Notes on Troubleshooting LC/MS Contamination
Contents
1. sss 16 HPLC Equipment as sources of contamination essere enne 18 Ghost DedkS Sce eee I At NIRE ETERNI EC Fa An 18 Purge Procedure for Surveyor MS Pump Pulse Dampener eee 19 LC MS Contamination General Discussion Chemical contamination is one of the more common problems with LC MS recommend using the highest purity chemicals available e Try Burdick and Jackson solvents and water These seem to be uniformly better than other suppliers Switch to this brand if you have contamination problems e recommend this high purity acetic acid Aldrich p n 38 012 1 double distilled in Teflon bottle 175 for 500 mL In my experience a chemical contamination problem with specific mass peaks is almost never deep inside the MS always prioritize like this HPLC pre or trap column HPLC column HPLC hardware Chemicals ESI or APCI probe Spray shield area Heated capillary reaming only removes blockages MS Tube lens skimmer MS Octopoles Multipoles 0 MS Trap 1 MS Dynode Sa Cee ore NS Mostly if 8 11 are dirty you will see random peaks not specific masses Identifying the source of contamination To Start 1 Is the contamination always there or does it elute as specific peaks during the HPLC run If the later the problem is most likely with the HPLC 2 Turn off flow If the noise disappears take the column out of the flow path and test again If the noise is st2. Contaminated water reversed phase Bill Mahn Page 18 Solution Use strong solvent flush after each run improve sample cleanup Improve sample cleanup Use mobile phase as injection solvent Prepare fresh daily use antioxidant Check water by varying equilibration time use HPLC grade water 8 11 2005 LC MS Contamination Purge Procedure for Surveyor MS Pump Pulse Dampener The following procedure should be used to periodically purge the Surveyor MS Pump as part of a comprehensive preventive maintenance program 1 Prepare a series of beakers containing 50ml each of the following water 5 nitric acid iso propanol and n hexane The water iso propanol and n hexane should all be HPLC grade Disconnect the 1 16th inch 316 SS solvent line from the pulse dampener outlet and plug the pulse dampener outlet fitting Locate the 1 16th inch PEEK line connecting the MS Pump s Liquid Displacement Assembly to the pulse dampener s inlet fitting Disconnect this line at the LDA outlet and place the free end of the tubing into the beaker containing HPLC grade water Attach a luer tipped priming syringe to the pulse dampener s purge valve Open the purge valve and draw 10ml of water through the pulse dampener Close the purge valve and empty the priming syringe into a suitable waste container Replace the beaker containing water with the beaker containing 5 nitric acid solution Reconnect the priming syringe to the purge val
3. LC MS Contamination e Using the syringe pump spray acetonitrile at a relatively high flow rate and cycle the heated capillary from 150 to 270 C two times Allow a few minutes before changing the temperature Disassemble and clean the ESI head Disassemble and clean the API stack Remove and clean the octopoles Disassemble and clean the Trap If the source of the contamination is the HPLC will leave you to deal with it e Test the mobile phase for contamination by infusing at 500 uL min with 500 uL syringe e To minimize contamination use fresh chemicals particularly acids Pentafluoropropionic Acid May stick on PEEK tubing and fittings Usually associate contamination with areas that are poorly swept by the flowing liquid e g unions If you have concerns about the pentafluoropropionic acid then use fused silica and steel unions The ferrules should be kel F PEEK tube nuts should be ok They do not touch the liquids What is the purity of the pentafluoropropionic acid always worry about purity of mobile phase additives Water If you see possible contamination use high grade bottled water Burdick amp Jackson HPLC grade Avoid ANY nanoPure or MilliQ water The equipment may not be maintained correctly 798 and 803 Contamination They are two main ions were seen one at 798 1 and 1 at 803 4 They both appeared at high organic during the LC MS run Because it was also seen without the column and trap it must ei
4. 1995 The general formula is Fe30 CH3COO 6 L x where L is a ligand from the mobile phase water or organic component acetonitrile solvent or water 538 could be 6 acetates with no additional ligand The 555 presumably has additional water 533 88 could be a chromium cluster similar to the iron 537 88 Stainless steel in the system contains both iron and chromium Potential sources in the ESI probe could be the ESI spray needle or the with the metal needle kit This is not common try replacing the needle Common Contaminants in Protein Peptide Analysis From Sequest Browser Bill Lane The lines have the following format MH z graphlabel SEQUENCE description after the number sign note that the first column is the calculated MH not the m z of the z need not be sorted graphlabel no longer optional will be displayed on the chromatogram marking the peak to which it corresponds SEQUENCE will be displayed as a hyperlink appended to the description put just X without the quotes for the SEQUENCE if not applicable entries were originally made with the experimental m z OBSERVED not calculated They should vary between exact amp average mass since they were determined by TSQ and LCQ over a long period of time Since tolerance was expected to be 1 0 no attempt has been made to correct these values MH z graphlabel SEQUENCE description after the number sign Type Il Cytoskeletal Keratin 1 amp 2 704 41 Keratin LDSELK Keratin Tyll
5. Cytoskel 1 2330 49 3 Keratin QISNLQQSISDAEQRGENALK Keratin Tyll Cytoskel 1 1394 56 2 Keratin TNAENEFVTIKK Keratin 67K Tyll Cytoskel 1 1141 5 2 Keratin DYOELMNTK if Keratin 67K Tyll Cytoskel 1 1371 7 2 Keratin LNDLEEALQQAK Keratin tYii cYTOSKEL 2 1475 8 2 Keratin FLEQQNQVLQTK Keratin 67K Tyll Cytoker 1 2 1476 70 2 Keratin WELLQQVDTSTR Keratin 67K Tyll Cytoker 1 1357 72 Keratin LNDLEDALQQAK Keratin 67K Tyll Cytoker 1 Bill Mahn Page 10 8 11 2005 LC MS Contamination 1179 62 Keratin YEELQITAGR Keratin 67K Tyll CytoKer 1 1265 62 Keratin TNAENEFVTIK Keratin 67K Tyll Cytoskel 1 1383 7 2 Keratin SLNNOFASFIDK Keratin 67K Tyll CytoKer 1 875 01 Keratin SLYNLGGSK i Keratin 67K Tyll CytoKer 1 1033 5 2 Keratin TLLEGEESR Keratin 67K Tyll CytoKer 1 973 52 Keratin IEISELNR Keratin 67K Tyll CytoKer 1 1302 7 2 Keratin SLDLDSIIAEVK Keratin 67K Tyll CytoSkel 1 1599 8 2 Keratin NNLNDLEDALOOAK Keratin 67K Tyll CytoSkel 1 1994 0 2 Keratin THNLEPYFESFINNLR Keratin 67K Tyll CytoKer 1 1657 8 2 Keratin SGGGFSSGSAGIINYQR Keratin 67K Tyll CytoKer 1 2501 23 Keratin SKAEAESLYQSKYEELQITAGR Keratin 67K Tyll CytoKer 1 2932 5 3 Keratin FLEQQNQVLQTKWELLQQVDTSTR Keratin 67K Tyll CytoKer 1 1340 62 Keratin SKAEAESLYQSK Keratin Tyll Cytoskel 1 2184 13 Keratin NKLNDLEDALQQAKEDLAR Keratin Tyll Cytoskel 1 2581 1 3 Keratin FSGECAPNVSVSVSTSHTTISGGGSR Keratin Tyl CytoSkel 1 Msx gt F 1765 7 2 Keratin R IFSSCGGGGGSFGAGGGFGSR Kera
6. NIDVLEGNEOFINAAR Trypsin Promega Cterm K to R change 2914 43 Trypsin LGEHNIDVLEGNEQFINAARIITHPN Trypsin Promega K to R change 3347 03 Trypsin LGEHNIDVLEGNEQFINAARIITHPNFNGN Trypsin Promega K to R change 1987 1 2 Trypsin TLDNDIMLIRLSSPATLN Trypsin Promega K to R change 1987 1 3 Trypsin TLDNDIMLIRLSSPATLN Trypsin Promega K to R change 2003 1 2 Trypsin TLDNDIFLIRLSSPATLN Trypsin Promega K to R change 2003 1 3 Trypsin TLDNDIFLIRLSSPATLN Trypsin Promega K to R change 1071 6 2 Trypsin IRLSSPATLN Trypsin Promega K to R change 1071 61 Trypsin IRLSSPATLN Trypsin Promega K to R change 1203 6 2 Trypsin TLDNDIMLIR Trypsin Promega K to R change 1219 6 2 Trypsin TLDNDIFLIR Trypsin Promega K to R change F Msx 1318 0 2 Trypsin TLDNDIMLIRL Trypsin Promega K to R change 1334 0 2 Trypsin TLDNDIFLIRL Trypsin Promega K to R change F Msx 1262 83 Trypsin LLHGVATVSLPR Trypsin wrong z VATVSLPR 805 42 Trypsin SAASLNSR Trypsin 805 41 Trypsin SAASLNSR Trypsin 515 001 Trypsin IQVR Trypsin 2283 23 Trypsin IITHPNFNGNTLDNDIMLIK Trypsin 2299 2 3 Trypsin IITHPNFNGNTLDNDIFLIK Trypsin F Msx 2311 23 Trypsin IITHPNFNGNTLDNDIMLIR Trypsin Promega K to R change 2327 23 Trypsin IITHPNFNGNTLDNDIFLIR Trypsin Promega K to R change F Msx 3338 03 Trypsin IITHPNFNGNTLDNDIMLIRLSSPATLNSR Trypsin Promega K to R change 3354 03 Trypsin IITHPNFNGNTLDNDIFLIRLSSPATLNSR Trypsin Promega K to
7. Notes on Troubleshooting LC MS Contamination Table of Contents General Discussion Ge cec e GG pn A oet ea eter e OE 2 Identifying the source of contamination ceesceceeseceseeeceeneeceeeeeceeaceceeneeceeaeeceeeeeseneeeees 2 HPEC troubleshooting ars Feet ie ttes RP lees 3 Contamination an the MS aienea Ee b cnet A D WERL FL GY 3 Phthalate Contamination 391 413 798 803 0 cecccccssssssecececcceceessrsecsecececeeeesensnteaeees 3 AA 5 Background Ramps Up With Gradient eese rennen enne eene 3 339 SerIeS AA AA 6 ETT Senes teste PUPIL NTG ete aq ee e n e 6 Se PS CTI ocio eiie hcm etu e IE tat cal AD c UM 6 613 and 122955 AUN AA AA SE LL Mas 6 Nanospray Peaks 371 445 and others nennen 8 PO SCESE 5 e Mee a a MN aaa T E 8 Pentatluoroprop omic Acidi ose y os Scene BNG ots ASA 9 Water ieu Gi use kas a Y hha sn NG NAN AGES in 9 798 and 803 Contamination 00001 111 aa 9 453 3 679 5 and 905 7 Contamination sessi eee 10 Peak Clusters at 21 17 35 52 icici cccccssssscscccccccccssssscsccccceccessssssscccccsecuseussssescssseeeees 10 Iron Acetate clusters 538 555 and 534 oo ccssesescecccccesessssscscceccessusesseeceesessseeenees 10 Common Contaminants in Protein Peptide Analysis eene 10 Removing Ultramark Contamination ceeecceescecesscecesececeeneeceeneeceeneeceeneeceeeeeeseneeeeeeeees 14 Trouble Shooting Excessive Noise In the LCO DUO and DECA
8. R change F Msx 3338 0 4 Trypsin IITHPNFNGNTLDNDIMLIRLSSPATLNSR Trypsin Promega K to R change 3354 04 Trypsin IITHPNFNGNTLDNDIFLIRLSSPATLNSR Trypsin Promega K to R change F Msx 2155 1 2 Trypsin IITHPNFNGNTLDNDIMLI Trypsin trunc 2155 1 3 Trypsin IITHPNFNGNTLDNDIMLI Trypsin trunc 1176 01 2 Trypsin TLDNDIMLIK Trypsin 1192 01 2 Trypsin TLDNDIFLIK Trypsin F Msx 1126 6 2 Trypsin IITHPNFNGN Trypsin 1023 51 Trypsin SSYPGQITGN Trypsin trunc 1023 5 2 Trypsin SSYPGQITGN Trypsin trunc 1020 51 Trypsin SIPYQVSLN Trypsin trunc 1020 5 2 Trypsin SIPYQVSLN Trypsin trunc 950 52 Trypsin YVNWIOO Trypsin sx F M F Msx Bill Mahn Page 12 8 11 2005 LC MS Contamination UNCHARACTERIZED POLYMER 44AMU SUBUNIT 801 71 2 X 801 7 series 44 ladder 1 845 65 2 X 801 7 series 44 ladder 2 889 72 2 X 801 7 series 44 ladder 3 933 87 2 X 801 7 series 44 ladder 4 977 87 2 X 801 7 series 44 ladder 5 1021 87 2 X 801 7 series 44 ladder 6 1065 61 2 X 801 7 series 44 ladder 7 1110 00 2 X 801 7 series 44 ladder 8 1154 13 2 X 801 7 series 44 ladder 9 1198 1 2 X 801 7 series 44 ladder 10 1242 1 2 X 801 7 series 44 ladder 11 1286 1 2 X 801 7 series 44 ladder 12 1330 1 2 X 801 7 series 44 ladder 13 1374 1 2 X 801 7 series 44 ladder 14 1418 1 2 X if 801 7 series 44 ladder 15 1462 4 2 X 801 7 series 44 ladder 16 1506 4 2
9. X 801 7 series 44 ladder 17 1550 5 2 X 801 7 series 44 ladder 18 1594 5 2 X 801 7 series 44 ladder 19 1638 5 2 X 801 7 series 44 ladder 20 523 1 X unknown UNCHARACTERIZED POLYMER 44AMU SUBUNIT 300 3 1 X 300 3 series 44 ladder 344 1 1 X 300 3 series 44 ladder 388 13 1 X 300 3 series 44 ladder 432 20 1 X 300 3 series 44 ladder 476 20 1 X 300 3 series 44 ladder 520 30 1 X 300 3 series 44 ladder 564 30 1 X 4 300 3 series 44 ladder 608 30 1 X 300 3 series 44 ladder 652 30 1 X 300 3 series 44 ladder 696 30 1 X 300 3 series 44 ladder 740 30 1 X 300 3 series 44 ladder 784 30 1 X 300 3 series 44 ladder 828 30 1 X 300 3 series 44 ladder 392 101ZX Zwit 316 monomer 783 401Z2X Zwit 316 dimer PERSISTENT TUNING SOLUTION IONS 922 5 1 U X Ultramark 1022 5 1 U X Ultramark 1122 5 1 U X Ultramark 1222 5 1 U X Ultramark 1322 5 1 U X Ultramark 1422 5 1 U X Ultramark 1522 5 1 U X Ultramark 1622 5 1 U X Ultramark ANGIOTENSIN STANDARDS ANGIOMIX 884 07 2 RVYVHPI RVYVHPI xx Angio 2 7 918 09 2 RVYVHPF RVYVHPF bo Angio 2 7 932 12 2 RVYIHPF RVYIHPF hu Angio 2 7 1283 50 3 DRVYVHPFHL DRVYVHPFHL bo Angio 1297 50 3 DRVYIHPFHL DRVYIHPFHL hu Angio 1297 50 2 DRVYIHPFHL DRVYIHPFHL hu Angio UNKNOWN ARTIFACTS NOT SEEN IN A WHILE 883 0 2 unk_x85 X 285 385 485 585 unk 869 0 2 unk_x85 X 285 385 485 585
10. a lot of that before we eliminated as much metal as possible We also saw 59 adducts onto larger acidic peptides 77 Series had been experiencing contamination on the Deca ca 77 u clusters mostly across the mass range This appears to have resolved itself upon replacing the heated capillary and seal kelrez the soft black one The seal was visibly chewed up don t know how this might related to the problem but at least it appears to be gone Contamination peaks related to a bad heated capillary o ring is a new one on me could postulate that the worn o ring was allowing leakage into the tube lens skimmer area and the leak was also leaching something out of the o ring Normally the o ring is a total block and any polymers in it would not enter the MS 74 Series Peaks at 536 610 and 684 have been seen with LCQDecaXP and LCQAdvantage and TSQ Quantum The contamination came from connecting the opaque silicon tube to the API source housing The proper tubing is Tygon The instrument parts kits contain both types of tubing The TSQ Quantum contains a 12 foot length of clear Tygon tube and a 2 5 foot length of opaque silicon tube The intended function of the silicon tube is to connect from the waste container P N 00301 57020 to the fume exhaust system If the silicon tube is instead connected to the API source housing background ions at m z 536 610 and 684 might be observed Solution Remove all silicon tubing from th
11. e solvent waste system 615 7 and 1229 8 CHAPS 615 7 is MH of CHAPS and 1229 8 is 2M H in the sample Removal can be tough Acetone precipitation removes the excess CHAPS but there may still be an appreciable amount remaining determined by the above ions in the mass spectrum CHAPS won t kill your SCX chromatography but it will chromatograph nicely on reversed phase and you ll get an intense ion at 615 m z MH LC MS system contamination by CHAPS Bill Mahn Page 6 8 11 2005 LC MS Contamination If the contamination came from your sample likely with CHAPS contamination Then all the bits that contacted the liquid sample should be thoroughly cleaned or replaced This means from the injector to the skimmer in the MS Details On the HPLC side recommend a complete replacement of the parts that were touched by the CHAPS This starts with the sample pickup needle continues through the injection loop to the high voltage tee and column and spray tip for nanospray You need to replace all at once For example expect that some CHAPS upstream from a new column may have already contaminated it On the MS side if this is ESI disassemble the probe and clean All fittings to the probe should be replaced Internally the ion transfer tube skimmer and tube lens are stainless and can be soaked in 30 nitric acid for cleaning Usually do not worry about contamination past the skimmer Very little material gets deeper into the MS You s
12. e the analyzer assembly then the multiplier Rinse in methanol and blow dry Blow off the anode mounted in top cover assembly While the multiplier is removed check to see if the anode is loose It should be hand tight Do not use any tools to tighten or loosen it Clean if necessary Reinstall multiplier and blow off the analyzer assembly before installation Cleaning the trap end caps ring electrode and quartz insulators 1 2 sonicate the parts separately first in acetone for one minute then in methanol for one minute If the insulators are cleaned a 24 hour pump down will be required or RF noise will occur If parts are contaminated or a heavy accumulation of sample is suspected analyzer parts stainless steel or plastic can be scrubbed with soap and water rinsed well with water then rinsed well with methanol Do not clean the multiplier with soap and water Methanol rinse only All parts can be sonicated except octopoles quadrupoles When sonicating parts keep the parts separate to minimize surface damage Bill Mahn Page 17 8 11 2005 LC MS Contamination HPLC Equipment as sources of contamination Autosampler e Could be bad rotor seal e Needle seal Pump e Pulse dampener e Reservoir filters Ghost Peaks Possible Cause Peaks from previous injection column contamination Unknown interferences in samples Upset equilibrium ion pair Problem Ghost peaks Oxidation of TFA peptide mapping
13. etergent containing samples on the system that could explain the background Also PEGs and other ethoxylated polymers give 44 ion series The PEG s could also be from the water or extracted polymer from plastic ware silicon coatings Example Noise is overwhelming the entire spectrum Every 44 there is a peak from 400 to 800 They see the problem when they have an LC input The impurity increased as the gradient went on Mobile phase is acetonitrile water Pattern of 75 from 1500 up to 1900 They changed the pump and the rest of the system components ESI probe Cleaned the spray shield needle nozzle changed the ESI capillary The have changed bottles of both acetonitrile and water She will fax some example data Also she will try spectra with cap on heated capillary cap off heated capillary no flow and LC flow on First pump was HP 1100 mixing from 2 different bottles Second pump was Beckman isocratic only so they pre mixed Infusing methanol get peaks at 812 817 1211 7 1610 8 level is E5 Noise is below 1 What are these Pumped in 50 50 acetonitrile water with the HPLC Got the same patterns that they had sent to me Not the same ones as with the infused methanol except for 812 which is still there Level is still E5 also The noise is about 15 She will try acetonitrile water as used with the Beckman pump through the syringe pump 9 9 97 New fax from her She saw the contamination with the acetonitrile water mixture a
14. hould also clean any areas that the spray might contact Be thorough Bill Mahn Page 7 8 11 2005 LC MS Contamination Nanospray Peaks 371 445 and others Peaks at 371 and 445 amu are commonly observed with the nanospray probe This may result from the silica A long troubleshoot for a source of contamination in Nanospray ended when the user replaced 97044 60290 HV Emmitter nanospray ay 141 the nanospray micro Tee The spectrum was 442 0 459 7 477 5 495 4 see picture The emitter Tee assembly had obviously become contaminated This piece could be hard to clean as the electrode side of the Tee cannot be taken apart and assembled again correctly ackground 2 9 RT 0 04 0 24 AV 8 ML 2 7868 T cFullms 150 00 2000 00 1004 903 Relative Abundance 510 5 77 B812 908 0 10594 1381 1 1488 1 1707 9 1860 8 136 Series Background contamination has a repeating sequence of 136 amu TFA sodium salt is 136 and there was a nice spectrum of peaks 136 units apart when the sample contained it Try removing TFA from the sample and or mobile phase TFA may be difficult to remove from the column so take off the column and flow mobile phase directly into the MS This could be from the chromatograph or internal to the LCQ Identify the source by infusing methanol with a clean syringe and a new piece of tubing If the source of the contamination is the LCQ then try the following Bill Mahn Page 8 8 11 2005
15. ight of noise spikes should be less than the low E4 range 1 If you suspect that there is chemical contamination from the HPLC mobile phase use a clean syringe and tubing to infuse the mobile phase components individually into the MS e g water then water acid then methanol or acetonitrile Most commonly the acid is the source of contamination 2 If the noise disappears when HPLC flow is turned off and you have eliminated contamination a Trim the sample tube and set it to the correct distance within the needle b If still noisy the cause could be chemical noise Change source conditions e g increase heated capillary temperature 10 or 20 C increase Sheath gas flow 10 or 20 units increase Auxiliary gas flow 10 units C If still noisy there is a possibility of a bad 8kv power supply HV cable or if this is an LCQ API 1 and spray current is high there may be moisture between HV connector and ESI probe metal can 3 If there is still excessive noise with the heated capillary capped the ESI source is not the problem a Go to Diagnostics Toggles and Detectors and turn off the Main RF 1 If noise is gone then clean the Trap End Caps Ring Electrode and Insulators 2 Inspect the Insulators for scratches deposits chips or cracks Inspect the RF feed through for deposits Replace parts if necessary 3 If still noisy go to next step b Go to Tune Multiplier and turn off the Dynode 4 If noise is gone then clean
16. ill there try these items 3 If you suspect that there is chemical contamination from the HPLC mobile phase use a clean syringe and tubing to infuse the mobile phase components individually into the MS e g water then water acid then methanol or acetonitrile Most commonly the acid is the source of contamination Bill Mahn Page 2 8 11 2005 LC MS Contamination HPLC troubleshooting Many times contaminant peaks occur during a gradient Try this procedure to locate the source of the contamination e Inject a solvent blank e Make a zero volume injection e Run a gradient with the injection valve or autosampler out of the liquid flow Contamination in the MS If the noise disappears when HPLC flow is turned off and you have eliminated contamination in the mobile phase Trim or replace the sample tube and set it to the correct distance within the needle Clean the ESI spray nozzle cone and needle Replace the Teflon needle seal behind the needle If still noisy the cause could be neutral chemical noise non ionized materials Change source conditions e g increase heated capillary temperature 10 or 20 C increase Sheath gas flow 10 or 20 units increase Auxiliary gas flow 10 units Other possibilities e Solvent reservoir pickup filters are common sources of contamination Usually the aqueous phase one will be the first to go e Offline solvent filters are common sources of contamination have seen serious contamination w
17. in FASFIDK Keratin Tyll CytoKer 8 1792 9 2 Keratin LEAELGNMQGLVEDFK Keratin Tyll CytoKer 8 1357 71 Keratin LNDLEDALQQAK Keratin Tyll 1476 8 7 Keratin FLEQQNKVLETK Keratin Tyll 1301 7 2 Keratin ALEEANADLEVK Keratin Tyl CytoSkel 14 16 17 1278 5 2 Keratin GSCGIGGGIGGGSSR Keratin Tyl 16 1036 5 2 Keratin IRDWYQR Keratin Tyl 14 16 Porcine Trypsin autolytic fragments observed 2211 103 Trypsin LGEHNIDVLEGNEQFINAAK Trypsin 2211 10 2 Trypsin LGEHNIDVLEGNEQFINAAK Trypsin 1567 6 2 Trypsin LGEHNIDVLEGNEO Trypsin trunc 2083 4 2 Trypsin LGEHNIDVLEGNEQFINAA Trypsin trunc 1940 92 Trypsin LGEHNIDVLEGNEGQFIN Trypsin trunc 1713 82 Trypsin LGEHNIDVLEGNEOF Trypsin trunc 1046 00 2 Trypsin LSSPATLNSR Trypsin 1046 00 1 Trypsin LSSPATLNSR Trypsin 802 41 Trypsin LSSPATLN Trypsin trunc 802 42 Trypsin LSSPATLN Trypsin trunc 842 87 2 Trypsin VATVSLPR Trypsin 842 87 1 Trypsin VATVSLPR Trypsin trypsin like artifact sequences below are best fit sequences and not intended to be rigorously determined sequence interpretations They are merely entered to flag these ions as possible contaminants 871 11 Trypsin QATVSLPR Trypsin like artifact 871 12 Trypsin QATVSLPR Trypsin like artifact 899 51 Trypsin VQTVSLPR Trypsin like artifact 899 52 Trypsin VQTVSLPR Trypsin like artifact 824 5 1 Trypsin PGVVSLPR Trypsin like 253 VSLPR 824 52 Trypsin PGVVSLPR Trypsin like 253 VSLPR 2239 1 3 Trypsin LGEH
18. ith the Nylon 0 22 um filters used to filter buffer solutions e You might want to think about the containers you are using for your acetonitrile We had a similar problem that we traced to using glass scintillation vials with polypropylene insert caps to make up our solvent After switching to a different type of glass vial the problem went away e Surveyor MS pump The pump has a pulse dampener which is not directly in the liquid flow It is Tee d off the flow A dampener containing a badly contaminated working fluid can leach contamination into the LC solvent A thorough cleaning procedure is listed at the end of this document Phthalate Contamination 391 413 798 803 391 protonated dioctyl phthalate M H 413 sodium adduct of dioctyl phthalate M Na 798 ammoniated dimer of dioctyl phthalate 2M NH4 803 sodiated dimer of dioctyl phthalate 2M Na This is usually from contaminated solvents It can concentrate on the column and elute during a gradient Follow the normal solvent checking procedures Bill Mahn Page 3 8 11 2005 LC MS Contamination Glassware run through a dishwasher often picks up phthalate contamination Remove this with a rinse of 30 nitric acid followed by a rinse with 2M NH4OH The APCI probe can retain this Baking the APCI will eliminate this problem Try 550 deg C for 15 min Bill Mahn Page 4 8 11 2005 LC MS Contamination 44 Series Possible polymer contamination If you have shot d
19. lse dampener outlet 25 Empty all beakers into a suitable waste container Bill Mahn Page 19 8 11 2005
20. nitrile then inject methanol and water and look for the Ultramark Another Technique Before the sensitivity test is run you must clean out the residual Ultramark with methanol or isopropanol Flush spray shield capillary and needle with alcohol then 1 uL syringe with methanol 2 Cool to 150C 3 Heat capillary to 270C 4 Repeat 2 times Must get rid of the Ultramark because it is an electron acceptor The APCI is a particular problem with Ultramark contamination because the heat causes the Ultramark to boil out of the contamination sites These areas must be cleaned thoroughly The areas of concern are the outer surface of the heated capillary the PEEK bushing between the heated capillary and the spray shield the o ring and the inner diameter of the spray shield Disassemble the API stack and clean these thoroughly with acetone and then methanol You might replace the bushing and o ring Bill Mahn Page 14 8 11 2005 LC MS Contamination Bill Mahn Page 15 8 11 2005 LC MS Contamination Trouble Shooting Excessive Noise In the LCQ DUO and DECA The purpose of this document is to help isolate the cause of noise and to provide possible causes and fixes The type of noise covered in this document is defined as random noise spikes seen in the spectrum The sources of noise covered are Chemical ESI source Main RF Dynode Multiplier and associated electronics and cabling The manufacturing specification for random noise spikes is He
21. sked her to try the water and the acetonitrile separately The water is from their nanopure system The acetonitrile is from Fisher She wants to clean the API but this is unlikely to be the problem Probably the source is a filter in the water system If the problem might be from solvent clusters try source CID at 10 to 20 to confirm clustering If every infused solvent shows contamination it may be due to some residual olyethylene glycols PEG s in the MS They could be in the API stack the spray shield area or in the ESI probe or all of the above Flush the spray shield capillary and needle with methanol acetonitrile or isopropyl alcohol This means around the spray shield area directly on the end of the heated capillary around the base of the heated capillary at the PEEK sleeve and all around the needle of the ESI probe Background Ramps Up With Gradient This can be contaminated organic phase or the high organic content can be eluting retained junk from the column Try Burdick and Jackson organic phase and a new column Bill Mahn Page 5 8 11 2005 LC MS Contamination 59 Series And what about a 59 ion series I m using acetic acid in a 5 concentration 59 Da is the mass of the acetate ion But how acetate interact with the peptide Is it possible to have acetate polymerization There is another explanation for the 59 ion series Polymers of 59 might be iron in some form presumably leaching from the steel in acid We saw
22. the Dynode and Shields Scratches on the Dynode are also a source of noise Inspect the Dynode feed through for deposits Replace parts if necessary 5 If still noisy possibly the Dynode power supply is bad or go to the next step C Go to Diagnostics Toggles and Detectors and turn off the Multiplier 6 i If the noise is gone rinse the multiplier with methanol and blow dry 7 ii If still noisy possibly the multiplier power supply is bad or go to the next step d Possible bad Top cover PCB Blue cable Acquisition pcb or loose or dirty or multiplier anode When finished turn on all devices turned off during trouble shooting Bill Mahn Page 16 8 11 2005 LC MS Contamination Cleaning Processes preferably done by a qualified service engineer Cleaning the Dynode 1 2 3 4 5 6 Remove the dynode power supply and then remove the dynode feed through from the manifold Loosen the set screw attaching the dynode to the shaft Sonicate the dynode in acetone for 1 minute then 1 minute in methanol Blow dry with clean dry gas Do not touch the dynode even with gloves especially when wet with solvents Handle with a kimwipe or a lint free paper Reassemble dynode onto the shaft and feed through blow off parts then insert into the manifold While the dynode is out clean the shields by sonicating them in methanol Reinstall them then reinstall the dynode Cleaning the multiplier 1 2 3 4 remov
23. ther be coming from the HPLC or MS This may be phthalate contamination 798 could be the gas phase dimer of di octylphthalate MW 390 plus ammonium 2M NH4 The m z 803 would then be 2M Na Sometimes this can be really hard to track down If it s coming from your HPLC it can concentrate on your column until you ramp the gradient to knock it off We recommend checking the solvents by infusion to determine if it s coming from your HPLC or if it s in one of your reagents e g TFA In this case the source was finally identified as the dishwasher used to clean the HPLC reservoir bottles These could be cleaned by rinsing well with 30 Nitric Acid followed after a water rinse by 2M NH4OH This has removed many odd artifacts from our LC MS MS runs Bill Mahn Page 9 8 11 2005 LC MS Contamination 453 3 679 5 and 905 7 Contamination Nylon HPLC solvent filters can produce nylon 6 6monomer peaks at masses of 226 Da a dimer 452 trimer 678 and tetramer 905 Da The contaminant is very hard to get rid off since it binds very well to C18 Peak Clusters at 21 17 35 52 21 is sodium 17 35 52 are various losses of ammonia and water if you look at the 17 ion you will probably see a small amount of the 18 as well Iron Acetate clusters 538 555 and 534 Mobile phases with acetic acid can react with iron in the LC MS system to produce Iron acetate clusters as established by ljames Dutky and Fales J Am Soc Mass Spec 6 1226
24. tin Tyll Cytoskel 1 1716 82 Keratin QISNLQQSISDAEQR Keratin Tyll Cytoskel 1 1277 7 2 Keratin LALDIEIATYR Keratin Tyll CytoKer 4 7 8 8 Type Il Cyto Keratin 7 1418 7 2 Keratin VDALNDEINFLR Keratin Tyll CytoKer 7 1453 8 2 Keratin EVTINQSLLAPLR Keratin Tyll CytoKer 7 1442 8 2 Keratin LPDIFEAQIAGLR Keratin Tyll CytoKer 7 Type l Cyto Keratin 9 809 41 Keratin LASYLDK Keratin Tyl Cytoskel 9 1060 6 2 Keratin TLLDIDNTR Keratin Tyl CytoKer 9 1066 0 2 Keratin STMQELNSR Keratin Tyl CytoKer 9 1315 7 2 Keratin LNDLEEALQQAK Keratin Tyl CytoSkel 9 1586 8 2 Keratin VQALEEANNDLENK Keratin Tyl Cytoskel 9 1190 6 2 Keratin QVLDNLTMEK Keratin Tyl Cytoskel 9 1307 7 2 Keratin IKFEMEQNLR Keratin Tyl Cytoskel 9 2171 02 Keratin SDLEMQYETLQEELMALK Keratin Tyl Cytoskel 9 1851 92 Keratin TLNDMRQEYEQLIAK s Keratin Tyl CytoKer 9 2565 88 3 Keratin EVTQLRHGVQELEIELQSQLSK Keratin Tyl Cytoskel 9 1350 7 2 Keratin I GLGGRGGSGGSYGR Keratin Tyl CytoKer 9 2902 7 3 Keratin NYSPYYNTIDDLKDQIVDLTVGNNK Keratin Tyl CytoKer 9 1791 7 2 Keratin GGSGGSYGGGGSGGGYGGGSGSR Keratin Tyl CytoKer 9 1837 92 Keratin HGVQELEIELQSQLSK Keratin Tyl CytoKer 9 2705 13 Keratin GGGGSFGYSYGGGSGGGFSASSLGGGFGGGSR Keratin Tyl CytoKer 9 1315 7 2 Keratin DQIVDLTVGNNK Keratin Tyl CytoKer 9 2510 1 1 Keratin EIETYHNLLEGGQEDFESSGA Keratin Tyl CytoKer 9 3223 2 3 Keratin GGSGGSHGGGSGFGGESGGSYGGGEEASGSGGGYGGGSGK Keratin Tyl CytoKer 9 Type I C
25. unk 983 5 2 unk_x85 X 285 385 485 585 unk BSA TRYPTIC PEPTIDES 1479 82 BSA LGEYGFONALIVR BSA 1567 72 BSA DAFLGSFLYEYSR BSA 1305 7 2 BSA HLVDEPQNLIK BSA Bill Mahn Page 13 8 11 2005 LC MS Contamination 1443 62 BSA YICDNQDTISSK BSA 1002 6 2 BSA LVVSTOTALA BSA cterm 1640 32 BSA KVPQVSTPTLVEVSR BSA 1640 3 3 BSA KVPQVSTPTLVEVSR BSA 1440 83 BSA RHPEYAVSVLLR BSA 1400 7 2 BSA TVMENFVAFVDK BSA 1072 62 BSA HCIAEVEK BSA 1567 72 BSA DAFLGSFLYEYSR BSA 1491 82 BSA FYAPELLYYANK BSA 732 51 BSA GLVLIAF BSA CASEIN TRYPTIC PEPTIDES 1384 7 2 Casein FFVAPFPEVFGK bo Casein alpha S1 1267 7 2 Casein YLGYLEQLLR bo Casein alpha S1 742 51 Casein GPFPILV bo Casein alpha S1 1251 7 2 Casein YIPIQYVLSR bo Casein alpha S1 PROTEASE I PRECURSOR API LYSYL ENDOPEPTIDASE 1830 8 2 API ACHLY SDQYGRVFTSWTGGGAAA Lys C 1367 7 2 API ACHLY VFTSWTGGGAAASR Lys C 2055 0 3 API ACHLY RVLGQLHGGPSSCSATGTNR Lys C 1366 7 2 API ACHLY RISNSTSPTSFVA Lys C 1064 6 2 API ACHLY DIIRAVGAYS Lys C Removing Ultramark Contamination Empty the syringe that contained the tuning solution Flush the syringe 3 times with HPLC grade acetone Set the sheath gas to 80 Manually inject acetone three times Open the source and squirt acetone clean squeeze bottle onto the end of the fused silica and on the heated capillary and the spray shield Thoroughly flush this area then wipe dry with Kim wipes Repeat this procedure with aceto
26. ve open the purge valve and draw 10ml of the nitric acid solution through the pulse dampener Close the purge valve and empty the priming syringe into a suitable waste container Replace the beaker containing nitric acid with the beaker containing water Reconnect the priming syringe open the purge valve and draw 10ml of water through the pulse dampener Close the purge valve and empty the priming syringe into a suitable waste container Replace the beaker containing water with the beaker containing iso propanol Reconnect the priming syringe open the purge valve and draw 10ml of iso propanol through the pulse dampener Close the purge valve and empty the priming syringe into a suitable waste container Replace the beaker containing iso propanol with the beaker containing n hexane Reconnect the priming syringe open the purge valve and draw 10ml of n hexane through the pulse dampener Close the purge valve and empty the priming syringe into a suitable waste container Replace the beaker containing n hexane with the beaker containing iso propanol Reconnect the priming syringe open the purge valve and draw 10ml of iso propanol through the pulse dampener Close the purge valve and empty the priming syringe into a suitable waste container Reconnect the 1 16th inch PEEK line going to the pulse dampener inlet to the LDA outlet Reconnect the 1 16th inch 316 SS solvent line from Surveyor Autosampler to the pu
27. yto Keratin 10 809 42 Keratin LASYLDK Keratin Tyl CytoSkel 10 1031 6 2 Keratin VLDELTLTK Keratin Tyl CytoSkel 10 1165 6 2 Keratin LENEIQTYR Keratin Tyl CytoSkel 10 1708 8 2 Keratin GSLGGGFSSGGFSGGSFSR Keratin Tyl CytoSkel 10 1798 1 2 Keratin NVQALEIELQSQLALK Keratin Tyl CytoSkel 10 1390 7 2 Keratin QSLEASLAETEGR Keratin Tyl Cytoskel 10 2872 4 3 Keratin NVSTGDVNVEMNAAPGVDLTQLLNNMR st Keratin Tyl CytoSkel 10 2082 9 2 Keratin AETECQNTEYQQLLDIK Keratin Tyl CytoSkel 10 1549 6 2 Keratin SGGGGGGGGCGGGGGVSSLR Keratin Tyl CytoSkel 10 2746 4 3 Keratin YCVQLSQIQAQISALEEQLQQIR Keratin Tyl CytoSkel 10 1493 7 2 Keratin SQYEQLAEQNRK Keratin Tyl CytoSkel 10 1003 5 2 Keratin SEITELRR Keratin Tyl CytoSkel 10 1262 6 2 Keratin SLLEGEGSSGGGGR Keratin Tyl CytoSkel 10 1381 62 Keratin ALEESNYELEGK Keratin Tyl CytoSkel 10 995 51 Keratin IKEWYEK Keratin Tyl CytoSkel 10 2904 4 3 Keratin NNSTGDVNVEMNAAPGVDLTGLLNNMR Keratin Tyl CytoSkel 10 2240 13 Keratin ADLEFQIESLTEELAYLKK Keratin Tyl CytoSkel 10 Msx gt F Type Il Cyto Keratin 8 1000 6 2 Keratin LOAEIEGLK Keratin Tyll CytoKer 8 1344 72 Keratin ASLEAAIADAEQR Keratin Tyll CytoKer 8 1419 7 2 Keratin LEGLTDEINFLR Keratin Tyll CytoKer 8 2109 02 Keratin ELQSQISDTSVVLSMDNSR Keratin Tyll CytoKer 8 Bill Mahn Page 11 8 11 2005 LC MS Contamination 1847 82 Keratin SNMDNMFESYINNLR Keratin Tyll CytoKer 8 1129 6 2 Keratin LSELEAALQR Keratin Tyll CytoKer 8 827 41 Kerat
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