Scientists aren’t perfect. Nobody is. In our Technical Tips, we can solve your most debilitating issues—from mechanics to methodology—together.
The only thing more frustrating than discovering a backpressure issue is diagnosing it. Trust me—you’re not alone!
There are three classes of backpressure deviations that most HPLC-users face:
● fluctuating backpressure, which is generally system related
● low backpressure, the most uncommon type of backpressure
● and high backpressure, the most common type of backpressure
A backpressure fluctuation in a repetitive pattern can often be traced to the pump. Usually, an air bubble is the culprit, which means a pump head needs to be purged. The flow will subsequently pulse—typically visible by removing the column, increasing the flow rate, and observing a pattern of eluent pulses that otherwise should be a smooth, streaming flow.
Backpressure fluctuation that is erratic, however, can often be traced to a leak in the system. The injection valve is a common component where a small leak could go unnoticed, as well as the connection to the column if not sufficiently or properly connected with the nut and ferrule.
Under gradient elution, ineffective gradient mixing can also be the source of erratically fluctuating backpressures.
Backpressures significantly lower than what are typically observed are a bit unusual and can often be traced to a simple oversight.
Be sure to check:
● column dimensions – is your internal diameter or particle size too large?
● mobile phase composition – are you using the correct type?
● flow rate – is it set too low?
If you’re running a gradient, you could possibly be pumping from the wrong lines with a higher percentage of organic than initially intended.
Backpressures significantly higher than expected are due to a blockage in the flow path.
If the backpressure is high upon initial installation prior to any injections, find the source of the blockage in the system and purge. If the column is found to be the source, examine the connective tubing and fittings to ensure a proper fit has been applied. Additionally, it is useful to know what solvents were run through the system previously, and ensure that incompatible solvents and buffers were thoroughly flushed out of the system lines.
If the backpressures increase upon successive sample injections, something from the sample is likely fouling the column inlet. For method development cases where the expectations have not been thoroughly characterized, a guard column can be particularly useful to protect the analytical column from fouling through unknowns, such as precipitation or chemical attack.
If the backpressure increases unexpectedly during a routine analysis that has already been thoroughly characterized, examining chromatographic symptoms associated with the increase in backpressure can be a clue as to the likely root cause.
Good luck, and happy analyzing!
More technical support:
• Request-a-Method™: Contact our Method Development Team for a tailored solution!
• Applications: We have thousands of them! Search by analyte name or structure.
• Free Guides and Posters: Download free guides and posters for troubleshooting, method development, and more.
• Product Videos: See the technology in action. Watch our product videos and demonstrations.
• The Technical Library: Check out our helpful collection of technical notes, posters, webinars, how-to videos, guides, and more.
• Interactive Web Tools: Simple, interactive tools to instantly find the right products and solutions.
• FAQ Portal: Find answers to popular technical questions here.
• Quality and Safety Documents: Download your CQA, C of A, and MSDS.
• Product Care and Use: Love your products and treat them right using these handy guides.
• Training & On-Site Support: We’ll come to you! Classroom training or in-lab demonstrations—you pick the topic.
• Troubleshooting: Did something go wrong? We’ll fix it!
• Chat With Us: Our Phenomenex technical experts (Phenoms) are ready to chat with you about method development and technical support.
• Better UHPLC Performance Starts NOW with Kinetex 1.7µm Core-Shell Columns [Brochure]
• Intact IgG Separations by HPLC/UHPLC
• Kinetex Care and Use Notes
• HPLC Column Care Guide
• HPLC Troubleshooting Guide