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©2012 Waters Corporation 1
Knowing your LC/MS system
- benchmark tests and
fundamental fault finding
Tore Caesar
3rd [Nordic MS Symposium], Jurmala 2012
©2012 Waters Corporation 2
Agenda
Performance tracking
Automatic system checks
Basic trouble shooting
Remote service
Questions
©2012 Waters Corporation 3
Performance tracking
Systems running routine analyses
– QC samples
– Internal standards
– Standards
Open Access Systems and systems with diverse projects
– Specific Test sample for a ”System Suitability Test”
– Not expensive, stable in solution, non-toxic and compatible with the
chemistry used.
©2012 Waters Corporation 4
Test Mix MS
Component Empirical
Formula
Exact Mass
(as [M+H]+)
Exact Mass
(as [M-H]-)
UV Absorbance
Acetaminophen C8H9NO2 152.0712 150.0555 257 nm
Caffeine C8H10N4O2 195.0882 193.0726 273 nm
Sulfaguanidine C7H10N4O2S 215.0603 213.0446
Sulfadimethoxi
ne
C12H14N4O4S 311.0814 309.0658
Val-Tyr-Val C19H29N3O5 380.2185 378.2029
Verapamil C27H38N3O4 455.2910 453.2753
Terfenadine C32H41NO2 472.3216 470.3059
Leucine
Enkephalin
C28H37N5O7 556.2771 554.2615
Reserpine C33H40N2O9 609.2812 607.2656
Erythromycin C37H67NO13 734.4691 732.4534
©2012 Waters Corporation 5
Test Mix MS
©2012 Waters Corporation 6
Test Compounds UV
Component UV Abs
2-Acetylfuran 254 nm
Caffeine 273 nm
Acetanilide 254 nm
Acetophenone 254 nm
Propiophenone 254 nm
Bytulparaben 254 nm
Benzophenone 254 nm
Valerophenone 254 nm
©2012 Waters Corporation 7
Evaporative Light Scattering Fluorescence
Test Compounds
Caffeine
– λmax 273 nm
Anthracene
– λEx 246 nm
– λEm 402 nm
©2012 Waters Corporation 8
What to look for
Retention time
– Pump, mobile phases, column temperature
Peak Area or Peak Height
– Injector, detector sensitivity
Signal to noise
– Background level, mobile phases, detector sensitivity
Blanks
– Carryover, contaminated mobile phase
Backpressure – absolute and ripple
– Clogged column / guard / prefilter / capillary, leak
©2012 Waters Corporation 9
TargetLynx Trendplot
©2012 Waters Corporation 10
Automatic system checks
”LC/MS system check” with IntelliStart
– Schedule a SST to run, process and report
– A report will tell the user if all specifications have been met
”QC Monitor” with TargetLynx
– Real time monitoring during batch
– Can take actions when specification is not met
o Reinject sample
o Pause sample list
o Skip to next batch
o Run shutdown
©2012 Waters Corporation 11
LC/MS system check - IntelliStart
©2012 Waters Corporation 12
LC/MS system check - IntelliStart
©2012 Waters Corporation 13
TargetLynx QC Monitor
©2012 Waters Corporation 14
TargetLynx QC Monitor
©2012 Waters Corporation 15
TargetLynx QC Monitor
©2012 Waters Corporation 16
TargetLynx QC Monitor
©2012 Waters Corporation 17
Basic trouble shooting
Retention time
– Is back pressure ”normal” across the gradient?
– Reverse the gradient (run from B to A)
– For a binary (HP mixing) pump, e.g. Acquity Classic, I-class
o Check that both pumps generate the same backpressure at the
same flow with the same eluent.
– For a low pressure mixing pump (Alliance, H-Class, 600...)
o GPV test Alliance, put A and B line in 100 ml cylinders, pump at
50:50 @ 5 ml/min. Stop when A reaches 75 ml. B should be 72-78
ml.
– Column temp, column fitted correctly in compartment, pre-heaters.
– Mobile phase correctly prepared? Organic content and additives.
– Pressure decay test – use MeOH or 50:50 MeOH:H2O
©2012 Waters Corporation 18
Basic trouble shooting
Peak Area / Peak Height
– Injector, possible to test other injection mode?
– More than one detector in system?
– MS
o Check spray
o Readbacks ok?
o Clean cone or entire source in ultrasonic bath
– Is sample ok?
– Reproducibility
o Inject from same vial
o Method related? Try another compound.
o Make a simple MS method
©2012 Waters Corporation 19
Basic trouble shooting
Signal to noise
– Is the peak response low or is the baseline noisy or elevated?
– UV and PDA are sensitive to bad mixing when one eluent has higher
absorbance. Compare with a pre-mixed eluent.
– PDA
o Dirty flow cell – clean according to protocol
o Dirty mobile phase / no clean eluent in flow cell during from start
o Lamp intensity down – try new lamp
– MS
o Contaminated mobile phase – prepare new, don’t forget stock
solutions. Higher grade chemicals.
o Dirty source – clean in ultrasonic bath
o Check spray
o Can longer dwell time be used?
©2012 Waters Corporation 20
Basic trouble shooting
Blanks
– Carry over?
o Make several blanks. Usually response decreases from one injection to the next.
o Try other strong wash, e.g. 25:25:25:25 of IPA:MeOH:ACN:H2O + 0.1-2% FA or NH4OH. Use both.
o Increase wash volumes
o Try other needle and loop materials or different injection mode
– Contaminated mobile phase?
o Run gradient without injection or bypass injector?
o Deliver 100% A a longer time before gradient to see if response increase
o Document “Controlling Contamination in UltraPerformance LC®/MS and HPLC/MS Systems” on waters.com
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Basic trouble shooting
Increased backpressure
– Column or pre-column clogged
– Correct column temperature
– Disconnect one thing at the time, starting at the end
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Connections Insight
– iAlert
– iHelp (iRequest on waters.com)
– iAssist
TeamViewer
– 3rd party software
– Remote desktop
Remote support
©2012 Waters Corporation 23
Thank You!