Instrument Maintenance - Waters

1 2012 Waters Corporation 1 Instrument Maintenance Biopharma LC Meeting Eschborn, 16th 17th October, 2012 Mario Boras Eoin Cosgrave Jean-Michel Plankeele 2012 Waters Corporation 2 Instrument Maintenance Suggested Topics Quaternary Solvent Manager Solvent filters Gradient mixers Leaving the system? Cleaning, decontamination Columns General guidelines Flow Cells General e-community web site General recommendations (solvents) Need to find a P/N? Reference material System preparation System Prep function System set up (solvents, wash solvents, ..) 2012 Waters Corporation 3 ACQUITY UPLC Online Community A ACQUITY UPLC Online Community Everything You Always Wanted to Know About.

2 UPLC Log on to Waters web site (Professional e-mail adress required) Quick Links (bottom right) > Waters Communities > ACQUITY UPLC Go to BSM / QSM serial number required for registration Provides all ACQUITY UPLC System users a wealth of UPLC-related information at their fingertips, including: Peer-reviewed journal publications and reviews UPLC application and technology discussions (no taboo !) Exclusive product previews and early research Latest application notes, white papers, and articles Educational webcasts, how-to videos UPLC basics and troubleshooting ACQUITY Users Meeting happenings from around the world 2012 Waters Corporation 4 ACQUITY UPLC Online Community Main page after Login Blog, discussion group Product preview How to videos 2012 Waters Corporation 5 ACQUITY UPLC Online Community Main page after Login Presentations Users guides, application notes White papers, Care and use 2012 Waters Corporation 6 New Challenges With UPLC Mobile Phase Preparation Smaller particles, columns (ID & length) and frits Typical HPLC conditions: Flow rate: ~ 1 ml/mn Column.

3 ~ 4 mm Inlet frit pore size: ~ 2 m Typical UPLC conditions: Flow rate: ~ ml/mn Column : ~ 2 mm Inlet frit pore size: ~ m Small particles which do not affect HPLC columns can affect UPLC columns Column lifetime can be reduced Pressure is higher and higher, column plugged Impurities (from solvent and sample) can be detected Bacterial growth can impact column lifetime 2012 Waters Corporation 7 ACQUITY UPLC Tips and Tricks Mobile Phase Preparation Always make sure that there are no insoluble particles in the eluents Nothing different from Use high quality solvents, buffers and additives. Organic solvents Usually High Grade organic solvents are filtered through a membrane (read the label on the bottle) JT Baker LC/MS Grade, Biosolve, Burdick & Jackson, Fisher Optima LC/MS Grade Water Milli-Q water is filtered through a m membrane (sterile) oBacterial contamination is a problem Milli-Q system, properly maintained Bottled water ?

4 2012 Waters Corporation 8 ACQUITY UPLC Tips and Tricks Mobile Phase Preparation Chemicals, reagents, powders Use high grade reagents Filter non volatile buffers ( m or m high quality membrane)! Use clean glassware (bottles and device for filtration) Who takes care of this ? How is it managed ? What is the quality of glass ? Is it compatible with the mobile phase (pH) ? Bottles must be capped, do not top-off the bottles Waters UPLC Media bottle kit (P/N EEE000394): High quality type 1 Class A borosilicate bottle kits One kit includes 7 bottles (4 x 1 l and 3 x 500 ml) All caps and pouring rings (colour coded) All bottles are printed with ACQUITY & Waters Logo, batch number and part number 2012 Waters Corporation 9 The Graphical Parts Locator Access To All Part Numbers From Waters Home Page Must be a registered user Can also be accessed from ACQUITY UPLC Community 2012 Waters Corporation 10 The Graphical Parts Locator Select the Instrument or the model 2012 Waters Corporation 11 The Graphical Parts Locator ACQUITY UPLC H-Class System Select the component of interest 2012 Waters Corporation 12 The Graphical Parts Locator ACQUITY UPLC H-Class System The P/N will be shown after the next mouse click on the spare parts 2012 Waters Corporation 13 Reference Material Care and Use Size Exclusion and Ion-Exchange Chromatography of Proteins using the

5 ACQUITY UPLC System, 715002147, REV. A Size Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System, 715002909, Rev A Controlling contamination in LC/MS and HPLC/MS Systems, 715001307 Improving the Lifetime of UPLC Size-Exclusion Chromatography Columns Using Short Guard Columns, Waters Technical Brief, 720004034en Guidelines for Routine Use and Maintenance of Ultra-Performance Size-Exclusion and Ion-Exchange Chromatography Systems , Waters Technical Brief, 720004182en Downloadable from 2012 Waters Corporation 14 ACQUITY UPLC Tips and Tricks System Preparation System preparation function (introduced with ) All steps required to condition the system combined in one function oPrime/Purge all pumps and all solvent lines oPrime/Purge all syringes oCharacterise sample loop and needle (SM-FL) Required if weak wash solvent was changed oEquilibrate system and chemistry with customized parameters (solvent, flow rate, temperature) oFrom console or included in the sample set (Empower) Refresh function oPrime solvents A1 and B1 (2 min.)

6 OPrime all syringes 2012 Waters Corporation 15 ACQUITY UPLC Tips and Tricks System Preparation System Preparation function In the console select Start up from main pull down menu Right other control pannels (run window) Prime solvents (QSM/BSM and SM) Characterize volumes / Needle seal Equilibrate 2012 Waters Corporation 16 ACQUITY UPLC Tips and Tricks System Preparation System Preparation function in progress: 2012 Waters Corporation 17 System Set Up Solvents (eluents) Configuration for AutoBlend Plus (mobile phases) Line A: Acidic salt (usually 100 mM) Line B: Basic salt (usually 100 mM) Line C: Salt (usually M, used for controlling ionic strength) Line D: Pure Water AutoBlend Plus is available for both H-Class and H-Class Bio Other solvents configured on the QSM Seal wash: Pure water + 5% organic solvent (bacterial growth!)

7 Purge: pure water with 5% organic solvent (solvent in the sample syringe) Wash solvent (in the sample manager; SM-FTN): For washing the external part of the needle. Prevents carry-over Must dissolve samples and contaminants Sample diluent is appropriate Initial mobile phase strength is appropriate 2012 Waters Corporation 18 System Set Up What If You Would Like To Use A SM-FL ACQUITY UPLC and ACQUITY I-Class can be configured with a SM-FL FL: Fixed loop Same rules apply for solvents (eluents) lines and seal wash 2 Wash solvents (SM-FL): Strong wash (to prevent carry over; internal part of needle & tubing assy): must dissolve sample & contaminants pure water Weak wash: for replacing the strong wash with a less elutive solvent.

8 Mobile phase A 2012 Waters Corporation 19 Quaternary Solvent Managers Stainless Steel & Titanium Solvent Filters Solvent filters (bottle filters, sinkers) can be the source of contamination Previous buffers Bacterial growth With high concentration salts use titanium filters P/N: 700005378 (SOLVENT FILTERS, Ti, 7pk) When contamination is suspected, can be washed Backflush with 70% organic solvent (IPA, EtOH or MeOH) in water 10 ml at minimum, with a syringe (luer-lock) Regular (SS) solvent filters P/N 700003615 (Filter, Solvent Bottle, SS, PKG 1) P/N 700003616 (Flter, Solvent Bottle, SS, PKG 7) 2012 Waters Corporation 20 Quaternary Solvent Managers Gradient Mixers Mixers are required for an optimum mixing of mobile phases The larger the mixer, the lower the baseline noise The larger the mixer, the larger the delay volume (system volume) Default: 100 l (P/N 700005258) Optional.

9 250 l (P/N 205000737) Column based gradient mixer for peptide mapping (H2O/AcN/TFA); P/N 205000403 Useable for applications up to 10000 psi Uses Zirconia beads (800 m), volume about 425 L 2012 Waters Corporation 21 Leaving The System? Short term over night Wash the system and column with water (remove all buffer and salts) Maintain a low flow rate with 100% water Turn off the lamp (from console or through a stand-by method) Long term Store column according specifications (care & use manual) Remove the column and replace with union Place all solvent lines in water (high quality) Flush all lines with water Place all lines in water with organic solvent (30% AcN, MeOH or IPA) Flush all lines then stop flow Switch off all components 2012 Waters Corporation 22 Preparation And Decontamination Washing Procedure Magic Mix: 25/25/25/25 H20/MeOH/ACN/ FA Passivation and decontamination of system 50.

10 50 methanol/water Isopropanol; flush with water 30:70 (v/v) phosphoric acid in water; flush with water 50% water; 49% methanol; 1% ammonium hydroxide 100% water Only for BSM/QSM and SM ! Disconnect column and detector ! Detectors (LG cells and fluorescence detector) 1% solution of formic acid or phosphoric acid More sophisticated (and longer !) procedures are available Controlling Contamination in UltraPerformance LC/MS and HPLC/MS Systems (PN 715001307) 2012 Waters Corporation 23 UV Detectors Which Flow-Cell? Teflon cell vs Titanium cell