Is high system volume causing broad peaks in your LC?
7 Dec 2023If you want to speed up the LC analysis or save solvent, try the following changes to your column:
- Select a smaller inner diameter (ID). This saves solvent due to the lower flow rate.
- Take a shorter column with smaller particles (the ID is usually reduced as well). This offers better separation performance, so a shorter column is usually sufficient.
- A faster flow rate is possible, accelerating analysis.
- Take a core-shell column.
- This creates sharper peaks, meaning a better separation performance without reducing particle size.
- A faster flow rate is possible, accelerating analysis.
If this reduces the column volume, the extra (pre) column volume (everything between injector and column, often wrongly called “dead volume”) comes into play at some point.
- Did you not get the hoped-for sharp peaks and separation performance on an older HPLC instrument and found that the peaks remained broad?
- This could be caused by the system (dead) volume. The example below shows what a difference it can make if you change the system volume (+ 22µL) in front of the column:
This shows...
- that, in reducing the column volume (rough rule of thumb: < 100 mm length, < 3 mm ID), the extra (pre) column volume starts playing a detrimental role in peak broadening.
- The effect is mainly seen in isocratic methods, as gradient elution can compensate some of the effect due to the band focusing on the column head. However, early eluting substances are susceptible to peak broadening even here.
- Generally, you cannot benefit from using modern, short, thin columns with small particles on an older system that has not yet been optimized for dead volume.
- However! For these oldies, you can use 5μm core-shell columns, e.g. with 3 mm ID and 100-150mm length. Due to the special structure of the particles (hard core, porous shell), it is possible to achieve separation efficiencies comparable to a fully porous 3μm particle which is usually a significant improvement.
However, this also shows that you need to be careful when installing capillaries between the injector and column. If you accidentally get a wrong capillary with a larger ID, this can result in broader peaks. In the example above, the higher system volume was simply achieved by installing an additional piece of capillary.
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- 22 μL of additional system volume can be achieved e.g. by installing approx. 10 cm of tubing with an ID of 0.5 mm (0.02") or approx. 40 cm of tubing with an ID of 0.25 mm (0.01").
- The ID of a capillary has a stronger influence on its volume then the length, as the radius is squared in the volume, the length only single (V = p r² L). Doubling the length of a capillary doubles its volume, doubling the ID quadruples its volume.
- Consequently, the ID of the connecting capillaries should be kept as small as possible, but not TOO small. It must be well balanced between practicability and performance.
- Please consider that the thinner the connecting capillaries are, the more easily they can be clogged, raising the backpressure.
- Working with UHPLC instrumentation means working with thin capillaries and high pressures. On a classic HPLC system with long columns with 5μm particles and 4.6 mm ID, you are not used to working as cleanly or particle-free as in UHPLC. Then problems with clogged capillaries and excessive pressure are more frequent.
- In LC, connection capillaries with ID 0.13 mm (0.005") or 0.18 mm (0.007") are used most frequently, followed (at a considerable distance) by 0.25 mm (0.01").
- Please consider that the thinner the connecting capillaries are, the more easily they can be clogged, raising the backpressure.