1. 1 The world leader in serving science I Orbi 5 - 2014 Performing nanoLC Doesn’t Have to be Wicked Hard

2. 2 Read Your System Manuals 90% of the questions asked are answered in the manual Example: use of LC/MS-grade solvents only. You may have been able to get away with LC Grade Solvents on your High Flow LC System—Don’t Try it on your nanoLC. YOU WILL REGRET IT!!!

3. 3 Keep Your Solvents Fresh—From the Manual Because the vapor pressures of formic acid, water, and acetonitrile differ, the solvent composition changes over time. Refresh the solvent to maintain a consistent solvent composition. Solvents will last several weeks, recommended replacement is 2-4 weeks to avoid large solvent composition changes or bacterial growth. Replace solvents rather than “top off” and time the replacements to fit in between sample sets. Particularly when using pre-mixed solvents, make sure that the solvents are always fresh, and have been properly prepared. After preparation, degass by ultrasonication, to remove the excess air that could have introduced from the mixing.

4. 4 Keep your nano-LC System On and Flowing at all times

5. 5 When a Sequence Queue Has Finished Set %B to a least 50% Allow MS to go to Standby or Keep Running—Your Preference If you keep flow rate the same as acquisition flow Ok to leave MS On OK to Set MS to Standby If you decrease flow rate Don’t go below 100 nl/min Set MS to go to Standby

6. 6 Setting Idle Flow on an EASY nanoLC 100

7. 7 Setting Idle Flow on an RSLC nano

8. 8 Good Habits—Form them NOW! Do not use and re-use fittings and sleeves Have ample ferrules, sleeves, and connectors on hand See nLC-Connection Guide for Part Numbers Consider replacing liquid junction semi-annually for high use, annually for moderate use Electrode corrodes and erodes over time Spray stability can become compromised Avoid Introducing Air to the System Never allow any solvent bottle to fall below 1/2 full Set a minimum pressure in your methods--just in case you don’t heed preceding bullet point--to keep air out of system when solvents run low De-gas your solvents

9. 9 NanoVipers Use them! Ensures a zero dead volume leak free system No more silica scraping rotors Do not over tighten connections (general guide line: fingertight + maximum an additional one eighth of a turn).

10. 10 EASY-nLC Maintenance Daily/Before LCMS Acquisition/Solvent Refresh Make sure Solvent A, B, and W3 are ¾ full. Refresh (NOT REFILL) if necessary. Check wash bottle (W4) and the plastic waste beaker in front of the solvent system compartment. Empty if necessary. Run the Purge Solvent scripts with 2x iterations for Pumps A, B, and S. Follow Purge with the Flush Air script until flush volumes on all pumps fall below 10 μL for the EASY-nLC II instrument or 12 μL for the EASY-nLC 1000 instrument. Quarterly/As Needed Run the Back Pressure script for both solvents. Run the Leaks script for “A+B” and for “System,” to check for and diagnose instrument leaks

11. 11 RSLC Nano Maintenance Daily Check All Solvent Levels (includes transfer vial in AS) Check/Empty Waste Containers Monthly/Replacing Solvents Purge Solvent A and Solvent B (20 minutes) Purge Flow Meter (45 minutes) Run Pressure Transducer Test Run Viscosity Measurement (optional) Performs a low level test on pump performance Pass, Pass with limitations or Fail Every six weeks, loading pump automatically performs an internal maintenance procedure when you initiate a purge cycle. When internal maintenance is complete, the purge cycle starts automatically If no purge cycle is initiated after another six weeks, a message appears in the Chromeleon Audit Trail, reminding you to start a purge cycle.

12. 12 Column Maintenance Condition Your New Columns You may need to start with low flow rates and build to desired flow rate to avoid over pressurizing your system Run at 50% for 20-30 minutes Equilibrate with Solvent A Store Columns in 25-50% Solvent B Remove and Cap Easy-Spray and nano-Viper Columns Other columns see: http://www.newobjective.com/support/tipstricks.shtml Wash and Equilibrate Columns with Appropriate Volume 3x column volume See next slide Do not Exceed Max Pressure or Temperature for column Set these slightly lower than max to give yourself a cushion www.thermoscientific.com/chromexpert Column Support:

13. 13 Know Your Column and Dead Volumes Tubing Volume (µl) Length (cm)25 µm ID50 µm ID75 µm ID100 µm ID150 µm ID 100.050.200.440.791.77 200.100.390.881.573.53 300.150.591.332.365.30 400.200.791.773.147.07 500.250.982.213.938.83 600.291.182.654.7110.60 700.341.373.095.5012.37 800.391.573.536.2514.13 900.441.773.987.0715.90 1000.491.964.427.8517.67 Vol (µl) = Length in mm * π * (ID in mm)/2) 2

14. 14 Something to Consider Ideal to keep your system under pressure the entire flow path Drops in pressure can cause out-gassing Out-gassing causes unstable spray Unstable spray results in poor LCMS data If Set-Up requires an emitter post-column Ensure emitter ID is less than column ID—recommend 20 µm Consider packing the emitter with 1-2 mm of resin Decreases out-gassing Pack it yourself New Objective will do it for you

15. 15 Establish A Baseline—Record Keeping and QCs Expected Column Pressure Range Pressure will vary from column to column, but you should establish a range Record Pressure Daily Aids in troubleshooting and knowing when it is time to change the column Use QCs Sets a baseline expectation you can always refer to—invaluable for troubleshooting Run daily and intermitted during sample acquisition Once LCMS method is established for QC don’t change it Simple Mixture like PRTC can be thought of as an LC QC Provides info on expected RT, Peak Quality and Carryover Changes from the expected indicate problems with the Column and/or LC Complex Sample like HeLa cell digest can be thought of as a whole platform QC More MS info like #of Peptides ID More indicative of your actual samples

16. 16 Because I Knew You Would Ask PRTC Part numbers PRTC mix, 5 pmol/µl, 200 µl: Pierce 88321 PRTC mix, 0.5 pmol/µl, 50 µl: Pierce 88320 HeLa Cell Digest Part numbers HeLa Protein Digest Standard, 20 µg: Pierce 88328 HeLa Protein Digest Standard, 5 x 20 µg: Pierce 88329

17. 17 nanoLC Connection Guide Problem: the connectivity between our nanoLC, nanoESI and consumables has been the cause of customer confusion and frustration Download: http://planetorbitrap.com/library?t=QTEyOTFlODg0OWE1NWYzYw%3D%3D&ke ywords=A1291#tab:keywords http://planetorbitrap.com/library?t=QTEyOTFlODg0OWE1NWYzYw%3D%3D&ke ywords=A1291#tab:keywords

18. 18 The Moral of the Story Is…. Good lcms grade solvents, zero dead volume connections, proper lc and column Maintenance all contribute to good liquid chromatography separation and quality nanospray Result: Excellent nanoLCMS Data