We call the introduction of our UHPLC and HPLC instruments to new operators a familiarization. It includes an LC instrument overview, guidelines for basic operation, best practices, maintenance, and much more.
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.
Best practices for HPLC & UHPLC instrument operation
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
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Contaminated check valves, e.g., through particles from solvent reservoirs, cause unstable system pressure
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Low-quality solvents typically lead to a certain detector baseline drift. With high-quality solvents, in turn, no detector baseline drift from solvent quality effects is detectable
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
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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
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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
For information about chromatography data software, mass spectrometry, and our training resources, visit HPLC Training & Education.
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.
HPLC columns, vials, caps, and septa selection guides
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.
LC Column selection guide
Take the challenge out of finding the correct or equivalent column. This guide will take you directly to the right product so you can start your method today.
We offer help with product selection and ordering, product applications and use, instrument support, field service fulfillment, depot repair, or finding the dealer or distributor for your product of interest.