Why Pressure Is Driving Liquid Chromatography Technology

[AnalyteGuru_KBA]

The word pressure is among scientific terms used in our daily life. At least once in life, you have pumped up a ball or balloon. Atmospheric pressure is a weather indicator, and as a car owner, you regularly check tire pressure. You feel the pressure build up in your ears while a plane ascends (which does not happen often nowadays due to quarantine and travel restrictions between countries), hiking in mountains, or diving deep in the water.

In the modern fast-paced world, we use the term “under pressure” immortalized by Queen and David Bowie¹­ to describe situations when somebody is trying to make you do something or a difficult situation that makes you worried or unhappy².

The fact is, beautiful diamonds formed from carbon under extreme pressure (ca. 50K bars) and temperature (ca. 1000 °C)³ make the relationship between pressure and diamonds a popular motivational and inspiring meme. So how did pressure become the driver for the technology development in one of the most used and powerful analytical techniques—liquid chromatography (LC).

After it was fully proven ca. 60-70 years ago that the efficiency of analyte separation in liquid chromatography is drastically improved by using smaller particles, the LC systems with gravity-driven solvent flow requiring hours to complete were transformed by engineers into high-tech instruments.

First, high-performance liquid chromatography (HPLC) pushing liquid at several hundred bars became a mainstream analytical technique. Now ultra-high performance liquid chromatography (UHPLC) can handle above 1000 bar backpressure and is a routine analytical method for many applications. Just imagine, 1000 bar (14503 psi) is ca. 1000 times more than atmospheric pressure and equals the pressure at the deepest oceanic trench on Earth—Mariana Trench⁴.

Now the questions come. Why do we need extreme pressure capabilities in liquid chromatography? What are the benefits of developing ultra-high pressure analytical-flow liquid-chromatography systems like Thermo Scientific™ Vanquish™ Horizon UHPLC System with industry-leading 1500 bar (22.000 psi) pumping performance or nano-flow Thermo Scientific™ EASY™-nLC 1200 system with 1200 bar and Intelligent Flow Control (IFC™) for constant pressure pump operation?

Here are examples of what ultra-high pressure permits:

• Smaller particles. The use of columns packed with small particles boosts efficiency and improves resolution. In theory, the same factor connects particle size and column length, so two times smaller particles give the same resolution as a column with double length. In practice, this means you reduce the analysis time while keeping a similar resolution using a shorter column


• Longer columns. The increase in column length promotes higher efficiency and sharper peaks. This feature is critical to achieving low detection and quantification limits and profiling complex samples in nanoLC-MS, pushing the limits of bottom-up proteomics with 75 cm long column⁷


• Smaller internal column diameter. The decrease in column internal diameter brings an exponential increase in sensitivity for concentration sensitive detectors⁶ and opens the door for ultra-sensitive analysis to the level of single cells


• Higher linear velocity. Increasing the flow while keeping dimensions of a column packed with small particles reduces analysis time and enables high-throughput separations, even for nano-, and capillary-flow LC⁷. Additionally, accelerated sample loading and column equilibration at maximum available pressure allow getting these steps completed much faster even for long columns⁸. This increase minimizes the overhead time and maximizes expensive mass-spectrometry utilization time

So, what is the significance? The revolutionary technology enabling ultra-high pressure capabilities for liquid chromatography brings higher separation performance, lower-solvent consumption, greater productivity, and higher sensitivity to detect and quantify trace compounds.

How will the future look and what will technology advancements bring? Can you imagine the benefits of LC systems with even higher-pressure capabilities?

1. “Queen - Under Pressure (Official Video)” https://youtu.be/a01QQZyl-_I


2. https://dictionary.cambridge.org/dictionary/english/pressure


3. “How diamonds are formed”, Cape Town Diamond Museum https://www.capetowndiamondmuseum.org/about-diamonds/formation-of-diamonds/


4. “The Mariana Trench Is 7 Miles Deep: What’s Down There?”, https://www.scientificamerican.com/article/the-mariana-trench-is-7-miles-deep-whats-down-there/


5. Pushing the Limits of Bottom-Up Proteomics with State-Of-The-Art Capillary UHPLC and Orbitrap Mass Spectrometry for Reproducible Quantitation of Proteomes, AN-639. https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/AN-639-LC-MS-Bottom-Up-Proteomics-A...


6. Capillary-flow LC-MS: combining high sensitivity, robustness, and throughput. TN-72277. https://assets.thermofisher.com/TFS-Assets/CMD/Technical-Notes/TN-72277-LC-MS-Capillary-Flow-TN72277...


7. Tailored high-throughput low-flow LC-MS methods for large sample cohort analysis. TN-73208. https://assets.thermofisher.com/TFS-Assets/CMD/Technical-Notes/tn-73208-lc-ms-large-sample-cohort-an...


8. EASY-nLC 1200. Leading in simplicity and performance. https://assets.thermofisher.com/TFS-Assets/CMD/brochures/BR-64590-LC-EASY-nLC-1200-BR64590-EN.pdf