Troubleshooting and How to Take Care of Your Column and HPLC

Troubleshooting and How to Take Care of Your Column and HPLC
Troubleshooting and How to
Take Care of Your Column and
HPLC System
SHIMADZU
Solutions for Science Since 1875
HPLC Troubleshooting
n
Pressure: too much or too little
Leaks: pump, autosampler, detector
Reproducibility: pump, autosampler
n
Column Care: Flushing and equilibration
n
n
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Solutions for Science Since 1875
Pump Troubleshooting
n
No pressure, or fluctuating pressure
Pump may not be completely full of liquid – check
solvent inlet line
n Air in check valve – always degas mobile phase!
n “Stuck” (κολλημένη) check valve – the pump may
have been idle (αδρανής) for too long and solvent
has dried inside the check valve.
Poor quality solvent: may contain resins that coat the
ball (σφαίρα) inside the check valve, and that film
won’t let the ball seat properly
n
SHIMADZU
Solutions for Science Since 1875
Pump Troubleshooting
n
High Pressure
n
n
Outlet frit (φρίτα, πορώδες φίλτρο) may be blocked with
particles from mobile phase or seal (φλάντζα) material
Leaks (διαρροές)
n
Damage to seal and/or plunger (έμβολο) due to several
factors
n Misaligned (μη ευθυγραμμισμένο) plunger
n Solvent incompatibility with seal material
n Salt crystal buildup from buffers – use
a rinse kit!
SHIMADZU
Solutions for Science Since 1875
Pump Troubleshooting
n
Retention Time Reproducibility
For a dual piston pump, only one side may be filled
with liquid – check solvent inlet lines
n Temperature change (may not be the pump’s fault)
n
n A 1o
shift in temperature can result in a 1-2% shift in
retention time
n Avoid drafty (μη θερμενόμενες) locations in the lab
n Use a column oven when possible
SHIMADZU
Solutions for Science Since 1875
Autosampler Troubleshooting
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High Pressure
n
Particulates from mobile phase, sample, pump may
be trapped (παγιδευτούν) in the inlet tubing or valve
n Filter mobile phase AND sample when possible
n
Leaks
n
Fittings may be loose on the valve
n Tighten (σφίξτε) fittings
(προσαρμογές) properly and
don’t exceed the pressure limit of the autosampler
SHIMADZU
Solutions for Science Since 1875
Autosampler Troubleshooting
n
Area % Reproducibility
Always degas rinse phase (απαέρωσε τη φάση
έκπλυσης) , and use some volume of liquid for
rinsing to keep all flow paths in the valves full of
liquid
n Make sure the needle stroke (κτύπημα της βελόνας) is
deep enough to draw sample from the vial
n Check for leaks on the valve fittings, and the
connection to the column inlet
n
SHIMADZU
Solutions for Science Since 1875
Detector Troubleshooting
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Spiky Baseline (γραμμή βάσης με οδοντώσεις)
n
Air bubble in flow cell – degas mobile phase!
n Put some restriction (συμπιεστή) on the
cell outlet, but not
too much! Tubing with 0.005” i.d. is fine.
n
Leaks
n
Cracked flow cell (ραγισμένη κυψελίδα)
n Don’t exceed the
n
pressure limit of the cell
Poor tubing connections
n Use the
proper fittings and tighten appropriately
SHIMADZU
Solutions for Science Since 1875
Column Care
n
n
Follow MER’s (κατασκευαστής) recommendations for
solvent compatibility (συμβατότητα), flow rate, and
pressure limits
Filter samples when possible
n
n
Use care when reversing (αντιστροφή) column flow
n
n
Particulates will build up on the inlet frit over time
Connect the outlet to waste, NOT inline with the detector to
prevent further contamination
Store columns in recommended solvents
SHIMADZU
Solutions for Science Since 1875
Troubleshooting Summary
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Throw away bad parts and columns.
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Leaks do not fix themselves.
n
If it doesn’t pass, you must degas.
SHIMADZU
Solutions for Science Since 1875
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