HPLC vs UHPLC (UPLC): what changes at 2.1 mm and sub-2 µm

First, the names. UPLC is a registered trademark of Waters. Agilent, Thermo and Shimadzu call the same technology UHPLC. This guide uses UHPLC, but everything here applies to a Waters ACQUITY as much as to an Agilent 1290, a Thermo Vanquish or a Shimadzu Nexera.

Agilent’s LC Handbook gives a clear definition. UHPLC is liquid chromatography above 400 bar, which was the usual maximum for HPLC systems for decades, with columns of particles smaller than 3 µm.

When you move from HPLC to UHPLC, two things are obvious: the runs are shorter and the pressure is higher. The rest of this guide is about the less obvious changes.

Why smaller particles need more pressure

A packed column separates better when the particles are smaller. The cost is pressure.

The Agilent handbook gives the pressure equation. Pressure rises with solvent viscosity, flow rate and column length. It falls with the column radius squared and the particle diameter squared. So at the same flow, half the particle size means four times the pressure.

That’s why UHPLC needed new pumps, injectors and fittings. The chromatography is the same.

Maximum pressure by system

From each vendor’s own documents:

Two things stand out. Some ratings depend on flow rate, like the Agilent 1290’s. And the line between “HPLC” and “UHPLC” isn’t sharp. Shimadzu’s page even says which particles each Nexera is for: sub-2 µm to 3 µm on the X3 and XS, 2 to 3 µm on the XR, above 3 µm on the lite.

The lowest-rated part sets your limit

A system is only as high-pressure as its weakest part. Agilent’s specification compendium shows this on one sheet. The 1290 multisampler is rated to 1300 bar, but with the large 900 µL injection head it’s limited to 400 bar.

Columns have their own rating too. Waters rates its ACQUITY columns to 1241 bar (18,000 psi), higher than the classic ACQUITY system itself.

The detector flow cell sits after the column, so it only sees the pressure after the column. But its limit is low. Agilent rates the 1290 DAD cartridge cells at 70 bar. Waters rates the ACQUITY flow cells at 69 bar, and warns not to connect anything after the cell that could push it over that.

Before you run a method on a system, check it in this order:

flowchart TD
    B(["Under the pump rating?"]) -->|"Yes"| C(["Under the injector rating?"])
    C -->|"Yes"| D(["Under the column rating?"])
    D -->|"Yes"| E(["Line after the detector clear?"])
    E -->|"Yes"| F["It can run"]
    B -->|"No"| X1["Won't run as written"]
    C -->|"No"| X2["Won't run as written"]
    D -->|"No"| X3["Won't run as written"]
    E -->|"No"| Y["Clear the outlet first"]

Check the pump rating at your flow rate, the injector with the head that’s fitted, and the flow cell’s own limit, about 70 bar.

The column holds very little liquid

A 2.1 mm column is small inside. The Waters column manual gives these empty volumes:

Waters’ H-Class guide puts a 4.6 × 150 mm HPLC column at about 2.5 mL. That’s more than ten times a 2.1 × 50.

Three things follow.

Use less injection volume. 20 µL is a small fraction of a 4.6 mm column. On a 0.2 mL column it’s a large fraction, and the peak shape shows it.

Tubing matters more. The Agilent handbook says the extra-column volume, everything outside the column, shouldn’t be more than 10% of the column volume. On a 4.6 mm column that’s easy. On a 2.1 × 50 mm column every short length of tubing counts.

Equilibration is quick, but check it. Waters asks for at least 10 column volumes of the new mobile phase. On a 2.1 × 50 that’s about 2 mL, or 4 minutes at 0.5 mL/min. Ion-pairing mobile phases can need 100 to 200 column volumes, and formate mobile phases can take longer.

Everything has to be cleaner

Smaller particles need smaller frits at each end of the column, and those frits trap any particles that get into the system. Both Agilent and Waters say to filter aqueous mobile phases through 0.2 µm for UHPLC. Agilent says 0.45 µm is fine for standard HPLC.

Agilent also recommends certified HPLC/MS grade solvents for UHPLC, and replacing buffers about every 2 days.

Buffers need one more step

Before you put a buffered mobile phase on the column, flush it with water and organic, at the same or lower organic content as the buffered mobile phase. The Waters column manual’s example: flush with 60% methanol in water before you switch to 60% methanol with 40% buffer.

Buffer salts dissolved in water can come out of solution when they meet a lot of organic solvent. Salt in a sub-2 µm packed bed is expensive, and it usually gets blamed on the column.

Example: starting a new column on a Waters ACQUITY

Each vendor has its own procedure. This one is from the Waters column manual. Check your own column’s instructions.

1. Purge any buffer out of the pump. Then connect the column inlet to the injector.
2. Flush with 100% methanol or acetonitrile. Start at 0.1 mL/min and go up to 0.5 mL/min over 5 minutes.
3. When solvent flows freely out of the column, stop the flow and connect the outlet to the detector. This keeps air out of the detector.
4. Bring the flow up slowly again. Wait for steady pressure and a steady baseline before you start.

Same parts, different names

If you move between vendors, the module names change:

The software doesn’t always match the hardware. I’ve worked on an Agilent 1200 stack running under Waters Empower.

If you’re deciding between them

If your system is an HPLC and the method is a UHPLC method, the column isn’t the problem. The pressure limit of the system is. A 2.1 mm, sub-2 µm column on a 400 bar system either runs at a flow rate that wastes the small particles, or it doesn’t run.

The other direction works. A UHPLC runs an HPLC column. The Waters operator’s guide says to set the pump’s high-pressure limit to the column’s rating, and to expect retention times to shift because the system volume is smaller.

Sources

• Agilent, The LC Handbook: the UHPLC definition (p. 31), the pressure equation (p. 10), extra-column volume (p. 44), filtration, frits and solvents (p. 116).
• Agilent InfinityLab LC Series Specification Compendium, manual 01200-90062 (2016): 1290 and 1260 pump and multisampler pressure ratings, 1290 DAD flow cell rating.
• Thermo Scientific Vanquish Pumps, product specifications 73056: Vanquish Horizon, Flex and Core pressure ratings.
• Shimadzu Nexera Series specifications page (shimadzu.com): Nexera pressure ratings and the particle sizes each model is for.
• Waters ACQUITY UPLC System Operator’s Guide, 71500082502 rev F: the system rating, the UPLC trademark, running HPLC columns on an ACQUITY.
• Waters ACQUITY and ACQUITY Premier Columns Care and Use Manual, 720008980: column rating, column volumes, equilibration, the buffer flush and the start-up steps.
• Waters ACQUITY UPLC H-Class System Guide, rev A: the 4.6 × 150 mm column volume.
• Waters ACQUITY UPLC PDA and eλ PDA Overview and Maintenance Guide, 715002209: the 69 bar flow cell rating.

Checked 3 October 2026. Ratings change between models and years, so check the specification for your own system.

---

Chromkit sells parts for HPLC and UHPLC systems from several vendors, and has two refurbished Waters ACQUITY UPLC systems in stock, one with a PDA detector and one with a TUV detector. The columns are here.