Some years ago I was a young university researcher having fun developing new LC-MS methods. I worked with passion and enthusiasm and did the best I could with my LC instruments, but I always avoided UHPLC methods.Now you may think “UHPLC is great! What happened to Giorgia? She must have been traumatized,” and actually you may be right. But before going into the details, let me give some background information to help you understand my reasons.
UHPLC (or Ultra High Performance Liquid Chromatography) is a sub-category of HPLC and is a separation technique based on the use of sub-2 μm particle stationary phases in conjunction with a low dispersion HPLC system capable of >600 bars (9000 psi) pressure. Stationary phase particle size is one of the most important factors in the van Deemter equation to increase column efficiency. For a given column length (L), the plate number (N) is inversely related to the particle size of the column packing. The smaller the particles, the higher the plate number and separation power.
N = c (L/dp)
Where N is the plate number, dp is the diameter the particle, c is a constant and L is the column length.
Since sub-2 μm particles are so small the diffusion path between the analytes and the stationary phase is shorter and the efficiency is higher.
Therefore, LC columns made with small particles can be shorter while maintaining efficiencies comparable to longer columns packed with larger particles. A shorter column means a faster separation can be achieved because separation time is proportional to column length. A shorter column run, at the same linear velocity as a longer column, also uses less solvent.
Another positive consequence of decreasing particle size is that the C-term tends to flatten out at higher linear velocities with minimum shifts toward the right on a van Deemter plot. Thus, small particles (<3 µm) allow for an increase in the flow reducing the analysis time without significantly lowering resolution between the peaks of interest.
Now after all this theory, let’s talk about the practice. What happens when you try to use a sub-2 μm particle column? You guessed it - the pressure will dramatically increase.
The change in pressure is inversely related to the square of the particle size. Smaller particles generate higher pressure that can be estimated based on the square of the ratio of the particle diameters:
∆ pressure = c (dp1/dp2)2
Where dp1 and dp2 are the diameter of the original and new particles, respectively.
You can easily calculate that under the same conditions of mobile phase, flow rate, column diameter and length, moving from 5 µm to sub 2- µm particles requires 6 times higher pressure.
And this is where all my problems started.
Have you ever tried to set up fluidic connections that work at 800-1000 bar or higher? Or worse, let someone with less experience than you try to do it?
At high pressure you typically have to work with metal capillaries with metal ferrules and install them with tools throughout the system including the head of the column. If you’ve had this experience you know you have to screw them just right to get them properly connected – tighten too much and you risk stripping the screw but if you don’t tighten enough you get leaks. More often than I would have liked, the capillary wasn’t tight enough and would start leaking in the middle of the night. The result? All my overnight measurements were gone. Needless to say, installing tubing for a UHPLC set up is an art and difficult in practice.
Now you can understand why I tried to avoid UHPLC.
After my time researching at the university I was introduced to Thermo Scientific™ Viper™ capillaries and my life changed! Have you heard of them? They are fingertight capillaries that are pressure stable up to 1500 bar (22,000 psi).
Viper capillaries make UHPLC possible for everyone – beginners to experts (no need to perfect the ‘art’ of tightening capillaries and metal ferrules). With Viper capillaries you can set up perfect fluidic connections every time without any tools or experience. Watch this video to see how easy it is to connect Viper capillaries.
Viper capillaries are available in different materials, lengths and IDs for analytical, micro, capillary and nano LC separations. With so many Viper options, there is always the right capillary for your application. For more information check the Product Specifications.
Besides Viper capillary being easy-to-use, they are also designed to virtually eliminate dead volumes that can cause peak broadening and negatively impact separation efficiency. See how Viper improves chromatography in this short animation.
Additional Information:
Thermo Fisher Scientific
United States
