a Check first with all valves in their Inject position blue a b Then check with all valves in their Load green positions 1 Check for leaks along the flow path of this pump 2 Check for air bubbles along the fluidics lines E If you notice a leak Make sure all nuts and ferrules are tightly in place and hold the PEEK tubing firmly Make sure the end cuts of all tubing are square sharp cuts perfectly perpendicular to the tubing axis and bottomed out in their respective ports Replace fittings and or pieces of tubing as needed If you observe some air bubbles Whirl the solvent bottles to let air bubbles escape Prime the pumps or flush them for a couple of minutes at a flow rate of 10 mL min with the diversionary valves open Resume your RapidFire run Agilent RapidFire High throughput MS Troubleshooting Guide 13 Symptom 4 The mass spectrometer signal is very low Actions Supporting actions Comments or Results 1 Check that the sample is getting See Symptom 1 The RapidFire system does sipped out of the plate not
13. ream of P2 instead of P1 Keep in mind that the lines that most commonly clog are the VIP1 V2P6 red tubing the V1P2 V1P5 beige loop Agilent RapidFire High throughput MS Troubleshooting Guide Symptom 8 Pump 3 is overpressuring This troubleshooting procedure consists of disconnecting one by one the segments of tubing downstream of the overpressuring P3 until the position of a clog can be determined Actions Supporting actions Comments or Results 1 You may need to change the cartridge a Turn off P3 and actuate all three nanovalves to the Load position green b Select the Load position from the System Tools gt Column Changer menu then click Go To c Take out the cartridge and insert a new one d Select the position of the new column from the System Tools gt Column Changer menu then click Go To e Ifthe back pressure of P3 returns to normal continue normal operation f Check that the back pressure of all three pumps remain low both when the valves are in Load position green and when they are in Inject position blue 2 You may need to replace the V2P2 MS_ a Disconnect the V2P2 MS red tubing at OD 1 32 red tubing MS input b Turn on P3 c Ifthe pressure of P3 is still high the red tubing is clogged Replace it 3 You may need to replace the MS input a Disconnect the red tubing at MS inject MS inject OD 1 16 red tubing b Turn on P3 c Ifthe pressure of P3 is still high the thick red tubing
14. seem to be sipping on page 9 2 Check that all pumps are turned on P3 in particular including the peristaltic pump P4 3 Check the back pressure of the e Ifthe pressures are Jower than normal check that no leak is occurring along pumps the fluidics lines Look for large air bubbles and get rid of them see Symptom 3 A pump is experiencing Underpressure on page 13 Ifthe pressures are higher than normal try to pinpoint the location of the clog s 4 Check that you are using MS methods Verify that you are filtering for the correct Q1 adequate for your assay and or 03 m z 5 Check that the MS plate is clean If needed remove the plate from the MS head and wipe it gently with methanol and clean Kimwipes 6 Check that you have inserted the Column affinity to the analytes cartridge adapted to your specific experiment Check that you are using the proper set of aqueous and organic solvents for your assay Consider changing method parameters can greatly influence MS signal quality and intensity Adding 0 1 TEA or TFA sometimes causes significant changes in MS signal quality and intensity The wash load time State 2 duration in the RapidFire Cycle Durations section of the RF MS software page and the flow rate of P1 The elution time State 3 together with the flow rate of P3 Add Blank Injections Between Wells on main RF MS software page to reduce carryover e Widen the resolution of quadrupol
15. the backpressure remains low then ramp up the flow rate incrementally 1 5 then 3 then 5 mL min Pump Settings Pump 1 Pump 2 Pump 3 Pump 4 M Active Active O V Active 1 5 mL min 1 25 mL min 1 25 mL min Augie 1 bars 1 bars 1 bars Connected to instrument Refresh Update O 10 Agilent RapidFire High throughput MS Troubleshooting Guide Actions Supporting actions Comments or Results d Switch V1 to Load green e Observe the backpressure of P1 If P1 overpressures a clog is likely to exist at V1P3 V1P4 or in the groove connecting these two ports See Symptom 5 A valve port or groove is clogged on page 15 for information on how to troubleshoot a valve port or groove that is clogged Agilent RapidFire High throughput MS Troubleshooting Guide 11 Symptom 2 The sip sensor does not detect liquid sample Actions Supporting actions Comments or Results 1 Verify that aspiration is occurring Follow Steps 1 4 of Symptom 1 The properly RapidFire system does not seem to be sipping on page 9 2 Check the aspiration time set in State 1 or aspiration time should be set your RF method rfcfg to 600 ms or greater 3 Check the digital output display of a With the vacuum on record the value The second value sipping liquid the sip sensor module located displayed when air is being sipped should be approximately 2x the first along the inside left wall of your b Hold a tube
16. tubing flush the sipper tube on page 8 The following tips apply to all flow diagrams e The three nanovalves V1 V2 and V3 have six color coded ports e Ports are connected by inner grooves which are shown as white segments on the flow diagrams These links are different in the Inject and Load positions For example When V1 is in the Inject state blue V1P6 and V1P1 are connected When V1 is in the Load state green V1P1 and V1P2 are connected Agilent RapidFire High throughput MS Troubleshooting Guide Figure 1 State 1 Aspirate Legend Port colors forall 3 valves pump2 wi vacuum sipsensor sipper W2 pump 3 cartridge Agilent RapidFire High throughput MS Troubleshooting Guide 3 Figure 2 State 2 Wash Load Legend Port colors forall 3 valves pump2 wi vacuum sipsensor sipper W2 pump 3 cartridge Agilent RapidFire High throughput MS Troubleshooting Guide Figure 3 State 3 Elute Legend Port colors forall 3 valves pump2 wi vacuum sipsensor sipper W2 pump 3 cartridge Agilent RapidFire High throughput MS Troubleshooting Guide 5 Figure 4 State 4 Re equilibrate Legend Port colors forall 3 valves pump2 w1 vacuum sipsensor sipper W2 pump 3 cartridge Agilent RapidFire High throughput MS Troubleshooting Guide Figure 5 Flush the sipper tube Legend Port colors forall 3 valves pump2 wi vacuum sipsensor sipper W2 pump 3 cartridge Agilent RapidFire High t
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