The question “What is the difference between running green stainless vs. red stainless HPLC tubing at 0.3 mL/min?” arose from the video HPLC Tubing and Fittings.
In this video I explored the differences between stainless steel and PEEK tubing, as well as the importance of choosing the right fittings and tubing diameter for optimal efficiency.
I explained that the choice of tubing diameter plays a crucial role in system efficiency. Green tubing (0.17 mm ID) is commonly used for standard flow rates. Red tubing (0.12 mm ID) is preferred for low-flow applications, such as LC-MS, to minimize peak broadening and efficiency loss.
This brings us to the core question: what is the functional difference between running green vs. red stainless steel tubing at 0.3 mL/min?
Green vs. Red Stainless Steel Tubing at 0.3 mL/min
In the Agilent color-coded tubing system, green tubing (0.17 mm ID) is the standard choice for most HPLC applications, especially at flow rates between 1.0 and 2.0 mL/min. At these flow rates, green tubing is optimal, and there is no benefit in switching to a smaller diameter. However, when operating at very low flow rates—such as 0.3 mL/min—green tubing is too large. This will lead to unnecessary dead volume and reduced efficiency.
At low flow rates, mobile phase velocity decreases, resulting in slower sweeping of the tubing. This causes dispersion and broadening of peaks, which negatively impacts resolution. Red tubing (0.12 mm ID) offers a smaller internal volume, reducing dispersion and preserving efficiency at low flow rates.
When to Switch to Red Tubing
If you are running at 0.3 mL/min, especially with a 2.1 mm ID column for LC-MS applications, I recommend switching to red tubing. To optimize performance:
- Replace all green tubing with red tubing throughout the system—from the injector to the detector.
- Minimize the length of tubing wherever possible to reduce extra dead volume.
- Ensure proper fittings—whether using stainless steel or PEEK—to maintain a good connection and prevent leaks.
For Non-Agilent Systems
If you’re not using Agilent equipment but still need to apply the same principle, follow these general guidelines:
- Replace 0.010” ID tubing (equivalent to green tubing) with 0.005” ID tubing (equivalent to red tubing).
- Shorten tubing lengths to improve system efficiency and reduce unnecessary internal volume.
Conclusion
At standard flow rates (1.0–2.0 mL/min), green stainless steel tubing is perfectly fine. However, when reducing flow rates to 0.3 mL/min, switching to red stainless steel tubing is essential to maintaining efficiency and minimizing peak broadening. If you’re running LC-MS at low flow rates, making this simple tubing adjustment will ensure sharper peaks and better system performance.
