When everyone first started working with liquid chromatography, I wonder if anyone else had the same question as me: why are the inner diameters of chromatography columns such odd, fragmented numbers like 4.6mm or 2.1mm, instead of more pleasing, rounded numbers like 5mm or 10mm?
As everyone may have noticed, most chromatography columns on the market have these strange inner diameter specifications. So… why is that exactly?
When learning the fundamental theory of liquid chromatography, we always hear a well-known name: Van Deemter curve. This curve has become one of the most classic theories in liquid chromatography separation.
The Van Deemter equation in the following figure:
The first term represents eddy diffusion,
The second term represents molecular diffusion,
The third term represents mass transfer resistance.
- The horizontal axis represents linear velocity.
- The vertical axis represents theoretical plate height.
The column efficiency increases with the flow rate until it reaches a maximum value, after which it decreases with increasing flow rate. The flow rate at which the chromatographic column achieves its maximum efficiency is called the optimum flow rate. The flow rate range in which the actual column efficiency is very close to the optimum efficiency is called the optimum flow rate range.
The linear velocity refers to the speed at which the chromatographic peak or band moves and is typically measured in cm/min. On the other hand, the flow rate refers to the volumetric flow rate of the liquid passing through the chromatography system and is typically measured in mL/min.
The conversion relationship between linear velocity and flow rate is given by the formula: volumetric flow rate = (linear velocity/60) × (π×d2/4), where d is the inner diameter of the chromatography column in cm. For a 5 μm particle size chromatography column, the optimal linear velocity is 1 mm/s (6 cm/min), which corresponds to a column flow rate of 1 mL/min for a 4.6 mm inner diameter column. This is why the recommended optimal flow rate for 4.6 mm inner diameter columns is 1 mL/min. For 3.0 mm, the corresponding flow rate is 0.4 mL/min, for 2.1 mm it is 0.2 mL/min, and for 1.0 mm it is 0.05 mL/min. To facilitate the input of integer flow rates when using liquid chromatography, the inner diameters of chromatography columns on the market are not integers.
From the van Deemter equation, it can be inferred that the smaller the particle diameter of the chromatographic packing material, the higher the column efficiency (i.e., the smaller the plate height). Each particle size corresponds to an optimal linear velocity, at which the highest column efficiency can be achieved.
The graph shows that for particle diameters below 2μm, the optimal speed range allowed for the highest efficiency is greatly expanded. This means that faster flow rates can be used without sacrificing efficiency. When using a 1.7μm particle size chromatography column (UPLC column), the column length can be reduced to one-third of a conventional HPLC column while achieving the same efficiency. Compared to HPLC, shorter separation times can be achieved at the same efficiency and separation power, or higher efficiency and separation power can be achieved at the same separation time.