HPLC Introduction

Use only solvents and additives that are compatible with all parts in the HPLC flow path and detection principle

• Using tetrahydrofuran, trichloromethane, ketones, dimethyl sulfoxide, or ammonium hydroxide as solvents can shorten lifetime of parts in the flow path, especially piston seals
• Using concentrated acids, such as sulfuric acid and nitric acid, or a mixture of hexane, ethyl acetate, and methanol, can shorten lifetime of certain wetted parts in the flow path
• Anhydrous methanol, particularly when formic acid or trifluoroacetic acid is present, can cause stress failures in titanium surfaces, so add at least 3% water to prevent this
• Avoid using 100% organic or aqueous solvents and add 5 to 10% organic to aqueous solvent​ or, respectively, add 5 to 10% aqueous to organic solvent
• Avoid using trifluoroacetic acid with mass spectrometry detection (max. 0.1% TFA and only if essentially required for the HPLC separation)
• Use only volatile buffers (e.g., ammonium acetate)​ with charged aerosol or mass spectrometry detection
• Never use methanol from aluminum reservoirs and, in general, avoid storage and usage of solvents in a container made from aluminum

At minimum, use HPLC grade solvents and additives for standard HPLC applications

• Use filtered, high-quality and high-purity solvents and additives as required by the application, for example, UHPLC-grade or LCMS-grade for UHPLC applications
• For gradient operation, use solvents and additives that are explicitly specified as gradient grade quality
• For gradient operation, adapt the concentration of additives in solvents to avoid base line drift
• If a fluorescence detector is used in the HPLC system, use fluorescence grade solvents
• Consider that bottled HPLC grade water often contains ionic contamination, hence, use higher quality especially for charged aerosol or mass spectrometry detection
• Always use high-quality water, e.g., bottled UHPLC-grade or LCMS-grade (0.2 µm filtered), especially for charged aerosol or mass spectrometry detection

Before starting the sample analysis

1. Start the detector module, e.g., turn on lamp(s) or gas flow(s)
2. Make sure the type and amount of solvent in your solvent bottles are appropriate for your application
3. Check the status of the seal wash system and the seal wash liquid level in your bottle; it is best to activate (start) the seal wash system
4. Start pump purging (all channels, including those not used for the application) and wait until pump purging is complete
5. Flush the entire chromatography system with the starting solvent of your application at a reasonable flow rate to rinse out any other solvent
6. Install the appropriate HPLC column for your application
7. Close all doors of the HPLC system
8. Warm up (or cool down) all temperature-controlled devices and wait until the respective (set) temperature has been reached and stabilized
9. Start the pump, observe the pressure increase and wait until pump pressure is stable (drift and ripple within reasonable limits for the application)
10. Monitor the detector signal and wait until the detector signal is stable (drift and noise within reasonable limits for the application)
11. Perform an autozero of the detector baseline
12. The system is ready for sample analysis if pump pressure and detector baseline are stable for a certain time frame
13. It is best to run an HPLC column test to verify performance and suitability

Always equilibrate the system before use

• After powering up the system (even after short-term shutdown)
• After changing the flow rate or temperature setpoints
• After changing a solvent type or HPLC column
• After a gradient run (to ensure column re-equilibration)

Pump best practices

• Use the rear seal wash system properly to prevent damages to pistons, piston seals, or support rings
• Use the degasser with reversed phase solvents
• Use normal phase solvents only with normal phase compatible HPLC systems and bypass the degasser with normal phase solvents
• Purge your pump regularly to remove air bubbles that may have become lodged in the flow path over time
• For durability, always drain the liquid from the silicone tubing of the purge valve once the purging process is complete
• For pumps with manual purging, turn the purge valve knob only with your fingers as overtightening may damage the purge valve seals
• After switching to another type of solvent, purge the pump and then flush the entire HPLC system (remove the HPLC column beforehand)
• Flush your HPLC system on a regular basis to avoid contamination, especially if you are using mobile phases containing salts or buffers

Autosampler best practices

• Perform a needle wash for lowest possible carry over with a needle wash liquid that is suitable for your application
• Replace the needle wash liquid daily when using 100% water
• Leave the autosampler closed during sample processing to avoid any interruptions
• Before you switch the injection valve, make sure that pump flow is turned on
• Make sure the needle unit is moved into service position and secure the needle unit for maintenance or transport

Column compartment best practices

• Use shortest capillary connections possible, at best the Viper capillary kit
• Do not mix stainless steel (SST) and MP35N Viper capillaries in the same HPLC system; nanoViper and PEEK Viper can be mixed with either SST or MP35N Viper capillaries
• Always take care of flow directions (e.g., as indicated on the column)
• Column temperature should remain constant during the entire analysis
• Consider using a pre-heater with UHPLC applications
• Always close the column compartment during operation
• Environmental conditions, such as draft or air conditioning, may extend the waiting period or even prevent the column compartment from entering the Ready state

UV detector best practices

• Do not turn off the UV lamp if you intend to use the detector again within 24 hours because an ignition reduces lamp lifetime
• Allow lamps and optics to reach a stable working temperature
• If the lamp is turned on, never leave the flow cell without a small flow (e.g., 0.1 mL/min)
• Consider influences of detection settings and flow cell characteristics (refer to the operating manual)
• Ensure that the operating conditions are suitable, e.g., a stable temperature, no air drafts, no vibrations and no mechanical shocks

HPLC operation best practices and hints

• Chromatographic hygiene:  How to prolong the lifetime of your HPLC Column
• LC Knowledge: HPLC Column care and general method development guides
• Universal tool-free connections for every LC and nano LC system:  Thermo Scientific Viper and nanoViper Fingertight Fitting System
• Tune your mixing volume for gradient generation:  Achieve Better Detection Limits
• Chromeleon CDS:  Intelligent Integration using Cobra and SmartPeaks
• Chromeleon CDS:  Advanced Internal Standard Techniques for Quantitation
• Chromeleon CDS:  Improving Peak Results by Reducing Solvent Strength Prior to Sample Injection
• Fluorescence detection optimization :  Extend the Lifetime of Your Xenon Flash Lamp
• Factors influencing charged aerosol detector performance:  Getting the Most Out of your Charged Aerosol Detector
• Charged aerosol detector method transfer and optimization:  From Earlier Model Corona Detectors to Corona Veo and Vanquish Charged Aerosol Detectors
• Compendium:  Successful and Stress-Free LC Method Transfers

Short-term (>24 hours to two weeks) shutdown procedure for standard HPLC systems

1. Prepare the column for storage. Refer to the column’s operating manual for appropriate storage conditions
2. Remove the columns from the system and plug the inlet and outlet ports to prevent the column material from drying out
3. Connect the LC tubing with an appropriate connector (e.g., Viper union)
4. Flush the system with a mixture of 10% methanol or acetonitrile in 90% deionized water mixture for at least 30 minutes at a flow rate of 1 mL/min
5. Remove all samples from the autosampler racks
6. Check waste containers and empty if required
7. Turn off all system components

Prolonged (two weeks or more) shutdown procedure for standard HPLC systems

1. Prepare the column for storage. Refer to the column's Operating Manual for the appropriate long-term storage conditions
2. Remove the columns from the system and plug the inlet and outlet ports to prevent the column material from drying out
3. Connect the LC tubing with an appropriate connector (e.g., Viper union)
4. Flush the system with a mixture of 10% methanol or acetonitrile in 90% deionized water mixture for at least 30 minutes at a flow rate of 1 mL/min
5. At a minimum, remove all reservoirs containing aqueous solvents, preferably all reservoirs containing organic solvents as well
6. Flush all solvent lines of the system with 100% isopropanol or methanol for at least 10 minutes at a flow rate of 1 mL/min
7. Remove all samples from the autosampler racks
8. Empty all waste containers
9. Turn off all system components

Shutdown procedures for nano flow LC systems

• Refer to the respective operating manual

General shutdown procedure for transport of LC systems and devices

1. Run the procedure for a prolonged shutdown that applies to your LC system (see above)
2. Make sure the system is thoroughly flushed for decontamination, especially if it has been in contact with toxic, flammable, corrosive, or radioactive substances
3. For Vanquish only: If a single LC device needs to be exchanged or repaired, refer to the operating manual in order to only remove its slide-in unit
4. Move the autosampler needle unit into service position and secure it for transportation
5. Turn off all LC devices with their main power switch
6. Remove all solvent reservoirs and solvent lines from the solvent rack
7. Remove all flow connections between the LC devices and all waste lines
8. Swap an analytical detector flow cell for a transport or dummy flow cell
9. Close open LC tubing (e.g., Viper capillaries, solvent lines) with appropriate end caps and open ports (e.g., valves, flow cells) with appropriate blind nuts
10. Remove all signal cables and power cords
11. Make sure all LC devices are secured and prepared for transportation as required (e.g., detector optical bench is secured, autosampler racks are removed, etc.)
12. For Vanquish only: Make sure the Vanquish System Base is unlocked for any LC system movement on the workbench
13. Depending on configuration and situation, remove solvent rack, detector, column compartment, autosampler, and pump as described in each operating manual
14. Refer to respective manuals for detailed and case-specific information on how to further prepare a specific LC device for transport or shipment