From HPLC to UHPLC: which components need to change?
Pressure, particle size and dead volume when switching to ultra-high performance
Switching from HPLC to UHPLC delivers faster analyses and better separation, but requires more than just a new column. UHPLC works with much smaller column particles and therefore at considerably higher pressures. All components in the flow path must be rated for this.
This article shows which components differ between HPLC and UHPLC, why these adjustments are technically necessary, and which mistakes are most often made in practice during the transition.
⏱ approx. 7 minutes reading time
Why UHPLC is more demanding
UHPLC uses column particles of approximately 1.7 to 2 micrometres instead of the 3 to 5 micrometres of classic HPLC. This has three direct consequences:
- Efficiency increases: more theoretical plates per column length, and therefore more separating power.
- The optimal flow velocity shifts to higher values, allowing faster analyses.
- The back pressure rises sharply, because smaller particles offer more resistance to the liquid flow.
This third consequence is the reason that all components must be reviewed. Whereas classic HPLC works up to approximately 400 bar, UHPLC goes towards 1,000 to 1,500 bar.
Component comparison: HPLC versus UHPLC
| Component | HPLC | UHPLC |
| Column particles | 3 to 5 micrometres | Approx. 1.7 to 2 micrometres |
| System pressure | Up to approx. 400 bar | Up to approx. 1,000 to 1,500 bar |
| Pump | Lower pressure, standard seals | High-pressure pump with reinforced seals |
| Injection valve / autosampler | Suitable for lower pressure | High-pressure resistant, very accurate injection |
| Capillaries and fittings | Larger internal volume acceptable | Very small internal volume; minimal dead volume |
| Detector cell | Larger cell volume possible | Smaller cell volume to counter extra-column broadening |
| Column oven | Standard temperature control | Faster and more accurate temperature control |
The three requirements for every component
When switching to UHPLC, every component in the flow path must meet three conditions.
1. Withstand higher pressure
The pump, injection valve, column connections and fittings must safely withstand the system pressure. PEEK fittings suitable up to approx. 400 bar are no longer sufficient at UHPLC pressures. Stainless steel fittings with matching ferrules are then necessary.
2. Cause less dead volume
The gain from smaller particles is immediately lost if the system has too much extra-column volume. With UHPLC using narrow columns (2.1 mm), the total dead volume must remain below a few microlitres. This calls for capillaries with a small internal diameter, correctly mounted fittings and a small detector cell.
3. Respond faster
UHPLC peaks are narrower and pass through the detector in a shorter time. The detector must therefore have a higher data sampling rate and a lower time constant. A detector with a slow response distorts the peak shape, no matter how well the column performs.
The most common mistake when switching
Installing a UHPLC column in a classic HPLC system. The column does deliver higher efficiency, but the system cannot reach the optimal flow because of its pressure limit, and the existing dead volume destroys the gain in peak shape. The result is an expensive column that performs barely any better.
The 6-port injection valve: why the design matters
The injection valve determines the shape of the sample band entering the column. A 6-port valve works with a rotating rotor that switches between two positions.
Load position: filling the sample loop
The mobile phase goes directly from the pump to the column. The sample loop is disconnected from the mobile phase flow. The sample is introduced into the loop via the injection port; excess sample leaves the loop via the waste port. The loop now contains an exactly known volume.
Inject position: injecting the sample
The rotor turns approximately 60 degrees, changing the liquid routes. The mobile phase is now passed through the sample loop and carries the sample to the column. The sample arrives as a compact plug.
Why six ports? The six ports make it possible to simultaneously let the mobile phase flow in and out, connect the column, connect the sample loop and connect the syringe port to waste. The rotor forms the correct connections between these ports internally.
Effect on your chromatography
A properly functioning injection valve gives a narrow and reproducible injection band, limits extra-column peak broadening and prevents variation in retention times and peak areas. A worn rotor seal is a common but easily overlooked cause of poor reproducibility.
Frequently asked questions about switching to UHPLC
Can I transfer my existing HPLC method directly to UHPLC?
Not one-to-one. You need to scale the method: the ratio between column length, particle size, flow and injection volume must be adjusted to maintain the same separation with a shorter analysis time. Many manufacturers offer calculation models for method scaling.
Can PEEK fittings be used with UHPLC?
For the low-pressure zone (reservoir to pump, after the detector), PEEK fittings remain perfectly usable. For the high-pressure zone between pump and column, stainless steel fittings are necessary because of the pressure. Some reinforced PEEK variants reach higher pressures, but always check the specification.
Why does my UHPLC system perform worse than expected?
The most common causes are too much extra-column volume, a detector cell that is too large for the column diameter, too low a data sampling rate, or capillaries with too large an internal diameter. Check the entire flow path from injector to detector.
