Troubleshooting and Supporting Windows® 7 in the Enterprise_06
2006MD 7 - Troubleshooting
Transcript of 2006MD 7 - Troubleshooting
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HPLC
Troubleshooting
A.NARENDER
Asst.Service Manager
Spinco Biotech Pvt Ltd.
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Daily Maintenance
Pre-analysis checks (1)
Filter mobile phase
Wait for mobile phase to reach room temperature
Purge all the flow lines with mobile phase
Replace mobile phases as necessary
If buffer is used as mobile phase, wash the back of plungerseal
If autosampler is used, verify that the autosampler rinse bottle is
full
Purge the autosampler rinse solution
Check for leaks
Check the pump pressure
Check the oven temperature
Perform a baseline check
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Daily Maintenance
Post-analysis checks
Wash the column
If the column is not going to be used for long time, take off it
from the system after washing, and cap both ends of the
column, store it in cool and dark place
Clean the flow line or the rest of the instrument, as necessary
Store the solvent reservoir filters in rinse solvent to prevent
them from drying out
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Outline
- Troubleshooting Process
- Categories of Problems
Pressure Issues
Baseline Issues
Peak Shape Issues
Retention Issues
- Troubleshooting Examples
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Troubleshooting Process
Gather the facts not theories.
Compare the performance obtained to the expected performance.
List possible causes.
Check the simplest things first its easier.
Work through the possible causes in a step-by-step manner
checking the outcome from any changes made.
As a last resort get help from elsewhere, for example your
instrument supplier help desk or your local technical support
department.
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Potential Sources of Problems
Mobile phase
Injector
Pump
Sample
Column and guard column
Detectors
Tubing and connectors
etc.
Analysts should simplify the problem source
chemistry or mechanical?
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Troubleshooting Guide
Main Troubles with HPLC1. Pressure
Low pressure
Pressure fluctuation
High pressure
2. Unstable Baseline
Drift/noisy
Spikes
Sawtooth
Cyclic
No change
3. Distorted Peak Shape
Broading
Tailing
Leading
Splitting
No peaks
4. Retention Time
Changing Retention
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Pressure Low pressure
Leaking in pumps, check
valves, seals or connectors
Pump cavitation
No pumping
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Possible causes :
1. Air bubbles in pump head
2. Leaks in system
3. Check valvesare dirty
4. Plunger seal is damaged
5. Plunger is spoilt.
Remedy:
1. Purge the pump2. Check for leaks in all the connections, especially new connection.
3. Clean check valves in ultrasonicator
4. Replace plunger seal
5. Replace plunger
Pressure Fluctuation in System
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High Pressure in System
Possible Causes
Clogging in
Detector cell Column
Pump
Mixer
Piping
Too much work ?
Too much nagging ?
Medical problem ?
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Locate the point of the pressure increases
Column or system?
Check the system methodically:
Disconnect column from detector, if the pressure problem goes way,
the problem is clogging of detector cell or piping to and from flow
cell.
If pressure persist, disconnect guard column and main column. If
pressure becomes normal, clean/replace main column.
If pressure persist, remove guard column. If pressure is normal,replace guard column.
Continue in this manner from auto-injector (if present) to mixer and
finally to pump until find out the high pressure spot.
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Possible reason:
Clogging in system. It may occur in any part of system.
Remedy :
If clogging in piping, it may be possible to flush out using
IPA or nitric acid at higher flow rate. In this case, do not
connect column or detector.
Clean or replace in-line filter.
High Pressure in System
Troubleshooting Guide
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Possible reason:
Column clogging/failure.
Accumulation of particulates and/or adsorbed substances on column
Remedy :
1.Clean guard column and column.
2.Replace filter frit on column if possible.
3.Flush the column reversely or repair the column (This is recommended
only as a last resort).
Precaution:
In these cases, it is always recommended to use guard column
whenever
possible. The guard columns are cheaper and can be easily replaced
when clogging or adsorption occurs.
High Pressure in Column
Troubleshooting Guide
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Precautions for Column
Buffer solution must be filtered
by 0.2 or 0.45 m membrane filter.
Sample must be filtered
by 0.2 or 0.45 m membrane filter.
Be sure do not jump from buffer to pure organic or from organic tobuffer. This can lead to buffer precipitation, plugging and pressureproblems.
Always use a washout, intermediate solvent.
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l ti (1)
Wash the column with suitable solvent
Open the column end, carefully remove the
filter/frit with the column in an upright
position, and sonicate it.
Change the filter/frit.
Wash the column reversely at low flow rate.
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C l m Clea i g
Washing the column will remove non-polars.
Buffered mobile phase:
must wash out the buffer with the same mobile phase minusthe buffer first
Aqueous organic solvent
Wash with Acetonitrile or methanol for at ~20 column
volumes.
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Column Cleaning
For reversed phase column
1. Wash with mobile phase without buffer salts
2. Wash with methanol or acetonitrile
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Solution (2)
only ~1 cm might be polluted
column
Last resort:
Remove the contaminated part and
repack with clean packing material.
Always use the same size and type of
material used originally in the column.
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Baseline Troubleshooting
Noisy baseline
Sawtooth
Drift
Cyclic
Spikes
Positive & negative peaks
Troubleshooting Guide
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Unstable Baseline
Contaminated column
Detector cell is dirty
System is contaminated
Weak detector lamp
Leaks
Unstable pumping
Mobile phase is not degassed properly
Air bubbles in pumps/detector
Electronic noise
Temperature is fluctuating
Wavelength is too low
More
en? Why is itnot flat?
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Spike Baseline
Bubbles in flow cell
Apply back pressure to the flow cell
Degas mobile phase
Clean flow cell with IPA
Pump pulsation
Check pump function
Install damper
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Baseline - Sawtooth
Bubbles
Mixing problem
Plugged lines
Electrical problems
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Temperature fluctuations
Mixing problem
Mobile phase was not degassed
Pump problem
Baseline - Cyclic
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Baseline no change
Faulty circuits
replace any faulty parts
Lamp is off
set lamp parameter to turn on lamp power
Range setting is not correct
enter an appropriate range value
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Peak Shape Issues
Peak broading
Peak tailing
Peak fronting
Peak splitting
Other asymmetry peaks
No Peaks
Many peak shape issues are combined
Troubleshooting Guide
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Peak Broading
Sample overload
Large extra system volume
Retention time is too long
Poor column efficiency
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Peak Tailing
T e fl -line ntain e tra ea l e
a le erl a ing
ili a- a e l n egra e at ig r ig
te erat re
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Peak Fr nting
a le erl a ing
C anneling in l n
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Sample is too diluted
Injection volume is too small
Detector lamp is off
Leaks
Pump is not pumping
Injector is damaged
Zoom setting of chromatogram is not correct
Column retains all compounds
Incorrect mobile phase/sample
o Peaks
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Changing Retention Time
Contamination was built up
Equilibration time is insufficient
Inconsistent mobile phase preparation
Inconsistent on-line mobile phase mixing
Selective evaporation of mobile phase component
Unstable column temperature
Leakage
Unstable pumping
Troubleshooting Guide
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Tips
Good habits
Log book record pressure and mobile phase
Wash the column after sample analysis
Read manual/instruction
If any problem happens
Observe the instrument carefully look, smell, and feel
Check the simple things first
The hardware is not easy to be broken
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Thank You!