We provide you with interactive HPLC introduction documents for your self-study. Our HPLC familiarization documents cover basic introduction and operation of our HPLC instruments, ensuring you have a positive start-up experience. Incomplete HPLC familiarization can lead to improper use of the instrument, so make sure your team successfully completes the tutorial. This introduction neither includes nor replaces any application, technical, or specialist training.​

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Thermo Scientific UltiMate 3000 HPLC & UHPLC Familiarization

Get an overview of UltiMate 3000 Sytstems​, best practices​, pumps, autosamplers, column compartments, optical detectors​, Corona Veo Detectors, and accessories.​

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Proper solvent quality, preparation, and handling are key to successful HPLC operation and results. Optimal system equilibration, handling, and preventative inspection are key to reproducible and reliable HPLC results. The recommendations describe best practices for using a standard LC instrument in general. For best practices for a specific HPLC instrument, refer to the associated familiarization document, as shown above, or to its operating manual.

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

Use only high purity solvents and additives without impurities and prevent any contamination

  • Particles entering the HPLC system can block capillaries and valves, increase wear, and damage the HPLC column or system
  • Especially with aqueous solvents, algae and other microorganisms can grow and deposit in the system and block the solvent line filters
  • Blocked capillaries and filters can cause increased or unstable system pressure
  • Consider that most additives and even bottled water have an expiry date after being opened
  • Consider possible reactivity of impurities with sample substances

Use only fresh, degassed and properly prepared solvents​ and solutions

  • Avoid the use of detergents when cleaning glassware (consider UV-absorbance of detergents)
  • Pay attention to the cleanliness and compatibility of the equipment used for solvent preparation, e.g., organic solvents can leach plasticizers and other chemical agents from low-quality plastic pipettes
  • When preparing salt solutions or buffers and preparation is complete, use membrane filtration (0.2 µm) to remove any particulate matter and to reduce microbial growth
  • To reduce the growth of algae, use amber glassware and use appropriate additives, for example, formic acid, for aqueous solvents

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
Incomplete solvent mixing is observed as high detector baseline noise in the chromatogram which reduces the limit of detection. With sufficient solvent mixing, in turn, no detector baseline ripples are detectable. The limit of detection is preserved.

Before filling a solvent reservoir, rinse the reservoir thoroughly with a high-purity solvent

  • Replace solvents at least on a weekly basis and, always, replace the solvent in the reservoir completely
  • Never add-up solvent to a reservoir as this can lead to solvent composition change and contamination
  • Never recycle or recirculate solvent to ensure piston seal life and avoid particulate matter in the system
  • Consider UV-absorbance of impurities​ in solvents and additives
  • Consider that trifluoroacetic acid or formic acid absorbs at 210 nm
  • Consider special properties of the solvents, such as viscosity, miscibility, boiling point, UV absorption, as well as the solubility of salts for all proportions during the gradient

In non-passivated vials and glassware, significant amounts of ionic leachables like borate, silicate, sodium or unknowns may be present (detected here with a charged aerosol detector).These ionic substances may react with sample analytes and may alter the analytical result in several ways.

Use only the appropriate solvent line filters as recommended by Thermo Fisher Scientific

  • Check the filter frits for permeability at regular intervals and replace them, as necessary
  • To avoid the formation of air bubbles in liquid lines, always place reservoirs in the solvent rack on top of the system stack
  • Rinse glassware used for charged aerosol detection or LCMS applications (including graduated cylinders) with LCMS grade solvents prior to use; it is best to store such glassware separately
  • When using water from water purification systems, all kinds of contamination can occur if the purification system is not properly and regularly maintained

Always use fresh solvents and replace them at regular intervals

  • With premixed solutions, be especially sure that they are properly prepared, filtered and fresh
  • Before filling a solvent reservoir, rinse it thoroughly with the solvent or solution to be stored
  • Always close open holes in reservoir caps with appropriate plugs
  • Always place the solvent containers in the solvent rack on top of the system to avoid air bubbles forming in the liquid lines
  • When changing to a different type of solvent, make sure that previous and new solvent are miscible
  • Before changing from a buffer or salt solution to organic solution, or vice versa, flush the system thoroughly with de-ionized water for about 10 minutes with at least 200 bar system backpressure
  • Purge the pump and then flush the solvent lines thoroughly with the new solvent
  • Ensure that solvent and seal wash lines are not kinked, pinched or squeezed anywhere in the flow path

Recommendations on HPLC vials, caps and well plates

  • Use vials, caps and well plates as required by the application, for example, LCMS-certified vials for charged aerosol or mass spectrometry detection
  • Do not use vial caps or well plate seals that are likely to result in material being punched out by the autosampler needle
  • Only use talcum-free, unfilled silicone or PTFE-based vial septa or well plate cover mats to prevent particles that may cause backpressure issues or blockages in the system
  • Do not use sealing and resealing tape with adhesive in the cavity area to avoid contamination, especially with the injection needle
  • Do not use aluminum sealing tape to prevent aluminum particles from being punched out and, e.g., get stuck in the needle seat
  • It is best to follow this selection help:  SureSTART Collection - Vials and well plates

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

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
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Vanquish 3D Product Tours

HPLC and UHPLC instruments are complex systems composed of multiple components. Are you curious to see what our Vanquish HPLC/UHPLC Systems look like on the inside?

Our interactive 3D LC product tours and virtual demos help you explore these systems in a self-guided manner. Embedded in each tour are videos and other detailed descriptions of key features and capabilities.

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Looking for enhanced resolution, speed, and consistency in your chromatography? Our extensive family of products offers a variety of LC columns to meet all separation needs, including improved resolution, enhanced sensitivity, faster analysis and consistent performance. We also focus on sample security and integrity. You'll find the right vials and closures to match your application, autosampler, and budget from our comprehensive portfolio.

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