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©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 waters.com-based ACQUITY UPLC® Online Community
– Everything You Always Wanted to Know About … UPLC
– Log on to Waters web site (Professional e-mail adress required)
– Quick Links (bottom right) > Waters Communities > ACQUITY UPLC
– Go to http://forums.waters.com/community/acquity_uplc
– 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 I.D.: ~ 4 mm
– Inlet frit pore size: ~ 2 µm
Typical UPLC conditions:
– Flow rate: ~ 0.6 ml/mn
– Column I.D.: ~ 2 mm
– Inlet frit pore size: ~ 0.2 µm
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 HPLC…
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 0.22 µm membrane (sterile)
o Bacterial contamination is a problem
– Milli-Q system, properly maintained
– Bottled water ?
©2012 Waters Corporation 8
ACQUITY UPLC™ Tips and Tricks Mobile Phase Preparation
Chemicals, reagents, powders
– Use high grade reagents
– Filter non volatile buffers (0.45 µm or 0.22 µm 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 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 www.waters.com
©2012 Waters Corporation 14
ACQUITY UPLC™ Tips and Tricks System Preparation
System preparation function (introduced with V1.20)
– All steps required to condition the system combined in one
function
o Prime/Purge all pumps and all solvent lines
o Prime/Purge all syringes
o Characterise sample loop and needle (SM-FL)
• Required if weak wash solvent was changed
o Equilibrate system and chemistry with customized parameters
(solvent, flow rate, temperature)
o From console or included in the sample set (Empower)
Refresh function
o Prime solvents A1 and B1 (2 min.)
o Prime 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 1.0 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!)
– 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
– i.e. pure water
– Weak wash: for replacing the strong wash with a less elutive
solvent. 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: 100µl (P/N 700005258)
Optional: 250µl (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/IPA/0.2% FA
Passivation and decontamination of system
– 50:50 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
Recommended