•ACQUITY UPLC Troubleshooting, Tips and Tricks. · ACQUITY UPLC™ Tips and Tricks...

•©2009 Waters Corporation | COMPANY CONFIDENTIAL•©2011 Waters Corporation

•ACQUITY UPLC Troubleshooting, Tips and Tricks.


AgendaAgenda

• General use, Recommendations

• System Preparation

• Injection Modes

• Carry-over

• Plumbing

• Cleaning procedures

• Vials

©2011 Waters Corporation 3

General use, Recommendations- Mobile Phase Preparation - Solvent Manager (BSM and QSM)- Sample Manager- Detectors (optical)- System Preparation


ACQUITY UPLC™ Tips and Tricks Mobile Phase Preparation

� Always make sure that there are no insoluble particles in the eluents— Nothing different from HPLC…

� Organic solvents— Usually High Grade organic solvents are prefiltered through a

membrane (read the label on the bottle)— If you need to filter, check out membrane incompatibility with

organic solvent(s).� Water

— Milli-Q water is filtered through a 0.22 µm membrane (sterile)— Avoid using 100% aqueous mobile phases – add 5% to 10%

organic modifier where possible.— Use clean glassware, bottles and for filtration (borosilicate

,Pyrex® or equivalent)


Recommended Guidelines of Use for the Acquity System

� Solvent Manager (BSM and QSM)— Do not block the degasser vent line

o Trim if necessary (Do not put the line in the waste bottle)— Keep all solvent lines primed

o Fill unused lines with water/methanol— Long term storage

o Flush with water, then organic solvent (IPA)— Keep seal wash primed !!!!

o 90-95% water— Re-prime solvent lines before starting

o Sys Prep or Start-up— Change aqueous mobile phases often – every 24 – 48 hr

o Remember: UPLC® flow rates are much lower, thusmobile phases are consumed at a lower rate and last longer


System equilibration BSM/QSM

•Compression / Decompression Ratio:•1 - 1.4 = Normal•1.4 -1.8 = Fair•> 1.8 = Possible Bubble

•Pressure ripple in psi for the past minute should normally be < 30 psi indicating that the BSM/QSM is operating well.



� Sample Manager— Use « ANSI-XXXX » format (plate) and pre-slit septa (vials)— Stay within the recommended limits for each injection mode

and loop size (see table on injections modes)— Characterize needle and loop volumes when changing weak

wash solvents as well as loops and needles— Always use the column stabilizer tube even if temperature is

not used— Use the correct aspiration speed (viscosity of the sample)— Defrost function


ACQUITY UPLC™ Tips and TricksRecommendations for Sample Manager

� Check your Weak Wash Solvent— Water or 95/5 water/AcN is fine (Partial Loop with Pressure assist !)— Same as your initial solvent conditions— No salt buffers in wash solvents

� Check your Strong Wash Solvent— Sample must be soluble to both weak and strong— Pure organic solvent (AcN, MeOH) is fine, but …— Try 70:20:10 Methanol/Water/IPA— Change proportions

o 25% each Methanol, Acetonitrile, Water, Isopropanol� Within a method, use ratio 3:1, weak wash to strong to ensure weak wash flushed the strong from needle and sample loop.

o Could observe peak retention shifts and deformity of early eluting peaks as an indicator of lack of weak wash amount



� Detectors (optical)— ALWAYS ensure the back pressure regulator is installed if UV

detector is last in line.— Flow cell is filled with transparent solvent(ACN or water) or

constant flow in cell before power on— At least 30 minutes equilibrate time— Choose a data rate to collect 25-50 points across the peak.— Recommended settings to start

o Data rate: 20 pts/sec or 40 pts/seco Filter time constant: Fast


ACQUITY UPLC™ Tips and TricksSystem Preparation

� Prime pumps - solvents— New mobile phases (replaced)

o Same as before: 4 min4 min

o Different as before: 7 min7 min (with intermmediate is needed)(with intermmediate is needed)

— Old mobile phases (continuing)

o System has not been working less than 4 hours: 1 min1 min

o More: 3 min3 min

— Seal Wash



� Injector priming (Prime Syringes)— New solutions (Weak&Strong Needle Wash): 10 cycles10 cycles

— Old, continuing: 4 cycles4 cycles



Equilibrate to Method



Characterize Volume— After sample needle replacement select “Characterize Seal” and

“Characterize needle and loop volumes”— After Weak wash solvent replacement or loop replacement

enter loop volume (Change...) and perform “Characterize needle and loop volumes”


Injection modes- overview- amount of sample used- recommended injection volumes


Injection modes:overview

••Reminder: Reminder:

••Partial loop (pressure assist) Partial loop (pressure assist) ••loop contains sample, compressed air loop contains sample, compressed air and and Weak needle washWeak needle wash

••Partial loop with needle overfill (PLUNO)Partial loop with needle overfill (PLUNO)••loop contains sample and mobile phaseloop contains sample and mobile phase

••Full loopFull loop: : ••loop contains sampleloop contains sample


Injection Modes: Amount of sample used

Injection volume

programmed

Injection volume

used On column

Partial loopPressure assist 1 µL 1 µL 1 µL

Partial loop needle overfill 1 µL 4 to 31 µL 1 µL

Full loop1 µL loop10 µL loop

1 µL 10 µL

*

5.8 µL 40 µL

Loop volume(see

characterization)

•Needle overfill flush


Injection Modes:Injection Modes:Recommended injection volumesRecommended injection volumes


Carry-over- definition- why?- specifications- tips & tricks


Carryover:definition

� What is carryover?— Analyte remaining from a previous injection

� How do you measure carryover?— Sample or standard followed by one or more blanks

� Does ACQUITY Sample Manager have a carryover specification?— Yes. 0.005% or 2.0 nL. Compound and detector specific.

� Possible Sources Of Carry-over — Column— Needles (Sample needle and piercing needle)— Sample loop— Connections in the injection valve


� You have to check if the two Peek connections located on the inject valve are correctly positioned. If not, some active product can stay on the dead volume created by bad connections.

� Injection pod & loops are replaced during PM

•Typical problem with bad peek connections•First blank 0.1 %•Second blank 0.01 %. •.

• With proper Peek connections•First blank <0.005 %•. •.

Carry-over: Check loop


� Check all connections— Sample loop, tubing from injection valve to column …

� Change gradient (final step with organic solvent)� Do not overfill the vials (cross contamination from the piercing

needle)� Optimize/change weak and strong solvents

— Adapt wash solvent with sample solvent— Magic mixture : H2O/ACN/MeOH/Isopropanol (25/25/25/25)— DMSO can be used

� Increase volumes (wash solvent)— Respect ratio strong/weak 1/3

� Add a blank injection(strong wash solvent) every 20 to 30 samples injected

� Use the stainless steel needle (less adsorption of very hydrophobic compounds).

Carry-over: Tips & Tricks


Plumbing


Passive Column Pre-Heater / Stabilizer � Passive column pre-heater reduces sensitivity to ambient

temperature swings and reduces / minimizes band spreading


Column Stabilizer Kit – 30, 50 & 100 mm columns

••Column Stabilizer Kit Column Stabilizer Kit –– 150 mm column150 mm column


Pre-heater SparesUse these part numbers when

replacing a pre-heater

� Column stabilizer kit: first generation 205000291 ASSY, TUBE, PRE-HEATER STABILIZER STD205000365 ASSY, TUBE, PRE-HEATER STABILIZER 150MM

� Column stabilizer kit: second generation fixed205000489 KIT, HTCH PRE-HEATER STABILIZER 50MM205000494 KIT, HTCH PRE-HEATER STABILIZER 150MM


ACQUITY UPLC™ H-Class Update Active Pre-Heater and Sleeve

•Waters Confidential and


Connections to Column Stabilizer tube


Finger Tight Fitting Spares� Use these part numbers when replacing any of the existing finger-tight fitting components


Band spreading, tailing


Cleaning procedures

� baseline noise/drift, � decreased sample energy levels, � calibration failure


Preparation And DecontaminationWashing Procedure

� Magic Mix: 25/25/25/25— H20/MeOH/ACN/IPA/0.2% FA

� Passivation and decontamination of system— 50:50 methanol/water — Isopropanol— 100% water — 30:70 (v/v) phosphoric acid:water — 100% water — 50:50 (v/v) methanol:water— Disconnect column and MS !

� Detectors (LG cells and fluorescence detector)— First with 1% solution of formic acid and then above

� More sophisticated (and longer !) procedures are available— “Controlling Contamination in UltraPerformance LC/MS and

HPLC/MS Systems” (PN 715001307)


Baseline problems

� Name some baseline, background problems— Spikes— Drop out— Periodic cycling— Increase or decrease in baseline


•Time

•Mixing of A and B mobile phases incomplete and additive, e.g. formic acid is not the same.•Period will change with a gradient.

•Variation in room temperature

•Check valve problem. Period is linked to the piston filling

•QUESTION: What is the time of each period or cycle?

•Air bubble

Baseline ProblemsWhat causes these baselines?


ACQUITY UPLC™ Platform UpdateGradient Mixers

� Use of 100 µl Gradient Mixer to improve the baseline in low UV detection (for ex 210 nm) with H20/MeOH or H20/ACN/TFA

� Three fonctions - T connections- On-line filter- Zirconium Bed suitable for basic pH

AU

0.00874

0.00876

0.00878

0.00880

0.00882

0.00884

0.00886

0.00888

0.00890

0.00892

0.00894

0.00896

0.00898

0.00900

0.00902

0.00904

0.00906

0.00908

0.00910

Minutes33.60 33.80 34.00 34.20 34.40 34.60 34.80 35.00 35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20

•With gradient Mixer 50 µl•Réf 700002911

AU

0.0013

0.0014

0.0015

0.0016

0.0017

0.0018

0.0019

0.0020

0.0021

0.0022

0.0023

Minutes25.20 25.40 25.60 25.80 26.00 26.20 26.40 26.60 26.80 27.00 27.20 27.40 27.60 27.80 28.00 28.20 28.40 28.60 28.80

•With gradient Mixer 100 µl•Réf 205000272


Vials


ACQUITY Sample ManagerRecommended Settings, Vials

� Recommended settings for the different vial formats (p57)— When the selected plate format is ANSI 48 vial 2-ml, the offset

is automatically turned to 2mm— Use “Advanced …” in the method (Sample Manager area) to

change the default settings


•Thank you very much for your attention

•ACQUITY UPLC Troubleshooting, Tips and Tricks. · ACQUITY UPLC™ Tips and Tricks...

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