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1/12/2018
1
Practical Training Basic: LC Theory for Troubleshooting
Autumn Breaud
Johns Hopkins University
LC Basics Part I:Demystifying Liquid Chromatography Separations and Hardware
Introduction to Liquid Chromatography
• What is HPLC?• High performance liquid chromatography
• Principles of HPLC• Stationary phase
• Mobile phase
To MS
Principles of HPLC
• Stationary phase + mobile phase + pump = separation
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Principles of HPLC
• Stationary Phase• Common column types
• Non‐polar• C18, C8, C4
• Polar
• Silica, amino, cyano
• Ion Exchange
• Cation and anion exchange
• Size‐exclusion
• Phenyl/alkyl
• Mixed mode
• Combinations of above media
• Mobile Phase• Aqueous: Water/Buffer
• Organic: Methanol/Acetonitrile
• Additives:
• Acetic acid
• Formic acid
• Ammonium formate
• Ammonium hydroxide
• Ammonium acetate
• TFA*
HPLC Hardware: The Missing Link(s)
HPLC Hardware: The Missing Link: Autosampler
To MS
Anatomy of an AS‐HPLC System
Autosampler Pump
Mobile Phases
Stationary Phase
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Anatomy of an AS‐HPLC System: AutosamplerSyringe and Injector Valve
Anatomy of an AS‐HPLC System: Pumps
Anatomy of an AS‐HPLC System: Common pump components• Mobile phase inlet
• Mobile phase outlet
• Pump head
• Piston
• Check valve
Anatomy of an AS‐HPLC System: Common pump components• Check valve
• Inlet and outlet pictured
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Anatomy of an AS‐HPLC System: Check Valves• What are they and why do I care?
• Common cause of HPLC run failure
• Stuck ball valve
• Debris in valve chamber
Slide Credit: M. Sagar Kishor Savale
Anatomy of an AS‐HPLC System: Valves
Anatomy of an AS‐HPLC System: Valves• Selection valves
• Bypass valves
• Injection valves• Common cause of HPLC run failure
Anatomy of an AS‐HPLC System: Valves• Stator face
• Rotor
• Rotor seal
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Variations on a Theme: The Many Faces of Liquid Chromatography• UHPLC
• Nanoflow/Microflow
• Multiplexing
• Multi‐dimensional separations
Variations on a Theme: UHPLC
HPLC
• Particle size larger
• 3‐10 um
• Longer run times
• Lower line pressure
• 100‐200 bar max
UHPLC
• Particle size smaller
• 0.75‐<2um
• Shorter run times
• Higher line pressure
• >500 bar max
• Improved resolution
• Lower sample and solvent volumes
Variations on a Theme: NanoflowChromatography
HPLC/UHPLC
‐typical flow: 200‐1000 uL/min
‐column ID: 0.5‐0.075mm
Nanoflow
‐typical flow <200 uL/min
‐column ID: 2‐4 mm1 min 4 min.
Channel
1
1 min .2 min .
2 min
Channel
2
MS Data
Acquisition Window
Idle Time Idle Time
Each channel connects to the detector during the
elution step
MS Fully Utilized 3 min 4 min etc.
Variations on a Theme: Multiplexing
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Variations on a Theme: MultiplexingVariations on a Theme: Multi‐dimensional Separations
LC Basics Part II:Troubleshooting and Staying Productive, or Up a Creek? Pick a Paddle
Troubleshooting LC Systems
• Diagnostic tools• Pressure trace analysis
• Check valve failures
• Empty sample/vial/well
• Air in LC lines
• Mobile phase composition errors
• Loss of pressure analysis• Locate your clog
• Lot check‐in logs• Use them or lose it
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25
Troubleshooting LC Systems: Pressure Trace AnalysisStep 1) Characterize a ‘healthy’ pressure trace:
Reverse Phase Pressure Trace:Normal Phase Pressure Trace:
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Troubleshooting LC Systems: Pressure Trace Analysis
Step 2) Learn to ‘read’ aberrant pressure traces
‐Check valve failure
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Troubleshooting LC Systems: Pressure Trace Analysis
Step 2) Learn to ‘read’ aberrant pressure traces
‐Sample vial/well empty or AS does not transfer sample volume
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Troubleshooting LC Systems: Pressure Trace Analysis
Step 2) Learn to ‘read’ aberrant pressure traces
‐Empty mobile phase line/s
Healthy pressure trace Aberrant pressure trace
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Normal Pressure Trace Problem Pressure Trace
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Troubleshooting LC Systems: Mobile Phase Composition Error
Step 2) Learn to ‘read’ aberrant pressure traces
‐Mobile phase B topped off before run
30
Troubleshooting LC Systems: No Pressure‐Find that Clog
Lot check‐in logs: Use them or lose it
• Consumables:
• columns
• mobile phases
• internal standard solution lots
31
Troubleshooting LC Systems: Lot Change Tracking
The Six Most Common LC Problems and How to Spot Them
• Column aging and degradation• Peak tailing, fronting,
broadening
• Changes in retention time
• Loss of signal
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The Six Most Common LC Problems and How to Spot Them• Clogging
Healthy pressure trace Aberrant pressure trace
The Six Most Common LC Problems and How to Spot Them• Check valve failure
• Could be air in the line/s
The Six Most Common LC Problems and How to Spot Them• Autosampler syringe failure
Normal Chromatogram Problem Chromatogram
January 12, 2018 36
RT: 0.00 - 3.90 SM: 7B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
Peak Area: 158640
1.73
2.420.89 2.571.050.30 3.01 3.20 3.793.641.48
NL:1.13E4TIC F: + c ESI sid=1.00 SRM ms2 353.150 [283.990-284.010] MS Cal_007
RT: 0.00 - 3.90 SM: 7B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
Peak Area: 2129282
2.17
2.923.42 3.571.290.920.36 1.640.55
NL:1.03E5TIC F: + c ESI sid=1.00 SRM ms2 353.150 [283.990-284.010] MS Cal_007
The Six Most Common LC Problems and How to Spot ThemIncorrect mobile phase composition
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January 12, 2018 37
The Six Most Common LC Problems and How to Spot ThemIncorrect mobile phase composition
Healthy pressure trace Aberrant pressure trace
The Six Most Common LC Problems and How to Spot Them• Best and most insidious problem:
The Six Most Common LC Problems and How to Spot Them• Best and most insidious problem:
The Six Most Common LC Problems and How to Spot Them• Best and most insidious problem:
• LC eluant on floor
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LC Basics Part III:Case Studies in Separations Sleuthing
Case Studies in LC Troubleshooting• Case Study 1:
• The case of the vanishing busulfanpeak
Case Studies in LC Troubleshooting• Case Study 1:
• The case of the vanishing busulfanpeak
• Pressure trace analysis
Healthy pressure trace Aberrant pressure trace
Case Studies in LC Troubleshooting• Case Study 1:
• The case of the vanishing busulfan peak
• Check valve replaced, new problem arises
Healthy pressure trace New aberrant pressure trace
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Case Studies in LC Troubleshooting• Case Study 1:
• The case of the vanishing busulfan peak • Check valve failure
• Gradient pressure profile still looks unusual
• Mobile phase A (aqueous) as loaded in position of mobile phase B• Mobile phase A lot check‐in procedure not completed, but bottle is labeled with new date
• ‘Marker test’
• No elution of analyte
• No pressure changes associated with solvent front or change in mobile phase surface tension from aqueous to organic
Case Studies in LC Troubleshooting
• Case Study 2:• The case of the intermittent missing antiretroviral peaks
Healthy pressure trace Aberrant pressure trace
Case Studies in LC Troubleshooting
• Case Study 2:• The case of the intermittent missing antiretroviral peaks
Case Studies in LC Troubleshooting
• Case Study 2:• The case of the intermittent missing antiretroviral peaks
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Case Studies in LC Troubleshooting
• Case Study 2:• The case of the intermittent missing antiretroviral peaks
Case Studies in LC Troubleshooting
• Case Study 2:• The case of the intermittent missing antiretroviral peaks
• Flat pressure profile upon injection is aberrant and associated with missing internal standard signal
• Change of AS syringe, needle, needle seal, injector rotor seal: still see intermittent signal loss
Case Studies in LC Troubleshooting
• Case Study 2:• The case of the intermittent missing antiretroviral peaks
• Change of bungee cord: no more intermittent loss of signal
• Pressure of injection was not high enough to overcome line pressure at moment of injection
• Optimized needle penetration height in injector port
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Speaker and Presentation Evaluation for Dr. Autumn BreaudSurvey Monkey