How to prolong instrument lifetime HPLC UHPLC

1. The world leader in serving scienceProprietary & Confidential
Borrowed from: Per Lassen Nielsen,
Field Service Engineer, Unity Lab Services
HPLC Tips & Tricks
- how to keep your instrument going

2. 2 Proprietary & Confidential
• Service plans
• Inputs from the Nordic Field service team
• Visually inspect your instrument
• High backpressure?
• Useful tools
• Control Charts
• Questions
Outline

3. 3 Proprietary & Confidential
Instrument Service Plans

4. 4 Proprietary & Confidential
The following slides may contain pictures
of disturbing character
Warning!

5. 5 Proprietary & Confidential
Inputs from the Nordic Field Service Engineers
”If possible use Thermo certified
consumables and spare parts
suitable for the instrument”
”Use the correct seal type e.g. reverse
phase or normal phase?”
”Occasionally open the column
oven to check if the system is still
leak free”
”To ensure best possible degassing,
always have liquid in all degasser
chambers”
”Visually inspect you system at
least once a week – it is surprising
how many issues can be resolved
by doing so”
”Sampler error: Needle drive:
Obstruction detected. Check that
racks are correctly installed.
Disable puncture offset if used.
Check alignment – used vials in
racks may give an indication to if
alignment is off center”

6. 6 Proprietary & Confidential
Inputs from the Nordic Field Service Engineers
”Always troubleshoot in the
direction of the flow path i.e.
Pump-> Sampler -> Column Oven
-> Detector”
”When troubleshooting, never test
more than one thing at a time”
”Make sure to log all steps and
outcome when troubleshooting”
”If you are running buffers in you
mobile phases. Check for
precipitating crystals around the
pump heads”
”If switching between applications
be aware of correct flushing in
between to avoid damaging system
and columns.”
”Keep the rear seal wash system
operational. It protects system and
prolong seal lifetime.”
”If you are not sure about solvent
miscibility and possibilities of
precipitation, try mixing in a
vial/beaker before putting on your
system.”
”If possible run a wash of your
system after an ended sequence”
”Always flush out a new column
(storage solution) to waste before
fully connecting to system”

7. 7 Proprietary & Confidential
Inputs from the Nordic Field Service Engineers – Viper capillaries
”The Viper Calibration Tool can
really helpful to reshape capillaries
and extend their re-usability – PN:
6040.2312”
”Viper capillaries are easy to use
and ensures zero dead volume
operation. Comes in various
lengths, ID’s and material. No tools
required.”
”Finger tight only will ensure that
Viper capillaries are not damaged.
No tools!”
”Protect Vipers not in use with black
rubber cover. This way dirt, particles
and damage can be avoided”
”Viper capillaries can be connected
with zero dead volume using Viper
union PN: 6040.2304. Connect so
capillaries meet in center of union.”

8. 8 Proprietary & Confidential
Visually inspect your instrument
Tip 1:
• Inspect.
• Clean.
• Remove capillaries as
needed and clean.
• Retighten.
• Check for tightness.
• Monitor.
• Replace as needed.
• $$$ Expensive repair $$$
• Downtime.
Tip 2:
Do not overtighten ! It might
damage system. Replacing a
capillary is cheaper and faster
than replacing a pump head.

9. 9 Proprietary & Confidential
Visually inspect your instrument
• In both cases: Did this just happen? Or did accumulate over time?
• Maybe only cosmetic for time being,
but how about long term?
• Low sensitivity on MS? Be aware of
what is connected to your LC…

10. 10 Proprietary & Confidential
Visually inspect your instrument
• Leak alarm will catch this, but maybe an aborted
sequence and re-running samples can be avoided…
• Change the eluent filters before they
cause more problems than they solve.

11. 11 Proprietary & Confidential
Tip 2:
Monitor instrument over time e.g. using a
log book.
High back pressure?
What affects back pressure?
• Solvent composition
• Flow rate
• Column
• Temperature
• Flow path e.g ID and length of capillaries
• In-line filters
Tip 1:
Know your system. Establish a baseline
without column under known conditions
(e.g. flow, solvent etc.)
Suggestion: Water at 1mL/min.
Tip 3:
Collect pump pressure in all instrument
methods.
Easier to troubleshoot
Predict and prevent

12. 12 Proprietary & Confidential
• Remove column. System pressure normal?
• If high pressure without column, Try to isolate where high pressure is coming from:
• Flow on (e.g. water at 1mL/min).
• Monitor backpressure e.g. manual data acquisition in Chromeleon.
• Undo or loosen connections one at the time starting from back of instrument (typically UV detector).
• Look for drops in pressure as you remove flow part of flow path (flow cell, capillaries etc.).
• If clog is identified, flush/clean/replace as appropriate.
• Consider backflush – why?
High back pressure - troubleshooting
Tip:
In case of clog, flush only relevant part.

13. 13 Proprietary & Confidential
This is a common place for clogs – why?
• A lot mechanical movement i.e. the rotor seal.
• Sample is introduced.
High back pressure in autosampler?
Be aware of:
• Sample/matrix vs. solvent – precipitation?
• Sampler alignment and height – especially
when using inserts.
• Multiple injections from same vial.
• Lids being used – masking tape is not a good
solution…

14. 14 Proprietary & Confidential
Based on split loop samplers:
High back pressure in autosampler – trouble shooting

15. 15 Proprietary & Confidential
With flow on, switch between
load and inject and observe
changes in back pressure.
(can be easily done from sampler panel)
High back pressure autosampler – troubleshooting
1. If high pressure only in inject, most likely a
clog in injection system i.e. loop, needle…
In most cases pump can be used to flush out a clog
- we typically have >600bars to flush with.
• Flush only part of flow path e.g. by connecting via
injection valve or with union.
• Consider backflush.
Example for WPS3000 split loop:
• Safety first: It is recommended to use
water for this.
• Open “Wellness” panel
• Click “Start change” under “Needle
Change”
• The needle will move to the middle of the
sampler and the valve to load.
• Move the capillary from port 6 on the
injection valve to port 4. This will allow
you to flush the sample loop and needle
using the pump.
• Keep some tissue or a beaker under the
needle. Start with a flow of e.g. 1 mL/min
and then increase to 5 or 10.
• In some cases a sudden drop in pressure
is seen when clog is removed see a
pressure drop the clog is flushed out.
• Similar operation can be used to flush the
needle port/capillary...
2. If high pressure only in load, most likely a clog in
rotor seal (or leak in inject flow path).

16. 16 Proprietary & Confidential
Useful tools – blind nut
• Can be used for pressure testing.
• Ensure liquid in flow path
• Flow off
• Use blind nut to block flow
• Build up pressure at low flow rates (e.g.
0.05uL/min
• Stop flow
• Monitor pressure drop
• Examples:
• Check for system tightness/leaks
• After replacing piston seals
• After servicing auto sampler i.e. replacing
needle, rotor seal or injection port.
• Isolate leak e.g. is system tight in both auto
sampler load and inject position.
PN: 6040.2303

17. 17 Proprietary & Confidential
The HPLC Syringe. Useful tool? Really?
• Can be connected to outlet of pump with silicone tubing.
With flow on, syringe can then help to get the flow going
e.g. if pump has been off for a while or check valves are
stuck.
• Can be used to flush check valves.
• Can be used to clean/flush connections of columns,
stators, pump heads etc.
• Can be used to fill syringe to ensure air free operation.
Useful tools – The HPLC syringe
Supplied with all our HPLC’s

18. 18 Proprietary & Confidential
Useful tools – backpressure coils
Why replace column with backpressure coil?
• Remove chemistry -> Easier to troubleshoot.
• Can be used for qualification (OQ/PQ).
Tip: Using Chromeleon build in PQ templates can be an easy way
to asses instrument. The flow and reproducibility test can be a
good starting point.
Viper restriction coil. Included in PN: 6044.0100
Nano Viper capillary for added backpressure.
Especially useful for UHPLC. Included in
PN: 6041.5125.
Filter to protect lightpipe flow cells. PN: 6042.5014

19. 19 Proprietary & Confidential
Log book
”Keeping a log can be very helpful
for history and troubleshooting”
”Logbooks only work if they are
used correctly by everyone”
”Include hardware changes in log
book e.g. to monitor how often you
need to replace specific parts”
Tip:
In Chromeleon you can log changing of
common parts like piston seals, needle,
syringe, rotor seal etc.

20. 20 Proprietary & Confidential
Control Chart in Chromeleon

21. 21 Proprietary & Confidential
Control Chart in Chromeleon
Other examples
could be:
• Asymmetry
• Theoretical plates
• Resolution
• Signal to Noise
• Back pressure
• Detector background
• Area
• Calculated Amount

22. 22 Proprietary & Confidential
Thank you for your attention.
Thank you – questions?
Any questions?