Agilent Best Practices For Using An Agilent LC System
Agilent Best Practices For Using An Agilent LC System
Agilent Best Practices For Using An Agilent LC System
Technical Note
This technical note describes best practices for using an Agilent LC system.
Housekeeping 2
Housekeeping
Filtering through nylon filters is not recommended for High Sensitivity LCMS.
NOTE
• Use brown bottles and fill them with the ACN volume to be consumed within 1-2 days to
prevent photochemical reactions and oxidation.
• Filtering is the preferred method to remove the insoluble fraction from samples and avoid
blockages in the system. If not possible, centrifuge samples vigorously and be careful not to
contaminate the supernatant with precipitates while decanting or aspirating it.
• Make sure the sample diluent is also free of particles.
Daily Tasks
• Replace solvents and solvent bottles for mobile phases based on water/buffer.
• Replace solvents and solvent bottles for organic mobile phase latest every second day.
• Check presence of seal wash solvent.
• Purge each channel with fresh solvent at 2.5 – 3 mL/min for 5 min before operation.
• Equilibrate the system with composition and flow rate of subsequent method.
Weekly Tasks
• Change seal wash solvent (10 % isopropanol in water) and bottle.
• Inspect solvent filters for dirt or blockages. Exchange if no flow is coming out of the solvent
line when removed from the degasser inlet.
Follow the instructions prompted on the screen when starting the Evacuation Mode.
NOTE
When mixing incompatible solvents, salts can precipitate at the point of mixing blocking the
NOTE downstream flow path and damaging parts.
Using the seal wash function is mandatory when installed and when using buffers and other
non-volatile solvents or additives that could deposit on pistons and seals. The seal wash function
regularly cleans these parts automatically.
• Position wash solvent bottle above and waste bottle below instrument.
• Do not refill seal wash solvent bottle, always use properly cleaned, fresh bottles.
• For pumps not equipped with seal wash sensor, test peristaltic pump.
• Touch the peristaltic pump with your finger to check that the pump is working, or confirm
seal wash solvent flow by watching seal wash solvent to drop out of the tubing.
Table 1 Seal wash dialog and operation
Figure 2 Seal wash settings (1290 Infinity Pumps) Figure 3 Seal wash settings (all 1260 pumps)
Figure 4 Context Menu (1290 Infinity Pumps) Figure 5 Context Menu (all 1260 pumps)
The seal wash pump is operating not only when the analytic pump is moving solvent, but also
when it is on standby or not-ready states. The seal wash function regularly cleans pistons and
seals from deposits automatically.
The seal wash sensor will constantly check the performance of the seal wash system and warn
the user in case an anomaly has been detected.
• Check seal wash tubing and filter for kinks, leaks, or blockages.
• Check for blockages of the waste tubings, make sure that the solvent waste can drain off
freely:
• If solvent waste is building up in the tube, the sensor does not perform correctly.
• Always use fresh wash solvent for the needle or seat backflush function.
• Place the wash solvent reservoir for needle wash or seat backflush into the solvent cabinet.
• Use an appropriate solvent based on the sample and mobile phase properties.
The composition of the needlewash solvent should be the most solubilizing compatible solvent
NOTE (your strongest diluent). Selecting wash solvents is part of the method development. A mixture of
50 – 100 % organic solvent in distilled water is a good choice for many applications.
• Check the drainage routing of the washport outlet into a waste container.
• Fill each vial with enough sample solution for all injections.
• Use Agilent recommended vials only.
• Do not overfill the vials, fill each vial up to 90 % only.
• Use pre-slitted septa when drawing large volumes or multiple times from the same vial.
• Filter, decant, or centrifuge sample to separate from insoluble solid.
• Take care that the sample solvents match the proposed mobile phase as closely as possible.
Multiwash
Multiwash option is designed for the low carryover and can be installed in any Agilent
Multisampler.
This option reduces carryover for critical samples by allowing:
• External needle wash with up to three different solvents
• Seat backflush with up to three different solvents
The use of the Multiwash option is not recommended with salt-containing mobile phases, as salt
will crystallize on the needle and needle seat. Due to the flow path of the Multiwash hydraulic box,
when the metering device moves to the home position before injection, the mobile phase will
come out from the needle tip and might fall into the seat. This is expected behavior, independent
of the Injection Path Cleaning settings, and does not indicate leakage.
If the Multiwash is installed on the system with salt-containing mobile phases, to avoid salt
build-up on the needle and the seat, it is recommended to flush the Multisampler daily with water
for 15 min and visually inspect the needle and seat. If necessary, use a lint-free tissue with distilled
water to manually clean the needle, seat, and other parts that still contain salt residue.
When should the Multiwash option be used?
• If the system is intended for reversed-phase applications only and no precipitating compounds
are present in the mobile phase.
• If the system is intended for applications with salt-containing mobile phases, the Multiwash
option is not recommended. If the option is however installed, crystallization on the needle and
the seat can take place. The following practices should be followed:
• Perform daily a 15 min flushing with water to remove salt residue.
• Perform external needle wash and seat backflush.
• Visually control needle/seat/washport for salt residue.
• Manually clean needle/seat/washport if necessary.
If these procedures are not followed, it will lead to needle and seat blockages.
NOTE
• If the system is intended for alternating applications with salt-containing mobile phases AND
reversed-phase applications, Multiwash option is not supported. There is a high risk that salt
residue will be continuously released and either interfere with reversed-phase chromatography
or even cause clogging of the system.
A considerable amount of solvent is aging in the lines of the Multiwash flow paths, which could
lead to elevated background in highly sensitive UV and MS measurements. To mitigate this, it is
recommended to prime solvents in each Multiwash line in use to the wash port and S1 line (initial
chromatographic condition) to the seat for at least 5 minutes before actual LC analysis is started.
Usually, the lifetime limiting factor for UHPLC columns is high backpressure. Particulate matter in
the sample is collected on the inlet frit of the column, which causes an increase in the
backpressure until the pressure limit of the system is reached. Using the inline filter is
recommended to prevent blockages of the column frit, especially when sample preparation does
not allow for filtering or the sample potentially forms precipitate.
The following modules can be equipped with an extra inline filter:
• Quaternary pumps (G7104A, G7104C, G4204A, G7131A, G7131C):
• Inline Filter Assembly (5067-5407)
• Inline Filter Assembly, Bio-Compatible (5720-0003)
• 1290 Infinity and 1290 Infinity II Agilent autosamplers (except G5668A):
• 1290 Infinity II Inline Filter Kit (5067-6189)
• Bio Infinity II Inline Filter Kit (5720-0020)
These inline filters with a nominal pore size of 0.3 µm protect the UHPLC column from clogging by
filtering particulate matter from samples or the UHPLC system.
Advantages of the inline filter:
• Very small internal volume
• Delay volume with rigid capillary 1.3 µL
• Delay volume with flexible capillary 1.6 µL
• Specified for working at high pressures (max. operating pressure 1300 bar)
Installing the inline filter into the pumpis recommended to protect the downstream system from
blockages in the following cases:
• When solvent combinations are used that can form precipitates after mixing,
• When running applications with buffers or additives while using columns with small particle
sizes
General hints for effective use of the inline filter:
• Filter solvents before use.
• Follow best practices.
• For G4204A, G7104A/C, and G7131A/C pumps, backflush the filter in the pump weekly (start
Flush Filter from the context menu).
• For inline filters installed on the sampler, exchange the filter frit (Frit 0.3 µm for inline filter, 5/pk
(5023-0271) or Frit 0.3 for inline filter, Bio-Compatible, 5/pk (5320-0022)) every 1000 injections
or when the back-pressure rises by 15 %.
Long-term storage of columns should always be in the appropriate storing solvent, for more
NOTE details on the column in use, see the User Guide inserted in the column package.
There is a safety period/wait time before a lamp can be re-ignited after it has been turned off.
NOTE
Agilent Bio-inert and Bio LC systems should not be subject to passivation or similar
C AU T I O N procedures
This can cause irreversible damage to the system's internal surfaces
Do not perform passivation or similar procedures on bio-inert and bio-compatible systems.
Flushing Procedure
This procedure should be used when salt-containing mobile phases are used. It has to be
performed regularly, at least once a week, or prior a long standby or off time, to remove salt
deposits from the flow path and surfaces in contact with the solvents. How to prepare the
system for shutdown see “Shut Down the System” on page 5.
The procedure is mandatory for switching from salt-containing mobile phase to reversed
phase applications (or any applications running with high organics), where the precipitation of
salt can occur.
• Flush the column with recommended storage solvent, be sure that this solvent is compatible
with current mobile phase and cannot cause precipitation.
• Replace the column with a union, replace the salt-containing solvent bottle with a new bottle of
HPLC-grade water at room temperature.
• Clean the bottle head assembly using lint-free wipes to minimize carryover of remaining salt
solution into the new water bottle.
• Autosampler: perform at least 15 min purge with water to remove salt residues from all lines,
both needle wash and seat backflush for Multiwash option, visually control
needle/seat/washport for salt residues, if necessary manually clean needle/seat/washport.
• Purge each pump channel that has pumped buffer separately, for at least 10 min at 5 mL/min.
• Flush the entire system flow path with water for at least 10 min at 2 mL/min. During this step,
switch the injection valve and the column selection valve (if installed) position every 1 min, and
repeat this until every position has been selected for at least 5 times.
• Replace water with fresh solvent bottles to minimize salt carryover.
Purge
Use the Purge function to:
• Fill the system with fresh or different solvent.
• Ensure that the new solvent is miscible with the previous solvent.
• Prevent damage to the degasser or pump by using an intermediate step with a co-miscible
solvent, if necessary.
• Remove air bubbles in tubes and pump heads.
• After the pump has been idle for a few hours or more (air may have diffused into the solvent
lines).
As soon as the purge procedure ends the module automatically switches to analytical conditions
again.
Condition
If micro air bubbles persist in the pump head, the overall pump performance may be
compromised and flow accuracy / precision may be negatively affected. This may be visible as
increased pressure and / or detector baseline ripple. A reliable indicator of such situation is a
strongly negative but slowly increasing tuning signal value (below –1). To remove the air
efficiently, the Condition function can be used. During conditioning, the pump is delivering flow
into the system (column) and the last used method settings, like flow, composition and max
pressure are used. It is not possible to perform sample analysis while conditioning.
Use a reasonable flow rate (for example 1.5 mL/min), composition setting (for example A: 50 % B:
50 %) and backpressure (>200 bar) to ensure efficient air bubble removal from all pump heads.
Condition the pump if you see:
• Excessive pressure ripple.
• Excessive composition ripple (baseline noise/mixing noise – noise level changes with the
composition), when you are sure that the solvent type is correctly set, and there is no evidence
of a leak in the pump.
Conditioning may be necessary:
• After a long period of standby
• After running out of solvent
• After service or repair
Prime
The Prime function is helpful if air has entered the pump heads and cannot be removed by
conditioning for 15 minutes. The module draws solvent at a high speed with all pump drives
simultaneously and dispenses it into the waste position of the automatic purge valve. This is done
20 times and is stressful to the valve and rotor seal. Therefore, it should be performed only as a
last resource, before forcefully filling the pump heads with a syringe or attempting to repair the
pump heads.
Use the Prime function to:
• Free a potentially stuck valve.
The described functions can be triggered from the driver interface:
• 1290 Infinity and 1290 Infinity II
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Agilent Technologies Inc. 2016-2021
Edition: 03/2021
Document No: SD-29000194 Rev. C.00