instrument Maintenance - Waters Biopharma_Instrume… · ©2012 Waters Corporation 2 Instrument Maintenance Suggested Topics Quaternary Solvent Manager –Solvent filters –Gradient

©2012 Waters Corporation 1

Instrument Maintenance

Biopharma LC Meeting

Eschborn, 16th – 17th October, 2012

Mario Boras

Eoin Cosgrave

Jean-Michel Plankeele


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, …)


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

http://forums.waters.com/community/acquity_uplc



Main page after Login

Blog, discussion group Product preview “How to” videos



Main page after Login

Presentations

Users guides, application notes

White papers, Care and use


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


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 ?


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


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


The Graphical Parts Locator

Select the instrument or the model


The Graphical Parts Locator ACQUITY UPLC H-Class System

Select the component of interest


The Graphical Parts Locator ACQUITY UPLC H-Class System

The P/N will be shown after the next mouse click on the spare

parts


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


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



“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



“System Preparation” function in progress:


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


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


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)


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


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


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)


UV Detectors Which Flow-Cell?

Teflon cell vs Titanium cell

Documents

instrument Maintenance - Waters Biopharma_Instrume… · ©2012 Waters Corporation 2 Instrument Maintenance Suggested Topics Quaternary Solvent Manager –Solvent filters –Gradient