HPLC Systems. Column Chromatography HPLC Modes HPLC – System Components.
VWR HPLC Troubleshooting.ppt HPLC Troubleshooting.pdf · Where to Begin? System flush (no column...
Transcript of VWR HPLC Troubleshooting.ppt HPLC Troubleshooting.pdf · Where to Begin? System flush (no column...
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HPLC Troubleshooting
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Course Outline
• Potential Sources
• Where to Begin?
• Baseline Troubleshooting
• Column Troubleshooting• Column Troubleshooting
• Other Issues
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Potential Sources of Chromatographic
Problems
• Mobile Phase
• Injector
• In -Line Filter
• Pump
• Guard Column
• Connecting Tubing• In -Line Filter
• Column
• Detector
• Connecting Tubing
and Fittings
• Integrator/Recorder
The Scientist/Analyst
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Where to Begin?
System flush (no column in-line)
• Check for little or no back pressure
• Inject blank – No baseline problems• Inject blank – No baseline problems
• Compare chromatograms
• Inspect baseline
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BASELINE TROUBLESHOOTING
Noisy baseline
Synchronous noise
Cyclic
Spikes
Synchronous noise
Asynchronous noise
Drift
Spikes
No peaks
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Baseline Troubleshooting
Noisy baseline
• Gas in mobile phase
• Degas mobile phase
• Leaks
• Find leak and repair
• Electronic noise• Electronic noise
• Remove source. Shield cables
• Weak detector lamp
• Replace lamp
• Sensitivity too high
• Lower sensitivity
• Detector cell dirty
• Flush with 6N nitric acid
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Baseline Troubleshooting
Synchronous Noise
ALMOST ALWAYS CAUSED BY THE PUMP
• Air in the pump head
• Prime pump and degas solvent
• Check valve problem
• Clean or replace
• Broken plunger
• Replace
• Mixing Problem
• Increase system volume
• Electrical noise
• Remove source
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Baseline Troubleshooting
Asynchronous Noise
• Bubbles
• Degas mobile phase
• Gas caught in detector
• Degas mobile phase. Put backpressure on cell
• Leaks• Leaks
• Find leak and repair
• Mixing problems
• Increase system volume
• Plugged lines
• Remove plug – flush system
• Electrical problems
• Remove source
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Baseline Troubleshooting
Baseline Drift
• Gradient – Solvent B absorbs more than Solvent A
• Try new mobile phase. Use baseline subtraction
• Compounds eluting off column
• Run strong solvent until baseline is stable
• Solvent compostion change (e.g. Evaporation)• Solvent compostion change (e.g. Evaporation)
• Ensure solvents are enclosed
• Solvent leaks
• Tighten, replace fittings
• Backpressure changes
• Filter solvents and samples. Samples may be too viscous
• Mixing problems
• Increase system volume
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Baseline Troubleshooting
Cyclic Baseline
• Temperature fluctuations
• Thermally insulate. Move away from ventilation. Increase cell temperature
• Mixing problems
• Increase system volume
• Gas in mobile phase• Gas in mobile phase
• Degas solvents
• Electrical problems
• Remove source
• Erratic pump
• Repair
• Plug
• Remove obstruction – flush system
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Baseline Troubleshooting
Spikes
• Bubbles
• Degas solvent
• Poor electrical connection, loose wiring
• Clean and tighten detector leads, check wiring
• Lamp relay trying to fire a dead lamp
• Replace lamp
• Electrical noise
• Remove source. Common sources – Switching valves, compressors, muffle furnaces,
fraction collectors, power conditioners, lighting
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Baseline Troubleshooting
No Peaks
• Injector not injecting
• Pump not pumping
• Dead detector
• Integrator/recorder/PC not connected correctly• Integrator/recorder/PC not connected correctly
• Gain setting too low
• Leaks
• Column retaining all compounds
• Bad or incorrect mobile phase
• Bad or incorrect standard or sample
• Incorrect guard column
INJECT ACETONE SOLUTION TO MAKE A PEAK
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Baseline Troubleshooting
Negative & Positive Peaks
• Some eluting compounds absorb less than solvent
• Use a different or cleaner solvent
• Air bubbles passing through cell
• Degas mobile phase
• All peaks are negative
• Change detector polarity
• Negative peaks with RI detector
• May be normal (peak direction is function of RI differential from mobile phase
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Baseline Troubleshooting
Poor Peak Shape• Contaminated in-line filter
• Replace frit
• Column dying• Replace column
• System void volume• System void volume• Check system tubing
• Contaminated guard column• Replace
• Incorrect or wrong solvent• Make new mobile phase. Consider ion pairing/suppression
• Column destroyed• pH <2 washes off functional group
• pH >8 dissolves silica base
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Column Troubleshooting
Common Problems
• Peak shape
• Retention time• Retention time
• Other
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Column Troubleshooting
Chromatographic problems may be related to:
• Instrument
• Sample
• Column
Two Major Problems are:
• Peak Shape/Width
• Retention Time Changes
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Column Troubleshooting
Peak Shape Problems
Most Common Problem in HPLC:
Distorted peaks will cause integration or resolution problem
Indication that optimal column performance is not being attained
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Column Troubleshooting
Peak Shape Problems
• Column Destroyed
• Secondary Interactions
• Incorrect Sample Solvent• Incorrect Sample Solvent
• Column Overload
• Mass Overload
• Volume Overload
• Other Extra-Column Effects
• Sampling Rate
• Time Constant
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Column Troubleshooting
All Peaks Affected
• COLUMN
• Connection
• Replace frit
double peaks
fronting peaks• Regenerate or replace column
• COLUMN DESTROYED
• pH <2 washes off functional group
• pH >8 dissolves silica base
fronting peaks
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Column Troubleshooting
Column Collapse
Well packed column
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Column Troubleshooting
Well packed column
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Column Troubleshooting
Column Collapse
Voided column
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Column Troubleshooting
Voided column
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Column Troubleshooting
Column Protection
Major cause of column deterioration is contamination.
Use of guard columns may increase column life-time to > 10,000 analyses
30mm Guard ColumnColumn
Coupler
30mm Guard ColumnColumn
Coupler
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Column Troubleshooting
Column Protection
Guard column should be regarded as a cost-effective sacrifice to extend
analytical column life-time
Should contain IDENTICAL packing material as the analytical columnShould contain IDENTICAL packing material as the analytical column
e.g. using a different C18, with different retention properties could actually
destroy the separation or impair protection
Well designed, well packed guard columns will actually IMPROVE the
analytical separation efficiency
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Column Troubleshooting
Column Protection
Other Techniques to Protect the Column:
• In-line Filter between the Injector and Column• In-line Filter between the Injector and Column
• Filtering of the Sample ( Doesn't Protect against Seal Shedding)
• Sample Cleanup through Solid Phase Extraction (SPE)
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Column Troubleshooting
Column Storage
Store in Mobile Phase for Short Periods of Time ( <72hrs.)
Store in Shipping Solvent for Longer Periods of TimeStore in Shipping Solvent for Longer Periods of Time
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Column Troubleshooting
Column Storage
• Column should be stored in solvent which manufacturer recommends
• For bonded phases, use organic solvent
• (eg. MeOH or ACN) Using non aqueous solvents minimizes • (eg. MeOH or ACN) Using non aqueous solvents minimizes
hydrolysis.
• Some bonded phases (CN) become unstable in polar organic mobile
phases.
• Storage in water or buffer is then okay.
• Worst mobile phase for CN column is CH3CN (Acetonitrile)
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Column Troubleshooting
Column Storage
Columns which may be stored in Water or Buffered Solvents:
• Ion exchangers
• Aqueous SEC packings• Aqueous SEC packings
However:
Prevent microbial growth by using 0.05% sodium azide in mobile phase
OR
Small quantity of organic solvent (acetonitrile 5% or methanol 10%)
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Column Troubleshooting
Column Storage
Columns which should be stored in Mobile Phase:
• Normal Phase • Normal Phase
• Organic SEC (GPC)
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Other Issues
Tailing
What Causes Tailing?
Mixed mode retention
Hydrophobic – interaction with bonded phase
Ion exchange – interaction with charged sitesIon exchange – interaction with charged sites
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Other Issues
Incorrect Sample Solvent
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Other Issues
Column/Volume Overload
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Other Issues
Column/Volume Overload
A good approach to a loading study is to increase the amount injected by a
factor of 2
This is fine enough to find the point of maximum load but course enough to
cover a large concentration quickly
It is often not necessary to perform measurements on the chromatogram. A
visual inspection is often sufficient
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Other Issues
Mass Overload
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Other Issues
Mass Overload
How to identify?
• Indicated by a rapid detector response
How to remedy the problem?
• Dilute the sample by 50% then re-inject
One can observe better resolution and slightly rounded leading edge of the peak
We then dilute the sample by 50% again until we observe a much more pronounced
leading edge of the peak of interest.
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Summary
• Assess all potential sources
• What is the baseline telling you?
• Treat your columns well
• Assess the suitability of your method• Assess the suitability of your method• Mobile phase composition etc.
• Ensure injection volume and sample concentration are
suitable
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HPLC Troubleshooting
Thank you for your attentionThank you for your attention