Agilent LC System Technical Note

Best Practices for Using an Agilent LC System

Technical Note

This technical note describes best practices for using an Agilent LC system.

Housekeeping 2

Daily / Weekly Tasks 3

Power Up / Shut Down the System 4

Recommendations for Degassers 6

Recommendations for Pumps 7

Recommendations for Samplers 11

Recommendations for Pumps and Samplers With Optional Inline Filter 13

Recommendations for Columns 14

Recommendations for Detectors 15

Recommendations for Bio-Compatible and Bio-Inert Systems 16

Additional Information for 1290 Infinity and 1290 Infinity II Pumps 17

Best Practices - Technical Note

Housekeeping

How to deal with solvents

• Use clean borosilicate glass bottles only.

• Rinse bottle with desired solvent before refilling it.

• Bottles can get contaminated with detergents from the dishwasher.

• Use solvent inlet filters to protect the system from incoming particles.

• Exchange water-based solvents daily.

• Algae growth may block the degasser or filters.

• Precipitation of insoluble salts may block filters or capillaries.

• Prepare solvent volume to be consumed within 1 – 2 days.

• Use only HPLC-grade solvents and water.

• All prepared organic solvents, mixtures and aqueous buffers must be filtered through

0.2 µm filter.

• Residues or contaminations may block filters or capillaries.

• Label bottles correctly with bottle content, filling date and expiry date.

• Reduce risk of algae growth: use brown bottles for aqueous solvents, avoid direct sunlight or

wrap the bottles in aluminium foil.

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CAUTI O N

Extra measures with Acetonitrile (ACN)

• ACN and other organic solvents can be filtered using a 0.2 µm PTFE filter membrane (e.g. 5191-4339).

Filtering through nylon filters is not recommended for High Sensitivity LCMS.

• Use brown bottles and fill them with the ACN volume to be consumed within 1-2 days to prevent photochemical reactions and oxidation.

How to prepare samples

Possible sample precipitation

 Ensure the sample is completely soluble in both sample diluent and mobile phase.

 Match the sample diluent as closely as possible to the initial mobile phase in order to avoid

precipitation, pressure spikes and solvent peaks on the chromatogram.

• 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.

Best Practices - Technical Note 2

Daily / Weekly Tasks

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.

CAUTI O N

Contamination of seal wash solvent

Damage to pistons and seals

 Do not recycle seal wash solvent.

 Exchange seal wash solvent weekly.

 Typical solvent consumption: 0.5 L per week.

 Use of Seal Wash Bottle Head Kit (5067-6131) is strongly recommended.

Best Practices - Technical Note 3

Power Up / Shut Down the System

Power Up the System

• Ensure that all modules are in an environment with stable temperature protected from direct

sun light.

• Switch on all modules and proceed to prepare them as described below:

Prepare the Pump

• Use fresh or different mobile phase (as required).

• Purge each channel with 2.5 – 3 mL/min for 5 min. Open the manual purge valve or use the

purge command, depending on the pump type.

Prepare the Sampler

• Avoid using needle wash and seat backflush with organic solvents with buffer applications, because this can cause salt precipitation in the needle seat.

• When using needle wash and/or seat backflush:

• Always use fresh solvents.

• Methanol, ACN, isopropanol, water, and their mixtures are good options.

• Do not use not miscible or precipitating wash solutions.

• When using a Sample Cooler / Sample Thermostat:

• Turn on cooler/thermostat and wait until the target temperature is reached and stable.

This can be done while the detector lamp warms up (see “Prepare the Detector” on page 4).

• Ensure that the vials contain enough sample solution for all injections.

Prepare the Detector

• Warm up the lamp for at least 1 h to guarantee baseline stability.

• For refractive index (RI) detectors only: switch on the heater and flush the reference and

sample sides with fresh solvent used for the current application.

Equilibrate the System

• While the detector lamp is warming up, equilibrate your system (column and detector included) using the composition of your application for at least 15 min, until pressure and detector baseline signal are stable.

Best Practices - Technical Note 4

Power Up / Shut Down the System

Shut Down the System

• Flush the column with the appropriate solvents and store it according to column manual

instructions (be sure that the flushing solvent is compatible with the solvent present in the system to avoid precipitation).

• Install a union or a restriction capillary and flush the system extensively with water, especially after using buffers. For details, see “Flushing Procedure” on page 16.

• Flush and store the system in 50 % methanol or 50 % isopropanol in water, without additives.

• Remove all samples from the autosampler and store them according to good laboratory

practices.

• Power off all modules.

Best Practices - Technical Note 5

Recommendations for Degassers

CAUTI O N

Liquid inside the degasser

In case a low boiling point solvent condensates or there is a leak, liquid can accumulate inside the degasser chambers, and this will compromise performance.

When this happens,

 Purge all solvent channels with isopropanol.

 Keep unused channels filled with isopropanol.

• Check compatibility of solvents with degasser and application

• Use the standalone standard degassers (G1322A or G7122A) for RI applications, flow rates

higher than 5 mL/min, with low boiling point solvents (<60 °C) and with hexane, tetrahydrofuran and any halogenated solvents.

• Use integrated or a standalone high-performance degasser (G4225A) for all other

applications.

• If enough vacuum for the optimal degassing performance cannot be reached or maintained (as indicated by yellow or red status LED in standalone degassers, or specific error messages on integrated degassers), power cycle the module.

• If, after power cycling, vacuum still cannot be reached or maintained on integrated degassers, use the Evacuation Mode, available in the instrument control screen of Agilent Lab Advisor.

Figure 1 Degasser Control for internal degassers in Agilent Lab Advisor

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Best Practices - Technical Note 6

Follow the instructions prompted on the screen when starting the Evacuation Mode.

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